WO1991019512A1 - Peptides antimicrobiens - Google Patents

Peptides antimicrobiens Download PDF

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Publication number
WO1991019512A1
WO1991019512A1 PCT/US1991/004414 US9104414W WO9119512A1 WO 1991019512 A1 WO1991019512 A1 WO 1991019512A1 US 9104414 W US9104414 W US 9104414W WO 9119512 A1 WO9119512 A1 WO 9119512A1
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Prior art keywords
alanine
peptides
hpqynqr
oligopeptide
activity
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PCT/US1991/004414
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English (en)
Inventor
James Travis
William M. Shafer
Neelesh Bangalore
Jan Pohl
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University Of Georgia Research Foundation, Inc.
Emory University
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Publication of WO1991019512A1 publication Critical patent/WO1991019512A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the field of this invention is the area of antimicrobial peptides with activity against a broad range of Gram-negative and Gram-positive bacteria and fungi.
  • the antimicrobial peptides of this invention are useful in pharmaceutical compositions for treatment or prevention of infections and for the treatment and/or prevention of gingivitis.
  • Microbes which invade the human body are challenged by several defense mechanisms.
  • the nature of the defense mechanisms which any given microbe faces depends on the genetic makeup and the physiologic state of the host as well as the portal of entry of the invading microorganism.
  • Host defenses include mechanical factors and chemical factors. Mechanical factors which help protect epithelial surfaces include the washing action of bodily fluids including tears, saliva and urine, trapping on mucous layers, removal by cilia and elimination by coughing, sneezing or desquamation. A further mechanical defense is offered by the physical integrity of the skin, although mucus membranes can be penetrated by some pathogens.
  • Chemical defense factors include the acidity of gastric secretions, unsaturated fatty acids on the skin which kill certain bacterial species, lysozyme in tears, saliva and nasal secretions, iron-binding proteins at the mucosal surface, transferrin in serum, and spermine in semen.
  • Secretions of the mucous membranes also contain antibodies, especially those of the IgA class. Microbial antagonism between different potentially pathogenic bacteria, fungi and yeast strains occurs at the level of competition for nutrients and through the production of inhibitory substances; this antagonism affords further protection to the host.
  • immunological factors i.e., antibodies
  • transferrin which chelates available iron on which microorganisms are dependent.
  • PMNs actively phagocytize particulates such as bacterial or fungal cells.
  • PMNs are the first class of phagocytic cells recruited to the site of infection or inflammation.
  • the PMNs contain azurophilic or primary granules, which contain lysosomal proteases, myeloperoxidase and certain antimicrobial proteins as well as lysozyme. Secondary granules within these cells contain alkaline phosphatase, lactoferrin and lysozyme.
  • Stores of glycogen within the PMNs provides for energy through glycolysis so that the cell can function in an anaerobic environment.
  • Adherence of a particle to the surface of a phagocytic cell initiates phagocytosis; the particle enters the cytoplasm in a phagocytic vacuole. This triggers a respiratory burst and the generation of microbicidal metabolites and the primary granule fuses with the phagocytic vacuole to form a digestive vacuole called the phagolysosome. Intracellular killing of the ingested microorganism occurs as a result of oxygen-dependent and oxygen-independent mechanisms.
  • the oxygen-dependent bactericidal halogenating system uses granule myeloperoxidase, hydrogen peroxide and chloride ion to kill bacteria and viruses via either halogenation or cellular or viral constituents or reactive oxygen intermediates. Oxygen-dependent killing can also proceed by the direct reduction of molecular oxygen via the cytochrome b-oxidase system (reviewed by Orkin (1989) Ann. Rev. Immunol.2*277-308). Oxygen-dependent killing mechanisms are reviewed by Beaman and Beaman (1984) Ann. Rev. Microbiol. 38:27-48.
  • the primary granules contain three major groups of antibacterial proteins.
  • the first group includes catalytically active proteins which are only weakly antibacterial when tested individually in purified form. Examples from this group include lysozyme, elastase and collagenase. These enzymes probably participate in the digestion of microorganisms killed by other mechanisms, but elastase, for example, is believed to potentiate killing by the halogenating system.
  • the second category of granule proteins includes those with catalytic activity and bactericidal activity which is independent of the catalytic activity. An example is the chymotrypsin-like neutral protease of human neutrophils.
  • a third group contains members which lack known catalytic activity; such a protein class has been purified from rabbit neutrophils. Included in this class are defensins and cationic antibacterial proteins.
  • Some of the cationic antibacterial proteins are of relatively high molecular weight (greater than about 25 kDa) and kill certain Gram negative bacteria such as Escherichia coli. Salmonella tvphimurium and Pseudomonas aeru inosa by damaging the cytoplasmic membrane, leading to increased membrane permeability.
  • Human bactericidal/permeability increasing protein (BPI) is a strongly basic protein with a molecular weight of about 59 kDa. It is believed that when bound to the outer membrane of susceptible bacterial cells, hydrophobic channels through the outer envelope are exposed, and as a secondary effect, there is a selective activation of autolytic enzymes including phospholipase and peptidoglycan hydrolases. Gram positive bacteria, certain Gram negative bacteria and fungi are not affected by this protein in vitro.
  • cationic proteins with fungicidal activity have been identified in alveolar acrophages. It is believed that cationic proteins are most efficient in killing phagocytized microorganisms in combination with other microbicidal defense mechanisms.
