US20140142028A1

US20140142028A1 - Targeted antimicrobial moieties

Info

Publication number: US20140142028A1
Application number: US13/768,839
Authority: US
Inventors: Randal H. Eckert; Daniel K. Yarbrough; Wenyuan Shi; Maxwell H. Anderson; Jian He; Chris Kaplan; Jee-Hyun Sim
Original assignee: University of California
Current assignee: University of California
Priority date: 2009-02-05
Filing date: 2013-02-15
Publication date: 2014-05-22
Also published as: US8389679B2; US20100316643A1; WO2010091294A9; WO2010091294A2

Abstract

This invention provides novel targeted antimicrobial compositions. In various embodiments chimeric moieties are provided comprising an antimicrobial peptide attached to a peptide targeting moiety that binds a bacterial strain or species.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 12/701,443, filed on Feb. 5, 2010, which claims priority to U.S. Provisional Application No. 61/150,287, filed Feb. 5, 2009; and to U.S. Provisional Application No. 61/224,825, filed Jul. 10, 2009, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

[Not Applicable]

FIELD OF THE INVENTION

The present invention relates to novel targeting peptides, novel antimicrobial peptides, chimeric moieties comprising novel targeting and/or novel antimicrobial peptides and uses thereof.

BACKGROUND OF THE INVENTION

Antibiotic research at the industrial level was originally focused on the identification of refined variants of already existing drugs. This resulted example, in the development of antibiotics such as newer penicillins, cephalosporins, macrolides, and fluoroquinolones.
However, resistance to old and newer antibiotics among bacterial pathogens is evolving rapidly, as exemplified by extended beta-lactamase (ESBL) and quinolone resistant gram-negatives, multi-resistant gonococci, methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant enterococci (VRE), penicillin non-susceptible pneumococci (PNSP) and macrolide resistant pneumococci and streptococci (see, e.g., Panlilo et al. (1992) Infect. Control Hosp. EpidemioL., 13: 582-586; Morris et al. (1995) Ann Intern Med., 123: 250-259, and the like). An overuse, or improper use, of antibiotics is believed to be of great importance for triggering and spread of drug resistant bacteria. Microbes have, in many cases, adapted and are resistant to antibiotics due to constant exposure and improper use of the drugs.
Drug resistant pathogens represent a major economic burden for health-care systems. For example, postoperative and other nosocomial infections will prolong the need for hospital care and increase antibiotic drug expenses. It is estimated that the annual cost of treating drug resistant infections in the United States is approximately $5 billion.

SUMMARY OF THE INVENTION

In certain embodiments, novel targeting moieties (e.g., peptides) that specifically/preferentially bind to microorganisms (e.g., certain bacteria, yeasts, fungi, molds, viruses, algae, protozoa, and the like) are provided. The targeting moieties can be attached to effectors (e.g., detectable labels, drugs, antimicrobial peptides, etc.) to form chimeric constructs for specifically/preferentially delivering the effector to and/or into the target organism. In certain embodiments novel antimicrobial peptides that can be used to inhibit (e.g., kill and/or inhibit growth and/or proliferation) of certain microorganisms (e.g., certain bacteria, yeasts, fungi, molds, viruses, algae, protozoa, and the like) are provided. Any targeting moiety disclosed herein can be attached to any one or more effector disclosed herein. Any targeting moiety disclosed herein can be attached to any one or more antimicrobial peptide disclosed herein.
In certain embodiments chimeric moieties are provided where the chimeric moieties comprise an effector attached to a peptide targeting moiety comprising or consisting of the amino acid sequence of a peptide found in Table 2 and/or Table 15. In certain embodiments the targeting peptide comprises or consists of amino acid or retro or inverso or retro-inverso sequence or beta sequence of a peptide found in Table 2 and/or Table 15. In certain embodiments the effector comprises a moiety selected from the group consisting of a detectable label, an antimicrobial peptide, an antibiotic, and a photosensitizer. In certain embodiments the effector comprises an antimicrobial peptide comprising the amino acid sequence of a peptide found in Table 2, and/or Table 8, and/or Table 9, and/or Table 10. In certain embodiments the effector comprises an antibiotic found in Table 7. In certain embodiments the effector comprises a photosensitizer. In certain embodiments the photosensitizer is selected from the group consisting of a porphyrinic macrocycle, a porphyrin, a chlorine, a crown ether, an acridine, an azine, a phthalocyanine, a cyanine, a psoralen, and a perylenequinonoid. In certain embodiments the photosensitizing agent is an agent shown in any of FIGS. 1-11.
Also provided is a chimeric construct comprising a targeting moiety attached to an antimicrobial peptide where the antimicrobial peptide comprises or consists of the amino acid or retro or inverso or retro-inverso sequence of a peptide found in Table 2. In certain embodiments the targeting moiety is a peptide that comprises or consists of the amino acid or retro or inverso or retro-inverso or beta sequence of a peptide found in Table 2, and/or Table 3, and/or Table 4, and/or Table 6, and/or Table 15. In certain embodiments, the targeting moiety comprises an antibody (e.g., an antibody identified in Table 5). In certain embodiments the targeting moiety is chemically conjugated to the effector directly or via a linker. In certain embodiments the targeting moiety is chemically conjugated to the effector via a linker comprising a polyethylene glycol (PEG). In certain embodiments the targeting moiety is chemically conjugated to the effector via a non-peptide linker found in Table 11. In certain embodiments the where the targeting moiety is linked to the effector via a peptide linkage. In certain embodiments the chimeric construct is a fusion protein. In certain embodiments the linker is a peptide linker found in Table 11. In certain embodiments the chimeric moiety is functionalized with a polymer (e.g., polyethylene glycol, a cellulose, a modified cellulose, etc.) to increase serum halflife.
Also provided are pharmaceutical compositions comprising the chimeric construct(s)/chimeric moieties described herein in a pharmaceutically acceptable carrier. In certain embodiments the composition is formulated as a unit dosage formulation. In certain embodiments the composition is formulated for administration by a modality selected from the group consisting of intraperitoneal administration, topical administration, oral administration, inhalation administration, transdermal administration, subdermal depot administration, and rectal administration.
Also provided is an antimicrobial composition comprising an isolated antimicrobial moiety comprising or consisting of the amino acid sequence of a peptide found in Table 2. In certain embodiments the peptide comprises or consists of the amino acid or retro or inverso or retro-inverso sequence or beta sequence of a peptide found in Table 2. In certain embodiments the peptide is a peptide selected from the group consisting of a peptide consisting of the amino acid sequence of a peptide found in Table 2 comprising all L residues, a peptide consisting of the amino acid sequence of a peptide found in Table 2 comprising a peptide found in Table comprising all D residues, a peptide comprising the inverse of an amino acid sequence found in Table 2, a peptide comprising the retro-inverso form of a peptide found in Table 2, a peptide found in Table 2 comprising a conservative substitution, and a peptide found in Table 2 comprising a substitution of a naturally occurring amino acid with a non-naturally occurring amino acid. In certain embodiments the peptide comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions.
Also provided is a composition comprising an isolated targeting moiety comprising or consisting of the amino acid sequence of a peptide found in Table 2 or Table 15. In certain embodiments the peptide comprises or consists of the amino acid or retro or inverso or retro-inverso sequence or beta sequence of a peptide found in Table 2 or Table 15. In certain embodiments the peptide is a peptide selected from the group consisting of a peptide consisting of the amino acid sequence of a peptide found in Table 2 or Table 15 comprising all L residues, a peptide consisting of the amino acid sequence of a peptide found in Table 2 comprising a peptide found in Table comprising all D residues, a peptide comprising the inverse of an amino acid sequence found in Table 2 or Table 15, a peptide comprising the retro-inverso form of a peptide found in Table 2 or Table 15, a peptide found in Table 2 or Table 15 comprising a conservative substitution, and a peptide found in Table 2 or Table 15 comprising a substitution of a naturally occurring amino acid with a non-naturally occurring amino acid. In certain embodiments the peptide comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions.
In certain embodiments methods are provided for inhibiting the growth and/or proliferation of a microorganism and/or a biofilm comprising a microorganism. The methods typically involve contacting the microorganism or biofilm with a composition comprising an antimicrobial peptide comprising or consisting of the amino acid sequence of a peptide found in Table 2 (e.g., the amino acid or retro or inverso or retro-inverso sequence or beta sequence of a peptide found in Table 2); and/or contacting the microorganism or biofilm with a composition comprising an antimicrobial moiety attached to a targeting peptide comprising or consisting of the amino acid sequence of a peptide found in Table 2 (e.g., the amino acid or retro or inverso or retro-inverso sequence or beta sequence of a peptide found in Table 2). In certain embodiments the microorganism or biofilm is a bacterium or a bacterial film. In certain embodiments the targeting peptide is chemically conjugated to the antimicrobial peptide. In certain embodiments the targeting peptide is linked directly to the antimicrobial peptide. In certain embodiments the targeting peptide is linked to the antimicrobial peptide via a linker comprising a polyethylene glycol. In certain embodiments the targeting peptide is linked to the antimicrobial peptide via a non-peptide linkage in Table 11. In certain embodiments the targeting peptide is linked to the antimicrobial peptide via a peptide linkage. In certain embodiments the targeting peptide linked to the antimicrobial peptide is a fusion protein. In certain embodiments the linker is a peptide linker in Table 11.
In various embodiments methods are provided for detecting a microorganism (e.g., bacteria, yeast, protozoan, virus, algae, fungi, etc.) or biofilm comprising the microorganism. The methods typically involve contacting the microorganism or biofilm with a composition comprising a detectable label attached to a targeting peptide comprising the amino acid sequence of a peptide comprising or consisting of the amino acid or retro or inverso or retro-inverso sequence of a peptide found in Table 2; and detecting the detectable label where the quantity and/or location of the detectable label is an indicator of the presence of the microorganism and/or biofilm film. In certain embodiments the microorganism or biofilm is a bacterium or a bacterial film. In certain embodiments the detectable label is a label selected from the group consisting of a radioactive label, a radio-opaque label, a fluorescent dye, a fluorescent protein, an enzymatic label, a colorimetric label, and a quantum dot.
In certain embodiments compositions are also provided comprising a photosensitizing agent attached to a targeting peptide where the targeting peptide comprising or consisting of the amino acid or retro or inverso or retro-inverso sequence of a peptide found in Table 2 or Table 15. In certain embodiments the photosensitizing agent is an agent selected from the group consisting of a porphyrinic macrocycle, a porphyrin, a chlorine, a crown ether, an acridine, an azine, a phthalocyanine, a cyanine, a psoralen, and a perylenequinonoid. In certain embodiments the photosensitizing agent is an agent shown in any of FIGS. 1-11. In certain embodiments the photosensitizing agent is attached to the targeting peptide by a non-peptide linker. In certain embodiments photosensitizing agent is attached to the targeting peptide by a linker comprising a polyethylene glycol (PEG). In certain embodiments the photosensitizing agent is attached to the targeting peptide by a non-peptide linker found in Table 11.
In certain embodiments methods are provided for inhibiting the growth or proliferation of a microorganism and/or a biofilm (e.g., a bacterium and/or a bacterial film), where the methods involve contacting the a microorganism and/or a biofilm with a composition comprising a photosensitizing agent attached to a targeting peptide as described herein. In certain embodiments the method further comprises exposing the microorganism or biofilm to a light source. In certain embodiments the microorganism is a microorganism selected from the group consisting of a bacterium, a yeast, a fungus, a protozoan, and a virus. In certain embodiments the biofilm comprises a bacterial film.
In certain embodiments chimeric moieties are provided wherein the chimeric moiety comprises multiple targeting moieties attached to each other. In certain embodiments the targeting moieties are directly attached to each other. In certain embodiments the targeting moieties are attached to each other via a peptide linker. In certain embodiments the targeting moieties are attached to each other via a non-peptide linker. In certain embodiments chimeric moieties are provided wherein the chimeric moiety comprises multiple effectors attached to each other. In certain embodiments the effectors are directly attached to each other. In certain embodiments the effectors are attached to each other via a peptide linker. In certain embodiments the effectors are attached to each other via a non-peptide linker.
In certain embodiments chimeric moieties are provided where the chimeric moiety comprises one or more targeting moieties attached to one or more effectors. In certain embodiments the chimeric moiety comprises one or more of the targeting moieties shown in Table 2, and/or Table 4 and/or Table 6, and/or Table 15 attached to a single effector. In certain embodiments the chimeric moiety comprises one or more effectors attached to a single targeting moiety. In certain embodiments the chimeric moiety comprises one or more effectors comprising one or more of the antimicrobial peptides shown in Table 2, and/or Table 8, and/or Table 9, and/or Table 10 attached to a single targeting moiety. In certain embodiments the chimeric moiety comprises multiple targeting moieties attached to multiple effectors.

DEFINITIONS

The term “peptide” as used herein refers to a polymer of amino acid residues typically ranging in length from 2 to about 50 residues. In certain embodiments the peptide ranges in length from about 2, 3, 4, 5, 7, 9, 10, or 11 residues to about 50, 45, 40, 45, 30, 25, 20, or 15 residues. In certain embodiments the peptide ranges in length from about 8, 9, 10, 11, or 12 residues to about 15, 20 or 25 residues. Where an amino acid sequence is provided herein, L-, D-, or beta amino acid versions of the sequence are also contemplated as well as retro, inversion, and retro-inversion isoforms. Peptides also include amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. In addition, the term applies to amino acids joined by a peptide linkage or by other, “modified linkages” (e.g., where the peptide bond is replaced by an α-ester, a β-ester, a thioamide, phosphonamide, carbomate, hydroxylate, and the like (see, e.g., Spatola, (1983) Chem. Biochem. Amino Acids and Proteins 7: 267-357), where the amide is replaced with a saturated amine (see, e.g., Skiles et al., U.S. Pat. No. 4,496,542, which is incorporated herein by reference, and Kaltenbronn et al., (1990) Pp. 969-970 in Proc. 11th American Peptide Symposium, ESCOM Science Publishers, The Netherlands, and the like)).
The term “residue”” as used herein refers to natural, synthetic, or modified amino acids. Various amino acid analogues include, but are not limited to 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine (beta-aminopropionic acid), 2-aminobutyric acid, 4-aminobutyric acid, piperidinic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4 diaminobutyric acid, desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, n-ethylglycine, n-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, n-methylglycine, sarcosine, n-methylisoleucine, 6-n-methyllysine, n-methylvaline, norvaline, norleucine, ornithine, and the like. These modified amino acids are illustrative and not intended to be limiting.
“β-peptides” comprise of “β amino acids”, which have their amino group bonded to the β carbon rather than the α-carbon as in the 20 standard biological amino acids. The only commonly naturally occurring β amino acid is β-alanine.
Peptoids, or N-substituted glycines, are a specific subclass of peptidomimetics. They are closely related to their natural peptide counterparts, but differ chemically in that their side chains are appended to nitrogen atoms along the molecule's backbone, rather than to the α-carbons (as they are in natural amino acids).
The terms “conventional” and “natural” as applied to peptides herein refer to peptides, constructed only from the naturally-occurring amino acids: Ala, Cys, Asp, Glu, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr. A compound of the invention “corresponds” to a natural peptide if it elicits a biological activity (e.g., antimicrobial activity) related to the biological activity and/or specificity of the naturally occurring peptide. The elicited activity may be the same as, greater than or less than that of the natural peptide. In general, such a peptoid will have an essentially corresponding monomer sequence, where a natural amino acid is replaced by an N-substituted glycine derivative, if the N-substituted glycine derivative resembles the original amino acid in hydrophilicity, hydrophobicity, polarity, etc. Thus, for example, the following pairs of peptides would be considered “corresponding”:

Ia.

(SEQ ID NO: 1)

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (Angiotensin II)

and

Ib.

(SEQ ID NO: 2)

Asp-Arg-Val*-Tyr-Ile*-His-Pro-Phe;

IIa.

(SEQ ID NO: 3)

Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (Bradykinin)

and

IIb:

(SEQ ID NO: 4)

Arg-Pro-Pro-Gly-Phe*-Ser*-Pro-Phe*-Arg;

IIIa:

(SEQ ID NO: 5)

Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-

Leu-Val-Thr (β-Endorphin);

and

IIIb:

(SEQ ID NO: 6)

Gly-Gly-Phe*-Met-Ser*-Ser-Glu-Lys*-Ser-Gln-Ser*-

Pro-Leu-Val*-Thr.

In these examples, “Val*” refers to N-(prop-2-yl)glycine, “Phe*” refers to N-benzylglycine, “Ser*” refers to N-(2-hydroxyethyl)glycine, “Leu*” refers to N-(2-methylprop-1-yl)glycine, and “Ile*” refers to N-(1-methylprop-1-yl)glycine. The correspondence need not be exact: for example, N-(2-hydroxyethyl)glycine may substitute for Ser, Thr, Cys, and Met. N-(2-methylprop-1-yl)glycine may substitute for Val, Leu, and Ile. Note in IIIa and IIIb above that Ser* is used to substitute for Thr and Ser, despite the structural differences: the sidechain in Ser* is one methylene group longer than that of Ser, and differs from Thr in the site of hydroxy-substitution. In general, one may use an N-hydroxyalkyl-substituted glycine to substitute for any polar amino acid, an N-benzyl- or N-aralkyl-substituted glycine to replace any aromatic amino acid (e.g., Phe, Trp, etc.), an N-alkyl-substituted glycine such as N-butylglycine to replace any nonpolar amino acid (e.g., Leu, Val, Ile, etc.), and an N-(aminoalkyl)glycine derivative to replace any basic polar amino acid (e.g., Lys and Arg).

A “compound antimicrobial peptide” or “compound AMP” refers to a construct comprising two or more AMPs joined together. The AMPs can be joined directly or through a linker. They can be chemically conjugated or, where joined directly together or through a peptide linker can comprise a fusion protein.
In certain embodiments, conservative substitutions of the amino acids comprising any of the sequences described herein are contemplated. In various embodiments one, two, three, four, or five different residues are substituted. The term “conservative substitution” is used to reflect amino acid substitutions that do not substantially alter the activity (e.g., antimicrobial activity and/or specificity) of the molecule. Typically conservative amino acid substitutions involve substitution one amino acid for another amino acid with similar chemical properties (e.g. charge or hydrophobicity). Certain conservative substitutions include “analog substitutions” where a standard amino acid is replaced by a non-standard (e.g., rare, synthetic, etc) amino acid differing minimally from the parental residue. Amino acid analogs are considered to be derived synthetically from the standard amino acids without sufficient change to the structure of the parent, are isomers, or are metabolite precursors. Examples of such “analog substitutions” include, but are not limited to, 1) Lys-Orn, 2) Leu-Norleucine, 3) Lys-Lys[TFA], 4) Phe-Phe[Gly], and 5) δ-amino butylglycine-ξ-amino hexylglycine, where Phe[gly] refers to phenylglycine (a Phe derivative with a H rather than CH₃component in the R group), and Lys[TFA] refers to a Lys where a negatively charged ion (e.g., TFA) is attached to the amine R group. Other conservative substitutions include “functional substitutions” where the general chemistries of the two residues are similar, and can be sufficient to mimic or partially recover the function of the native peptide. Strong functional substitutions include, but are not limited to 1) Gly/Ala, 2) Arg/Lys, 3) Ser/Tyr/Thr, 4) Leu/Ile/Val, 5) Asp/Glu, 6) Gln/Asn, and 7) Phe/Trp/Tyr, while other functional substitutions include, but are not limited to 8) Gly/Ala/Pro, 9) Tyr/His, 10) Arg/Lys/His, 11) Ser/Thr/Cys, 12) Leu/Ile/Val/Met, and 13) Met/Lys (special case under hydrophobic conditions). Various “broad conservative substations” include substitutions where amino acids replace other amino acids from the same biochemical or biophysical grouping. This is similarity at a basic level and stems from efforts to classify the original 20 natural amino acids. Such substitutions include 1) nonpolar side chains: Gly/Ala/Val/Leu/Ile/Met/Pro/Phe/Trp, and/or 2) uncharged polar side chains Ser/Thr/Asn/Gln/Tyr/Cys. In certain embodiments broad-level substitutions can also occur as paired substitutions. For example, any hydrophilic neutral pair [Ser, Thr, Gln, Asn, Tyr, Cys]+[Ser, Thr, Gln, Asn, Tyr, Cys] can may be replaced by a charge-neutral charged pair [Arg, Lys, His]+[Asp, Glu]. The following six groups each contain amino acids that are typical conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K), Histidine (H); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
In certain embodiments, targeting peptides, antimicrobial peptides, and/or STAMPs compromising at least 80%, preferably at least 85% or 90%, and more preferably at least 95% or 98% sequence identity with any of the sequences described herein are also contemplated. The terms “identical” or percent “identity,” refer to two or more sequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. With respect to the peptides of this invention sequence identity is determined over the full length of the peptide. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman & Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson & Lipman (1988) Proc. Natl. Acad. Sci., USA, 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection.
The term “specificity” when used with respect to the antimicrobial activity of a peptide indicates that the peptide preferentially inhibits growth and/or proliferation and/or kills a particular microbial species as compared to other related and/or unrelated microbes. In certain embodiments the preferential inhibition or killing is at least 10% greater (e.g., LD₅₀is 10% lower), preferably at least 20%, 30%, 40%, or 50%, more preferably at least 2-fold, at least 5-fold, or at least 10-fold greater for the target species.
“Treating” or “treatment” of a condition as used herein may refer to preventing the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof.
The term “consisting essentially of” when used with respect to an antimicrobial peptide (AMP) or AMP motif as described herein, indicates that the peptide or peptides encompassed by the library or variants, analogues, or derivatives thereof possess substantially the same or greater antimicrobial activity and/or specificity as the referenced peptide. In certain embodiments substantially the same or greater antimicrobial activity indicates at least 80%, preferably at least 90%, and more preferably at least 95% of the anti microbial activity of the referenced peptide(s) against a particular bacterial species (e.g., S. mutans).
The term “porphyrinic macrocycle” refers to a porphyrin or porphyrin derivative. Such derivatives include porphyrins with extra rings ortho-fused, or orthoperifused, to the porphyrin nucleus, porphyrins having a replacement of one or more carbon atoms of the porphyrin ring by an atom of another element (skeletal replacement), derivatives having a replacement of a nitrogen atom of the porphyrin ring by an atom of another element (skeletal replacement of nitrogen), derivatives having substituents other than hydrogen located at the peripheral (meso-, .beta.-) or core atoms of the porphyrin, derivatives with saturation of one or more bonds of the porphyrin (hydroporphyrins, e.g., chlorins, bacteriochlorins, isobacteriochlorins, decahydroporphyrins, corphins, pyrrocorphins, etc.), derivatives obtained by coordination of one or more metals to one or more porphyrin atoms (metalloporphyrins), derivatives having one or more atoms, including pyrrolic and pyrromethenyl units, inserted in the porphyrin ring (expanded porphyrins), derivatives having one or more groups removed from the porphyrin ring (contracted porphyrins, e.g., corrin, corrole) and combinations of the foregoing derivatives (e.g. phthalocyanines, porphyrazines, naphthalocyanines, subphthalocyanines, and porphyrin isomers). Certain porphyrinic macrocycles comprise at least one 5-membered ring.
As used herein, an “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_L) and variable heavy chain (V_H) refer to these light and heavy chains respectively.
Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′₂, a dimer of Fab which itself is a light chain joined to V_H—C _H1 by a disulfide bond. The F(ab)′₂may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab′)₂dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab′ fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies, including, but are not limited to, Fab′₂, IgG, IgM, IgA, scFv, dAb, nanobodies, unibodies, and diabodies.
In certain embodiments antibodies and fragments of the present invention can be bispecific. Bispecific antibodies or fragments can be of several configurations. For example, bispecific antibodies may resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions). In various embodiments bispecific antibodies can be produced by chemical techniques (Kranz et al. (1981) Proc. Natl. Acad. Sci., USA, 78: 5807), by “polydoma” techniques (see, e.g., U.S. Pat. No. 4,474,893), or by recombinant DNA techniques. In certain embodiments bispecific antibodies of the present invention can have binding specificities for at least two different epitopes, at least one of which is an epitope of a microbial organism. The microbial binding antibodies and fragments can also be heteroantibodies. Heteroantibodies are two or more antibodies, or antibody binding fragments (e.g., Fab) linked together, each antibody or fragment having a different specificity.
The term “STAMP” refers to Specifically Targeted Anti-Microbial Peptides. An MH-STAMP is a STAMP bearing two or more targeting domains (i.e., a multi-headed STAMP).
In various embodiments the amino acid abbreviations shown in Table 1 are used herein.

TABLE 1

Amino acid abbreviations.

Abbreviation

Name

	3 Letter	1 Letter

Alanine	Ala	A
βAlanine (NH₂—CH₂—CH₂—COOH)	βAla
Arginine	Arg	R
Asparagine	Asn	N
Aspartic Acid	Asp	D
Cysteine	Cys	C
Glutamic Acid	Glu	E
Glutamine	Gln	Q
Glycine	Gly	G
Histidine	His	H
Homoserine	Hse	—
Isoleucine	Ile	I
Leucine	Leu	L
Lysine	Lys	K
Methionine	Met	M
Methionine sulfoxide	Met (O)	—
Methionine methylsulfonium	Met (S—Me)	—
Norleucine	Nle	—
Phenylalanine	Phe	F
Proline	Pro	P
Serine	Ser	S
Threonine	Thr	T
Tryptophan	Trp	W
Tyrosine	Tyr	Y
Valine	Val	V
episilon-aminocaproic acid	Ahx	J
(NH²—(CH₂)₅—COOH)
4-aminobutanoic acid	gAbu
(NH₂—(CH₂)₃—COOH)
tetrahydroisoquinoline-3-		O
carboxylic acid
Lys(N(epsilon)-trifluoroacetyl)		K[TFA]
α-aminoisobutyric acid	Aib	B

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows some illustrative porphyrins (compounds 92-99) suitable for use as targeting moieties and/or antimicrobial effectors.

FIG. 2 shows some illustrative porphyrins (compounds 100-118) suitable for use as targeting moieties and/or antimicrobial effectors.

FIG. 3 shows some illustrative porphyrins (in particular phthalocyanines) (compounds 119-128) suitable for use as targeting moieties and/or antimicrobial effectors.

FIG. 4 illustrates the structures of two phthalocyanines, Monoastral Fast Blue B and Monoastral Fast Blue G suitable for use as targeting moieties and/or antimicrobial effectors.

FIG. 5 illustrates certain azine photosensitizers suitable for use as targeting moieties and/or antimicrobial effectors in the compositions and methods described herein.

FIG. 6 shows illustrative cyanine suitable for use as targeting moieties and/or antimicrobial effectors in the compositions and methods described herein.

FIG. 7 shows illustrative psoralen (angelicin) photosensitizers suitable for use as targeting moieties and/or antimicrobial effectors in the compositions and methods described herein.

FIG. 8 shows illustrative hypericin and the perylenequinonoid pigments suitable for use as targeting moieties and/or antimicrobial effectors in the compositions and methods described herein.

FIG. 9 shows illustrative acridines suitable for use as targeting moieties and/or antimicrobial effectors in the compositions and methods described herein.

FIG. 10 illustrates the structure of the acridine Rose Bengal.

FIG. 11 illustrates various crown ethers suitable for use as targeting moieties and/or antimicrobial effectors in the compositions and methods described herein.

FIG. 12 schematically shows some illustrative configurations for chimeric constructs described herein. A: Shows a single targeting moiety T1 attached to a single effector E1 by a linker/spacer L. B: Shows multiple targeting moieties T1, T2, T3 attached directly to each other and attached by a linker L to a single effector E1. In various embodiments T1, T2, and T3, can be domains in a fusion protein. C: Shows multiple targeting moieties T1, T2, T3 attached to each other by linkers L and attached by a linker L to a single effector E1. In various embodiments T1, T2, and T3, can be domains in a fusion protein. D: Shows a single targeting moiety T1 attached by a linker L to multiple effectors E1, E2, and E3 joined directly to each other. E: Shows a single targeting moiety T1 attached by a linker L to multiple effectors E1, E2, and E3 joined to each other by linkers L. F: Shows multiple targeting moieties joined directly to each other and by a linker L to multiple effectors joined to each other by linkers L. G: Shows multiple targeting moieties joined to each other by linkers L and by a linker L to multiple effectors joined to each other by linkers L. In various embodiments T1, T2, and T3, and/or E1, E2, and E3 can be domains in a fusion protein. H: Illustrates a branched configuration where multiple targeting moieties are linked to a single effector. I: Illustrates a dual branched configuration where multiple targeting moieties are linked to multiple effectors. J: Illustrates a branched configuration where multiple targeting moieties are linked to multiple effectors where the effectors are joined to each other in a linear configuration.

FIG. 13 illustrates various MH-STAMPs used in Example 1. The design, sequence, and observed mass (m/z) for M8(KH)-20 (SEQ ID NO:7), BL(KH)-20 (SEQ ID NO:8), and M8(BL)-20 (SEQ ID NO:9.

FIGS. 14A and 14B show HPLC and MS spectra of M8(KH)-20. The quality of the completed MH-STAMP was analyzed by HPLC (FIG. 14A) and MALDI mass spectroscopy (FIG. 14B). At UV absorbance 215 nm (260 and 280 nm are also plotted), a single major product was detected by HPLC (* retention volume 11.04 mL). After fraction collection, the correct mass (m/z) for single-charged M8(KH)-20, 4884.91 (marked by *), was observed for this peak. Y-axis: 14A, mAU miliabsorbance units; 14B, percent intensity.

FIG. 15A-15E show growth inhibitory activity of MH-STAMPs. Monocultures of S. mutans (FIG. 15A); P. aeruginosa (FIG. 15B); S. epidermidis (FIG. 15C); S. aureus (FIG. 15D); or E. coli (FIG. 15E); were treated with peptides (as indicated in the figure) for 10 min. Agent was then removed and fresh media returned. Culture recovery was measured over time (OD600). Plots represent the average of at least 3 independent experiments with standard deviations.

FIG. 16 illustrates the selective activity of dual-targeted and single-targeted MH-STAMPs in mixed culture. A mixture of P. aeruginosa (Pa), S. mutans (Sm), E. coli (Ec), and S. epidermidis (Se) planktonic cells were mixed with MH-STAMPs (as indicated in the figure) and treated 24 h. After incubation, cfu/mL of remaining constituent species were quantitated after plating to selective media. * indicates under 200 surviving cfu/mL recovered.

FIG. 17 depicts the results of minimal inhibitory concentration (MIC) assays, conducted against S. mutans and a panel of bacteria, including two oral streptococci, S. sanguinis and S. sobrinus, to gauge Library 1 STAMP antimicrobial activity and S. mutans-selectivity.

FIG. 18 shows the results of MIC assays utilizing targeting peptide 2_—1 conjugated to one of five AMPs, RWRWRWF (2c-4), FKKFWKWFRRF (B-33), IKQLLHFFQRF (B-38), RWRRLLKKLHHLLH (α-11), LQLLKQLLKLLKQF (a-7); attached at the C- or N-terminus, and a linker selected from L1, SGG (L2), L3 and LC. MIC assays were conducted against a panel of bacteria, including S. mutans (S. mu), S. gordonii (S. gor), S. sanguinis (S. san), and S. mitis (S. mit), to gauge differences in activity between attaching the targeting peptide to the C- or N-terminus of the AMP region or differences in activity between the linker used.

FIG. 19 shows the killing kinetics of selected peptides against oral streptococci. All tested bacteria, (A) S. mitis (Smit), (B) S. gordonii (Sgor), (C) S. sanguinis (Ssan), and (D) S. mutans (Smut), were treated with peptide solutions at 25 m/mL for 30 s to 2 h and survivors plated. Data represent an average of three independent experiments.

FIG. 20 illustrates the inhibitory activity of STAMPs against S. mutans biofilms. S mutans monoculture biofilms were grown and exposed to a 25 m/mL of STAMP, unmodified parental AMP or oral antiseptic (for 1 min), washed, and replenished with fresh medium. Biofilm recovery was monitored after 4 h by A600. Data represent an average of three experiments.

DETAILED DESCRIPTION

In various embodiments this invention is based on the development of a method for the rapid identification and synthesis of small peptides from sequenced genomes for the purposes of quickly screening these molecules for diverse biological activities. Previously, small secreted peptides had to be identified by direct collection (purification of spent microbial growth medium or biological tissue/fluid), or by identification through differential microarray analysis or as a by-product of genetic operon characterization. Both are multi-step processes that terminate at peptide identification; several separate enterprises are required to synthesize or sequence any peptides for characterization. Additionally, because of their small size and lack of protein domain homologies, small peptides are frequently overlooked when describing biological systems, though many organisms contain numerous peptide-sized open reading frames (ORFs).
Genomic sequence analysis tools already in the public domain and high-throughput solid phase peptide chemistry were used to rapidly identify and synthesize biologically active peptides. Using sequences and sometimes annotated genomes, genomes were scanned, by hand or with a search algorithm, for ORFs predicted to encode peptides of less than for example, 50-60 amino acids (ignoring small tRNA ORFs or other well characterized genes).
Once noted, these peptides were batch synthesized on a multiplex peptide synthesizer, yielding 5-10 mg of each peptide that could readily be screened for biological activity (e.g., binding activity and/or antimicrobial activity.
Accordingly, in certain embodiments, this invention pertains to the identification of novel peptides (see, e.g., Table 2) that specifically or preferentially bind particular microorganisms (e.g., bacteria) and/or that have antimicrobial activity. In certain embodiments these peptides can be attached to effectors (e.g., drugs, labels, etc.) and used as targeting moieties thereby providing a chimeric moiety that preferentially or specifically delivers the effector to a target microorganism, a population of target microorganisms, a microbial film comprising the target microorganism(s), a biofilm comprising the target microorganism(s), and the like.
In certain embodiments these peptides can be exploited for their antimicrobial activity to inhibit the growth or proliferation of a microorganism and/or to inhibit the formation and/or growth of a biofilm comprising the microorganism. These antimicrobial peptides can be used alone, in conjunction with other agents (e.g., antibacterial agents), and/or they can be coupled to a targeting moiety to provide a chimeric moiety that preferentially directs the antimicrobial peptide to a target tissue and/or to a target organism (e.g., a bacterium, a population of target microorganisms, a microbial film, a biofilm, and the like).
In certain embodiments the antimicrobial peptides and/or the chimeric moieties described herein can be formulated with a pharmaceutically acceptable carrier to form a pharmacological composition.
The amino acid sequences of illustrative peptides of this invention having antimicrobial and/or targeting activity against methicillin resistant Streptococcus mutans, Streptococcus pyogenes, and/or Treponema denticola is shown in Table 2. As with the other peptides described herein, it will be recognized that these peptides can comprise all “L” form residues, all “D” form residues, mixtures of “L” and “D” residues, and beta peptide sequences. It will also be appreciated in addition to the D-form and L-form and beta-peptide sequences this invention also contemplates retro and retro-inverso forms of each of these peptides. In retro forms, the direction of the sequence is reversed. In inverse forms, the chirality of the constituent amino acids is reversed (i.e., L form amino acids become D form amino acids and D form amino acids become L form amino acids). In the retro-inverso form, both the order and the chirality of the amino acids is reversed. Thus, for example, a retro form of the of the peptide Smu11 (MKNLIETVEKFLTYSDEKLEELAKKNQALREEISRQKSK, SEQ ID NO:11) has the sequence KSKQRSIEERLAQNKKALEELKEDSYTLFKEVTEILNKM (SEQ ID NO:10). Where the Smu11 peptide comprises all L amino acids, the inverse form will comprise all D amino acids and the retro-inverso (retro-inverse) form will have the sequence of SEQ ID NO:10 and comprise all D form amino acids. Also contemplated are peptides having the amino acid sequences or retro amino acid sequences of the peptides in Table 2 (or the other tables shown herein) and comprising one, two, three, four, five, six, seven, eight, nine, or ten conservative substitutions, but retaining substantially the same binding and/or antimicrobial activity. Also contemplated are peptides having the amino acid sequences or retro amino acid sequences of the peptides in Table 2 (or other tables herein) and comprising one, two, three, four, five, six, seven, eight, nine, or ten deletions, but retaining substantially the same binding and/or antimicrobial activity.
In various embodiments, chimeric moieties comprising one or more of the targeting peptides found in Table 2 attached to one or more effectors (e.g., antimicrobial peptides as described herein) are contemplated. In various embodiments, one or more of the antimicrobial peptides found in Table 2 used as effectors or attached to one or more targeting moieties (e.g., targeting peptides, targeting antibodies, and the like) to form chimeric moieties are contemplated.

TABLE 2

Peptides having antimicrobial and/or targeting activity
against one or more of the following: Streptococcus mutans,
Streptococcus pyogenes, and Treponema denticola.

		SEQ
ID	Amino Acid Sequence	ID NO

Smu11	MKNLIETVEKFLTYSDEKLEELAKKNQALREEISRQKSK		11

Smu.18	GGRAGRIKKLSQKEAEPFEN	12

Smu41	MFIRSKLRRVDFSGVRRGNKHFLLDKLLITLVK	13

Smu68	MSALFYDTLAAIWISIAGVDARWGH		14

Smu150	GGKVSGGEAVAAIGICATASAAIGGLAGATLVTPYCVGTWGLI		15
	RSH

Smu151	DKQAADTFLSAVGGAASGFTYCASNGVWHPYILAGCAGVGA
	16
	VGSVVFPH

Smu223c	GGKYLFLASKTKEYFKSHFREIMIDV	17

Smu225c	MFISFVDCIQNIEKIEKELLKIGITDIQINQDAGWLY	18

Smu277	YLTEIEGEGLGLGICLGLVGFAGGFAHGVVQGAGVGTAIEPGY	19
	GTIIGALVDGVGQDLIYGGAGFAAGYSL

Smu283	GGFDVKGVAASYLAMGTAALGGLACTTPVGAVLYLGAEVCA
	20
	GAAVIYYGAN

Smu299c	GGGLYDGANGYAYRDSQGHWAYKVTKTPAQALTDVVVNSW	21
	ASGAASFAAYA

Smu390	GGRSSYNGFSKICFLKIEHFGSYSYQGR
	22

Smu423	GGGMIRCALGTAGSAGLGFVGGMGAGTVTLPVVGTVSGAAL
	23
	GGWSGAAVGAATFC

Smu427	MEKTYHIDGLKCQGCADNVTKRFSELKKVNDVKVDLDKKEV
	24
	RITGNPSKWSLKRALKGTNYELGAEI

Smu444	MSVGMGVIERGSFDFSASAILQKRETKCLKNKPFT
	25

Smu451	MKMRAGQVVFIYKLILVLLFYVLQKLFDLKKGCF	26

Smu529	MLPDSALDERIKGRVSAKNSSLLSALIKKLALIIFIG	27

Smu616	GGVATYGAATMGLCAVSGPIGWGLGGAYLLTCAAAGGMIGY	28
	GAATLD

Smu750c	MLSNVLSRSVVSPNVDIPNSMVILSPLLISISNYH	29

Smu812	MAILTFFMALLFTYLKEKAQILYWPLFLHLMFYFVTA		30

Smu1018	MNTNDLLQAFELMGLGMAGVFIVLGILYIVAELLIKIFPVNN		31

Smu1047c	MVHLFSFVKLIYYDIMKYSIEEKVFFESPVGEIIQ
	32

Smu1131c	GGYATAKLTQTKPTMPKNVKKGTPPKGAPEDTPPNGNSNDSS		33
	QSDSDSDSNSSNTNSNSSITNG

Smu1231c	MDKKRVIERIKSFSLRDEVIHFGELCIYWGK	34

Smu1232c	GGKNKLVMSDLRQQVTDMGFVNVKTYINSGNLFFQSDCPRAN		35
	ISSRFEQFFADHYPFV

Smu1358	MNNAYRWFFRLTLADNMHRFTTYGKEWQLSFPK	36

Smu1359	MKLAGIEKKINLSKRRKLYLENSSLLINYVKVNMSY	37

Smu1368	MTEILNFLIAVHDDRKNWKIKHCLSNSSFDFLCSPDSSR
	38

Smu1369	MPVQKALHVVSAYATDLGICYDQVVTVMIREVKTQLYQIY	39

Smu1372c	GGSYQQVYSWVRKFKKDGINGLLDRRGKGLESKLISVVMVAI		40
	VLRPV

Smu1504c	GGMSSGWLSDDFWLKSAIPLLKKRLAKWNETL	41

Smu1505c	MAYSLTFQNPNDNLTDEEVAKYMEKITKALTEKIGAEVR	42

Smu1719c	MNTFLWILLVIIALLAGLVGGTFIARKQMEKYLEENPPLNEDVI	43
	RNMMSQMGQKPSEAKVQQVVRQMNKQQKAAKAKAKKKK

Smu1750c	MIFNRRKFFQYFGLSKEAMVEHIQPFILDIWQIHLF	44

Smu1752c	GGWLNAISLYGRIG	45

Smu1768c	GGHKQLVIEPLVSQNDQLSLIESLSGILSDSETVDVKDYRSERK	46
	EERLKKYESLT

Smu1808c	MTTTQKTYLHIIRELENQDIDLIMRSLTSLT	47

Smu1813	MPMTYCGSPRRTDLAVITDEELGQTLEVINHWPRNV
	48

Smu1882c	GGATDGEIIANRMLQGKATKGEITMYTWNIIQNGWVNSLVSW	49
	GIGGYNSSIGYSAQGNRGFSNYPYDVSMDSDNSSSSSNTTGGY
	VNYNQSFNSGW

Smu1889c	GGLAGAGTGAAVSAPAAEGGGLGPIAGAAIGWDLGAISGAGL	50
	GWANFCQ

Smu1895c	GGMTWAEIGAIVGATIGSFYIPNPVIVPFRVR
	51

Smu1896c	GGFGWDSIWRGFKCVAGTAGTIGTGALGGSATGGLTLPIIGHV	52
	SGGIIGGISGAGVGIASFC

Smu1899	MLSISQRTDRVIVMDKGKIIEEGTHSELIAANGFYHHLFNK	53

Smu1902c	GGAFYQRKENVISLDPREWLGFNVTEK	54

Smu1903c	GGNIFEYFLE	55

Smu1905c	GGGRAPRCAALVGASIYDGLAVVGDPVGVAMAAGTIAAGSFC	56

Smu1906c	GGCSWKGADKAGFSGGVGGLIGAGGNPVGGVLGIAGGLDAY	57
	GELVGGN

Smu1907	MEQNILNGSYFVLNGKNAKFLLEIDKLTLPDKLATLPVPHQVR	58

Smu1914c	GGGRGWNCAAGIALGAGQGYMATAGGTAFLGPYAIGTGAFG	59
	AIAGGIGGALNSCG

Smu1915	GGSGSLSTFFRLFNRSFTQALGK
	60

Smu1948	GGVFSVLKHTTWPTRKQSWHDFISILEYSAFFALVIFIFDKLLTL	61
	GLAELLKRF

Smu1968c	GGSVLGKHALFILLKAGFKAYELAGAFEGWKGMHLPTEKC	62

Smu1972c	GGLVMNDETIYLFTYENGQISYEEDKRDCSKNV	63

Smu2105	MRFLKDELSVSVRLQEKSIEALPFRTKIEIEIESDNQIKTL		64

Smu2106c	GGASGEKILEKLIHERKCQLTQNRQIVLKTDLNNLMKDFYK	65

Smu2121c	GGIILAKAADLAEIERIISEDPFKINEIANYDIIEFCPTKSSKAFEK		66
	VLK

Uses of Targeting Moieties.

When exploited for their targeting activity, the novel targeting peptides described herein (see, e.g., Table 2) can be used to preferentially or specifically deliver an effector to a microorganism (e.g., a bacterium, a fungus, a protozoan, a yeast, an algae, etc.), to a bacterial film comprising the microorganism, to a biofilm comprising the microorganism, and the like. Where the effector comprises an epitope tag and/or a detectable label, binding of the targeting moiety provides an indication of the presence and/or location, and/or quantity of the target (e.g., target microorganism). Thus targeting moieties are thus readily adapted for use in in vivo diagnostics, and/or ex vivo assays. Moreover, because of small size and good stability, the microorganism binding peptides are well suited for microassay systems (e.g., microfluidic assays (Lab on a Chip), microarray assays, and the like).
In certain embodiments the microorganism binding peptides (targeting peptides) can be attached to an effector that has antimicrobial activity (e.g., an antimicrobial peptide, an antibacterial and/or antifungal, a vehicle that contains an antibacterial or antifungal, etc. In various embodiments these chimeric moieties can be used in vivo, or ex vivo to preferentially inhibit or kill the target organism(s).
In certain embodiments the targeting peptides can be used in various pre-targeting protocols. In pre-targeting protocols, a chimeric molecule is utilized comprising a primary targeting species (e.g. a microorganism-binding peptide) that specifically binds the desired target (e.g. a bacterium) and an effector that provides a binding site that is available for binding by a subsequently administered second targeting species. Once sufficient accretion of the primary targeting species (the chimeric molecule) is achieved, a second targeting species comprising (i) a diagnostic or therapeutic agent and (ii) a second targeting moiety, that recognizes the available binding site of the primary targeting species, is administered.
An illustrative example of a pre-targeting protocol is the biotin-avidin system for administering a cytotoxic radionuclide to a tumor. In a typical procedure, a monoclonal antibody targeted against a tumor-associated antigen is conjugated to avidin and administered to a patient who has a tumor recognized by the antibody. Then the therapeutic agent, e.g., a chelated radionuclide covalently bound to biotin, is administered. The radionuclide, via its attached biotin is taken up by the antibody-avidin conjugate pretargeted at the tumor. Examples of pre-targeting biotin/avidin protocols are described, for example, in Goodwin et al., U.S. Pat. No. 4,863,713; Goodwin et al. (1988) J. Nucl. Med. 29: 226; Hnatowich et al. (1987) J. Nucl. Med. 28: 1294; Oehr et al. (1988) J. Nucl. Med. 29: 728; Klibanov et al. (1988) J. Nucl. Med. 29: 1951; Sinitsyn et al. (1989) J. Nucl. Med. 30: 66; Kalofonos et al. (1990) J. Nucl. Med. 31: 1791; Schechter et al. (1991) Int. J. Cancer 48:167; Paganelli et al. (1991) Cancer Res. 51: 5960; Paganelli et al. (1991) Nucl. Med. Commun. 12: 211; Stickney et al. (1991) Cancer Res. 51: 6650; and Yuan et al. (1991) Cancer Res. 51:3119.
It will be recognized that the tumor-specific antibody used for cancer treatments can be replaced with a microorganism binding peptide of the present invention and similar pre-targeting strategies can be used to direct labels, antibiotics, and the like to the target organism(s).
Three-step pre-targeting protocols in which a clearing agent is administered after the first targeting composition has localized at the target site also have been described. The clearing agent binds and removes circulating primary conjugate which is not bound at the target site, and prevents circulating primary targeting species (antibody-avidin or conjugate, for example) from interfering with the targeting of active agent species (biotin-active agent conjugate) at the target site by competing for the binding sites on the active agent-conjugate. When antibody-avidin is used as the primary targeting moiety, excess circulating conjugate can be cleared by injecting a biotinylated polymer such as biotinylated human serum albumin. This type of agent forms a high molecular weight species with the circulating avidin-antibody conjugate which is quickly recognized by the hepatobiliary system and deposited primarily in the liver.
Examples of these protocols are disclosed, e.g., in PCT Application No. WO 93/25240; Paganelli et al. (1991) Nucl. Med. Comm., 12: 211-234; Oehr et al. (1988) J. Nucl. Med., 29: 728-729; Kalofonos et al. (1990) J. Nucl. Med., 31: 1791-1796; Goodwin et al. (1988) J. Nucl. Med., 29: 226-234; and the like).
These applications of microorganism binding peptides of this invention are intended to be illustrative and not limiting. Using the teaching provided herein, other uses will be recognized by one of skill in the art.

Uses of Antimicrobial Peptides.

When exploited for their antimicrobial activity, the novel antimicrobial peptides described herein (see, e.g., Table 2) can be used to inhibit the growth and/or proliferation of a microbial species and/or the growth or proliferation of a biofilm comprising the microbial species. In various embodiments the peptides can be formulated individually, in combination with each other, in combination with other antimicrobial peptides, and/or in combination with various antibacterial agents to provide antimicrobial reagents and/or pharmaceuticals.
In various embodiments, the antimicrobial peptides described herein can be formulated individually, in combination with each other, in combination with other antimicrobial peptides, and/or in combination with various antibiotic (e.g., antibacterial) agents in “home healthcare” formulations. Such formulations include, but are not limited to toothpaste, mouthwash, tooth whitening strips or solutions, contact lens storage, wetting, or cleaning solutions, dental floss, toothpicks, toothbrush bristles, oral sprays, oral lozenges, nasal sprays, aerosolizers for oral and/or nasal application, wound dressings (e.g., bandages), and the like.
In various embodiments the antimicrobial peptides described herein can be formulated individually, in combination with each other, in combination with other antimicrobial peptides, and/or in combination with various antibiotic (e.g., antibacterial) agents in various cleaning and/or sterilization formulations for use in the home, workplace, clinic, or hospital.
In certain embodiments the antimicrobial peptides described herein are attached to one or more targeting moieties to specifically and/or to preferentially deliver the peptide(s) to a target (e.g. a target microorganism, biofilm, bacterial film, particular tissue, etc.).
Other possible uses of the targeting and/or antimicrobial peptides disclosed herein include, but are not limited to biofilm dispersal, biofilm retention, biofilm formation, anti-biofilm formation, cell agglutination, induction of motility or change in motility type, chemoattractant or chemorepellent, extracellular signal for sporogenesis or other morphological change, induction or inhibition of virulence gene expression, utilized as extracellular scaffold, adhesin or binding site, induction or suppression of host immune response, induction or suppression of bacterial/fungal antimicrobial molecule production, quorum-sensing, induction of swarming behavior, apoptosis or necrosis inducing in eukaryotic cells, affecting control of or inducing the initiation of cell cycle in eukaryotes, in archaea or prokaryotes, induces autolysis or programmed cell death, inhibition of phage/virus attachment or replication, evasion of innate immunity, induction or inhibition of genetic transformation or transduction competence, induction or inhibition of pilus-mediated conjugation, induction or inhibition of mating behavior in bacteria and fungi, induction or inhibition of nodule formation or metabolic compartmentalization, metal, ion, or nutrient binding, acquisition or inhibition of metal, ion, or nutrient binding and acquisition, and the like.
These applications of the peptides described herein are intended to be illustrative and not limiting. Using the teaching provided herein, other uses will be recognized by one of skill in the art.

Design and Construction of Chimeric Moieties.

In various embodiments this invention provides chimeric moieties comprising one or more targeting moieties attached to one or more effectors. The targeting moieties are typically selected to preferentially bind to a target microorganism (e.g., bacteria, virus, fungi, yeast, alga, protozoan, etc.), or group of microorganisms (e.g., gram-negative or gram-positive bacteria, particular genus, particular species, etc.) In certain embodiments the targeting moiety comprises one or more of the targeting peptides shown in Table 2, and/or Table 4 and/or Table 6. In certain embodiments the targeting moiety comprises non-peptide moieties (e.g., antibodies, receptor, receptor ligand, lectin, and the like).
The effector typically comprises one or more moieties whose activity is to be delivered to the target microorganism(s), and/or to a biofilm comprising the target microorganism(s), and/or to a cell or tissue comprising the target microorganism(s), and the like. In certain embodiments the targeting moiety comprises one or more antimicrobial peptide(s) as described herein (see, e.g., antimicrobial peptides in Table 2, and/or Table 8, and/or Table 9, and/or Table 10), an antibiotic (including, but not limited to a steroid antibiotic) (see, e.g., Table 7), a detectable label, a porphyrin, a photosensitizing agent, an epitope tag, a lipid or liposome, a nanoparticle, a dendrimer, and the like.
In certain embodiments one or more targeting moieties are attached to a single effector (see, e.g. FIG. 12). In certain embodiments one or more effectors are attached to a single targeting moiety. In certain embodiments multiple targeting moieties are attached to multiple effectors. The targeting moietie(s) can be attached directly to the effector(s) or through a linker. Where the targeting moiety and the effector comprise peptides the chimeric moiety can be a fusion protein.
Targeting Moieties.
In various embodiments this invention provides targeting moieties that preferentially and/or specifically bind to a microorganism (e.g., a bacterium, a fungus, a yeast, etc.). One or more such targeting moieties can be attached to one or more effectors to provide chimeric moieties that are capable of delivering the effector(s) to a target (e.g., a bacterium, a fungus, a yeast, a biofilm comprising the bacterium or fungus or yeast, etc.).
In various embodiments, targeting moieties include, but are not limited to peptides that preferentially bind particular microorganisms (e.g., bacteria, fungi, yeasts, protozoa, algae, viruses, etc.) or groups of such microorganisms, antibodies that bind particular microorganisms or groups of microorganisms, receptor ligands that bind particular microorganisms or groups of microorganisms, porphyrins (e.g., metalloporphyrins), lectins that bind particular microorganisms or groups of microorganisms, and the like. As indicated, it will be appreciated that references to microorganisms or groups of microorganism include bacteria or groups of bacteria, viruses or groups of viruses, yeasts or groups of yeasts, protozoa or groups of protozoa, viruses or groups of viruses, and the like.
Targeting Peptides.
In certain embodiments the chimeric constructs described herein comprise a targeting moiety that is or comprises a targeting peptide. Typically the targeting peptides bind particular bacteria, and/or fungi, and/or yeasts, and/or algae, and/or viruses and/or bind particular groups of bacteria, and/or groups of fungi, and/or groups of yeasts, and/or groups of algae. The targeting peptides provided can be used to effectively deliver one or more effectors to or into a target microorganism. Illustrative targeting peptides include, but are not limited to the targeting peptides found in Table 2.
Other suitable targeting peptides include the peptides that have been identified as binding to particular target organisms as shown in Table 4 and/or Table 4.

TABLE 3

Illustrative list of novel targeting peptides.

