Classifications

• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
• • 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

• Anurans synthesize polypeptides with a broad spectrum of antimicrobial activity through the granular glands present in their skin.
• the bioactive peptides are released into skin secretions in a holocrine fashion upon stress or injury and protect against invasion by pathogenic microorganisms.
• the amphibian antimicrobial peptides are generally synthesized as members of structurally-related families and examples include magainins from Xenopus laevis, bombinins from Bombina variegata and Bombina orientalis, dermaseptins from Phyllomedusa sauvagii and Phyllomedusa bicolor, buforins from Bufo bufo gargarizans, and caerins from Litoria chloris and Litoria splendida.
• the members of a particular family have distinct spectra of antimicrobial activity and it has been speculated that this molecular diversity is important in protecting the animal from invasion by a wide array of different microorganisms.
• Frogs from the genus Rana constitute an extremely diverse and widely distributed group with an estimated 250 species worldwide and at least 36 species having been identified in North America (12 delete). Analysis of skin secretions and/or skin extracts of different species of Ranid frogs has led to the following characterizations: 1) gaegurins and rugosins from R. rugosa, 2) brevinins from ?. brevipoda porsa, R. esculenta and ?. sphenocephala, 3) esculentins from R. esculenta, 4) ranalexin and ranatuerins from R. catesbeiana and 5) temporins and ranatuerin IT from R. temporaria.
• Peptides of the brevinin family have also been isolated from an extract of gastric tissue from R. esculenta. Further examples of such peptides are discussed in: 1) Lazarus, L.H., and Attila, M., "The toad, ugly and venomous, wears yet a precious jewel in his skin,” Prog. Neurobiol, 41, 473- 507 (1993);
• the present invention has as its goal the isolation and characterization of amphibian antimicrobial peptides to assess their value as taxonomic and phylogenetic markers.
• a further goal of the present invention is the study of seven different classes of Anuran peptides with antimicrobial activity which are obtained from extracts of the skins of six closely related species of North American frogs of the genus Rana: the spotted frog R. luteiventris, the Rio Grande leopard frog R. berlandieri,_grcen frog R. clamitans, the pig frog R. grylio. and the northern leopard frog R. pipiens and the North American bullfrog. R catesbeiana.
• the present invention which is directed to seven families of peptides obtained from North American frogs (families of Rana peptides), to purified Rana compositions containing these peptides, to purified antibacterial extracts from Rana skin, to modified Rana peptides, to truncated Rana peptides, to pharmaceutical compositions containing Rana peptides or modified forms thereof, and to methods for treatment of bacterial infections using Rana peptides or modified forms thereof.
• the invention is directed to seven families of Rana peptides shown below in Table 1. These Rana peptides have antibacterial activity and are selected from any of the following seven peptide famihes.
• the Rana peptides may be in the form of their C-terminus carboxylic acids, or modified as C- terminus amides or C-terminus esters.
• the amides may be a simple amide (CONH2), which is the isolated form for the Temporin-A and -B Famihes, or amides derived from Cl to CIO primary, secondary or tertiary aliphatic or aromatic amines.
• the esters (COOR wherein the R group is the alcohol residue) are derived from Cl to CIO aliphatic or aromatic alcohols.
• the preferred C- terminus forms for the Temporin-A and Temporin-B famihes are the simple amides.
• the single letters designate amino acid residues according to accepted convention except that in the formulas for the peptides designated with a Family name followed by the term "mod", the letters X, B, Z, U, O, J' and J, which are not conventional single letter designations of amino acid residues, are used as symbols to indicate a substitution of either any of the amino acid residues occurring at the same position within the other peptides of the same family or a conservative substitution of an amino acid residue for the amino acid substitution of an amino acid residue at the same position for any of the peptides within the same family of peptides.
• the X means that at the 3 position, the amino acid residue can be L, I or a conservative substitution therefor, at the 16 position the amino acid residue can be L, F or a conservative substitution therefor, and at the 21 position the amino acid residue can be I, N or a conservative substitution therefor.
• conservative substitution means substitution of an amino acid residue by another that has the same side chain ionicity, basicity, acidity, lipophilicity or hydrogen bonding character as the residue being replaced.