  • the final pH of the phagolysosome is not known, it is thought to be about 6.0; it is also unknown whether the concentrations of the polyamines spermine and spermidine within the phagolysosome are bactericidal (Elsbach and Weiss (1983) supra) .
  • Cathepsin G is a granule protein with chymotrypsin-like activity; it is also known as chymotrypsin- like cationic protein.
  • Cat G (Odeberg and Olsson (1975) J.
  • defensins are active against a broad spectrum of Gram positive bacteria, Gram negative bacteria and fungi (Shafer et al. (1986) Infect. Immun. 54.:184-188; Shafer et al. (1988) Infect. Immun. 56:51-53; Drazin and Lehrer (1977) Infect. Immun. .17:382-388; Ganz et al. (1986) Semin. Respir. Infect. 1:107-117).
  • defensins are relatively small polypeptides of about 3-4 kDa, which are rich in cysteine and arginine.
  • a 4 kDa defensin (HNP-4) and a 29 kDa polypeptide named azurocidin were purified and shown to possess broad spectrum antimicrobial activity.
  • Defensins as a class have activity against some bacteria, fungi and viruses. They are also reported to have cytotoxic activity against transformed cells. Selsted et al. (1985) J. Clin. Invest. 26:1436-1439, presents a sequence comparison of human and rabbit defensins. The defensins are believed to have molecular conformations, stabilized by cystine infrastructure, which are essential for biological activity.
  • Granzymes are a family of serine proteases in the granules of cytoly ic lymphocytes. Proteolytic enzymes are believed to function xn cell-mediated cytoxicity; some of the genes have been cloned, and sequence information is available. Within the granzyme family there is at least 38% amino acid sequence identity. Human lymphocyte protease has 73% amino acid sequence identity to mouse granzyme B (Jenne and Tschopp (1988) Immunol. Reviews 103:53-71) .
  • Gabay et al. (1989) supra. has reported antibacterial activity of a number of proteins isolated from human PMNs, including cathepsin G and elastase, and has given the amino terminal sequence of those and other proteins.
  • the N-terminal five amino acids of elastase and Cat G are identical; further sequences have significant homology.
  • Another class of antimicrobial polypeptides are those Icnown as magainins; at least five proteins can be isolated from the skin of the African clawed frog (Xenopus laevis) .
  • the natural proteins are active against a broad range of microorganisms including bacteria, fungi and protozoans (Zasloff (1987) Proc. Natl. Acad. Sci. USA 84:5449-5453) .
  • the broad spectrum antimicrobial activity is present in synthetic peptides and in certain truncated analogs of the natural proteins. Derivatives of about 19 to about 23 amino acids have antibacterial activity as measured using Escherichia coli.
  • the configuration of the bioactive peptides can be modeled as an amphophilic alpha-helix and are sufficiently long to span a lipid bilayer. (Zasloff et al. (1988) Proc, Natl. Acad. Sci. USA j$5.:910-913) . Spanning a lipid bilayer is believed to require at least 20 amino acid residues in an alpha-helical configuration (Kaiser and Kennedy (1987) Ann. Rev. Biophys. Chem. 15:562-581) .
  • the sequence of a representative magainin peptide is GIGKFLHSAKKFKAFVGEIMN (Zasloff et al. (1988) supra) .
  • the antimicrobial peptides of the present invention are comprised of from about five to about twenty amino acids joined in a linear array by peptide bonds.
  • the peptides comprise the amino acid sequences IIGGR, IVGGR and HPQYNQR.
  • the peptides of the sequences IIGGR and HPQYNQR are particularly preferred.
  • a consensus sequence has been formulated: HX 1 X 2 X 3 X 4 X 5 X 6 , where X 1 is proline, histidine or alanine; X 2 is aspartic acid, asparagine, glutamic acid, glutamine, alanine, serine, threonine, isoleucine, valine, histidine, tyrosine, arginine, methionine oxide or methionine sulfone; X 3 is tyrosine, phenylalanine, tryptophan or beta-naphthyl-alanine; X 4 is asparagine or alanine; X 5 is glutamine, proline, N-methyl alanine, or alanine; and X 6 is
  • X. is proline, alanine or_histidine?_ X-, is one of asparagine, aspartic acid, glutamine, glutamic acid or alanine; X 3 is tyrosine or phenylanine; X 4 is asparagine or alanine; X 5 is proline, glutamine, alanine or N-methyl alanine; and X 6 is lysine, arginine, alanine, -OH or -NH 2 .
  • a peptide fitting the consensus sequence preferably has at position 3 (as X 2 above) a nonbulky, hydrophilic amino acid capable of hydrogen bonding, glutamine or proline at position 6 (as X 5 above) and lysine or arginine at position 7 (as X 6 above) .
  • amino acid 3 is glutamine, alanine, glutamate, asparagine, or aspartate.
  • Antimicrobial peptide sequences whose sequences fall within the consensus sequence include, but are not limited to, HPQYNQR, HPAYNPK, HPAYNPR and HPAYNQR.
  • HPQYNQR Additional antimicrobial peptide sequences related to HPQYNQR include, but are not limited to, HPQYAQR, HPQYNQA, HPQYNAR, HPAYNPR, HAQYNQR, HHQYNQR and HPQYNQ and RHPQYNQR. These peptides possess microbicidal activity for Gram positive and Gram negative bacteria.
  • a further object of the invention is to provide therapeutic compositions, suitable for human, veterinary, agricultural or pharmaceutical use, comprising one or more of the antimicrobial peptides of the present invention and a suitable pharmacological carrier.
