INHIBITORS OF PLA2

Abstract: The present invention relates to a three-dimensional structure which inhibits the activity of PLA2. In one particular aspect the structure has a conformation and polarity such that it binds to (i) at least one amino acid in the N-terminal helix region of PLA2; and/or (ii) at least one amino acid in the region spanning residues (70-77) of PLA2. In another aspect the structure is a peptide comprising D-amino acids which has a sequence corresponding to the reverse sequence of a region between residues (69 and 75) of PLA2.

Description: Inhibitors of PLA Z Field of the Invention The present invention relates to three dimensional structures which inhibit the activity of phospholipases (PLA 2 s), In another aspect the present invention relates to a peptide based on the sequence KYSLF which comprises D-amino acids. In addition, the present invention relates to pharmaceutical compostions including as the active ingredients these structures, and to methods of treatment involving administration of these structures, Background of the Invention Secretory phospholipases A 2 (sPLA 2 s) are a family of calcium dependent 14kD enzymes, that catalyse the hydrolysis of the sn-2 fatty acyl ester bond of phospholipids (1). These enzymes, which were first described as components of snake venoms and later in mammals, are classified into two major classes, Type I and Type II, based on their primary structures. Type I PLA 2 of mammalian origin is mainly found in the pancreas (2) while the Type II enzyme is stored in secretory granules in blood platelets, macrophage and neutrophils (3, 4) and in tissues, is localised in mast cells, paneth cells and chondrocytes (5, 6). It is also found in fluids derived from patients with inflammatory conditions (7, 8) and is induced in several cell types in response to inflammatory stimuli (5). Type II PLA 2 has therefore been implicated in the pathogenesis of several inflammatory diseases in humans such as rheumatoid arthritis and septic shock (56,57). Murine, inhibitory monoclonal antibodies raised against synovial PLA 2 have demonstrated pre-clinical efficacy. Accordingly, there is interest in the development of compositions which inhibit the enzymatic activity of PLA 2 . Despite differences in their primary sequences (approx. 30% homology only), crystal structures of PLA 2 s from bovine pancreas (Type 1) and human synovial fluid (Type II) are almost superimposable and, not surprisingly, the active sites from both types of enzymes are virtually identical (9). Asp 99 and His 48 form an essential catalytic dyad in the fashion of serine proteases (10). Tyr 52 and Tyr 73 appear to be associated with these two residues via a hydrogen bonding network, but there is evidence (11) that Tyr 52 is not essential for the catalytic reaction. From structure-function studies of Type I and II enzymes, the first eight residue at the N-terminus together with Tyr 69, based on the Renetseder et al numbering system (see Figure ll)(12,12a), are thought to play a functional role in interfacial binding of the enzyme to aggregated substrate (13). Both chemical modification (14) and site-directed mutagenesis (15-17) studies have shown that the N-terminal residue is crucial for activity. Further studies with Type I PLA 2 7 - 18) have shown that significant alterations of the invariant hydrophobic face of the N- terminal amphipathic helix (residues 2, 5 or 9) are detrimental to enzyme activity. Residue 10 is necessary for interfacial binding and changes in other residues have modest or no effect on catalytic activity. Production of the fully active enzyme depends on conformational changes of the N-terminal helical region which occur, firstly, on interfacial binding to the phospholipid micelle and secondly on substrate binding at the active site (19). NMR studies of porcine pancreatic PLA 2 indicated that the first few residues of the N-terminus are not -helical in solution, but become helical and rigid on formation of a ternary complex with micelles or a substrate analogue (20-22). X-ray crystallographic studies showed that the N- terminal and β-loop region (residues 62-73) are held by hydrogen bonding in the active enzyme conformation and form the catalytic network involving His 48, Tyr 52, Tyr 73 and Asp 99 (23-26). High affinity inhibitors of the human Type II PLA 2 have been identified which bind either reversibly or irreversibly. Reversible competitive inhibitors of PLA 2 have been derived from phospholipid analogues. These substrate analogues interact with the catalytic residues in the enzyme's active site thereby perturbing substrate binding. These inhibitors have been designed to emulate the putative tetrahedral intermediate that forms during hydrolysis of the substrate (24,27,28) or are based on non-hydrolyzable phospholipid analogues (29,30). Schevitz et al (31) have generated a potent and selective inhibitor of human type II PLA 2 which binds at the active site of the enzyme. The initial lead compound was identified by large scale screening of library compounds and then further improved by minimising the interactions of other substrate analogues in the active site. Manoalide, a natural product derived from sponge, and its analogues have been demonstrated to possess anti-inflammatory properties associated with inhibition of PLA 2 (32). These compounds inhibit PLA 2 by a mechanism that does not directly involve the catalytic site. The inhibitory reaction is irreversible and involves covalent modification of specific lysine residue of the enzyme (33). There are a number of cases where knowledge of protein structure has contributed to the design of new therapeutic agents and/or an understanding of the action of existing agents. Examples of these include influenza virus neuraminidase (34), HIV protease (for a selected sample see reference 35), purine nucleoside phosphorylase (36) and thymidylate synthase (37). The use of peptides as lead structures in the development of non-peptidic drugs is therefore achievable. Modern computational technologies have improved these drug development strategies. Design strategies for peptide leads have been previously described (38). In order to understand how an enzyme binds an inhibitor, automated docking methods are invaluable where there is a knowledge of the enzyme structure from X-ray crystallographic (39) or