• Examples include isoleucine (I), leucine (L), alanine (A), valine (N), phenyl analine (F), proline (P) and glycine (G) as an interchangeable group; lysine (K), histidine (H) and arginine (R) as an interchangeable group, serine (S), tyrosine (Y) and threonine (T) as an interchangeable group; cysteine (C) and methionine (M)as an interchangeable group asparagine ( ⁇ ), glutamine (Q) and tryptophan (W) as an interchangeable group, and aspartic acid (D) and glutamic acid (E) as an interchangeable group.
• each dashed line constitutes a spacer instead of an amino acid residue.
• the 5 dashed lines of Ranatuerin-2 stand in the place of 5 amino acid residues so that the A residue following the dashed lines is counted in the 19 th position for the purpose of placing the amino acid residues of the individual peptides of the family into homologous alignment.
• the 4 and 8 dashed lines of the Ranatuerin-2Ca and Ranatuerin-2Cb peptides respectively place the A residue following those lines into the 19 th position.
• Esculentin-2L GI SLFTGGIKALGKTLFKMAGKAGAEHLACKAT QC (SEQ ID NO: 22) Esculentin-2B GLFSILRGAAKFAS GLGKDLTKLGVDLVACKISKQC (SEQ ID NO: 23) Esculentin-2P GFSSIFRGVAKFASKGLGKDLARLGVNLVACKISKQC (SEQ ID NO: 24) Esculentin-2 mod GZZSZZZGZZKZZZKZLZKZZZZZGZZZZZQC (SEQ ED NO: 25) Brevinin-1 family
• Temporin B Family as the C-terminus carboxamide Temporin-lLa VLPLISMALGKLL (SEQ ID NO: 55) Temporin-lLb NFLGTLINLAKKIM (SEQ ID NO: 56) Temporin-lGa SILPT ⁇ VSFLSKFV (SEQ ID NO: 57) Temporin-lGb SILPTIVSFLSKFL (SEQ ID NO: 58) Temporin-lGc SILPT ⁇ VSFLTKFIL (SEQ ID NO: 59) Temporin-lGd FIIPLIASFLSKFIL (SEQ ID NO: 60) Temporin-B mod JJJJ ⁇ JSFLJKJJL (SEQ ID NO: 61)
• the purified compositions of the invention includes any composition containing one or more of the foregoing peptides obtained from a natural source such as Rana skin or obtained from a synthetic source.
• the modified Rana peptides of the invention include any of the foregoing Rana peptides designated as Ranatuerin-1 mod, Ranatuerin-2 mod, Esculentin-2 mod, Brevinin- 1 mod, Brevinin-2 mod, Temporin-A mod and Temporin-B mod wherein the symbols X, B, Z, U, O, J' and J indicate substitutions of amino acid residues as discussed above.
• the modified Rana peptides of the invention also include the C-terminus amides and esters described above, which are derived from Cl to CIO aliphatic or aromatic amines or alcohols.
• Further modified Rana peptides include those wherein one or more of the amino acid residues valine (V), alanine (A), lysine (K), and glutamic acid (E) are substituted for the glycine (G) residues.
• Further modified Rana peptides of the invention include any of the foregoing Rana peptides wherein arginine (R) is substituted for one or more of the lysine (K) residues.
• Additional modified Rana peptides contain modified side chains so that any of the Rana peptides with amino acid residues having functionalized side chains can be modified with amidation or esterification groups.
• Examples include those in which the epsilon amino group of a lysine (K) residue has been coupled to a C 10 to C 20 fatty acid such a palmitate or glycosylated sugar, those in which the epsilon carboxy group of aspartic (D) or glutamic (E) acid residues or hydrolyzed glutamine (Q) residue have been coupled to fatty alcohols or glycosylated sugars, or those in which the epsilon hydroxyl group of threonine (T), tyrosine (Y), or serine (S) residues have been coupled to fatty acids or glycosylated sugars.
• K lysine
• ranatuerin 1 having any of these side chain modifications, where the appropriate amino acid residue is present, is preferred.
• the modified Rana peptides include those in which a truncated fragment is synthetically condensed with a non-natural peptide or peptide sequence optionally containing heterocyclic organic moieties such as a beta or gamma amino acid, an aliphatic diamine, an aliphatic or aromatic dicarboxylic acid, pyridine carboxylic acid, aromatic diols and the like.
• the truncated versions of Rana peptides include the fragments formed by cleaving at any of the glycine residues or between any repeat residues, and fragments formed by N-terminal deletion of pairs of amino acid residues. Further truncated versions include those that are cleaved at the single or multiple dash symbols.