  • Such therapeutic compositions can be formulated as understood in the art, e.g., for topical or aerosol application, for controlling and/or preventing infection by Gram positive or Gram negative bacteria, fungi or protozoans.
  • the antimicrobial peptides of the present invention are used in the treatment of infections by Gram-negative or Gram-positive bacteria.
  • the antimicrobial peptides of the present invention when used in therapeutic compositions, will not have significant immunogenic activity.
  • compositions contain a therapeutically effective amount of an antimicrobial peptide.
  • a therapeutically effective amount of an antimicrobial peptide can be readily determined according to methods Icnown in the art.
  • Pharmaceutical compositions are formulated to contain the therapeutically effective amount of an antimicrobial peptide and a pharmaceutically acceptable carrier appropriate for the route of administration (topical, gingival, intravenous, aerosol, local injection) as known to the art.
  • the composition comprises a therapeutically effective amount of an antimicrobial peptide and an agriculturally acceptable carrier suitable for the organism (e.g., plant) to be treated.
  • the antimicrobial peptide will have an ED 50 in vitro less than about 10 " ⁇ .
  • the skilled artisan can readily determine a therapeutically effective amount against a target bacterial strain, for example, based on the ED 50 using the methods disclosed herein and the teachings of the art.
  • compositions may be administered by topical, dental rinse, aerosol or intravenous application, or by local injection for the control or prevention of infection or control of tumor cell growth, by any means known to the art.
  • the IIGGR-related antimicrobial peptides of the present invention may also be used to kill or control the growth of tumor cells or virus-infected cells.
  • these peptides will be particularly useful when coupled to antibodies or other molecules which are specific for the target tumor cell or virus-infected cell so that the peptide acts specifically on the tumor or virus-infected cell.
  • Figure 1 illustrates the bactericidal activity of clostripain digests of human neutrophil cathepsin G and elastase. Proteinases were digested as described in the
  • Each data point represents the mean of three samples with ⁇ 5% variance between each.
  • Figure 2 shows Reverse Phase-HPLC (RP-HPLC) fractionation of cathepsin G peptides obtained from clostripain fragmentation.
  • Low Mr peptides obtained by gel filtration chromatography were lyophilized, dissolved in 250 microliters of 0.1 % (v/v) TFA and subjected to RP-HPLC as described in the Examples.
  • Peptides were eluted in several peaks (1-10) . These were further purified, separately, by RP-HPLC as described in Methods. Only those in peaks 6 and 7 were found to exert antibacterial action in vitro against __[___ aureus and __!____ gonorrhoeae.
  • Figure 2 (A) cathepsin G digest fractionation
  • Figure 2 (B) rechromatography of peak 6
  • Figure 2 (C) rechromatography of peak 7.
  • Figure 3 illustrates the bactericidal activity of synthetic peptides derived from cathepsin G. Synthetic peptides (0 - 100 ug) were tested against __ _ gonorrhoeae and S. aureus as described in the Examples. Each data point represents the mean of three determinations from three separate experiments using the same lot of synthetic peptide.
  • Figure 3 (A) peptides IIGGR vs IVGGR;
  • Figure 3 (B) peptides HPQYNQR s APQYNQR.
  • Figure 4 illustrates the pH Dependency for the bactericidal activity of HPQYNQR. Aliquots of peptide (100 ug) were assayed for antibacterial activity in incubation with either of the two test bacteria at different pH values, as described in the Examples. Each data point is the average of two determinations from three separate experiments.
  • Figure 5 compares the bactericidal capacities of Cat G synthetic peptides and certain antibiotics.
  • IjN. gonorrhoeae strain FA 102 was exposed to 5.0 x 10 "4 M of IIGGR, HPQYNQR, penicillin G, chloramphenicol, tetracycline, streptomycin, kanamycin, and a mixture of three partially purified human defensins in HBSS (Ph 7.5). After 30 min at 37°C the samples were plated onto GCB agar, incubated for 48 hr, and colonies counted. There were no gonococcal colonies after 48 hr of incubation.
  • Figure 6 illustrates the bactericidal action of Cat G synthetic peptides (HPQYNQR, Ac-HPQYNQR) (see Table 2) , and human granzyme B-derived peptide HPAYNPK) under conditions of different pH, Figure 6 (Panel A) and ionic strength.
  • Figure 6 (Panel B)
  • Figure 6 (Panel C)
  • the synthetic peptides (5xl0 "4 M) and purified, enzymatically inactive Cat G 3"5 (1.8x10 " ⁇ ), were tested against S_ s . aureus in HBSS modified to different pH (A) and ionic strengths (B) as described previously.
  • Figure 7 illustrates the antibacterial activity of increasing concentrations of HPQYNQR against Sj_ aureus 8436-5 in LB broth culture. Viable cell counts (measured as CFU/ml) were taken at intervals.
  • (- ⁇ -) is the control culture without peptide
  • (-V-) is 0.05 ⁇ g/ml
  • (- ⁇ -) is 0.25 ⁇ g/ml
  • (-0-) is 1 ⁇ g/ml HPQYNQR.
  • Figure 8 illustrates the effects of increasing concentrations of HPAYNPK in LB broth cultures on viable cell counts (measured as CFU/ml) of S_ s . aureus 8325-4 over a 24 hr period.
  • (- ⁇ -) is the control without peptide
  • (-V-) represents 0.05 ⁇ g/ml
  • (-0-) represents 1 ⁇ g/ml APQYNPK, respectively.
