WO1998006743A1

WO1998006743A1 - Acylated peptide cytolytic peptide inhibitors

Info

Publication number: WO1998006743A1
Application number: PCT/AU1997/000511
Authority: WO
Inventors: Donald Edward Rivett; Dean Raymond Hewish; Jerome Werkmeister; Cyril Curtis Curtain; Barry Ross Matthews; Jonathan Bayldon Baell
Original assignee: Commonwealth Scientific And Industrial Research Organisation; Biomolecular Research Institute
Priority date: 1996-08-13
Filing date: 1997-08-13
Publication date: 1998-02-19
Also published as: CA2263789A1; AUPO161196A0; EP1012172A1; EP1012172A4; JP2001505541A

Abstract

The present invention relates to hybrid molecules and novel peptides which inhibit cytolytic compounds such as melittin. The invention also relates to pharmaceutical compositions including these inhibitors and to methods of inhibiting cytolytic compounds.

Description

ACYLATED PEPTTDE CYTOLYTIC PEPTIDE INHIBITORS HELD OF THE INVENTION

The present invention relates to unique peptide/fatty acid hybrid molecules and to novel peptides. In particular, the invention relates to peptide/fatty acid hybrid molecules which inhibit cytolytic peptides such as melittin. The invention also relates to pharmaceutical compositions including these inhibitors and to methods of inhibiting cytolytic peptides. BACKGROUND OF THE INVENTION Melittin is a 26 residue peptide in which the first 20 residues form an amphipathic helix with a bend, or hinge, in the region of a proline residue at position 14, Schroder et al 1971, although the bend is much more prominent in structures deduced by X-ray crystallography than from NMR, Terwillinger and Eisenberg 1982. A number of theories for the mechanism by which melittin induces cell lysis have been proposed (see review by

Dempsey 1990), all of which have included interaction of the helical portion of the molecule with the cell membrane, with the 6 C-terminal hydrophilic residues (strongly basic region) probably being associated with the phospholipid head groups of the lipid bilayer. Sequential reduction in length of the basic C-terminal sequence leads to a gradual loss in activity. Werkmeister et al 1993.

Using a synthetic peptide combinational library, Blondelle et al 1993. identified the peptide Ac-IVIFDC-NH₂ as an active inhibitor of melittin haemolysis. Furthermore they demonstrated that increasing the concentration of melittin resulted in a reduction of the activity of the inhibitor, suggesting that the inhibition was the result of peptide binding to melittin, rather than to the cell membrane. The authors concluded that the results suggested a mechanism of inhibition involving hydrophobic interactions between the peptides and melittin that prevented melittin from interacting with the membrane.

The present inventors have now determined a novel formula which defines compounds which have the ability to inhibit the activity of lytic peptides such as mellitin. Compounds synthesised according to this formula have demonstrated the ability to inhibit melittin-induced haemolysis and melittin-induced lysis of CEM T cell lymphoma cells using the 90° light scatter parameter of the flow cytometer as described by eston et al. 1994. Melittin is considered to be a convenient model for a typical cytolytic peptide.

SUMMARY OF THE INVENTION

Accordingly, in a first aspect the present invention provides a compound of the formula:

R_rA-B-X wherein

R, is a hydrophobic group of substantially the same size and charge as a fatty acid acyl group with a carbon chain of 3 to 19 carbon atoms,

A is absent or a hydrophobic amino acid, B is an amino acid, and X is an amino acid, with the proviso that if A is phenylalanine, B is aspartic acid and X is cysteine or cysteic acid, then R is not a fatty acid acyl group with a carbon chain of 8 carbon atoms.

In a preferred embodiment R- is a fatty acid acyl group with a carbon chain of 3 to 19 carbon atoms. R- may be a substituted or unsubstituted fatty acyl group and may be saturated or unsaturated, cyclic or acyclic. The fatty acyl group preferably has a carbon chain of 6 to 19 carbon atoms and more preferably from 9 to 16 carbon atoms.

The fatty acid may terminate in an amino group (eg. II-amino- undecanoic acid), an aromatic ring (eg. cinnamic acid), a hydroxylated aromatic ring (eg caffeic acid), a cyclopentene ring (eg. Chaulmoogric acid) or a hydroxy group.

The fatty acyl group may also be a branched chain fatty acid. Preferably, the branched chain fatty acid is phytanic acid. The branched chain fatty acid is preferable in cases where it is desirable to reduce the toxicity of the molecule. In a preferred embodiment. A is an aromatic amino acid. The aromatic amino acid may be selected from phenylalanine, tryptophan, tyrosine and phenylglycine.