• truncated versions of ranatuerin 1 include those containing amino acid residues 1-9, the fragment containing amino acid residues 16 - 25, N-terminally deleted fragments such as ranatuerin 1 (3-25), (5-25), (7- 25), (9-25), (11-25), (13-25), (15-25), (17-25) and (19-25).
• the pharmaceutical composition of the invention includes the combination of one or more Rana peptides with a pharmaceutical carrier rendering the composition suitable for an appropriate route of administration to a patient.
• the method of treatment of the invention involves administration of an effective amount of the pharmaceutical composition by an appropriate route to a patient suffering from a target bacterial infection.
• FIG. 1 presents a graph of a gel permeation chromatography of an extract of the skin of Rana luteiventris.
• FIGS. 2 A, B present graphs of a reverse-phase HPLC of the antimicrobial activity of R. luteiventris skin.
• FIGS. 3 A, B present graphs of the chromatographic results of a partial purification of the fractions from R luteiventris skin having antimicrobial activity.
• FIGS. 4 A, B and C present graphs of the chromatographic results of a purification of ranatuerin 2La, ranatuerin 2Lb and esculentin 2L.
• FIG. 5 presents gel permeation chromatography on Sephadex G-25 of an extract of the skin of Rana catesbeiana after partial purification on Sep-Pak cartridges.
• the fractions in zone A contained ranatuerins 1-5 and the fractions in zone B contained ranatuerins 6-9.
• FIGS. 6 A and 6B present reversed-phase HPLC on a semipreparative Nydac C-18 column.
• FIG. 6 A presents the pooled fractions from zone A in Fig. 5 and Fig. 6B presents the pooled fractions from zone B in FIG. 5.
• the dashed line shows the concentration of acetronitrile in the eluting solvent and the bars show the fractions containing the ranatuerins indicated.
• the present invention is directed to a systematic investigation of extracts of the skin of adult Ranid frogs and characterization of all the peptides with inhibitory activity towards bacteria.
• the skins of frogs of the genus Rana produce a diverse array of peptides with antimicrobial activity.
• a comparison of the amino acid sequences of these peptides demonstrates that they may be arranged in families on the basis of structural similarity. Attempts to catalogue the antimicrobial peptides are hampered by the absence of a systematic terminology and by the fact that peptides clearly belonging to the same structurally family have been given different names.
• the C-terminal region of peptides of the brevinin-1, ranalexin, brevinin- 2, esculentin-1, esculentin-2 and ranatuerin-1 families contains a cystine-bridged cyclic lieptapeptide whereas the corresponding region of peptides of the ranatuerin-2 family contains a cystine-bridged cyclic hexapeptide.
• the temporin family peptides lack the cyclic region and their primary structures have been poorly conserved. All members of the family contain between 10-14 amino acid residues and terminate in a C-terminally ( ⁇ -amidated amino acid residue.
• the present invention establishes that extracts of the skins of six closely- related North American frogs of the genus Rana contain antimicrobial peptides belonging to 5 of the known families: brevinin-1, ranatuerin-2, esculentin-2, ranalexin and temporin. These peptides classified by using the initial letter of the species name to indicate their origin (L for luteiventris, B for berlandieri and P fox pipiens, C for clamitans and G for grylio.
• the catesbeiana peptides are regarded as the "parent" and so are not given a letter.) Where two or more members of the same family are produced by the one species, the peptides are differentiated by the letters a, b, c, etc. as in the examples of ranatuerin-2La and ranatuerin-2Lb.
• the peptides of the brevinin-1, esculentin-2 and ranatuerin-2 families isolated in the present study show a broad spectrum of antimicrobial activity inhibiting the growth of a gram positive bacterium (Staphylococcus aureus), a gram negative bacterium (Escherichia coli) and a yeast (Candida albicans).
• Staphylococcus aureus a gram positive bacterium
• Escherichia coli gram negative bacterium
• yeast Candidadida albicans
• ranatuerin 4 contains limited structural similarity, including the presence of a C-terminal heptapeptide ring, to the previously characterized gaegurins (Park, J.M., Jung, J.E., and Lee, B.J., Biochem. Biophys. Res. Commun. 205, 948-954 (1994)); and brevinins (Morikawa, N., Hagiwara, K., and Nakajima, T., Biochem. Biophys. Res.