  • an oligopeptide is composed of from about three to about twenty amino acids linked together by peptide bonds in a linear array.
  • the peptide may be in a linear conformation or it may assume secondary structure.
  • a cyclic peptide derivative can also have antimicrobial activity, and thus is a functional equivalent of the antimicrobial peptides of the present invention. Sequences are conventionally given from the amino terminus to the carboxyl terminus.
  • the peptides of the present invention have antimicrobial activity by themselves or when coupled to another molecule so long as the peptides are positioned such that they can come into effective contact with a microorganism or target tumor or virus-infected cell.
  • Boc tert-butyloxycarbonyl
  • HLE • human leukocyte elastase
  • Antimicrobial activity refers to the ability of a compound, such as an oligopeptide of the present invention, to kill at least one species selected from the group consisting of Gram positive bacteria, Gram negative bacteria, fungi, and protozoans.
  • Sensitive Gram positive bacteria include, but are not limited to, Staphylococcus aureus. Streptococcus pneumoniae. Streptococcus pyogenes and Listeria monocytogenes Typ 4b.
  • Sensitive Gram negative bacteria include, but are not limited to, Escherichia coli, Neisseria gonorrhoeae. E. coli Neumann TP, Klebsiella sp.
  • Sensitive fungi can include, but are not limited to, Candida albicans.
  • Antimicrobial activity can also refer to the ability to kill or inhibit the growth of other cells, in particular, those which are tumor cells or virus- infected cells.
  • the antimicrobial peptides of the present invention are oligopeptides which possess antimicrobial activity as defined herein. These antimicrobial peptides may contain modifications, such as acetylation, provided that the antimicrobial activity is not destroyed. Chemical modifications which will not destroy antimicrobial activity are those which do not substantially decrease the hydrophilicity of the antimicrobial peptide and those which are not bulky hydrophobic chemical groups. Modified peptides with antimicrobial activity are functionally equivalent to the antimicrobial peptides of the present invention.
  • modified peptides with antimicrobial activity include, but are not limitedto, (l-methyl-H)QYNQR, (3-methyl-H)PQYNQR, (Ac-H)PQYNQR and HPAYNA M K.
  • Antibacterial pharmaceutical compositions comprise a pharmaceutically acceptable carrier and one or more antibacterial peptides of the present invention.
  • Such antimicrobial pharmaceutical compositions may be formulated in ways, as understood in the art, for use for topical application, for gingival application (for gingivitis or periodontal disease) or for local or systemic injection.
  • the peptides of the present invention can be incorporated in effective amounts in a dental rinse for application to the buccal area, or they may be incorporated in other suitable compositions for topical application.
  • the antibacterial peptides of the present invention may also be incorporated in effective amounts in chewing gum, lozenges for sucking, toothpowder or toothpaste.
  • the antibacterial peptides of the present invention may comprise from 0.1% to 90% of such compositions. It will be understood that a composition for systemic injection will comprise an antimicrobial peptide, e.g. an antibacterial peptide such as HPQYNQR, in a therapeutically effective amount or a therapeutically effective amount of an antimicrobial peptide can be conjugated to an antibody or any other compound as understood in the art with specificity for the target cell type.
  • an antimicrobial peptide e.g. an antibacterial peptide such as HPQYNQR
  • the Cat G protein was analyzed to determine whether the same portions of the protein were responsible for the enzymatic and antibacterial activity.
  • Human Cat G was purified and digested with the proteolytic enzyme clostripain. Peptides resulting-from that-digestion were purified and individually tested for antibacterial and enzymatic activity. None of the peptides tested exhibited the chymotrypsin-like activity of the intact molecule.
  • two Cat G-derived peptides exhibited antibacterial activity using Staphylococcus aureus or Neisseria gonorrhoeae as the indicator organism. Those peptides were IIGGR (peptide 1; amino acids 1-5) and HPQYNQR (peptide 2; amino acids 77-83) . Similar antibacterial activities were observed for synthetic peptides identical in sequence to the above-noted peptides.
  • Figure 4(A) illustrates killing of S. aureus and N_j_ gonorrhoeae by these two peptide
  • ED 50 is the concentration of an antimicrobial agent which kills or otherwise inhibits the growth 50% of the input indicator microorganism or cell under particular test conditions.
  • Antimicrobial activity of the Cat G-derived peptides was found to be dependent on assay temperature, pH and ionic strength. As for full length Cat G, effective killing of S. aureus by the synthetic peptides was optimal at 37°C with activity from about 30 to about 39 * C, and was optimal at pH values of 7.0-7.5 for the range of pH values tested (Results for HPQYNQR are shown in Figs. 4 and 6B) . The antimicrobial action of these peptides was optimal in standard HBSS (0.12 M NaCl) ; conditions of increased ionic strength (0.17-0.21 M NaCl) completely inhibited the antibacterial action (Fig. 6B) except for peptides HPAYNPK or Ac-HPQYNQR.
  • peptides IIGGR and HPQYNQR were tested for synergistic activity. Under optimal conditions of temperature, pH and ionic strength, IIGGR and HPQYNQR together were about twice as active against S ⁇ . aureus than when tested individually, but still less active than native Cat G or a clostripain digest of Cat G.
  • a synthetic peptide consisting of the peptides 1 and 2 joined by a bridge (AIR) had activity similar to that of HPQYNQR alone (ED 50 5.0 x 10 *5 M) . Thus, the peptides together were less effective against ⁇ ___.