In a further preferred embodiment, B is a hydrophilic amino acid. Preferably, B is a positively or negatively charged amino acid. Preferably, B is an amino acid selected from asparagine, glutamine, aspartic acid and glutamic acid. In a further preferred embodiment, X is a hydrophobic amino acid or a sulphur containing amino acid. X may be an amino acid selected from cysteine, cystine, cysteic acid, methionine, penicillamine and a cysteine derivative in which the -SH group is blocked (e.g. by acetamidomethyl). In a further preferred embodiment, X is an amino acid of the general formula: NH₂-CH(CH₂)R-COOH where R is H, (CH₂)nCH₃, CH(CH₃)₂, CH(CH₃)C₂H₅ and n is 0 to 3. In a preferred embodiment X is isoleucine. In a further preferred embodiment the compound is selected from

(C₁₄)-Phe-Asp-Cys- NH_Z, (C₁₄)-Phe-Asn-Cys- NH₂, (C₁₄)-Asn-Cys- NH₂, (C₁₄)- Phe-Asp-CyS0₃H- NH₂, (C₁₄)-Phe-Asp-CySCMC- NH₂, (C₆)-Phe-Asp-Cys- NH₂, (C_g)-Phe-Asp-Cys- NH₂, (C₁₀)-Phe-Asp-Cys- NH₂, (C₁₂)-Phe-Asp-Cys- NH₂, (C₁₃)-Phe-Asp-Cys- NH₂, (C₁₆)-Phe-Asp-Cys- NH₂, and (C_lβ)-Phe-Asn- Abu- NH_Z.

In a second aspect the present invention provides a peptide of the formula Ac-IVIWDC-NH₂, Ac-IVIFDC(Acm)-NH₂, Ac-IVIFDS-NH₂, Ac- IVIFDV-NH₂, Ac-XVIGDC-NH₂, Ac-IVIFNC-NH₂, IVIFNC-NH₂, Ac-IVIFDM- NH₂, (Ac-IVIFD)₂K-NH₂, Ac-IVILDC-NH₂₎ Ac-(NorLeu)VI(pg)DC-NH2. Ac- IVIFN-Abu-NH₂, Ac-IVIFN-Abu-NH₂ or Ac-LL NFI-NH₂.

The compounds or peptides of the present invention may be incorporated into larger molecules where the larger molecules substantially retain the activity of the compouns or peptides of the present invention. For example, the compounds or peptides of the invention may be in dimeric form. The compounds or peptides of the present invention may also be conjugated or attached to molecules, such as carrier molecules, where the conjugation or attachment does not significantly affect the abililty of the compound or peptide to inhibit the cytolytic activity of compounds such as melittin. The conjugated or attached molecules may be peptides or single amino acids.

It will be appreciated by those skilled in the art that a number of modifications may also be made to the compounds of the present invention without deleteriously affecting the biological activity of the peptide. This may be achieved by various changes, such as insertions and substitutions, either conservative or non-conservative in the peptide sequence where such changes do not substantially decrease the ability of the compounds to inhibit the cytolytic activity of compounds such as melittin.

Modifications of the peptides contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the peptides.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with

NaBH₄; amidation with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5 '-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as

2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy- 5-bitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form 3- nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N- carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine,

4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6- aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid; 2-thienyl alanine and/or D-isomers of amino acids. The peptides of the present invention may be synthesised using techniques well known to those skilled in this field. For example, the peptides may be synthesised using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled "Peptide Synthesis" by Atherton and Sheppard which is included in a publication entitled "Synthetic Vaccines" edited by Nicholson and published by Blackwell Scientific Publications. Preferably a solid phase support is utilised which may be polystyrene gel beads wherein the polystyrene may be cross-linked with a small proportion of divinylbenzene (e.g. 1%) which is further swollen by lipophilic solvents such as dichloromethane or more polar solvents such as dimethylformamide (DMF). The polystyrene may be functionalised with chloromethyl or anionomethyl groups. Alternatively, cross-linked and functionalised polydimethyl-acrylamide gel is used which may be highly solvated and swollen by DMF and other dipolar aprolic solvents. Other supports can be utilised based on polyethylene glycol which is usually grafted or otherwise attached to the surface of inert polystyrene beads. In a preferred form, use may be made of commercial solid supports or resins which are selected from PAL-PEG, PAK-PEG, KA, KR or TGR.

In solid state synthesis, use is made of reversible blocking groups which have the dual function of masking unwanted reactivity in the α- amino, carboxy or side chain functional groups and of destroying the dipolar character of amino acids and peptides which render them inactive. Such functional groups can be selected from t-butyl esters of the structure RCO- OCMe₃-CO-NHR which are known as t-butoxy carboxyl or ROC derivatives. Use may also be made of the corresponding benzyl esters having the structure RCO-OCH₂-C₆H₅ and urethanes having the structure C6H5CH₂O CO-NHR which are known as the benzyloxycarbonyl or Z-derivatives. Use may also be made of derivatives of fluorenyl methanol and especially the fluorenyl-methoxy carbonyl or Fmoc group. Each of these types of protecting group is capable of independent cleavage in the presence of one other so that frequent use is made, for example, of BOC-benzyl and Fmoc- tertiary butyl protection strategies.