• ranalexin shows potent cytolytic activity towards S. aureus (minimal inhibitory concentration of 4 ⁇ g/ml), it would not have been missed during the investigation of the bullfrog skin extracts. It is probable, therefore, that the expression of the antimicrobial peptides in bullfrog skin is developmentally regulated.
• the amino acid sequence of the potent Rana peptides can also form the basis for modified versions exhibiting higher potency and with specificity towards pathogenic microorganisms that have developed resistance to commonly used antibiotics.
• one modification focuses upon the presence of a cystine bridge at the C-terminal end of the brevinin-1, ranalexin, brevinin-2, esculentin- 1, esculentin-2 and ranatuerin-1 families and the ranatuerin-2 family.
• Synthesis of modified Rana peptides containing the substitutions Cys ⁇ Ala and Cys ⁇ Ser provides peptides that will have no cysteine bridges.
• modified Rana peptides will show inhibitor activity against Escherichia coli, Staphylococcus aureus and Candida albicans.
• other modified ranatuerins can be formulated through an understanding of the mechanism by which antimicrobial peptides are cytotoxic.
• Antimicrobial peptides such as the Rana peptides have a propensity to form amphipathic ⁇ -helical structures and their mechanism of action is believed to involve a direct interaction with the fatty acyl chains in the cell membrane leading to disruption of normal membrane function responsible for osmotic balance.
• the presence of cationic residues (particularly lysine) in antimicrobial peptides is believed to destroy the ionic gradient across cell membranes by forming ion channels.
• SMLSVLKNLGKVGLGFVACKINKQC (SEQ ID NO: 62) .vAAAAAA-TBBBBBBBAAATTTTTT
• modified Rana peptides is constructed by substituting one or more of the ⁇ -helix-extending residues (hydrogen bonding residues) for the lipophilic amino acid residues within the helix regions of an individual Rana peptide, especially those within the C- terminus region of the peptides.
• Other amino acid residues can be substituted as follows:
• Ala is substituted for Gly in order to replace the ⁇ -sheet region in the central portion of the peptide with an extended ⁇ -helix.
• the increased ⁇ -helical character of the peptide promotes interaction with the cell membrane of the targeted microorganism;
• Val is substituted for Gly in order to stabilize the ⁇ -sheet region in the central portion of the peptide
• Ala or another lipophilic amino acid residue is substituted for Asn in order to promote ⁇ -helical character;
• Glu is substituted for Gly in order to promote ⁇ -helical character.
• Modified Rana peptides also include those Rana peptide Family modified peptides with the formulas containing the symbols X, B, Z, U, O, J' and J as 5 discussed in the Summary of the Invention.
• modified Rana peptides can also prepared by substituting one or more of the lysine residues by arginine. This substitution will increase the cationic character of the peptide so as to promote its ability to affect the cellular membrane ionic gradient.
• modified Rana peptides includes truncated Rana peptides lacking redundant segments of the original molecules, and protease- resistant modified Rana peptides containing D-amino acid residues and/or unnatural amino acids and/or side chain modified amino acids.
• the Rana peptides can also be modified by substitution of one or more of the amino acid
• Rana peptides 15 residues valine (V), alanine (A), lysine (K), and glutamic acid (E) for the glycine (G) residues.
• Further modified Rana peptides include those wherein arginine (R) is substituted for one or more of the lysine (K) residues.
• the truncated versions of Rana peptides include the fragments cleaved on the N side of the any of the glycine residues or between repeat residues as well as N-terminally deleted
• Any of the Rana peptides can be modified with amino acid residues having modified side chains such as those in which the epsilon amino group of a lysine (K) residue has been coupled to a fatty acid such a palmitate or glycosylated sugar, those in which the epsilon carboxy group of aspartic (D) or glutamic (E) acid residues or a hydrolyzed glutamine (Q) residue have been
• modified Rana peptides include those in which a truncated version is synthetically condensed with a non-natural peptide or peptide sequence optionally containing
• heterocychc organic moieties such as a beta or gamma amino acid, an ahphatic diamine, an aliphatic or aromatic dicarboxylic acid, pyridine carboxylic acid, aromatic diols and the like.
• the Rana peptides and modified versions can be obtained by automated peptide synthesis using such techniques as the "Merrifeld” technique, the recombinant DNA techniques involving insertion, expression and isolation, and by extraction techniques from any source in nature (for naturally occurring Rana peptides).