  • IIGGR and HPQYNQR were compared with other known antimicrobial agents. As shown in Fig. 5, these peptides exhibited activities roughly comparable to those of a pool of defensins and to the clinically useful antibiotics chloramphenicol and tetracycline (The test concentration of each compound was 5.0 x 10 * ⁇ ) . For the heptapeptides, this corresponds to abc ⁇ t 100 micrograms per ml. Chloramphenicol was more effective at this concentration than the defensin pool or the test peptides. At this concentration peptides from Cat G were more active than tetracycline, streptomycin, kanamycin or the defensin pool.
  • the active peptide sequences IIGGR and HPQYNQR were used to search known protein sequences of other cytotoxic serine proteases present in the cytolytic lymphocytes of mice and humans for homologies.
  • Proteins containing the IIGGR sequence at their N-termini include Cat G and eosinophil cationic protein (Gleich et al. (1986) Proc. Natl. Acad. Sci. USA 82:3146-3150).
  • Human leukocyte elastase (Gabay et al. (1989) supra r azurocidin (Sinha et al. (1987) Proc. Natl. Acad. Sci. USA 84:2228-2232) and CAP 37 (Pereira et al.
  • IVGGR Human leukocyte elastase does not have antimicrobial activity in vitro.
  • CCCP I and human lymphocyte granzymes B, D, E and G begin with IIGGH while RMCP II begins with the sequence IIGGV
  • IIGGR of Cat G is located on the interior of the protein because it forms a salt bridge with Asp-102 in the catalytic triad (using chymotrypsin nomenclature) . This hypothesis is consistent with the x-ray crystallographic structure of chymase
  • HPAYNPK corresponding to an internal region of human leukocyte protease, possesses broad spectrum antimicrobial activity in vitro.
  • the homologous sequences of mouse granzymes A, C, E, F and G do not possess such activity (see Table 5) .
  • HPQYNQR were tested for antimicrobial activity.
  • the results of testing variant sequences of the HPQYNQR peptide sequence for antimicrobial activity has led to the formulation of a consensus sequence for an antimicrobial peptide: HX 1 X 2 X 3 X 4 X 5 X 6 .
  • X. is proline, alanine or histidine
  • X 2 is one of asparagine, aspartic acid, glutamine, glutamic acid or alanine
  • X 3 is tyrosine or phenylanine
  • X 4 is asparagine or alanine
  • X 5 is proline, glutamine, alanine or N-methyl alanine
  • X 6 is lysine, arginine, alanine, -OH or -NH 2 .
  • Antimicrobial peptide sequences whose sequences fall within the consensus sequence include, but are not limited to, HPQYNQR, HPAYNPK, HPAYNPR and HPAYNQR. Additional antimicrobial peptide sequences related to HPQYNQR include, but are not limited to, HPQYAQR, HPQYNQA, HPQYNAR, HPAYNPR, HAQYNQR HPQYNQ, HHQYNQR and HPAYNA H K.
  • Antimicrobial synthetic peptide HPQYNOR, its derivatives with single alanine substitutions at each position, and a control peptide (HPQKNTY) were synthesized on an Applied Biosystem Model 403A peptide synthesizer (0.1 mmol scale) using phenylacetamidomethy1 or p-methylbenzyhydrylamine polystyrene resins (Applied Biosystems, Inc.) and tert-butyloxycarbonyl (Boc)-protected amino acids.
  • the data are presented as % survival + SEM and represent results from at least 3 separate experiments for each peptide with S___ aureus as the indicator organism.
  • Peptides corresponding to sequences withinmouse granzymes A,B,C,D,E,F and G and human granzymes A and B were synthesized. As shown in Table 4, only the mouse granzyme-derived synthetic peptide corresponding to granzyme B had some antibacterial activity. The synthetic peptides corresponding to human granzyme B, but not A, had significant antibacterial activity.
  • the human granzyme B-derived peptide differs from the Cat G peptide at positions 3, 6 and 7. To test how these differences affected antibacterial activity, variants of the Cat G and granzyme B peptides containing some of these heterologous amino acids or with alanine substitution at position 3 or 6 were synthesized and tested (Table 5) . Placement of the Ala-3 residue in the Cat G only had a slight effect on antibacterial action (Tables 2 and 4) . Conversely placement of the Gln-3 residue in the Granzyme B peptide severely inhibited antibacterial activity. The Pro-6 residue of the granzyme B peptide also appeared to be crucial, because the replacement of Pro-6 with Alanine abolished microbicidal activity.
  • Peptide sequences for mouse granzymes D and E are identical. Human granzyme B variants are designated
  • the antibacterial action of the granzyme B-derived peptide was found to be optimal at slightly basic pH (Fig. 6a) although, like the acetylated derivative of HPQYNQR, significant antibacterial activity was observed at a pH of 6.0.
  • the antibacterial action of human granzyme B was also sensitive to increasing concentrations of NaCl in the incubation mixture.
  • the Cat G-derived peptide HPQYNQR and its acetylated derivative retained antibacterial activity even at the highest NaCl concentration (0.21 M) tested (Fig. 6B) .
  • peptides containing an N-terminal five amino acid extension (RRAIRHPQYNQR) or a C- terminal five amino acid extension (HPOYNQRTIQIND) were synthesized and tested. There was no significant change in the ED 50 relative to that of HPQYNQR.
  • HPQYNQR-related peptides containing an additional amino acid residue at the amino-terminus were compared to HPQYNQR with respect to antimicrobial activity using the agar diffusion assay.