			SEQ ID
ID	Target(s)	Targeting Peptide Sequence	NO

1T-1	S. mutans	YVNYNQSFNSGW	67

1T-2	S. mutans	NIFEYFLE	68
	S. sanguinis
	S. gordonii
	S. mitis
	S. oralis
	V. atypica
	Lactobacillus casei
	Saliva-derived biofilms

1T-3	S. mutans	VLGIAGGLDAYGELVGGN	69
	S. gordonii

1T-4	S. mutans	LDAYGELVGGN	70
	S. gordonii
	S. sanguinis
	S. oralis
	V. atypica
	L. casei

1T-5	S. mutans	LGPIAGAAIGWDLGAISGAGL	71
	S. sanguinis	GWANFCQ

1T-6	S. mutans	KFINGVLSQFVLERK	72

1T-7	Myxococcus xanthus	SQRIIEPVKSPQPYPGFSVS	73

1T-8	M. xanthus	FSVAACGEQRAVTFVLLIEDLI	74

1T-9	M. xanthus	WAWAESPRCVSTRSNIHALAF	75
		RVEVAALT

1T-10	M. xanthus	SPAGLPGDGDEA	76

1T-11	S. mutans	RISE	77
	S. epidermidis
	P. aeruginosa

1T-12	Corynebacterium	FGNIFKGLKDVIETIVKWTAAK	78
	xerosis
	Corynebacterium
	striatum
	S. epidermidis
	S. mutans

1T-13	S. aureus	FRSPCINNNSLQPPGVYPAR	79
	S. epidermidis
	P. aeruginosa

1T-14	S. mutans	ALAGLAGLISGK	80
	S. aureus
	S. epidermidis
	C. xerosis

1T-15	S. mutans	DVILRVEAQ	81

1T-16	P. aeruginosa	IDMR	82

1T-17	S. mutans	NNAIVYIS	83

1T-18	S. aureus	YSKTLHFAD	84
	S. epidermidis
	C. striatum
	P. aeruginosa

1T-19	S. aureus	PGAFRNPQMPRG	85
	S. epidermidis
	P. aeruginosa

1T-20	S. mutans	PALVDLSNKEAVWAVLDDHS	86
	P. aeruginosa

1T-21	S. mutans	YVEEAVRAALKKEARISTEDTP	87
	P. aeruginosa	VNLPSFDC

1T-22	S. epidermidis	VPLDDGTRRPEVARNRDKDRED	88
	P. aeruginosa

1T-23	S. mutans	PALVDLSNKEAVWAVLDDHS	89
	P. aeruginosa

1T-24	P. aeruginosa	EEAEEKLAEVSQAVKRLVR	90

1T-25	S. aureus	VGLDVSVLVLFFGLQLLSVLLG	91
	S. epidermidis	AMIR
	C. xerosis
	C. striatum
	P. aeruginosa

1T-26	S. mutans	LTILPTTFFAIIVPILAVAFIAYSG	92
	S. aureus	FKIKGIVEHKDQW
	S. epidermidis
	Corynebacterium
	jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-27	S. mutans	ALFVSLEQFLVVVAKSVFALCH	93
	S. aureus	SGTLS
	S. epidermidis
	C. jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-28	P. aeruginosa	VSRDEAMEFIDREWTTLQPAG	94
		KSHA

1T-29	S. mutans	GSVIKKRRKRMSKKKHRKMLR	95
	S. aureus	RTRVQRRKLGK
	S. epidermidis
	C. jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-30	S. aureus	GKAKPYQVRQVLRAVDKLETR	96
	S. epidermidis	RKKGGR
	C. xerosis
	C. striatum
	P. aeruginosa

1T-31	S. mutans	NATGTDIGEVTLTLGRFS	97
	P. aeruginosa

1T-32	S. mutans	VSFLAGWLCLGLAAWRLGNA	98

1T-33	S. aureus	VRTLTILVIFIFNYLKSISYKLKQ	99
	S. epidermidis	PFENNLAQSMISI
	C. jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-34	S. aureus	AFWLNILLTLLGYIPGIVHAVYI	100
	S. epidermidis	IAKR
	C. jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-35	P. aeruginosa	EICLTLVFPIRGSYSEAAKFPVPI	101
		HIVEDGTVELPK

1T-36	S. aureus	VYRHLRFIDGKLVEIRLERK	102
	S. epidermidis
	C. jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-37	S. mutans	YIVGALVILAVAGLIYSMLRKA	103
	S. aereus
	S. epidermidis
	C. jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-38	S. mutans	VMFVLTRGRSPRPMIPAY	104
	S. aereus
	S. epidermidis
	C. jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-39	S. mutans	FGFCVWMYQLLAGPPGPPA	105
	P. aeruginosa

1T-40	S. mutans	QRVSLWSEVEHEFR	106
	P. aeruginosa

1T-41	S. mutans	KRGSKIVIAIAVVLIVLAGVWVW	107
	S. aureus
	S. epidermidis
	C. jeikeium
	C. striatum
	P. aeruginosa

1T-42	S. aureus	TVLDWLSLALATGLFVYLLVA	108
	S. epidermidis	LLRADRA
	C. xerosis
	C. striatum
	P. aeruginosa

1T-43	C. jeikium	DRCLSVLSWSPPKVSPLI	109
	P. aeruginosa

1T-44	S. mutans	DPALADFAAGMRAQVRT	110
	S. aureus
	S. epidermidis
	C. jeikeium
	C. striatum
	P. aeruginosa

1T-45	S. aureus	WTKPSFTDLRLGFEVTLYFANR	111
	S. epidermidis
	C. striatum
	P. aeruginosa

1T-46	S. aureus	FSFKQRVMFRKEVERLR	112
	S. epidermidis
	C. jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-47	S. mutans	VIKISVPGQVQMLIP	113
	S. epidermidis
	P. aeruginosa

1T-48	S. aureus	KLQVHHGRATHTLLLQPPLCA	114
	S. epidermidis	PGTIR
	C. jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-49	S. aureus	SLVRIHDQQPWVTRGAFIDAAR	115
	S. epidermidis	TCS
	C. jeikeium
	P. aeruginosa

1T-50	P. aeruginosa	HSDEPIPNILFKSDSVH	116

1T-51	S. aureus	GKPKRMPAEFIDGYGQALLAGA	117
	P. aeruginosa

1T-52	S. aureus	DEYPAKLPLSDKGATEPRRH	118
	C. xerosis
	P. aeruginosa

1T-53	P. aeruginosa	SDILAEMFEKGELQTLVKDAA	119
		AKANA

1T-54	S. epidermidis	RWVSCNPSWRIQ	120
	C. xerosis
	C. striatum
	P. aeruginosa

1T-55	C. xerosis	NHKTLKEWKAKWGPEAVESW	121
	P. aeruginosa	ATLLG

1T-56	C. xerosis	LALIGAGIWMIRKG	122
	P. aeruginosa

1T-57	P. aeruginosa	RLEYRRLETQVEENPESGRRPM	123
		RG

1T-58	P. aeruginosa	CDDLHALERAGKLDALLSA	124

1T-59	S. aureus	AVGNNLGKDNDSGHRGKKHR	125
	S. epidermidis	KHKHR
	P. aeruginosa

1T-60	S. aureus	YLTSLGLDAAEQAQGLLTILKG	126
	S. epidermidis
	C. jeikeium
	C. striatum
	P. aeruginosa

1T-61	P. aeruginosa	HATLLPAVREAISRQLLPALVP	127
		RG

1T-62	S. epidermidis	GCKGCAQRDPCAEPEPYFRLR	128
	P. aeruginosa

1T-63	S. aureus	EPLILKELVRNLFLFCYARALR	129
	S. epidermidis
	C. jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-64	S. aureus	QTVHHIHMHVLGQRQMHWPPG	130
	S. epidermidis
	C. jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-65	S. mutans	HARAAVGVAELPRGAAVEVEL	131
	S. aureus	IAAVRP
	S. epidermidis
	C. jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-66	S. mutans	DTDCLSRAYAQRIDELDKQYA	132
	S. aureus	GIDKPL
	S. epidermidis
	C. jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-67	S. aureus	GQRQRLTCGRVSGCSEGPSREA	133
	S. epidermidis	AR
	C. jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-68	S. mutans	GGTKEIVYQRG	134
	S. aureus
	C. jeikeium
	C. xerosis
	C. striatum
	P. aeruginosa

1T-69	S. mutans	ILSQEADRKKLF	135
	P. aeruginosa

1T-70	S. aureus	NRQAQGERAHGEQQG	136
	C. jeikeium
	P. aeruginosa

1T-71	P. aeruginosa	KIDTNQWPPNKEG	137

1T-72	P. aeruginosa	EPTDGVACKER	138

1T-73	Streptococcus	GWWEELLHETILSKFKITKALE	139
	pneumoniae	LPIQL

1T-74	S. pneumoniae	DIDWGRKISCAAGVAYGAIDG	140
		CATTV

1T-75	S. pneumoniae	GVARGLQLGIKTRTQWGAATG	141
		AA

1T-76	S. pneumoniae	EMRLSKFFRDFILWRKK	142

1T-77	S. pneumoniae	EMRISRIILDFLFLRKK	143

1T-78	S. pneumoniae	FFKTIFVLILGALGVAAGLYIEK	144
		NYIDK

1T-79	S. pneumoniae	FGTPWSITNFWKKNFNDRPDF	145
		DSDRRRY

1T-80	S. pneumoniae	GGNLGPGFGVIIP	146

1T-81	S. pneumoniae	AIATGLDIVDGKFDGYLWA	147

1T-82	S. pneumoniae	FGVGVGIALFMAGYAIGKDLR	148
		KKFGKSC

1T-83	S. pneumoniae	QKPRKNETFIGYIQRYDIDGNG	149
		YQSLPCPQN

1T-84	S. pneumoniae	FRKKRYGLSILLWLNAFTNLVN	150
		SIHAFYMTLF

1T-85	A. naeslundii, F. nucleatum,	VMASLTWRMRAASASLPTHSR	151
	P. gingivalis	TDA
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-86	S. mitis, S. oralis, S. salivarious	HRKNPVLGVGRRHRAHNVA	152

1T-87	S. mitis, S. mutans, S. oralis	EAVGQDLVDAHHP	153

1T-88	Unanalyzed	GRLVLEITADEVKALGEALAN	154
		AKI

1T-89	S. mitis, S. mutans	HEDDKRRGMSVEVLGFEVVQH	155
		EE

1T-90	S. gordonii, S. mitis, S. mutans,	RNVIGQVL	156
	S. oralis, S. sanguinis

1T-91	S. mitis, S. mutans, S. oralis,	TSVRPGAAGAAVPAGAAGAA	157
	S. sanguinis	GAGWRWP

1T-92	S. mitis, S. mutans	GQDEGQRRAGVGEGQGVDG	158

1T-93	S. epidermidis, S. gordonii,	AMRSVNQA	159
	S. mitis, S. mutans,
	S. oralis, S. sanguinis

1T-94	S. mitis, S. mutans, S. oralis	DQVAHSGDMLVQARRRDS	160

1T-95	S. gordonii, S. mitis, S. mutans,	GHLLRVGGRVGGVGGVAGAC	161
	S. oralis, S. sanguinis	AQPFGGQ

1T-96	S. gordonii, S. mitis, S. mutans,	VAGACAQPFGGQ	162
	S. oralis, S. sanguinis

1T-97	A. naeslundii, F. nucleatum,	GVAERNLDRITVAVAIIWTITIV	163
	P. gingivalis	GLGLVAKLG
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-98	A. naeslundii, F. nucleatum,	VRSAKAVKALTAAGYTGELVN	164
	P. gingivalis	VSGGMKAWLGQ
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-99	S. gordonii, S. mitis, S. mutans,	MKAWLGQ	165
	S. oralis, S. sanguinis

1T-100	S. gordonii, S. mitis, S. mutans	LDPLEPRIAPPGDRSHQGAPAC	166
		HRDPLRGRSARDAER

1T-101	A. naeslundii, P. gingivalis	RLRVGRATDLPLTSFAVGVVR	167
	S. epidermidis,	NLPDAPAH
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. sanguinis

1T-102	A. naeslundii, F. nucleatum,	WKRLWPARILAGHSRRRMRW	168
	P. gingivalis	MVVWRYFAAT
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-103	A. naeslundii, F. nucleatum,	AQFYEAIITGYALGAGQRIGQL	169
	P. gingivalis
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. sanguinis

1T-104	S. mitis	RAVAAHLQGRHHGHQVRRQR	170
		HGQR

1T-105	S. epidermidis, S. gordonii,	GEGLPPPVLHLPPPRMSGR	171
	S. mitis, S. mutans,
	S. oralis

1T-106	S. gordonii, S. mitis, S. mutans,	DALRRSRSQGRRHR	172
	S. oralis, S. salivarious

1T-107	A. naeslundii S. epidermidis,	SPVPRFTAVGGVSRGSP	173
	S. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-108	S. gordonii, S. mitis, S. mutans,	WGPLGPERPLW	174
	S. oralis, S. salivarious,
	S. sanguinis

1T-109	A. naeslundii S. epidermidis,	VTTNVRQGAGS	175
	S. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-110	A. naeslundii, P. gingivalis	LAAKTAVCVGRAFM	176
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. sanguinis

1T-111	A. naeslundii, F. nucleatum,	GRLSRREEDPATSIILLRGAYR	177
	P. gingivalis	MAVF
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-112	S. gordonii	SDNDGKLILGTSQ	178

1T-113	S. mitis	HGAHQRTGQRLHHHRGRTVSG	179
		CRQNPVAGVDPDEHR

1T-114	A. naeslundii, P. gingivalis	RQAPGPGLVTITAACSAPGSRSR	180
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. sanguinis

1T-115	A. naeslundii, F. nucleatum,	LLIERFSNHH	181
	P. gingivalis
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-116	A. naeslundii, P. gingivalis	MILHRRRDR	182
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-117	S. mutans	GPGVVGPAPFSRLPAHALNL	183

1T-118	A. naeslundii, F. nucleatum,	TASPPAPSDQGLRTAFPATLLIA	184
	P. gingivalis	LAALARISR
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-119	S. gordonii, S. mitis, S. mutans,	SPATQKAPTRAQPSRAPVQDC	185
	S. oralis	GDGRPTAAPDDVERLSPR

1T-120	A. naeslundii, F. nucleatum,	DVRDRVDLAGADLCAAHATR	186
	P. gingivalis
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-121	S. gordonii, S. mitis, S. mutans,	FAKETGFGIGGAQEGWWIIADI	187
	S. oralis, S. salivarious,	YGPNPF
	S. sanguinis

1T-122	S. mitis	GAIPDPVTHRVDWEEDHQTRP	188
		SR

1T-123	S. gordonii	LVRRNAVAGRSDGLAGAEQLD	189
		LVRLQGVL

1T-124	S. mitis, S. mutans, S. oralis	LFDERNKIA	190

1T-125	S. epidermidis, S. gordonii,	DAITGGNPPLSDTDGLRP	191
	S. mutans, S. oralis

1T-126	S. gordonii, S. mitis, S. mutans	QGLARPVLRRIPL	192

1T-127	A. naeslundii, F. nucleatum,	YDPVPKRKNKNSEGKREE	193
	P. gingivalis,
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-128	A. naeslundii, P. gingivalis	SGSAIRMLEIATKMLKR	194
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-129	A. naeslundii, P. gingivalis	YDKYIKYLSIQPPFIVYFI	195
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-130	A. naeslundii, F. nucleatum,	QKIIDMSKFLFSLILFIMIVVIYI	196
	P. gingivalis	GKSIGGYSAIVSSIMLELDTVLY
	S. epidermidis,	NKKIFFIYK
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-131	A. naeslundii, F. nucleatum,	DEVWKMLGI	197
	P. gingivalis,
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-132	A. naeslundii, F. nucleatum,	YSKKLFEYFYFIIFILIRYLIFYKI	198
	P. gingivalis	IQNKNYYINNIAYN
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-133	A. naeslundii, P. gingivalis	YFIKDDNEALSKDWEVIGNDL	199
	S. epidermidis,	KGTIDKYGKEFKVR
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-134	A. naeslundii, F. nucleatum,	SRLVREIKKKCRKS	200
	P. gingivalis
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-135	A. naeslundii, P. gingivalis	FESLLPQATKKIVNNKGSKINKIF	201
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-136	A. naeslundii, F. nucleatum,	ELLTQIRLALLYSVNEW	202
	P. gingivalis,
	S. epidermidis, S. gordonii,
	S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-137	A. naeslundii, F. nucleatum,	PLNFYRAVKENRLPLSEKNIND	203
	P. gingivalis	FTNIKLKVSPKLINLLQESSIFY
	S. epidermidis,	NFSPKKRNTN
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-138	A. naeslundii, F. nucleatum,	YPNEYCIFLENLSLEELKEIKAI	204
	P. gingivalis	NGETLNLEEIINERKNLKD
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-139	A. naeslundii S. gordonii,	AVAGAAVGALLGNDARSTAV	205
	S. mitis, S. mutans, S. oralis	GAAIGGALGAGAGELTKNK

1T-140	A. naeslundii, F. nucleatum,	IKGTIAFVGEDYVEIRVDKGVK	206
	P. gingivalis	LTFRKSAIANVINNNQQ
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-141	F. nucleatum, P. gingivalis	KKFIILLFILVQGLIFSATKTLSD	207
	S. epidermidis,	IIAL
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. sanguinis

1T-142	A. naeslundii, F. nucleatum,	FTQGIKRIVLKRLKED	208
	P. gingivalis,
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-143	A. naeslundii, F. nucleatum,	MPKRHYYKLEAKALQFGLPFA	209
	P. gingivalis	YSPIQLLK
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-144	A. naeslundii, F. nucleatum,	IIELHPKSWTQDWRCSFL	210
	P. gingivalis,
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-145	S. mitis, S. mutans, S. oralis	VEAGKRNISLENIEKISKGLGISI	211
		SELFKYIEEGEDKIG

1T-146	A. naeslundii, F. nucleatum,	RNSADNQTKIDKIRIDISLWDE	212
	P. gingivalis,	HLNIVKQGK
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-147	A. naeslundii, F. nucleatum,	GVENRRFYERDVSKVSMMTSE	213
	P. gingivalis,	AVAPRGGSK
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-148	A. naeslundii, F. nucleatum,	IVELDDTTILERALSMLGEANA	214
	P. gingivalis,
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-149	A. naeslundii, F. nucleatum,	SVRAVKPIDETVARHFPGDFIVN	215
	P. gingivalis,
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-150	A. naeslundii, F. nucleatum,	YINRRLKKAFSDADIKEAPAEF	216
	P. gingivalis,	YEELRRVQYV
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-151	A. naeslundii, F. nucleatum,	SVRAVKPIDEIVAWHFPGDFIVN	217
	P. gingivalis,
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-152	A. naeslundii, F. nucleatum,	YVSADESAYNHIVTDDIPLADR	218
	P. gingivalis	RIEAVQQ
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-153	A. naeslundii, F. nucleatum,	YIACPGYFY	219
	P. gingivalis
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-154	P. gingivalis	YFSFLEIVGMARR	220

1T-155	A. naeslundii, F. nucleatum,	LKLAFGVYPFQAMSQSDTAVS	221
	P. gingivalis	ERNVLWR
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-156	A. naeslundii, F. nucleatum,	GRFQISIRGEEKSKVKVQGKGT	222
	P. gingivalis,	FTDRNTT
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-157	A. naeslundii, F. nucleatum,	RRFRKTTENREKSKNKKAVLG	223
	P. gingivalis,	LSTTSTASY
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-158	A. naeslundii, F. nucleatum,	WENKPSPLGSIKKLQGLVYRLI	224
	P. gingivalis	GYRHFWV
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-159	P. gingivalis	IFSLHHFALICSEMGTFAVSKRA		225
		KYKWEVL

1T-160	A. naeslundii, F. nucleatum,	AQYKYINKLLN	226
	P. gingivalis,
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-161	A. naeslundii, F. nucleatum,	NKVLQVEVMWDGSVVGRPAG	227
	P. gingivalis	VISIKSSKKG
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-162	A. naeslundii, F. nucleatum,	QKAKEESDRKAAVSYNGFHRV	228
	P. gingivalis,	NVVSIPK
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-163	A. naeslundii, F. nucleatum,	MENILIYIPMVLSPFGSGILLFLG	229
	P. gingivalis	KDRRYML
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-164	A. naeslundii, F. nucleatum,	KKSHSQGKRKLKDLNSAYKID	230
	P. gingivalis	NQLHYALR
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-165	A. naeslundii, F. nucleatum,	CYDSFDFSIFVTFANRMKLSVGS	231
	P. gingivalis
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-166	A. naeslundii, F. nucleatum,	AQSAGQIKRKSKVRIHV	232
	P. gingivalis
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-167	A. naeslundii, F. nucleatum,	SRMSEHSPAGLVFEVGPMDKG	233
	P. gingivalis	SFIILDSYHPTVKK
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-168	A. naeslundii, F. nucleatum,	ELHRIMSTEKIGAVTKMNFDTA	234
	P. gingivalis	PIMSILPIDIYPKEVGIGS
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-169	A. naeslundii, F. nucleatum,	FARVRRLHQNRILTQPLTNLKY	235
	P. gingivalis	CLRQPIYSD
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-170	P. gingivalis	AYGKVFSMDIMLSENDKLIVLR	236
		ISHHSAWH

1T-171	A. naeslundii, F. nucleatum,	SVRAVKPIDKTVARHFPGDFIVN	237
	P. gingivalis
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-172	A. naeslundii, F. nucleatum,	FEGLKNLLGDDII	238
	P. gingivalis
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-173	A. naeslundii, F. nucleatum,	LFRKEDQEHVLL	239
	P. gingivalis
	S. gordonii, S. mitis,
	S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-174	A. naeslundii, F. nucleatum,	SGGSDTDGSSSGEPGSHSGDL	240
	P. gingivalis,
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-175	A. naeslundii, F. nucleatum,	GEPGSHSGDL	241
	P. gingivalis,
	S. epidermidis, S. gordonii,
	S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-176	A. naeslundii, P. gingivalis	PVGDIMSGFLRGANQPRFLLDH	242
	S. epidermidis,	ISFGS
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-177	P. gingivalisS. gordonii,	GTNVPTQILGYSREERFDYEPA	243
	S. mitis, S. mutans, S. oralis,	PEQR
	S. salivarious, S. sanguinis

1T-178	A. naeslundii, F. nucleatum,	LLASHPERLSLGVFFVYRVLHL	244
	P. gingivalis	LLENT
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-179	A. naeslundii, F. nucleatum,	TCYPLIQRKTDRAYEA	245
	P. gingivalis,
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-180	A. naeslundii, F. nucleatum,	VVFGGGDRLV	246
	P. gingivalis,
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-181	A. naeslundii, F. nucleatum,	YGKESDP	247
	P. gingivalis,
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-182	A. naeslundii, F. nucleatum,	LTASICRQWNDNSTPYQR	248
	P. gingivalis,
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-183	A. naeslundii, F. nucleatum,	PLRSFVAEKAEHAFRVVRIADF	249
	P. gingivalis	DFGHS
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-184	A. naeslundii, F. nucleatum,	ALLVLNLLLMQFFFGKNM	250
	P. gingivalis,
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-185	A. naeslundii, F. nucleatum,	HYHFLLEFGFHKGDYLE	251
	P. gingivalis,
	T. denticolaS. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-186	S. sanguinis	LAKKNQALREEISRQKSK	252

1T-187	S. sanguinis	RAGRIKKLSQKEAEPFEN	253

1T-188	S. sanguinis	HRKDVYKK	254

1T-189	F. nucleatum, S. sanguinis	FIRSKLRRVDFSGVRRGNKHFL	255
		LDKLLITLVK

1T-190	A. naeslundii, F. nucleatum,	IQIIVNAFVEKDKTGAVIEVLYA	256
	P. gingivalis	SNNHEKVKAKYEELVAIS
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-191	F. nucleatum, S. sanguinis	SALFYDTLAAIWISIAGVDARW	257
		GH

1T-192	S. sanguinis	ILVLLALQVELDSKFQY	258

1T-193	S. sanguinis	LMIFDKHANLKYKYGNRSFGV	259
		EAIM

1T-194	F. nucleatum, S. sanguinis	LAGATLVTPYCVGWGLIRSH	260

1T-195	Unanalyzed	AASGFTYCASNGVWHPY	261

1T-196	F. nucleatum, S. sanguinis	KPEKEKLDTNTLMKVVNKALS	262
		LFDRLLIKFGA

1T-197	A. naeslundii, F. nucleatum,	TEILNFLITVCADRENWKIKHG	263
	P. gingivalis	LSDSVLLIFFARFTGAEYW
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-198	P. gingivalisS. epidermidis,	MPVSKKRYMLSSAYATALGIC	264
	S. gordonii,	YGQVATDEKESEITAIPDLLDY
	S. mitis, S. mutans, S. oralis,	LSVEEYLL
	S. sanguinis

1T-199	S. sanguinis	RAGRIKKLSQKEAEPFEN	265

1T-200	A. naeslundii, F. nucleatum	MRFKRFDRDYALSGDNVFEVL	266
	S. epidermidis,	TASCDVIERNLSYREMCGLMQ
	S. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-201	S. sanguinis	KRKHENVIVAEEMRVIKN	267

1T-202	A. naeslundii, F. nucleatum,	LCRLEKLCKQFLRQDKVVTYY	268
	P. gingivalis	LMLPYKRAIEAFYQELKERS
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-203	A. naeslundii, F. nucleatum,	YPFCLATVDHLPEGLSVTDYER	269
	P. gingivalis	VQRLVSQFLLNKEER
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-204	F. nucleatum, S. sanguinis	KYLFLASKTKEYFKSHFREIMI	270
		DV

1T-205	A. naeslundii, F. nucleatum,	FISFVDCIQNIEKIEKELLKIGIT	271
	P. gingivalis	DIQINQDAGWLY
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-206	S. sanguinis	AGFAAGYSL	272

1T-207	F. nucleatum, S. sanguinis	SPLEKYGTGSMTALTFLLGCCL	273
		LVLSKKSR

1T-208	Unanalyzed	KRKRWAILTLFLAGLGAVGIVL	274
		ATF

1T-209	F. nucleatum, S. sanguinis	WSGAAVGAATFC	275

1T-210	S. sanguinis	SVGMGVIERGSFDFSASAILQK	276
		RETKCLKNKPFT

1T-211	S. sanguinis	AEPIIKVTEG	277

1T-212	S. sanguinis	LLSALIKKLALIIFIG	278

1T-213	S. sanguinis	AYLLTCAAAGGMIGYGAATLD	279

1T-214	S. sanguinis	MIGYGAATLD	280

1T-215	S. sanguinis	VCFKDISVFLSPFRGQEVLFCG	281
		KAKHSLIYVIGT

1T-216	S. sanguinis	FFLNVIAIRIPHF	282

1T-217	F. nucleatum, S. sanguinis	MLSNVLSRSVVSPNVDIPNSMV	283
		ILSPLLISISNYH

1T-218	F. nucleatum, S. sanguinis	KLIFAALGLVFLLIGLRDSRSK	284

1T-219	S. sanguinis	RNINVSATFITEKSLV	285

1T-220	A. naeslundii, F. nucleatum,	AILTFFMALLFTYLKEKAQILY	286
	P. gingivalis	WPLFLHLMFYFVTA
	S. epidermidis,
	S. gordonii, S. oralis, S. salivarious,
	S. sanguinis

1T-221	A. naeslundii, F. nucleatum,	DIGRIIGKKGRTITAIRSIVYSVP	287
	P. gingivalis	TQGKKVRLVIDEK
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-222	F. nucleatum, S. sanguinis	RIEASLISAIMFSMFNAIVKFLQK	288

1T-223	A. naeslundii, F. nucleatum,	NQKMEINSMTSEKEKMLAGHF	289
	P. gingivalis	HNEANFAVIFKYSLFYNFF
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-224	A. naeslundii, F. nucleatum,	VHLFSFVKLIYYDIMKYSIEEK	290
	P. gingivalis	VFFESPVGEIIQ
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-225	A. naeslundii, F. nucleatum,	RRSLGNSASFAEWIEYIRYLHYI	291
	P. gingivalis	IRVQFIHFFSKNKKI
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-226	A. naeslundii, F. nucleatum	KLQEKQIDRNFERVSGYSTYRA	292
	S. epidermidis,	VQAAKAKEKGFISLEN
	S. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-227	S. sanguinis	RFEQFFADHYPFV	293

1T-228	A. naeslundii, F. nucleatum,	IFKLFEEHLLYLLDAFYYSKIFR	294
	P. gingivalis	RLKQGLYRRKEQPYTQDLFRM
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-229	S. gordonii, S. oralis, S. sanguinis	LLINYVKVNMSY	295

1T-230	A. naeslundii, F. nucleatum,	EFLEKFKVLKQPRKANNISKNR	296
	P. gingivalis	VAMIFLTIHKSRGFLSSPY
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-231	S. gordonii, S. mitis, S. mutans,	FDFLCSPDSSR	297
	S. oralis, S. salivarious

1T-232	S. sanguinis	AYSLTFQNPNDNLTDEEVAKY	298
		MEKITKALTEKIGAEVR

1T-233	A. naeslundii, P. gingivalis	TDQELEHLIVTELESKRLDFTYS	299
	S. epidermidis,	KDITEFFDEAFPEYDQNY
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-234	A. naeslundii, F. nucleatum,	DNFYLILKMEERGKSKKTSQTR	300
	P. gingivalis	GFRAFFDIIRKKIKKEDGK
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-235	S. sanguinis	GWLSDDFWLKSAIPLLKKRLA	301
		KWNETL

1T-236	A. naeslundii, F. nucleatum,	PVQKALHVVSAYATDLGICYD	302
	P. gingivalis	QVVTVMIREVKTQLYQIY
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. sanguinis

1T-237	S. sanguinis	EDPVPNHFTLRRNKKEKPSKS	303

1T-238	A. naeslundii, F. nucleatum,	IFNRRKFFQYFGLSKEAMVEHI	304
	P. gingivalis	QPFILDIWQIHLF
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-239	A. naeslundii S. gordonii,	ADDLLNKRLTDLIMENAETVK	305
	S. mitis, S. mutans, S. oralis,	TIDLDNSD
	S. sanguinis

1T-240	A. naeslundii, F. nucleatum,	VILGNGISNIAQTLGQLPNIAW	306
	P. gingivalis	VWIYMVLIAALLEESNVC
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-241	S. sanguinis	TQKTYLHIIRELENQDIDLIMRS	307
		LTSLT

1T-242	F. nucleatum, S. sanguinis	KQVQNTTLIICGTVLLGILFKSY	308
		LKSQKSV

1T-243	A. naeslundii, P. gingivalis	SENIARFAAAFENEQVVSYAR	309
	S. epidermidis,	WFRRSWRGSGSSSRF
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-244	A. naeslundii, P. gingivalis	MTWAEIGAIVGATIGSFYIPNPV	310
	S. epidermidis,	IVPFRVR
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. sanguinis

1T-245	F. nucleatum, S. sanguinis	IIGGISGAGVGIASFC	311

1T-246	S. sanguinis	ISGAGVGIASFC	312

1T-247	A. naeslundii, F. nucleatum,	LSISQRTDRVIVMDKGKIIEEGT	313
	P. gingivalis	HSELIAANGFYHHLFNK
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-248	S. sanguinis	IGGALNSCG	314

1T-249	F. nucleatum, S. sanguinis	VFSVLKHTTWPTRKQSWHDFIS	315
		ILEYSAFFALVIFIFDKLLTLGLA
		ELLKRF

1T-250	S. mitis, S. mutans, S. oralis	LVQGDTILIENHVGTPVKDDGK	316
		DCLIIREADVLAVVND

1T-251	S. mitis, S. oralis, S. sanguinis	LVMNDETIYLFTYENGQISYEE	317
		DKRDCSKNV

1T-252	F. nucleatum, S. sanguinis	MKKNLKRFYALVLGFIIGCLFV	318
		SILIFIGY

1T-253	A. naeslundii, F. nucleatum,	KTKESLTQQEKKFLKDYDRKS	319
	P. gingivalis	LHHFRDILTYCFILDKLTNK
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-254	S. sanguinis	RFLKDELSVSVRLQEKSIEALPF	320
		RTKIEIEIESDNQIKTL

1T-255	A. naeslundii, F. nucleatum,	LFIVEYKDKASVPGEIDNTYVE	321
	P. gingivalis	SYTYSDILTEKTLIRYFD
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-256	S. sanguinis	KGKSLMPLLKQINQWGKLYL	322

1T-257	A. naeslundii, F. nucleatum,	IILAKAADLAEIERIISEDPFKIN	323
	P. gingivalis	EIANYDIIEFCPTKSSKAFEKVLK
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-258	A. naeslundii, F. nucleatum,	TINIDDKVLDYLKKINSKAITID	324
	P. gingivalis,	LIGCAS
	T. denticola, S. mitis, S. mutans,
	S. oralis

1T-259	F. nucleatum, P. gingivalis,	EKLKKILLKLAVCGKAWYTL	325
	T. denticola,
	S. mitis, S. mutans, S. oralis,
	S. sanguinis

1T-260	A. naeslundii, P. gingivalis	NILYFIHDENQWEPQKAEIFRG	326
	S. epidermidis,	SIKHCAWLSS
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. sanguinis

1T-261	F. nucleatum S. mutans,	SFEKNKIENNLKIAQAYIYIKPK	327
	S. oralis S. sanguinis	PRICQA

1T-262	A. naeslundii, F. nucleatum,	LSLPLIVLTKSI	328
	P. gingivalis
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-263	A. naeslundii, F. nucleatum,	FIAVSFTGNPATFKLVIGCKADN	329
	P. gingivalis
	S. epidermidis,
	S. gordonii, S. mitis, S. oralis,
	S. salivarious, S. sanguinis

1T-264	S. sanguinis	LEGKFYMAEDFDKTPECFKDYV	330

1T-265	A. naeslundii, F. nucleatum,	GMFENLLMINFQIMNDLKIEIV	331
	P. gingivalis	VKDRICAV
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-266	S. sanguinis	RAGTWLVVDEIR	332

1T-267	A. naeslundii, F. nucleatum,	RIKEERKNRSYKFFIWRLFDEK	333
	P. gingivalis,	TGFI
	T. denticola, S. mitis, S. mutans,
	S. oralis S. sanguinis

1T-268	F. nucleatum S. mutans,	PITPKKEKCGLGTYAPKNPVFS	334
	S. oralis S. sanguinis	KSRV

1T-269	F. nucleatum S. mutans,	PLYVAAVEKINTAKKH	335
	S. oralis S. sanguinis

1T-270	F. nucleatum S. mutans,	VHEFDIQKILQNR	336
	S. oralis S. sanguinis

1T-271	A. naeslundii, F. nucleatum,	FLIQKFLLIKTFPPYRKKYVVIV	337
	P. gingivalis	SQTGTA
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-272	F. nucleatum S. mutans,	QLAPIDKQLKAVKKIAFYESES	338
	S. oralis S. sanguinis	TAAKAVTVA

1T-273	F. nucleatum, P. gingivalis,	YNEPNYKWLESYKIYKQRCED	339
	T. denticola,	RTGMYYTEET
	S. mitis, S. mutans, S. oralis

1T-274	F. nucleatum S. mutans,	ETTTEINAIKLHRIKQRSPQGTR	340
	S. oralis S. sanguinis	RVN

1T-275	A. naeslundii, F. nucleatum,	QVLKNFSISRRYKINNPFFKILL	341
	P. gingivalis,	FIQLRTL
	T. denticola, S. epidermidis,
	S. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-276	A. naeslundii, F. nucleatum,	ILTLLILGSIGFFILKIKLKLGRF	342
	P. gingivalis
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. sanguinis

1T-277	A. naeslundii, F. nucleatum,	IYYMRFVNKPLEKTFFKI	343
	P. gingivalis,
	T. denticola S. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-278	A. naeslundii, F. nucleatum,	SINSSAGIQPHCLSSSFVLRTKH	344
	P. gingivalis,	CFY
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-279	A. naeslundii, F. nucleatum,	FVLRTKHCFY	345
	P. gingivalis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-280	A. naeslundii, F. nucleatum,	TNNKNKVIIKAIKFKNKDFINL	346
	P. gingivalis,	DLFIYRR
	T. denticola S. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-281	A. naeslundii, F. nucleatum,	KYEKLTKENLFIRNSGNMCVFI	347
	P. gingivalis	YFLFFG
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-282	F. nucleatum, P. gingivalis,	ISLVFPAYT	348
	S. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-283	A. naeslundii, F. nucleatum,	LCTKLEDKQRGRIPAELFIISPIK	349
	P. gingivalis,	ILERNDAL
	T. denticola, S. epidermidis,
	S. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-284	A. naeslundii, F. nucleatum,	FQYYFSLKRV	350
	P. gingivalis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-285	A. naeslundii, F. nucleatum,	FFPYYLADFYKQLKFLNEYQT	351
	P. gingivalis,	KNKDKVVEFK
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-286	S. sanguinis	LGFFNNKADLVKADTERDNRM	352
		SSLKIKDL

1T-287	P. gingivalis, T. denticola	KGYPLPFQYRLNNH	353
	S. gordonii, S. mitis,
	S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-288	F. nucleatum, S. gordonii,	RWVGGEPSADIYLSAKDTKT	354
	S. salivarious,
	S. sanguinis

1T-289	F. nucleatum, P. gingivalis,	EPSADIYLSAKDTKT	355
	S. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. sanguinis

1T-290	A. naeslundii, F. nucleatum,	IINQLNLILLRLMEILIL	356
	P. gingivalis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-291	A. naeslundii, F. nucleatum,	DMKIIKLYIKILSFLFIKYCNKK	357
	P. gingivalis,	LNSVKLKA
	T. denticola, S. mitis, S. mutans,
	S. oralis

1T-292	A. naeslundii, F. nucleatum,	IINQLNLILLRLMEILIL	358
	P. gingivalis
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-293	A. naeslundii, F. nucleatum,	HVEDCFLLSNARTTAIHGRANP	359
	P. gingivalis	ARGEPRTRSE
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-294	T. denticola	YDKIADGVFKIGKRGVL	360

1T-295	S. mitis, S. salivarious,	KYKLKKIIL	361
	S. sanguinis

1T-296	A. naeslundii, F. nucleatum,	EYSQQSFKAKPCSERGVLSP	362
	P. gingivalis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-297	A. naeslundii, F. nucleatum,	RSLRLNNALTKLPKLWYNRIKE	363
	T. denticola,	AFYAYNDYDK
	S. mitis, S. mutans, S. oralis

1T-298	A. naeslundii, F. nucleatum,	ILNKKPKLPLWKLGKNYFRRF	364
	P. gingivalis,	YVLPTFLA
	T. denticola S. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-299	A. naeslundii, F. nucleatum	SMLTSFLRSKNTRSLKMYKDV	365
	S. epidermidis,	HF
	S. gordonii,
	S. mitis, S. mutans, S. oralis,
	S. salivarious, S. sanguinis

1T-300	A. naeslundii, F. nucleatum,	PLIISKAQIKMSGDILGSCFKLF	366
	P. gingivalis	YLRPFF
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-301	F. nucleatum, S. gordonii,	SKLPRVLDASLKL	367
	S. sanguinis

1T-302	A. naeslundii, P. gingivalis	IIIILPKIYLVCKTV	368
	S. epidermidis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-303	A. naeslundii, F. nucleatum,	LDYENMDCKKRIRI	369
	P. gingivalis,
	S. gordonii, S. mitis, S. mutans,
	S. oralis, S. salivarious,
	S. sanguinis

1T-304	P. gingivalis	STAGEASRRTASEASRRTAAKL	370
		RG

TT-305	F. nucleatum	ARNALNMRDVPVDAAIIGIIDG	371
		MDEE

TT-306	F. nucleatum	KILNEAEGKLLKVIEKNGEIDIE	372
		EI

TT-307	F. nucleatum	NGDKKAKEELDKWDEVIKELN	373
		IQF

TT-308	F. nucleatum	GLVIIPNLIALIILFSQVRQQTKD	374
		YFSNPKLSSR

TT-309	F. nucleatum	EPLPLTKYDKKDTEMKKVFKEI	375
		LAGKVGYEKEEE

TT-310	F. nucleatum	TKLKKNNKLLSAKKENTLHTK	376
		DK

TT-311	S. mutans, S. sobrinus	AIFDAMHNL	377

As described above, in certain embodiments of the present invention, the targeting moiety can comprise targeting peptide capable of binding, specifically binding, or preferentially binding to a microorganism, e.g., a target microbial organism. In one embodiment, the targeting peptide be identified via screening peptide libraries. For example, a phage display peptide library can be screened against a target microbial organism or a desired antigen or epitope thereof. Any peptide identified through such screening can be used as a targeting peptide for the target microbial organism. Illustrative additional targeting peptides are shown in Table 4.

TABLE 4

Additional illustrative targeting moieties.

Targeting Moiety/		SEQ ID
Organism	Structure/sequence	NO

LPSB-1	RGLRRLGRRGLRRLGR	378

Phob-1	KPVLPVLPVLPVL	379

LPSB-2	VLRIIRIAVLRIIRIA	380

LPTG-1	LPETGGSGGSLPETG	381

α-1	RAHIRRAHIRR	382

ANION-1	DEDEDDEEDDDEEE	383

PHILIC-1	STMCGSTMCGSTMCG	384

SA5.1/S. aureus	VRLPLWLPSLNE	385

SA5.3/S. aureus	ANYFLPPVLSSS	386

SA5.4/S. aureus	SHPWNAQRELSV	387

SA5.5/S. aureus	SVSVGMRPMPRP	388

SA5.6/S. aureus	WTPLHPSTNRPP	389

SA5.7/S. aureus	SVSVGMKPSPRP	390

SA5.8/S. aureus	SVSVGMKPSPRP	391

SA5.9/S. aureus	SVPVGPYNESQP	392

SA5.10/S. aureus	WAPPLFRSSLFY	393

SA2.2/S. aureus	WAPPXPXSSLFY	394

SA2.4/S. aureus	HHGWTHHWPPPP	395

SA2.5/S. aureus	SYYSLPPIFHIP	396

SA2.6/S. aureus	HFQENPLSRGGEL	397

SA2.7/S. aureus	FSYSPTRAPLNM	398

SA2.8/S. aureus	SXPXXMKXSXXX	399

SA2.9/S. aureus	VSRHQSWHPHDL	400

SA2.10/S. aureus	DYXYRGLPRXET	401

SA2.11/S. aureus	SVSVGMKPSPRP	402

S. aureus/Consensus	V/Q/H-P/H-H-E-F/Y-K/H-H/A-L/H-X-X-K/R-P/L	403

DH5.1/E. coli	KHLQNRSTGYET	404

DH5.2/E. coli	HIHSLSPSKTWP	405

DH5.3/E. coli	TITPTDAEMPFL	406

DH5.4/E. coli	HLLESGVLERGM	407

DH5.5/E. coli	HDRYHIPPLQLH	408

DH5.6/E. coli	VNTLQNVRHMAA	409

DH5.7/E. coli	SNYMKLRAVSPF	410

DH5.8/E. coli	NLQMPYAWRTEF	411

DH5.9/E. coli	QKPLTGPHFSLI	412

CSP/S. mutans	SGSLSTFFRLFNRSFTQALGK	413

CSPC18/S. mutans	LSTFFRLFNRSFTQALGK	414

CSPC16/S. mutans	TFFRLFNRSFTQALGK	415

CSPM8/S. mutans	TFFRLFNR	416

KH/Pseudomonas spp	KKHRKHRKHRKH	417
(US 2004/0137482)

cCF10	LVTLVFV	418

AgrD1	YSTCDFIM	419

AgrD2	GVNACSSLF	420

AgrD3	YINCDFLL	421

NisinA	ITSISLCTPGCKTGALMGCNMRTATCIICSIIIVSK	422

PlnA	KSSAYSLQMGATAIKQVKKLFKKWGW	423

S3L1-5	WWYNWWQDW	424

Penetratin	RQIKIWFWNRRMKWKK*	425

Tat	EHWSYCDLRPG	426

Pep-1N	KETWWETWWTEW	427

Pep27	MRKEFHNVLSSGQLLADKRPARDYNRK	428

HABP35	LKQKIKHVVKLKVVVKLRSQLVKRKQN	429

HABP42 (all D)	STMMSRSHKTRSHHV	430

HABP52	GAHWQFNALTVRGGGS	431

Hi3/17	KQRTSIRATEGCLPS	432

α-E. coli peptide	QEKIRVRLSA	433

Salivary Receptor	QLKTADLPAGRDETTSFVLV*	434
Adhesion Fragment

S1 (Sushi frag.)	GFKLKGMARISCLPNGQWSNFPPKCIRECAMVSS	435
(LPS binding)

S3 (Sushi frag.)	HAEHKVKIGVEQKYGQFPQGTEVTYTCSGNYFLM	436
(LPS binding)

MArg.1	AMDMYSIEDRYFGGYAPEVG	437
(Mycoplasma infected
cell line binding peptide

BPI fragment
1	ASQQGTAALQKELKRIKPDYSDSFKIKH	438
(LPS binding)
6,376,462

BPI fragment 2	SSQISMVPNVGLKFSISNANIKISGKWKAQKRFLK	439
(LPS binding)
6,376,462

BPI fragment 3	VHVHISKSKVGWLIQLFHKKIESALRNK	440
(LPS binding)
6,376,462

LBP fragment 1	AAQEGLLALQSELLRITLPDFTGDLRIPH	441
(LPS binding)
6,376,462

LBP fragment 2	HSALRPVPGQGLSLSISDSSIRVQGRWKVRKSFFK	442
(LPS binding)
6,376,462

LBP fragment 3	VEVDMSGDLGWLLNLFHNQIESKFQKV	443
(LPS binding)
6,376,462

B. anthracis spore	ATYPLPIR	444
binding
(WO/1999/036081)

Bacillus spore binding	peptides of 5-12 amino acids containing the sequence	445
(WO/1999/036081)	Asn-His-Phe-Leu
	peptides of 5-12 amino acids containing the sequence	446
	Asn-His-Phe-Leu-Pro
	Thr-Ser-Glu-Asn-Val-Arg-Thr (TSQNVRT)	447
	A peptide of formula Thr-Tyr-Pro-X-Pro-X-Arg	448
	(TYPXPXR) where X is a Ile, Val or Leu.
	A peptide having the sequence TSQNVRT.	449
	A peptide having the sequence TYPLPIR	450

LPS binding peptide 1	TFRRLKWK	451
(6,384,188)

LPS BP 2 (6,384,188)	RWKVRKSFFKLQ	452

LPS BP 3 (6,384,188)	KWKAQKRFLKMS	453

Pseudomonas pilin	KCTSDQDEQFIPKGCSK	454
binding peptide
(5,494,672)

Patents and patent publications disclosing the referenced antibodies are identified in the table.

In certain embodiments the targeting moieties can comprise other entities, particularly when utilized with an antimicrobial peptide as described, for example, in Table 2. Illustrative targeting moieties can include a polypeptide, a peptide, a small molecule, a ligand, a receptor, an antibody, a protein, or portions thereof that specifically interact with a target microbial organism, e.g., the cell surface appendages such as flagella and pili, and surface exposed proteins, lipids and polysaccharides of a target microbial organism.
Targeting Antibodies.
In certain embodiments the targeting moieties can comprise one or more antibodies that bind specifically or preferentially a microorganism or group of microorganisms (e.g., bacteria, fungi, yeasts, protozoa, viruses, algae, etc.). The antibodies are selected to bind an epitope characteristic or the particular target microorganism(s). In various embodiments such epitopes or antigens are typically is gram-positive or gram-negative specific, or genus-specific, or species-specific, or strain specific and located on the surface of a target microbial organism. The antibody that binds the epitope or antigen can direct an anti-microbial peptide moiety or other effector to the site. Furthermore, in certain embodiments the antibody itself can provide anti-microbial activity in addition to the activity provided by effector moiety since the antibody may engage an immune system effector (e.g., a T-cell) and thereby elicit an antibody-associated immune response, e.g., a humoral immune response.
Antibodies that bind particular target microorganisms can be made using any methods readily available to one skilled in the art. For example, as described in U.S. Pat. No. 6,231,857 (incorporated herein by reference) three monoclonal antibodies, i.e., SWLA1, SWLA2, and SWLA3 have been made against S. mutans. Monoclonal antibodies obtained from non-human animals to be used in a targeting moiety can also be humanized by any means available in the art to decrease their immunogenicity and increase their ability to elicit anti-microbial immune response of a human. Illustrative microorganisms and/or targets to which antibodies may be directed are shown, for example, in Tables 5.
Various forms of antibody include, without limitation, whole antibodies, antibody fragments (e.g., (Fab′)₂, Fab′, etc.), single chain antibodies (e.g., scFv), minibodies, Di-miniantibody, Tetra-miniantibody, (scFv)₂, Diabody, scDiabody, Triabody, Tetrabody, Tandem diabody, VHH, nanobodies, affibodies, unibodies, and the like.
Methods of making such antibodies are well known to those of skill in the art. In various embodiments, such methods typically involve providing the microorganism, or a component thereof for use as an antigen to raise an immune response in an organism or for use in a screening protocol (e.g., phage or yeast display).
For example, polyclonal antibodies are typically raised by one or more injections (e.g. subcutaneous or intramuscular injections) of the target microorganism(s) or components thereof into a suitable non-human mammal (e.g., mouse, rabbit, rat, etc.).
If desired, the immunizing microorganism or antigen derived therefrom can be administered with or coupled to a carrier protein by conjugation using techniques that are well-known in the art. Such commonly used carriers which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The coupled peptide is then used to immunize the animal (e.g. a mouse or a rabbit).
The antibodies are then obtained from blood samples taken from the mammal. The techniques used to develop polyclonal antibodies are known in the art (see, e.g., Methods of Enzymology, “Production of Antisera With Small Doses of Immunogen: Multiple Intradermal Injections”, Langone, et al. eds. (Acad. Press, 1981)). Polyclonal antibodies produced by the animals can be further purified, for example, by binding to and elution from a matrix to which the peptide to which the antibodies were raised is bound. Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies see, for example, Coligan, et al. (1991) Unit 9, Current Protocols in Immunology, Wiley Interscience).
In certain embodiments the antibodies produced will be monoclonal antibodies (“mAb's”). The general method used for production of hybridomas secreting mAbs is well known (Kohler and Milstein (1975) Nature, 256:495
Antibody fragments, e.g. single chain antibodies (scFv or others), can also be produced/selected using phage display and/or yeast display technology. The ability to express antibody fragments on the surface of viruses that infect bacteria (bacteriophage or phage) or yeasts makes it possible to isolate a single binding antibody fragment, e.g., from a library of greater than 10¹⁰nonbinding clones. To express antibody fragments on the surface of phage (phage display) or yeast, an antibody fragment gene is inserted into the gene encoding a phage surface protein (e.g., pIII) and the antibody fragment-pIII fusion protein is displayed on the phage surface (McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133-4137).
Since the antibody fragments on the surface of the phage or yeast are functional, phage bearing antigen binding antibody fragments can be separated from non-binding phage by antigen affinity chromatography (McCafferty et al. (1990) Nature, 348: 552-554). Depending on the affinity of the antibody fragment, enrichment factors of 20 fold-1,000,000 fold are obtained for a single round of affinity selection.
Human antibodies can be produced without prior immunization by displaying very large and diverse V-gene repertoires on phage (Marks et al. (1991) J. Mol. Biol. 222: 581-597.
In certain embodiments, nanobodies can be used as targeting moieties. Methods of making V_hH (nanobodies) are also well known to those of skill in the art. The Camelidae heavy chain antibodies are found as homodimers of a single heavy chain, dimerized via their constant regions. The variable domains of these camelidae heavy chain antibodies are referred to as V_HHdomains or V_HH, and can be either used per se as nanobodies and/or as a starting point for obtaining nanobodies. Isolated V_HHretain the ability to bind antigen with high specificity (see, e.g., Hamers-Casterman et al. (1993) Nature 363: 446-448). In certain embodiments such V_HHdomains, or nucleotide sequences encoding them, can be derived from antibodies raised in Camelidae species, for example in camel, dromedary, llama, alpaca and guanaco. Other species besides Camelidae (e.g. shark, pufferfish) can produce functional antigen-binding heavy chain antibodies, from which (nucleotide sequences encoding) such naturally occurring V_HHcan be obtained, e.g. using the methods described in U.S. Patent Publication US 2006/0211088.
In various embodiments, for use in therapy, human proteins are preferred, primarily because they are not as likely to provoke an immune response when administered to a patient. Comparisons of camelid V_HHwith the V_Hdomains of human antibodies reveals several key differences in the framework regions of the camelid V_HHdomain corresponding to the V_H/V_Linterface of the human V_Hdomains. Mutation of these human residues to V_HHresembling residues has been performed to produce “camelized” human V_Hdomains that retain antigen binding activity, yet have improved expression and solubility.
Libraries of single V_Hdomains have also been derived for example from V_Hgenes amplified from genomic DNA or from mRNA from the spleens of immunized mice and expressed in E. coli (Ward et al. (1989) Nature 341: 544-546) and similar approaches can be performed using the V_Hdomains and/or the V_Ldomains described herein. The isolated single VH domains are called “dAbs” or domain antibodies. A “dAb” is an antibody single variable domain (V_Hor V_L) polypeptide that specifically binds antigen. A “dAb” binds antigen independently of other V domains; however, as the term is used herein, a “dAb” can be present in a homo- or heteromultimer with other V_Hor V_Ldomains where the other domains are not required for antigen binding by the dAb, i.e., where the dAb binds antigen independently of the additional V_Hor V_Ldomains.
As described in U.S. Patent Publication US 2006/0211088 methods are known for the cloning and direct screening of immunoglobulin sequences (including but not limited to multivalent polypeptides comprising: two or more variable domains—or antigen binding domains—and in particular V_Hdomains or V_HHdomains; fragments of V_L, V_Hor V_HHdomains, such as CDR regions, for example CDR3 regions; antigen-binding fragments of conventional 4-chain antibodies such as Fab fragments and scFv's, heavy chain antibodies and domain antibodies; and in particular of V_Hsequences, and more in particular of V_HHsequences) that can be used as part of and/or to construct such nanobodies.
Methods and procedures for the production of VHH/nanobodies can also be found for example in WO 94/04678, WO 96/34103, WO 97/49805, WO 97/49805 WO 94/25591, WO 00/43507 WO 01/90190, WO 03/025020, WO 04/062551, WO 04/041863, WO 04/041865, WO 04/041862, WO 04/041867, PCT/BE2004/000159, Hamers-Casterman et al. (1993) Nature 363: 446; Riechmann and Muyldermans (1999) J. Immunological Meth., 231: 25-38; Vu et al. (1997) Molecular Immunology, 34(16-17): 1121-1131; Nguyen et al. (2000) EMBO J., 19(5): 921-930; Arbabi Ghahroudi et al. (19997) FEBS Letters 414: 521-526; van der Linden et al. (2000) J. Immunological Meth., 240: 185-195; Muyldermans (2001) Rev. Molecular Biotechnology 74: 277-302; Nguyen et al. (2001) Adv. Immunol. 79: 261, and the like, which are all incorporated herein by reference.
In certain embodiments the antibody targeting moiety is a unibody. Unibodies provide an antibody technology that produces a stable, smaller antibody format with an anticipated longer therapeutic window than certain small antibody formats. In certain embodiments unibodies are produced from IgG4 antibodies by eliminating the hinge region of the antibody. Unlike the full size IgG4 antibody, the half molecule fragment is very stable and is termed a uniBody. Halving the IgG4 molecule left only one area on the UniBody that can bind to a target. Methods of producing unibodies are described in detail in PCT Publication WO2007/059782, which is incorporated herein by reference in its entirety (see, also, Kolfschoten et al. (2007) Science 317: 1554-1557).
Affibody molecules are class of affinity proteins based on a 58-amino acid residue protein domain, derived from one of the IgG-binding domains of staphylococcal protein A. This three helix bundle domain has been used as a scaffold for the construction of combinatorial phagemid libraries, from which Affibody variants that target the desired molecules can be selected using phage display technology (see, e.g., Nord et al. (1997) Nat. Biotechnol. 15: 772-777; Ronmark et al. (2002) Eur. J. Biochem., 269: 2647-2655.). Details of Affibodies and methods of production are known to those of skill (see, e.g., U.S. Pat. No. 5,831,012 which is incorporated herein by reference in its entirety).
It will also be recognized that antibodies can be prepared by any of a number of commercial services (e.g., Berkeley antibody laboratories, Bethyl Laboratories, Anawa, Eurogenetec, etc.).
Illustrative antibodies that bind various microorganisms are shown in Table 5.