Reference also should be made to a condensing agent to link the amino and carboxy groups of protected amino acids or peptides. This may be done by activating the carboxy group so that it reacts spontaneously with a free primary or secondary amine. Activated esters such as those derived from p-nitrophenol and pentafluorophenyl may be used for this purpose. Their reactivity may be increased by addition of catalysts such as 1- hydroxybenzotriazole. Esters of triazine DHBT (as discussed on page 215- 216 of the abovementioned Nicholson reference) also may be used. Other acylating species are formed in situ by treatment of the carboxylic acid (i.e. the Na-protected amino acid or peptide) with a condensing reagent and are reacted immediately with the amino component (the carboxy or C-protected amino acid or peptide). Dicyclohexylcarbodiimide, the BOP reagent (referred to on page 216 of the Nicholson reference), O'Benzotriazole-N, N, N'N'-tetra methyl-uronium hexaflurophosphate (HBTU) and its analogous tetrafluroborate are frequently used condensing agents.

The attachment of the first amino acid to the solid phase support may be carried out using BOC-amino acids in any suitable manner. In one method BOC amino acids are attached to chloromethyl resin by warming the triethyl ammonium salts with the resin. Fmoc-amino acids may be coupled to the p-alkoxybenzyl alcohol resin in similar manner. Alternatively, use may be made of various linkage agents or "handles" to join the first amino acid to the resin. In this regard, p-hydroxymethyl phenylactic acid linked to aminomethyl polystyrene may be used for this purpose. In order to generate the hydrophic group-peptide molecule, the hydrophobic group may be added to the peptide chain using similar techniques. For example, fatty acid molecules may be added to a dipeptide or peptide chain in the same way as that of the Fmoc protected amino acids. The present inventors have also determined and describe herein a molecular model of the inhibitor compounds of the present invention. In view of this information, a person skilled in the art will be able to design non-peptide structures which, in three dimensional terms mimic the compounds of the present invention. It is believed that these non-peptide structures will also mimic the physiological effects of the compounds of the present invention. It is intended that such non-peptide structures are also included within the scope of the present invention.

Accordingly, it is envisaged that the present invention extends to a structure the three dimensional form of which substantially corresponds to the three dimensional form of the compounds of the first or second aspects of the present invention, wherein the structure is capable of inhibiting the lytic activity of cytolytic peptides. In a third aspect the present invention provides a pharmaceutical composition including a compound or structure according to the first, second or third aspect of the present invention and a pharmaceutically acceptable carrier. In a fourth aspect the present invention provides a method of inhibiting a cytolytic peptide which method includes administering to a subject in need thereof an effective amount of a compound or structure according to the first, second or third aspect of the present invention.

It will be appreciated by persons skilled in the art that the compounds of the present invention are essentially amphipathic molecules. It is envisaged that these amphipathic molecules will inhibit cytolytic peptides such as melittin.

It will be understood that an aromatic amino acid is an amino acid which contains a heterocyclic structure, the heterocyclic structure having the ability to undergo electrophilic substitution reactions. Aromatic amino acids therefore include tyrosine, phenylalanine, phenylglycine and tryptophan.

The abbreviations used herein are as follows:

X L-Norleucine

G L-Phenylglycine

Ac Acetyl

Acm acetamidomethyl

(CH)2-C14 2-hydroxy Myristic acid

Chau Chaulmoogric Acid

Cin Cinnamic acid abu Aminobutyric acid pg Phenylglycine

(d) D- amino acids

H- Free amino group

-OH Free carboxyl group

DETAILED DESCRIPTION OF THE INVENTION

In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following examples and Figures in which:- Figure 1: Flow cytometric analysis of the effect of melittin and inhibitors. CEM cells were analysed before (A) or after (B) melittin treatment. Density plots depict propidium iodide penetration (fluorescence) on the verticle axis and forward angle light scatter on the horizontal axis. Cell numbers are indicated by grey shading density. The effect of peptide inhibitors 31 (C) and 38 (D) are shown. In these assays, the inhibitors were added to the cells before the addition of melittin as in Methods. Quadrants were set manually such that 90% of normal undamaged cells were located in quadrant 4. Figure 2: Ultraviolet fluorescence spectra of the tryptophan residue in melittin. Excitation wavelength 290 NM, temperature 24°C. In each plot the solid line is the spectrum of melittin alone, the broken line that of melittin + a two molar excess of IVIFDC and the dotted line that of melittin + an equimolar spin-labelled IVIFDC. In plot A the solvent was me thanol/ water, 60/40: in plot B, pH 7.4,

0.05 M Tris: in plot C, pH7.4, 0.1 Tris, 1.5 M NaCl: in plot D, pH 7.4. 2.0 M phosphate.

Figure 3: EPR spectra of spin-labelled IVIFDC. A = SL-IVIFDC in pH 7.4. 0.05 M Tris buffer. B = SL-IVIFDC in the buffer + equimolar melittin. Spectrum taken at 28^υC, end to end width of spectrum 0.08 mT. Figure 4: Ball and stick model of melittin from the coordinates of Terwillinger and Eisenberg (3).