  • L-arginine resulted in nearly full antimicrobial activity.
  • D- arginine, L-citrilline, L-nitroarginine, lysine, alanine, cysteine or glutamate at the amino-terminus caused a loss of activity (See Table 6) .
  • arginine is the amino acid preceding the HPQYNQR sequence. It is postulated that bacterial proteases may process full length Cat G, and perhaps RHPQYNQR, to yield HPQYNQR.
  • Example 1.5 Agar diffusion assay for antimicrobial activity was performed as described in Example 1.5, with each peptide dissolved at a concentration of 1 mg/ l, and with S. aureus as the indicator microorganism. Structures for D- arginine, L-citrulline and L-nitroarginine are given below.
  • Figure 7 illustrates the effects of increasing concentrations of HPQYNQR on growing cultures of S ⁇ . aureus 8325-4. Concentrations of 0.25 and 1 microgram/ml effectiely inhibited growth and killed a signigicant proportion of the viable cells.
  • Figure 8 shows the effects of increasing concentrations of HPAYNPK on S___ aureus 8325-4 cultures.
  • This peptide was less effective than the HPQYNQR peptide in the inhibition of bacterial growth, and viable cell numbers were incresing after 24 hr of incubation after the initial decline for the 1 microgram/ml treatment.
  • the input bacteria were reduced to about 4 x 10 5 / ⁇ al an to 100 ⁇ g/ml of the same peptides. These data are shown in Table 8. After 24 hr, no viable bacteria were detected in the sample containing HPQYNQR:
  • HPQYNQR appeared more effective than HPAYNPK, which ⁇ sequence is derived from human granzyme B, against all bacterial tested except the Pseudomonas Walter TP strain tested, where neither was effective.
  • HPQYNQR composed of
  • 35 D-amino acids had no detectable antimicrobial activity against any bacterium tested to date. Antimicrobial activity was also observed for HPQYNQR against Streptococcus pneumoniae. Streptococcus pyogenes. Neisseria meningitidis. Neisseria gonorrhoeae and Proteus mirabilis.
  • Porphyromonas gingivalis (formerly called Bacteroides gingivalis) was very
  • HPQYNQR was also tested against two clinical isolates of Bacteroides fragilis.
  • B. fra ⁇ ilis is a bacterium normally present in the bowel. It is often associated with bacteremia associated with ruptured appendices.
  • B_ fragilis ATCC49417 which is believed to be nonencapsulated, was sensitive to HPQYNQR.
  • B___ fragilis A7436 strain did not appear to be sensitive to HPQYNQR.
  • HPAYNPK corresponds to an amino acid sequence within Human Granzyme B.
  • D-(HPQYNQR) is composed of the D-isomers of histidine, proline, glutamine, tyrosine, asparagine and arginine. D-(HPQYNQR) had no apparent bactericidal activity, while HPQYNQR had 4-8 fold greater activity than HPAYNPR against sensitive strains.
  • MIC determination for the antibacterial peptides were repeated using peptides dissolved in distilled water which had been stored frozen at -20"C before use. MICs of the antimicrobial peptides were compared to those of ciprofloxacin (CIPRO) and ampicillin (AMPI) .
  • HPQYNQR The effects of HPQYNQR were also determined on resting S. aureus cells (suspended in PBS) .
  • Binding Protein 3 (PBP3) was correlated with a reduction in sensitivity to HPQYNQR and that presence of ⁇ -lactamase was also correlated in IL. coli with decreased sensitivity to HPQYNQR. Further experiments with other IL. coli strains suggested that the ompF porin, which mediates movement of certain compounds through the outer membrane, was involved in transport of HPQYNQR to its target, which was not established. We postulate that the target for lethal action in _£____ coli is Penicillin Binding Protein 3, which is analogous to PBP2 in N. gonorrheae.
  • PBP3 in E___ coli is a bifunctional enzyme involved in cell wall synthesis; it has transglycosylase and transpeptidase activity. Inhibition of the transpeptidase domain by Penicillin G___ in growing cells leads to cell lysis.
  • HPQYNQR mimics penicillin-like compounds by binding to an active site of _ coli PBP3. It is likely that ⁇ -lactamase may confer resistance to HPQYNQR in certain strains by competing for binding sites.
  • results of variant (HPQYNQR) peptides suggested that the N-terminal histidine and the internal tyrosine residues were important in generating antimicrobial activity.
  • a substitution of alanine for histidine, D-histidine for the L-isomer or the attachment of a 2, 4-dinitrophenyl group to the imidazole group of L-histidine resulted in a loss of activity.
  • a substitution of alanine for tyrosine at position 4 led to a loss of microbicidal activity (see Table 4) .
  • HPQYNQR was modified by the addition of an acetyl or a heptanoyl group to the N- terminal amino group. Acetylation did not affect activity, while the heptanoyl derivative lacked antimicrobial activity.
  • the antibacterial activity of the HLP-derived peptide cannot be readily explained using the Cat G sequence (HPQYNQR) as a canonical model.
  • the alanine residue at position 3 and the proline at position 6 of the HLP-derived peptide are not conservative amino acid changes, although the lysine for arginine at position 7 is a conservative change.
  • the ability of a position 7 substitution variant (alanine for arginine) of the Cat G sequence to retain significant killing activity might suggest that this is not a key position in determining activity.
  • An alanine substitution at position 3 decreased activity of the Cat G-related peptide.