• semi-synthetic Rana peptides can be obtained by preparation of the peptide sequence using any of the foregoing techniques and coupling the peptide sequence to the chemical moiety by esterification, amidation, Schiff base formation under appropriate side chain protection if needed.
• These techniques are well known in the art. For example, a Merrifeld technique is described in US Patent No.
• DNA sequence such as the 19-25 region of ranatuerin 1 or analogous regions of the other Rana peptide DNA sequences can be synthesized for use as a primer in the RACE (rapid amplification of cDNA 3' end) protocol using the polymerase chain reaction. Amplification products can be cloned into the XhoI EcoRV restriction site of the BlueScript vector (Stratagene). The clone can be labeled by random priming and then used to screen the cDNA to obtain a full-length clone. Positive clones can be selected and analyzed by nucleotide sequencing.
• the Rana peptides and their modified and truncated forms as well as the compositions of the present invention may be administered either alone or in combination with a pharmaceutically-acceptable carrier, by topical, oral, anal, ocular, buccal, nasal, intramuscular, subcutaneous, intravenous, or parenteral routes.
• a pharmaceutically-acceptable carrier by topical, oral, anal, ocular, buccal, nasal, intramuscular, subcutaneous, intravenous, or parenteral routes.
• the ultimate choice of route, formulation and dose is made by the attending physician or veterinarian and is based upon the patient's or animal's unique condition.
• the usual dosage for administration to humans lies in the range of approximately 50-2000 mg. P.O. per day, and preferably in about one to four doses where the dose is based upon the activity of pure Rana peptide.
• the usual dosage for administration to small animals lies in the same approximate range as that for humans.
• the usual dosage is higher per unit of body weight so that the dose given lies in the range of about 20 to 20,000 mg. P.O. per day.
• This dosage may vary somewhat with the weight of the subject (human or animal) being treated; in general, about 1-40 mg./kg. of body weight per day can be employed for humans and small animals while about 1 to 400 mg./kg. of body weight per day can be employed for large animals.
• the Rana peptides of this invention can be combined with inert pharmaceutical excipients such as lactose, oil, mannitol and starch, and formulated into dosage forms such as elixirs, liquids, ointments, lotions, IV fluids, alcohol, tablets, capsules, and the like.
• inert pharmaceutical excipients such as lactose, oil, mannitol and starch
• dosage forms such as elixirs, liquids, ointments, lotions, IV fluids, alcohol, tablets, capsules, and the like.
• these peptides can be formulated with an inert, parenterally acceptable vehicle such as water, saline, sesame oil, ethanol buffered aqueous medium, propylene glycol and the like.
• these peptides can be formulated with waxes, oils, buffered aqueous medium, and the like.
• the antibacterial activity of the Rana peptides as well as the modified and truncated forms thereof likely will show inhibition action against the following strains of bacteria and fungi.
• Drag-resistant strains of this "harmless" organism are responsible for twice as many cases of nosocomial infections as any other microbe, particularly hospital-acquired urinary tract infections.
• E.faecalis is also responsible for urinary tract infections and frequently invades heart valves and prosthetic devices of hospitalized patients.
• the organism is frequently introduced into body orifices by contaminated catheters and other invasive instruments and proliferates in respirators and renal dialysis units
• Klebsiella pneumoniae The organism readily grows in glucose solutions used for intravenous therapy leading to deaths from bacteremia. Pneumonia caused by Klebsiella has a 50% mortality rate in spite of treatment with existing antibiotics.
• the organism is responsible for 80% of all bacterial pneumonias and is particularly prevalent in institutions housing old people such as nursing homes and geriatric wards. In young people infection of the middle ear leads to meningitis and otitis media.
• This pathogen is responsible for the increasing incidence of acute bacterial meningitis in children and is often transmitted in day-care settings. Even with prompt diagnosis and aggressive treatment with existing antibiotics, 33% of children sustain residual disability.
• Staphylococcus sp. in hospitals is generally antibiotic resistant and poses a particular problem in nurseries, delivery rooms and burn units.
• S. saprophyticus causes urinary-tract infections particularly in sexually active young women and in immunosuppressed patients.
• Antibiotic therapy or severe immunosuppression often precedes extensive invasion by C. albicans and is particularly common in burn patients and in recipients of renal, heart and bone marrow transplants.