Figure 5: Diagrams showing relative configuration of modelled inhibitor peptide (IVIFDC) to melittin protein; (a) & (b), relative position of peptide within melittin; the cysteine residue of the peptide (Cysl) does not appear to interact with the melittin surface as it projects away from the protein; (c) & (d), peptide hydrophobic residue He 1, Val 2 and He 3 are in close proximity to protein hydrophobic residues He 20, Leu 16 and Leu 13 respectively, and are therefore likely to be involved in protein-peptide interactions; furthermore. Phe 4 of the peptide is adjacent to Trp 19 of the melittin protein. EXAMPLES

Materials and Methods Synthesis of inhibitors Peptides and fatty acid/peptide compounds were synthesised using an Applied Biosystems 430A Peptide Synthesiser coupled with the FastMoc strategy. Fatty acids were added similarly after the first two or three amino acids were coupled to the resin. RINK resin was used as the substrate, producing the peptide derivatives as amides on cleavage with trifluoroacetic acid. The cysteine side chain was protected by trityl, except in the case where a final protected cysteine was required where acetamidomethyl (Acm) was used. In all cases where cysteine was used (except in the case of the Acm-protected cysteine), all of the peptides were found to be dimeric (disulfides). The Fmoc protected amino acids and the RINK resin were supplied by Auspep Pty Ltd. The peptide derivatives were purified by reverse-phase HPLC(C₁₈) and their integrity confirmed by amino acid analysis and electrospray mass spectrometry. Haemolvtic Assay

The haemolytic effect of melittin in the presence of the inhibitor analogues was determined over the range of 20:1 to 1:1 inhibitor to melittin (w/w). The amount of melittin used was determined from a concentration titration curve and a quantity equal to a limiting concentration was used to allow a more sensitive means of assessing inhibition with the peptides. Peptides were dissolved in dimethyl sulphoxide (DMSO) at 5 mg/ml and diluted our in phosphate buffered saline (PBS). The putative inhibitor peptides were titrated in duplicate by two-fold dilutions (50μl) in 96-well U- bottomed microtitre plates (Nunc, Denmark) and pre-incubated with either 5 or 10 μg/ml final concentration of melittin (50μl) for 1 h at 22°C. After this incubation, 0.6% suspension of washed human red blood cells (lOOμl) were added for a further 1 hour. Plates were centrifuged at 150 x g for 5 min, and 100 μl aliquot's were transferred to a 96-well polyvinylchloride plate (Dynatech Laboratories, Alexandria, Va). Haemolysis was assessed by measurement of optical density at 405 nm with an automatic EAR 400 SF ELISA plate reader (SLT Lab instruments, Groedig/Salzburg, Austria). The percentage of haemolysis was calculated by comparison with absorbances from a buffer blank ("no lysis" control) and a sample treated with 0.1% Triton X-100 ("maximum lysis" control). The degree of peptide inhibition of melittin haemolysis was calculated by comparing the percentage of homologous in the presence and absence of peptide inhibitors. Flow Cytometrv

To further assay the effects of cytolytic peptides on cells, flow cytometry was utilised as described by Weston et al (1994). Flow cytometry uses analysis of the scattering of laser from cells moving in a fluid stream to give information about the state of the cells. Light scattering in the direction of the laser beam (forward scatter) is an indication of cell size. Light scattered at right angle to the beam (side scatter) is an indication of cell granularity, which increases in damaged cells (Shapiro, 1995). In addition, fluorescent molecules can be added to the cells and their specific fluorescence, when excited by the laser beam can be measured by light emission at right angles to the laser beam. This fluorescence can give information about the composition or state of the cells, depending on the specific fluorescent dye used.

CEM human lymphoma cells were used in these experiments. The cells were grown in RPMl 1640 medium containing 10% foetal calf serum. The cells were washed with phosphate buffered saline and resuspended in the same buffer at a density of 1 x 10⁶ cells/ml.

Flow cytometry was performed using a Coulter EPICS® flow cytometer. Illumination was carried out using a 488 nm argon ion laser, 0.25 ml of cell suspension was placed in the cytometer tubes and 1 μl of propidium iodide (1 mg/ml) was added. Inhibitors, dissolved to 5 mg/ml dimethly sulphoxide were then added. After approximately 5000 data points has been collected, the tube was removed, melittin was added to 5 μg and data collection was continued. Weston et al (1994) showed that cells change their 90° light scattering properties when exposed to melittin. We have measured the extent of these changes by but have concentrated our analyses on two additional parameters. The first is measurement of cell membrane integrity by exclusion of the dye propidium iodide. Cells with intact membranes do not take up this dye and do not become fluorescently stained. Cells with damaged membranes take up the dye, which binds to their nucleic acids and becomes brightly fluorescent. Cell viability can therefore be measured at the same time as light scattering and is indicated by the proportion of cells that are not fluorescent in the presence of the propidium iodide. The proportion of cells with forward light scatter below a defined limit could be used as a reliable index of melittin induced changes and a two dimensional plot of forward scatter versus propidium iodide penetration was found to give a good representation of melittin action and inhibitor effect. EPR Spectroscopy

EPR spectra were collected at 20°C using a Varian E109 X-band spectrometer. Centre field was 0.344T, the sweep width 8 mT and the frequency 9.6800000 Ghz. Quantitation of spectra, sample handling and other conditions were as described by Gordon and Curtain (1988). Ultraviolet Fluorescence Spectroscopy