  • Cat G and human neutrophil elastase were purified from extracts of human PMN granules as described previously (Baugh and Travis (1976) Biochemistry .15:836-841). After purification, each enzyme was stored at -20"C in 50 mM sodium acetate (pH 5.5) 0.5 M NaCl prior to use.
  • Cat G 600 micrograms was incubated with purified clostripain (Sigma Chemical Company, St. Louis, MO) at a molar ratio of 50:1 in 50 mM Tris-HCl (pH 7.5) 10 mM CaCl 2 0.16 M NaCl 2.5 mM Dithiothreitol at 37°C for 24 hours. During the incubation period samples were removed at various times and assayed for enzymatic and/or bactericidal activity.
  • Clostripain (Mr 30 kDa) and undigested Cat G were separated from the degradation peptides by loading the 24 h digestion mixture (1.0 ml) on a Sephadex G-50TM (Pharmacia Fine Chemicals, Piscataway, New Jersey) column which was equilibrated with 1.0 M NH 4 0H. Absorbance at both 220nm and 280nm was monitored and those fractions containing peptides were pooled, lyophilized and dissolved in 0.1% trifluoroacetic acid (TFA) .
  • TFA trifluoroacetic acid
  • the peptide mixture was then applied to a reverse phase HPLC (RP-HPLC) C-18-10 column which had been previously equilibrated with 0.1% TFA. Bound peptides were then eluted using a linear gradient of acetonitrile (0 to 70% (v/v) in 0.1% TFA at a flow rate of 0.5 ml/min. Fractions were lyophilized for subsequent testing in antimicrobial assays. Fractions with bactericidal activity were reapplied to the RP-HPLC C-18-10 column. The bound peptides were eluted with a non-linear gradient of acetonitrile (0 to 50%) in 0.1% TFA with a flow rate of 0.5 ml/h.
  • RP-HPLC reverse phase HPLC
  • Neisseria gonorrhoeae strain FA 102 and Staphylococcus aureus strain 8325-4 were the test bacteria used; these strains have been described previously (Shafer et al. (1986) supra; Shafer and Onunka (1989) J. Gen. Microbiol. 135:825-830) .
  • N. gonorrhoeae were passaged on clear typing agar as non-piliated, transparent variants. For testing cultures were grown with shaking at 37 ⁇ C in GC broth containing glucose, iron and sodium bicarbonate supplements. ______ aureus was grown at 37 ⁇ C with shaking in LB broth.
  • Peptides were dissolved in HBSS (pH 7.5) and added in various amounts (0 to 100 micrograms) to sterile mi ⁇ rotiter wells. After UV sterilization of the wells, 0.1 ml samples of the bacteria were added and the volumes in each well were adjusted with HBSS to 0.2 ml. The bacteria-peptide mixtures were incubated at 37 ⁇ C for 45-60 min. For I . gonorrhoeae. incubation was carried out under an atmosphere of 5% C0 2 .
  • Viability was determined after incubation by plating 10 and 100 microliter samples on LB agar for S_ a . aureus and on GCB agar for __ ⁇ ____ gonorrhoeae. All assays were done in duplicate or triplicate, and the results given are the means of three independent experiments. The % survival of the test bacteria was calculated as 100 x (# CFU in the presence of peptide)/(# CFU in the absence of peptide) ; standard error of the mean for each data point was never greater than 5%.
  • Penicillin G, tetracycline, chloramphenicol, streptomycin and kanamycin All from Sigma Chemical Company, St. Louis, MO
  • Penicillin G, tetracycline, chloramphenicol, streptomycin and kanamycin All from Sigma Chemical Company, St. Louis, MO
  • All antibiotic solutions were stored at -20°C.
  • This preparation was provided by Dr. John Spitznagel, Emory University School of Medicine, Atlanta, GA) .
  • the preparation was obtained by Sephadex G-75 TM chromatography of a crude acid extract of human PMN granules (Shafer et al. (1988) Infect. Immun. 56:51-53) .
  • the pooled defensins were dialyzed against 4 liters of distilled water at 4°C using dialysis tubing with an exclusion limit of 3 kDa.
  • Example 1.5 Agar Diffusion Assay for Antimicrobial Activity Potentially microbicidal peptides were dissolved in sterile distilled water at a concentration of 1 mg/ml.
  • S. aureus 8325-4 and ______ aureus 133 were inoculated from an agar slant culture to LB broth and grown overnight at 37°C. The culture was then diluted 1:100 into fresh LB broth and incubated 4 h at 37 ⁇ C to produce a mid-logarithimic culture. The midlog culture was used to inoculate the surface of an LB agar plate by dipping a sterile cotton swab into the culture and then evenly swabbing the entire surface of the plate. The plate was then incubated 1 h at 37 ⁇ C. Then wells in the agar were made by removing agar plugs with the wide end of a sterile Pasteur pipette. 100 ⁇ l aliquots of peptide solutions at various concentrations and a sterile distilled water control are added to the wells.
  • the plates were incubated overnight at 37°C and then examined for zones of inhibition, which were measured if present.
  • Oligopeptides were synthesizedusing anApplied Biosystems Model 430A peptide synthesizer (0.1 mmol scale) using phenylacetamidomethyl (Pam) or p-methylbenzyhydrylamine polystyrene resins (Applied Biosystems, Inc.) and tert- butyloxycarbonyl (Boc)-protected amino acids (Applied Biosystems, Inc. or Bachem, Inc., Torrance, CA) . Boc-Arg(Tos) , Boc-His(Z) and Boc-Tyr(Br-Z) were used for the incorporation of Arg, His and Tyr respectively. All amino acids except glycine used in this work have the L configuration unless otherwise noted.