• Skin secretions containing very high concentrations of antimicrobial peptides, can easily be collected from Ranid frogs by non-invasive procedures (mild electrical stimulation or subcutaneous injection of epinephrine) so that identification of the species does not necessitate sacrifice of the animal.
• Molecular techniques have been used previously to facilitate identification and to study phylogenetic relationships among Ranid frogs. For example, analysis of the nucleotide sequences of mitochondrial and genomic DNA has been used to investigate the evolutionary history of specimens of the closely related species of European water frogs, R. ridibunda, R. esculenta and R. lessonae.
• the frozen skins (R. luteiventris 45 g; R. berlandieri 66 g; R. clamitans 125g; R. grylio; 96 g; R. pipiens 58 g) were separately extracted by homogenization in ethanol/0.7 M HC1 (3:1 v/v; 10 ml/g ) at 0°C using a Waring blender. The homogenates were stirred for 2 h at 0°C and centrifuged (4000 x g for 30 min at 4°C).
• Ethanol was removed from the supernatants under reduced pressure and, after further centrifugation (4000 x g for 30 min at 4°C), the extracts were separately pumped onto 8 Sep-Pak C-18 cartridges (Waters Associates, Milford, MA, U.S.A.) connected in series at a flow rate of 2 ml/min. Bound material was eluted with acetonitrile/water/trifluoroacetic acid (70.0:29.9:0.1, v/v/v) and freeze-dried.
• Ethanol was removed from the supernatant under reduced pressure and, after further centrifugation (4000 x g for 30 min at 4 °C), the extract was pumped onto 8 Sep- Pak C-18 cartridges (Waters Associates) connected in series at a flow rate of 2 ml/min. Bound material was eluted with acetonitrile/ water/ trifluoroacetic acid ((70.0:29.9:0.1, v/v/v) and freeze-dried.
• the column was eluted at a flow rate of 48 ml/h and fractions (8 ml) were collected. Absorbance was measured at 280 mn. The ability of aliquots (50 ⁇ l) of the fractions to inhibit the growth of S. aureus was determined as described in the previous section. Fractions containing maximum activity (denoted by the bars in Fig. 1) were pooled and injected onto a (25 x 1 cm) Vydac 218TP510 C-18 reverse- phase HPLC column (Separations Group, Hesperia, CA, USA) equilibrated with 0.1% (v/v) trifluoroacetic acid/water at a flow rate of 2 ml/min.
• the concentration of acetonitrile in the eluting solvent was raised to 21% (v/v) over 10 min and to 49% (v/v) over 60 min using linear gradients. Absorbance was monitored at 214 nm and 280 nm and fractions (1 min) were collected. The fractions containing antimicrobial activity were successively rechromatographed on (250 x 4.6 mm) Vydac 214TP54 (C-4) and Vydac 219TP54 (phenyl) reverse- phase HPLC columns. The concentration of acetonitrile in the eluting solvent was raised from 21% to 55% over 40 min and the flow rate was 1.5 ml/min. The purification of the R.
• catesbeiana frog skin extract was accomplished in similar fashion. After partial purification on Sep-Pak cartridges, the extract was redissolved in 1% (v/v) trifluoroacetic acid/water (5 ml) and chromatographed on a (100 x 2.6 cm) column of Sephadex G-25 (Pharmacia Biotech) equilibrated with 1 M acetic acid. The column was eluted at a flow rate of 48 ml/h and fractions (8 ml) were collected. Absorbance was measured at 280 nm. The antimicrobial activity of aliquots of the fractions was determined. Fractions containing maximum activity (denoted by the bars in Fig.
• zone A contained esculentin-2L, ranatuerin-2La, and ranatuerin-2Lb and zone B contained brevinin-1 La, brevinin-1 Lb, temporin- 1 La, temporin-lLb and temporin-lLc.
• the antimicrobial peptides were purified to near homogeneity, as assessed by symmetrical peak shape, by a final chromatography on a Vydac phenyl column as shown in Figs. 4 (A- C).
• the final yields of pure peptides were: ranatuerin 2La, 4050 nmol; ranatuerin 2Lb, 320 nmol; and esculentin-2L, 740 nmol.
• brevinin-lPa 1050 nmol
• brevinin-lPb (1110 nmol
• brevinin-lPc 64 nmol
• brevinin-lPd 315 nmol
• brevinin-lPe 8 nmol
• esculentin-2P (12 nmol
• ranatuerin-2P 690 nmol
• temporin- IP 205 nmol
• ranatuerin-lC (570nmol), ranalexin-lC (715 nmol), ranatuerin-2Ca (30 nmol), ranatuerin-2Cb (110 nmol), Temporin- ICa (240 nmol), temporin- lCb (qnm), temporin- lCc (qnm).