Tryptophan fluorescence was measured at 20°C in a Perkin-Elmer MPF3 fluorescence spectrophotometer using an excitation wavelength of 290 nm. Density Gradient Centrifugation

Large unilamellar vesicles (LUV) were prepared by dispersing dried films of dipalmitoyl phosphatidyl choline (Sigma Mo) in buffer (5mM Hepes, 100 mM NaCl, pH7.4), freezing and thawing the dispersion 10 times and extruding 10 times through two stacked 0.4 μm pore size polycarbonate filters (Nucleopore. Pleasanton Ca) as described by Hope et al. (1985). in order to determine the distribution of the peptides between LUV and buffer, density gradient centrifugation was carried out in Metrizamide™ (Nyegaard, Oslo, Norway) buffer (pH 7.4, 5mM HEPES, 100 mM NaCl) in polycarbonate centrifuge tubes as described by Cornell et al (1988). Six 1ml fractions were collected and their densities determined refractometrically (Rickwood and Birnie. 1975) at 20°C. Melittin (1 mole/100 lipid) was mixed with the LUV and layered on top of a preformed Metrizamide™ density gradient (p= 1.00- 1.28) which was then centrifuged for 245 min at 28000 g. The experiment was then repeated by a 4/1 molar ration of the inhibitor peptide. Ac-IVIFDC- NH₂, was added to the melittin before it was mixed with the LUV. In order to determine whether the inhibitor interacted with the LUV the spin labelled inhibitor was added in a 4/100 lipid/peptide molar ratio to the LUV before centrifugation. The distribution of labelled inhibitor in the gradient fractions was determined by EPR spectroscopy. Molecular Modellinfi

The molecular model of the inhibitor peptide was constructed within GEMM (version 7.89, Cammisa, J., Kim, J.R. and Lee, B.K., 1993). The initial coordinates of this structure were saved and imported into the Sculpt modelling system (Surles et al., 1994), which allows continual energy- minimization of a protein. After modelling the peptide within Sculpt, the coordinates were saved and imported back into GEMM. The coordinates for melittin (Protein Data Bank Gopher://PDB.BNL.GOV:70/ll/file 2MLT.FULL) were also imported into the GEMM environment and both proteins were configured so as to show possible interactions between key amino acid residues. All modelling was performed on as SGI Indy workstation with a lOOmhz R4600PC processor. Results and Discussion Flow Cytometry The changes in forward angle light scatter and propidium iodide penetration induced by melittin are shown in Fig. 1. In this figure the percentages of cells in the various quadrants are characteristic of the effects of the peptides. In a normal cell population, the majority cells have measurements that lie in quadrant 4, but on melittin action the bulk of the cells move to quadrants 1 and 3 indicating increased propidium iodide fluorescence and decreased forward light scatter respectively. It was found that the inhibitory peptides reduced both of these changes, and the reduction increased with increasing excess of inhibitory peptide over melittin concentration (Fig. 1). Inhibition of the effect of melittin on propidium iodide penetration was indicated by a reduced percentage of cells in quadrants 1 and 2 and inhibition of melittin's action on forward light scatter was indicated by a decrease in the percentage of cells in quadrant 3. In most cases, it was found that the relative reduction of propidium iodide penetration was usually similar to the relative decrease in forward light scatter, but some peptides differed slightly in their abilities to affect these two parameters. However, the overall effectiveness of the peptides correlated with their relative efficiency in inhibiting melittin-induced haemolysis. Some peptides showed low toxicity at high concentrations, indicated by changes in the measured parameters, or aggregated the cells, indicated by a large increase in both forward and 90° light scatter. Density Gradient Centrifugation

The results of these experiments using the inhibitory peptide AcIVIFDC-NH₂ are given in Table I. Since the liposomes float on top of the tube (p < 1) and peptides band at p= 1.24 we concluded that the inhibitor acted by preventing the melittin from binding to the LUV. It is therefore likely that the mechanism of inhibition of the action of melittin on biological membranes involves the formation of an inhibitor-melittin complex that cannot enter the lipid bilayer. In the experiments where spin labelled inhibitor was added to LUV only 1.5% of the spins were found to be associated with the LUV, 95% being associated with the p= 1.24 fraction. This distribution suggested that the inhibitor, in its spin-labelled form at least, did not react with the vesicles, confirming the conclusion of Blondelle et al. (1993) that it did not react with the membrane.

Table I: Tryptophan (324nm) fluorescence of Metrizamide™ density gradient fractions of melittin and the inhibitory peptide Ac-IVIFDC-NH₂ in the presence of dipalmitlyl phosphatidyl choline large unilamellar vesicles

Density Melittin fluorescence alone Melittin fluorescence + inhibitor

< 1.00 7.8 1.6

1.06 2.3 1.3

1.12 0.1 0.8

1.18 0.075 1.2

1.24 0.8 6.7

1.28 0.5 1.4

EPR and Ultraviolet Florescence Spectroscopy The EPR spectrum of the spin label on the inhibitor shows that the label is freely rotating with a slight degree of anisotropy. Such a spectrum is to be expected with a label attached through a 2 carbon spacer to a small peptide. The fact that the spectrum is unchanged by the addition of melittin (Fig. 2) indicates that the N-terminal end of the peptide is still capable of free rotation after the inhibitor is bound to the melittin.