TABLE 5

Illustrative antibodies that bind target microorganisms.

	Source	Antibody

	7,195,763	Polyclonal/monoclonal binds specific Gram(+)
		cell wall repeats
	6,939,543	Antibodies against G(+) LTA
	7,169,903	Antibodies against G(+) peptidoglycan
	6,231,857	Antibody against S. mutans (Shi)
	5,484,591	Gram(−) binding antibodies
	US 2007/0231321	Diabody binding to Streptococcus surface
		antigen I/II
	US
2003/0124635	Antibody against S. mutans
	US
2006/0127372	Antibodies to Actinomyces naeslundii,
		Lactobacillus casei
	US
2003/0092086	Antibody to S. sobrinus

In addition, antibodies (targeting moieties) that bind other microorganisms can readily be produced using, for example, the methods described above.
Porphyrins.
In certain embodiments porphyrins, or other photosensitizing agents, can be used as targeting moieties in the constructs described herein. In particular, metalloporphyrins, particularly a number of non-iron metalloporphyrins mimic heme in their molecular structure and are actively accumulated by bacteria via high affinity heme-uptake systems. The same uptake systems can be used to deliver antibiotic-porphyrin and antibacterial-porphyrin conjugates. Illustrative targeting porphyrins suitable for this purpose are described in U.S. Pat. No. 6,066,628 and shown herein, for example, in FIGS. 1 and 2.
For example, certain artificial (non-iron) metalloporphyrins (MPs) (Ga-IX, Mn-IX,) are active against Gram-negative and Gram-positive bacteria and acid-fast bacilli (e.g., Y. enterocolitica, N. meningitides, S. marcescens, E. coli, P. mirabills, K. pneumoniae, K. oxytoca, Ps. aeruginosa, C. freundii, E. aerogenes, F. menigosepticum, S. aureus, B. subtilis, S. pyogenes A, E. faecalis, M. smegmatis, M. bovis, M. tuber., S. crevisiae) as described in Tables 1-5 of U.S. Pat. No. 6,066,628. These MPs can be used as targeting moieties against these microorganisms.
Similarly, some MPs are also growth-inhibitory against yeasts, indicating their usefulness targeting moieties to target Candida species (e.g., Candida albicans, C. krusei, C. pillosus, C. glabrata, etc.) and other mycoses including but not limited to those caused by as Trichophyton, Epidermophyton, Histoplasma, Aspergillus, Cryptococcus, and the like.
Porphyrins, and other photosensitizers, also have antimicrobial activity. Accordingly, in certain embodiments, the porphyrins, or other photosensitizers, can be used as effectors (e.g., attached to targeting peptides as described herein). In various embodiments the porphyrins or other photosensitizers can provide a dual functionality, e.g., as a targeting moiety and an antimicrobial and can be attached to a targeting peptide and/or to an antimicrobial peptide as described herein.
Illustrative porphyrins and other photosensitizers are shown in FIGS. 1-11 and described in more detail in the discussion of effectors below.
Pheromones.
In certain embodiments, pheromones from microorganisms can be used as targeting moieties. Illustrative pheromones from bacteria and fungi are shown in Table 6. In certain embodiments, chimeric moieties are contemplated comprising a targeting moiety comprising or consisting of the amino acid sequence (or retro or retro-inverso or beta) sequence of a peptide shown in Table 6 attached to one or more of the peptides shown in Table 2.

TABLE 6

Illustrative bacterial and fungal pheromones utilizable as targeting moieties.

Bacterial Pheromones
Locus tag	Product	Sequence	SEQ ID

gi\|1041118\|dbj\|BAA11198.1\|	iPD1 [Enterococcus	MKQQKKHIAALLF	455
	faecalis]	ALILTLVS

gi\|1113947\|gb\|AAB35253.1\|	iAM373sex pheromone	SIFTLVA	456
	inhibito [Enterococcus
	faecalis, Peptide, 7 aa]

gi\|115412\|sp\|P13268.1\|CAD1_ENTFA	Sex pheromone CAD1	LFSLVLAG	457

gi\|116406\|sp\|P11932.1\|CIA_ENTFA	Sex pheromone cAM373	AIFILAS	458
	(Clumping-inducing
	agent) (CIA)

gi\|117240\|sp\|P13269.1\|CPD1_ENTFA	Sex pheromone cPD1	FLVMFLSG	459

gi\|12056953\|gb\|AAG48144.1\|AF322594_1	putative peptide	DSIRDVSPTFNKIRR	460
	pheromone PrcA	WFDGLFK
	[Lactobacillus paracasei]

gi\|123988\|sp\|P24803.1\|IAD1_ENTFA	Sex pheromone inhibitor	MSKRAMKKIIPLIT	461
	determinant precursor	LFVVTLVG
	(iAD1)

gi\|126362994\|emb\|CAM35812.1\|	precursor of pheromone	KDEIYWKPS	462
	peptide ComX [Bacillus
	amyloliquefaciens FZB42]

gi\|1587088\|prf\|\|2205353A	pheromone	YSTCDFIM	463

gi\|15900442\|ref\|NP_345046.1\|	peptide pheromone BlpC	GLWEDLLYNINRY	464
	[Streptococcus	AHYIT
	pneumoniae TIGR4]

gi\|1617436\|emb\|CAA66791.1\|	competence pheromone	DIRHRINNSIWRDIF	465
	[Streptococcus gordonii]	LKRK

gi\|1617440\|emb\|CAA66786.1\|	competence pheromone	DVRSNKIRLWWEN	466
	[Streptococcus gordonii]	IFFNKK

gi\|18307870\|gb\|AAL67728.1\|AF456134_2	ComX pheromone	PTTREWDG	467
	precursor [Bacillus
	mojavensis]

gi\|18307874\|gb\|AAL67731.1\|AF456135_2	ComX pheromone	LQIYTNGNWVPS	468
	precursor [Bacillus
	mojavensis]

gi\|29377808\|ref\|NP_816936.1\|	sex pheromone inhibitor	MSKRAMKKIIPLIT	469
	determinant [Enterococcus	LFVVTLVG
	faecalis V583]

gi\|3342125\|gb\|AAC27522.1\|	putative pheromone	GAGKNLIYGMGYG	470
	[Enterococcus faecium]	YLRSCNRL

gi\|41018893\|sp\|P60242.1\|CSP1_STRPN	Competence-stimulating	EMRLSKFFRDFILQ	471
	peptide type 1 precursor	RKK
	(CSP-1)

gi\|57489126\|gb\|AAW51333.1\|	PcfP [Enterococcus	WSEIEINTKQSN	472
	faecalis]

gi\|57489152\|gb\|AAW51349.1\|	PrgT [Enterococcus	HISKERFEAY	473
	faecalis]

gi\|58616083\|ref\|YP_195761.1\|	UvaF [Enterococcus	KYKCSWCKRVYTL	474
	faecalis]	RKDHRTAR

gi\|58616111\|ref\|YP_195802.1\|	PcfP [Enterococcus	WSEIEINTKQSN	475
	faecalis]

gi\|58616132\|ref\|YP_195769.1\|	PrgQ [Enterococcus	MKTTLKKLSRYIA	476
	faecalis]	VVIAITLIFI

gi\|58616137\|ref\|YP_195772.1\|	PrgT [Enterococcus	HISKERFEAY	477
	faecalis]

gi\|6919848\|sp\|O33689.1\|CSP_STROR	Competence-stimulating	DKRLPYFFKHLFSN	478
	peptide precursor (CSP)	RTK

gi\|6919849\|sp\|O33666.1\|CSP2_STRMT	Competence-stimulating	EMRKPDGALFNLF	479
	peptide precursor (CSP)	RRR

gi\|6919850\|sp\|O33668.1\|CSP3_STRMT	Competence-stimulating	EMRKSNNNFFHFL	480
	peptide precursor (CSP)	RRI

gi\|6919851\|sp\|O33672.1\|CSP1_STRMT	Competence-stimulating	ESRLPKIRFDFIFPR	481
	peptide precursor (CSP)	KK

gi\|6919852\|sp\|O33675.1\|CSP4_STRMT	Competence-stimulating	EIRQTHNIFFNFFKRR	482
	peptide precursor (CSP)

gi\|6919853\|sp\|O33690.1\|CSP2_STROR	Competence-stimulating	DWRISETIRNLIFPR	483
	peptide precursor (CSP)	RK

gi\|999344\|gb\|AAB34501.1\|	cOB1bacterial sex	VAVLVLGA	484
	pheromone [Enterococcus
	faecalis, Peptide, 8 aa]

gi\|18307878\|gb\|AAL67734.1\|AF456136_2	ComX pheromone	FFEDDKRKSFI	485
	precursor [Bacillus
	subtilis]

gi\|18307882\|gb\|AAL67737.1\|AF456137_2	ComX pheromone	FFEDDKRKSFI	486
	precursor [Bacillus
	subtilis]

gi\|28272731\|emb\|CAD65660.1\|	accessory gene regulator	MKQKMYEAIAHLF	487
	protein D, peptide	KYVGAKQLVMCC
	pheromone precursor	VGIWFETKIPDELRK
	[Lactobacillus plantarum
	WCFS1]

gi\|28379890\|ref\|NP_786782.1\|	accesory gene regulator	MKQKMYEAIAHLF	488
	protein D, peptide	KYVGAKQLVMCC
	pheromone precursor	VGIWFETKIPDELRK
	[Lactobacillus plantarum
	WCFS1]

gi\|57489105\|gb\|AAW51312.1\|	PrgF [Enterococcus	VVAYVITQVGAIRF	489
	faecalis]

gi\|58616090\|ref\|YP_195779.1\|	PrgF [Enterococcus	VVAYVITQVGAIRF	490
	faecalis]

gi\|58616138\|ref\|YP_195762.1\|	PrgN [Enterococcus	LLKLQDDYLLHLE	491
	faecalis]	RHRRTKKIIDEN

gi\|57489117\|gb\|AAW51324.1\|	PcfF [Enterococcus	EDIKDLTDKVQSLN	492
	faecalis]	ALVQSELNKLIKRK
		DQS

gi\|57489119\|gb\|AAW51326.1\|	PcfH [Enterococcus	WFLDFSDWLSKVP	493
	faecalis]	SKLWAE

gi\|58616102\|ref\|YP_195792.1\|	PcfF [Enterococcus	EDIKDLTDKVQSLN	494
	faecalis]	ALVQSELNKLIKRK
		DQS

gi\|58616104\|ref\|YP_195794.1\|	PcfH [Enterococcus	WFLDFSDWLSKVP	495
	faecalis]	SKLWAE

Fungi			496

gi\|1127585\|gb\|AAA99765.1\|	mfa1 gene product	MLSIFAQTTQTSAS	497
		EPQQSPTAPQGRDN
		GSPIGYSSCVVA

gi\|1127592\|gb\|AAA99771.1\|	mfa2 gene product	MLSIFETVAAAAPV	498
		TVAETQQASNNEN
		RGQPGYYCLIA

gi\|11907715\|gb\|AAG41298.1\|	pheromone precursor	PSLPSSPPSLLPPLPL	499
	MFalpha1D	LKLLATRRPTLVG
	[Cryptococcus neoformans	MTLCV
	var. neoformans]

gi\|13810235\|emb\|CAC37424.1\|	M-factor precursor Mfm1	MDSMANSVSSSSV	500
	[Schizosaccharomyces	VNAGNKPAETLNK
	pombe]	TVKNYTPKVPYMC
		VIA

gi\|14269436\|gb\|AAK58071.1\|AF378295_1	peptide mating pheromone	MDTFTYVDLAAVA	501
	precursor Bbp2-3	AAAVADEVPRDFE
	[Schizophyllum	DQITDYQSYCIIC
	commune]

gi\|14269440\|gb\|AAK58073.1\|AF378297_1	peptide mating pheromone	SNVHGWCVVA	502
	precursor Bbp2-1
	[Schizophyllum
	commune]

gi\|1813600\|gb\|AAB41859.1\|	pheromone precursor	NTTAHGWCVVA	503
	Bbp1(1) [Schizophyllum
	commune]

gi\|24940428\|emb\|CAD56313.1\|	a-pheromone	MQPSTVTAAPKDK	504
	[Saccharomyces	TSAEKKDNYIIKGV
	paradoxus]	FWDPACVIA

gi\|27549492\|gb\|AAO17258.1\|	pheromone phb3.1	GPTWWCVNA	505
	[Coprinopsis cinerea]

gi\|27549494\|gb\|AAO17259.1\|	pheromone phb3.2	SGPTWFCIIQ	506
	[Coprinopsis cinerea]

gi\|27752314\|gb\|AAO19469.1\|	pheromone protein a	FTAIFSTLSSSVASK	507
	precursor [Cryptococcus	TDAPRNEEAYSSG
	neoformans var. grubii]	NSP

gi\|2865510\|gb\|AAC02682.1\|	MAT-1 pheromone	MFSIFAQPAQTSVS	508
	[Ustilago hordei]	ETQESPANHGANP
		GKSGSGLGYSTCV
		VA

gi\|3023372\|sp\|P78742.1\|BB11_SCHCO	RecName: Full = Mating-	NTTAHGWCVVA	509
	type pheromone BBP1(1);
	Flags: Precursor

gi\|3025079\|sp\|P56508.1\|SNA2_YEAST	RecName: Full = Protein	SDDNYGSLA	510
	SNA2

gi\|37626077\|gb\|AAQ96360.1\|	pheromone precursor Phb3	NGLTFWCVIA	511
	B5 [Coprinopsis cinerea]

gi\|37626081\|gb\|AAQ96362.1\|	pheromone precursor	PSWFCVIA	512
	Phb3.2 B45 [Coprinopsis
	cinerea]

gi\|37626083\|gb\|AAQ96363.1\|	pheromone precursor	ASWFCTIA	513
	Phb3.1 B47 [Coprinopsis
	cinerea]

gi\|37961432\|gb\|AAP57503.1\|	Ste3-like pheromone	PHHKIANASDKRR	514
	receptor [Thanatephorus	RMYFEIFMCAVL
	cucumeris]

gi\|400250\|sp\|P31962.1\|MFA1_USTMA	RecName: Full = A1-	MLSIFAQTTQTSAS	515
	specific pheromone;	EPQQSPTAPQGRDN
	AltName: Full = Mating	GSPIGYSSCVVA
	factor A1

gi\|400251\|sp\|P31963.1\|MFA2_USTMA	RecName: Full = A2-	MLSIFETVAAAAPV	516
	specific pheromone;	TVAETQQASNNEN
	AltName: Full = Mating	RGQPGYYCLIA
	factor A2

gi\|41209131\|gb\|AAR99617.1\|	lipopeptide mating	SLTYAWCVVA	517
	pheromone precursor
	Bap2(3) [Schizophyllum
	commune]

gi\|41209146\|gb\|AAR99650.1\|	lipopeptide mating	TSMAHAWCVVA	518
	pheromone precursor
	Bap3(2) [Schizophyllum
	commune]

gi\|41209149\|gb\|AAR99653.1\|	lipopeptide mating	GYCVVA	519
	pheromone precursor
	Bbp2(8) [Schizophyllum
	commune]

gi\|46098187\|gb\|EAK83420.1\|	MFA1_USTMA A1-	MLSIFAQTTQTSAS	520
	SPECIFIC PHEROMONE	EPQQSPTAPQGRDN
	(MATING FACTOR A1)	GSPIGYSSCVVA
	[Ustilago maydis 521]

gi\|546861\|gb\|AAB30833.1\|	M-factor mating	MDSMANTVSSSVV	521
	pheromone	NTGNKPSETLNKT
	[Schizosaccharomyces	VKNYTPKVPYMCV
	pombe]	IA

gi\|5917793\|gb\|AAD56043.1\|AF184069_1	pheromone Mfa2	MFSLFETVAAAVK	522
	[Ustilago hordei]	VVSAAEPEHAPTNE
		GKGEPAPYCIIA

gi\|6014618\|gb\|AAF01424.1\|AF186389_1	Phb3.2.42 [Coprinus	LTWFCVIA	523
	cinereus]

gi\|68266363\|gb\|AAY88882.1\|	putative pheromone	LREKRRRRWFEAF	524
	receptor STE3.4	MGFGL
	[Coprinellus disseminatus]

gi\|71012805\|ref\|XP_758529.1\|	A1-specific pheromone	MLSIFAQTTQTSAS	525
	[Ustilago maydis 521]	EPQQSPTAPQGRDN
		GSPIGYSSCVVA

gi\|72414834\|emb\|CAI59748.1\|	mating factor a1.3	MDALTLFAPVSLG	526
	[Sporisorium reilianum]	AVATEQAPVDEER
		PNRQTFPWIGCVVA

gi\|72414854\|emb\|CAI59758.1\|	mating factor a2.1	MFIFESVVASVQAV	527
	[Sporisorium reilianum]	SVAEQDQTPVSEG
		RGKPAVYCTIA

gi\|1127587\|gb\|AAA99767.1\|	rba1 gene product	PWMSLLFSFLALLA	528
		LILPKLSKDDPLGL
		TRQPR

gi\|151941959\|gb\|EDN60315.1\|	pheromone-regulated	ASISLIMEGSANIEA	529
	membrane protein	VGKLVWLAAALPL
	[Saccharomyces cerevisiae	AFI
	YJM789]

gi\|3025095\|sp\|Q07549.1\|SNA4_YEAST	Protein SNA4	ARNVYPSVETPLLQ	530
		GAAPHDNKQSLVE
		SPPPYVP

gi\|73921293\|sp\|Q08245.3\|ZEO1_YEAST	RecName: Full = Protein	FLKKLNRKIASIFN	531
	ZEO1; AltName:
	Full = Zeocin resistance
	protein 1

gi\|74644573\|sp\|Q9P305.3\|IGO2_YEAST	RecName: Full = Protein	DSISRQGSISSGPPP	532
	IGO2	RSPNK

Effectors.
Any of a wide number of effectors can be coupled to targeting moieties as described herein to preferentially deliver the effector to a target organism and/or tissue. Illustrative effectors include, but are not limited to detectable labels, small molecule antibiotics, antimicrobial peptides, porphyrins or other photosensitizers, epitope tags/antibodies for use in a pretargeting protocol, microparticles and/or microcapsules, nanoparticles and/or nanocapsules, “carrier” vehicles including, but not limited to lipids, liposomes, dendrimers, cholic acid-based peptide mimics or other peptide mimics, steroid antibiotics, and the like.
Detectable Labels.
In certain embodiments chimeric moieties are provided comprising a targeting moiety (e.g. as described in Table 2) attached directly or through a linker to a detectable label. Such chimeric moieties are effective for detecting the presence and/or quantity, and/or location of the microorganism(s) to which the targeting moiety is directed. Similarly these chimeric moieties are useful to identify cells and/or tissues and/or food stuffs and/or other compositions that are infected with the targeted microorganism(s).
Detectable labels suitable for use in such chimeric moieties include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Illustrative useful labels include, but are not limited to, biotin for staining with labeled streptavidin conjugates, avidin or streptavidin for labeling with biotin conjugates fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like, see, e.g., Molecular Probes, Eugene, Oreg., USA), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ⁹⁹Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ^113mIn, ⁹⁷Ru, ⁶²Cu, 641Cu, ⁵²Fe, ^52mMn, ⁵¹Cr, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, ¹¹¹Ag, and the like), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), various colorimetric labels, magnetic or paramagnetic labels (e.g., magnetic and/or paramagnetic nanoparticles), spin labels, radio-opaque labels, and the like. Patents teaching the use of such labels include, for example, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
It will be recognized that fluorescent labels are not to be limited to single species organic molecules, but include inorganic molecules, multi-molecular mixtures of organic and/or inorganic molecules, crystals, heteropolymers, and the like. Thus, for example, CdSe—CdS core-shell nanocrystals enclosed in a silica shell can be easily derivatized for coupling to a biological molecule (Bruchez et al. (1998) Science, 281: 2013-2016). Similarly, highly fluorescent quantum dots (zinc sulfide-capped cadmium selenide) have been covalently coupled to biomolecules for use in ultrasensitive biological detection (Warren and Nie (1998) Science, 281: 2016-2018).
In various embodiments spin labels are provided by reporter molecules with an unpaired electron spin which can be detected by electron spin resonance (ESR) spectroscopy. Illustrative spin labels include organic free radicals, transitional metal complexes, particularly vanadium, copper, iron, and manganese, and the like. Exemplary spin labels include, for example, nitroxide free radicals.
Means of detecting such labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence, e.g., by microscopy, visual inspection, via photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing appropriate substrates for the enzyme and detecting the resulting reaction product. Finally, simple colorimetric labels may be detected simply by observing the color associated with the label.
Antibiotics.
In certain embodiments chimeric moieties are provided comprising a targeting moiety (e.g. as described in Table 2) attached directly or through a linker to a small molecule antibiotic and/or to a carrier (e.g., a lipid or liposome, a polymer, etc.) comprising a small molecule antibiotic (e.g., an antibiotics shown in Table 7).

TABLE 7

Illustrative antibiotics for use in the chimeric moieties described herein.

Class	Generic Name	Brand Name

Aminoglycosides	Amikacin	Amikin
	Gentamicin	Garamycin
	Kanamycin	Kantrex
	Neomycin
	Netilmicin	Netromycin
	Streptomycin
	Tobramycin	Nebcin
	Paromomycin	Humatin
Carbacephem	Loracarbef	Lorabid
Carbapenems	Ertapenem	Invanz
	Doripenem	Finibax
	Imipenem/Cilastatin	Primaxin
	Meropenem	Merrem
Cephalosporins	Cefadroxil	Duricef
(First generation)	Cefazolin	Ancef
	Cefalotin or Cefalothin	Keflin
	Cefalexin	Keflex
Cephalosporins	Cefaclor	Ceclor
(Second	Cefamandole	Mandole
generation)	Cefoxitin	Mefoxin
	Cefprozil	Cefzil
	Cefuroxime	Ceftin, Zinnat
Cephalosporins	Cefixime	Suprax
(Third generation)	Cefdinir	Omnicef
	Cefditoren	Spectracef
	Cefoperazone	Cefobid
	Cefotaxime	Claforan
	Cefpodoxime
	Ceftazidime	Fortaz
	Ceftibuten	Cedax
	Ceftizoxime
	Ceftriaxone	Rocephin
Cephalosporins	Cefepime	Maxipime
(Fourth
generation)
Cephalosporins	Ceftobiprole
(Fifth generation)
Glycopeptides	Teicoplanin
	Vancomycin	Vancocin
Macrolides
Azithromycin	Zithromax
Clarithromycin	Biaxin
Dirithromycin
Erythromycin	Erythocin, Erythroped
Roxithromycin
Troleandomycin
Telithromycin	Ketek
Monobactams
	Aztreonam
Penicillins	Amoxicillin	Novamox, Amoxil
	Ampicillin
	Azlocillin
	Carbenicillin
	Cloxacillin
	Dicloxacillin
	Flucloxacillin	Floxapen
	Mezlocillin
	Meticillin
	Nafcillin
	Oxacillin
	Penicillin
	Piperacillin
	Ticarcillin
Polypeptides	Bacitracin
	Colistin
	Polymyxin B
Quinolones	Mafenide
	Prontosil (archaic)
	Sulfacetamide
	Sulfamethizole
	Sulfanilimide (archaic)
	Sulfasalazine
	Sulfisoxazole
	Trimethoprim Trimethoprim-	Bactrim
	Sulfamethoxazole (Co-
	trimoxazole) (TMP-SMX)
Tetracyclines	Demeclocycline
	Doxycycline	Vibramycin
	Minocycline	Minocin
	Oxytetracycline	Terracin
	Tetracycline	Sumycin
Cationic steroid	squalamine
antibiotics	CSA-8
	CSA-11
	CSA-13
	CSA-15
	CSA-25
	CSA-46
	CSA-54
	CSA-90
	CSA-97
Others	Arsphenamine	Salvarsan
	Chloramphenicol	Chloromycetin
	Clindamycin	Cleocin
	Lincomycin
	Ethambutol
	Fosfomycin
	Fusidic acid	Fucidin
	Furazolidone
	Isoniazid
	Linezolid	Zyvox
	Metronidazole	Flagyl
	Mupirocin	Bactroban
	Nitrofurantoin	Macrodantin,
		Macrobid
	Platensimycin
	Pyrazinamide
	Quinupristin/Dalfopristin	Syncercid
	Rifampin or Rifampicin
	Tinidazole

Porphyrins and Non-Porphyrin Photosensitizers.
In certain embodiments, porphyrins and other photosensitizers can be used as targeting moieties and/or as effectors in the methods and compositions of this invention. A photosensitizer is a drug or other chemical that increases photosensitivity of the organism (e.g., bacterium, yeast, fungus, etc.). As targeting moieties the photosensitizers (e.g., porphyrins) are preferentially uptaken by the target microorganisms and thereby facilitate delivery of the chimeric moiety to the target microorganism.
As effectors, photosensitizers can be useful in photodynamic antimicrobial chemotherapy (PACT). In various embodiments PACT utilizes photosensitizers and light (e.g., visible, ultraviolet, infrared, etc.) in order to give a phototoxic response in the target organism(s), often via oxidative damage.
Currently, the major use of PACT is in the disinfection of blood products, particularly for viral inactivation, although more clinically-based protocols are used, e.g. in the treatment of oral infection or topical infection. The technique has been shown to be effective in vitro against bacteria (including drug-resistant strains), yeasts, viruses, parasites, and the like.
Attaching a targeting moiety (e.g., a targeting peptide as shown in Table 2) to the photosensitizer, e.g., as described herein, provides a means of specifically or preferentially targeting the photosensitizer(s) to particular species or strains(s) of microorganism.
A wide range of photosensitizers, both natural and synthetic are known to those of skill in the art (see, e.g., Wainwright (1998) J. Antimicrob. Chemotherap. 42: 13-28). Photosensitizers are available with differing physicochemical make-up and light-absorption properties. In various embodiments photosensitizers are usually aromatic molecules that are efficient in the formation of long-lived triplet excited states. In terms of the energy absorbed by the aromatic-system, this again depends on the molecular structure involved. For example: furocoumarin photosensitizers (psoralens) absorb relatively high energy ultraviolet (UV) light (c. 300-350 nm), whereas macrocyclic, heteroaromatic molecules such as the phthalocyanines absorb lower energy, near-infrared light.
Illustrative photosensitizers include, but are not limited to porphyrinic macrocyles (especially porphyrins, chlorines, etc., see, e.g., FIGS. 1 and 2). In particular, metalloporphyrins, particularly a number of non-iron metalloporphyrins mimic heme in their molecular structure and are actively accumulated by bacteria via high affinity heme-uptake systems. The same uptake systems can be used to deliver antibiotic-porphyrin and antibacterial-porphyrin conjugates. Illustrative targeting porphyrins suitable for this purpose are described in U.S. Pat. No. 6,066,628 and shown herein in FIGS. 1 and 2.
For example, certain artificial (non-iron) metalloporphyrins (MPs) (Ga-IX, Mn-IX,) are active against Gram-negative and Gram-positive bacteria and acid-fast bacilli (e.g., Y enterocolitica, N. meningitides, S. marcescens, E. coli, P. mirabills, K. pneumoniae, K. oxytoca, Ps. aeruginosa, C. freundii, E. aerogenes, F. menigosepticum, S. aureus, B. subtilis, S. pyogenes A, E. faecalis, M. smegmatis, M. bovis, M. tuber., S. crevisiae) as described in Tables 1-5 of U.S. Pat. No. 6,066,628. These MPs can be used as targeting moieties against these microorganisms.
Similarly, some MPs are also growth-inhibitory against yeasts, indicating their usefulness targeting moieties to target Candida species (e.g., Candida albicans, C. krusei, C. pillosus, C. glabrata, etc.) and other mycoses including but not limited to those caused by as Trichophyton, Epidermophyton, Histoplasma, Aspergillus, Cryptococcus, and the like.
Other photosensitizers include, but are not limited to cyanines (see, e.g., FIG. 6) and phthalocyanines (see, e.g., FIG. 4), azines (see, e.g., FIG. 5) including especially methylene blue and touidine blue, hypericin (see, e.g., FIG. 8), acridines (see, e.g., FIG. 9) including especially Rose Bengal (see, e.g., FIG. 10), crown ethers (see, e.g., FIG. 11), and the like.
In certain embodiments the photosensitizers are toxic or growth inhibitors without light activation. For example, some non-iron metalloporphyrins (MPs) (see, e.g., FIGS. 1 and 2 herein) possess a powerful light-independent antimicrobial activity. In addition, haemin, the most well known natural porphyrin, possesses a significant antibacterial activity that can augmented by the presence of physiological concentrations of hydrogen peroxide or a reducing agent.
Typically, when activated by light, the toxicity or growth inhibition effect is substantially increased. Typically, they generate radical species that affect anything within proximity. In certain embodiments to get the best selectivity from targeted photosensitizers, anti-oxidants can be used to quench un-bound photosensitizers, limiting the damage only to cells where the conjugates have accumulated due to the targeting peptide. The membrane structures of the target cell act as the proton donors in this case.
In typical photodynamic antimicrobial chemotherapy (PACT) the targeted photosensitizer is “activated by the application of a light source (e.g., a visible light source, an ultraviolet light source, an infrared light source, etc.). PACT applications however need not be limited to topical use. Regions of the mouth, throat, nose, sinuses are readily illuminated. Similarly regions of the gut can readily be illuminated using endoscopic techniques. Other internal regions can be illumined using laparoscopic methods or during other surgical procedures. For example, in certain embodiments involving the insertion or repair or replacement of an implantable device (e.g., a prosthetic device) it contemplated that the device can be coated or otherwise contacted with a chimeric moiety comprising a targeting moiety attached to a photosensitizer as described herein. During the surgical procedure and/or just before closing, the device can be illuminated with an appropriate light source to activate the photosensitizer.
The targeted photosensitizers and uses thereof described herein are illustrative and not to be limiting. Using the teachings provided herein, other targeted photosensitizers and uses thereof will be available to one of skill in the art.
Antimicrobial Peptides.
In certain embodiments the chimeric moieties described herein include one or more antimicrobial peptides (e.g., certain peptides shown in Table 2 and/or Table 10) as effectors. Thus, for example, in certain embodiments, where the peptides described in Table 2 are exploited for their targeting ability, chimeric moieties are contemplated comprising one or more of the targeting peptides of Table 2 attached to one or more of the antimicrobial peptides of Table 10. In certain embodiments chimeric moieties are contemplated comprising one or more of the targeting peptides of Table 2 attached to one or more of the antimicrobial peptides of Table 2. In certain embodiments chimeric moieties are contemplated comprising other targeting moieties (e.g., porphyrins, antibodies, etc.) attached to one or more of the antimicrobial peptides of Table 2.
Antimicrobial peptides (also called host defense peptides) are an evolutionarily conserved component of the innate immune response and are found among all classes of life. Unmodified, these peptides are potent, broad spectrum antibiotics which demonstrate potential as novel therapeutic agents. Antimicrobial peptides have been demonstrated to kill Gram-negative and Gram-positive bacteria (including strains that are resistant to conventional antibiotics), mycobacteria (including Mycobacterium tuberculosis), enveloped viruses, and fungi.
Naturally-occurring antimicrobial peptides are typically short peptides, generally between 12 and 50 amino acids. These peptides often include two or more positively charged residues provided by arginine, lysine or, in acidic environments, histidine, and frequently a large proportion (generally >50%) of hydrophobic residues (see, e.g., Papagianni et al. (2003) Biotechnol Adv 21: 465; Sitaram and Nagaraj (2002) Curr Pharm Des 8: 727; Dun et al. (2006) Biochim. Biophys. Acta 1758: 1408-1425).
Frequently the secondary structures of these molecules follow 4 themes, including i) α-helical, ii) β-stranded due to the presence of 2 or more disulfide bonds, iii) β-hairpin or loop due to the presence of a single disulfide bond and/or cyclization of the peptide chain, and iv) extended. Many of these peptides are unstructured in free solution, and fold into their final configuration upon partitioning into biological membranes. The ability to associate with membranes is a definitive feature of antimicrobial peptides although membrane permeabilisation is not necessary. These peptides have a variety of antimicrobial activities ranging from membrane permeabilization to action on a range of cytoplasmic targets.
The modes of action by which antimicrobial peptides kill bacteria is varied and includes, but is not limited to disrupting membranes, interfering with metabolism, and targeting cytoplasmic components. In many cases the exact mechanism of killing is not known.
In various embodiments one or more antimicrobial peptides are used alone (e.g., as broad spectrum antimicrobials) and/or are provided as effectors attached to one or more targeting moieties thereby providing a narrower spectrum (directed) antimicrobial. In certain embodiments one or more antimicrobial peptides are provided as effectors attached to one or more targeting moieties and/or one or more effectors thereby providing a component of a multiple effector composition/strategy.
Suitable antimicrobial peptides for this use include, but are not limited to the antimicrobial peptides found in Table 2, and/or Table 9, and/or Table 10.

TABLE 8

Novel antimicrobial peptides.

ID	Structure/sequence	SEQ ID NO

K-1	GLGRVIGRLIKQIIWRR	533

K-2	VYRKRKSILKIYAKLKGWH	534

K-3	AFYQRKENVISLDPREWLGFNVTEK	535

K-4	DKKRVIERIKSFSLRDEVIHFGELCIYWGK	536

K-5	RSSYNGFSKICFLKIEHFGSYSYQGR	537

K-6	WLNAISLYGRIG	538

K-7	NYRLVNAIFSKIFKKKFIKF	539

K-8	KIL K FLF K KVF	540

K-9	FI RK FLK KW LL	541

K-10	KLFKFLRKHLL	542

K-11	KIL K FLF K QVF	543

K-12	KIL K KLF K FVF	544

K-13	GIL K KLF T KVF	545

K-14	L R K FL H K LF	546

K-15	L R KNL R WLF	547

K-16	FI RK FLQ KLHL	548

K-17	FTRKFLKFLHL	549

K-18	KKFKKFKVLKIL	550

K-19	LLKLLKLKKLKF	551

In certain embodiments peptides that induce alterations in phenotype or other biological activities can also be used as antimicrobial effector moieties. Illustrative alternative peptides are shown in Table 9.

TABLE 9

Novel growth phenotype-inducing or peptides
with other activities.

			SEQ
			ID
ID	Organism, effect	Structure/sequence	NO

G-1	S. mutans: Ca2+ bindng	DSSQSDSDSDSNSSNTNSNSSITNG	552

G-2	S. mutans: biofilm	LPGTLHIQAEFPVQLEAGSLIQIFD	553
	structure

G-3	S. mutans: biofilm	LACTTPVGAVLYLGAEVCAGAAVI	554
	structure	YYGAN

G-4	S. mutans:	EIPIQLANDLANYYDISLDSIFFW	555
	Biofilm structure

G-5	M. xanthus:	RDMTVAGKRPNFLIITTDEE	556
	Altered cell morphology

G-6	M. xanthus:	NTSIVCAVTFAPIKEVPLLWRAGLT	557
	Altered cell morphology	LRSRQS

G-7	M. xanthus:	QAKVEREVERDLVYTLRRLCDPSG	558
	Altered cell morphology	SERTK

G-8	S. mutans:	PRMIDIISFHGCHGDHQVWTDPQAT	559
	Altered biofilm structure	ALPR

Other illustrative antimicrobial peptides include, but are not limited to the AMPs of Table 10.

TABLE 10

Other illustrative antimicrobial peptides. AP numbers refer to ID
in antimicrobial peptide database (http://aps.unmc.edu/AP/main.php).

		SEQ ID
Effector	Structure/Sequence	No

AP00274	1BH4, Circulin A	GIPCGESCVWIPCISAALGCSCKNKVC	560
	(CirA, plant	YRN
	cyclotides, XXC, ZZHp)

AP00036	1BNB, Beta-defensin 1	DFASCHTNGGICLPNRCPGHMIQIGICF	561
	(cow)	RPRVKCCRSW

AP00047	1BNB, Bovine	GPLSCGRNGGVCIPIRCPVPMRQIGTC	562
	neutrophil beta-defensin	FGRPVKCCRSW
	12 (BNBD-12, cow)

AP00428	1C01, MiAMP1	SAFTVWSGPGCNNRAERYSKCGCSAI	563
	(Macadamia integrifolia	HQKGGYDFSYTGQTAALYNQAGCSG
	antimicrobial peptide 1,	VAHTRFGSSARACNPFGWKSIFIQC
	plant)

AP00154	1CIX, Tachystatin A2	YSRCQLQGFNCVVRSYGLPTIPCCRGL	564
	(Horseshoe crabs,	TCRSYFPGSTYGRCQRY
	Crustacea, BBS)

AP00145	1CW5,	VNYGNGVSCSKTKCSVNWGQAFQER	565
	Carnobacteriocin B2	YTAGINSFVSGVASGAGSIGRRP
	(CnbB2, class IIA
	bacteriocin, bacteria)

AP00153	1CZ6, Androctonin	RSVCRQIKICRRRGGCYYKCTNRPY	566
	(scorpions)

AP00152	1D6X, Tritrpticin	VRRFPWWWPFLRR	567
	(synthetic)

AP00201	1D7N, Mastoparan	INLKALAALAKKIL	568
	(insect)

AP00140	1D9J, CecropinA-	KWKLFKKIGIGKFLHSAKKF	569
	Magainin2 hybrid
	(synthetic)

AP00178	1DFN, human alpha	DCYCRIPACIAGERRYGTCIYQGRLW	570
	Defensin HNP-3	AFCC
	(human neutrophil
	peptide-3, HNP3,
	human defensin, ZZHh)

AP01153	1DQC, Tachycitin	YLAFRCGRYSPCLDDGPNVNLYSCCS	571
	(horseshoe crabs,	FYNCHKCLARLENCPKGLHYNAYLK
	Crustacea, BBS)	VCDWPSKAGCT

AP00437	1DUM, Magainin 2	GIGKYLHSAKKFGKAWVGEIMNS	572
	analog (synthetic)

AP00451	1E4S, Human beta	DHYNCVSSGGQCLYSACPIFTKIQGTC	573
	defensin 1 (HBD-1,	YRGKAKCCK
	human defensin)

AP00149	1EWS, Rabbit kidney	MPCSCKKYCDPWEVIDGSCGLFNSKY	574
	defensin 1 (RK-1)	ICCREK

AP00141	1F0E, CecropinA-	KWKLFKKIPKFLHSAKKF	575
	Magainin2 Hybrid (P18,
	synthetic)

AP00142	1F0G, CecropinA-	KLKLFKKIGIGKFLHSAKKF	576
	Magainin2 Hybrid
	(synthetic)

AP00143	1F0H, CecropinA-	KAKLFKKIGIGKFLHSAKKF	577
	Magainin2 Hybrid
	(synthetic)

AP00524	1FD4, Human beta	GIGDPVTCLKSGAICHPVFCPRRYKQI	578
	defensin 2 (HBD-2,	GTCGLPGTKCCKKP
	human defensin, ZZHh)

AP00438	1FJN, Mussel Defensin	GFGCPNNYQCHRHCKSIPGRCGGYCG	579
	MGD-1	GWHRLPCTCYRCG

AP00155	1FRY, SMAP-29	RGLRRLGRKIAHGVKKYGPTVLRIIRI	580
	(SMAP29, sheep	AG
	cathelicidin)

AP00150	1G89, Indolicidin (cow	ILPWKWPWWPWRR	581
	cathelicidin, BBN,
	ZZHa)

AP00156	1GR4, Microcin J25,	VGIGTPISFYGGGAGHVPEYF	582
	linear (MccJ25,
	bacteriocin, bacteria)

AP00151	1HR1, Indolicidin P to	ILAWKWAWWAWRR	583
	A mutant (synthetic)

AP00196	1HU5, Ovispirin-1	KNLRRIIRKIIHIIKKYG	584
	(synthetic)

AP00197	1HU6, Novispirin G10	KNLRRIIRKGIHIIKKYG	585
	(synthetic)

AP00198	1HU7, Novispirin T7	KNLRRITRKIIHIIKKYG	586
	(synthetic)

AP00445	1HVZ, Monkey RTD-1	GFCRCLCRRGVCRCICTR	587
	(rhesus theta-defensin-1,
	minidefensin-1, animal
	defensin, XXC, BBS,
	lectin, ZZHa)

AP00103	1i2v, Heliomicin variant	DKLIGSCVWGAVNYTSDCNGECLLRG	588
	(Hel-LL, synthetic)	YKGGHCGSFANVNCWCET

AP00216	1ICA, Phormia defensin	ATCDLLSGTGINHSACAAHCLLRGNR	589
	A (insect defensin A)	GGYCNGKGVCVCRN

AP01224	1Jo3, Gramicidin B	VGALAVVVWLFLWLW	590
	(bacteria)

AP01225	1jo4, Gramicidin C	VGALAVVVWLYLWLW	591
	(bacteria)

AP00191	1KFP, Gomesin (Gm,	ECRRLCYKQRCVTYCRGR	592
	Spider, XXA)

AP00283	1KJ6, Huamn beta	GIINTLQKYYCRVRGGRCAVLSCLPKE	593
	defensin 3 (HBD-3,	EQIGKCSTRGRKCCRRKK
	human defensin, ZZHh)

AP00147	1KV4, Moricin (insect,	AKIPIKAIKTVGKAVGKGLRAINIASTA	594
	silk moth)	NDVFNFLKPKKRKA

AP00227	1L4V, Sapecin (insect,	ATCDLLSGTGINHSACAAHCLLRGNR	595
	flesh fly)	GGYCNGKAVCVCRN

AP01161	1L9L, Human	GRDYRTCLTIVQKLKKMVDKPTQRSV	596
	granulysin (huGran)	SNAATRVCTRGRSRWRDVCRNFMRR
		YQSRVIQGLVAGETAQQICEDLRLCIP
		STGPL

AP00026	1LFC, Lactoferricin B	FKCRRWQWRMKKLGAPSITCVRRAF	597
	(LfcinB, cow, ZZHa)

AP00193	1M4F, human LEAP-1	DTHFPICIFCCGCCHRSKCGMCCKT	598
	(Hepcidin 25)

AP00499	1MAG, Gramicidin A	VGALAVVVWLWLWLW	599
	(gA, bacteria)

AP00403	1MM0, Termicin	ACNFQSCWATCQAQHSIYFRRAFCDR	600
	(termite defensin, insect	SQCKCVFVRG
	defensin)

AP00194	1MMC, Ac-AMP2	VGECVRGRCPSGMCCSQFGYCGKGP	601
	(plant defensin, BBS)	KYCGR

AP01206	1MQZ, Mersacidin	CTFTLPGGGGVCTLTSECIC	602
	(bacteria)

AP00429	1NKL, Porcine NK-	GYFCESCRKIIQKLEDMVGPQPNEDTV	603
	Lysin (pig)	TQAASQVCDKLKILRGLCKKIMRSFL
		RRISWDILTGKKPQAICVDIKICKE

AP00633	log7, Sakacin P/	KYYGNGVHCGKHSCTVDWGTAIGNI	604
	Sakacin 674 (SakP,	GNNAAANWATGGNAGWNK
	class IIA bacteriocin,
	bacteria)

AP00195	1PG1, Protegrin 1	RGGRLCYCRRRFCVCVGR	605
	(Protegrin-1, PG-1, pig
	cathelicidin, XXA,
	ZZHa, BBBm)

AP00928	1PXQ, Subtilosin A	NKGCATCSIGAACLVDGPIPDFEIAGA	606
	(XXC, class I	TGLFGLWG
	bacteriocin, Gram-
	positive bacteria)

AP00480	1Q71, Microcin J25	VGIGTPIFSYGGGAGHVPEYF	607
	(cyclic MccJ25, class I
	microcins, bacteriocins,
	Gram-negative bacteria,
	XXC; BBP)

AP00211	1RKK, Polyphemusin I	RRWCFRVCYRGFCYRKCR	608
	(crabs, Crustacea)

AP00430	1T51, IsCT (Scorpion)	ILGKIWEGIKSLF	609

AP00731	1ut3, Spheniscin-2	SFGLCRLRRGFCARGRCRFPSIPIGRCS	610
	(Sphe-2, penguin	RFVQCCRRVW
	defensin, avian
	defensin)

AP00013	1VM5, Aurein 1.2	GLFDIIKKIAESF	611
	(frog)

AP00214	1WO1, Tachyplesin I	KWCFRVCYRGICYRRCR	612
	(crabs, Crustacea, XXA,
	ZZHa)

AP00644	1xc0, Pardaxin 4	GFFALIPKIISSPLFKTLLSAVGSALSSS	613
	(Pardaxin P-4, Pardaxin	GGQE
	P4, Pa4, flat fish)

AP00493	1XKM, Distinctin (two	NLVSGLIEARKYLEQLHRKLKNCKV	614
	chains for stability and
	transport? frog)

AP00420	1XV3, Penaeidin-4d	HSSGYTRPLRKPSRPIFIRPIGCDVCYGI	615
	(penaeidin 4, shrimp,	PSSTARLCCFRYGDCCHL
	Crustacea)

AP00035	1YTR, Plantaricin A	KSSAYSLQMGATAIKQVKKLFKKWGW	616
	(PlnA, bacteriocin,
	bacteria)

AP00166	1Z64, Pleurocidin (fish)	GWGSFFKKAAHVGKHVGKAALTHYL	617

AP00780	1Z6V, Human	GRRRRSVQWCAVSQPEATKCFQWQR	618
	lactoferricin	NMRKVRGPPVSCIKRDSPIQCIQA

AP00549	1ZFU, Plectasin (fungi,	GFGCNGPWDEDDMQCHNHCKSIKGY	619
	fungal defensin)	KGGYCAKGGFVCKCY

AP00177	1ZMH, human alpha	CYCRIPACIAGERRYGTCIYQGRLWAF	620
	Defensin HNP-2	CC
	(human neutrophil
	peptide-2, HNP2,
	human defensin, ZZHh)

AP00179	1ZMM, human alpha	VCSCRLVFCRRTELRVGNCLIGGVSFT	621
	Defensin HNP-4	YCCTRVD
	(human neutrophil
	peptide-4, HNP4,
	human defensin)

AP00180	1ZMP, human alpha	QARATCYCRTGRCATRESLSGVCEISG	622
	Defensin HD-5 (HD5,	RLYRLCCR
	human defensin)

AP00181	1ZMQ, human alpha	STRAFTCHCRRSCYSTEYSYGTCTVM	623
	Defensin HD-6 (HD6,	GINHRFCCL
	human defensin)

AP00399	1ZRW, Spinigerin	HVDKKVADKVLLLKQLRIMRLLTRL	624
	(insect, termite)

AP01157	1ZRX, Stomoxyn	RGFRKHFNKLVKKVKHTISETAHVAK	625
	(insect)	DTAVIAGSGAAVVAAT

AP00637	2A2B, Curvacin A/	ARSYGNGVYCNNKKCWVNRGEATQS	626
	sakacin A (CurA, SakA,	IIGGMISGWASGLAGM
	class IIA bacteriocin,
	bacteria)

AP00558	2B68, Cg-Def	GFGCPGNQLKCNNHCKSISCRAGYCD	627
	(Crassostrea gigas	AATLWLRCTCTDCNGKK
	defensin, oyster
	defensin, animal
	defensin)

AP01154	2B9K, LCI (bacteria)	AIKLVQSPNGNFAASFVLDGTKWIFKS	628
		KYYDSSKGYWVGIYEVWDRK

AP01005	2DCV, Tachystatin B1	YVSCLFRGARCRVYSGRSCCFGYYCR	629
	(BBS, horseshoe crabs)	RDFPGSIFGTCSRRNF

AP01006	2DCW, Tachystatin B1	YITCLFRGARCRVYSGRSCCFGYYCR	630
	(BBS, horseshoe crabs)	RDFPGSIFGTCSRRNF

AP00275	2ERI, Circulin B (CirB,	CGESCVFIPCISTLLGCSCKNKVCYRN	631
	plant cyclotides, XXC,	GVIP
	ZZHp)

AP00707	2f3a, LLAA (LL-37-	RLFDKIRQVIRKF	632
	derived aurein 1.2
	analog, retro-FK13,
	synthetic)

AP00708	2fbs, FK-13 (FK13,	FKRIVQRIKDFLR	633
	NMR-discovered LL-37
	core peptide, XXA,
	ZZHs, synthetic)

AP00088	2G9L, Gaegurin-4	GILDTLKQFAKGVGKDLVKGAAQGV	634
	(Gaegurin 4, frog)	LSTVSCKLAKTC

AP01011	2G9P, Latarcin 2a	GLFGKLIKKFGRKAISYAVKKARGKH	635
	(Ltc2a, BBM, spider)

AP00612	2GDL, Fowlicidin-2	LVQRGRFGRFLRKIRRFRPKVTITIQGS	636
	(chCATH-2, bird	ARFG
	cathelicidin, chicken
	cathelicidin, BBL)

AP00402	2GL1, VrD2 (Vigna	KTCENLANTYRGPCFTTGSCDDHCKN	637
	radiata defensin 2, plant	KEHLRSGRCRDDFRCWCTRNC
	defensin, mung bean)

AP00285	2GW9, Cryptdin-4	GLLCYCRKGHCKRGERVRGTCGIRFL	638
	(Crp4, animal defensin,	YCCPRR
	alpha, mouse)

AP00613	2hfr, Fowlicidin-3	RVKRFWPLVPVAINTVAAGINLYKAI	639
	(chCATH-3, bird	RRK
	cathelicidin, chicken
	cathelicidin)

AP01007	2JMY, CM15	KWKLFKKIGAVLKVL	640
	(Synthetic)

AP00728	2jni, Arenicin-2 (marine	RWCVYAYVRIRGVLVRYRRCW	641
	polychaeta, BBBm)

AP00473	2jos, Piscidin 1 (fish)	FFHHIFRGIVHVGKTIHRLVTG	642

AP01151	2JPJ, Lactococcin G-a	GTWDDIGQGIGRVAYWVGKALGNLS	643
	(chain a, class IIb	DVNQASRINRKKKH
	bacteriocin, bacteria.
	For chain b, see info)

AP00757	2jpy, Phylloseptin-H2	FLSLIPHAINAVSTLVHHF	644
	(PLS-H2, Phylloseptin-
	2, PS-2) (XXA, frog)

AP00546	2jq0, Phylloseptin-1	FLSLIPHAINAVSAIAKHN	645
	(Phylloseptin-H1, PLS-
	H1, PS-1, XXA, frog)

AP00758	2jq1, Phylloseptin-3	FLSLIPHAINAVSALANHG	646
	(Phylloseptin-H3, PLS-
	H3, PS-3) (XXA, frog)

AP00727	2jsb, Arenicin-1 (marine	RWCVYAYVRVRGVLVRYRRCW	647
	polychaeta, BBBm)

AP00592	2k10, Ranatuerin-2CSa	GILSSFKGVAKGVAKDLAGKLLETLK	648
	(frog)	CKITGC

AP00485	2K38, Cupiennin 1a	GFGALFKFLAKKVAKTVAKQAAKQG	649
	(spider)	AKYVVNKQME

AP00310	2K6O, Human LL-37	LLGDFFRKSKEKIGKEFKRIVQRIKDFL	650
	(LL37, human	RNLVPRTES
	cathelicidin; released by
	proteinase 3 from its
	precursor in neutrophils;
	FALL-39; BBB, BBM,
	BBP, BBW, BBD, BBL,
	ZZHh)

AP00199	2LEU, Leucocin A	KYYGNGVHCTKSGCSVNWGEAFSAG	651
	(LeuA, class IIa	VHRLANGGNGFW
	bacteriocin, bacteria)

AP00144	2MAG, Magainin 2	GIGKFLHSAKKFGKAFVGEIMNS	652
	(frog)

AP00146	2MLT, Melittin (insect,	GIGAVLKVLTTGLPALISWIKRKRQQ	653
	ZZHa)

AP01010	2PCO, Latarcin 1 (Ltc1,	SMWSGMWRRKLKKLRNALKKKLKG	654
	BBM, spider)	EK

AP00176	2PM1, human alpha	ACYCRIPACIAGERRYGTCIYQGRLW	655
	Defensin HNP-1	AFCC
	(human neutrophil
	peptide-1, HNP1,
	human defensin, ZZHh)