• ranalexin- 1G 50 nmol
• ranatuerin-lG 70 nmol
• ranatuerin-2G 55 nmol
• temporin-lGa 180 nmol.
• chromatography of pooled fraction from zone B led to the separation of activity into three fractions subsequently shown to contain ranatuerins 6+ 7, ranatuerin 8 and rantuerin 9 (Fig. 6B).
• the peptides were purified to near homogeneity, as assessed by a symmetrical peak shape, by chromatography on analytical Vydac phenyl and C-8 columns.
• Amino acid compositions were determined by precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccimrnidyl carbamate using a Waters AccQ Tag system with fluorescence detection and separation of the amino acid derivatives by reverse-phase HPLC. Hydrolysis in 5.7 M HCl (24 h at 110°C) of approximately 1 nmol of peptide was carried out. The primary structures of the peptides were determined by automated Edman degradation using an Applied Biosystems model 471 A sequenator modified for on-line detection of phenylthiohydantoin amino acids under gradient elution conditions.
• Electrospray mass spectrometry was carried out using a Perkin Elmer Sciex API 150EX single qua ⁇ rupole instrument. The accuracy of mass determinations was ⁇ 0.02%.
• MICs Minimal inhibitory concentrations of the peptides were determined by a standard microdilution method using 96-well microliter cell- culture plates as previously described. Serial dilutions of the peptides in Mueller-Hinton broth (50 ⁇ L) were incubated with an inoculum (50 ⁇ L of 10 3 CFU/mL) from an overnight culture of Escherichia coli (ATCC 25922) and Staphylococcus aureus (NCTC 8325), for 18 h at 37°C in an humidified atmosphere of 5% CO- in air. Incubations with Candida albicans (ATCC 90028) were carried out in RPMI 1640 medium for 48 h at 35°C.
• Ranatuerins 1-9 tested at a concentration of 20 ⁇ g/ml, showed no detectable hemolytic activity towards human erythrocytes.
• Ranatuerin-1 (SMLSVLKNLG 10 KVGLGLVACK 20 INKQC) (SEQ ID NO: 8), isolated from the skin of the American bullfrog Rana catesbeiana, shows antimicrobial activity against the gram-negative bacterium, Escherichia coli, the gram-positive bacterium Staphylococcus aureus, and the yeast, Candida albicans.
• the peptide is predicted to comprise three structural domains: alpha- helix (residues 1- 8), beta-sheet (residues 11- 16) and beta-turn (residues 20-25). Substitution of Cys 19 and Cys25 by Ser residues had only a minor effect on potency demonstrating that the disulfide bridge is not necessary for activity, but deletion of the cyclic heptapeptide region produced an inactive analog.
• the crude synthetic peptides were purified to near homogeneity by chromatography on a 1 x 25-cm Vydac 218TP510 C-18 reversed-phase HPLC column (Separations Group, Hesperia, CA) equilibrated with 20 acetonitrile/water/ trifluoroacetic acid at a flow rate of 2 ml/min.
• the concentration of acetonitrile in the eluting solvent was raised to 49% over 60 min using linear gradients.
• Absorbance was measured at 214 and 280 nm and the major peak in the chromatogram was collected by hand.
• the synthetic peptides were characterized by automated Edman degradation using an Applied Biosystems model 471 A sequenator modified for on-line detection of phenylthiohydantoin amino acids under gradient elution conditions and by electrospray mass spectrometry using a Perkin Elmer Sciex API 150EX single quadrapole instrument. The accuracy of mass determinations was Bl 0.02%.
• MICs Minimal inhibitory concentrations of the peptides were determined by a standard microdilution method using 96- well microtiter cell- culture plates as previously described. Serial dilutions of the peptides in Mueller- Hinton broth (50 mL) were incubated with an inoculum (50 mL of 103 CFU/mL) from an overnight culture of Escherichia coli (ATCC 25922) and Staphylococcus aureus (NCTC 8325), for 18 h at 37 0C in a humidified atmosphere of 5% C02 in air. Incubations with Candida albicans (ATCC 90028) were carried out in RPMI 1640 medium for 48 h at 35 OC.

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