In all solvents both the spin labelled and unlabelled inhibitor caused a distinct blue shift in the ultraviolent emission spectrum of the melittin tryptophan residue (Fig. 3) suggesting that the inhibitor was binding close to this residue when the melittin was in a variety of conformations: α-helical for methanol/water, random in low ionic strength buffer and tetrameric at high molar phosphate. The spin labelled inhibitor only gave significant quenching of the spectrum in the methanol/water solvent, suggesting that the N-terminal spin label was only able to contact the tryptophan when the melittin was in the α-helical configuration. Molecular Modelling

Using the secondary structure of melittin as derived from the coordinates of Terwillinger and Eisenberg (1982), Fig. 4, it is expected that the strongly hydrophobic inhibitor interacts with the hydrophobic side of the amphiphilic helix. The fluorescence results suggest that binding of the inhibitor probably involves the tryptophan residue of melittin. This requirement can be accommodated by the observation that all inhibitor peptides have hydrophobics as the first four residues. Phenylalanine, tryptophan and phenylglycine are all effective to various degrees in position 4. Tryptophan or another aromatic at position 19 is known to be crucial for the activity of melittin (Habermann and Kowallek, 1970; Blondelle and Houghten, 1991; 1991a) and it is possible that the inhibitor masks this residue. The activity of peptides 31, 35, 38, 31B and 47 supports this hypothesis as the aspartic acid residue would be adjacent to the lys/arg region. However this is obviously not greatly important for inhibition as the substitution of asparagine for aspartic acid has no deleterious effect. It is not surprising that the introduction of a hydrophilic residue (serine) in position 3 destroys activity as it would interfere with the hydrophobic interaction. Fig. 5 shows the relative position of the inhibitor superimposed by modelling on the Terwillinger and Eisenberg plot of the melittin structure, assuming interaction of the phenylalanine residue of the inhibitor with the tryptophan melittin. The I V I section of the inhibitor lies adjacent to the hydrophobic area of melittin defined by the residues V8. L9, L13, L16 and 120. The aspartic acid residue is in the vicinity of K23 and R24 where polarity and hydrophilicity would be expected to encourage interaction. In this model of the melittin/inhibitor complex the sixth residue is distant from the melittin backbone and thus does not appear to be directly involved in interaction.

Inhibition of Haemolysis

Compounds were assayed for their inhibitory effects on melittin haemolysis at various ratios of peptide to toxin (w/w) ranging from 20:1 to 1.2:1. A summary of the inhibitory effects of a number of compounds on melittin haemolysis at a concentration of 10 μg/ml is shown in Table II. A summary of results obtained to date is shown in Table III. Peptides with a free cysteine-SH are readily oxidised to form dimers. Accordingly, a number of the peptides tested were in dimeric form. The initial peptide sequence tested (peptide 31), for example, was confirmed to be a dimer and showed 100% inhibition at a ratio of peptide to toxin of 10:1. Dimerisation using a terminal lysine instead of cysteine (peptide 39) was also effective in inhibiting haemolysis. Dimerisation was not critical, as the monomeric form of peptide 31 (peptide 31B) caused comparable inhibition to the dimeric form.

What appears to be critical for the peptide inhibitory activity is that the first three amino acids are hydrophobic. As shown in Table II, substitution of isoleucine with serine in peptides 32, 36 and 37 resulted in complete loss of activity.