AP01158	2RLG, RP-1 (synthetic)	ALYKKFKKKLLKSLKRL	656

AP00102	8TFV, Thanatin (insect)	GSKKPVPIIYCNRRTGKCQRM	657

AP00995	A58718, Carnocin UI49	GSEIQPR	658
	(bacteria)

AP01002	AAC18827, Mutacin III	KSWSLCTPGCARTGSFNSYCC	659
	(mutacin 1140, bacteria)

AP00987	ABI74601, Arasin 1	SRWPSPGRPRPFPGRPKPIFRPRPCNCY	660
	(Crustacea)	APPCPCDRW

AP01000	CAA63706, variacin	GSGVIPTISHECHMNSFQFVFTCCS	661
	(lantibiotic, class I
	bacteriocin, bacteria)

AP00361	O15946, Lebocin 4	DLRFWNPREKLPLPTLPPFNPKPIYID	662
	(insect, silk moth)	MGNRY

AP00343	O16825, Andropin	VFIDILDKMENAIHKAAQAGIGIAKPIE	663
	(insect, fruit fly)	KMILPK

AP00417	O17513, Ceratotoxin D	SIGTAVKKAVPIAKKVGKVAIPIAKAV	664
	(insect, fly)	LSVVGQLVG

AP00435	O18494, Styelin C (sea	GWFGKAFRSVSNFYKKHKTYIHAGLS	665
	squirt, tunicate, XXA)	AATLL

AP00330	O18495, Styelin D (Sea	GWLRKAAKSVGKFYYKHKYYIKAA	666
	squirt, tunicate, XXA)	WQIGKHAL

AP00331	O18495, Styelin E (Sea	GWLRKAAKSVGKFYYKHKYYIKAA	667
	squirt, tunicate, XXA)	WKIGRHAL

AP01001	O54329, Mutacin II	NRWWQGVVPTVSYECRMNSWQHVF	668
	(lantibiotic, mutacin H-	TCC
	29B, J-T8, class I
	bacteriocin, bacteria)

AP00342	O81338, Antimicrobial	AKCIKNGKGCREDQGPPFCCSGFCYR	669
	peptide 1 (plant)	QVGWARGYCKNR

AP00373	O96059, Moricin 2	AKIPIKAIKTVGKAVGKGLRAINIASTA	670
	(insect)	NDVFNFLKPKKRKH

AP00449	P01190, Melanotropin	SYSMEHFRWGKPV	671
	alpha (Alpha-MSH)

AP00187	P01376,	VVCACRRALCLPRERRAGFCRIRGRIH	672
	CORTICOSTATIN III	PLCCRR
	(MCP-1, rabbit
	neutrophil peptide 1,
	NP-1) (animal defensin,
	alpha-defensin, rabbit)

AP00188	P01377,	VVCACRRALCLPLERRAGFCRIRGRIH	673
	CORTICOSTATIN IV	PLCCRR
	(MCP-2, rabbit
	neutrophil defensin
2,
	NP-2, animal defensin,
	rabbit)

AP00049	P01505, Bombinin	GIGALSAKGALKGLAKGLAEHFAN	674
	(toad)

AP00139	P01507, Cecropin A	KWKLFKKIEKVGQNIRDGIIKAGPAVA	675
	(insect, ZZHa)	VVGQATQIAK

AP00128	P01509, Cecropin B	KWKIFKKIEKVGRNIRNGIIKAGPAVA	676
	(insect, silk moth)	VLGEAKAL

AP00131	P01511, Cecropin D	WNPFKELERAGQRVRDAIISAGPAVA	677
	(insect, moth)	TVAQATALAK

AP00136	P01518, Crabrolin	FLPLILRKIVTAL	678
	(insect, XXA)

AP00183	P04142, Cecropin B	RWKIFKKIEKMGRNIRDGIVKAGPAIE	679
	(insect)	VLGSAKAI

AP00448	P04205, Mastoparan M	INLKAIAALAKKLL	680
	(MP-M, insect, XXA)

AP00234	P06833,	SDEKASPDKHHRFSLSRYAKLANRLA	681
	Seminalplasmin (SPLN,	NPKLLETFLSKWIGDRGNRSV
	calcium transporter
	inhibitor, caltrin, cow)

AP00314	P07466, Rabbit	VFCTCRGFLCGSGERASGSCTINGVRH	682
	neutrophil peptide 5	TLCCRR
	(NP-5, animal defensin,
	alpha-defensin)

AP00189	P07467, Rabbit	VSCTCRRFSCGFGERASGSCTVNGVR	683
	neutrophil peptide 4	HTLCCRR
	(NP-4)

AP00186	P07468,	GRCVCRKQLLCSYRERRIGDCKIRGV	684
	CORTICOSTATIN II	RFPFCCPR
	(Rabbit neutrophil
	peptide 3b (NP-3b,
	rabbit)

AP00185	P07469,	ICACRRRFCPNSERFSGYCRVNGARY	685
	CORTICOSTATIN I	VRCCSRR
	(rabbit)

AP00217	P07469, Rabbit	GICACRRRFCPNSERFSGYCRVNGAR	686
	neutrophil defensin 3a	YVRCCSRR
	(NP-3a, animal
	defensin, alpha-
	defensin)

AP00067	P07493, Bombolitin II	SKITDILAKLGKVLAHV	687
	(insect, bee)

AP00068	P07494, Bombolitin III	IKIMDILAKLGKVLAHV	688
	(insect, bee)

AP00069	P07495, Bombolitin IV	INIKDILAKLVKVLGHV	689
	(insect, bee)

AP00070	P07496, Bombolitin V	INVLGILGLLGKALSHL	690
	(insect, bee)

AP00236	P07504, Pyrularia	KSCCRNTWARNCYNVCRLPGTISREIC	691
	thionin (Pp-TH, plant)	AKKCDCKIISGTTCPSDYPK

AP00230	P08375, Sarcotoxin IA	GWLKKIGKKIERVGQHTRDATIQGLGI	692
	(insect, flesh	AQQAANVAATAR

AP00231	P08376, Sarcotoxin IB	GWLKKIGKKIERVGQHTRDATIQVIG	693
	(insect, flesh	VAQQAANVAATAR

AP00232	P08377, Sarcotoxin IC	GWLRKIGKKIERVGQHTRDATIQVLGI	694
	(insect, flesh	AQQAANVAATAR

AP00066	P10521, Bombolitin I	IKITTMLAKLGKVLAHV	695
	(insect, bee)

AP00206	P10946, Lantibiotic	WKSESLCTPGCVTGALQTCFLQTLTC	696
	subtilin (class I	NCKISK
	bacteriocin, bacteria)

AP00312	P11477, Cryptdin-2	LRDLVCYCRARGCKGRERMNGTCRK	697
	(Crp2, animal defensin,	GHLLYMLCCR
	alpha, mouse)

AP00205	P13068, Nisin A	ITSISLCTPGCKTGALMGCNMKTATC	698
	(lantibiotic, class I	HCSIHVSK
	bacteriocin, bacteria)

AP00215	P14214, Tachyplesin II	RWCFRVCYRGICYRKCR	699
	(crabs, Crustacea)

AP00212	P14216, Polyphemusin	RRWCFRVCYKGFCYRKCR	700
	II (crabs, Crustacea,
	XXA, ZZHa.
	Derivatives: T22)

AP00134	P14661, Cecropin P1	SWLSKTAKKLENSAKKRISEGIAIAIQ	701
	(pig)	GGPR

AP00011	P14662, Bactericidin B2	WNPFKELERAGQRVRDAVISAAPAVA	702
	(insect)	TVGQAAAIARG

AP00032	P14663, Bactericidin B-	WNPFKELERAGQRVRDAIISAGPAVA	703
	3 (insect)	TVGQAAAIARG

AP00033	P14664, Bactericidin B-	WNPFKELERAGQRVRDAIISAAPAVA	704
	4 (insect)	TVGQAAAIARG

AP00034	P14665, Bactericidin B-	WNPFKELERAGQRVRDAVISAAAVAT	705
	5P (insect)	VGQAAAIARG

AP00125	P14666, Cecropin	RWKIFKKIEKVGQNIRDGIVKAGPAV	706
	(insect, silk moth)	AVVGQAATI

AP00002	P15450, ABAECIN	YVPLPNVPQPGRRPFPTFPGQGPFNPKI	707
	(insect, honeybee)	KWPQGY

AP00505	P15516, human Histatin	DSHAKRHHGYKRKFHEKHHSHRGY	708
	5 (ZZHs; derivatives
	Dh-5)

AP00520	P15516, human Histatin 3	DSHAKRHHGYKRKFHEKHHSHRGYR	709
		SNYLYDN

AP00523	P15516, human Histatin 8	KFHEKHHSHRGY	710

AP00226	P17722, Royalisin	VTCDLLSFKGQVNDSACAANCLSLGK	711
	(insect, honeybee)	AGGHCEKVGCICRKTSFKDLWDKRF

AP00213	P18252, Tachyplesin III	KWCFRVCYRGICYRKCR	712
	(horseshoe crabs,
	Crustacea)

AP00233	P18312, Sarcotoxin ID	GWIRDFGKRIERVGQHTRDATIQTIAV	713
	(insect, flesh	AQQAANVAATLKG

AP00207	P19578, Lantibiotic	TAGPAIRASVKQCQKTLKATRLFTVS	714
	PEP5 (class I	CKGKNGCK
	bacteriocin, bacteria)

AP00009	P19660, BACTENECIN	RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPI	715
	5 (bac5, cow	RPPFRPPLGPFP
	cathelicidin)

AP00010	P19661, BACTENECIN	RRIRPRPPRLPRPRPRPLPFPRPGPRPIP	716
	7 (bac7, cow	RPLPFPRPGPRPIPRPLPFPRPGPRPIPRPL
	cathelicidin)

AP00200	P21564, Mastoparan B	LKLKSIVSWAKKVL	717
	(MP-B, insect, XXA)

AP00005	P21663, Andropin	VFIDILDKVENAIHNAAQVGIGFAKPF	718
	(insect, fly)	EKLINPK

AP00008	P22226, Cyclic	RLCRIVVIRVCR	719
	dodecapeptide (cow
	cathelicidin)

AP01205	P23826, Lactocin S	STPVLASVAVSMELLPTASVLYSDVA	720
	(XXD3, bacteria)	GCFKYSAKHHC

AP00239	P24335, XPF (the	GWASKIGQTLGKIAKVGLKELIQPK	721
	xenopsin precursor
	fragment, African
	clawed frog)

AP00235	P25068, Bovine tracheal	NPVSCVRNKGICVPIRCPGSMKQIGTC	722
	antimicrobial peptide	VGRAVKCCRKK
	(TAP, cow)

AP00418	P25230, CAP18 (rabbit	GLRKRLRKFRNKIKEKLKKIGQKIQGF	723
	cathelicidin, BBL)	VPKLAPRTDY

AP00203	P25403, Mj-AMP1	QCIGNGGRCNENVGPPYCCSGFCLRQ	724
	(MjAMP1, plant	PGQGYGYCKNR
	defensin)

AP00202	P25404, Mj-AMP2	CIGNGGRCNENVGPPYCCSGFCLRQP	725
	(MjAMP2, plant	NQGYGVCRNR
	defensin)

AP00138	P28310, Cryptdin-3	LRDLVCYCRKRGCKRRERMNGTCRK	726
	(Crp3, animal defensin,	GHLMYTLCCR
	alpha, mouse)

AP00184	P28794, MBP-1 (plant)	RSGRGECRRQCLRRHEGQPWETQEC	727
		MRRCRRRG

AP00050	P29002, Bombinin-like	GIGASILSAGKSALKGLAKGLAEHFAN	728
	peptide 1 (BLP-1, toad)

AP00051	P29003, Bombinin-like	GIGSAILSAGKSALKGLAKGLAEHFAN	729
	peptide 2 (BLP-2, toad)

AP00052	P29004, Bombinin-like	GIGAAILSAGKSALKGLAKGLAEHF	730
	peptide 3 (BLP-3, XXA,
	toad)

AP00053	P29005, Bombinin-like	GIGAAILSAGKSIIKGLANGLAEHF	731
	peptide 4 (BLP-4, toad)

AP00634	P29430, Pediocin PA-1/	KYYGNGVTCGKHSCSVDWGKATTCII	732
	AcH (PedPA1, class IIA	NNGAMAWATGGHQGNHKC
	bacteriocin, bacteria)

AP00204	P29559, Nisin Z	ITSISLCTPGCKTGALMGCNMKTATC	733
	(lantibiotic, class I	NCSIHVSK
	bacteriocin, bacteria)

AP00130	P29561, Cecropin C	GWLKKLGKRIERIGQHTRDATIQGLGI	734
	(insect, fly)	AQQAANVAATAR

AP00001	P31107,	GLWSKIKEVGKEAAKAAAKAAGKAA	735
	ADENOREGULIN	LGAVSEAV
	(Dermaseptin B2,
	Dermaseptin-B2, DRS-
	B2, DRS B2, frog)

AP00228	P31529, Sapecin B	LTCEIDRSLCLLHCRLKGYLRAYCSQQ	736
	(insect, flesh fly)	KVCRCVQ

AP00229	P31530, Sapecin C	ATCDLLSGIGVQHSACALHCVFRGNR	737
	(insect, flesh fly)	GGYCTGKGICVCRN

AP00218	P32195, Protegrin 2	RGGRLCYCRRRFCICV	738
	(PG-2, pig cathelicidin)

AP00219	P32196, Protegrin 3	RGGGLCYCRRRFCVCVGR	739
	(PG-3, pig cathelicidin)

AP00073	P32412, Brevinin-1E	FLPLLAGLAANFLPKIFCKITRKC	740
	(frog)

AP00080	P32414, Esculentin-1	GIFSKLGRKKIKNLLISGLKNVGKEVG	741
	(frog)	MDVVRTGIDIAGCKIKGEC

AP00074	P32423, Brevinin-1	FLPVLAGIAAKVVPALFCKITKKC	742
	(frog)

AP00075	P32424, Brevinin-2	GLLDSLKGFAATAGKGVLQSLLSTAS	743
	(frog)	CKLAKTC

AP00175	P34084, Macaque	DSHEERHHGRHGHHKYGRKFHEKHH	744
	histatin (M-Histatin 1,	SHRGYRSNYLYDN
	primate, monkey)

AP00006	P35581, Apidaecin IA	GNNRPVYIPQPRPPHPRI	745
	(insect, honeybee)

AP00007	P35581, Apidaecin IB	GNNRPVYIPQPRPPHPRL	746
	(insect, honeybee)

AP00414	P36190, Ceratotoxin A	SIGSALKKALPVAKKIGKIALPIAKAA	747
	(insect, fly)	LP

AP00415	P36191, Ceratotoxin B	SIGSAFKKALPVAKKIGKAALPIAKAA	748
	(insect, fly)	LP

AP00172	P36193, Drosocin	GKPRPYSPRPTSHPRPIRV	749
	(insect)

AP00170	P37362, Pyrrhocoricin	VDKGSYLPRPTPPRPIYNRN	750
	(insect)

AP00635	P38577, Mesentericin	KYYGNGVHCTKSGCSVNWGEAASAG	751
	Y105 (MesY105, class	IHRLANGGNGFW
	IIA bacteriocin,
	bacteria)

AP00636	P38579,	AISYGNGVYCNKEKCWVNKAENKQA	752
	Carnobacteriocin BM1	ITGIVIGGWASSLAGMGH
	(CnbBM1, PiscV1b,
	class IIA bacteriocin,
	bacteria)

AP00209	P39080, Peptide PGQ	GVLSNVIGYLKKLGTGALNAVLKQ	753
	(frog)

AP00513	P39084, Ranalexin	FLGGLIKIVPAMICAVTKKC	754
	(frog)

AP00071	P40835, Brevinin-1EA	FLPAIFRMAAKVVPTIICSITKKC	755
	(frog)

AP00072	P40836, Brevinin-1EB	VIPFVASVAAEMQHVYCAASRKC	756
	(frog)

AP00076	P40837, Brevinin-2EA	GILDTLKNLAISAAKGAAQGLVNKAS	757
	(frog)	CKLSGQC

AP00077	P40838, Brevinin-2EB	GILDTLKNLAKTAGKGALQGLVKMA	758
	(frog)	SCKLSGQC

AP00078	P40839, Brevinin-2EC	GILLDKLKNFAKTAGKGVLQSLLNTA	759
	(frog)	SCKLSGQC

AP00079	P40840, Brevinin-2ED	GILDSLKNLAKNAGQILLNKASCKLSG	760
	(frog)	QC

AP00081	P40843, Esculentin-1A	GIFSKLAGKKIKNLLISGLKNVGKEVG	761
	(frog)	MDVVRTGIDIAGCKIKGEC

AP00082	P40844, Esculentin-1B	GIFSKLAGKKLKNLLISGLKNVGKEVG	762
	(frog)	MDVVRTGIDIAGCKIKGEC

AP00083	P40845, Esculentin-2A	GILSLVKGVAKLAGKGLAKEGGKFGL	763
	(frog)	ELIACKIAKQC

AP00084	P40846, Esculentin-2B	GIFSLVKGAAKLAGKGLAKEGGKFGL	764
	(ES2B_RANES, frog)	ELIACKIAKQC

AP00299	P46156, Chicken	GRKSDCFRKSGFCAFLKCPSLTLISGK	765
	gallinacin 1 (Gal 1,	CSRFYLCCKRIW
	avian beta-defensin,
	bird)

AP00300	P46157, Gallinacin 1	GRKSDCFRKNGFCAFLKCPYLTLISGK	766
	alpha (avian beta-	CSRFHLCCKRIW
	defensin, Bird),

AP00298	P46158, Chicken	LFCKGGSCHFGGCPSHLIKVGSCFGFR	767
	gallinacin 2 (Gal 2,	SCCKWPWNA
	avian beta-defensin,
	bird)

AP00037	P46160, Beta-defensin 2	VRNHVTCRINRGFCVPIRCPGRTRQIG	768
	(cow)	TCFGPRIKCCRSW

AP00038	P46161, Beta-defensin 3	QGVRNHVTCRINRGFCVPIRCPGRTRQ	769
	(cow)	IGTCFGPRIKCCRSW

AP00039	P46162, Beta-defensin 4	QRVRNPQSCRWNMGVCIPFLCRVGM	770
	(cow)	RQIGTCFGPRVPCCRR

AP00040	P46163, Beta-defensin 5	QVVRNPQSCRWNMGVCIPISCPGNMR	771
	(cow)	QIGTCFGPRVPCCRRW

AP00041	P46164, Beta-defensin 6	QGVRNHVTCRIYGGFCVPIRCPGRTR	772
	(cow)	QIGTCFGRPVKCCRRW

AP00042	P46165, Beta-defensin 7	QGVRNFVTCRINRGFCVPIRCPGHRRQ	773
	(cow)	IGTCLGPRIKCCR

AP00043	P46166, Beta-defensin 8	VRNFVTCRINRGFCVPIRCPGHRRQIG	774
	(cow)	TCLGPQIKCCR

AP00044	P46167, Beta-defensin 9	QGVRNFVTCRINRGFCVPIRCPGHRRQ	775
	(cow)	IGTCLAPQIKCCR

AP00045	P46168, Beta-defensin	QGVRSYLSCWGNRGICLLNRCPGRMR	776
	10 (cow)	QIGTCLAPRVKCCR

AP00046	P46169, Beta-defensin	GPLSCRRNGGVCIPIRCPGPMRQIGTC	777
	11 (cow)	FGRPVKCCRSW

AP00048	P46171, Bovine beta-	SGISGPLSCGRNGGVCIPIRCPVPMRQI	778
	defensin 13 (cow)	GTCFGRPVKCCRSW

AP00350	P48821, Enbocin	PWNIFKEIERAVARTRDAVISAGPAVR	779
	(insect, moth)	TVAAATSVAS

AP00173	P49112, GNCP-2	RCICTTRTCRFPYRRLGTCLFQNRVYT	780
	(Guinea pig neutrophil	FCC
	cationic peptide 2)

AP00369	P49930, PMAP-23	RIIDLLWRVRRPQKPKFVTVWVR	781
	(PMAP23, pig
	cathelicidin)

AP00370	P49931, PMAP-36	VGRFRRLRKKTRKRLKKIGKVLKWIP	782
	(PMAP36, pig	PIVGSIPLGCG
	cathelicidin)

AP00371	P49932, PMAP-37	GLLSRLRDFLSDRGRRLGEKIERIGQKI	783
	(PMAP37, pig	KDLSEFFQS
	cathelicidin)

AP00220	P49933, Protegrin 4	RGGRLCYCRGWICFCVGR	784
	(PG-4, pig cathelicidin)

AP00221	P49934, Protegrin 5	RGGRLCYCRPRFCVCVGR	785
	(PG-5, pig cathelicidin)

AP00346	P50720, Hyphancin IIID	RWKIFKKIERVGQNVRDGIIKAGPAIQ	786
	(Fall webworm, insect)	VLGTAKAL

AP00347	P50721, Hyphancin IIIE	RWKFFKKIERVGQNVRDGLIKAGPAI	787
	(Fall webworm, insect)	QVLGAAKAL

AP00348	P50722, Hyphancin IIIF	RWKVFKKIEKVGRNIRDGVIKAGPAI	788
	(Fall webworm, insect)	AVVGQAKAL

AP00349	P50723, Hyphancin IIIG	RWKVFKKIEKVGRHIRDGVIKAGPAIT	789
	(Fall webworm, insect)	VVGQATAL

AP00281	P51473, mCRAMP	GLLRKGGEKIGEKLKKIGQKIKNFFQK	790
	(mouse cathelicidin;	LVPQPEQ
	derivatives: CRAMP
	18)

AP00366	P54228, BMAP-27	GRFKRFRKKFKKLFKKLSPVIPLLHLG	791
	(BMAP27, cow
	cathelicidin, ZZHs,
	derivatives BMAP-18
	and BMAP-15)

AP00367	P54229, BMAP-28	GGLRSLGRKILRAWKKYGPIIVPIIRIG	792
	(BMAP28, cow
	cathelicidin)

AP00450	P54230, Cyclic	RICRIIFLRVCR	793
	dodecapeptide (sheep
	cathelicidin)

AP00359	P54684, Lebocin 1/2	DLRFLYPRGKLPVPTPPPFNPKPIYIDM	794
	(insect, silk moth)	GNRY

AP00360	P55796, Lebocin 3	DLRFLYPRGKLPVPTLPPFNPKPIYIDM	795
	(insect, silk moth)	GNRY

AP00307	P55897, Buforin I (toad)	AGRGKQGGKVRAKAKTRSSRAGLQF	796
		PVGRVHRLLRKGNY

AP00308	P55897, Buforin II	TRSSRAGLQFPVGRVHRLLRK	797
	(toad)

AP00240	P56226, Caerin 1.1	GLLSVLGSVAKHVLPHVVPVIAEHL	798
	(frog, ZZHa)

AP00241	P56227, Caerin 1.2	GLLGVLGSVAKHVLPHVVPVIAEHL	799
	(frog)

AP00242	P56228, Caerin 1.3	GLLSVLGSVAQHVLPHVVPVIAEHL	800
	(frog)

AP00243	P56229, Caerin 1.4	GLLSSLSSVAKHVLPHVVPVIAEHL	801
	(frog)

AP00244	P56230, Caerin 1.5	GLLSVLGSVVKHVIPHVVPVIAEHL	802
	(frog)

AP00245	P56231, Caerin 1.6	GLFSVLGAVAKHVLPHVVPVIAEK	803
	(frog)

AP00246	P56232, Caerin 1.7	GLFKVLGSVAKHLLPHVAPVIAEK	804
	(frog)

AP00249	P56233, Caerin 2.1	GLVSSIGRALGGLLADVVKSKGQPA	805
	(frog)

AP00250	P56234, Caerin 2.2	GLVSSIGRALGGLLADVVKSKEQPA	806
	(frog)

AP00251	P56236, Caerin 2.4	GLVSSIGKALGGLLADVVKTKEQPA	807
	(frog)

AP00252	P56236, Caerin 2.5	GLVSSIGRALGGLLADVVKSKEQPA	808
	(frog)

AP00253	P56238, Caerin 3.1	GLWQKIKDKASELVSGIVEGVK	809
	(frog)

AP00254	P56238, Caerin 3.2	GLWEKIKEKASELVSGIVEGVK	810
	(frog)

AP00255	P56240, Caerin 3.3	GLWEKIKEKANELVSGIVEGVK	811
	(frog)

AP00256	P56241, Caerin 3.4	GLWEKIREKANELVSGIVEGVK	812
	(frog)

AP00257	P56242, Caerin 4.1	GLWQKIKSAAGDLASGIVEGIKS	813
	(frog)

AP00258	P56243, Caerin 4.2	GLWQKIKSAAGDLASGIVEAIKS	814
	(frog)

AP00259	P56244, Caerin 4.3	GLWQKIKNAAGDLASGIVEGIKS	815
	(frog)

AP00434	P56249, Frenatin 3	GLMSVLGHAVGNVLGGLFKS	816
	(frog)

AP00272	P56386, Murine beta-	DQYKCLQHGGFCLRSSCPSNTKLQGT	817
	defensin 1 (mBD-1,	CKPDKPNCCKS
	mouse)

AP00368	P56425, BMAP-34	GLFRRLRDSIRRGQQKILEKARRIGERI	818
	(BMAP34, cow	KDIFRG
	cathelicidin)

AP00273	P56685, Buthinin	SIVPIRCRSNRDCRRFCGFRGGRCTYA	819
	(Sahara scorpion)	RQCLCGY

AP00282	P56872,	SIPCGESCVFIPCTVTALLGCSCKSKVC	820
	Cyclopsychotride A	YKN
	(CPT, plant cyclotides,
	XXC)

AP00094	P56917, Temporin A	FLPLIGRVLSGIL	821
	(XXA, frog)

AP00096	P56918, Temporin C	LLPILGNLLNGLL	822
	(XXA, frog)

AP00097	P56920, Temporin E	VLPIIGNLLNSLL	823
	(XXA, frog)

AP00098	P56921, Temporin F	FLPLIGKVLSGIL	824
	(XXA, frog)

AP00100	P56923, Temporin K	LLPNLLKSLL	825
	(XXA, frog)

AP00295	P56928, eNAP-2 (horse)	EVERKHPLGGSRPGRCPTVPPGTFGHC	826
		ACLCTGDASEPKGQKCCSN

AP00101	P57104, Temporin L	FVQWFSKFLGRIL	827
	(XXA, frog)

AP00095	P79874, Temporin B	LLPIVGNLLKSLL	828
	(XXA, frog)

AP00099	P79875, Temporin G	FFPVIGRILNGIL	829
	(XXA, frog)

AP00413	P80032, Coleoptericin	SLQGGAPNFPQPSQQNGGWQVSPDLG	830
	(insect)	RDDKGNTRGQIEIQNKGKDHDFNAG
		WGKVIRGPNKAKPTWHVGGTYRR

AP00396	P80054, PR-39 (PR39,	RRRPRPPYLPRPRPPPFFPPRLPPRIPPG	831
	pig cathelicidin)	FPPRFPPRFP

AP00182	P80154, Insect defensin	GFGCPLDQMQCHRHCQTITGRSGGYC	832
		SGPLKLTCTCYR

AP00444	P80223, Corticostatin	GICACRRRFCLNFEQFSGYCRVNGAR	833
	VI (CS-VI) (animal	YVRCCSRR
	defensin, rabbit)

AP00208	P80230, Peptide 3910	RADTQTYQPYNKDWIKEKIYVLLRRQ	834
	(pig)	AQQAGK

AP00157	P80277, Dermaseptin-	ALWKTMLKKLGTMALHAGKAALGA	835
	S1 (Dermaseptin S1,	AADTISQGTQ
	DRS S1, DRS-S1, frog)

AP00158	P80278, Dermaseptin-	ALWFTMLKKLGTMALHAGKAALGA	836
	S2 (Dermaseptin S2,	AANTISQGTQ
	DRS S2, DRS-S2, frog)

AP00159	P80279, Dermaseptin-	ALWKNMLKGIGKLAGKAALGAVKKL	837
	S3 (Dermaseptin S3,	VGAES
	DRS S3, DRS-S3, frog)

AP00160	P80280, Dermaseptin-	ALWMTLLKKVLKAAAKALNAVLVG	838
	S4 (Dermaseptin S4,	ANA
	DRS S4, DRS-S4, frog)

AP00161	P80281, Dermaseptin-	GLWSKIKTAGKSVAKAAAKAAVKAV	839
	S5 (Dermaseptin S5,	TNAV
	DRS S5, DRS-S5, frog)

AP00293	P80282, Dermaseptin-	AMWKDVLKKIGTVALHAGKAALGA	840
	B1 (DRS-B1, DRS B1,	VADTISQ
	frog)

AP00264	P80389, Chicken	GRKSDCFRKSGFCAFLKCPSLTLISGK	841
	Heterophil Peptide 1	CSRFYLCCKRIR
	(CHP1, bird, animal)

AP00265	P80390, Chicken	GRKSDCFRKNGFCAFLKCPYLTLISGL	842
	Heterophil Peptide 2	CSFHLC
	(CHP2, bird, animal)

AP00266	P80391, Turkey	GKREKCLRRNGFCAFLKCPTLSVISGT	843
	Heterophil Peptide 1	CSRFQVCC
	(THP1, turkey)

AP00267	P80392, Turkey	LFCKRGTCHFGRCPSHLIKVGSCFGFR	844
	Heterophil Peptide 2	SCCKWPWDA
	(THP2, bird, anaimal)

AP00269	P80393, Turkey	LSCKRGTCHFGRCPSHLIKGSCSGG	845
	Heterophil Peptide 3
	(THP3, bird, animal)

AP00085	P80395, Gaegurin-1	SLFSLIKAGAKFLGKNLLKQGACYAA	846
	(Gaegurin 1, frog)	CKASKQC

AP00086	P80396, Gaegurin-2	GIMSIVKDVAKNAAKEAAKGALSTLS	847
	(Gaegurin 2, frog)	CKLAKTC

AP00087	P80397, Gaegurin-3	GIMSIVKDVAKTAAKEAAKGALSTLS	848
	(Gaegurin 3, frog)	CKLAKTC

AP00089	P80399, Gaegurin-5	FLGALFKVASKVLPSVFCAITKKC	849
	(Gaegurin 5, frog)

AP00090	P80400, Gaegurin-6	FLPLLAGLAANFLPTIICKISYKC	850
	(Gaegurin 6, frog)

AP00362	P80408, Metalnikowin I	VDKPDYRPRPRPPNM	851
	(insect)

AP00363	P80409, Metalnikowin	VDKPDYRPRPWPRPN	852
	IIA (insect)

AP00364	P80410, Metalnikowin	VDKPDYRPRPWPRNMI	853
	IIB (insect)

AP00365	P80411, Metalnikowin	VDKPDYRPRPWPRPNM	854
	III (insect)

AP00632	P80569, Piscicolin 126/	KYYGNGVSCNKNGCTVDWSKAIGIIG	855
	Piscicocin Via	NNAAANLTTGGAAGWNKG
	(PiscV1a, Pisc126, class
	IIA bacteriocin,
	bacteria)

AP01003	P80666, Mutacin B-	FKSWSFCTPGCAKTGSFNSYCC	856
	Ny266 (bacteria)

AP00276	P80710, Clavanin A	VFQFLGKIIHHVGNFVHGFSHVF	857
	(urochordates, sea
	squirts, and sea pork,
	tunicate)

AP00277	P80711, Clavanin B	VFQFLGRIIHHVGNFVHGFSHVF	858
	(Sea squirt, tunicate)

AP00278	P80712, Clavanin C	VFHLLGKIIHHVGNFVYGFSHVF	859
	(Sea squirt, tunicate)

AP00279	P80713, Clavanin D	AFKLLGRIIHHVGNFVYGFSHVF	860
	(Sea squirt, tunicate)

AP00280	P80713, Clavanin D	LFKLLGKIIHHVGNFVHGFSHVF	861
	(Sea squirt, tunicate)

AP00294	P80930, eNAP-1 (horse)	DVQCGEGHFCHDQTCCRASQGGACC	862
		PYSQGVCCADQRHCCPVGF

AP00400	P80952, Skin peptide	YPPKPESPGEDASPEEMNKYLTALRH	863
	tyrosine-tyrosine (skin-	YINLVTRQRY
	PYY, SPYY, frog)

AP00091	P80954, Rugosin A	GLLNTFKDWAISIAKGAGKGVLTTLS	864
	(frog)	CKLDKSC

AP00092	P80955, Rugosin B	SLFSLIKAGAKFLGKNLLKQGAQYAA	865
	(frog)	CKVSKEC

AP00093	P80956, Rugosin C	GILDSFKQFAKGVGKDLIKGAAQGVL	866
	(frog)	STMSCKLAKTC

AP00392	P81056, Penaeidin-1	YRGGYTGPIPRPPPIGRPPLRLVVCAC	867
	(shrimp, Crustacea)	YRLSVSDARNCCIKFGSCCHLVK

AP00393	P81057, Penaeidin-2a	YRGGYTGPIPRPPPIGRPPFRPVCNACY	868
	(shrimp, Crustacea)	RLSVSDARNCCIKFGSCCHLVK

AP00394	P81058, Penaeidin-3a	QVYKGGYTRPIPRPPPFVRPLPGGPIGP	869
	(shrimp, Crustacea)	YNGCPVSCRGISFSQARSCCSRLGRCC
		HVGKGYS

AP00247	P81251, Caerin 1.8	GLFKVLGSVAKHLLPHVVPVIAEK	870
	(frog)

AP00248	P81252, Caerin 1.9	GLFGVLGSIAKHVLPHVVPVIAEK	871
	(frog, ZZHa)

AP00126	P81417, Cecropin A	GGLKKLGKKLEGVGKRVFKASEKAL	872
	(insect, mosquito)	PVAVGIKALG

AP00169	P81437, Formaecin 2	GRPNPVNTKPTPYPRL	873
	(insect, ants)

AP00168	P81438, Formaecin 1	GRPNPVNNKPTPHPRL	874
	(insect, ants)

AP00296	P81456, Fabatin-1	LLGRCKVKSNRFHGPCLTDTHCSTVC	875
	(plant defensin)	RGEGYKGGDCHGLRRRCMCLC

AP00297	P81457, Fabatin-2	LLGRCKVKSNRFNGPCLTDTHCSTVC	876
	(plant defensin)	RGEGYKGGDCHGLRRRCMCLC

AP01215	P81463, European	FVPYNPPRPYQSKPFPSFPGHGPFNPKI	877
	bumblebee abaecin	QWPYPLPNPGH
	(insect)

AP01214	P81464, Apidaecin	GNRPVYIPPPRPPHPRL	878
	(insect)

AP00440	P81465, defensin	VTCFCRRRGCASRERHIGYCRFGNTIY	879
	HANP-1 (hamster)	RLCCRR

AP00441	P81466, defensin	CFCKRPVCDSGETQIGYCRLGNTFYRL	880
	HANP-2 (hamster)	CCRQ

AP00442	P81467, defensin	VTCFCRRRGCASRERLIGYCRFGNTIY	881
	HANP-3 (hamster)	GLCCRR

AP00439	P81468, defensin	VTCFCKRPVCDSGETQIGYCRLGNTF	882
	HANP-4 (hamster)	YRLCCRQ

AP00328	P81469, Styelin A (Sea	GFGKAFHSVSNFAKKHKTA	883
	squirt, tunicate, XXA)

AP00329	P81470, Styelin B (Sea	GFGPAFHSVSNFAKKHKTA	884
	squirt, tunicate, XXA)

AP00492	P81474, Misgurin (fish)	RQRVEELSKFSKKGAAARRRK	885

AP00165	P81485, Dermaseptin-	ALWKNMLKGIGKLAGQAALGAVKTL	886
	B3 (Dermaseptin B3,	VGAE
	DRS-B3, DRS B3, frog)

AP00163	P81486, Dermaseptin-	ALWKDILKNVGKAAGKAVLNTVTDM	887
	B4 (Dermaseptin B4,	VNQ
	DRS-B4, DRS B4,
	DRS-TR1, IRP, frog)

AP00162	P81487, Dermaseptin-	GLWNKIKEAASKAAGKAALGFVNEMV	888
	B5 (Dermaseptin B5,
	DRS-B5, DRS B5, frog)

AP00164	P81488, Dermaseptin-	ALWKTIIKGAGKMIGSLAKNLLGSQA	889
	B9 (Dermaseptin B9,	QPES
	DRS-B9, DRS DRG3,
	frog)

AP00167	P81565, Phylloxin	GWMSKIASGIGTFLSGMQQ	890
	(phylloxin-B1, PLX-B1,
	XXA, frog)

AP00291	P81568, Defensin D5	MFFSSKKCKTVSKTFRGPCVRNAN	891
	(So-D5) (plant defensin)

AP00290	P81569, Defensin D4	MFFSSKKCKTVSKTFRGPCVRNA	892
	(So-D4) (plant defensin)

AP00289	P81570, Defensin D3	GIFSSRKCKTVSKTFRGICTRNANC	893
	(So-D3) (plant defensin)

AP00288	P81572, Defensin D1	TCESPSHKFKGPCATNRNCES	894
	(So-D1) (plant defensin)

AP00292	P81573, Defensin D7	GIFSSRKCKTPSKTFKGYCTRDSNCDT	895
	(So-D7) (plant defensin)	SCRYEGYPAGD

AP00270	P81591, Pn-AMP	QQCGRQASGRLCGNRLCCSQWGYCG	896
	(PnAMP, plant	STASYCGAGCQSQCRS
	defensin)

AP00412	P81592, Acaloleptin A1	SLQPGAPNVNNKDQPWQVSPHISRDD	897
	(insect)	SGNTRTDINVQRHGENNDFEAGWSK
		VVRGPNKAKPTWHIGGTHRW

AP00433	P81605, human	SSLLEKGLDGAKKAVGGLGKLGKDA	898
	Dermcidin (DCD-1)	VEDLESVGKGAVHDVKDVLDSV

AP00332	P81612, Mytilin A	GCASRCKAKCAGRRCKGWASASFRG	899
	(Blue mussel)	RCYCKCFRC

AP00333	P81613, Mytilin B	SCASRCKGHCRARRCGYYVSVLYRG	900
	(Blue mussel)	RCYCKCLRC

AP00334	P81613, Moronecidin	FFHHIFRGIVHVGKTIHKLVTG	901
	(fish)

AP00351	P81835, Citropin 1.1	GLFDVIKKVASVIGGL	902
	(amphibian, frog)

AP00352	P81840, Citropin 1.2	GLFDIIKKVASVVGGL	903
	(amphibian, frog)

AP00353	P81846, Citropin 1.3	GLFDIIKKVASVIGGL	904
	(amphibian, frog)

AP00338	P81903, Histone H2B-	PDPAKTAPKKGSKKAVTKA	905
	1(HLP-1) (fish)

AP00271	P82018, ChBac5 (Goat	RFRPPIRRPPIRPPFNPPFRPPVRPPFRPP	906
	cathelicidin)	FRPPFRPPIGPFP

AP00316	P82027, Uperin 2.1	GIVDFAKKVVGGIRNALGI	907
	(amphibian, toad)

AP00317	P82028, Uperin 2.2	GFVDLAKKVVGGIRNALGI	908
	(amphibian, toad)

AP00318	P82029, Uperin 2.3	GFFDLAKKVVGGIRNALGI	909
	(amphibian, toad)

AP00319	P82030, Uperin 2.4	GILDFAKTVVGGIRNALGI	910
	(amphibian, toad)

AP00320	P82031, Uperin 2.5	GIVDFAKGVLGKIKNVLGI	911
	(amphibian, toad)

AP00323	P82032, Uperin 3.1	GVLDAFRKIATVVKNVV	912
	(amphibian, toad)

AP00326	P82035, Uperin 4.1	GVGSFIHKVVSAIKNVA	913
	(amphibian, toad)

AP00321	P82039, Uperin 2.7	GIIDIAKKLVGGIRNVLGI	914
	(amphibian, toad)

AP00322	P82040, Uperin 2.8	GILDVAKTLVGKLRNVLGI	915
	(amphibian, toad)

AP00324	P82042, Uperin 3.5	GVGDLIRKAVSVIKNIV	916
	(amphibian, toad)

AP00325	P82042, Uperin 3.6	GVIDAAKKVVNVLKNLP	917
	(amphibian, toad)

AP00327	P82050, Uperin 7.1	GWFDVVKHIASAV	918
	(amphibian, frog)

AP00260	P82066, Maculatin 1.1	GLFVGVLAKVAAHVVPAIAEHF	919
	(XXA, frog, ZZHa)

AP00261	P82067, Maculatin 1.2	GLFVGLAKVAAHNNPAIAEHFQA	920
	(XXA, frog)

AP00262	P82068, Maculatin 2.1	GFVDFLKKVAGTIANVVT	921
	(frog)

AP00263	P82069, Maculatin 3.1	GLLQTIKEKLESLESLAKGIVSGIQA	922
	(frog)

AP00345	P82104, Caerin 1.10	GLLSVLGSVAKHVLPHVVPVIAEKL	923
	(frog)

AP00456	P82232, Brevinin-1T	VNPIILGVLPKFVCLITKKC	924
	(frog)

AP00459	P82233, Brevinin-1TA	FITLLLRKFICSITKKC	925
	(frog)

AP00457	P82234, Brevinin-2TC	GLWETIKNFGKKFTLNILHKLKCKIGG	926
	(frog)	GC

AP00458	P82235, Brevinin-2TD	GLWETIKNFGKKFTLNILHNLKCKIGG	927
	(frog)	GC

AP00397	P82238, Salmocidin 2A	SGFVLKGYTKTSQ	928
	(fish, trout)

AP00398	P82239, Salmocidin 2B	AGFVLKGYTKTSQ	929
	(fish, trout)

AP00055	P82282, Bombinin H1	IIGPVLGMVGSALGGLLKKI	930
	(frog)

AP00056	P82284, Bombinin H4	LIGPVLGLVGSALGGLLKKI	931
	(frog, XXA, XXD)

AP00057	P82285, Bombinin H5	IIGPVLGLVGSALGGLLKKI	932
	(frog, XXD)

AP00419	P82286, Bombinin-like	GIGASILSAGKSALKGFAKGLAEHFAN	933
	peptides 2 (amphibian,
	toad)

AP00137	P82293, Cryptdin-1	LRDLVCYCRTRGCKRRERMNGTCRK	934
	(Crp1, animal defensin,	GHLMYTLCCR
	alpha, mouse)

AP00443	P82317, defensin	ACYCRIPACLAGERRYGTCFYMGRV	935
	RMAD-2 (monkey)	WAFCC

AP00012	P82386, Aurein 1.1	GLFDIIKKIAESI	936
	(amphibian, frog)

AP00014	P82388, Aurein 2.1	GLLDIVKKVVGAFGSL	937
	(amphibian, frog)

AP00015	P82389, Aurein 2.2	GLFDIVKKVVGALGSL	938
	(amphibian, frog)

AP00016	P82390, Aurein 2.3	GLFDIVKKVVGAIGSL	939
	(XXA, amphibian, frog)

AP00017	P82391, Aurein 2.4	GLFDIVKKVVGTIAGL	940
	(XXA, amphibian, frog)

AP00018	P82392, Aurein 2.5	GLFDIVKKVVGAFGSL	941
	(XXA, amphibian, frog)

AP00019	P82393, Aurein 2.6	GLFDIAKKVIGVIGSL	942
	(XXA, amphibian, frog)

AP00020	P82394, Aurein 3.1	GLFDIVKKIAGHIAGSI	943
	(XXA, amphibian, frog)

AP00021	P82395, Aurein 3.2	GLFDIVKKIAGHIASSI	944
	(XXA, amphibian, frog)

AP00022	P82396, Aurein 3.3	GLFDIVKKIAGHIVSSI	945
	(XXA, amphibian, frog)

AP00376	P82414, Ponericin G1	GWKDWAKKAGGWLKKKGPGMAKA	946
	(ants)	ALKAAMQ

AP00377	P82415, Ponericin G2	GWKDWLKKGKEWLKAKGPGIVKAA	947
	(ants)	LQAATQ

AP00378	P82416, Ponericin G3	GWKDWLNKGKEWLKKKGPGIMKAA	948
	(ants)	LKAATQ

AP00379	P82417, Ponericin G4	DFKDWMKTAGEWLKKKGPGILKAA	949
	(ants)	MAAAT

AP00380	P82418, Ponericin G5	GLKDWVKIAGGWLKKKGPGILKAAM	950
	(ants)	AAATQ

AP00381	P82419, Ponericin G6	GLVDVLGKVGGLIKKLLP	951
	(ants)

AP00382	P82420, Ponericin G7	GLVDVLGKVGGLIKKLLPG	952
	(ants)

AP00383	P82421, Ponericin L1	LLKELWTKMKGAGKAVLGKIKGLL	953
	(ants)

AP00384	P82422, Ponericin L2	LLKELWTKIKGAGKAVLGKIKGLL	954
	(ants)

AP00386	P82423, Ponericin W1	WLGSALKIGAKLLPSVVGLFKKKKQ	955
	(ants)

AP00387	P82424, Ponericin W2	WLGSALKIGAKLLPSVVGLFQKKKK	956
	(ants)

AP00388	P82425, Ponericin W3	GIWGTLAKIGIKAVPRVISMLKKKKQ	957
	(ants)

AP00389	P82426, Ponericin W4	GIWGTALKWGVKLLPKLVGMAQTKKQ	958
	(ants)

AP00390	P82427, Ponericin W5	FWGALIKGAAKLIPSVVGLFKKKQ	959
	(ants)

AP00391	P82428, Ponericin W6	FIGTALGIASAIPAIVKLFK	960
	(ants)

AP00303	P82651, Tigerinin-1	FCTMIPIPRCY	961
	(frog)

AP00304	P82652, Tigerinin-2	RVCFAIPLPICH	962
	(frog)

AP00305	P82653, Tigerinin-3	RVCYAIPLPICY	963
	(frog)

AP00301	P82656, Hadrurin	GILDTIKSIASKVWNSKTVQDLKRKGI	964
	(scorpion)	NWVANKLGVSPQAA

AP00113	P82740,	GLLSGLKKVGKHVAKNVAVSLMDSL	965
	RANATUERIN 1T	KCKISGDC
	(frog)

AP00114	P82741,	SMLSVLKNLGKVGLGFVACKINKQC	966
	RANATUERIN 1
	(Ranatuerin-1, frog)

AP00115	P82742,	GLFLDTLKGAAKDVAGKLEGLKCKIT	967
	RANATUERIN 2	GCKLP
	(Ranatuerin-2, frog)

AP00116	P82780,	GFLDIINKLGKTFAGHMLDKIKCTIGT	968
	RANATUERIN 3	CPPSP
	(Ranatuerin-3, frog)

AP00117	P82819,	FLPFIARLAAKVFPSIICSVTKKC	969
	RANATUERIN 4
	(Ranatuerin-4, frog)

AP00405	P82821,	FISAIASMLGKFL	970
	RANATUERIN 6 (frog)

AP00406	P82822,	FLSAIASMLGKFL	971
	RANATUERIN 7 (frog)

AP00407	P82823,	FISAIASFLGKFL	972
	RANATUERIN 8 (frog)

AP00408	P82824,	FLFPLITSFLSKVL	973
	RANATUERIN 9 (frog)

AP00461	P82825, Brevinin-1LA	FLPMLAGLAASMVPKLVCLITKKC	974
	(frog)

AP00462	P82826, Brevinin-1LB	FLPMLAGLAASMVPKFVCLITKKC	975
	(frog)

AP00118	P82828,	GILDSFKGVAKGVAKDLAGKLLDKLK	976
	RANATUERIN 2La	CKITGC
	(Ranatuerin-2La, frog)

AP00119	P82829,	GILSSIKGVAKGVAKNVAAQLLDTLK	977
	RANATUERIN 2Lb	CKITGC
	(Ranatuerin-2Lb, frog)

AP00109	P82830, Temporin-1La	VLPLISMALGKLL	978
	(Temporin 1La, frog)

AP00110	P82831, Temporin-1Lb	NFLGTLINLAKKIM	979
	(Temporin 1Lb, frog)

AP00111	P82832, Temporin-1Lc	FLPILINLIHKGLL	980
	(Temporin 1Lc, frog)

AP00463	P82833, Brevinin-1BA	FLPFIAGMAAKFLPKIFCAISKKC	981
	(frog)

AP00464	P82834, Brevinin-1BB	FLPAIAGMAAKFLPKIFCAISKKC	982
	(frog)

AP00465	P82835, Brevinin-1BC	FLPFIAGVAAKFLPKIFCAISKKC	983
	(frog)

AP00466	P82836, Brevinin-1BD	FLPAIAGVAAKFLPKIFCAISKKC	984
	(frog)

AP00467	P82837, Brevinin-1BE	FLPAIVGAAAKFLPKIFCVISKKC	985
	(frog)

AP00468	P82838, Brevinin-1BF	FLPFIAGMAANFLPKIFCAISKKC	986
	(frog)

AP00120	P82840,	GLLDTIKGVAKTVAASMLDKLKCKIS	987
	RANATUERIN 2B	GC
	(Ranatuerin-2B, frog)

AP00469	P82841, Brevinin-1PA	FLPIIAGVAAKVFPKIFCAISKKC	988
	(frog)

AP00460	P82842, Brevinin-1PB	FLPIIAGIAAKVFPKIFCAISKKC	989
	(frog)

AP00470	P82843, Brevinin-1PC	FLPIIASVAAKVFSKIFCAISKKC	990
	(frog)

AP00471	P82844, Brevinin-1PD	FLPIIASVAANVFSKIFCAISKKC	991
	(frog)

AP00472	P82845, Brevinin-1PE	FLPIIASVAAKVFPKIFCAISKKC	992
	(frog)

AP00121	P82847,	GLMDTVKNVAKNLAGHMLDKLKCKI	993
	RANATUERIN 2P	TGC
	(Ranatuerin-2P, frog)

AP00112	P82848, Temporin-1P	FLPIVGKLLSGLL	994
	(Temporin 1P, frog)

AP00452	P82871, Brevinin-1SY	FLPVVAGLAAKVLPSIICAVTKKC	995
	(frog)

AP00122	P82875, Ranatuerin-1C	SMLSVLKNLGKVGLGLVACKINKQC	996
	(Ranatuerin 1C, frog)

AP00514	P82876, Ranalexin-1Ca	FLGGLMKAFPALICAVTKKC	997
	(frog)

AP00515	P82877, Ranalexin-1Cb	FLGGLMKAFPAIICAVTKKC	998
	(frog)

AP00124	P82878, Ranatuerin-2Ca	GLFLDTLKGAAKDVAGKLLEGLKCKI	999
	(Ranatuerin 2Ca, frog)	AGCKP

AP00123	P82879, Ranatuerin-	GLFLDTLKGLAGKLLQGLKCIKAGCKP	1000
	2Cb (Ranatuerin 2Cb,
	frog)

AP00104	P82880, Temporin-1Ca	FLPFLAKILTGVL	1001
	(Temporin 1Ca, frog)

AP00105	P82881, Temporin-1Cb	FLPLFASLIGKLL	1002
	(Temporin 1Cb, frog)

AP00106	P82882, Temporin-1Cc	FLPFLASLLTKVL	1003
	(Temporin 1Cc, frog)

AP00107	P82883, Temporin-1Cd	FLPFLASLLSKVL	1004
	(Temporin 1Cd, frog)

AP00108	P82884, Temporin-1Ce	FLPFLATLLSKVL	1005
	(Temporin 1Ce, frog)

AP00453	P82904, Brevinin-1SA	FLPAIVGAAGQFLPKIFCAISKKC	1006
	(frog)

AP00454	P82905, Brevinin-1SB	FLPAIVGAAGKFLPKIFCAISKKC	1007
	(frog)

AP00455	P82906, Brevinin-1SC	FFPIVAGVAGQVLKKIYCTISKKC	1008
	(frog)

AP00996	P82907, Lichenin	ISLEICAIFHDN	1009
	(bacteria)

AP00302	P82951, Hepcidin (fish)	GCRFCCNCCPNMSGCGVCCRF	1010

AP00058	P83080, Maximin 1	GIGTKILGGVKTALKGALKELASTYAN	1011
	(toad)

AP00059	P83081, Maximin 2	GIGTKILGGVKTALKGALKELASTYVN	1012
	(toad)

AP00060	P83082, Maximin 3	GIGGKILSGLKTALKGAAKELASTYLH	1013
	(toad, ZZHa)

AP00061	P83083, Maximin 4	GIGGVLLSAGKAALKGLAKVLAEKYAN	1014
	(toad)

AP00062	P83084, Maximin 5	SIGAKILGGVKTFFKGALKELASTYLQ	1015
	(toad)

AP00063	P83085, Maximin 6	ILGPVISTIGGVLGGLLKNL	1016
	(toad)

AP00064	P83086, Maximin 7	ILGPVLGLVGNALGGLIKNE	1017
	(toad)

AP00065	P83087, Maximin 8	ILGPVLSLVGNALGGLLKNE	1018
	(toad)

AP00355	P83171, Ginkbilobin	ANTAFVSSAHNTQKIPAGAPFNRNLR	1019
	(Chinese plant)	AMLADLRQNAAFAG

AP00475	P83188, Pseudin 1	GLNTLKKVFQGLHEAIKLINNHVQ	1020
	(frog)

AP00476	P83189, Pseudin 2	GLNALKKVFQGIHEAIKLINNHVQ	1021
	(frog)

AP00477	P83190, Pseudin 3	GINTLKKVIQGLHEVIKLVSNHE	1022
	(frog)

AP00478	P83191, Pseudin 4	GINTLKKVIQGLHEVIKLVSNHA	1023
	(frog)

AP00410	P83287, Oncorhyncin	SKGKKANKDVELARG	1024
	III (fish)

AP00357	P83305, Japonicin-1	FFPIGVFCKIFKTC	1025
	(amphibian, frog)

AP00358	P83306, Japonicin-2	FGLPMLSILPKALCILLKRKC	1026
	(amphibian, frog)