  • Peptides were cleaved from the resin and deprotected in liquid HF/E-cresol/dimethyl sulfide (0:1:0.5) at -10 ⁇ C for 90 min.
  • the resins were washed with cold diethyl ether, and the peptides were extracted into 1.0 M acetic acid and lyophilized.
  • Peptides were then purified by RP-HPLC on an AquaporeTM RP-300 C18 silica column (1x10 cm, Applied Biosystems, Inc.) using a linear gradient of acetonitrile in 0.1% TFA.
  • the purity of each synthetic peptide preparation was confirmed by microbore HPLC on C18 slides (1x250 mm, Applied Biosystems, Inc.) sequencing, as described above. All peptides were stored in the lyophilized form at 4°C prior to use in the antibacterial assays.
  • Boc-Cys (4-methoxyl benzyl) is used to incorporate cysteine residues.
  • Boc-His (methyl) was incorporated in a manual mode on a 0.02 mmol scale using the N,N'-dicyclohexylcarbodiimide/1-hydroxybenzotriazole coupling protocol.
  • the antimicrobial peptides of the present invention will be useful for research and, when formulated into therapeutic compositions, will be useful in the treatment of infections, especially bacterial infections.
  • the antimicrobial peptides can be administered by any mechanism known to the art, as appropriate for a particular type of infection.

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Abstract

Peptides présentant une activité antimicrobienne comparable à celle de certains antibiotiques connus. Ces peptides sont apparentés en séquence à des séquences d'aminoacides dans Cathepsin G. IIGGR et IIGGR et agissent contre à la fois des souches bactériennes Gram négatif et Gram positif. HPQYNYR et certains peptides apparentés agissent aussi contre, à la fois, des bactéries Gram positif et Gram négatif, y compris, mais pas exclusivement, les souches de Escherichia coli, Neisseria gonorrheae, Proteus vulgaris, Staphylococcus aureus, Listeria monocytogenes et Porphyromonas gingivalis. Les peptides selon la présente invention seront utiles dans des compositions pharmaceutiques servant à traiter et à prévenir des infections.
PCT/US1991/004414 1990-06-21 1991-06-21 Peptides antimicrobiens WO1991019512A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5428016A (en) * 1991-03-13 1995-06-27 Morinaga Milk Industry Co., Ltd. Antimicrobial peptide and antimicrobial agent
EP0665754A1 (fr) * 1992-10-02 1995-08-09 Emory University Peptides antimicrobiens
WO2009033822A2 (fr) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Utilisation d'un peptide en tant qu'agent thérapeutique
US9346865B2 (en) 2004-12-15 2016-05-24 The Regents Of The University Of Colorado, A Body Corporate Antimicrobial peptides and methods of use
US9352015B2 (en) 2009-06-05 2016-05-31 The Regents Of The University Of Colorado, A Body Corporate Antimicrobial peptides
WO2018073416A1 (fr) * 2016-10-20 2018-04-26 Lysando Ag Nouveaux agents antimicrobiens contre les bactéries enterococcus
WO2023066184A1 (fr) * 2021-10-20 2023-04-27 庄国昇 Utilisation d'oligopeptide dans le traitement de l'inflammation gingivale, de l'atrophie gingivale et dans la réparation de la muqueuse buccale

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4725576A (en) * 1983-12-29 1988-02-16 Research Foundation Of State University Of New York Fungicidal polypeptide compositions containing L-histidine and methods for use therefore

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4725576A (en) * 1983-12-29 1988-02-16 Research Foundation Of State University Of New York Fungicidal polypeptide compositions containing L-histidine and methods for use therefore

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, Vol. 26, issued 1987, SALVESEN et al., "Molecular Cloning of Human Cathepsin G: Structural Similarity to Most Cell and Cytotoxic T Lymphocyte Proteinase", pages 2289-2293. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Vol. 86, issued July 1989, GABAY et al., "Antibiotic Proteins of Human Polymorphonuclear Leukocytes", pages 5610-5614. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5428016A (en) * 1991-03-13 1995-06-27 Morinaga Milk Industry Co., Ltd. Antimicrobial peptide and antimicrobial agent
EP0665754A1 (fr) * 1992-10-02 1995-08-09 Emory University Peptides antimicrobiens
EP0665754A4 (fr) * 1992-10-02 1996-06-12 Univ Emory Peptides antimicrobiens.
US9346865B2 (en) 2004-12-15 2016-05-24 The Regents Of The University Of Colorado, A Body Corporate Antimicrobial peptides and methods of use
WO2009033822A2 (fr) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Utilisation d'un peptide en tant qu'agent thérapeutique
WO2009033822A3 (fr) * 2007-09-11 2009-05-22 Mondobiotech Lab Ag Utilisation d'un peptide en tant qu'agent thérapeutique
US9352015B2 (en) 2009-06-05 2016-05-31 The Regents Of The University Of Colorado, A Body Corporate Antimicrobial peptides
WO2018073416A1 (fr) * 2016-10-20 2018-04-26 Lysando Ag Nouveaux agents antimicrobiens contre les bactéries enterococcus
WO2023066184A1 (fr) * 2021-10-20 2023-04-27 庄国昇 Utilisation d'oligopeptide dans le traitement de l'inflammation gingivale, de l'atrophie gingivale et dans la réparation de la muqueuse buccale

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