Table II

PEPTIDE SEQUENCE % INHIBITION OF HAEMOLYSIS

RATIO (Inhibitor:Mβlittin, W/Wl

20 10 5 2.5 1.2

31 Ac-IVIFDC-NH2 100 100 90 59 37

31B Ac-IVIFDC(Acm)-NH2 100 93 80 21 10

310 Ac-IVIFDC(S03H)-NH2 5 5 0 0 0

32 Ac-LISWIC-NH2 0 0 0 0 0

320 Ac-LISWIC(S03H)-NH2 10 9 0 0 0

35 Ac-IVIWDC-NH2 100 72 50 45 30

350 Ac-IVIWDC(S03H)-NH2 10 10 2 2 0

36 Ac-IVSWDC-NH2 0 0 0 0 0

37 AoLISWDC-NH2 0 0 0 0 0

38 Ac-IVILDC-NH2 100 100 100 100 92

380 Ac-IVILDC(S03H)-NH2 42 36 11 6 0

39 Ac-2(IVIFD)K-NH2 93 67 36 13 10

40 Ac-(NorLβu)VI(pg)DC-NH2 100 95 96 81 21

400 Ac-(NorLeu)VI(pg)DC(S03H)-NH2 3 53 36 25 0

43 H-IVIFD-NH2 0 0 0 0 0

47 Ac-IVIFDM-NH2 100 100 100 100 41

AP5 (C16)FDC-NH2 100 100 100 92 57

60 (C14)FDC-NH2 92 96 95 74 50

60A (C14)FDC(S03H)-NH2 - 66 100 48 0

60B (C14)FDC(SCMC)-NH2 100 100 92 90 48

AP6 (C13)FDC-NH2 60 95 98 89 78

AP4 (C12)FDC-NH2 89 98 94 89 52

65 (C11)FDC-NH2 16 23 13 35 -

AP3 (C10)FDC-NH2 100 100 95 94 68

AP2 (C9)FDC-NH2 100 63 57 62 55

62 (C8)FDC-NH2 0 0 0 0 0

AP (C6)FDC-NH2 41 33 73 73 73

73 (C14)NC-NH2 80 58 35 26 17

74 (C14)FNC-NH2 98 96 83 50 40

JB-APl (C9)FDS-NH2 0 0 4 4 4 JB-AP2 AC-IVIFDS-NH2 100 85 29 18 12

AP7 (C9)WNC-NH2 58 28 12 4 4

AP8 AC-IVIFDV-NH2 100 92 73 41 21

AP9 Ac-IVIFNAbu-NH2 96 90 75 42 24

AP10 (H)-IVIFNC-NH2 45 41 38 19 11

AP1 (H)-IVIFNAbu-NH2 51 30 22 15 9

AP12 AC-IVIFNC-NH2 100 98 91 63 45

API 3 Ac-IVIFDC-(OH) 100 100 100 100 100

AP14 (Cl6)FNAbu-(OH) 100 100 97 89 58

AP15 (chaulmoogric acid)-FNAbu-(OH) 0 21 83 74 23

AP16 (cinnamic acid)-FNAb u-(OH) 8 11 14 17 12

CC12 AC-LLLHNM-NH2 5 5 12 0 0

69 Ac-(NLeu)3(Pg)N(Pen- Acm)-NH2

100 100 99 88 55

Table HI

Peptide Sequence Molecular Inhibition

Number Weight

API C6-FDC-NH2 481 +/-

AP2 C9-FDC-NH2 523 +

AP3 C10-FDC-NH2 537 + +

AP4 C12-FDC-NH2 565 +

AP5 C16-FDC-NH2 621 + +

AP6 C13-FDC-NH2 592 +

AP7 C9-WNC-NH2 561 +/-

AP8 Ac-IVIFDV-NH2 746 +

AP9 Ac-IVIFN-Abu-NH2 730 +

AP10 IVIFNC-NH2 707 +/^■

AP11 IVIFN-Abu-NH2 688 +/-

AP12 Ac-IVIFNC-NH2 748 +

AP13 Ac-IVIFDC-OH 751 + +

AP14 Cl6-FN-Abu-NH2 602 + +

AP15 Chau-FN-Abu-OH 626 +/-

ΛP16 Cin-FN-Abu-OH 494 +/-

31 Λc-IVIFDC-NH2 749 + +

31b Ac-IVIFDC(-Acm)-NH2 820 +

31o Ac-IVIFDC(-S03H)-NH2 -

32 Ac-LISWIC-NH2 774 -

32o Ac-LISWIC(-S03H)-NH2 -

35 Ac-IVIWDC-NH2 + +

35o Ac-IVIWDC(-S03H)-NH2 -

36 Ac-IVSWDC-NH2 776 -

37 Ac-LISWDC-NH2 776 -

38 Ac-IVILDC-NH2 715 + +

38o Λc-IVILDC(-S03H)-NH2 +/-

39 (Ac-IVIFD)2K-NH2 1404 +

40 Ac-(NorLeu)VI(pg)DC-NH2 + +

40o Ac-(NorLeu)VI(pg)DC(-SO3H)-NH2 + 43 H-IVIFD-NH2 646 -

47 Ac-IVIFDM-NH2 777 + +

54 Ac-IVIFDC(-Acm)-NH2 (d) 820 +

60 C14-FDC-NH2 592 +

60a C14-FDC(-S03H)-NH2 +

60b Cl4-FDC(-SCMC)-NH2 + +

62 C8-FDC-NH2 509 -

62o C8-FDC(-S03H)-NH2 -

65 C11-FDC-NH2 553 +/-

69 Ac-(NorLeu)3(pg)N(penicillamine)(-Acm)-NH2 758 + +

73 C14-NC-NH2 444 +

74 C14-FNC-NH2 591 + +

84 H-LLLFNI-OH 732 + +

84Ac Ac-LLLFNI-OH +

CC12 Ac-LLLHN -NH2 780 - jB-APl (C9)FDS-NH2 -

JB-AP2 Ac-IVIFDS-NH2 734 +

7-10b (OH)2-Cl4-cystamine 400 +/-

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

REFERENCES:

Blondelle, S.E.. Simpkins, L.R. and Houghton, R.A (1993) Peptides 1992, 761-762, CH. Schneider and A.N Eberie (Eds), ESCOM Science Publishers BV.