AP00385	P83312, Parabutoporin	FKLGSFLKKAWKSKLAKKLRAKGKE	1027
	(scorpion)	MLKDYAKGLLEGGSEEVPGQ

AP00374	P83313, Opistoporin 1	GKVWDWIKSTAKKLWNSEPVKELKN	1028
	(scorpion)	TALNAAKNLVAEKIGATPS

AP00375	P83314, Opistoporin 2	GKVWDWIKSTAKKLWNSEPVKELKN	1029
	(scorpion)	TALNAAKNFVAEKIGATPS

AP00336	P83327, Histone H2A	AERVGAGAPVYL	1030
	(fish)

AP00335	P83338, Histone H6-	PKRKSATKGDEPA	1031
	like protein (fish)

AP00411	P83374, Oncorhyncin II	KAVAAKKSPKKAKKPAT	1032
	(fish)

AP00999	P83375, Serracin-P 43 kDa	DYHHGVRVL	1033
	subunit (bacteria)

AP00284	P83376, Dolabellanin	SHQDCYEALHKCMASHSKPFSCSMKF	1034
	B2 (sea hare)	HMCLQQQ

AP00998	P83378, Serracin-P 23 kDa	ALPKKLKYLNLFNDGFNYMGVV	1035
	subunit
	(bacteriocin, bacteria)

AP00129	P83403, Cecropin	GWLKKIGKKIERVGQNTRDATVKGLE	1036
	(insect, moth)	VAQQAANVAATVR

AP00127	P83413, Cecropin A	RWKVFKKIEKVGRNIRDGVIKAAPAIE	1037
	(insect, moth)	VLGQAKAL

AP00372	P83416, Virescein	GKIPIGAIKKAGKAIGKGLRAVNIAST	1038
	(insect)	AHDVYTFFKPKKRH

AP00356	P83427, Heliocin	QRFIHPTYRPPPQPRRPVIMRA	1039
	(insect)

AP00409	P83428, Locustin	ATTGCSCPQCIIFDPICASSYKNGRRGF	1040
	(insect)	SSGCHMRCYNRCHGTDYFQISKGSKCI

AP00339	P83545, Chrysophsin-1	FFGWLIKGAIHAGKAIHGLIHRRRH	1041
	(Red sea bream, madai)

AP00340	P83546, Chrysophsin-2	FFGWLIRGAIHAGKAIHGLIHRRRH	1042
	(Red sea bream, madai)

AP00341	P83547, Chrysophsin-3	FIGLLISAGKAIHDLIRRRH	1043
	(Red sea bream, madai)

AP01004	P84763, Thuricin-S	DWTAWSALVAAACSVELL	1044
	(bacteria)

AP00553	P84868, Sesquin (plant,	KTCENLADTY	1045
	ZZHp)

AP00132	Q06589, Cecropin 1	GWLKKIGKKIERVGQHTRDATIQTIAV	1046
	(insect, fly)	AQQAANVAATAR

AP00135	Q06590, Cecropin 2	GWLKKIGKKIERVGQHTRDATIQTIGV	1047
	(insect fly)	AQQAANVAATLK

AP00416	Q17313, Ceratotoxin C	SLGGVISGAKKVAKVAIPIGKAVLPVV	1048
	(insect, fly)	AKLVG

AP00171	Q24395, Metchnikowin	HRHQGPIFDTRPSPFNPNQPRPGPIY	1049
	(insect)

AP00354	Q27023, Tenecin 1	VTCDILSVEAKGVKLNDAACAAHCLF	1050
	(insect)	RGRSGGYCNGKRVCVCR

AP00401	Q28880, Lingual	GFTQGVRNSQSCRRNKGICVPIRCPGS	1051
	antimicrobial peptide	MRQIGTCLGAQVKCCRRK
	(LAP, beta defensin,
	cow)

AP00224	Q62713, RatNP-3 (rat)	CSCRTSSCRFGERLSGACRLNGRIYRL	1052
		CC

AP00225	Q62714, RatNP-4 (rat)	ACYCRIGACVSGERLTGACGLNGRIY	1053
		RLCCR

AP00223	Q62715, RatNP-2 (rat)	VTCYCRSTRCGFRERLSGACGYRGRI	1054
		YRLCCR

AP00222	Q62716, RatNP-1 (rat)	VTCYCRRTRCGFRERLSGACGYRGRI	1055
		YRLCCR

AP00174	Q64365, GNCP-1	RRCICTTRTCRFPYRRLGTCIFQNRVY	1056
	(Guinea pig neutrophil	TFCC
	cationic peptide 1)

AP00311	Q90W78, Galensin	CYSAAKYPGFQEFINRKYKSSRF	1057
	(frog)

AP00395	Q95NT0, Penaeidin-4a	HSSGYTRPLPKPSRPIFIRPIGCDVCYGI	1058
	(shrimp, Crustacea)	PSSTARLCCFRYGDCCHR

AP00423	Q962B0, Penaeidin-3n	QGYKGPYTRPILRPYVRPVVSYNACT	1059
	(shrimp, Crustacea)	LSCRGITTTQARSCSTRLGRCCHVAKG
		YS

AP00422	Q962B1, Penaeidin-3m	QGCKGPYTRPILRPYVRPVVSYNACT	1060
	(shrimp, Crustacea)	LSCRGITTTQARSCCTRLGRCCHVAK
		GYS

AP00421	Q963C3, Penaeidin-4C	YSSGYTRPLPKPSRPIFIRPIGCDVCYGI	1061
	(shrimp, Crustacea)	PSSTARLCCFRYGDCCHR

AP00210	Q99134, PGLa (African	GMASKAGAIAGKIAKVALKAL	1062
	clawed frog, XXA)

AP00054	Q9DET7, Bombinin-	GIGGALLSAGKSALKGLAKGLAEHFAN	1063
	like peptide 7 (BLP-7,
	toad)

AP00315	Q9PT75, Dermatoxin	SLGSFLKGVGTTLASVGKVVSDQFGK	1064
	(Two-colored leaf frog)	LLQAGQ

AP00133	Q9Y0Y0, Cecropin B	GGLKKLGKKLEGVGKRVFKASEKAL	1065
	(insect, mosquito)	PVLTGYKAIG

AP00004	Ref, Ct-AMP1	NLCERASLTWTGNCGNTGHCDTQCR	1066
	(CtAMP1, C. ternatea-	NWESAKHGACHKRGNWKCFCYFDC
	antimicrobial peptide 1,
	plant defensin)

AP00027	Ref, hexapeptide	RRWQWR	1067
	(synthetic)

AP00529	Ref, Lantibiotic Ericin S	WKSESVCTPGCVTGVLQTCFLQTITC	1068
	(bacteria)	NCHISK

AP00306	Ref, Tigerinin-4 (frog)	RVCYAIPLPIC	1069

AP00309	Ref, Human KS-27	KSKEKIGKEFKRIVQRIKDFLRNLVPR	1070
	(KS27 from LL-37)

AP00344	Ref, Apidaecin II	GNNRPIYIPQPRPPHPRL	1071
	(honeybee, insect)

AP00424	Ref, XT1 (frog)	GFLGPLLKLAAKGVAKVIPHLIPSRQQ	1072

AP00425	Ref, XT 2 (frog)	GCWSTVLGGLKKFAKGGLEAIVNPK	1073

AP00426	Ref, XT 4 (frog)	GVFLDALKKFAKGGMNAVLNPK	1074

AP00427	Ref, XT 7 (frog)	GLLGPLLKIAAKVGSNLL	1075

AP00431	Ref, human LLP 1	RVIEVVQGACRAIRHIPRRIRQGLERIL	1076

AP00432	Ref, human LLP	RIAGYGLRGLAVIIRICIRGLNLIFEIIR	1077

AP00447	Ref, Anoplin (insect)	GLLKRIKTLL	1078

AP00474	Ref, Piscidin 3 (fish)	FIHHIFRGIVHAGRSIGRFLTG	1079

AP00481	Ref, Kaliocin-1	FFSASCVPGADKGQFPNLCRLCAGTG	1080
	(synthetic)	ENKCA

AP00482	Ref, Thionin mutation	KSCCRNTWARNCYNVCRLPGTISREIC	1081
	(synthetic)	AKKCRCKIISGTTCPSDYPK

AP00484	Ref, Stomoxyn (insect,	RGFRKHFNKLVKKVKHTISETAHVAK	1082
	fly)	DTAVIAGSGAAVVAAT

AP00486	Ref, Cupiennin 1b	GFGSLFKFLAKKVAKTVAKQAAKQG	1083
	(spider)	AKYIANKQME

AP00487	Ref, Cupiennin 1c	GFGSLFKFLAKKVAKTVAKQAAKQG	1084
	(spider)	AKYIANKQTE

AP00488	Ref, Cupiennin 1D	GFGSLFKFLAKKVAKTVAKQAAKQG	1085
	(spider)	AKYVANKHME

AP00489	Ref, Hipposin (fish)	SGRGKTGGKARAKAKTRSSRAGLQFP	1086
		VGRVHRLLRKGNYAHRVGAGAPVYL

AP00923	Ref, Carnobacteriocin	AISYGNGVYCNKEKCWVNKAENKQA	1087
	B1 (XXO, class IIa	ITGIVIGGWASSLAGMGH
	bacteriocin, bacteria)

AP00496	Ref, HP 2-20 (synthetic)	AKKVFKRLEKLFSKIQNDK	1088

AP00497	Ref, Maximin H5 (toad)	ILGPVLGLVSDTLDDVLGIL	1089

AP00498	Ref, rCRAMP (rat	GLVRKGGEKFGEKLRKIGQKIKEFFQ	1090
	cathelicidin)	KLALEIEQ

AP00500	Ref, S9-P18 (synthetic)	KWKLFKKISKFLHLAKKF	1091

AP00501	Ref, L9-P18 (synthetic)	KWKLFKKILKFLHLAKKF	1092

AP00502	Ref, Clavaspirin (sea	FLRFIGSVIHGIGHLVHHIGVAL	1093
	squirt, tunicate)

AP00503	Ref, human P-113D	AKRHHGYKRKFH	1094

AP00504	Ref, human MUC7 20-	LAHQKPFIRKSYKCLHKRCR	1095
	Mer

AP00507	Ref, Nigrocin 2 (frog)	GLLSKVLGVGKKVLCGVSGLC	1096

AP00508	Ref, Nigrocin 1 (frog)	GLLDSIKGMAISAGKGALQNLLKVAS	1097
		CKLDKTC

AP00509	Ref, human Calcitermin	VAIALKAAHYHTHKE	1098

AP00510	Ref, Dicynthaurin (sea	ILQKAVLDCLKAAGSSLSKAAITAIYN	1099
	peach)	KIT

AP00511	Ref, KIGAKI	KIGAKIKIGAKIKIGAKI	1100
	(synthetic)

AP00516	Ref, Lycotoxin I	IWLTALKFLGKHAAKHLAKQQLSKL	1101
	(spider)

AP00517	Ref, Lycotoxin II	KIKWFKTMKSIAKFIAKEQMKKHLGGE	1102
	(spider)

AP00518	Ref, Ib-AMP3 (plant	QYRHRCCAWGPGRKYCKRWC	1103
	defensin, balsam)

AP00519	Ref, Ib-AMP4 (plant	EWGRRCCGWGPGRRYCRRWC	1104
	defensin, balsam)

AP00521	Ref, Dhvar4 (synthetic)	KRLFKKLLFSLRKY	1105

AP00522	Ref, Dhvar5 (synthetic)	LLLFLLKKRKKRKY	1106

AP00525	Ref, Maximin H2 (toad)	ILGPVLSMVGSALGGLIKKI	1107

AP00526	Ref, Maximin H3 (toad)	ILGPVLGLVGNALGGLIKKI	1108

AP00527	Ref, Maximin H4 (toad)	ILGPVISKIGGVLGGLLKNL	1109

AP00528	Ref, Anionic peptide	DDDDDD	1110
	SAAP (sheep)

AP00530	Ref, Lantibiotic Ericin	VLSKSLCTPGCITGPLQTCYLCFPTFA	1111
	A (bacteria)	KC

AP00531	Ref, Kenojeinin I (sea	GKQYFPKVGGRLSGKAPLAAKTHRRL	1112
	skate)	KP

AP00532	Ref, Lunatusin (plant,	KTCENLADTFRGPCFATSNC	1113
	ZZHp)

AP00533	Ref, Fallaxin (frog)	GVVDILKGAAKDIAGHLASKVMNKL	1114

AP00534	Ref, Tu-AMP 2	KSCCRNTTARNCYNVCRIPG	1115
	(TuAMP2, thionin-like
	antimicrobial peptides,
	plant defensin, tulip)

AP00535	Ref, Pilosulin 1 (Myr b	GLGSVFGRLARILGRVIPKVAKKLGPK	1116
	I) (Australian ants)	VAKVLPKVMKEAIPMAVEMAKSQEE
		QQPQ

AP00536	Ref, Luxuriosin (insect)	SVRTQDNAVNRQIFGSNGPYRDFQLS	1117
		DCYLPLETNPYCNEWQFAYHWNNAL
		MDCERAIYHGCNRTRNNFITLTACKN
		QAGPICNRRRH

AP00537	Ref, SAMP H1 (fish,	AEVAPAPAAAAPAKAPKKKAAAKPK	1118
	Atlantic salmon)	KAGPS

AP00538	Ref, Halocidin (dimer	WLNALLHHGLNCAKGVLA	1119
	Hal18 + Hal15)
	(tunicate)

AP00539	Ref, AOD (American	GFGCPWNRYQCHSHCRSIGRLGGYCA	1120
	oyster defensin, animal	GSLRLTCTCYRS
	defensin)

AP00540	Ref, Pentadactylin	GLLDTLKGAAKNVVGSLASKVMEKL	1121
	(frog)

AP00541	Ref, Polybia-MPI	IDWKKLLDAAKQIL	1122
	(insect, social wasp)

AP00542	Ref, Polybia-CP (insect,	ILGTILGLLKSL	1123
	social wasp)

AP00543	Ref, Ocellatin-1 (XXA,	GVVDILKGAGKDLLAHLVGKISEKV	1124
	frog)

AP00544	Ref, Ocellatin-2 (XXA,	GVLDIFKDAAKQILAHAAEKQI	1125
	frog)

AP00545	Ref, Ocellatin-3 (frog)	GVLDILKNAAKNILAHAAEQI	1126

AP00548	Ref, CMAP 27 (chicken	RFGRFLRKIRRFRPKVTITIQGSARFG	1127
	myeloid antimicrobial
	peptide 27, bird
	cathelicidin, chicken
	cathelicidin)

AP00550	Ref, Tu-AMP-1	KSCCRNTVARNCYNVCRIPGTPRPVC	1128
	(TuAMP 1, thionin-like	AATCDCKLITGTKCPPGYEK
	antimicrobial peptides,
	plant defensin, tulip)

AP00551	Ref, Combi-2	FRWWHR	1129
	(synthetic)

AP00552	Ref, Maximin 9 (frog)	GIGRKFLGGVKTTFRCGVKDFASKHLY	1130

AP00554	Ref, S1 moricin (insect)	GKIPVKAIKKAGAAIGKGLRAINIAST	1131
		AHDVYSFFKPKHKKK

AP00555	Ref, Parasin I (catfish)	KGRGKQGGKVRAKAKTRSS	1132

AP00556	Ref, Kassinatuerin-1	GFMKYIGPLIPHAVKAISDLI	1133
	(frog)

AP00557	Ref, Fowlicidin-1	RVKRVWPLVIRTVIAGYNLYRAIKKK	1134
	(chCATH-1, bird
	cathelicidin, chicken
	cathelicidin)

AP00559	Ref, Eryngin	ATRVVYCNRRSGSVVGGDDTVYYEG	1135
	(mushroom, fungi)

AP00560	Ref, Dendrocin (plant,	TTLTLHNLCPYPVWWLVTPNNGGFPII	1136
	bamboo)	DNTPVVLG

AP00561	Ref, Coconut antifungal	EQCREEEDDR	1137
	peptide (plant)

AP00562	Ref, Pandinin 1 (African	GKVWDWIKSAAKKIWSSEPVSQLKG	1138
	scorpion)	QVLNAAKNYVAEKIGATPT

AP00563	Ref, White cloud bean	KTCENLADTFRGPCFATSNCDDHCKN	1139
	defensin (plant	KEHLLSGRCRDDFRCWCTRNC
	defensin)

AP00564	Ref, Dybowskin-1	FLIGMTHGLICLISRKC	1140
	(frog)

AP00565	Ref, Dybowskin-2	FLIGMTQGLICLITRKC	1141
	(frog)

AP00566	Ref, Dybowskin-3	GLFDVVKGVLKGVGKNVAGSLLEQL	1142
	(frog)	KCKLSGGC

AP00567	Ref, Dybowskin-4	VWPLGLVICKALKIC	1143
	(frog)

AP00568	Ref, Dybowskin-5	GLFSVVTGVLKAVGKNVAKNVGGSL	1144
	(frog)	LEQLKCKKISGGC

AP00569	Ref, Dybowskin-6	FLPLLLAGLPLKLCFLFKKC	1145
	(frog)

AP00570	Ref, Pleurain-A1 (frog)	SIITMTKEAKLPQLWKQIACRLYNTC	1146

AP00571	Ref, Pleurain-A2 (frog)	SIITMTKEAKLPQSWKQIACRLYNTC	1147

AP00574	Ref, Esculentin-IGRa	GLFSKFAGKGIKNLIFKGVKHIGKEVG	1148
	(frog)	MDVIRTGIDVAGCKIKGEC

AP00575	Ref, Brevinin-2GRa	GLLDTFKNLALNAAKSAGVSVLNSLS	1149
	(frog)	CKLSKTC

AP00576	Ref, Brevinin-2GRb	GVLGTVKNLLIGAGKSAAQSVLKTLS	1150
	(frog)	CKLSNDC

AP00577	Ref, Brevinin-2GRc	GLFTLIKGAAKLIGKTVAKEAGKTGLE	1151
	(frog)	LMACKITNQC

AP00578	Ref, Brevinin-1GRa	FLPLLAGLAANFLPKIFCKITKKC	1152
	(frog)

AP00579	Ref, Nigrocin-2GRa	GLLSGILGAGKHIVCGLSGLC	1153
	(frog)

AP00580	Ref, Nigrocin-2GRb	GLFGKILGVGKKVLCGLSGMC	1154
	(frog)

AP00581	Ref, Nigrocin-2GRc	GLLSGILGAGKNIVCGLSGLC	1155
	(frog)

AP00582	Ref, Brevinin-2GHa	GFSSLFKAGAKYLLKSVGKAGAQQLA	1156
	(frog)	CKAANNCA

AP00583	Ref, Brevinin-2GHb	GVITDALKGAAKTVAAELLRKAHCKL	1157
	(frog)	TNSC

AP00584	Ref, Guentherin (frog)	VIDDLKKVAKKVRRELLCKKHHKKLN	1158

AP00585	Ref, Brevinin-2GHc	SIWEGIKNAGKGFLVSILDKVRCKVA	1159
	(frog)	GGCNP

AP00586	Ref, Temporin-GH	FLPLLFGAISHLL	1160
	(frog)

AP00587	Ref, Brevinin-2TSa	GIMSLFKGVLKTAGKHVAGSLVDQLK	1161
	(frog)	CKITGGC

AP00588	Ref, Brevinin-1TSa	FLGSIVGALASALPSLISKIRN	1162
	(frog)

AP00589	Ref, Temporin-1TSa	FLGALAKIISGIF	1163
	(frog)

AP00593	Ref, Brevinin-1CSa	FLPILAGLAAKIVPKLFCLATKKC	1164
	(frog)

AP00594	Ref, Temporin-1CSa	FLPIVGKLLSGLL	1165
	(frog)

AP00595	Ref, Temporin-1CSb	FLPIIGKLLSGLL	1166
	(frog)

AP00596	Ref, Temporin-1CSc	FLPLVTGLLSGLL	1167
	(frog)

AP00597	Ref, Temporin-1CSd	NFLGTLVNLAKKIL	1168
	(frog)

AP00598	Ref, Temporin-1SPb	FLSAITSLLGKLL	1169
	(frog)

AP00599	Ref, Brevinin-2-related	GIWDTIKSMGKVFAGKILQNL	1170
	(frog)

AP00600	Ref, Odorranain-HP	GLLRASSVWGRKYYVDLAGCAKA	1171
	(frog)

AP00601	Ref, Brevinin-1DYa	FLSLALAALPKFLCLVFKKC	1172
	(frog)

AP00602	Ref, Brevinin-1DYb	FLSLALAALPKLFCLIFKKC	1173
	(frog)

AP00603	Ref, Brevinin-1DYc	FLPLLLAGLPKLLCLFFKKC	1174
	(frog)

AP00607	Ref, Brevinin-2DYb	GLFDVVKGVLKGAGKNVAGSLLEQL	1175
	(frog)	KCKLSGGC

AP00608	Ref, Brevinin-2DYc	GLFDVVKGVLKGVGKNVAGSLLEQL	1176
	(frog)	KCKLSGGC

AP00609	Ref, Brevinin-2DYd	GIFDVVKGVLKGVGKNVAGSLLEQLK	1177
	(frog)	CKLSGGC

AP00610	Ref, Brevinin-2DYe	GLFSVVTGVLKAVGKNVAKNVGGSL	1178
	(frog)	LEQLKCKISGGC

AP00611	Ref, Temporin-1DYa	FIGPIISALASLFG	1179
	(frog)

AP00615	Ref, Palustrin-1b (frog)	ALFSILRGLKKLGNMGQAFVNCKIYK	1180
		KC

AP00616	Ref, Palustrin-1c (frog)	ALSILRGLEKLAKMGIALTNCKATKKC	1181

AP00617	Ref, Palustrin-1d (frog)	ALSILKGLEKLAKMGIALTNCKATKKC	1182

AP00619	Ref, Palustrin-2b (frog)	GFFSTVKNLATNVAGTVIDTLKCKVT	1183
		GGCRS

AP00620	Ref, Palustrin-2c (frog)	GFLSTVKNLATNVAGTVIDTLKCKVT	1184
		GGCRS

AP00621	Ref, Palustrin-3a (frog)	GIFPKIIGKGIKTGIVNGIKSLVKGVGM	1185
		KVFKAGLNNIGNTGCNEDEC

AP00622	Ref, Palustrin-3b (frog)	GIFPKIIGKGIKTGIVNGIKSLVKGVGM	1186
		KVFKAGLSNIGNTGCNEDEC

AP00624	Ref, human ALL-38 (an	ALLGDFFRKSKEKIGKEFKRIVQRIKD	1187
	LL-37 analog released	FLRNLVPRTES
	from its precursor
	hCAP-18 by gastricsin
	in vivo)

AP00625	Ref, human KR-20	KRIVQRIKDFLRNLVPRTES	1188
	(KR20 from LL-37)

AP00626	Ref, human KS-30	KSKEKIGKEFKRIVQRIKDFLRNLVPR	1189
	(KS30 from LL-37)	TES

AP00627	Ref, human RK-31	RKSKEKIGKEFKRIVQRIKDFLRNLVP	1190
	(RK31 from LL-37)	RTES

AP00628	Ref, human LL-23	LLGDFFRKSKEKIGKEFKRIVQR	1191
	(LL23 from LL-37)

AP00629	Ref, human LL-29	LLGDFFRKSKEKIGKEFKRIVQRIKDFLR	1192
	(LL29 from LL-37)

AP00630	Ref, Amoeba peptide	GEILCNLCTGLINTLENLLTTKGAD	1193
	(protozoan para

AP00631	Ref, Mundticin	KYYGNGVSCNKKGCSVDWGKAIGIIG	1194
	(bacteria)	NNSAANLATGGAAGWSK

AP00638	Ref, Citropin 2.1 (frog)	GLIGSIGKALGGLLVDVLKPKL	1195

AP00639	Ref, Citropin 2.1.3	GLIGSIGKALGGLLVDVLKPKLQAAS	1196
	(frog)

AP00640	Ref, Maculatin 1.3	GLLGLLGSVVSHVVPAIVGHF	1197
	(frog)

AP00641	Ref, Pardaxin 1	GFFALIPKIISSPLFKTLLSAVGSALSSS	1198
	(Pardaxin P-1, Pardaxin	GEQE
	P1, Pa1, flat fish)

AP00642	Ref, Pardaxin 2	GFFALIPKIISSPIFKTLLSAVGSALSSS	1199
	(Pardaxin P-2, Pardaxin	GGQE
	P2, Pa2, flat fish)

AP00643	Ref, Pardaxin 3	GFFAFIPKIISSPLFKTLLSAVGSALSSS	1200
	(Pardaxin P-3, Pardaxin	GEQE
	P3, Pa3, flat fish)

AP00645	Ref, Pardaxin 5	GFFAFIPKIISSPLFKTLLSAVGSALSSS	1201
	(Pardaxin P-5, Pardaxin	GDQE
	P5, Pa5, flat fish)

AP00647	Ref, Brevinin-1PLb	FLPLIAGLAANFLPKIFCAITKKC	1202
	(frog)

AP00648	Ref, Brevinin-1PLc	FLPVIAGVAAKFLPKIFCAITKKC	1203
	(frog)

AP00649	Ref, Esculentin-1PLa	GLFPKINKKKAKTGVFNIIKTVGKEAG	1204
	(frog)	MDLIRTGIDTIGCKIKGEC

AP00650	Ref, Esculentin-1PLb	GIFTKINKKKAKTGVFNIIKTIGKEAG	1205
	(frog)	MDVIRAGIDTISCKIKGEC

AP00651	Ref, Esculentin-2PLa	GLFSILKGVGKIALKGLAKNMGKMGL	1206
	(frog)	DLVSCKISKEC

AP00652	Ref, Ranatuerin-2PLa	GIMDTVKNVAKNLAGQLLDKLKCKIT	1207
	(frog)	AC

AP00653	Ref, Ranatuerin-2PLb	GIMDTVKNAAKDLAGQLLDKLKCRIT	1208
	(frog)	GC

AP00654	Ref, Ranatuerin-2PLc	GLLDTIKNTAKNLAVGLLDKIKCKMT	1209
	(frog)	GC

AP00655	Ref, Ranatuerin-2PLd	GIMDSVKNVAKNIAGQLLDKLKCKIT	1210
	(frog)	GC

AP00656	Ref, Ranatuerin-2PLe	GIMDSVKNAAKNLAGQLLDTIKCKIT	1211
	(frog)	AC

AP00657	Ref, Ranatuerin-2PLf	GIMDTVKNAAKDLAGQLDKLKCRITGC	1212
	(frog)

AP00658	Ref, Temporin-1PLa	FLPLVGKILSGLI	1213
	(frog)

AP00659	Ref, Ranatuerin 5 (frog)	FLPIASLLGKYL	1214

AP00661	Ref, Esculentin-2L	GILSLFTGGIKALGKTLFKMAGKAGA	1215
	(frog)	EHLACKATNQC

AP00662	Ref, Esculentin-2B	GLFSILRGAAKFASKGLGKDLTKLGV	1216
	(ESC2B-RANBE, frog)	DLVACKISKQC

AP00663	Ref, Esculentin-2P	GFSSIFRGVAKFASKGLGKDLARLGV	1217
	(frog)	NLVACKISKQC

AP00664	Ref, Peptide A1 (frog)	FLPAIAGILSQLF	1218

AP00665	Ref, Peptide B9 (frog)	FLPLIAGLIGKLF	1219

AP00666	Ref, PG-L (frog)	EGGGPQWAVGHFM	1220

AP00667	Ref, PG-KI (frog)	EPHPDEFVGLM	1221

AP00668	Ref, PG-KII (frog)	EPNPDEFVGLM	1222

AP00669	Ref, PG-KIII (frog)	EPHPNEFVGLM	1223

AP00670	Ref, PG-SPI (frog)	EPNPDEFFGLM	1224

AP00660	Ref, Pandinin 2 (African	FWGALAKGALKLIPSLFSSFSKKD	1225
	scorpion)

AP00671	Ref, PG-SPII (frog)	EPNPNEFFGLM	1226

AP00673	Ref, Lantibiotic Ericin S	WKSESVCTPGCVTGVLQTCFLQTITC	1227
	(bacteria	NCHISK

AP00674	Ref, Lantibiotic Ericin	VLSKSLCTPGCITGPLQTCYLCFPTFA	1228
	A (bacteria	KC

AP00675	Ref, Human beta	FELDRICGYGTARCRKKCRSQEYRIGR	1229
	defensin 4 (HBD-4,	CPNTYACCLRKWDESLLNRTKP
	HBD4, human defensin)

AP00676	Ref, RL-37 (RL37,	RLGNFFRKVKEKIGGGLKKVGQKIKD	1230
	monkey cathelicidin)	FLGNLVPRTAS

AP00677	Ref, CAP11 (Guinea pig	GLRKKFRKTRKRIQKLGRKIGKTGRK	1231
	cathelicidin)	VWKAWREYGQIPYPCRI

AP00678	Ref, Canine cathelicidin	RLKELITTGGQKIGEKIRRIGQRIKDFF	1232
	(K9CATH) (dog)	KNLQPREEKS

AP00679	Ref, Esculentin 2VEb	GLFSILKGVGKIAIKGLGKNLGKMGL	1233
	(frog)	DLVSCKISKEC

AP00680	Ref, SMAP-34 (sheep	GLFGRLRDSLQRGGQKILEKAERIWC	1234
	cathelicidin)	KIKDIFR

AP00681	Ref, OaBac5 (sheep	RFRPPIRRPPIRPPFRPPFRPPVRPPIRPP	1235
	cathelicidin)	FRPPFRPPIGPFP

AP00682	Ref, OaBac6 (sheep	RRLRPRHQHFPSERPWPKPLPLPLPRP	1236
	cathelicidin)	GPRPWPKPLPLPLPRPGLRPWPKPL

AP00683	Ref, OaBac7.5 (sheep	RRLRPRRPRLPRPRPRPRPRPRSLPLPR	1237
	cathelicidin)	PQPRRIPRPILLPWRPPRPIPRPQIQPIPR
		WL

AP00684	Ref, OaBac11 (sheep	RRLRPRRPRLPRPRPRPRPRPRSLPLPR	1238
	cathelicidin)	PKPRPIPRPLPLPRPRPKPIPRPLPLPRP
		RPRRIPRPLPLPRPRPRPIPRPLPLPQPQ
		PSPIPRPL

AP00685	Ref, Ranatuerin 2VEb	GIMDTVKGVAKTVAASLLDKLKCKIT	1239
	(frog)	GC

AP00686	Ref, eCATH-1 (horse	KRFGRLAKSFLRMRILLPRRKILLAS	1240
	cathelicidin)

AP00687	Ref, eCATH-2 (horse	KRRHWFPLSFQEFLEQLRRFRDQLPFP	1241
	cathelicidin)

AP00688	Ref, eCATH-3 (horse	KRFHSVGSLIQRHQQMIRDKSEATRH	1242
	cathelicidin)	GIRIITRPKLLLAS

AP00689	Ref, Prophenin-1 (pig	AFPPPNVPGPRFPPPNFPGPRFPPPNFP	1243
	cathelicidin)	GPRFPPPNFPGPRFPPPNFPGPPFPPPIFP
		GPWFPPPPPFRPPPFGPPRFP

AP00690	Ref, Prophenin-2 (pig	AFPPPNVPGPRFPPPNVPGPRFPPPNFP	1244
	cathelicidin)	GPRFPPPNFPGPRFPPPNFPGPPFPPPIFP
		GPWFPPPPPFRPPPFGPPRFP

AP00691	Ref, HFIAP-1 (hagfish	GFFKKAWRKVKHAGRRVLDTAKGV	1245
	cathelicidin)	GRHYVNNWLNRYR

AP00692	Ref, HFIAP-3 (hagfish	GWFKKAWRKVKNAGRRVLKGVGIH	1246
	cathelicidin)	YGVGLI

AP00693	Ref, Trout cath (fish	RICSRDKNCVSRPGVGSIIGRPGGGSLI	1247
	cathelicidin)	GRPGGGSVIGRPGGGSPPGGGSFNDEF
		IRDHSDGNRFA

AP00694	Ref, MRP (melittin-	AIGSILGALAKGLPTLISWIKNR	1248
	related peptide)

AP00695	Ref, Temporin-1TGa	FLPILGKLLSGIL	1249
	(frog)

AP00696	Ref, Dahlein 1.1 (frog)	GLFDIIKNIVSTL	1250

AP00697	Ref, Dahlein 1.2 (frog)	GLFDIIKNIFSGL	1251

AP00698	Ref, Dahlein 4.1 (frog)	GLWQLIKDKIKDAATGFVTGIQS	1252

AP00699	Ref, Dahlein 4.2 (frog)	GLWQFIKDKLKDAATGLVTGIQS	1253

AP00700	Ref, Dahlein 4.3 (frog)	GLWQFIKDKFKDAATGLVTGIQS	1254

AP00701	Ref, Dahlein 5.1 (frog)	GLLGSIGNAIGAFIANKLKP	1255

AP00702	Ref, Dahlein 5.2 (frog)	GLLGSIGNAIGAFIANKLKPK	1256

AP00703	Ref, Dahlein 5.3 (frog)	GLLASLGKVLGGYLAEKLKP	1257

AP00704	Ref, Dahlein 5.4 (frog)	GLLGSIGKVLGGYLAEKLKPK	1258

AP00705	Ref, Dahlein 5.5 (frog)	GLLASLGKVLGGYLAEKLKPK	1259

AP00706	Ref, Dahlein 5.6 (frog)	GLLASLGKVFGGYLAEKLKPK	1260

AP00709	Ref, Mytilus defensin	GFGCPNDYPCHRHCKSIPGRAGGYCG	1261
	(mytilin) A (mollusc)	GAHRLRCTCYR

AP00711	Ref, Mussel defensin	GFGCPNNYACHQHCKSIRGYCGGYC	1262
	MGD2	AGWFRLRCTCYRCG

AP00712	Ref, scorpion defensin	GFGCPLNQGACHRHCRSIRRRGGYCA	1263
		GFFKQTCCYRN

AP00713	Ref, Androctonus	GFGCPFNQGACHRHCRSIRRRGGYCA	1264
	defensin	GLFKQTCTCYR

AP00714	Ref, Orinthodoros	GYGCPFNQYQCHSHCSGIRGYKGGYC	1265
	defensin A (soft ticks)	KGTFKQTCKCY

AP00715	Ref, VaD1 (plant	RTCMKKEGWGKCLIDTTCAHSCKNR	1266
	defensin)	GYIGGNCKGMTRTCYCLVNC

AP00722	Ref, Cryptonin (insect,	GLLNGLALRLGKRALKKIIKRLCR	1267
	cicada)

AP00723	Ref, Decoralin (insect)	SLLSLLRKLIT	1268

AP00724	Ref, RTD-2 (rhesus	RCLCRRGVCRCLCRRGVC	1269
	theta-defensin-2,
	minidefensin, XXC,
	BBS, lectin, ZZHa)

AP00725	Ref, RTD-3 (rhesus	RCICTRGFCRCICTRGFC	1270
	theta-defensin-3,
	minidefensin, XXC,
	BBS, lectin, ZZHa)

AP00726	Ref, Combi-1	RRWWRF	1271
	(synthetic)

AP00748	Ref, Gm pro-rich pept1	DIQIPGIKKPTHRDIIIPNWNPNVRTQP	1272
	(insect)	WQRFGGNKS

AP00749	Ref, Gm anionic pept 1	EADEPLWLYKGDNIERAPTTADHPILP	1273
	(insect)	SIIDDVKLDPNRRYA

AP00750	Ref, Gm pro-rich pept 2	EIRLPEPFRFPSPTVPKPIDIDPILPHPWS	1274
	(insect)	PRQTYPIIARRS

AP00752	Ref, Gm defensin-like	DKLIGSCVWGATNYTSDCNAECKRR	1275
	peptide (insect)	GYKGGHCGSFWNVNCWCEE

AP00753	Ref, Gm	VQETQKLAKTVGANLEETNKKLAPQI	1276
	apolipophoricin (insect)	KSAYDDFVKQAQEVQKKLHEAASKQ

AP00754	Ref, Gm anionic pept2	ETESTPDYLKNIQQQLEEYTKNFNTQV	1277
	(insect)	QNAFDSDKIKSEVNNFIESLGKILNTE
		KKEAPK

AP00755	Ref, Gm cecropin D-	ENFFKEIERAGQRIRDAIISAAPAVETL	1278
	like pept, insect	AQAQKIIKGGD

AP00756	Ref, Dermaseptin-B6	ALWKDILKNAGKAALNEINQLVNQ	1279
	(DRS-B6, DRS B6,
	XXA, frog)

AP00759	Ref, Phylloseptin-O1	FLSLIPHAINAVSTLVHHSG	1280
	(PLS-O1, Phylloseptin-
	4, PS-4, XXA, frog)

AP00760	Ref, Phylloseptin-O2	FLSLIPHAINAVSAIAKHS	1281
	(PLS-O2, Phylloseptin-
	5, PS-5, XXA, frog)

AP00761	Ref, Phylloseptin-6	SLIPHAINAVSAIAKHF	1282
	(Phylloseptin-H4, PLS-
	H4, PS-6, XXA, frog)

AP00762	Ref, Phylloseptin-7	FLSLIPHAINAVSAIAKHF	1283
	(Phylloseptin-H5, PLS-
	H5, PS-7, XXA, frog)

AP00763	Ref, Dermaseptin DPh-1	GLWSTIKNVGKEAAIAAGKAALGAL	1284
	(XXA, frog)

AP00764	Ref, Dermaseptin-S9	GLRSKIWLWVLLMIWQESNKFKKM	1285
	(DRS-S9, DRS S9, frog)

AP00765	Ref, Human salvic	MHDFWVLWVLLEYIYNSACSVLSATS	1286
		SVSSRVLNRSLQVKVVKITN

AP00766	Ref, Gassericin A	IYWIADQFGIHLATGTARKLLDAMAS	1287
	(XXC, XXD2, class IV	GASLGTAFAAILGVTLPAWALAAAGA
	bacteriocin, Gram-	LGATAA
	positive bacteria)

AP00767	Ref, Circularin A (XXC,	VAGALGVQTAAATTIVNVILNAGTLV	1288
	class IV bacteriocin,	TVLGIIASIASGGAGTLMTIGWATFKA
	Gram-positive bacteria)	TVQKLAKQSMARAIAY

AP00768	Ref, Closticin 574	PNWTKIGKCAGSIAWAIGSGLFGGAK	1289
	(bacteria)	LIKIKKYIAELGGLQKAAKLLVGATT
		WEEKLHAGGYALINLAAELTGVAGIQ
		ANCF

AP00769	Ref, Caerin 1.11 (XXA,	GLLGAMFKVASKVLPHVVPAITEHF	1290
	frog)

AP00770	Ref, Maculatin 1.4	GLLGLLGSVVSHVLPAITQHL	1291
	(XXA, frog)

AP00771	Ref, Magainin 1 (frog)	GIGKFLHSAGKFGKAFVGEIMKS	1292

AP00772	Ref, Oxyopinin 1	FRGLAKLLKIGLKSFARVLKKVLPKA	1293
	(spider)	AKAGKALAKSMADENAIRQQNQ

AP00773	Ref, Oxyopinin 2a	GKFSVFGKILRSIAKVFKGVGKVRKQF	1294
	(spider)	KTASDLDKNQ

AP00774	Ref, Oxyopinin 2b	GKFSGFAKILKSIAKFFKGVGKVRKGF	1295
	(spider)	KEASDLDKNQ

AP00775	Ref, Oxyopinin 2c	GKLSGISKVLRAIAKFFKGVGKARKQ	1296
	(spider)	FKEASDLDKNQ

AP00776	Ref, Oxyopinin 2d	GKFSVFSKILRSIAKVFKGVGKVRKGF	1297
	(spider)	KTASDLDKNQ

AP00777	Ref, NRC-1 (XXA, fish,	GKGRWLERIGKAGGIIIGGALDHL	1298
	gene predicted)

AP00778	Ref, NRC-2 (XXA, fish,	WLRRIGKGVKIIGGAALDHL	1299
	gene predicted)

AP00779	Ref, NRC-3 (XXA, fish,	GRRKRKWLRRIGKGVKIIGGAALDHL	1300
	gene predicted)

AP00781	Ref, NRC-5 (XXA, fish,	FLGALIKGAIHGGRFIHGMIQNHH	1301
	gene predicted)

AP00782	Ref, NRC-6 (XXA, fish,	GWGSIFKHGRHAAKHIGHAAVNHYL	1302
	gene predicted)

AP00783	Ref, NRC-7 (XXA, fish,	RWGKWFKKATHVGKHVGKAALTAYL	1303
	gene predicted)

AP00784	Ref, NRC-10 (XXA,	FFRLLFHGVHHVGKIKPRA	1304
	fish, gene predicted)

AP00785	Ref, NRC-11 (XXA,	GWKSVFRKAKKVGKTVGGLALDHYL	1305
	fish, gene predicted)

AP00786	Ref, NRC-12 (XXA,	GWKKWFNRAKKVGKTVGGLAVDHYL	1306
	fish, gene predicted)

AP00787	Ref, NRC-13 (XXA,	GWRLLLKKAEVKTVGKLALKHYL	1307
	fish, gene predicted)

AP00788	Ref, NRC-14 (XXA,	AGWGSIFKHIFKAGKFIHGAIQAHND	1308
	fish, gene predicted)

AP00789	Ref, NRC-15 (XXA,	GFWGKLFKLGLHGIGLLHLHL	1309
	fish, gene predicted)

AP00790	Ref, NRC-16 (XXA,	GWKKWLRKGAKHLGQAAIK	1310
	fish, gene predicted)

AP00791	Ref, NRC-17 (XXA,	GWKKWLRKGAKHLGQAAIKGLAS	1311
	fish, gene predicted)

AP00792	Ref, NRC-19 (XXA,	FLGLLFHGVHHVGKWIHGLIHGHH	1312
	fish, gene predicted)

AP00793	Ref, Bombinin H2	IIGPVLGLVGSALGGLLKKI	1313
	(XXA, frog)

AP00794	Ref, Bombinin H3 (frog,	IIGPVLGMVGSALGGLLKKI	1314
	XXD, XXA)

AP00795	Ref, Bombinin H7 (frog,	ILGPILGLVSNALGGLL	1315
	XXD, XXA)

AP00796	Ref, Bombinin GH-1L	IIGPVLGLVGKPLESLLE	1316
	(XXA, toad)

AP00797	Ref, Bombinin GH-1D	IIGPVLGLVGKPLESLLE	1317
	(toad, XXD, XXA)

AP00807	Ref, Enterocin E-760	NRWYCNSAAGGVGGAAGCVLAGYV	1318
	(bacteriocin, bacteria)	GEAKENIAGEVRKGWGMAGGFTHNK
		ACKSFPGSGWASG

AP00808	Ref, hepcidin (fish)	CRFCCRCCPRMRGCGLCCRF	1319

AP00809	Ref, hepcidin TH1-5	GIKCRFCCGCCTPGICGVCCRF	1320
	(fish)

AP00810	Ref, hepcidin TH2-3	QSHLSLCRWCCNCCRSNKGC	1321
	(fish)

AP00811	Ref, human LEAP-2	MTPFWRGVSLRPIGASCRDDSECITRL	1322
		CRKRRCSLSVAQE

AP00812	Ref, Enkelytin (cow)	FAEPLPSEEEGESYSKEPPEMEKRYGG	1323
		FM

AP00732	Ref, Spheniscin-1	SFGLCRLRRGSCAHGRCRFPSIPIGRCS	1324
	(Sphe-1, avian defensin)	RFVQCCRRVW

AP00733	Ref, Organgutan	LLGDFFRKAREKIGEEFKRIVQRIKDFL	1325
	ppyLL-37 (Great Ape,	RNLVPRTES
	primate cathelicidin)

AP00734	Ref, Gibbon hmdSL-37	SLGNFFRKARKKIGEEFKRIVQRIKDF	1326
	(hylobatidae, primate	LQHLIPRTEA
	cathelicidin)

AP00735	Ref, pobRL-37	RLGNFFRKAKKKIGRGLKKIGQKIKDF	1327
	(cercopithecidae,	LGNLVPRTES
	primate cathelicidin)

AP00736	Ref, cjaRL-37 (primate	RLGDILQKAREKIEGGLKKLVQKIKDF	1328
	cathelicidin)	FGKFAPRTES

AP00737	Ref, Plasticin PBN2KF	GLVTSLIKGAGKLLGGLFGSVTG	1329
	(XXA, DRP-PBN2,
	frog)

AP00738	Ref, Plasticin ANCKF	GLVTGLLKTAGKLLGDLFGSLTG	1330
	(XXA, synthetic)

AP00739	Ref, Plasticin PD36KF	GVVTDLLKTAGKLLGNLFGSLSG	1331
	(XXA, synthetic)

AP00740	Ref, Plasticin PD36K	GVVTDLLKTAGKLLGNLVGSLSG	1332
	(XXA, synthetic)

AP00741	Ref, Chicken	PITYLDAILAAVRLLNQRISGPCILRLR	1333
	cathelicidin-B1 (bird	EAQPRPGWVGTLQRRREVSFLVEDGP
	cathelicidin)	CPPGVDCRSCEPGALQHCVGTVSIEQ
		QPTAELRCRPLRPQ

AP00742	Ref, Chicken gallinacin	MRILYLLLSVLFVVLQGVAGQPYFSSP	1334
	4 (Gal 4)	IHACRYQRGVCIPGPCRWPYYRVGSC
		GSGLKSCCVRNRWA

AP00743	Ref, Chicken gallinacin	MKILCFFIVLFVAVHGAVGFSRSPRYH	1335
	7 (Gal 7)	MQCGYRGTFCTPGKCPYGNAYLGLC
		RPKYSCCRWL

AP00744	Ref, Chicken gallinacin	MQILPLLFAVLLLMLRAEPGLSLARGL	1336
	9 (Gal 9)	PQDCERRGGFCSHKSCPPGIGRIGLCS
		KEDFCCRSRWYS

AP00745	Ref, Chicken LEAP-2	MTPFWRGVSLRPVGASCRDNSECITM	1337
	(cLEAP-2)	LCRKNRCFLRTASE

AP00814	Ref, Caerulein	GLGSILGKILNVAGKVGKTIGKVADA	1338
	precursor-related	VGNKE
	fragment Ea (CPRF-Ea,
	frog)

AP00815	Ref, Caerulein	GLGSFLKNAIKIAGKVGSTIGKVADAI	1339
	precursor-related	GNKE
	fragment Eb (CPRF-Eb,
	frog)

AP00816	Ref, Caerulein	GLGSFFKNAIKIAGKVGSTIGKVADAI	1340
	precursor-related	GNKE
	fragment Ec (CPRF-Ec,
	frog)

AP00817	Ref, Temporin-1Oa	FLPLLASLFSRLL	1341
	(frog)

AP00818	Ref, Temporin-1Ob	FLPLIGKILGTIL	1342
	(frog)

AP00819	Ref, Temporin-1Oc	FLPLLASLFSRLF	1343
	(frog)

AP00820	Ref, Temporin-1Od	FLPLLASLFSGLF	1344
	(frog)

AP00821	Ref, Brevinin-20a (frog)	GLFNVFKGLKTAGKHVAGSLLNQLK	1345
		CKVSGGC

AP00822	Ref, Brevinin-20b (frog)	GIFNVFKGALKTAGKHVAGSLLNQLK	1346
		CKVSGEC

AP00824	Ref, Temporin-1Gb	SILPTIVSFLSKFL	1347
	(XXA, frog)

AP00825	Ref, Temporin-1Gc	SILPTIVSFLTKFL	1348
	(XXA, frog)

AP00826	Ref, Temporin-1Gd	FILPLIASFLSKFL	1349
	(XXA, frog)

AP00827	Ref, Ranatuerin-1Ga	SMISVLKNLGKVGLGFVACKVNKQC	1350
	(frog)

AP00829	Ref, Ranalexin-1G	FLGGLMKIIPAAFCAVTKKC	1351
	(frog)

AP00830	Ref, Ranatuerin-2G	GLLLDTLKGAAKDIAGIALEKLKCKIT	1352
	(frog)	GCKP

AP00831	Ref, Odorranain-NR	GLLSGILGAGKHIVCGLTGCAKA	1353
	(frog)

AP00832	Ref, Maximin H1	ILGPVISTIGGVLGGLLKNL	1354
	(XXA, toad)

AP00834	Ref, G. mellonella	KVNANAIKKGGKAIGKGFKVISAAST	1355
	moricin-like peptide A	AHDVYEHIKNRRH
	(Gm-mlpA, insect)

AP00835	Ref, G. mellonella	GKIPVKAIKKGGQIIGKALRGINIASTA	1356
	moricin-like peptide B	HDIISQFKPKKKKNH
	(Gm-mlpB, insect)

AP00836	Ref, G. mellonella	KVPIGAIKKGGKIIKKGLGVIGAAGTA	1357
	moricin-like peptide C1	HEVYSHVKNRH
	(Gm-mlpC1, insect)

AP00837	Ref, G. mellonella	KVPIGAIKKGGKIIKKGLGVLGAAGTA	1358
	moricin-like peptide C2	HEVYNHVRNRQ
	(Gm-mlpC2, insect)

AP00838	Ref, G. mellonella	KVPIGAIKKGGKIIKKGLGVIGAAGTA	1359
	moricin-like peptide C3	HEVYSHVKNRQ
	(Gm-mlpC3, insect)

AP00839	Ref, G. mellonella	KVPVGAIKKGGKAIKTGLGVVGAAGT	1360
	moricin-like peptide	AHEVYSHIRNRH
	C4/C5 (Gm-mlpC4/C5,
	insect)

AP00840	Ref, G. mellonella	KGIGSALKKGGKIIKGGLGALGAIGTG	1361
	moricin-like peptide D	QQVYEHVQNRQ
	(Gm-mlpD, insect)

AP00841	Ref, Enterocin A (EntA,	TTHSGKYYGNGVYCTKNKCTVDWAK	1362
	class IIA bacteriocin,	ATTCIAGMSIGGFLGGAIPGKC
	i.e. pediocin-like
	peptide, bacteria)

AP00842	Ref, Divercin V41	TKYYGNGVYCNSKKCWVDWGQASG	1363
	(DvnV41, class IIa	CIGQTVVGGWLGGAIPGKC
	bacteriocin, pediocin-
	like peptide, bacteria.
	DvnRV41 is the
	recombinant form)

AP00843	Ref, Divergicin M35	TKYYGNGVYCNSKKCWVDWGTAQG	1364
	(class IIa bacteriocin,	CIDVVIGQLGGGIPGKGKC
	pediocin-like peptide,
	bacteria)

AP00844	Ref, Coagulin	KYYGNGVTCGKHSCSVDWGKATTCII	1365
	(bacteriocin, pediocin-	NNGAMAWATGGHQGTHKC
	like peptide, bacteria)

AP00845	Ref, Listeriocin 743A	KSYGNGVHCNKKKCWVDWGSAISTI	1366
	(class IIa bacteriocin,	GNNSAANWATGGAAGWKS
	pediocin-like peptide,
	bacteria)

AP00846	Ref, Mundticin KS	KYYGNGVSCNKKGCSVDWGKAIGIIG	1367
	(enterocin CRL35,	NNSAANLATGGAAGWKS
	mundticin ATO6,
	mundticin QU2, class
	IIa bacteriocin,
	pediocin-like peptide,
	bacteria)

AP00847	Ref, Sakacin 5X	KYYGNGLSCNKSGCSVDWSKAISIIGN	1368
	(Sak5X, class IIa	NAVANLTTGGAAGWKS
	bacteriocin, pediocin-
	like peptide, bacteria)

AP00848	Ref, Leucocin C (class	KNYGNGVHCTKKGCSVDWGYAWAN	1369
	IIa bacteriocin,	IANNSVMNGLTGGNAGWHN
	pediocin-like peptide,
	bacteria)

AP00849	Ref, Lactococcin	TSYGNGVHCNKSKCWIDVSELETYKA	1370
	MMFII (class IIa	GTVSNPKDILW
	bacteriocin, pediocin-
	like peptide, bacteria)

AP00850	Ref, Sakacin G (SakG,	KYYGNGVSCNSHGCSVNWGQAWTC	1371
	class IIa bacteriocin,	GVNHLANGGHGVC
	pediocin-like peptide,
	bacteria)

AP00851	Ref, Plantaricin 423	KYYGNGVTCGKHSCSVNWGQAFSCS	1372
	(class IIa bacteriocin,	VSHLANFGHGKC
	pediocin-like peptide,
	bacteria)

AP00852	Ref, Plantaricin C19	KYYGNGLSCSKKGCTVNWGQAFSCG	1373
	(class IIa bacteriocin,	VNRVATAGHHKC
	pediocin-like peptide,
	bacteria)

AP00853	Ref, Enterocin P (EntP,	ATRSYGNGVYCNNSKCWVNWGEAK	1374
	class IIa bacteriocin,	ENIAGIVISGWASGLAGMGH
	pediocin-like peptide,
	bacteria)

AP00854	Ref, Bacteriocin 31	ATYYGNGLYCNKQKCWVDWNKASR	1375
	(Bac 31, Bac31, class	EIGKIIVNGWVQHGPWAPR
	IIa bacteriocin,
	pediocin-like peptide,
	bacteria)

AP00855	Ref, MSI-78 (XXA,	GIGKFLKKAKKFGKAFVKILKK	1376
	synthetic)

AP00856	Ref, MSI-594 (XXA,	GIGKFLKKAKKGIGAVLKVLTTGL	1377
	synthetic)

AP00857	Ref, Catestatin (human	SSMKLSFRARAYGFRGPGPQL	1378
	CHGA(352-372),
	human Cst)

AP00858	Ref, Temporin D (XXA,	LLPIVGNLLNSLL	1379
	frog)

AP00859	Ref, Temporin H (XXA,	LSPNLLKSLL	1380
	frog)

AP00861	Ref, Brevinin-ALb	FLPLAVSLAANFLPKLFCKITKKC	1381
	(frog)

AP00862	Ref, Brevinin 1E (frog)	FLPLLAGLAANFLPKIFCKITKRC	1382

AP00863	Ref, Temporin-ALa	FLPIVGKLLSGLSGLL	1383
	(XXA, frog)

AP00864	Ref, Temporin 1ARa	FLPIVGRLISGLL	1384
	(XXA, frog)

AP00865	Ref, Temporin 1AUa	FLPIIGQLLSGLL	1385
	(XXA, Temporin-
	1AUa) (frog)

AP00866	Ref, Temporin 1Bya	FLPIIAKVLSGLL	1386
	(XXA, Temporin-1Bya,
	frog)

AP00867	Ref, Temporin 1Ec	FLPVIAGLLSKLF	1387
	(XXA, frog)

AP00869	Ref, Temporin 1Ja	ILPLVGNLLNDLL	1388
	(XXA, Temporin-1Ja,
	frog)

AP00873	Ref, Temporin 1Pra	ILPILGNLLNGLL	1389
	(XXA, frog)

AP00874	Ref, Temporin 1VE	FLPLVGKILSGLI	1390
	(XXA, frog)

AP00875	Ref, Temporin 1Va	FLSSIGKILGNLL	1391
	(XXA, frog)

AP00876	Ref, Temporin 1Vb	FLSIIAKVLGSLF	1392
	(XXA, frog)

AP00877	Ref, Brevinin-1Ja (frog)	FLGSLIGAAIPAIKQLLGLKK	1393

AP00878	Ref, Brevinin-1BYa	FLPILASLAAKFGPKLFCLVTKKC	1394
	(frog)

AP00884	Ref, Ixosin-B (tick)	QLKVDLWGTRSGIQPEQHSSGKSDVR	1395
		RWRSRY

AP00885	Ref, Brevinin-1BYb	FLPILASLAAKLGPKLFCLVTKKC	1396
	(frog)

AP00886	Ref, Brevinin-1BYc	FLPILASLAATLGPKLLCLITKKC	1397
	(frog)

AP00887	Ref, Brevinin-2BYa	GILSTFKGLAKGVAKDLAGNLLDKFK	1398
	(frog)	CKITGC

AP00888	Ref, Brevinin-2BYb	GIMDSVKGLAKNLAGKLLDSLKCKIT	1399
	(frog)	GC

AP00891	Ref, Pilosulin 3 (Myr b	IIGLVSKGTCVLVKTVCKKVLKQG	1400
	III)(ants)

AP00892	Ref, Pilosulin 4 (Myr b	PDITKLNIKKLTKATCKVISKGASMCK	1401
	IV)(ants)	VLFDKKKQE

AP00893	Ref, Pilosulin 5 (Myr b	DVKGMKKAIKGILDCVIEKGYDKLAA	1402
	III)(ants)	KLKKVIQQLWE

AP00894	Ref, Ocellatin 4 (XXA,	GLLDFVTGVGKDIFAQLIKQI	1403
	frog)

AP00895	Ref, OH-CATH (snake	KRFKKFFKKLKNSVKKRAKKFFKKPR	1404
	cathelicidin, reptile	VIGVSIPF
	cathelicidin, or elapid
	cathelicidins)

AP00896	Ref, BF-CATH (snake	KRFKKFFKKLKKSVKKRAKKFFKKPR	1405
	cathelicidin)	VIGVSIPF

AP00897	Ref, NA-CATH (snake	KRFKKFFKKLKNSVKKRAKKFFKKPK	1406
	cathelicidin)	VIGVTFPF

AP00898	Ref, Temporin-1Sa	FLSGIVGMLGKLF	1407
	(XXA, frog)

AP00899	Ref, Temporin-1Sb	FLPIVTNLLSGLL	1408
	(XXA, frog)

AP00900	Ref, Temporin-1Sc	FLSHIAGFLSNLF	1409
	(XXA, frog)

AP00913	Ref, Ib-AMP1	EWGRRCCGWGPGRRYCVRWC	1410
	(IbAMP1, plant
	defensin)

AP00914	Ref, Ib-AMP2	QYGRRCCNWGPGRRYCKRWC	1411
	(IBAMP2, plant
	defensin)

AP00915	Ref, Ee-CBP (EeCBP,	QQCGRQAGNRRCANNLCCSQYGYCG	1412
	plant defensin, hevein-	RTNEYCCTSQGCQSQCRRCG
	type, E. europaeus
	chitin-binding protein)

AP00916	Ref, Pa-AMP1	AGCIKNGGRCNASAGPPYCCSSYCFQI	1413
	(PaAMP1, plant	AGQSYGVCKNR
	defensin, C6 type)

AP00917	Ref, Pa-AMP2	ACIKNGGRCVASGGPPYCCSNYCLQI	1414
	(PaAMP2, plant	AGQSYGVCKKH
	defensin, C6 type)

AP00924	Ref, Ornithodoros	GYGCPFNQYQCHSHCRGIRGYKGGY	1415
	defensin B (soft ticks)	CTGRFKQTCKCY

AP00925	Ref, Ornithodoros	GYGCPFNQYQCHSHCSGIRGYKGGYC	1416
	defensin C (soft ticks)	KGLFKQTCNCY

AP00926	Ref, Ornithodoros	GFGCPFNQYECHAHCSGVPGYKGGY	1417
	defensin D (soft ticks)	CKGLFKQTCNCY

AP00927	Ref, Butyrivibriocin	IYFIADKMGIQLAPAWYQDIVNWVSA	1418
	AR10 (XXC, class IV	GGTLTTGFAIIVGVTVPAWIAEAAAAF
	bacteriocin, gram-	GIASA
	positive bacteria)

AP00929	Ref, AS-48 (enterocin 4,	ASLQFLPIAHMAKEFGIPAAVAGTVIN	1419
	XXC, class IV	VVEAGGWVTTIVSILTAVGSGGLSLLA
	bacteriocin or class IId	AAGRESIKAYLKKEIKKKGKRAVIAW
	bacteriocin, Gram-
	positive bacteria)

AP00930	Ref, Reutericin 6 (XXC,	IYWIADQFGIHLATGTARKLLDAMAS	1420
	XXD1, class IV	GASLGTAFAAILGVTLPAWALAAAGA
	bacteriocin, Gram-	LGATAA
	positive bacteria)

AP00931	Ref, Uberolysin (XXC,	LAGYTGIASGTAKKVVDAIDKGAAAF	1421
	class IV bacteriocin,	VIISIISTVISAGALGAVSASADFIILTVK
	Gram-positive bacteria)	NYISRNLKAQAVIW

AP00932	Ref, Acidocin B (XXC,	IYWIADQFGIHLATGTARKLLDAVAS	1422
	class IV bacteriocin,	GASLGTAFAAILGVTLPAWALAAAGA
	Gram-positive bacteria)	LGATAA

AP00980	Ref, Phormia defensin B	ATCDLLSGTGINHSACAAHCLLRGNR	1423
	(insect defensin B)	GGYCNRKGVCVCRN

AP00990	Ref, Pth-St1 (plant	RNCESLSHRFKGPCTRDSN	1424
	defensin)

AP00991	Ref, Snakin-1 (StSN1,	GSNFCDSKCKLRCSKAGLADRCLKYC	1425
	plant defensin)	GICCEECKCVPSGTYGNKHECPCYRD
		KKNSKGKSKCP

AP00992	Ref, Snakin-2 (StSN2,	YSYKKIDCGGACAARCRLSSRPRLCN	1426
	plant defensin)	RACGTCCARCNCVPPGTSGNTETCPC
		YASLTTHGNKRKCP

AP00993	Ref, So-D2 (S. oleracea	GIFSSRKCKTPSKTFKGICTRDSNCDTS	1427
	defensin D2, plant	CRYEGYPAGDCKGIRRRCMCSKPC
	defensin)

AP00994	Ref, So-D6 (S. oleracea	GIFSNMYARTPAGYFRGP	1428
	defensin D6, plant
	defensin)

AP00997	Ref, Nisin Q	ITSISLCTPGCKTGVLMGCNLKTATCN	1429
	(lantibiotic,	CSVHVSK
	bacteriocins, bacteria)

AP01008	Ref, Tachystatin A1	YSRCQLQGFNCVVRSYGLPTIPCCRGL	1430
	(BBS, horseshoe crabs)	TCRSYFPGSTYGRCQRF

AP01009	Ref, Tachystatin C	DYDWSLRGPPKCATYGQKCRTWSPR	1431
	(BBS, horseshoe crabs)	NCCWNLRCKAFRCRPR

AP01012	Ref, Latarcin 3a (Ltc3a,	SWKSMAKKLKEYMEKLKQRA	1432
	XXA, BBM, spider)

AP01013	Ref, Latarcin 3b (Ltc3b,	SWASMAKKLKEYMEKLKQRA	1433
	XXA, BBM, spider)

AP01014	Ref, Latarcin 4a (Ltc4a,	GLKDKFKSMGEKLKQYIQTWKAKF	1434
	XXA, BBM, spider)

AP01015	Ref, Latarcin 4b (Ltc4b,	SLKDKVKSMGEKLKQYIQTWKAKF	1435
	XXA, BBM, spider)

AP01016	Ref, Latarcin 5 (Ltc5,	GFFGKMKEYFKKFGASFKRRFANLKK	1436
	XXA, BBM, spider)	RL

AP01018	Ref, Latarcin 6a (Ltc6a,	QAFQTFKPDWNKIRYDAMKMQTSLG	1437
	BBM, spider)	QMKKRFNL

AP01019	Ref, Latarcin 7 (Ltc7,	GETFDKLKEKLKTFYQKLVEKAEDLK	1438
	BBM, spider)	GDLKAKLS

AP01049	Ref, Kalata B2 (plant	VCGETCFGGTCNTPGCSCTWPICTRD	1439
	cyclotides, XXC)	GLP

AP01141	Ref, Cryptdin-6 (Crp6,	LRDLVCYCRARGCKGRERMNGTCRK	1440
	animal defensin, alpha,	GHLLYMLCCR
	mouse)

AP01142	Ref, Rabbit kidney	KPYCSCKWRCGIGEEEKGICHKFPIVT	1441
	defensin RK-2 (animal	YVCCRRP
	defensin, alpha-
	defensin)

AP01146	Ref, Gallinacin 6 (Gal6,	DTLACRQSHGSCSFVACRAPSVDIGTC	1442
	Gal-6, avian beta	RGGKLKCCKWAPSS
	defensin, bird)

AP01147	Ref, Gallinacin 8 (Gal8,	DTVACRIQGNFCRAGACPPTFTISGQC	1443
	Gal-8, avian beta	HGGLLNCCAKIPAQ
	defensin, bird)

AP01148	Ref, Gallinacin 3 (Gal3,	IATQCRIRGGFCRVGSCRFPHIAIGKCA	1444
	Gal-3, avian beta	TFISCCGRAY
	defensin, bird)

AP01152	Ref, Lactococcin Q	SIWGDIGQGVGKAAYWVGKAMGNM	1445
	(class IIb bacteriocin,	SDVNQASRINRKKKH
	bacteria, chain a. For
	chain b, see Info)

AP01155	Ref, Enterocin 1071	ESVFSKIGNAVGPAAYWILKGLGNMS	1446
	(Ent1071A, class IIb	DVNQADRINRKKH
	bacteriocin, bacteria;
	chain B is Enterocin
	1071B or Ent1071B, see
	info)

AP01156	Ref, Plantaricin S (chain	NKLAYNMGHYAGKATIFGLAAWALLA	1447
	a, class IIb bacteriocin,
	bacteria)

AP01159	Ref, Hinnavin II (Hin II,	KWKIFKKIEHMGQNIRDGLIKAGPAV	1448
	XXA, insect)	QVVGQAATIYK

AP01160	Ref, NK-2 (synthetic,	KILRGLCKKIMRSFLRRISWDILTGKK	1449
	XXA)

AP01167	Ref, Plantaricin NC8	LTTKLWSSWGYYLGKKARWNLKHPY	1450
	(PLNC8, chain a, class	VQF
	IIb bacteriocin, bacteria.
	For chain b, see Info)

AP01168	Ref, Carnocyclin A (a	LVAYGIAQGTAEKVVSLINAGLTVGSI	1451
	circular bacteriocin,	ISILGGVTVGLSGVFTAVKAAIAKQGI
	XXC, bacteria)	KKAIQL

AP01169	Ref, Lactacin F (LafX,	NRWGDTVLSAASGAGTGIKACKSFGP	1452
	class IIb bacteriocin,	WGMAICGVGGAAIGGYFGYTHN
	bacteria. For LafA, see
	Info)

AP01170	Ref, Brochocin C	YSSKDCLKDIGKGIGAGTVAGAAGGG	1453
	(BrcC, chain BrcA,	LAAGLGAIPGAFVGAHFGVIGGSAACI
	class IIb bacteriocin,	GGLLGN
	bacteria. For BrcB, see
	Info)

AP01171	Ref, Thermophilin 13	YSGKDCLKDMGGYALAGAGSGALW	1454
	(chain a ThmA, 2-chain	GAPAGGVGALPGAFVGAHVGAIAGG
	class IIb bacteriocin,	FACMGGMIGNKFN
	bacteria. For chain B
	ThmB, see Info)

AP01172	Ref, ABP-118 (chain a:	KRGPNCVGNFLGGLFAGAAAGVPLGP	1455
	Abp118alpha, class IIb	AGIVGGANLGMVGGALTCL
	bacteriocin, bacteria.
	For chain b:
	Abp118beta, see Info)

AP01173	Ref, Salivaricin P (chain	KRGPNCVGNFLGGLFAGAAAGVPLGP	1456
	a: Sln1; class IIb	AGIVGGANLGMVGGALTCL
	bacteriocin, bacteria.
	For chain b: Sln2, see
	Info)

AP01174	Ref, Mutacin IV (chain	KVSGGEAVAAIGICATASAAIGGLAG	1457
	a: NlmA, class IIb	ATLVTPYCVGTWGLIRSH
	bacteriocin, bacteria.
	For chain b:NLmB, see
	Info)

AP01175	Ref, Lactocin 705	GMSGYIQGIPDFLKGYLHGISAANKH	1458
	(chain a: Lac705alpha;	KKGRLGY
	class IIb bacteriocin,
	bacteria. For chain b:
	Lac705beta, see Info)

AP01176	Ref, Cytolysin (CylLS,	TTPACFTIGLGVGALFSAKFC	1459
	bacteria; Chain B:
	CylLL)

AP01177	Ref, Plantaricin EF	FNRGGYNFGKSVRHVVDAIGSVAGIL	1460
	(chain a: PlnE, class IIb	KSIR
	bacteriocin, bacteria.
	Chain b: PlnF)

AP01178	Ref, Plantaricin JK	GAWKNFWSSLRKGFYDGEAGRAIRR	1461
	(chain a: PlnJ; class IIb
	bacteriocin, bacteria.
	Chain b: PlnK)

AP01179	Ref, Enterocin SE-K4	NGVYCNKQKCWVDWSRARSEIIDRG	1462
	(class IIa bacteriocin,	VKAYVNGFTKVLGGIGGR
	bacteria)

AP01180	Ref, Acidocin J1132	NPKVAHCASQIGRSTAWGAVSGA	1463
	(class IIb bacteriocin,
	bacteria)

AP01181	Ref, Curvaticin L442	AYPGNGVHCGKYSCTVDKQTAIGNIG	1464
	(class IIa bacteriocin,	NNAA
	bacteria)

AP01182	Ref, Bacteriocin 32	FTPSVSFSQNGGVVEAAAQRGYIYKK	1465
	(Bac 32, class IIa	YPKGAKVPNKVKMLVNIRGKQTMRT
	bacteriocin, bacteria)	CYLMSWTASSRTAKYYYYI

AP01183	Ref, Bacteriocin 43	ATYYGNGLYCNKEKCWVDWNQAKG	1466
	(Bac 43, bacteriocin,	EIGKIIVNGWVNHGPWAPRR
	bacteria)

AP01184	Ref, Bacteriocin T8	ATYYGNGLYCNKEKCWVDWNQAKG	1467
	(Bac T8, class IIa	EIGKIIVNGWVNHGPWAPRR
	bacteriocin, bacteria)

AP01185	Ref, Enterocin B (EntB,	ENDHRMPNNLNRPNNLSKGGAKCGA	1468
	bacteriocin, bacteria)	AIAGGLFGIPKGPLAWAAGLANVYSK
		CN

AP01186	Ref, Acidocin A	KTYYGTNGVHCTKKSLWGKVRLKNV	1469
	(bacteriocin, bacteria)	IPGTLCRKQSLPIKQDLKILLGWATGA
		FGKTFH

AP01187	Ref, Enterocin Q (EntQ,	MNFLKNGIAKWMTGAELQAYKKKY	1470
	class IIc bacteriocin,	GCLPWEKISC
	leaderless, i.e. no signal
	peptide, bacteria)

AP01188	Ref, Enterocin EJ97	MLAKIKAMIKKFPNPYTLAAKLTTYEI	1471
	(EntEJ97, class IIc	NWYKQQYGRYPWERPVA
	bacteriocin, leaderless,
	i.e. no signal peptide,
	bacteria)

AP01189	Ref, Enterocin RJ-11	APAGLVAKFGRPIVKKYYKQIMQFIG	1472
	(EntRJ-11, class IIc	EGSAINKIIPWIARMWRT
	bacteriocin, leaderless,
	i.e. no signal sequence,
	bacteria)

AP01190	Ref, Enterocin L50 (old	MGAIAKLVAKFGWPIVKKYYKQIMQ	1473
	name: pediocin L50,	FIGEGWAINKIIEWIKKHI
	EntL50A, a two-chain
	class IIc bacteriocin,
	leaderless, i.e. no signal
	peptide, bacteria. The
	sequence of EntL50B is
	provided in Info)

AP01191	Ref, MR10 (MR10A,	MGAIAKLVAKFGWPIVKKYYKQIMQ	1474
	class IIc bacteriocin,	FIGEGWAINKIIDWIKKHI
	leaderless, i.e. no signal
	peptide, bacteria. For
	the sequence of chain b,
	see Info)

AP01192	Ref, Halocin S8 (HalS8,	SDCNINSNTAADVILCFNQVGSCALCS	1475
	microhalocin,	PTLVGGPVP
	archaeocins, archeae)

AP01193	Ref, Halocin C8	DIDITGCSACKYAAGQVCTIGCSAAG	1476
	(HalC8, microhalocins,	GFICGLLGITIPVAGLSCLGFVEIVCTV
	archaeocins, archaea)	ADEYSGCGDAVAKEACNRAGLC

AP01194	Ref, Lacticin 3147	CSTNTFSLSDYWGNNGAWCTLTHEC	1477
	(chain A1, a two-chain	MAWCK
	lantibiotic, bacteriocin,
	bacteria. The sequence
	of chain A2 is given in
	Info; XXD3)

AP01195	Ref, Salivaricin A	KRGSGWIATITDDCPNSVFVCC	1478
	(SalA, lantibiotic,
	bacteriocin, bacteria)

AP01196	Ref, Microcin E492	ATYYGNGLYCNKEKCWVDWNQAKG	1479
	(MccE492, class IIb	EIGKIIVNGWVNHGPWAPRR
	microcins, bacteriocin,
	bacteria; BBM; u-
	MccE492, siderophore
	peptide, BBI, XXG)

AP01197	Ref, Hiracin JM79	ATYYGNGLYCNKEKCWVDWNQAKG	1480
	(HirJM79, a Sec-	EIGKIIVNGWVNHGPWAPRR
	dependent class II
	bacteriocin, bacteria)

AP01198	Ref, Thermophilin 9	LSCDEGMLAVGGLGAVGGPWGAAV	1481
	(BlpDst, class IIb	GVLVGAALYCF
	bacteriocin, bacteria.
	beta-chains: BlpUst,
	BlpEst, BapFst)

AP01199	Ref, Penocin A (PenA,	KYYGNGVHCGKKTCYVDWGQATASI	1482
	class IIa bacteriocin,	GKIIVNGWTQHGPWAHR
	bacteria)

AP01200	Ref, Salivaricin B	GGGVIQTISHECRMNSWQFLFTCCS	1483
	(SalB, lantibotic,
	bacteriocin, bacteria)

AP01201	Ref, Lacticin 481	KGGSGVIHTISHECNMNSWQFVFTCCS	1484
	(lantibiotic, class I
	bacteriocin, bacteria)

AP01202	Ref, Bacteriocin J46	KGGSGVIHTISHEVIYNSWNFVFTCCS	1485
	(BacJ46, bacteriocin,
	bacteria)

AP01203	Ref, Nukacin A (NucA,	KKKSGVIPTVSHDCHMNSFQFVFTCCS	1486
	Nukacin ISK-1,
	NukISK-1, bacteriocin,
	bacteria)

AP01204	Ref, Streptococcin A-	GKNGVFKTISHECHLNTWAFLATCCS	1487
	FF22 (LANTIBIOTIC,
	class I bacteriocin,
	bacteria)

AP01210	Ref, Jelleine-I	PFKLSLHL	1488
	(honeybees, insect,
	XXA)

AP01211	Ref, Jelleine-II	TPFKLSLHL	1489
	(honeybees, insect,
	XXA)

AP01212	Ref, Jelleine-III	EPFKLSLHL	1490
	(honeybees, insect,
	XXA)

AP01213	Ref, Hymenoptaecin	EFRGSIVIQGTKEGKSRPSLDIDYKQR	1491
	(honeybees, insect	VYDKNGMTGDAYGGLNIRPGQPSRQ
	defensin, XXcooh)	HAGFEFGKEYKNGFIKGQSEVQRGPG
		GRLSPYFGINGGFRF

AP01216	Ref, Ascaphin-1 (frog,	GFRDVLKGAAKAFVKTVAGHIAN	1492
	XXA)

AP01218	Ref, Ascaphin-3 (frog)	GFRDVLKGAAKAFVKTVAGHIANI	1493

AP01220	Ref, Ascaphin-5 (frog)	GIKDWIKGAAKKLIKTVASNIANQ	1494

AP01222	Ref, Ascaphin-7 (frog)	GFKDWIKGAAKKLIKTVASSIANQ	1495

AP01223	Ref, Ascaphin-8 (frog,	GFKDLLKGAAKALVKTVLF	1496
	XXA)

AP01226	Ref, Microcin C7	MRTGNAD	1497
	(MccC7, microcin C51,
	MccC51, class I
	microcins, bacteriocins,
	bacteria. Others: MccA;
	XXamp; BBPe)

AP01227	Ref, Microcin B17	VGIGGGGGGGGGGSCGGQGGGCGGC	1498
	(MccB17, class I	SNGCSGGNGGSGGSGSHI
	microcins, bacteriocins,
	Gram-negative bacteria;
	BBPe)

AP01228	Ref, Microcin V (MccV,	ASGRDIAMAIGTLSGQFVAGGIGAAA	1499
	(old name) Colicin V,	GGVAGGAIYDYASTHKPNPAMSPSGL
	ColV; class II	GGTIKQKPEGIPSEAWNYAAGRLCNW
	microcins, bacteriocins,	SPNNLSDVCL
	Gram-negative bacteria)

AP01229	Ref, Microcin L (MccL,	GDVNWVDVGKTVATNGAGVIGGAFG	1500
	class IIa microcins,	AGLCGPVCAGAFAVGSSAAVAALYD
	bacteriocins, Gram-	AAGNSNSAKQKPEGLPPEAWNYAEG
	negative bacteria)	RMCNWSPNNLSDVCL

AP01230	Ref, Microcin M	DGNDGQAELIAIGSLAGTFISPGFGSIA	1501
	(MccM, class IIb	GAYIGDKVHSWATTATVSPSMSPSGI
	microcins, bacteriocins,	GLSSQFGSGRGTSSASSSAGSGS
	Gram-negative bacteria)

AP01231	Ref, Microcin H47	GGAPATSANAAGAAAIVGALAGIPGG	1502
	(MccH47, class IIb	PLGVVVGAVSAGLTTGIGSTVGSGSA
	microcins, bacteriocins,	SSSAGGGS
	Gram-negative bacteria)

AP01232	Ref, Microcin I47	MNLNGLPASTNVIDLRGKDMGTYIDA	1503
	(MccI47, class IIb	NGACWAPDTPSIIMYPGGSGPSYSMSS
	microcins, bacteriocins,	STSSANSGS
	Gram-negative bacteria)

	Aibellin	*Ac U A U A U A Q U F U G U U P V U	1504
		U E E
		[NHC(CH2Ph)HCH2NHCH2CH2]OH

	Alamethicin_F-30	*Ac U P U A U A Q U V U G L U P V U	1505
		U E Q F OH

	Alamethicin_F-50	*Ac U P U A U A Q U V U G L U P V U	1506
		U Q Q F OH

	Alamethicin_II	*Ac U P U A U U Q U V U G L U P V U	1507
		U E Q F OH

	Ampullosporin	*Ac W A U U L U Q U U U Q L U Q L	1508
		OH

	Ampullosporin_B	*Ac W A U U L U Q A U U Q L U Q L	1509
		OH

	Ampullosporin_C	*Ac W A U U L U Q U A U Q L U Q L	1510
		OH

	Ampullosporin_D	*Ac W A U U L U Q U U A Q L U Q L	1511
		OH

	Ampullosporin_E1	*Ac W A U U L U Q A U U Q L A Q L	1512
		OH

	Ampullosporin_E2	*Ac W A U U L U Q U A A Q L U Q L	1513
		OH

	Ampullosporin_E3	*Ac W A U U L U Q U U A Q L A Q L	1514
		OH

	Ampullosporin_E4	*Ac W A U U L U Q A A U Q L U Q L	1515
		OH

	Antiamoebin_I	*Ac F U U U J G L U U O Q J O U P F	1516
		OH

	Antiamoebin_II	*Ac F U U U J G L U U O Q J P U P F	1517
		OH

	Antiamoebin_III	*Ac F U U U U G L U U O Q J O U P F	1518
		OH

	Antiamoebin_IV	*Ac F U U U J G L U U O Q J O U P F	1519
		OH

	Antiamoebin_V	*Ac F U U U J A L U U O Q J O U P F	1520
		OH

	Antiamoebin_VI	*Ac F U U U U G L U U O Q U O U P F	1521
		OH

	Antiamoebin_VII	*Ac F A U J U G L U U O Q J O U P F	1522
		OH

	Antiamoebin_VIII	*Ac F U U U J G L U U O Q U O U P F	1523
		OH

	Antiamoebin_IX	*Ac F U A U J G L U U O Q J O U P F	1524
		OH

	Antiamoebin_X	*Ac F U U U J G L J U O Q U O U P F	1525
		OH

	Antiamoebin_XI	*Ac F U U U U A L U U O Q J O U P F	1526
		OH

	Antiamoebin_XII	*Ac F U U U U G L A U O Q J O U P F	1527
		OH

	Antiamoebin_XIII	*Ac V U U U U G L U U O Q J O U P F	1528
		OH

	Antiamoebin_XIV	*Ac V U U U V G L U U O Q J O U P F	1529
		OH

	Antiamoebin_XV	*Ac L U U U U G L U U O Q J O U P F	1530
		OH

	Antiamoebin_XVI	*Ac L U U U J G L U U O Q J O U P F	1531
		OH

	Atroviridin_A	*Ac U P U A U A Q U V U G L U P V U	1532
		U Q Q F OH

	Atroviridin_B	*Ac U P U A U A Q U V U G L U P V U	1533
		J Q Q F OH

	Atroviridin_C	*Ac U P U A U U Q U V U G L U P V U	1534
		J Q Q F OH

	Bergofungin_A	*Ac V U U U V G L U U O Q J O U F	1535
		OH

	Bergofungin_B	*Ac V U U U V G L V U O Q U O U F	1536
		OH

	Bergofungin_C	*Ac V U U U V G L U U O Q U O U F	1537
		OH

	Bergofungin_D	*Ac V U U V G L U U O Q U O U F OH	1538

	Boletusin	*Ac F U A U J L Q G U U A A U P U U	1539
		U Q W OH

	Cephaibol_A	*Ac F U U U U G L J U O Q J O U P F	1540
		OH

	Cephaibol_A2	*Ac F U U U U A L J U O Q J O U P F	1541
		OH

	Cephaibol_B	*Ac F U U U J G L J U O Q J O U P F	1542
		OH

	Cephaibol_C	*Ac F U U U U G L J U O Q U O U P F	1543
		OH

	Cephaibol_D	*Ac F U U U U G L U U O Q U O U P F	1544
		OH

	Cephaibol_E	*Ac F U U U U G L U U O Q J O U P F	1545
		OH

	Cephaibol_P	*Ac F J Q U I T U L U O Q U O U P F S	1546
		OH

	Cephaibol_Q	*Ac F J Q U I T U L U P Q U O U P F S	1547
		OH

	Cervinin_1	*Ac L U P U L U P A U P V L OH	1548

	Cervinin_2	*Ac L U P U L U P A U P V L OCOCH3	1549

	Chrysospermin_A	*Ac F U S U U L Q G U U A A U P U U	1550
		U Q W OH

	Chrysospermin_B	*Ac F U S U U L Q G U U A A U P J U U	1551
		Q W OH

	Chrysospermin_C	*Ac F U S U J L Q G U U A A U P U U U	1552
		Q W OH

	Chrysospermin_D	*Ac F U S U J L Q G U U A A U P J U U	1553
		Q W OH

	Clonostachin	*Ac U O L J O L J O U J U O J I	1554
		O[CH(CH(OH)CH2OH)CH(OH)CH(OH)CH2]OH

	Emerimicin_II_A	*Ac W I Q U I T U L U O Q U O U P F	1555
		OH

	Emerimicin_II_B	*Ac W I Q J I T U L U O Q U O U P F	1556
		OH

	Emerimicin_III	*Ac F U U U V G L U U O Q J O U F OH	1557

	Emerimicin_IV	*Ac F U U U V G L U U O Q J O A F OH	1558

	Harzianin_HB_I	*Ac U N L I U P J L U P L OH	1559

	Harzianin_HC_I	*Ac U N L U P S V U P U L U P L OH	1560

	Harzianin_HC_III	*Ac U N L U P S V U P J L U P L OH	1561

	Harzianin_HC_IX	*Ac U N L U P A I U P J L U P L OH	1562

	Harzianin_HC_VI	*Ac U N L U P A V U P U L U P L OH	1563

	Harzianin_HC_VIII	*Ac U N L U P A V U P J L U P L OH	1564

	Harzianin_HC_VIII	*Ac U N L U P A V U P J L U P L OH	1565

	Harzianin_HC_X	*Ac U Q L U P A V U P J L U P L OH	1566

	Harzianin_HC_XI	*Ac U N L U P S I U P U L U P L OH	1567

	Harzianin_HC_XII	*Ac U N L U P S I U P J L U P L OH	1568

	Harzianin_HC_XIII	*Ac U Q L U P S I U P J L U P L OH	1569

	Harzianin_HC_XIV	*Ac U N L U P A I U P U L U P L OH	1570

	Harzianin_HC_XV	*Ac U Q L U P A I U P J L U P L OH	1571

	Harzianin_HK_VI	*Ac U N I I U P L L U P L OH	1572

	Harzianin_PCU4	*Ac U N L U P S I U P U L U P V OH	1573

	Helioferin_A	*Fa P ZZ A U I I U U AAE	1574

	Helioferin_B	*Fa P ZZ A U I I U U AMAE	1575

	Heptaibin	*Ac F U U U V G L U U O Q U O U F	1576
		OH

	Hypelcin_A	*Ac U P U A U U Q L U G U U U P V U	1577
		U Q Q L OH

	Hypelcin_A_I	*Ac U P U A U U Q U L U G U U P V U	1578
		U Q Q L OH

	Hypelcin_A_II	*Ac U P U A U A Q U L U G U U P V U	1579
		U Q Q L OH

	Hypelcin_A_III	*Ac U P U A U U Q U L U G U U P V U	1580
		U Q Q [C7H16NO]