Cornell, B.A., Weir, L.E. and Separovic, F. (1988) Eur. Biophys. J. 16, 113-119.

Dempsey, C. E (1990) BIOCHEM. BIOPHYS. ACTA 1031, 143-161

Gordon, L.M. and Curtain, C.C. (1988) Advances in Membrane Fluidity (Aloia, R.C., Curtain, C.C, and Gordon, L.M. eds.), Alan R. Liss. New York, Vol. 1. Pp. 25-88.

Rickwood, D. and Birnie, G.D. (1975) FEBS Lett. 50, 102-110.

Schroder, E., Lubke, K., Lehman, M and Beetz, I. (1971) EXPERIMENTIA 27, 764-765.

Shapiro, H.M. (1994) Practical Flow Cytometry (3rd Edition) Wiley-Liss New York.

Surles, M.C., Richardson, J.S.,. Richardson, D.C, and Brooks, F.P. Ir. (1994). Protein Sci. 3, 198-210.

Terwillinger, T.C and Eisenberg, D (1982) J. BIOL. CHEM 257, 6016-6022.

Werkmeister, J.A., Kirkpatrick, A., McKenszie, J.A. and Rivett, D.E. (1993) BIOCHEM.BIOPHYS. ACTA 1157, 50-54.

Weston, K.M., Alsalami, M. and Raison, R.L (1994) CYTOMETRY 15, 141- 147.

Claims

Claims:

1. A compound of the formula:

R_rA-B-X wherein

Rj is a hydrophobic group of substantially the same size and charge as a fatty acid acyl group with a carbon chain of 3 to 19 carbon atoms,

A is absent or a hydrophobic amino acid, B is an amino acid, and

X is an amino acid, with the proviso that if A is phenylalanine, B is aspartic acid and X is cysteine or cysteic acid, then R_! is not a fatty acid acyl group with a carbon chain of 8 carbon atoms.

2. A compound according to claim 1 wherein R_t is a fatty acid acyl group with a carbon chain of 3 to 19 carbon atoms,

3. A compound according to claim 1 or claim 2 in which R- is a fatty acid acyl group with a carbon chain of 6 to 19 carbon atoms.

4. A compound according to any one of claims 1 to 3 in which R, is a fatty acid acyl group with a carbon chain of 10 to 16 carbon atoms.

5. A compound according to any one of claims 1 to 4 in which A is an aromatic amino acid.

6. A compound according to claim 5 in which A is selected from phenylalanine, tryptophan, tyrosine and phenylglycine.

7. A compound according to any one of claims 1 to 6 in which B is a hydrophilic amino acid.

8. A compound according to claim 7 in which B is an amino acid with a positive or negative charge.

9. A compound according to claim 8 in which B is selected from asparagine, glutamine, aspartic acid and glutamic acid.

10. A compound according to any one of claims 1 to 9 in which X is a hydrophobic amino acid.

11. A compound according to any one of claims 1 to 9 in which X is an amino acid of the general formula:

NH₂-CH(CH₂)R-COOH wherein R is H, (CH₂)nCH₃, CH(CH₃)₂, or CH(CH₃)C₂H₅ and n is 0 to 3.

12. A compound according to claim 11 in which X is isoleucine.

13. A compound according to any one of claims 1 to 9 in which X is a sulphur containing amino acid.

14. A compound according to claim 13 in which X is selected from cysteine, cystine, cysteic acid, methionine, penicillamine and a cysteine derivative in which the -SH group is blocked (e.g. by acetamidomethyl).

15. A compound selected from (C₁₄)-Phe- Asp-Cys- NH₂, (C₁₄)-Phe-Asn- Cys- NH₂, (C₁₄)-Asn-Cys- NH₂, (C₁₄)-Phe-Asp-CyS0₃H- NH₂, (C₁₄)-Phe-Asp- CySCMC- NH₂, (C_g) -Phe- Asp-Cys- NH₂, (C₁₀)-Phe-Asp-Cys- NH₂, (C₁₂)-Phe- Asp-Cys- NH₂, (C₁₃)-Phe-Asp-Cys- NH₂, (C₁₆)-Phe-Asp-Cys- NH₂, and (C₁₆)- Phe-Asn-Abu- NH₂.

16. A peptide selected from Ac-IVIWDC-NH₂, Ac-IVIFDC(Acm)-NH₂, Ac- IVIFDS-NH₂, Ac-rVIFDV-NH₂, Ac-XVIGDC-NH₂, Ac-rVIFNC-NH₂, IVIFNC- NH₂, Ac-IVIFDM-NH₂, (Ac-IVIFD)₂K-NH₂, Ac-IVILDC-NH₂, Ac-

(NorLeu)VI(pg)DC-NH2, Ac-IVIFN-Abu-NH₂, Ac-IVIFN-Abu-NH₂ and Ac- LLLNFI-NH₂.

17. A pharmaceutical composition including a compound or structure according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

18. A method of inhibiting a cytolytic peptide which method includes administering to a subject in need thereof an effective amount of a compound or structure according to any one of claims 1 to 16.