	Hypelcin_A_IV	*Ac U P U A U U Q U I U G U U P V U	1581
		U Q Q L OH

	Hypelcin_A-III	*Ac U P U A U U Q U L U G U U P V U	1582
		J Q Q L OH

	Hypelcin_A-IX	*Ac U P U A U U Q U I U G U U P V U J	1583
		Q Q L OH

	Hypelcin_A-V	*Ac U P U A U U Q U L U G U U P V U	1584
		U Q Q I OH

	Hypelcin_A-VI	*Ac U P U A U A Q U L U G U U P V U	1585
		U Q Q I OH

	Hypelcin_A-VII	*Ac U P U A U A Q U L U G U U P V U	1586
		J Q Q L OH

	Hypelcin_A-VIII	*Ac U P U A U A Q U I U G U U P V U	1587
		U Q Q L OH

	Hypelcin_B_I	*Ac U P U A U U Q U L U G U U P V U	1588
		U E Q L OH

	Hypelcin_B_II	*Ac U P U A U A Q U L U G U U P V U	1589
		U E Q L OH

	Hypelcin_B_III	*Ac U P U A U U Q U L U G U U P V U	1590
		J E Q L OH

	Hypelcin_B_IV	*Ac U P U A U U Q U I U G U U P V U	1591
		U E Q L OH

	Hypelcin_B_V	*Ac U P U A U U Q U L U G U U P V U	1592
		U E Q I OH

	Hypomurocin_A_I	*Ac U Q V V U P L L U P L OH	1593

	Hypomurocin_A_II	*Ac J Q V V U P L L U P L OH	1594

	Hypomurocin_A_III	*Ac U Q V L U P L I U P L OH	1595

	Hypomurocin_A_IV	*Ac U Q I V U P L L U P L OH	1596

	Hypomurocin_A_V	*Ac U Q I I U P L L U P L OH	1597

	Hypomurocin_A_Va	*Ac U Q I L U P L I U P L OH	1598

	Hypomurocin_B_I	*Ac U S A L U Q U V U G U U P L U U	1599
		Q V OH

	Hypomurocin_B_II	*Ac U S A L U Q U V U G U U P L U U	1600
		Q L OH

	Hypomurocin_B_IIIa	*Ac U A A L U Q U V U G U U P L U U	1601
		Q V OH

	Hypomurocin_B_IIIb	*Ac U S A L U Q J V U G U U P L U U	1602
		Q V OH

	Hypomurocin_B_IV	*Ac U S A L U Q U V U G J U P L U U	1603
		Q V OH

	Hypomurocin_B_V	*Ac U S A L U Q U V U G J U P L U U	1604
		Q L OH

	Leu1_Zervamicin	*Ac L I Q J I T U L U O Q U O U P F OH	1605

	Longibrachin_A_I	*Ac U A U A U A Q U V U G L U P V U	1606
		U Q Q F OH

	Longibrachin_A_II	*Ac U A U A U A Q U V U G L U P V U	1607
		J Q Q F OH

	Longibrachin_A_III	*Ac U A U A U U Q U V U G L U P V U	1608
		U Q Q F OH

	Longibrachin_A_IV	*Ac U A U A U U Q U V U G L U P V U	1609
		J Q Q F OH

	Longibrachin_B_II	*Ac U A U A U A Q U V U G L U P V U	1610
		U E Q F OH

	Longibrachin_B_III	*Ac U A U A U A Q U V U G L U P V U	1611
		J E Q F OH

	LP237_F5	*Oc U P Y U Q Q U Zor Q A L OH	1612

	LP237_F7	*Ac U P F U Q Q U U Q A L OH	1613

	LP237_F8	*Oc U P F U Q Q U Zor Q A L OH	1614

	NA_VII	*Ac U A A U J Q U U U S L U OCH3	1615

	Paracelsin_A	*Ac U A U A U A Q U V U G U U P V U	1616
		U Q Q F OH

	Paracelsin_B	*Ac U A U A U A Q U L U G U U P V U	1617
		U Q Q F OH

	Paracelsin_C	*Ac U A U A U U Q U V U G U U P V U	1618
		U Q Q F OH

	Paracelsin_D	*Ac U A U A U U Q U L U G U U P V U	1619
		U Q Q F OH

	Paracelsin_E	*Ac U A U A U A Q U L U G U A P V U	1620
		U Q Q F OH

	Peptaibolin	*Ac L U L U F OH	1621

	Peptaivirin_A	*Ac F U A U J L Q G U U A A U P J U U	1622
		Q W OH

	Peptaivirin_B	*Ac F U S U J L Q G U U A A U P J U U	1623
		Q F OH

	Polysporin_A	*Ac U P U A U U Q U V U G V U P V U	1624
		U Q Q F OH

	Polysporin_B	*Ac U P U A U U Q U V U G L U P V U	1625
		U Q Q F OH

	Polysporin_C	*Ac U P U A U U Q U I U G L U P V U	1626
		U Q Q F OH

	Polysporin_D	*Ac U P U A U U Q U I U G L U P V U	1627
		V Q Q F OH

	Pseudokinin_KLIII	*Ac U N I I U P L L U P NH2	1628

	Pseudokinin_KLVI	*Ac U N I I U P L V	1629
		hydroxyketopiperazine

	Samarosporin_I	*Ac F U U U V G L U U O Q J O A F OH	1630

	Samarosporin_II	*Ac F U U U V G L U U O Q J O U F OH	1631

	Saturnisporin_SA_I	*Ac U A U A U A Q U L U G U U P V U	1632
		U Q Q F OH

	Saturnisporin_SA_II	*Ac U A U A U A Q U L U G U U P V U	1633
		J Q Q F OH

	Saturnisporin_SA_III	*Ac U A U A U U Q U L U G U U P V U	1634
		U Q Q F OH

	Saturnisporin_SA_IV	*Ac U A U A U U Q U L U G U U P V U	1635
		J Q Q F OH

	Stilbellin_I	*Ac F U U U V G L U U O Q J O A F OH	1636

	Stilbellin_II	*Ac F U U U V G L U U O Q J O U F OH	1637

	Stilboflavin_A_1	*Ac U P U A U A Q U V U G U U P V U	1638
		U E Q V OH

	Stilboflavin_A_2	*Ac U P U A U A Q U L U G U U P V U	1639
		U E Q V OH

	Stilboflavin_A_3	*Ac U P U A U U Q U V U G U A P V U	1640
		U E Q L OH

	Stilboflavin_A_4	*Ac U P U A U A Q U L U G U U P V U	1641
		U E Q L OH

	Stilboflavin_A_5	*Ac U P U A U U Q U L U G U U P V U	1642
		U E Q V OH

	Stilboflavin_A_6	*Ac U P U A U A Q U L U G U U P V U	1643
		U E Q J OH

	Stilboflavin_A_7	*Ac U P U A U U Q U L U G U U P V U	1644
		U E Q I OH

	Stilboflavin_B_1	*Ac U P U A U A Q U V U G U U P V U	1645
		U Q Q V OH

	Stilboflavin_B_2	*Ac U P U A U A Q U L U G U U P V U	1646
		U Q Q V OH

	Stilboflavin_B_3	*Ac U P U A U A Q U V U G U U P V U	1647
		U Q Q L OH

	Stilboflavin_B_4	*Ac U P U A U A Q U L U G U U P V U	1648
		U Q Q L OH

	Stilboflavin_B_5	*Ac U P U A U U Q U L U G U U P V U	1649
		U Q Q V OH

	Stilboflavin_B_6	*Ac U P U A U U Q U V U G U U P V U	1650
		U Q Q V OH

	Stilboflavin_B_7	*Ac U P U A U U Q U L U G U U P V U	1651
		U Q Q L OH

	Stilboflavin_B_8	*Ac U P U A U U Q U V U G U U P V U	1652
		U Q Q L OH

	Stilboflavin_B_9	*Ac U P U A U U Q U L U G U U P V U	1653
		U Q Q I OH

	Stilboflavin_B_10	*Ac U P U A U U Q U V U G U U P V U	1654
		U Q Q I OH

	Suzukacillin	*Ac U A U A U A Q U U U G L U P V U	1655
		U Q Q F OH

	Trichobrachin_A-I	*Ac U N L L U P L U U P L OH	1656

	Trichobrachin_A-II	*Ac U N L L U P V L U P V OH	1657

	Trichobrachin_A-III	*Ac U N V L U P L L U P V OH	1658

	Trichobrachin_A-IV	*Ac U N L V U P L L U P V OH	1659

	Trichobrachin_B-I	*Ac U N L L U P V U V P L OH	1660

	Trichobrachin_B-II	*Ac U N V L U P L U V P L OH	1661

	Trichobrachin_B-III	*Ac U N L V U P L U V P L OH	1662

	Trichobrachin_B-IV	*Ac U N L L U P L U V P V OH	1663

	Trichocellin_TC-A-I	*Ac U A U A U A Q U L U G U U P V U	1664
		U Q Q F OH

	Trichocellin_TC-A-II	*Ac U A U A U A Q U L U G U U P V U	1665
		J Q Q F OH

	Trichocellin_TC-A-III	*Ac U A U A U A Q U I U G U U P V U	1666
		U Q Q F OH

	Trichocellin_TC-A-IV	*Ac U A U A U A Q U I U G U U P V U	1667
		J Q Q F OH

	Trichocellin_TC-A-V	*Ac U A U A U A Q U L U G L U P V U	1668
		U Q Q F OH

	Trichocellin_TC-A-VI	*Ac U A U A U A Q U L U G L U P V U	1669
		J Q Q F OH

	Trichocellin_TC-A-VII	*Ac U A U A U A Q U I U G L U P V U	1670
		U Q Q F OH

	Trichocellin_TC-A-VIII	*Ac U A U A U A Q U I U G L U P V U J	1671
		Q Q F OH

	Trichocellin_TC-B-I	*Ac U A U A U A Q U L U G U U P V U	1672
		U E Q F OH

	Trichocellin_TC-B-II	*Ac U A U A U A Q U L U G U U P V U	1673
		J E Q F OH

	Trichodecenin_TD_I	*(Z)-4-decenoyl G G L U G I L OH	1674

	Trichodecenin_TD_II	*(Z)-4-decenoyl G G L U G L L OH	1675

	Trichogin_A_IV	*Oc U G L U G G L U G I L OH	1676

	Trichokindin_Ia	*Ac U S A U U Q J L U A U U P L U U	1677
		Q I OH

	Trichokindin_Ib	*Ac U S A U J Q U L U A U U P L U U	1678
		Q I OH

	Trichokindin_IIa	*Ac U S A U U Q U L U A J U P L U U	1679
		Q I OH

	Trichokindin_IIb	*Ac U S A U J Q J L U A U U P L U U Q	1680
		L OH

	Trichokindin_IIIa	*Ac U S A U U Q J L U A J U P L U U Q	1681
		L OH

	Trichokindin_IIIb	*Ac U S A U J Q U L U A J U P L U U Q	1682
		L OH

	Trichokindin_IV	*Ac U S A U J Q J L U A U U P L U U Q	1683
		I OH

	Trichokindin_Va	*Ac U S A U U Q J L U A J U P L U U Q	1684
		I OH

	Trichokindin_Vb	*Ac U S A U J Q U L U A J U P L U U Q	1685
		I OH

	Trichokindin_VI	*Ac U S A U J Q J L U A J U P L U U Q	1686
		L OH

	Trichokindin_VII	*Ac U S A U J Q J L U A J U P L U U Q I	1687
		OH

	Trichokonin_Ia	*Ac U A U A U A Q U V U G L A P V U	1688
		U Q Q F OH

	Trichokonin_Ib	*Ac U G U A U A Q U V U G L U P V U	1689
		U Q Q F OH

	Trichokonin_IIa	*Ac U A U A U A Q U V U G L U P A U	1690
		U Q Q F OH

	Trichokonin_IIb	*Ac A A U A U A Q U V U G L U P V U	1691
		U Q Q F OH

	Trichokonin_IIc	*Ac U A A A U A Q U V U G L U P V U	1692
		U Q Q F OH

	Trichokonin_V	*Ac U A U A U Q U V U G L U P V U U	1693
		Q Q F OH

	Trichokonin_VII	*Ac U A U A U A Q U V U G L U P V U	1694
		J Q Q F OH

	Trichokonin_VIII	*Ac U A U A U U Q U V U G L U P V U	1695
		U Q Q F OH

	Trichokonin_IX	*Ac U A U A U A Q U V U G L U P V U	1696
		J Q Q F OH

	Tricholongin_BI	*Ac U G F U U Q U U U S L U P V U U	1697
		Q Q L OH

	Tricholongin_BII	*Ac U G F U U Q U U U S L U P V U J Q	1698
		Q L OH

	Trichopolyn_I	*Fa P ZZ A U U I A U U AMAE	1699

	Trichopolyn_II	*Fa P ZZ A U U V A U U AMAE	1700

	Trichopolyn_III	*Fa P ZZ A U U I A U A AMAE	1701

	Trichopolyn_IV	*Fa P ZZ A U U V A U A AMAE	1702

	Trichopolyn_V	*Fa′ P ZZ A U U I A U U AMAE	1703

	Trichorovin_TV_Ia	*Ac U N V Lx U P Lx Lx U P V OH	1704

	Trichorovin_TV_Ib	*Ac U N V V U P Lx Lx U P Lx OH	1705

	Trichorovin_TV_IIa	*Ac U N V V U P Lx Lx U P Lx OH	1706

	Trichorovin_TV_IIb	*Ac U N Lx V U P Lx Lx U P V OH	1707

	Trichorovin_TV_IIIa	*Ac U Q V V U P Lx Lx U P Lx OH	1708

	Trichorovin_TV_IIIb	*Ac U Q V Lx U P Lx Lx U P V OH	1709

	Trichorovin_TV_IVa	*Ac U Q V V U P Lx Lx U P Lx OH	1710

	Trichorovin_TV_IVb	*Ac U Q Lx V U P Lx Lx U P V OH	1711

	Trichorovin_TV_IVc	*Ac U N V Lx U P Lx Lx U P Lx OH	1712

	Trichorovin_TV_IXa	*Ac U Q V Lx U P Lx Lx U P Lx OH	1713

	Trichorovin_TV_IXb	*Ac U Q Lx Lx U P Lx Lx U P V OH	1714

	Trichorovin_TV_Va	*Ac U N V Lx U P Lx Lx U P Lx OH	1715

	Trichorovin_TV_Vb	*Ac U N Lx Lx U P Lx Lx U P V OH	1716

	Trichorovin_TV_VIa	*Ac U N V Lx U P Lx Lx U P Lx OH	1717

	Trichorovin_TV_VIb	*Ac U N Lx Lx U P Lx Lx U P V OH	1718

	Trichorovin_TV_VIIa	*Ac U N Lx V U P Lx Lx U P Lx OH	1719

	Trichorovin_TV_VIIb	*Ac U Q V Lx U P Lx Lx U P V OH	1720

	Trichorovin_TV_VIII	*Ac U Q V Lx U P Lx Lx U P Lx OH	1721

	Trichorovin_TV_Xa	*Ac U Q Lx V U P Lx Lx U P Lx OH	1722

	Trichorovin_TV_Xb	*Ac U N Lx Lx U P Lx Lx U P Lx OH	1723

	Trichorovin_TV_XIIa	*Ac U N I I U P L L U P I OH	1724

	Trichorovin_TV_XIIb	*Ac U N Lx Lx U P Lx Lx U P L OH	1725

	Trichorovin_TV_XIII	*Ac U Q Lx Lx U P Lx Lx U P Lx OH	1726

	Trichorovin_TV_XIV	*Ac U Q Lx Lx U P Lx Lx U P Lx OH	1727

	Trichorozin_I	*Ac U N I L U P I L U P V OH	1728

	Trichorozin_II	*Ac U Q I L U P I L U P V OH	1729

	Trichorozin_III	*Ac U N I L U P I L U P L OH	1730

	Trichorozin_IV	*Ac U Q I L U P I L U P L OH	1731

	Trichorzianine_TA_IIIc	*Ac U A A U U Q U U U S L U P V U I	1732
		Q Q W OH

	Trichorzianine_TB_IIa	*Ac U A A U U Q U U U S L U P L U I Q	1733
		E W OH

	Trichorzianine_TB_IIIc	*Ac U A A U U Q U U U S L U P V U I	1734
		Q E W OH

	Trichorzianine_TB_IVb	*Ac U A A U J Q U U U S L U P V U I Q	1735
		E W OH

	Trichorzianine_TB_Vb	*Ac U A A U U Q U U U S L U P L U I Q	1736
		E F OH

	Trichorzianine_TB_VIa	*Ac U A A U J Q U U U S L U P L U I Q	1737
		E F OH

	Trichorzianine_TB_VIb	*Ac U A A U U Q U U U S L U P V U I	1738
		Q E F OH

	Trichorzianine_TB_VII	*Ac U A A U J Q U U U S L U P V U I Q	1739
		E F OH

	Trichorzin_HA_I	*Ac U G A U U Q U V U G L U P L U U	1740
		Q L OH

	Trichorzin_HA_II	*Ac U G A U U Q U V U G L U P L U J	1741
		Q L OH

	Trichorzin_HA_III	*Ac U G A U J Q U V U G L U P L U U	1742
		Q L OH

	Trichorzin_HA_V	*Ac U G A U J Q U V U G L U P L U J Q	1743
		L OH

	Trichorzin_HA_VI	*Ac U G A U J Q J V U G L U P L U J Q	1744
		L OH

	Trichorzin_HA_VII	*Ac U G A U J Q V V U G L U P L U J Q	1745
		L OH

	Trichorzin_MA_I	*Ac U S A U U Q U L U G L U P L U U	1746
		Q V OH

	Trichorzin_MA_II	*Ac U S A U J Q U L U G L U P L U U Q	1747
		V OH

	Trichorzin_MA_III	*Ac U S A U J Q J L U G L U P L U U Q	1748
		V OH

	Trichorzin_PA_II	*Ac U S A U J Q U V U G L U P L U U	1749
		Q W OH

	Trichorzin_PA_IV	*Ac U S A U J Q J V U G L U P L U U Q	1750
		W OH

	Trichorzin_PA_V	*Ac U S A J J Q U V U G L U P L U U Q	1751
		W OH

	Trichorzin_PA_VI	*Ac U S A U J Q U V U G L U P L U U	1752
		Q F OH

	Trichorzin_PA_VII	*Ac U S A J J Q U V U G L U P L U U Q	1753
		W OH

	Trichorzin_PA_VIII	*Ac U S A U J Q J V U G L U P L U U Q	1754
		F OH

	Trichorzin_PA_IX	*Ac U S A J J Q U V U G L U P L U U Q	1755
		F OH

	Trichorzin_PAU4	*Ac U S A U U Q U V U G L U P L U U	1756
		Q W OH

	Trichosporin_TS-B-1a-1	*Ac U A G U A U Q U Lx A A Vx A P V	1757
		U Vx Q Q F OH

	Trichosporin_TS-B-1a-2	*Ac U A G A U U Q U Lx A A Vx A P V	1758
		U Vx Q Q F OH

	Trichosporin_TS-B-1b	*Ac U A G A U U Q U Lx U G Lx A P V	1759
		U A Q Q F OH

	Trichosporin_TS-B-1d	*Ac U A S A U U Q U Lx U G Lx A P V	1760
		U U Q Q F OH

	Trichosporin_TS-B-1e	*Ac U A G A U U Q U Lx U G Lx U P V	1761
		U U Q Q F OH

	Trichosporin_TS-B-1f	*Ac U A S A U U Q U Lx U G Lx U P V	1762
		U U Q Q F OH

	Trichosporin_TS-B-1g	*Ac U A G A U U Q U Lx U G Lx A P V	1763
		U U Q Q F OH

	Trichosporin_TS-B-1h	*Ac U A G A U U Q U Lx U G Lx U P V	1764
		U Vx Q Q F OH

	Trichosporin_TS-B-Ia	*Ac U A S A U U Q U L U G L U P V U	1765
		U Q Q F OH

	Trichosporin_TS-B-IIIa	*Ac U A A A U U Q U L U G L U P V U	1766
		U Q Q F OH

	Trichosporin_TS-B-IIIb	*Ac U A A A U U Q U I U G L U P V U	1767
		A Q Q F OH

	Trichosporin_TS-B-IIIc	*Ac U A A A A U Q U I U G L U P V U	1768
		U Q Q F OH

	Trichosporin_TS-B-IIId	*Ac U A A A U U Q U V U G L U P V U	1769
		U Q Q F OH

	Trichosporin_TS-B-IVb	*Ac U A A A U U Q U L U G L U P V U	1770
		J Q Q F OH

	Trichosporin_TS-B-IVc	*Ac U A U A U U Q U V U G L U P V U	1771
		U Q Q F OH

	Trichosporin_TS-B-IVd	*Ac U A A A U U Q U V U G L U P V U	1772
		J Q Q F OH

	Trichosporin_TS-B-V	*Ac U A A A U U Q U I U G L U P V U	1773
		U Q Q F OH

	Trichosporin_TS-B-VIa	*Ac U A U A U U Q U I U G L U P V U	1774
		U Q Q F OH

	Trichosporin_TS-B-VIb	*Ac U A A A U U Q U I U G L U P V U J	1775
		Q Q F OH

	Trichotoxin_A-40	*Ac U G U L U E U U U A U U P L U J	1776
		Q V OH

	Trichotoxin_A-40_I	*Ac U G U L U Q U U A A U U P L U U	1777
		E V OH

	Trichotoxin_A-40_II	*Ac U G U L U Q U U U A A U P L U U	1778
		E V OH

	Trichotoxin_A-40_III	*Ac U G U L U Q U U A A U U P L U J	1779
		E V OH

	Trichotoxin_A-40_IV	*Ac U G U L U Q U U U A U U P L U U	1780
		E V OH

	Trichotoxin_A-40_V	*Ac U G U L U Q U U U A U U P L U J	1781
		E V OH

	Trichotoxin_A-40_Va	*Ac U A U L U Q U U U A U U P L U U	1782
		E V OH

	Trichotoxin_A-50_E	*Ac U G U L U Q U U U A A U P L U U	1783
		Q V OH

	Trichotoxin_A-50_F	*Ac U G U L U Q U U A A A U P L U J	1784
		Q V OH

	Trichotoxin_A-50_G	*Ac U G U L U Q U U U A A U P L U J	1785
		Q V OH

	Trichotoxin_A-50_H	*Ac U A U L U Q U U U A A U P L U J	1786
		Q V OH

	Trichotoxin_A-50_I	*Ac U G U L U Q U U U A U U P L U J	1787
		Q V OH

	Trichotoxin_A-50_J	*Ac U A U L U Q U U U A U U P L U J	1788
		Q V OH

	Trichovirin-Ia	*Ac U G A L A Q Vx V U G U U P L U	1789
		U Q L OH

	Trichovirin-Ib	*Ac U G A L U Q A V U G J U P L U U	1790
		Q L OH

	Trichovirin-IIa	*Ac U G A L A Q U V U G J U P L U U	1791
		Q L OH

	Trichovirin-IIb	*Ac U G A L U Q U V U G U U P L U U	1792
		Q L OH

	Trichovirin-IIc	*Ac U G A L U Q Vx V U G U U P L U	1793
		U Q L OH

	Trichovirin-IIIa	*Ac U G A L U Q J V U G U U P L U U	1794
		Q L OH

	Trichovirin-IIIb	*Ac U G A L J Q J U U G U U P L U U Q	1795
		L OH

	Trichovirin-IVa	*Ac U G A L J Q J V U G U U P L U U Q	1796
		L OH

	Trichovirin-IVb	*Ac U G A L U Q U V U G J U P L U U	1797
		Q L OH

	Trichovirin-V	*Ac U G A L U Q J V U G J U P L U U Q	1798
		L OH

	Trichovirin-VIa	*Ac U G A L U Q J L U G J U P L U U Q	1799
		L OH

	Trichovirin-VIb	*Ac U G A L J Q J V U G J U P L U U Q	1800
		L OH

	Trikoningin_KA_V	*Ac U G A U I Q U U U S L U P V U I Q	1801
		Q L OH

	Trikoningin_KB_I	*Oc U G V U G G V U G I L OH	1802

	Trikoningin_KB_II	*Oc J G V U G G V U G I L OH	1803

	Tylopeptin_A	*Ac W V U J A Q A U S U A L U Q L	1804
		OH

	Tylopeptin_B	*Ac W V U U A Q A U S U A L U Q L	1805
		OH

	XR586	*Ac W J Q U I T U L U P Q U O J P F G	1806
		OH

	Zervamicin_A-1-16	*Boc W I A U I V U L U P A U P U P F	1807
		OCH3

	Zervamicin_ZIA	*Ac W I E J V T U L U O Q U O U P F	1808
		OH

	Zervamicin_ZIB	*Ac W V E J I T U L U O Q U O U P F	1809
		OH

	Zervamicin_ZIB′	*Ac W I E U I T U L U O Q U O U P F	1810
		OH

	Zervamicin_ZIC	*Ac W I E J I T U L U O Q U O U P F	1811
		OH

	Zervamicin_ZII-1	*Ac W I Q U V T U L U O Q U O U P F	1812
		OH

	Zervamicin_ZII-2	*Ac W I Q U I T U V U O Q U O U P F	1813
		OH

	Zervamicin_ZII-3	*Ac W V Q U I T U L U O Q U O U P F	1814
		OH

	Zervamicin_ZII-4	*Ac W I Q J V T U L U O Q U O U P F	1815
		OH

	Zervamicin_ZII-5	*Ac W I Q J I T U V U O Q U O U P F	1816
		OH

	Zervamicin_ZIIA	*Ac W I Q U I T U L U O Q U O U P F	1817
		OH

	Zervamicin_ZIIB	*Ac W I Q J I T U L U O Q U O U P F	1818
		OH

	CAMEL135 (CAM135)	GWRLIKKILRVFKGL	1819

	Novispirin G2	KNLRIIRKGIHIIKKY*	1820

	B-33	FKKFWKWFRRF	1821

	B-34	LKRFLKWFKRF	1822

	B-35	KLFKRWKHLFR	1823

	B-36	RLLKRFKHLFK	1824

	B-37	FKTFLKWLHRF	1825

	B-38	IKQLLHFFQRF	1826

	B-39	KLLQTFKQIFR	1827

	B-40	RILKELKNLFK	1828

	B-41	LKQFVHFIHRF	1829

	B-42	VKTLLHIFQRF	1830

	B-43	KLVEQLKEIFR	1831

	B-44	RVLQEIKQILK	1832

	B-45	VKNLAELVHRF	1833

	B-46	ATHLLHALQRF	1834

	B-47	KLAENVKEILR	1835

	B-48	RALHEAKEALK	1836

	B-49	FHYFWHWFHRF	1837

	B-50	LYHFLHWFQRF	1838

	B-51	YLFQTWQHLFR	1839

	B-52	YLLTEFQHLFK	1840

	B-53	FKTFLQWLHRF	1841

	B-54	IKTLLHFFQRF	1842

	B-55	KLLQTFNQIFR	1843

	B-56	TILQSLKNIFK	1844

	B-57	LKQFVKFIHRF	1845

	B-58	VKQLLKIFNRF	1846

	B-59	KLVQQLKNIFR	1847

	B-60	RVLNQVKQILK	1848

	B-61	VKKLAKLVRRF	1849

	B-62	AKRLLKVLKRF	1850

	B-63	KLAQKVKRVLR	1851

	B-64	RALKRIKHVLK	1852

	1C-1	RRRRWWW	1853

	1C-2	RRWWRRW	1854

	1C-3	RRRWWWR	1855

	1C-4	RWRWRWR	1856

	2C-1	RRRFWWR	1857

	2C-2	RRWWRRF*	1858

	2C-3	RRRWWWF*	1859

	2C-4	RWRWRWF*	1860

	3C-1	RRRRWWK	1861

	3C-2	RRWWRRK	1862

	3C-3	RRRWWWK	1863

	3C-4	RWRWRWK	1864

	4C-1	RRRKWWK	1865

	4C-2	RRWKRRK	1866

	4C-3	RRRKWWK	1867

	4C-4	RWRKRWK	1868

	a-3	LHLLHQLLHLLHQF*	1869

	a-4	AQAAHQAAHAAHQF*	1870

	a-5	KLKKLLKKLKKLLK	1871

	a-6	LKLLKKLLKLLKKF*	1872

	a-7	LQLLKQLLKLLKQF*	1873

	a-8	AQAAKQAAKAAKQF*	1874

	a-9	RWRRWWRHFHHFFH*	1875

	a-10	KLKKLLKRWRRWWR	1876

	a-11	RWRRLLKKLHHLLH*	1877

	a-12	KLKKLLKHLHHLLH*	1878

	BD-1	FVF RHK WVW KHR FLF	1879

	BD-2	VFI HRH VWV HKH VLF	1880

	BD-3	WR WR AR WR WR LR WR F	1881

	BD-4	WR IH LR AR LH VK FR F	1882

	BD-5	LR IH AR FK VH IR LK F	1883

	BD-6	FH IK FR VH LK VR FH F	1884

	BD-7	FH VK IH FR LH VK FH F	1885

	BD-8	LH IH AH FH VH IH LH F	1886

	BD-9	FK IH FR LK VH IR FK F	1887

	BD-10	FK AH IR FK LR VK FH F	1888

	BD-11	LK AK IK FK VK LK IK F	1889

	BD-12	WIW KHK FL HRH FLF	1890

	BD-13	VFL HRH VI KHK LVF	1891

	BD-14	FL HKH VL RHR IVF	1892

	BD-15	VF KHK IV HRH ILF	1893

	BD-16	FLF KH LFL HR IFF	1894

	BD-17	LF KH ILI HR VIF	1895

	BD-18	FL HKH LF KHK LF	1896

	BD-19	VF RHR FI HRH VF	1897

	BD-20	FI HK LV HKH VLF	1898

	BD-21	VL RH LF RHR IVF	1899

	BD-22	LV HK LIL RH LLF	1900

	BD-23	VF KR VLI HK LIF	1901

	BD-24	IV RK FLF RHK VF	1902

	BD-25	VL KH VIA HKR LF	1903

	BD-26	FI RK FLF KH LF	1904

	BD-27	VI RH VWV RK LF	1905

	BD-28	FLF RHR F RHR LVF	1906

	BD-29	LFL HKH A KHK FLF	1907

	BD-30	F KHK F KHK FIF	1908

	BD-31	L RHR L RHR LIF	1909

	BD-32	LIL K FLF K FVF	1910

	BD-33	VLI R ILV R VIF	1911

	BD-34	F RHR F RHR F	1912

	BD-35	L KHK L KHK F	1913

	BD-36	F K F KHK LIF	1914

	BD-37	L R L RHR VLF	1915

	BD-38	F K FLF K FLF	1916

	BD-39	L R LFL R WLF	1917

	BD-40	F K FLF KHK F	1918

	BD-41	L R LFL RHR F	1919

	BD-42	F K FLF K F	1920

	BD-43	L R LFL R F	1921

	AA-1	HHFFHHFHHFFHHF*	1922

	AA-2	FHFFHHFFHFFHHF*	1923

	AA-3	KLLK-GAT-FHFFHHFFHFFHHF	1924

	AA-4	KLLK-FHFFHHFFHFFHHF	1925

	AA-5	FHFFHHFFHFFHHFKLLK	1926

	RIP	YSPWTNF*	1927

Abreviations:
U - Aminoisobutyric Acid (Aib);
J - Isovaline (Iva);
O - Hydroxyproline (Hyp);
Z - Ethylnorvaline (EtNor);
x or xx means L or I at that position;
Ac - optionally acetylated N-term;
OH, OCH3 - optional C-term;
Alkane long chains are noted in brackets;
*optionally amidated C-terminus.

A number of antimicrobial peptides are also disclosed in U.S. Pat. Nos. 7,271,239, 7,223,840, 7,176,276, 6,809,181, 6,699,689, 6,420,116, 6,358,921, 6,316,594, 6,235,973, 6,183,992, 6,143,498, 6,042,848, 6,040,291, 5,936,063, 5,830,993, 5,428,016, 5,424,396, 5,032,574, 4,623,733, which are incorporated herein by reference for the disclosure of particular antimicrobial peptides.
Ligands.
In certain embodiments the effector can comprise one or more ligands, epitope tags, and/or antibodies. In certain embodiments preferred ligands and antibodies include those that bind to surface markers on immune cells. Chimeric moieties utilizing such antibodies as effector molecules act as bifunctional linkers establishing an association between the immune cells bearing binding partner for the ligand or antibody and the target microorganism(s).
The term “epitope tag” or “affinity tag” are used interchangeably herein, and as used refers to a molecule or domain of a molecule that is specifically recognized by an antibody or other binding partner. The term also refers to the binding partner complex as well. Thus, for example, biotin or a biotin/avidin complex are both regarded as an affinity tag. In addition to epitopes recognized in epitope/antibody interactions, affinity tags also comprise “epitopes” recognized by other binding molecules (e.g. ligands bound by receptors), ligands bound by other ligands to form heterodimers or homodimers, His₆bound by Ni-NTA, biotin bound by avidin, streptavidin, or anti-biotin antibodies, and the like.
Epitope tags are well known to those of skill in the art. Moreover, antibodies specific to a wide variety of epitope tags are commercially available. These include but are not limited to antibodies against the DYKDDDDK (SEQ ID NO:1928) epitope, c-myc antibodies (available from Sigma, St. Louis), the HNK-1 carbohydrate epitope, the HA epitope, the HSV epitope, the His₄(SEQ ID NO:1929), His_s(SEQ ID NO:1930), and His₆(SEQ ID NO:1931) epitopes that are recognized by the His epitope specific antibodies (see, e.g., Qiagen), and the like. In addition, vectors for epitope tagging proteins are commercially available. Thus, for example, the pCMV-Tag1 vector is an epitope tagging vector designed for gene expression in mammalian cells. A target gene inserted into the pCMV-Tag1 vector can be tagged with the FLAG® epitope (N-terminal, C-terminal or internal tagging), the c-myc epitope (C-terminal) or both the FLAG (N-terminal) and c-myc (C-terminal) epitopes.
Lipids and Liposomes.
In certain embodiments the effectors comprise one or more microparticles or nanoparticles that can be loaded with an effector agent (e.g., a pharmaceutical, a label, etc.). In certain embodiments the microparticles or nanoparticles are lipidic particles. Lipidic particles are microparticles or nanoparticles that include at least one lipid component forming a condensed lipid phase. Typically, a lipidic nanoparticle has preponderance of lipids in its composition. Various condensed lipid phases include solid amorphous or true crystalline phases; isomorphic liquid phases (droplets); and various hydrated mesomorphic oriented lipid phases such as liquid crystalline and pseudocrystalline bilayer phases (L-alpha, L-beta, P-beta, Lc), interdigitated bilayer phases, and nonlamellar phases (see, e.g., The Structure of Biological Membranes, ed. by P. Yeagle, CRC Press, Bora Raton, Fla., 1991). Lipidic microparticles include, but are not limited to a liposome, a lipid-nucleic acid complex, a lipid-drug complex, a lipid-label complex, a solid lipid particle, a microemulsion droplet, and the like. Methods of making and using these types of lipidic microparticles and nanoparticles, as well as attachment of affinity moieties, e.g., antibodies, to them are known in the art (see, e.g., U.S. Pat. Nos. 5,077,057; 5,100,591; 5,616,334; 6,406,713; 5,576,016; 6,248,363; Bondi et al. (2003) Drug Delivery 10: 245-250; Pedersen et al., (2006) Eur. J. Pharm. Biopharm. 62: 155-162, 2006 (solid lipid particles); U.S. Pat. Nos. 5,534,502; 6,720,001; Shiokawa et al. (2005) Clin. Cancer Res. 11: 2018-2025 (microemulsions); U.S. Pat. No. 6,071,533 (lipid-nucleic acid complexes), and the like).
A liposome is generally defined as a particle comprising one or more lipid bilayers enclosing an interior, typically an aqueous interior. Thus, a liposome is often a vesicle formed by a bilayer lipid membrane. There are many methods for the preparation of liposomes. Some of them are used to prepare small vesicles (d<0.05 micrometer), some for larger vesicles (d>0.05 micrometer). Some are used to prepare multilamellar vesicles, some for unilamellar ones. Methods for liposome preparation are exhaustively described in several review articles such as Szoka and Papahadjopoulos (1980) Ann. Rev. Biophys. Bioeng., 9: 467, Deamer and Uster (1983) Pp. 27-51 In: Liposomes, ed. M. J. Ostro, Marcel Dekker, New York, and the like.
In various embodiments the liposomes include a surface coating of a hydrophilic polymer chain. “Surface-coating” refers to the coating of any hydrophilic polymer on the surface of liposomes. The hydrophilic polymer is included in the liposome by including in the liposome composition one or more vesicle-forming lipids derivatized with a hydrophilic polymer chain. In certain embodiments, vesicle-forming lipids with diacyl chains, such as phospholipids, are preferred. One illustrative phospholipid is phosphatidylethanolamine (PE), which contains a reactive amino group convenient for coupling to the activated polymers. One illustrative PE is distearoyl PE (DSPE). Another example is non-phospholipid double chain amphiphilic lipids, such as diacyl- or dialkylglycerols, derivatized with a hydrophilic polymer chain.
In certain embodiments a hydrophilic polymer for use in coupling to a vesicle forming lipid is polyethyleneglycol (PEG), preferably as a PEG chain having a molecular weight between 1,000-10,000 Daltons, more preferably between 1,000-5,000 Daltons, most preferably between 2,000-5,000 Daltons. Methoxy or ethoxy-capped analogues of PEG are also useful hydrophilic polymers, commercially available in a variety of polymer sizes, e.g., 120-20,000 Daltons.
Other hydrophilic polymers that can be suitable include, but are not limited to polylactic acid, polyglycolic acid, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses, such as hydroxymethylcellulose or hydroxyethylcellulose.
Preparation of lipid-polymer conjugates containing these polymers attached to a suitable lipid, such as PE, have been described, for example in U.S. Pat. No. 5,395,
The liposomes can, optionally be prepared for attachment to one or more targeting moieties described herein. Here the lipid component included in the liposomes would include either a lipid derivatized with the targeting moiety, or a lipid having a polar-head chemical group, e.g., on a linker, that can be derivatized with the targeting moiety in preformed liposomes, according to known methods.
Methods of functionalizing lipids and liposomes with affinity moieties such as antibodies are well known to those of skill in the art (see, e.g., DE 3,218,121; Epstein et al. (1985) Proc. Natl. Acad. Sci., USA, 82:3688 (1985); Hwang et al. (1980) Proc. Natl. Acad. Sci., USA, 77: 4030; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324, all of which are incorporated herein by reference).
Polymeric Microparticles and/or Nanoparticles.
In certain embodiments the effector(s) comprise polymeric microparticles and/or nanoparticles and/or micelles.
Microparticle and nanoparticle-based drug delivery systems have considerable potential for treatment of various microorganisms. Technological advantages of polymeric microparticles or nanoparticles used as drug carriers are high stability, high carrier capacity, feasibility of incorporation of both hydrophilic and hydrophobic substances, and feasibility of variable routes of administration, including oral application and inhalation. Polymeric nanoparticles can also be designed to allow controlled (sustained) drug release from the matrix. These properties of nanoparticles enable improvement of drug bioavailability and reduction of the dosing frequency.
Polymeric nanoparticles are typically micron or submicron (<1 μm) colloidal particles. This definition includes monolithic nanoparticles (nanospheres) in which the drug is adsorbed, dissolved, or dispersed throughout the matrix and nanocapsules in which the drug is confined to an aqueous or oily core surrounded by a shell-like wall. Alternatively, in certain embodiments, the drug can be covalently attached to the surface or into the matrix.
Polymeric microparticles and nanoparticles are typically made from biocompatible and biodegradable materials such as polymers, either natural (e.g., gelatin, albumin) or synthetic (e.g., polylactides, polyalkylcyanoacrylates), or solid lipids. In the body, the drug loaded in nanoparticles is usually released from the matrix by diffusion, swelling, erosion, or degradation. One commonly used material is poly(lactide-co-glycolide) (PLG).
Methods of fabricating and loading polymeric nanoparticles or microparticles are well known to those of skill in the art. Thus, for example, Matsumoto et al. (1999) Intl. J. Pharmaceutics, 185: 93-101 teaches the fabrication of poly(L-lactide)-poly(ethylene glycol)-poly(L-lactide) nanoparticles, Chawla et al. (2002) Intl. J. Pharmaceutics 249: 127-138, teaches the fabrication and use of poly(ε-caprolactone) nanoparticles delivery of tamifoxen, and Bodmeier et al. (1988) Intl. J. Pharmaceutics, 43: 179-186, teaches the preparation of poly(D,L-lactide) microspheres using a solvent evaporation method. ° “Intl. J. Pharmaceutics, 1988, 43, 179-186. Other nanoparticle formulations are described, for example, by Williams et al. (2003) J. Controlled Release, 91: 167-172; Leroux et al. (1996) J. Controlled Release, 39: 339-350; Soppimath et al. (2001) J. Controlled Release, 70:1-20; Brannon-Peppas (1995) Intl. J. Pharmaceutics, 116: 1-9; and the like.
Peptide Preparation.
The peptides described herein can be chemically synthesized using standard chemical peptide synthesis techniques or, particularly where the peptide does not comprise “D” amino acid residues, the peptide can be recombinantly expressed. Where the “D” polypeptides are recombinantly expressed, a host organism (e.g. bacteria, plant, fungal cells, etc.) can be cultured in an environment where one or more of the amino acids is provided to the organism exclusively in a D form. Recombinantly expressed peptides in such a system then incorporate those D amino acids.
In certain embodiments, D amino acids can be incorporated in recombinantly expressed peptides using modified amino acyl-tRNA synthetases that recognize D-amino acids.
In certain embodiments the peptides are chemically synthesized by any of a number of fluid or solid phase peptide synthesis techniques known to those of skill in the art. Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is a preferred method for the chemical synthesis of the polypeptides of this invention. Techniques for solid phase synthesis are well known to those of skill in the art and are described, for example, by Barany and Merrifield (1963) Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield et al. (1963) J. Am. Chem. Soc., 85: 2149-2156, and Stewart et al. (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill.
In one embodiment, the peptides can be synthesized by the solid phase peptide synthesis procedure using a benzhyderylamine resin (Beckman Bioproducts, 0.59 mmol of NH₂/g of resin) as the solid support. The COOH terminal amino acid (e.g., t-butylcarbonyl-Phe) is attached to the solid support through a 4-(oxymethyl)phenacetyl group. This is a more stable linkage than the conventional benzyl ester linkage, yet the finished peptide can still be cleaved by hydrogenation. Transfer hydrogenation using formic acid as the hydrogen donor can be used for this purpose.
It is noted that in the chemical synthesis of peptides, particularly peptides comprising D amino acids, the synthesis usually produces a number of truncated peptides in addition to the desired full-length product. Thus, the peptides are typically purified using, e.g., HPLC.
D-amino acids, beta amino acids, non-natural amino acids, and the like can be incorporated at one or more positions in the peptide simply by using the appropriately derivatized amino acid residue in the chemical synthesis. Modified residues for solid phase peptide synthesis are commercially available from a number of suppliers (see, e.g., Advanced Chem Tech, Louisville; Nova Biochem, San Diego; Sigma, St Louis; Bachem California Inc., Torrance, etc.). The D-form and/or otherwise modified amino acids can be completely omitted or incorporated at any position in the peptide as desired. Thus, for example, in certain embodiments, the peptide can comprise a single modified acid, while in other embodiments, the peptide comprises at least two, generally at least three, more generally at least four, most generally at least five, preferably at least six, more preferably at least seven or even all modified amino acids. In certain embodiments, essentially every amino acid is a D-form amino acid.
As indicated above, the peptides and/or fusion proteins of this invention can also be recombinantly expressed. Accordingly, in certain embodiments, the antimicrobial peptides and/or targeting moieties, and/or fusion proteins of this invention are synthesized using recombinant expression systems. Generally this involves creating a DNA sequence that encodes the desired peptide or fusion protein, placing the DNA in an expression cassette under the control of a particular promoter, expressing the peptide or fusion protein in a host, isolating the expressed peptide or fusion protein and, if required, renaturing the peptide or fusion protein.
DNA encoding the peptide(s) or fusion protein(s) described herein can be prepared by any suitable method as described above, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis.
This nucleic acid can be easily ligated into an appropriate vector containing appropriate expression control sequences (e.g. promoter, enhancer, etc.), and, optionally, containing one or more selectable markers (e.g. antibiotic resistance genes).
The nucleic acid sequences encoding the peptides or fusion proteins described herein can be expressed in a variety of host cells, including, but not limited to, E. coli, other bacterial hosts, yeast, fungus, and various higher eukaryotic cells such as insect cells (e.g. SF3), the COS, CHO and HeLa cells lines and myeloma cell lines. The recombinant protein gene will typically be operably linked to appropriate expression control sequences for each host. For E. coli this can include a promoter such as the T7, trp, or lambda promoters, a ribosome binding site and preferably a transcription termination signal. For eukaryotic cells, the control sequences can include a promoter and often an enhancer (e.g., an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc.), and a polyadenylation sequence, and may include splice donor and acceptor sequences.
The plasmids can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation for E. coli and calcium phosphate treatment or electroporation for mammalian cells. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes.
Once expressed, the recombinant peptide(s) or fusion protein(s) can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, (1982) Protein Purification, Springer-Verlag, N.Y.; Deutscher (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y.). Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred.
One of skill in the art would recognize that after chemical synthesis, biological expression, or purification, the peptide(s) or fusion protein(s) may possess a conformation substantially different than desired native conformation. In this case, it may be necessary to denature and reduce the peptide or fusion protein and then to cause the molecule to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art (see, e.g., Debinski et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992) Anal. Biochem., 205: 263-270). Debinski et al., for example, describes the denaturation and reduction of inclusion body proteins in guanidine-DTE. The protein is then refolded in a redox buffer containing oxidized glutathione and L-arginine.
One of skill would recognize that modifications can be made to the peptide(s) and/or fusion protein(s) proteins without diminishing their biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.
Protecting Groups.
While the various peptides (e.g., targeting peptides, antimicrobial peptides, STAMPs) described herein may be shown with no protecting groups, in certain embodiments they can bear one, two, three, four, or more protecting groups. In various embodiments, the protecting groups can be coupled to the C- and/or N-terminus of the peptide(s) and/or to one or more internal residues comprising the peptide(s) (e.g., one or more R-groups on the constituent amino acids can be blocked). Thus, for example, in certain embodiments, any of the peptides described herein can bear, e.g., an acetyl group protecting the amino terminus and/or an amide group protecting the carboxyl terminus. One example of such a protected peptide is the 1845L6-21 STAMP having the amino acid sequence KFINGVLSQFVLERKPYPKLFKFLRKHLL* (SEQ ID NO:1953), where the asterisk indicates an amidated carboxyl terminus. Of course, this protecting group can be can be eliminated and/or substituted with another protecting group as described herein.
Without being bound by a particular theory, it was discovered that addition of a protecting group, particularly to the carboxyl and in certain embodiments the amino terminus can improve the stability and efficacy of the peptide.
A wide number of protecting groups are suitable for this purpose. Such groups include, but are not limited to acetyl, amide, and alkyl groups with acetyl and alkyl groups being particularly preferred for N-terminal protection and amide groups being preferred for carboxyl terminal protection. In certain particularly preferred embodiments, the protecting groups include, but are not limited to alkyl chains as in fatty acids, propionyl, formyl, and others. Particularly preferred carboxyl protecting groups include amides, esters, and ether-forming protecting groups. In one preferred embodiment, an acetyl group is used to protect the amino terminus and an amide group is used to protect the carboxyl terminus. These blocking groups enhance the helix-forming tendencies of the peptides. Certain particularly preferred blocking groups include alkyl groups of various lengths, e.g., groups having the formula: CH₃—(CH₂)_n—CO— where n ranges from about 1 to about 20, preferably from about 1 to about 16 or 18, more preferably from about 3 to about 13, and most preferably from about 3 to about 10.
In certain embodiments, the protecting groups include, but are not limited to alkyl chains as in fatty acids, propionyl, formyl, and others. Particularly preferred carboxyl protecting groups include amides, esters, and ether-forming protecting groups. In one embodiment, an acetyl group is used to protect the amino terminus and/or an amino group is used to protect the carboxyl terminus (i.e., amidated carboxyl terminus). In certain embodiments blocking groups include alkyl groups of various lengths, e.g., groups having the formula: CH₃—(CH₂)_n—CO— where n ranges from about 3 to about 20, preferably from about 3 to about 16, more preferably from about 3 to about 13, and most preferably from about 3 to about 10.
In certain embodiments, the acid group on the C-terminal can be blocked with an alcohol, aldehyde or ketone group and/or the N-terminal residue can have the natural amide group, or be blocked with an acyl, carboxylic acid, alcohol, aldehyde, or ketone group.
Other protecting groups include, but are not limited to Fmoc, t-butoxycarbonyl (t-BOC), 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, xanthyl (Xan), trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), benzyloxy (BzlO), benzyl (Bzl), benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), 1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA).
Protecting/blocking groups are well known to those of skill as are methods of coupling such groups to the appropriate residue(s) comprising the peptides of this invention (see, e.g., Greene et al., (1991) Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, Inc., Somerset, N.J.). In an illustrative embodiment, for example, acetylation is accomplished during the synthesis when the peptide is on the resin using acetic anhydride. Amide protection can be achieved by the selection of a proper resin for the synthesis. For example, a rink amide resin can be used. After the completion of the synthesis, the semipermanent protecting groups on acidic bifunctional amino acids such as Asp and Glu and basic amino acid Lys, hydroxyl of Tyr are all simultaneously removed. The peptides released from such a resin using acidic treatment comes out with the n-terminal protected as acetyl and the carboxyl protected as NH₂and with the simultaneous removal of all of the other protecting groups.
While amino acid sequences are disclosed herein, amino acid sequences comprising, one or more protecting groups, e.g., as described above (or any other commercially available protecting groups for amino acids used, e.g., in boc or fmoc peptide synthesis) are also contemplated.
Peptide Circularization.
In certain embodiments the peptides described herein (e.g., AMPs, compound AMPs, STAMPs, etc.) are circularized/cyclized to produce cyclic peptides. Cyclic peptides, as contemplated herein, include head/tail, head/side chain, tail/side chain, and side chain/side chain cyclized peptides. In addition, peptides contemplated herein include homodet, containing only peptide bonds, and heterodet containing in addition disulfide, ester, thioester-bonds, or other bonds.
The cyclic peptides can be prepared using virtually any art-known technique for the preparation of cyclic peptides. For example, the peptides can be prepared in linear or non-cyclized form using conventional solution or solid phase peptide syntheses and cyclized using standard chemistries. Preferably, the chemistry used to cyclize the peptide will be sufficiently mild so as to avoid substantially degrading the peptide. Suitable procedures for synthesizing the peptides described herein as well as suitable chemistries for cyclizing the peptides are well known in the art.
In various embodiments cyclization can be achieved via direct coupling of the N- and C-terminus to form a peptide (or other) bond, but can also occur via the amino acid side chains. Furthermore it can be based on the use of other functional groups, including but not limited to amino, hydroxy, sulfhydryl, halogen, sulfonyl, carboxy, and thiocarboxy. These groups can be located at the amino acid side chains or be attached to their N- or C-terminus.
Accordingly, it is to be understood that the chemical linkage used to covalently cyclize the peptides of the invention need not be an amide linkage. In many instances it may be desirable to modify the N- and C-termini of the linear or non-cyclized peptide so as to provide, for example, reactive groups that may be cyclized under mild reaction conditions. Such linkages include, by way of example and not limitation amide, ester, thioester, CH₂—NH, etc. Techniques and reagents for synthesizing peptides having modified termini and chemistries suitable for cyclizing such modified peptides are well-known in the art.
Alternatively, in instances where the ends of the peptide are conformationally or otherwise constrained so as to make cyclization difficult, it may be desirable to attach linkers to the N- and/or C-termini to facilitate peptide cyclization. Of course, it will be appreciated that such linkers will bear reactive groups capable of forming covalent bonds with the termini of the peptide. Suitable linkers and chemistries are well-known in the art and include those previously described.
Cyclic peptides and depsipeptides (heterodetic peptides that include ester (depside) bonds as part of their backbone) have been well characterized and show a wide spectrum of biological activity. The reduction in conformational freedom brought about by cyclization often results in higher receptor-binding affinities. Frequently in these cyclic compounds, extra conformational restrictions are also built in, such as the use of D- and N-alkylated-amino acids, α,β-dehydro amino acids or α,α-disubstituted amino acid residues.
Methods of forming disulfide linkages in peptides are well known to those of skill in the art (see, e.g., Eichler and Houghten (1997) Protein Pept. Lett. 4: 157-164).
Reference may also be made to Marlowe (1993) Biorg. Med. Chem. Lett. 3: 437-44 who describes peptide cyclization on TFA resin using trimethylsilyl (TMSE) ester as an orthogonal protecting group; Pallin and Tam (1995) J. Chem. Soc. Chem. Comm. 2021-2022) who describe the cyclization of unprotected peptides in aqueous solution by oxime formation; Algin et al. (1994) Tetrahedron Lett. 35: 9633-9636 who disclose solid-phase synthesis of head-to-tail cyclic peptides via lysine side-chain anchoring; Kates et al. (1993) Tetrahedron Lett. 34: 1549-1552 who describe the production of head-to-tail cyclic peptides by three-dimensional solid phase strategy; Tumelty et al. (1994) J. Chem. Soc. Chem. Comm. 1067-1068, who describe the synthesis of cyclic peptides from an immobilized activated intermediate, where activation of the immobilized peptide is carried out with N-protecting group intact and subsequent removal leading to cyclization; McMurray et al. (1994) Peptide Res. 7: 195-206) who disclose head-to-tail cyclization of peptides attached to insoluble supports by means of the side chains of aspartic and glutamic acid; Hruby et al. (1994) Reactive Polymers 22: 231-241) who teach an alternate method for cyclizing peptides via solid supports; and Schmidt and Langer (1997) J. Peptide Res. 49: 67-73, who disclose a method for synthesizing cyclotetrapeptides and cyclopentapeptides.
These methods of peptide cyclization are illustrative and non-limiting. Using the teaching provide herein, other cyclization methods will be available to one of skill in the art.
Joining Targeting Moieties to Effectors.
Chemical Conjugation.
Chimeric moieties are formed by joining one or more of the targeting moieties described herein to one or more effectors. In certain embodiments the targeting moieties are attached directly to the effector(s) via naturally occurring reactive groups or the targeting moiety and/or the effector(s) can be functionalized to provide such reactive groups.
In various embodiments the targeting moieties are attached to effector(s) via one or more linking agents. Thus, in various embodiments the targeting moieties and the effector(s) can be conjugated via a single linking agent or multiple linking agents. For example, the targeting moiety and the effector can be conjugated via a single multifunctional (e.g., bi-, tri-, or tetra-) linking agent or a pair of complementary linking agents. In another embodiment, the targeting moiety and the effector are conjugated via two, three, or more linking agents. Suitable linking agents include, but are not limited to, e.g., functional groups, affinity agents, stabilizing groups, and combinations thereof.
In certain embodiments the linking agent is or comprises a functional group. Functional groups include monofunctional linkers comprising a reactive group as well as multifunctional crosslinkers comprising two or more reactive groups capable of forming a bond with two or more different functional targets (e.g., labels, proteins, macromolecules, semiconductor nanocrystals, or substrate). In some preferred embodiments, the multifunctional crosslinkers are heterobifunctional crosslinkers comprising two or more different reactive groups.
Suitable reactive groups include, but are not limited to thiol (—SH), carboxylate (COOH), carboxyl (—COOH), carbonyl, amine (NH₂), hydroxyl (—OH), aldehyde (—CHO), alcohol (ROH), ketone (R₂CO), active hydrogen, ester, sulfhydryl (SH), phosphate (—PO₃), or photoreactive moieties. Amine reactive groups include, but are not limited to e.g., isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes and glyoxals, epoxides and oxiranes, carbonates, arylating agents, imidoesters, carbodiimides, and anhydrides. Thiol-reactive groups include, but are not limited to e.g., haloacetyl and alkyl halide derivates, maleimides, aziridines, acryloyl derivatives, arylating agents, and thiol-disulfides exchange reagents. Carboxylate reactive groups include, but are not limited to e.g., diazoalkanes and diazoacetyl compounds, such as carbonyldiimidazoles and carbodiimides. Hydroxyl reactive groups include, but are not limited to e.g., epoxides and oxiranes, carbonyldiimidazole, oxidation with periodate, N,N′-disuccinimidyl carbonate or N-hydroxylsuccimidyl chloroformate, enzymatic oxidation, alkyl halogens, and isocyanates. Aldehyde and ketone reactive groups include, but are not limited to e.g., hydrazine derivatives for schiff base formation or reduction amination. Active hydrogen reactive groups include, but are not limited to e.g., diazonium derivatives for mannich condensation and iodination reactions. Photoreactive groups include, but are not limited to e.g., aryl azides and halogenated aryl azides, benzophenones, diazo compounds, and diazirine derivatives.
Other suitable reactive groups and classes of reactions useful in forming chimeric moieties include those that are well known in the art of bioconjugate chemistry. Currently favored classes of reactions available with reactive chelates are those which proceed under relatively mild conditions. These include, but are not limited to, nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions), and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed in, for example, March (1985) Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York, Hermanson (1996) Bioconjugate Techniques, Academic Press, San Diego; and Feeney et al. (1982) Modification of Proteins; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, D.C.
In certain embodiments, the linking agent comprises a chelator. For example, the chelator comprising the molecule, DOTA (DOTA=1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecane), can readily be labeled with a radiolabel, such as Gd³⁺ and ⁶⁴Cu, resulting in Gd³⁺-DOTA and ⁶⁴Cu-DOTA respectively, attached to the targeting moiety. Other suitable chelates are known to those of skill in the art, for example, 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA) derivatives being among the most well known (see, e.g., Lee et al. (1997) Nucl Med. Biol. 24: 2225-23019).
A “linker” or “linking agent” as used herein, is a molecule that is used to join two or more molecules. In certain embodiments the linker is typically capable of forming covalent bonds to both molecule(s) (e.g., the targeting moiety and the effector). Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. In certain embodiments the linkers can be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine). However, in certain embodiments, the linkers will be joined to the alpha carbon amino and carboxyl groups of the terminal amino acids.
A bifunctional linker having one functional group reactive with a group on one molecule (e.g., a targeting peptide), and another group reactive on the other molecule (e.g., an antimicrobial peptide), can be used to form the desired conjugate. Alternatively, derivatization can be performed to provide functional groups. Thus, for example, procedures for the generation of free sulfhydryl groups on peptides are also known (See U.S. Pat. No. 4,659,839).
In certain embodiments the linking agent is a heterobifunctional crosslinker comprising two or more different reactive groups that form a heterocyclic ring that can interact with a peptide. For example, a heterobifunctional crosslinker such as cysteine may comprise an amine reactive group and a thiol-reactive group can interact with an aldehyde on a derivatized peptide. Additional combinations of reactive groups suitable for heterobifunctional crosslinkers include, for example, amine- and sulfhydryl reactive groups; carbonyl and sulfhydryl reactive groups; amine and photoreactive groups; sulfhydryl and photoreactive groups; carbonyl and photoreactive groups; carboxylate and photoreactive groups; and arginine and photoreactive groups. In one embodiment, the heterobifunctional crosslinker is SMCC.
Many procedures and linker molecules for attachment of various molecules to peptides or proteins are known (see, e.g., European Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. (1987) Cancer Res. 47: 4071-4075). Illustrative linking protocols are provided herein in Examples 2 and 3.
Fusion Proteins.
In certain embodiments where the targeting moiety and effector are both peptides or both comprise peptides, the chimeric moiety can be chemically synthesized or recombinantly expressed as a fusion protein (i.e., a chimeric fusion protein).
In certain embodiments the chimeric fusion proteins are synthesized using recombinant DNA methodology. Generally this involves creating a DNA sequence that encodes the fusion protein, placing the DNA in an expression cassette under the control of a particular promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein.
DNA encoding the fusion proteins can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862; and the solid support method of U.S. Pat. No. 4,458,066.
Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. One of skill would recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences can be obtained by the ligation of shorter sequences.
Alternatively, subsequences can be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments can then be ligated to produce the desired DNA sequence.
In certain embodiments, DNA encoding fusion proteins of the present invention may be cloned using DNA amplification methods such as polymerase chain reaction (PCR). Thus, for example, the nucleic acid encoding a targeting antibody, a targeting peptide, and the like is PCR amplified, using a sense primer containing the restriction site for NdeI and an antisense primer containing the restriction site for HindIII. This produces a nucleic acid encoding the targeting sequence and having terminal restriction sites. Similarly an effector and/or effector/linker/spacer can be provided having complementary restriction sites. Ligation of sequences and insertion into a vector produces a vector encoding the fusion protein.
While the targeting moieties and effector(s) can be directly joined together, one of skill will appreciate that they can be separated by a peptide spacer/linker consisting of one or more amino acids. Generally the spacer will have no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of the spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity.
The nucleic acid sequences encoding the fusion proteins can be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines. The recombinant protein gene will be operably linked to appropriate expression control sequences for each host. For E. coli this includes a promoter such as the T7, trp, or lambda promoters, a ribosome binding site and preferably a transcription termination signal. For eukaryotic cells, the control sequences will include a promoter and preferably an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.
The plasmids can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation for E. coli and calcium phosphate treatment or electroporation for mammalian cells. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes.
Once expressed, the recombinant fusion proteins can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes (1982) Protein Purification, Springer-Verlag, N.Y.; Deutscher (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y.). Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically.
One of skill in the art would recognize that after chemical synthesis, biological expression, or purification, the fusion protein may possess a conformation substantially different than the native conformations of the constituent polypeptides. In this case, it may be necessary to denature and reduce the polypeptide and then to cause the polypeptide to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art (See, Debinski et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al. (1992) Anal. Biochem., 205: 263-270).
One of skill would recognize that modifications can be made to the fusion proteins without diminishing their biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids placed on either terminus to create conveniently located restriction sites or termination codons.
As indicated above, in various embodiments a peptide linker/spacer is used to join the one or more targeting moieties to one or more effector(s). In various embodiments the peptide linker is relatively short, typically less than about 10 amino acids, preferably less than about 8 amino acids and more preferably about 3 to about 5 amino acids. Suitable illustrative linkers include, but are not limited to PSGSP ((SEQ ID NO:1932), ASASA (SEQ ID NO: 1933), or GGG (SEQ ID NO: 1934). In certain embodiments longer linkers such as (GGGGS)₃(SEQ ID NO:1935) can be used. Illustrative peptide linkers and other linkers are shown in Table 11.

TABLE 11

Illustrative peptide and non-peptide linkers.

	SEQ
Linker	ID NO:

AAA	1936

GGG	1937

SGG	1938

GGSGGS	1939

SAT	1940

PYP	1941

PSPSP	1942

ASA	1943

ASASA	1944

PSPSP	1945

KKKK	1946

RRRR	1947

(Gly₄Ser)₃	1948

GGGG	1954

GGGGS	1955

GGGGS GGGGS	1956

GGGGS GGGGS GGGGS GGGGS	1957

GGGGS GGGGS GGGGS GGGGS GGGGS	1958

GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS	1959

2-nitrobenzene or O-nitrobenzyl

Nitropyridyl disulfide

Dioleoylphosphatidylethanolamine (DOPE)

S-acetylmercaptosuccinic acid

1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetracetic acid (DOTA)

β-glucuronide and β-glucuronide variants

Poly(alkylacrylic acid)

Benzene-based linkers (for example: 2,5-
Bis(hexyloxy)-1,4-bis[2,5-bis(hexyloxy)-
4-formyl-phenylenevinylene]benzene)
and like molecules

Disulfide linkages

Poly(amidoamine) or like dendrimers
linking multiple target and killing peptides
in one molecule

Carbon nanotubes

Hydrazone and hydrazone variant linkers

PEG of any chain length

Succinate, formate, acetate butyrate,
other like organic acids

Aldols, alcohols, or enols

Peroxides

alkane or alkene groups of any chain length

One or more porphyrin or dye molecules
containing free amide and carboxylic acid
groups

One or more DNA or RNA nucleotides,
including polyamine and polycarboxyl-
containing variants

Inulin, sucrose, glucose, or other single,
di or polysaccharides

Linoleic acid or other polyunsaturated
fatty acids

Variants of any of the above linkers
containing halogen or thiol groups

(all amino-acid-based linkers could be L, D, combinations of L and D forms, β-form, PEG backbone, and the like)

Multiple Targeting Moieties and/or Effectors.
As indicated above, in certain embodiments, the chimeric moieties described herein comprise multiple targeting moieties attached to a single effector or multiple effectors attached to a single targeting moiety, or multiple targeting moieties attached to multiple effectors.
Where the chimeric construct is a fusion protein this is easily accomplished by providing multiple domains that are targeting domains attached to one or more effector domains. FIG. 12 schematically illustrates a few, but not all, configurations. In various embodiments the multiple targeting domains and/or multiple effector domains can be attached to each other directly or can be separated by linkers (e.g., amino acid or peptide linkers as described above).
When the chimeric construct is a chemical conjugate linear or branched configurations (e.g., as illustrated in FIG. 12) are readily produced by using branched or multifunctional linkers and/or a plurality of different linkers.

III. Administration and Formulations.

Pharmaceutical Formulations.
In certain embodiments, the antimicrobial peptides and/or the chimeric constructs (e.g., targeting moieties attached to antimicrobial peptide(s), targeting moieties attached to detectable label(s), etc.) are administered to a mammal in need thereof, to a cell, to a tissue, to a composition (e.g., a food, etc.). In various embodiments the compositions can be administered to detect and/or locate, and/or quantify the presence of particular microorganisms, microorganism populations, biofilms comprising particular microorganisms, and the like. In various embodiments the compositions can be administered to inhibit particular microorganisms, microorganism populations, biofilms comprising particular microorganisms, and the like.
These active agents (antimicrobial peptides and/or chimeric moieties) can be administered in the “native” form or, if desired, in the form of salts, esters, amides, prodrugs, derivatives, and the like, provided the salt, ester, amide, prodrug or derivative is suitable pharmacologically, i.e., effective in the present method(s). Salts, esters, amides, prodrugs and other derivatives of the active agents can be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th Ed. N.Y. Wiley-Interscience.
Methods of formulating such derivatives are known to those of skill in the art. For example, the disulfide salts of a number of delivery agents are described in PCT Publication WO 00/059863 which is incorporated herein by reference. Similarly, acid salts of therapeutic peptides, peptoids, or other mimetics, and can be prepared from the free base using conventional methodology that typically involves reaction with a suitable acid. Generally, the base form of the drug is dissolved in a polar organic solvent such as methanol or ethanol and the acid is added thereto. The resulting salt either precipitates or can be brought out of solution by addition of a less polar solvent. Suitable acids for preparing acid addition salts include, but are not limited to both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. An acid addition salt can be reconverted to the free base by treatment with a suitable base. Certain particularly preferred acid addition salts of the active agents herein include halide salts, such as may be prepared using hydrochloric or hydrobromic acids. Conversely, preparation of basic salts of the active agents of this invention are prepared in a similar manner using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or the like. In certain embodiments basic salts include alkali metal salts, e.g., the sodium salt, and copper salts.
For the preparation of salt forms of basic drugs, the pKa of the counterion is preferably at least about 2 pH lower than the pKa of the drug. Similarly, for the preparation of salt forms of acidic drugs, the pKa of the counterion is preferably at least about 2 pH higher than the pKa of the drug. This permits the counterion to bring the solution's pH to a level lower than the pHmax to reach the salt plateau, at which the solubility of salt prevails over the solubility of free acid or base. The generalized rule of difference in pKa units of the ionizable group in the active pharmaceutical ingredient (API) and in the acid or base is meant to make the proton transfer energetically favorable. When the pKa of the API and counterion are not significantly different, a solid complex may form but may rapidly disproportionate (i.e., break down into the individual entities of drug and counterion) in an aqueous environment.
Preferably, the counterion is a pharmaceutically acceptable counterion. Suitable anionic salt forms include, but are not limited to acetate, benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate (embonate), phosphate and diphosphate, salicylate and disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide, valerate, and the like, while suitable cationic salt forms include, but are not limited to aluminum, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, zinc, and the like.
In various embodiments preparation of esters typically involves functionalization of hydroxyl and/or carboxyl groups that are present within the molecular structure of the active agent. In certain embodiments, the esters are typically acyl-substituted derivatives of free alcohol groups, i.e., moieties that are derived from carboxylic acids of the formula RCOOH where R is alky, and preferably is lower alkyl. Esters can be reconverted to the free acids, if desired, by using conventional hydrogenolysis or hydrolysis procedures.
Amides can also be prepared using techniques known to those skilled in the art or described in the pertinent literature. For example, amides may be prepared from esters, using suitable amine reactants, or they may be prepared from an anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine.
In various embodiments, the active agents identified herein are useful for parenteral, topical, oral, nasal (or otherwise inhaled), rectal, or local administration, such as by aerosol or transdermally, for detection and/or quantification, and or localization, and/or prophylactic and/or therapeutic treatment of infection (e.g., microbial infection). The compositions can be administered in a variety of unit dosage forms depending upon the method of administration. Suitable unit dosage forms, include, but are not limited to powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectibles, implantable sustained-release formulations, lipid complexes, etc.
The active agents (e.g., antimicrobial peptides and/or chimeric constructs) described herein can also be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition. Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s). Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, protection and uptake enhancers such as lipids, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers.
Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s). Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, protection and uptake enhancers such as lipids, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers.
Other physiologically acceptable compounds, particularly of use in the preparation of tablets, capsules, gel caps, and the like include, but are not limited to binders, diluent/fillers, disentegrants, lubricants, suspending agents, and the like.
In certain embodiments, to manufacture an oral dosage form (e.g., a tablet), an excipient (e.g., lactose, sucrose, starch, mannitol, etc.), an optional disintegrator (e.g. calcium carbonate, carboxymethylcellulose calcium, sodium starch glycollate, crospovidone etc.), a binder (e.g. alpha-starch, gum arabic, microcrystalline cellulose, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose, cyclodextrin, etc.), and an optional lubricant (e.g., talc, magnesium stearate, polyethylene glycol 6000, etc.), for instance, are added to the active component or components (e.g., active) and the resulting composition is compressed. Where necessary the compressed product is coated, e.g., known methods for masking the taste or for enteric dissolution or sustained release. Suitable coating materials include, but are not limited to ethyl-cellulose, hydroxymethylcellulose, polyoxyethylene glycol, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, and Eudragit (Rohm & Haas, Germany; methacrylic-acrylic copolymer).
Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives that are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art would appreciate that the choice of pharmaceutically acceptable carrier(s), including a physiologically acceptable compound depends, for example, on the route of administration of the active agent(s) and on the particular physio-chemical characteristics of the active agent(s).
In certain embodiments the excipients are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well-known sterilization techniques. For various oral dosage form excipients such as tablets and capsules sterility is not required. The USP/NF standard is usually sufficient.
In certain therapeutic applications, the compositions of this invention are administered, e.g., topically administered or administered to the oral or nasal cavity, to a patient suffering from infection or at risk for infection or prophylactically to prevent dental caries or other pathologies of the teeth or oral mucosa characterized by microbial infection in an amount sufficient to prevent and/or cure and/or at least partially prevent or arrest the disease and/or its complications. An amount adequate to accomplish this is defined as a “therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the active agents of the formulations of this invention to effectively treat (ameliorate one or more symptoms in) the patient.
The concentration of active agent(s) can vary widely, and will be selected primarily based on activity of the active ingredient(s), body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Concentrations, however, will typically be selected to provide dosages ranging from about 0.1 or 1 mg/kg/day to about 50 mg/kg/day and sometimes higher. Typical dosages range from about 3 mg/kg/day to about 3.5 mg/kg/day, preferably from about 3.5 mg/kg/day to about 7.2 mg/kg/day, more preferably from about 7.2 mg/kg/day to about 11.0 mg/kg/day, and most preferably from about 11.0 mg/kg/day to about 15.0 mg/kg/day. In certain preferred embodiments, dosages range from about 10 mg/kg/day to about 50 mg/kg/day. In certain embodiments, dosages range from about 20 mg to about 50 mg given orally twice daily. It will be appreciated that such dosages may be varied to optimize a therapeutic and/or phophylactic regimen in a particular subject or group of subjects.
In certain embodiments, the active agents of this invention are administered to the oral cavity. This is readily accomplished by the use of lozenges, aersol sprays, mouthwash, coated swabs, and the like.
In certain embodiments, the active agent(s) of this invention are administered topically, e.g., to the skin surface, to a topical lesion or wound, to a surgical site, and the like.
In certain embodiments the active agents of this invention are administered systemically (e.g., orally, or as an injectable) in accordance with standard methods well known to those of skill in the art. In other preferred embodiments, the agents, can also be delivered through the skin using conventional transdermal drug delivery systems, i.e., transdermal “patches” wherein the active agent(s) are typically contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the drug composition is typically contained in a layer, or “reservoir,” underlying an upper backing layer. It will be appreciated that the term “reservoir” in this context refers to a quantity of “active ingredient(s)” that is ultimately available for delivery to the surface of the skin. Thus, for example, the “reservoir” may include the active ingredient(s) in an adhesive on a backing layer of the patch, or in any of a variety of different matrix formulations known to those of skill in the art. The patch may contain a single reservoir, or it may contain multiple reservoirs.
In one embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. Alternatively, the drug-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form. The backing layer in these laminates, which serves as the upper surface of the device, preferably functions as a primary structural element of the “patch” and provides the device with much of its flexibility. The material selected for the backing layer is preferably substantially impermeable to the active agent(s) and any other materials that are present.
Other formulations for topical delivery include, but are not limited to, ointments, gels, sprays, fluids, and creams. Ointments are semisolid preparations that are typically based on petrolatum or other petroleum derivatives. Creams containing the selected active agent are typically viscous liquid or semisolid emulsions, often either oil-in-water or water-in-oil. Cream bases are typically water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also sometimes called the “internal” phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant. The specific ointment or cream base to be used, as will be appreciated by those skilled in the art, is one that will provide for optimum drug delivery. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing.
As indicated above, various buccal, and sublingual formulations are also contemplated.
In certain embodiments, one or more active agents of the present invention can be provided as a “concentrate”, e.g., in a storage container (e.g., in a premeasured volume) ready for dilution, or in a soluble capsule ready for addition to a volume of water, alcohol, hydrogen peroxide, or other diluent.
While the invention is described with respect to use in humans, it is also suitable for animal, e.g., veterinary use. Thus certain preferred organisms include, but are not limited to humans, non-human primates, canines, equines, felines, porcines, ungulates, largomorphs, and the like.
The foregoing formulations and administration methods are intended to be illustrative and not limiting. It will be appreciated that, using the teaching provided herein, other suitable formulations and modes of administration can be readily devised.
Home Health Care Product Formulations.
In certain embodiments, one or more of the antimicrobial peptides (AMPs) and/or chimeric moieties described herein are incorporated into healthcare formulations, e.g., for home use. Such formulations include, but are not limited to toothpaste, mouthwash, tooth whitening strips or solutions, contact lens storage, wetting, or cleaning solutions, dental floss, toothpicks, toothbrush bristles, oral sprays, oral lozenges, nasal sprays, aerosolizers for oral and/or nasal application, wound dressings (e.g., bandages), and the like.
The formulation of such health products is well known to those of skill, and the antimicrobial peptides and/or chimeric constructs are simply added to such formulations in an effective dose (e.g., a prophylactic dose to inhibit dental carie formation, etc.).
For example, toothpaste formulations are well known to those of skill in the art. Typically such formulations are mixtures of abrasives and surfactants; anticaries agents, such as fluoride; tartar control ingredients, such as tetrasodium pyrophosphate and methyl vinyl ether/maleic anhydride copolymer; pH buffers; humectants, to prevent dry-out and increase the pleasant mouth feel; and binders, to provide consistency and shape (see, e.g., Table 12). Binders keep the solid phase properly suspended in the liquid phase to prevent separation of the liquid phase out of the toothpaste. They also provide body to the dentifrice, especially after extrusion from the tube onto the toothbrush.

TABLE 12

Typical components of toothpaste.

	Ingredients	Wt %

	Humectants	40-70
	Water	0-50
	Buffers/salts/tartar	0.5-10
	control
	Organic thickeners	0.4-2
	(gums)
	Inorganic thickeners	0-12
	Abrasives	10-50
	Actives (e.g., triclosan)	0.2-1.5
	Surfactants	0.5-2
	Flavor and sweetener	0.8-1.5

	Fluoride sources provide 1000-15000 ppm fluorine.

Table 13 lists typical ingredients used in formulations; the final combination will depend on factors such as ingredient compatibility and cost, local customs, and desired benefits and quality to be delivered in the product. It will be recognized that one or more antimicrobial peptides and/or chimeric constructs described herein can simply be added to such formulations or used in place of one or more of the other ingredients.

TABLE 13

List of typical ingredients

					Tartar
	Inorganic				Control
Gums	Thickeners	Abrasives	Surfactants	Humectants	Ingredient

Sodium	Silica	Hydrated	Sodium	Glycerine	Tetrasodium
carboxymethyl	thickeners	silica	lauryl sulfate		pyrophosphate
cellulose
Cellulose ethers	Sodium	Dicalcium	Sodium N-	Sorbitol	Gantrez S-70
	aluminum	phosphate	lauryl
	silicates	digydrate	sarcosinate
Xanthan Gum	Clays	Calcium	Pluronics	Propylene	Sodium tri-
		carbonate		glycol	polyphosphate
Carrageenans		Sodium		Xylitol
		bicarbonate
Sodium alginate		Calcium	Sodium	Polyethylene
		pyrophosphate	lauryl	glycol
			sulfoacetate
Carbopols		Alumina

One illustrative formulation described in U.S. Pat. No. 6,113,887 comprises (1) a water-soluble bactericide selected from the group consisting of pyridinium compounds, quaternary ammonium compounds and biguanide compounds in an amount of 0.001% to 5.0% by weight, based on the total weight of the composition; (2) a cationically-modified hydroxyethylcellulose having an average molecular weight of 1,000,000 or higher in the hydroxyethylcellulose portion thereof and having a cationization degree of 0.05 to 0.5 mol/glucose in an amount of 0.5% to 5.0% by weight, based on the total weight of the composition; (3) a surfactant selected from the group consisting of polyoxyethylene polyoxypropylene block copolymers and alkylolamide compounds in an amount of 0.5% to 13% by weight, based on the total weight of the composition; and (4) a polishing agent of the non-silica type in an amount of 5% to 50% by weight, based on the total weight of the composition. In certain embodiments, the antimicrobial peptide(s) and/or chimeric construct(s) described herein can be used in place of the bactericide or in combination with the bactericide.
Similarly, mouthwash formulations are also well known to those of skill in the art. Thus, for example, mouthwashes containing sodium fluoride are disclosed in U.S. Pat. Nos. 2,913,373, 3,975,514, and 4,548,809, and in US Patent Publications US 2003/0124068 A1, US 2007/0154410 A1, and the like. Mouthwashes containing various alkali metal compounds are also known: sodium benzoate (WO 9409752); alkali metal hypohalite (US 20020114851A1); chlorine dioxide (CN 1222345); alkali metal phosphate (US 2001/0002252 A1, US 2003/0007937 A1); hydrogen sulfate/carbonate (JP 8113519); cetylpyridium chloride (CPC) (see, e.g., U.S. Pat. No. 6,117,417, U.S. Pat. No. 5,948,390, and JP 2004051511). Mouthwashes containing higher alcohol (see, e.g., US 2002/0064505 A1, US 2003/0175216 A1); hydrogen peroxide (see, e.g., CN 1385145); CO₂gas bubbles (see, e.g., JP 1275521 and JP 2157215) are also known. In certain embodiments, these and other mouthwash formulations can further comprise one or more of the AMPs or compound AMPs of this invention.
Contact lens storage, wetting, or cleaning solutions, dental floss, toothpicks, toothbrush bristles, oral sprays, oral lozenges, nasal sprays, and aerosolizers for oral and/or nasal application, and the like are also well known to those of skill in the art and can readily be adapted to incorporate one or more antimicrobial peptide(s) and/or chimeric construct(s) described herein.
The foregoing home healthcare formulations and/or devices are meant to be illustrative and not limiting. Using teaching provided herein, the antimicrobial peptide(s) and/or chimeric construct(s) described herein can readily be incorporated into other products.

IV. Kits.

In another embodiment this invention provides kits for the inhibition of an infection and/or for the treatment and/or prevention of dental caries in a mammal. The kits typically comprise a container containing one or more of the active agents (i.e., the antimicrobial peptide(s) and/or chimeric construct(s)) described herein. In certain embodiments the active agent(s) can be provided in a unit dosage formulation (e.g., suppository, tablet, caplet, patch, etc.) and/or may be optionally combined with one or more pharmaceutically acceptable excipients.
In certain embodiments the kits comprise one or more of the home healthcare product formulations described herein (e.g., toothpaste, mouthwash, tooth whitening strips or solutions, contact lens storage, wetting, or cleaning solutions, dental floss, toothpicks, toothbrush bristles, oral sprays, oral lozenges, nasal sprays, aerosolizers for oral and/or nasal application, and the like).
In certain embodiments kits are provided for detecting and/or locating and/or quantifying certain target microorganisms and/or cells or tissues comprising certain target microorganisms, and/or prosthesis bearing certain target microorganisms, and/or biofilms comprising certain target microorganisms. In various embodiments these kits typically comprise a chimeric moiety comprising a targeting moiety and a detectable label as described herein and/or a targeting moiety attached to an affinity tag for use in a pretargeting strategy as described herein.
In addition, the kits optionally include labeling and/or instructional materials providing directions (i.e., protocols) for the practice of the methods or use of the “therapeutics” or “prophylactics” or detection reagents of this invention. Certain instructional materials describe the use of one or more active agent(s) of this invention to therapeutically or prophylactically to inhibit or prevent infection and/or to inhibit the formation of dental caries. The instructional materials may also, optionally, teach preferred dosages/therapeutic regiment, counter indications and the like.
While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1

Design and Activity of a “Dual-Targeted” Antimicrobial Peptide

Numerous reports have indicated the important role of human normal flora in the prevention of microbial pathogenesis and disease. Evidence suggests that infections at mucosal surfaces result from the outgrowth of subpopulations or clusters within a microbial community, and are not linked to one pathogenic organism alone. In order to preserve the protective normal flora while treating the majority of infective bacteria in the community, a tunable therapeutic is necessary that can discriminate between benign bystanders and multiple pathogenic organisms. Here we describe the proof-of-principle for such a multi-targeted antimicrobial: a multiple-headed specifically-targeted antimicrobial peptide (MH-STAMP). The completed MH-STAMP, M8(KH)-20, displays specific activity against targeted organisms in vitro (Pseudomonas aeruginosa and Streptococcus mutans) and can remove both species from a mixed planktonic culture with little impact against untargeted bacteria. These results demonstrate that a functional, dual-targeted molecule can be constructed from wide-spectrum antimicrobial peptide precursor.

Introduction

For nearly 30 years antimicrobial peptides (AMPs) have been rigorously investigated as alternatives to small molecule antibiotics and potential solutions to the growing crisis of antibiotic resistant bacterial infections [1, 2]. Numerous reports have characterized potential AMPs from natural sources, and a great body of work has been carried out designing “tailor-made” AMPs due to the approachable nature of solid-phase peptide synthesis (SPPS) [3, 4]. Several examples of the latter have shown remarkable activities in vitro against fungi, Gram-positive and Gram-negative bacteria, as well as some enveloped viruses [5].
Unlike small molecule antibiotics that may lose activity when their basic structures are modified even incrementally, peptides are a convenient canvas for molecular alteration. AMPs can be optimized through the incorporation of more or less hydrophobic or charged amino acids, which has been shown to affect selectivity for Gram-positive, Gram-negative or fungal membranes [6, 7]. Additionally, lysine residues can be utilized to improve AMP activity per μM. In this approach, multiple AMP chains can be attached to a single peptide scaffold through branching from lysine epsilon-amines [8, 9]. AMP activity can be specifically tuned through the attachment of a targeting peptide region, as described for a novel class of molecules, the specifically-targeted antimicrobial peptides, or STAMPs [10, 11]. These chimeric molecules can consist of functionally independent targeting and killing moieties within a linear peptide sequence. A pathogenic bacterium recognized (i.e. bound) by the targeting peptide can be eliminated from a multi-species community with little impact to bystander normal flora. As an extension of this concept, we hypothesized that a STAMP could be constructed with multiple targeting peptide “heads” attached to a single AMP by utilizing a central lysine residue branch point. Potentially, targeting “heads” could be specific for the same pathogen, or have different binding profiles. Utilizing the former approach, microbial resistance evolution linked to a targeting peptide could be inhibited or reduced, as no single microbial population would have the genetic diversity necessary to mutate multiple discrete targeting peptide receptors in one cell [12].
Multi-headed STAMP (MH-STAMP) molecules with differing bacterial targets may have appeal in treating poly-microbial infections, or where it may be advantageous to remove a cluster of biofilm constituents without utilizing several distinct molecules; for example in the simultaneously treatment of dental caries and periodontitis, or in the eradication of the Propionibacteria spp. and Staphylococcus spp. involved in acne and skin infections, respectively.
In this example, we present the proof-of-principle design, synthesis and in vitro activity of such a MH-STAMP, M8(KH)-20. Previously, we identified two functional STAMP targeting domains, one with specific recognition of the cariogenic pathogen S. mutans [10], and the other with Pseudomonas spp.-level selectivity [13]. Conjoined to a normally wide-spectrum linear AMP, we observed antimicrobial effects directed specifically to P. aeruginosa and S. mutans in vitro. Additionally, treatment of mixed bacterial communities with the multi-headed MH-STAMP resulted in the specific eradication of the target organisms with little impact on bystander population levels.

Materials and Methods

Bacterial Strains and Growth Conditions
P. aeruginosa ATCC 15692, Klebsiella pneumoniae KAY 2026 [14], Escherichia coli DH5α (pFW5, spectinomycin resistance) [15], Staphylococcus aureus Newmann [16], and Staphylococcus epidermidis ATCC 35984 were cultivated under aerobic conditions at 37° C. with vigorous shaking Aerobic Gram-negative organisms were grown in Lauri-Bertaini (LB) broth and Gram-positive bacteria in Brain-heart infusion (BHI) broth. Streptococcus mutans JM11 (spectinomycin resistant, UA140 background) was grown in Todd-Hewitt (TH) broth under anaerobic conditions (80% N2, 15% CO₂, 5% H2) at 37° C. [17]. All bacteria were grown overnight to an OD600 of 0.8-1.0 prior to appropriate dilution and antimicrobial testing.
Synthesis of Multi-Head STAMP Peptides
Conventional solid-phase peptide synthesis (SPPS) methodologies were utilized for the construction of all peptides shown in FIG. 13 (Symphony Synthesizer, PTI, Tucson, Ariz.). Chemicals, amino acids, and synthesis resins were purchased from Anaspec (San Jose, Calif.). BD2.20 (FIRKFLKKWLL (SEQ ID NO:1949), amidated c-terminus, mw 1491.92), an antimicrobial peptide developed in our laboratory with robust antimicrobial activity against a number of bacterial species (Table 14), served as the root sequence to which differing targeting peptides were attached: Firstly, BD2.20 was synthesized by SPPS (Rink-Amide-MBHA resin, 0.015 mmol), followed by the stepwise coupling of a functionalized alkane (NH₂(CH₂)₇COOH), and an Fmoc-protected Lys (side-chain protected with 4-methyltrityl (Mtt)) to the N-terminus. Standard SPPS methods were then employed for the step-wise addition of the S. mutans targeting peptide M8 plus a tri-Gly linker region (TFFRFLNR-GGG (SEQ ID NO:1950)) to the N-terminal of the central Lys. After assembly of Fmoc-M8-GGG-K(Mtt)-(CH₂)₇CO-BD2.20 (SEQ ID NO:1951), the Fmoc group was removed with 25% piperidine in DMF and the N-terminal was re-protected with an acetyl group with Ac₂O/DIEA (1:1, 20 molar excess) for 2 hours. The Mtt-protected amino group of the central Lys was then selectively exposed with 2% TFA in DCM (1.5 mL) for 15 minutes (three cycles of 5 min). The resulting product was reloaded into the synthesizer and the peptide sequence built from the Lys side-chain was completed with standard Fmoc SPPS methods. As shown in FIG. 13, the completed MH-STAMP M8(KH)-20 contained the side-chain peptide KH (Pseudomonas spp.-targeting, KKHRKHRKHRKH-GGG (SEQ ID NO:1952)), while in MH-STAMP M8(BL)-20 a peptide with no bacterial binding (data not shown), BL-1 (DAANEA-GGG), was utilized. BL(KH)-20 was constructed identically to M8(KH)-20, utilizing BL-1 in place of M8 (FIG. 13).

TABLE 14

MICs of MH-STAMPs and component peptides.

MIC (μM)

	P. aeruginosa	E. coli	K. pneumoniae	S. mutans	S. epidermidis	S. aureus

BD2.20	14.4 ± 4.40	5.47 ± 1.41	2.98 ± 0.47	2.86 ± 0.60	5.11 ± 1.58	5.625 ± 1.29
M8(KH)-	11.95 ± 3.32	2.72 ± 0.59	3.13	6.25	3.13	5.64 ± 1.07
20
M8(BL)-	50	5.97 ± 0.94	6.88 ± 1.98	6.25	6.25	18.05 ± 6.58
20
BL(KH)-	27.5 ± 7.90	6.25	6.25	6.25	6.25	6.25
20

Average MIC with standard deviation, n = 10 assays.

Synthesis progression was monitored by the ninhydrin test, and completed peptides cleaved from the resin with 95% TFA utilizing appropriate scavengers, and precipitated in methyl tert-butyl ether. Purification and MH-STAMP quality was confirmed by HPLC (Waters, Milford, Mass.) using a linear gradient of increasing mobile phase (acetonitrile 10 to 90% in water with 0.1% TFA) and a Waters XBridge BEH 130 C18 column (4.6×100 mm, particle size 5 μm). Absorbance at 215 nm was utilized as the monitoring wavelength, though 260 and 280 nm were also collected. LC spectra were analyzed with MassLynx Software v.4.1 (Waters). Matrix-assisted laser desorption ionization (MALDI) mass spectroscopy was utilized to confirm correct peptide mass (Voyager System 4291, Applied Biosystems) [18].
MIC Assay
Peptides were evaluated for basic antimicrobial activity by broth microdilution, as described previously [10, 11]. Briefly, 1×10⁵cfu/mL bacteria were diluted in TH (S. mutans), or Mueller-Hinton (MH) broth (all other organisms) and distributed to 96-well plates. Serially-diluted (2-fold) peptides were then added and the plates incubated at 37° C. for 18-24 h. Peptide MIC was determined as the concentration of peptide that completely inhibited organism growth when examined by eye (clear well). All experiments were conducted 10 times.
Post-Antibiotic Effect Assay
The activity and selectivity of MH-STAMPs after a 10 min incubation was determined by growth retardation experiments against targeted and untargeted bacteria in monocultures, as described previously [10, 11]. Cells from overnight cultures were diluted to ˜5×106 cfu/mL in MH (or TH with 1% sucrose for S. mutans), normalized by OD600 0.05-0.1 and seeded to 96-well plates. Cultures were then grown under the appropriate conditions for 2 h (3 h for S. mutans) prior to the addition of peptides for 10 min. Plates were then centrifuged at 3000×g for 5 min, the supernatants discarded, fresh medium returned (MH or TH without sucrose for S. mutans), and incubation resumed. Bacterial growth after treatment was then monitored over time by OD600.
Microbial Population Shift Assay
Mixed planktonic populations of P. aeruginosa, E. coli, S. epidermidis, and S. mutans were utilized to examine the potential of MH-STAMPs to direct species composition within a culture after treatment. Samples were prepared containing: ˜6×10⁴cfu/mL S. mutans, ˜2×10⁴cfu/mL E. coli, ˜2×10⁴cfu/mL S. epidermidis, and ˜0.5×10⁴cfu/mL P. aeruginosa in BHI (mixed immediately before peptide addition). Peptide (10 μM) or mock-treatment (1×PBS) was then added and samples were incubated at 37° C. for 24 h under anaerobic conditions (80% N2, 15% CO₂, 5% H2). After incubation, samples were serially diluted (1:10) in 1×PBS and aliquots from each dilution were then spotted to agar plates selective for each species in the mixture: TH plus 800 μg/mL spectinomycin (S. mutans), LB plus 25 μg/mL ampicillin (P. aeruginosa), LB plus 200 μg/mL spectinomycin (E. coli), and mannitol salt agar (MSA, S. epidermidis) in order to quantitate survivors from each species. Plates were then incubated 37° C. under aerobic conditions (TH plates were incubated anaerobically) and colonies counted after 24 h to determine survivors. Expected colony morphologies were observed for each species when plated on selective media. Gram stains and direct microscopic observation (from select isolated colonies) were undertaken to confirm species identity (data not shown). The detection limit of the assay was 200 cfu/mL.

Results

Design and Synthesis of Multi-Headed STAMPs
We constructed a prototype MH-STAMP from the well-established targeting peptides KH (specific to Pseudomonas spp) and M8 (specific for Streptococcus mutans). The wide-spectrum antimicrobial peptide BD2.20 was utilized as the base AMP for all MH171 STAMP construction. BD2.20 is a novel synthetic AMP with a cationic and amphipathic residue arrangement, which has robust MICs against a variety of Gram-negative and Gram-positive organisms (Table 14). For the synthesis of MH-STMAP M8(KH)-20 (construct presented in FIG. 13), BD2.20 and a Lys (Mtt-protected side-chain) residue were joined via an activated alkane spacer, followed by addition of the M8 targeting peptide to the N-terminus of the product. Selective deprotection of the central Lys(Mtt) side chain was then undertaken and the KH targeting peptide attached. The correct molecular mass (4888.79) and ˜90% purity was confirmed by HPLC and MALDI mass spectrometry (FIG. 14).
The non-binding “blank” targeting peptide BL-1 was incorporated into the synthesis scheme in place of KH or M8 to construct variant MH-STAMPs possessing a single functional targeting head: M8(BL)-20 and BL(KH)-20. The correct MW and acceptable purity were observed for these MH-STAMPs (FIG. 13, data not shown).
General Antimicrobial Activity of Multi-Head Constructs
After synthesis, the completed MH-STAMPs were evaluated for general antimicrobial activity by MIC against a panel of bacteria. As shown in Table 14, the MH-STAMP constructs M8(KH)-20, BL(KH)-20, and M8(BL)-20 were found to have similar activity profiles to that of BD2.20 for the organisms examined (less than two titration steps in 10-fold difference). Additionally, we observed a difference in general susceptibility between P. aeruginosa and the other organisms tested, suggesting this bacterium is more resistant to BD2.20. Overall, these data indicate that the addition of the targeting domains to the base sequence was tolerated and did not completely inhibit the activity of the antimicrobial peptide.
Peptide selectivity could not be determined utilizing these methods, as STAMPs and their parent AMP molecules often display similar MICs, but have radically different antimicrobial kinetics and selectivity due to increased specific-killing mediated by the targeting regions [10, 11]. Therefore, we performed different experiments to test for antimicrobial selectivity and functional MH-STAMP construction.
3.3 Selectivity and Post-Antibiotic Effect of MH-STAMP Constructs
MH-STAMP antimicrobial kinetics was ascertained utilizing a variation of the classical post-antibiotic effect assay, which measures the ability of an agent to affect an organism's growth after a short exposure period. Monocultures of MH-STAMP-targeted and untargeted organisms were exposed to M8(KH)-20, M8(BL)-20, BL(KH)-20, or unmodified BD2.20, then allowed to recover. As shown in FIG. 15A, S. mutans growth was effectively retarded by M8-containing constructs (M8(KH)-20, M8(BL)-20), but was not altered by a MH-STAMP construct lacking this region (BL(KH)-20). Similarly, the growth of the other targeted bacterium, P. aeruginosa, was inhibited in a KH-dependant manner (FIG. 15B). In comparison, the non-targeted bacteria E. coli, S. aureus, and S. epidermidis were not inhibited by treatment with any MH-STAMP and were only inhibited by the base antimicrobial peptide BD2.20, which displayed robust antimicrobial activity against all examined strains. These results indicate that MH-STAMPs containing KH or M8 targeting domains have activity against P. aeruginosa or S. mutans, respectively, and not other bacteria. Furthermore, replacement of the targeting region with a non-binding peptide abolishes specific activity.
Ability of MH-STAMPs to Direct a “Population Shift” within a Mixed Species Population
We hypothesized that potential MH-STAMP dual-functionality could affect a particular set of bacteria within a mixed population, thereby promoting the outgrowth of non-targeted organisms and “shifting” the constituent makeup. To examine this possibility, defined mixed populations of planktonic cells were treated continuously and the make-up of the community examined after 24 h. As shown in FIG. 16, treatment with the wide spectrum AMP BD2.20 resulted in a significant loss of recoverable cfu/mL after 24 h from all species in the mixture. Treatment with M8(KH)-20 was found to alter this pattern; we observed ˜1×10⁵cfu/mL surviving E. coli and S. epidermidis, but did not recover S. mutans or P. aeruginosa cfu/mL. In BL(KH)-20 treated samples, P. aeruginosa cfu/mL were not observed, though we recovered higher than input cfu/mL from S. mutans and unchanged numbers of S. epidermidis and E. coli. In samples exposed to M8(BL)-20, S. mutans recoverable cfu/mL were greatly reduced compared to input cfu/mL, while other species were not affected or affected to a lesser extent. Interestingly, these results suggest that M8(KH)-20, M8(BL)-20, and BL(KH)-20 retain their ability to affect organisms recognized by the targeting regions present, even within a mixed population of bacteria.

DISCUSSION

Our results indicate that we have successfully constructed a STAMP with dual antimicrobial specificities controlled by the targeting peptides present in the molecule; KH for Pseudomonas spp, M8 for S. mutans. In a closed multi-species system (FIG. 16), the dual specificity of M8(KH)-20 was readily discernable: the population of the culture “shifted” away from targeted organisms after MH-STAMP treatment. The targeted bacteria were eliminated and the population of untargeted organisms increased, to varying degrees, above-input cfu/mL. Additionally, interruption of KH or M8 in the MH-STAMP construct with the non-binding peptide BL-1 resulted in the expected elimination of only one targeted species. These results support the hypothesis that functional MH-STAMPs could be constructed from a wide-spectrum AMP base.
The emergence of metagenomics and the development of more sensitive molecular diagnostics has driven an increase in the understanding of human-associated microbial ecologies and host-microbe interactions [19-21]. At mucosal surfaces, it has become clear that our bodies harbor an abundance of residential flora which may impact innate and humoral immunity, nutrient availability, protection against pathogens, and even host physiology [22-25]. Furthermore, findings have indicated that shifts in the diversity of normal flora are associated with negative clinical consequences; for example the overgrowth of S. mutans in the oral cavity during cariogenesis (linked to the uptake of sucrose) or the antibiotic-assisted colonization of the intestine by Clostridium difficle [26, 27]. Other population shifts may be linked to axilla odor (Corynebacteria spp) [28, 29], or even host obesity. Given the quantity and diversity of microbes present, pathogenesis at mucosal surfaces is not likely to be associated with the overgrowth of a single strain or species. More often, it is a population shift resulting in the predominance of two or more species; for example the persistence of Burkholderia cepacia and P. aeruginosa in cystic fibrosis airway or Treponema denticola and Porphymonas gingivalis and other “red cluster” organisms in gingivitis [30, 31]. In many cases (such as the latter) these species may have only distant phylogenetic relationships and display differential susceptibilities to antibiotic therapies resulting in persistent disease progression despite treatment [32, 33]. Currently, available treatments for infections of mucosal surfaces are largely non-specific (traditional small-molecule antibiotics, mechanical removal), and thus are not effective in retaining flora or shifting the constituent balance back to a health-associated composition [34]. There is a need for a therapeutic treatment that can selectively target multiple pathogens, regardless of their phylogenetic relationship, and MH-STAMPs can help achieve this goal.
In monoculture experiments (FIG. 15), our results suggest that M8 or KH inclusion in the MH-STAMP drove activity towards S. mutans or P. aeruginosa, but also that the presence of a targeting domain reduced the activity of the parent AMP BD2.20 against untargeted organisms. In contrast, the results of our MIC assays (Table 14) indicate little difference in activity between BD2.20 and any MH-STAMP. Against untargeted organisms, the M8 and KH regions are likely to have a negative, but not completely inhibitory, impact on BD2.20 activity. Given the long duration of activity and the lower inoculum size in the MIC assay (compared with experiments in FIG. 15), it is likely that all BD2.20-containing peptides could reach equal levels of growth inhibition, despite large and target-specific differences in antimicrobial speed. This pattern of results was also observed when comparing MICs of targeted and untargeted organisms utilizing STAMPs against S. mutans and Pseudomonas mendocina [10, 11].
Although more rigorous studies and a more medically relevant combination of pathogen targets is desirable, these findings indicate that it is possible to design an antimicrobial peptide-based therapeutic with multiple and defined fidelities in vitro. MH-STAMPs may help improve human health through the promotion of healthy microbial constituencies.

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11. Eckert R, Qi F, Yarbrough D K, He J, Anderson M H, Shi W. Adding selectivity to antimicrobial peptides: rational design of a multidomain peptide against Pseudomonas spp. Antimicrob Agents Chemother 2006; 50:1480-8.
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13. Eckert R, Brady K M, Greenberg E P, Qi F, Yarbrough D K, He J, et al. Enhancement of Antimicrobial Activity against Pseudomonas aeruginosa by Coadministration of G10KHc and Tobramycin Antimicrob Agents Chemother 2006; 50:3833-8.
14. Sprenger G A, Lengeler J W. L-Sorbose metabolism in Klebsiella pneumoniae and Sor+ derivatives of Escherichia coli K-12 and chemotaxis toward sorbose. J Bacteriol 1984; 157:39-45.
15. Podbielski A, Spellerberg B, Woischnik M, Pohl B, Lütticken R. Novel series of plasmid vectors for gene inactivation and expression analysis in group A streptococci (GAS). Gene 1996; 177:137-47.
16. Duthie E S, Lorenz L L. Staphylococcal coagulase; mode of action and antigenicity. J Gen Microbiol 1952; 6:95-107.
17. Merritt J, Kreth J, Qi F, Sullivan R, Shi W. Non-disruptive, real-time analyses of the metabolic status and viability of Streptococcus mutans cells in response to antimicrobial treatments. J Microbiol Methods 2005; 61:161-70.
18. Anderson R C, Rehders M, Yu P L. Antimicrobial fragments of the pro-region of cathelicidins and other immune peptides. Biotechnol Lett 2008; 30:813-8.
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22. Metges C C. Contribution of Microbial Amino Acids to Amino Acid Homeostasis of the Host. J Nutr 2000; 130:1857 S-64.
23. Sears C L. A dynamic partnership: Celebrating our gut flora. Anaerobe 2005; 11:247-251.
24. Lievin-Le Moal V, Servin A L. The Front Line of Enteric Host Defense against Unwelcome Intrusion of Harmful Microorganisms: Mucins, Antimicrobial Peptides, and Microbiota Clin Microbiol Rev 2006; 19:315-37.
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Example 2

Synthesis of Peptide-Porphyrin Conjugate

The mixture of coupling reagent HATU (5 eq. excess, 10 mg) and purpurin-18 (MW 564, 5 eq excess, 15 mg) in 600 mL dry dichloromethane (DCM):DMF:dimethylsulphoxide (DMSO) (1:1:1 (v/v)) was added to the peptide resin (1 molar equivalent, 15 mg) which was swelled by placing in minimal DMF for 30 min prior to reaction. 26 μL (10 molar equivalents) DIPEA was then added to the reaction flask to initiate the reaction. The reaction mixture was protected with argon and stirred at room temperature for 3 h.
After finishing, the reaction mixture was then passed down a sintered glass filtered vial and extensively washed with DMF and DCM to remove all waste reagents. The resin was then dried overnight in vacuum, and cleaved with 1 ml of trifluoroacetic acid (TFA)/thioanisole/water/EDT (10/0.5/0.5/025) for 2 hr at room temperature, and the cleavage solution was precipitated with 10 mL methyl-tert butyl ether. The precipitate was washed twice with the same amount of ether.

Example 3

Synthesis of Peptide-CSA Conjugate

To the fully protected peptide (solution of B43-GGG (FIDSFIRSF-GGG, 0.025 mmol) and tri-Boc-CSA-15 (0.0125 mol) in 300 μL DMF, DCC (7.7 mg), HOBt (5.1 mg) and 13 μL DIEA were added in iced-bath. After stirred at room temperature for four days, the reaction mixture was poured into 5 ml water and extracted with chloroform (5×3 mL). The CHCl₃extract was evaporated under vacuum and dried in a lyophilizer overnight. The dried CHCl₃extracts was then dissolved in 1 mL DCM followed by added 1 mL of TFA in iced-bath. The reaction mixture was further stirred at room temperature for 2 hours and precipitated with methyl tert-butyl ether (10 mL). The precipitate was further washed once with the same amount ether and dried in vacuum.

Example 4

Systemically Designed STAMPS Against S. Mutans

We previously reported a novel strategy of “targeted-killing” with the design of narrow-spectrum molecules known as specifically-targeted antimicrobial peptides (STAMPs). Construction of these molecules requires the identification and the subsequent utilization of two conjoined yet functionally independent peptide components: the targeting and killing regions. In this study, we sought to design and synthesize a large number of STAMPs targeting Streptococcus mutans, the primary etiological agent of human dental caries, in order to identify candidate peptides with increased killing speed and selectivity when compared with their unmodified antimicrobial peptides (AMP) precursors. We hypothesized that a combinatorial approach utilizing a set number of AMP, targeting, and linker regions, would be an effective method for the identification of STAMPs with the desired level of activity. STAMPs composed of the S. mutans binding peptide 2_—1 and the AMP PL-135 displayed selectivity at minimal inhibitory concentrations after incubation for 18-24 h. STAMPs where PL-135 was replaced by the B-33 killing domain exhibited both selectivity and rapid killing within one minute of exposure. These results suggest that potent and selective STAMP molecules can be designed and improved via a tunable “building-block” approach.

Introduction

Pathogenic microorganisms have been a continuous source of human suffering and mortality throughout the course of human history and have spurred the clinical development of novel therapeutics. Even today, the overall burden of infectious disease remains high, constituting a leading (and rising) cause of death worldwide (12, 13). The conventional medical response to bacterial infections, small molecule antibiotics, have become less effective against emerging pathogens due to the evolution of drug-resistance stemming in part from the misuse of antibiotics (11). To counter the rapid progression of antibiotic resistance there is an urgent need for the development of novel lead compounds for clinical applications.
Our strategy for creating new antibacterial agents is based on the addition of a targeting peptide to an existing broad-spectrum antimicrobial peptide, thereby generating a specifically-targeted antimicrobial peptide (STAMP) selective for a particular bacterial species or strain. A completed STAMP consists of conjoined but functionally independent targeting and killing regions, separated by a small flexible linker, all within a linear peptide sequence. The STAMP targeting region drives enhancement of antimicrobial activity by increasing binding to the surface of a targeted pathogen, utilizing specific determinants such as overall membrane hydrophobicity and charge, pheromone receptors, etc., which in turn leads to increased selective accumulation of the killing moiety (6, 7).
As both the killing and targeting regions of the STAMP are linear peptides, we approached the design process using a tunable combinatorial methodology where, for example, the targeting peptide component is held constant, while a number of killing peptides are conjoined utilizing a variety of linker molecules, or vise versa, in order to generate a library of related STAMPs. Previously, we successfully demonstrated a pilot version of this approach when constructing G10KHc (6), a STAMP with Pseudomonas-spp selective activity, and when designing C16G2 (7), a STAMP specific for Streptococcus mutans, the leading causative agent of human tooth decay. In this study, synthetic targeting and antimicrobial peptide libraries were utilized as building blocks to generate a number of novel STAMPs with high S. mutans-selective activity. STAMPs designed by these methods were then improved through tuning the linker and killing peptides present to yield completed lead STAMP molecules that demonstrated activity against S. mutans biofilms.

Materials and Methods

Reagents.
Wang resin, Rink-MBHA resin, p-Benzyloxybenzyl alcohol resin (100-200 mesh), 9-fluorenylmethoxycarbonyl (Fmoc) amino acids, N-hydroxybenzotriazole hydrate (HOBT) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were obtained from Anaspec (San Jose, Calif.). All other solvents and reagents were purchased from Fisher Scientific (Pittsburgh, Pa.) at HPLC or peptide synthesis grade.
Bacterial Growth
All S. mutans (UA159) (1), Streptococcus gordonii Challis (DL 1), Streptococcus sobrinus ATCC 33478, and Streptococcus sanguinis NY101 strains were grown in Brain Heart Infusion (BHI) medium at 37° C. under anaerobic conditions (80% N₂, 10% CO₂, 10% H₂) (6). Pseudomonas aeruginosa (PAK) (17), and Escherichia coli W3110 (20), were cultured in Luria-Bertani (LB) medium under an aerobic atmosphere at 37° C. Methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus faecium (VRE) were grown in BHI under aerobic conditions at 37° C. (4).
Peptide Syntheses and Purification.
Peptides were synthesized using standard solid phase (Fmoc) chemistry with an Apex 396 peptide synthesizer (AAPPTec, Louisville, Ky.) at a 0.01 mM scale. N-terminal deblocking during linear peptide synthesis was conducted with 0.6 mL of 25% (v/v) piperidine in dimethylformamide (DMF), followed by agitation for 27 min and wash cycles of dichlormethane DCM (1×1 mL) and NMP (7×0.8 mL). Subsequent amino acid coupling cycles were conducted with a mixture of Fmoc-protected amino acid (5 eq), HOBT (5 eq), HBTU (5 eq), DIEA (10 eq) in DMF (0.1 mL), and NMP (0.2 mL) with agitation for 45 min. The washing cycle was repeated before the next round of deprotection and coupling. After synthesis, peptides were washed in methanol and dried 24 h. Protected peptide was cleaved with 1 mL of trifluoroacetic acid (TFA)/thioanisole/water/1,2-ethanedithiol (10/0.5/0.5/025) for 3 h at room temperature and the resultant peptide solution was precipitated in methyl tert-butyl ether.
Analytical and preparative HPLC was conducted, as described previously (5, 9), to purify each peptide to 80-90%. Correct peptide mass was confirmed by matrix-assisted laser desorption/ionization mass spectroscopy (MALDI, Voyager instrument, Applied Biosystems), as described previously (9). Measurements were made in linear, positive ion mode with an α-cyano-4-hydroxycinnamic acid matrix (data not shown).
Acetylated and benzolated peptides derivatives (Ac135(L1)2_—1 and bz135(L1)2_—1, respectively) were synthesized by the same method used for N-terminal fluorescent labeling, described previously (7). Briefly, acetylation (acetic anhydrous) or benzyl addition (benzoatic acid) was undertaken after assembly of the linear sequence by adding to the resin (10 eq in DIEA) for 2 h.
Binding of Targeting Peptides. The binding of peptides 1_—2, 1_—3, 1_—4, 1_—6, 2_—1, and 3_—1 was assessed by fluorescent microscopy. S. mutans UA159 was grown overnight and diluted 1:5000 in fresh TH with 1% sucrose before seeding to 96-well plates (flat bottom). Biofilms were grown 24 h and the spent medium replaced with 1×PBS containing 25 μM peptide. After 5 min incubation at room temperature, the supernatants were removed and the biofilms washed 2× with 1×PBS and imaged by bright-field and fluorescence microscopy (Nikon E400). Digital images were collected and analyzed for semiquantitative binding assessments with the manufacturers supplied software (SPOT, Diagnostics) (6).
Minimum Inhibitory Concentrations (MICS) of Peptides.
Peptide MIC was determined by broth microdilution (6, 15). Briefly, two-fold serial dilutions of each peptide were prepared with 50% BHI medium+50% sterile water (for oral streptococci; all other bacteria were diluted in 1× Mueller-Hinton broth) at a volume of 100 μL per well in 96-well flat-bottom microtiter plates. The concentration of peptide for the first test round ranged from 500 to 2 μg/mL or 125 to 1 μg/mL. If activity was detected, a second round of MIC tests was conducted with concentrations of 62.5 to 0.5 μg/mL. In either case, the microtiter plate was inoculated with a bacterial cell suspension to a final concentration of ˜1×10⁵cells per mL and the plates were incubated at 37° C. for 16-20 h under the appropriate conditions. After incubation, absorbance at 600 nm (A₆₀₀) was measured using a microplate UV-Vis spectrophotometer (Model 3550, BioRad, Hercules, Calif.) to assess cell growth. The MIC endpoint was calculated as the lowest concentration of antibacterial agent that completely inhibited growth or that produced at least 90% reduction in turbidity when compared with that of a peptide-free control. At least 10 independent tests were conducted per peptide. For peptides insoluble in aqueous solutions, stock solutions were prepared in methanol or ethanol and appropriate solvent controls were utilized. Cell growth was not affected by 5% (v/v) methanol or ethanol, as described previously (10).
Peptide Killing Kinetics.
To determine antimicrobial kinetics and specificity, assays similar to traditional time-kill experiments were performed, as described previously (6, 7). Briefly, overnight bacterial cultures were diluted in BHI to A₆₀₀0.08 and peptides were added as indicated. Aliquots were then removed at various intervals and diluted 1:50 in BHI and kept on ice until plating on appropriate growth medium. After 24 hours incubation, colonies were counted and the surviving cfu/mL determined. All assays were repeated at least 3 times and the average recovered cfu/mL were presented with standard deviations. Statistical analysis was conducted utilizing an unpaired Student's t-test.
Biofilm Growth Inhibitory Assays.
STAMPs were tested for anti-biofilm activity as described previously (6). Briefly, overnight cultures of S. mutans were diluted 1:50 in Todd-Hewitt (TH) broth medium supplemented with 0.5% sucrose and 100 μL of bacterial suspension was added to each well of a 96-well microtiter plate. After centrifugation, bacteria were then incubated under anaerobic conditions at 37° C. for 4 h. Supernatants were then removed and replaced with 25 μM peptide in 1×PBS for 30 s to 1 min, followed by removal, washing, and replacement with 100 μL fresh TH broth (without sucrose). Plates were then incubated at 37° C. under anaerobic conditions and the bacterial recovery monitored by recording A₆₀₀after 4 h incubation. An unpaired Student's t-test was utilized for statistical analysis.

Results

STAMPs consist of 3 regions: one targeting and one antimicrobial, connected via a flexible linker. In this report, we conjoined examples of each domain with a variety of linkers to construct a pool of initial STAMP candidates. These peptides were then evaluated for anti-S. mutans activity and selectivity, their designs improved, and the lead STAMPs evaluated against S. mutans biofilms.
Selection of Components and Initial STAMP Library Design.
As described elsewhere, we generated several novel S. mutans-specific binding peptides, including 2_—4, (previously S3L1-10 (FIKDFIERF)) and 1_—5, (previously S3L1-5 (WWYNWWQDW)) (7). From these base sequences, residues differing in hydrophobicity and/or charge were substituted at defined positions to yield a series of related targeting sequences that were then evaluated for binding to S. mutans biofilms. Several of the 1_—5 variants, and one of the 2_—4 variants, were found to retain biofilm binding (data not shown). These sequences (1_—2; 1_—3; 1_—4; 1_—6; 2_—1; 3_—1), as well as 1_—5, were regarded in the present study as the pool of S. mutans targeting peptides for STAMP construction (shown in Table 15). For the antimicrobial component, we selected PL-135, a peptide based on an AMP isolated from tunicates (19), for the initial round of design (Library 1). We hypothesized that linker regions and attachment orientation would exert an influence on STAMP activity. Therefore, we initially conjugated each potential targeting peptide to the N or C terminus of PL-135 through six different linkers, as shown in Table 15 (GGG, designated L1; SAT, L3; ASASA, L5; PYP, L7; PSGSP, L8; PSPSP, L9) leading to the synthesis of 84 STAMPs.

TABLE 15

STAMP constituent regions utilized
in STAMP Library 1.

S. mutans targeting
peptides	Linker

No.	Sequence	peptides (name)	Killing peptide

1_2	WWHSWWSTW	GGG (L1)	FHFHLHF* (PL-135)

1_3	WWSYWWTQW	SAT (L3)

1_4	WWKDWWERW	ASASA (L5)

1_5	WWYNWWQDW	PYP (L7)

1_6	WWQDWWNEW	PSGSP (L8)

2_1	FIKHFIHRF	PSPSP (L9)

3_1	LIKHILHRL

*amidated C-terminus

Antimicrobial Activity of Initial STAMP Library.
To roughly gauge Library 1 STAMP antimicrobial activity and S. mutans-selectivity, MIC (minimal inhibitory concentration) assays were conducted against S. mutans and a panel of bacteria, including two oral streptococci, S. sanguinis and S. sobrinus (FIG. 17). STAMPs containing 2_—1 conjoined to the C-terminus of PL-135 (135(L1)2_—1, 135(L3)2_—1, 135(L5)2_—1, 135(L7)2_—1, 135(L8)2_—1, 135(L9)2_—1), or 3_—1 conjoined to the N-terminus of PL-135 (135(L1)3_—1) were found to be active against S. mutans at concentrations lower than 100 μg/mL. These peptides were more active (2-4 2-fold dilution steps) against S. mutans than against the other oral streptococci or non-oral organisms tested. In contrast, native PL-135 had similar MICs against all strains examined (FIG. 17).
Impact of Linker and Terminal Modification on STAMP Antimicrobial Activity.
We sought to investigate the impact of linker length or type, and N-terminal modification on the activity of STAMP 135(L1)2_—1 from Library 1. As shown in Table 16, altered components included: 1) new linkers GGGG (G₄) to GGGGGGG (G₇), AAA (L2), AGA (L10), GAGAG (L11), 8-aminocaprylic acid (LC); 2) increased N-terminal aromacity (benzoic acid addition); 3) acetylation of N-terminus; or 4) component sequence inversion (135i(L1)2_—1i). We observed MICs for these Library 2 STAMPs that were similar to, or 2-fold less-active, than that of the base STAMP 135(L1)2_—1 (FIG. 17), suggesting that the linker, termini and inversion alterations did not lead to improved activity against S. mutans, as measured by these means.

TABLE 16

Minimal inhibitory concentration of (MIC)
of Library 2 STAMPs

No.	Sequence^a	S. mutans (MIC)^b

135(L1)2_1	FHFHLHFGGGFIKHFIHRF	16

135(G₄)2_1	FHFHLHFGGGGFIKHFIHRF	16

135(G₅)2_1	FHFHLHFGGGGGFIKHFIHRF	16

135(G₆)2_1	FHFHLHFGGGGGGFIKHFIHRF	32

135(G₇)2_1	FHFHLHFGGGGGGGFIKHFIHRF	32

135(L11)2_1	FHFHLHFSGSFIKHFIHRF	32

135(L12)2_1	FHFHLHFGSGSGFIKHFIHRF	32

Ac135(L1)2_1	Ac-FHFHLHFGGGFIKHFIHRF	32

bz135(L1)2_1	benzoate-FHFHLHFGGGFIKHFIHRF	32

135(L2)2_1	FHFHLHFAAAFIKHFIHRF	16

135(L13)2_1	FHFHLHFAGAFIKHFIHRF	32

135(L14)2_1	FHFHLHFGAGAGFIKHFIHRF	32

2_1i(L1)135i	FRHIFHKIFGGGFHLHFHF		16

135i(L1)2_1i	FHLHFHFGGGFRHIFHKIF		16

135(LC)2_1	FHFHLHF-[NH(CH₂)₇CO]-FIKHFIHRF	64

^aall C termini amidated
^bMIC (μg/mL) data from all S. mutans isolates tested, with a minimum of three independent trials.

Further Potential of PL-135-Based STAMPs.
Antiseptic mouthrinses, such as Listerine® (Warner-Lambert, Morris Plains, N.J.), are rapid-acting non-selective bactericidal agents that can inactivate viable bacteria within seconds of contact (3). In order for STAMPs to be useful mouthrinse ingredients, the antimicrobial kinetics must approach this scale. Therefore, the killing kinetics of our Library 1 and 2 STAMPs from Table 15 and FIG. 17 were evaluated (data not shown). The results indicate that these PL-135-containing STAMPs, although selective for S. mutans when measured by MIC, are not rapid killers of this bacterium in vitro, requiring several hours of exposure for observable antimicrobial activity. Therefore, we sought to improve our STAMP pool by substituting alternative killing domains for S. mutans STAMP construction.
Tuning the Design of 2_—1-Containing STAMPs.
To improve the identification of rapid-killing S. mutans STAMPs, we conjugated 2_—1 with five AMPs selected from our previous studies (10), to construct Library 3: RWRWRWF (2c-4), FKKFWKWFRRF (B-33), IKQLLHFFQRF (B-38), RWRRLLKKLHHLLH (α-11), LQLLKQLLKLLKQF (a-7); attached at the C- or N-terminus. The linkers selected were L1, SGG (L2), L3 and LC. As shown in FIG. 18, MIC results indicate little difference in activity between constructs where the targeting peptide was attached to the N or C terminus of the AMP region, and little difference between linkers employed. It was also apparent that these STAMPs were more active against S. mutans relative to the other oral and non-oral bacteria tested: peptide 2_—1(L1)B33 demonstrated the lowest MIC range of 4 to 8 μg/ml, which was a 2-4 fold improvement over the MIC for the killing peptide alone (10). Taken together, these data suggest that Library 3 STAMPs can effectively inhibit the growth of S. mutans at generally-improved potencies when compared to PL-135-containing STAMPs in Libraries 1 and 2.
STAMP Killing Kinetics Against Oral Bacteria.
Since the MIC assay measures antimicrobial activity after overnight incubation, large differences in killing rates between STAMPs and parental AMPs may be obscured in this assay, especially when the target organism is susceptible to the AMP (7, 9). To assess any significant selectivity and short-term antimicrobial activity of the Library 3 STAMPs, time-kill assays were performed against a variety of oral bacteria. Against the targeted bacterium S. mutans (examples shown in FIG. 19D), the STAMPs acted significantly faster than the killing peptide alone within 5 min of treatment (p<0.001 comparing B-33 alone vs. 2_—1(L1)B33, or 2C-4 vs. either 2C-4-containing STAMP). In contrast, other oral streptococci, such as S. mitis and S. gordonii, were less affected by STAMP treatments (FIG. 19A-C). Peptide 2_—1(L1)B33 exhibited the fastest killing kinetics and best selectivity: killing was observed even when cells were treated for as little as 30 s, a timescale more appropriate for oral cavity therapeutic applications. As expected from their wide-spectra of activities (10), parental AMPs 2C-4 and B-33 had similar levels of activity against the strains examined.
Inhibition of Biofilm Growth.
Although rapid and selective killing of S. mutans monocultures was apparent from the data shown in FIG. 19, we sought to determine whether these STAMPs would make suitable antimicrobial agents in the oral cavity, where multi-species biofilms known as dental plaque predominate (16, 18). To investigate, S. mutans biofilms were treated with STAMPs and the post-antibiotic effect was observed after 4 h of biofilm recovery. As shown in FIG. 20, STAMPs 2_—1(L1)2C-4, 2_—1(L6)₂C-4, and 2_—1(L1)B33 were found to significantly inhibit (p<0.001) the formation of biofilms when cells were treated with the peptide for 1 min at 25 μg/ml, compared to mock-treated biofilms or biofilms treated with untargeted AMP. Similar antimicrobial effects were observed for Listerine and Chlorhexidine. These results suggest that STAMP treatment persists after peptide removal at a level similar to established wide-spectrum oral antiseptics.

DISCUSSION AND CONCLUSION

In this report, we present a novel strategy for the design and synthesis of STAMPs with activity against the oral pathogen S. mutans. Successful design was achieved through a tunable, building-block approach that utilized various combinations of antimicrobial, targeting, and linker STAMP peptide regions. Our results demonstrate that less-efficacious STAMPs could be improved when alternative killing regions were substituted in the design. While it remains unclear whether the alternative moieties were more active, or simply more conjugation tolerant, this process resulted in STAMPs that displayed killing kinetics against biofilms consistent with oral therapeutic applications. Additionally, more understanding was gained regarding AMP or targeting peptide orientation dependencies, impact of linker regions, and appropriate targeting peptide choice, which could positively impact future anti-S. mutans STAMP design and refinement.
From the data presented here, it is difficult to determine the precise mechanism for the S. mutans-selectivity. Previous studies with STAMPs have indicated that the enhanced selectivity for the targeted strain is due to the binding of the targeting peptide moiety (6, 7). Although detailed binding analysis was not conducted in this study, our results suggest that a similar targeting-mediated killing is occurring here: targeting peptides, independently selected for STAMP construction on the basis of their S. mutans-binding abilities, were required to enhance AMP antimicrobial activity and selectivity.
The data presented suggest that PL-135 may be inhibited by conjugation to other peptide subunits, as unmodified PL-135 displayed activity against S. mutans that was 2 to 4-fold higher than in progeny STAMPs, as shown in FIG. 17 and Table 16 (however, these constructs were selective for S. mutans). The unusually small size of this AMP may impart a severe restriction on amino acid additions, especially if the mode of action depends on sequence-dependent self-association on the target-cell membrane, or binding to a discrete intracellular bacterial target (2). Our results suggest that AMPs with quicker “base” antimicrobial kinetics (such as B-33 or 2C-4), and higher tolerance for conjugations, should be selected for the design of STAMPs with optimal levels of enhanced killing kinetics and selectivity.
Outside of PL-135-containing examples (and potentially some α-7 and α-11 molecules, see FIG. 18), STAMPs in this report displayed no difference in activity between oppositely-oriented N and C-terminal AMP-targeting conjugations, suggesting that the optimal arrangement of STAMP domains in likely AMP-specific, and depends on which least affects the antimicrobial mechanism. For example, the Pseudomonas spp-specific STAMPs G10KHc and G10KHn (oriented target-killing and killing-target, respectively) both bind specifically to the target bacterium surface, but only G10KHc has significant membrane disruption activity (5, 7).
Interestingly, 2_—1 and 3_—1 containing STAMPs were active against S. mutans, and the constructs with any other targeting peptide in Table 15, were not. Targeting peptides 1_—2 through 1_—6 are strongly hydrophobic, compared with 2_—1 and 3_—1 (8), and it may be possible that this characteristic limits the dissociation of these molecules from the hydrophobic components of the S. mutans cell wall, resulting in their inhibitory affect on AMPs when conjugated, similarly to some strong LPS-binding AMPs (14). However, the systematic building-block design strategy employed allowed us to generate a diverse array of STAMPs, allowing us to identify useful compounds despite these stumbling blocks.
In conclusion, this report details the rational design of S. mutans-selective STAMPs with enhanced antimicrobial killing kinetics and selectivity when compared to untargeted AMPs. The S. mutans-selective STAMPs were constructed using a tunable, combinatorial approach that generated a diverse number of STAMP sequences for antimicrobial evaluation and improvement; a process may serve as an example for the systematic development of novel selective antimicrobial agents. We propose that these STAMPs could be useful in the design of therapeutics against oral or other mucosal pathogens, where the high diversity of “probiotic” beneficial microflora limits the effectiveness of broad-spectrum antimicrobial agents.

REFERENCES

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It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A chimeric moiety, said moiety comprising:

an effector attached to a peptide targeting moiety the amino acid sequence of said targeting moiety comprising the sequence GGSGSLSTFFRLFNRSFTQALGK (SEQ ID NO:60).

2. The chimeric moiety of claim 1, wherein the amino acid sequence of said targeting moiety is GGSGSLSTFFRLFNRSFTQALGK (SEQ ID NO:60).

3. The chimeric moiety of claim 1, wherein said effector comprises a moiety selected from the group consisting of a detectable label, an antimicrobial peptide, an antibiotic, and a photosensitizer.

4. The chimeric moiety of claim 1, wherein said effector comprises an antimicrobial peptide comprising the amino acid sequence of a peptide found in Table 2, Table 8, Table 9, or Table 10 or any antimicrobial peptide disclosed herein.

5. The chimeric moiety of claim 1, wherein said effector comprises an antibiotic found in Table 7.

6-14. (canceled)

15. The chimeric moiety of claim 1, wherein said targeting moiety is chemically conjugated to said effector.

16. The chimeric moiety of claim 15, wherein said targeting moiety is chemically conjugated to said effector via a linker.

17. The chimeric moiety of claim 15, wherein said targeting moiety is chemically conjugated to said effector via a linker comprising a polyethylene glycol (PEG).

18. The chimeric moiety of claim 15, wherein said targeting moiety is chemically conjugated to said effector via a non-peptide linker found in Table 11.

19. The chimeric moiety of claim 1, wherein said targeting moiety is linked directly to said effector.

20. The chimeric moiety of claim 1, wherein said targeting moiety is linked to said effector via a peptide linkage.

21. The chimeric moiety of claim 20, wherein the chimeric moiety is a fusion protein.

22. The chimeric moiety of claim 21, wherein said peptide linkage comprises a peptide linker found in Table 11.

23. The chimeric moiety of claim 20, wherein said chimeric moiety is functionalized with a polymer to increase serum halflife.

24. The chimeric moiety of claim 21, wherein said polymer comprises polyethylene glycol and/or a cellulose or modified cellulose.

25. A pharmaceutical composition comprising a chimeric moiety of claim 1 in a pharmaceutically acceptable carrier.

26. The composition of claim 25, wherein said composition is formulated as a unit dosage formulation.

27. The composition of claim 25, wherein said composition is formulated for administration by a modality selected from the group consisting of intraperitoneal administration, topical administration, oral administration, inhalation administration, transdermal administration, subdermal depot administration, and rectal administration.

28-45. (canceled)

46. The chimeric moiety of claim 1, wherein said effector comprises a photosensitizing agent.

47. (canceled)

48. The chimeric moiety of claim 46, wherein said photosensitizing agent is an agent selected from the group consisting of a porphyrinic macrocycle, a porphyrin, a chlorine, a crown ether, an acridine, an azine, a phthalocyanine, a cyanine, a psoralen, and a perylenequinonoid.

49. The chimeric moiety of claim 46, wherein said photosensitizing agent is an agent shown in any of FIGS. 1-11.

50. The chimeric moiety of claim 46, wherein said photosensitizing agent is attached to said targeting peptide by a non-peptide linker.

51. The chimeric moiety of claim 46, wherein said photosensitizing agent is attached to said targeting peptide by a linker comprising a polyethylene glycol (PEG).

52. The chimeric moiety of claim 46, wherein said photosensitizing agent is attached to said targeting peptide by a non-peptide linker found in Table 11.

53-58. (canceled)