nuclear magnetic resonance methods (40). The interactions are fundamental to molecules and can be described by energy terms. These methods, or variations thereof, in the drug design process are generally referred to as "structure-based drug design" (41). Many more methods become viable as computational technology grows. There are existing computer technologies for probing the target molecule for docking sites of lead compounds. Compounds resulting from such computations can be used as a scaffold of essential features with which to search databases for improved inhibitory structures. The technologies overlap because of their synergy (42-47). The present inventors have now applied these technologies to the lead peptide FLSYK and its target molecule human type II PLA 2 . The present inventors have previously demonstrated that the pentapeptide FLSYK inhibits the enzyme activity of Type II phospholipase A 2 (WO 93/01215). It was postulated that this inhibition is mediated by the peptide binding to the amino terminal residues of the enzyme and blocking the reaction either by blocking substrate access to the hydrophobic channel or by distorting the structure sufficiently to prevent correct orientation of the substrate. Summary of the Invention The present inventors have now identified the regions of PLA 2 which interact with the peptide FLSYK. Further, the present inventors have identified the components of FLSYK required for inhibition of PLA 2 . Unlike most previously identified PLA 2 inhibitors, the FLSYK peptide does not interact with the active site of PLA 2 . The identification of the PLA Z contact regions and inhibitory components of FLSYK enable the design of representative pharmacophore structures which may form the basis for lead structures in the development of new PLA 2 inhibitors. Accordingly, in a first aspect the present invention provides a structure not comprised solely of naturally occurring amino acids which has a conformation and polarity such that the structure binds to (i) at least one amino acid in the N-terminal helix region of PLA 2 ; and/or (ii) at least one amino acid in the region spanning amino acid residues 70-77 of PLA 2 ; wherein binding of the structure to regions (i) and (ii) inhibits the enzymatic activity of PLA 2 . By "naturally occurring amino acids" we mean the L-amino acids Tyr, Gly, Phe, Met, Ala, Ser, He, Leu, Thr, Val, Pro, Lys, His, Gin, Glu, Trp, Arg, Asp, Asn and Cys. In a preferred embodiment the structure binds to (i) at least one amino acid in the N-terminal helix region of PLA 2 ; and (ii) at least one amino acid in the region spanning amino acid residues 70-77 of PLA 2 ; In a further preferred embodiment the structure is a non-peptide structure. By "non-peptide structure" we mean any structure other than a compound which consists entirely of naturally occurring amino acids linked by peptide bonds. The term therefore includes within its scope chimeric molecules which include peptide portions, and molecules in which, for example, the amide linker between the amino acid side chains is replaced with an organic link with similar properties. In a preferred embodiment of the invention the PLA 2 is human PLA 2 . In a further preferred embodiment the non-peptide structure binds to at least one amino acid, and more preferably at least two amino acids, in the N-terminal helix region selected from the group consisting of Asnl, Val3, Asn4, His6, Arg7, LyslO and Leul2. In a further preferred embodiment the non-peptide structure has substantially the same spatial geometry and polarity as the peptide FLSYK wherein the peptide FLSYK is in a conformation which allows the peptide to bind to (i) at least one amino acid in the N-terminal helix region of PLA 2 selected from Asnl, Val3, Asn4, His6, Arg7, LyslO and Leul2; and/or (ii) at least one amino acid in the region spanning amino acid residues 70-77 of PLA 2 . The spatial geometry and polarity of the peptide FLSYK which allows the peptide to bind to the N-terminal helix region and the region spanning residues 70-77 may be described as follows. The spatial geometry is delineated by the hydrogen bonding network provided by the backbone of the residues in FLSYK to residues 1,3,4,7,72,73,74 and 75 of PLA 2 . The spatial geometry may be maintained by altering the side chain moieties as follows: Position 1 (a) Hydrophobic group, can be longer than Phe: and/or (b) Aromatic residue better than linear side chain, e.g. naphthylalanine. Position 2 (a) Hydrophobic group, preferably Leu; and/or (b) Chain of reasonable length side chain, preferably to interact with Arg 7 (e.g. unbranched nor-Leu). Position 3 (a) Polar group on side chain (for H-bonding); and/or (b) Peptide chain able to fold around this residue. Position 4 (a) Hydrophobic group larger than Phe (e.g. 2- naphthylalanine); and/or (b) Rotation around the N-Cα bond not restricted; and/or (c) Nor-Leu may be effective because of probable interaction with Val 3. Position 5 (a) Positive charged group with at least 3 methylene groups on side chain. Preferred configurations of the FLSYK peptide exhibit closely related conformations in which the termini are in proximity (e.g. within hydrogen bonding distance or as in a cyclic peptide formed from covalent links between the termini). Most preferred configurations are as follows: (i) The aromatic ring of tyrosine of FLSYK is stacked over and juxtaposed to the ring of Phe 75 of human PLA2. The hydroxyl of the ring is also in proximity to the main chain of FLSYK. Leucine makes hydrophobic contact at Arg 7. The configuration of FLSYK may be as depicted in Figure 4A. (ii) The phenylalanine contacts Arg 7, Tyr 73 and Lys 74. the tyrosine contacts Val 3 and lysine contacts Ser 72. The configuration of FLSYK may be as depicted in Figure 4B. (iii) The backbone at tyrosine contacts Asp 4, phenylalanine contacts Ser 72 and Tyr 73. Leucine and serine contact the Arg 7, as does phenylalanine. Lysine is in contact with Val 3. The configuration of FLSYK may be as depicted in Figure 4C. The present inventors have also determined scaffold structures for use in the design of organic-based inhibitors of PLA 2 . The scaffold structures were determined by plotting the phi-psi angles of the α-carbon atoms of cyclic peptide FLSYR on a Ramachandran Plot. These scaffold structures provide a template on which reactive groups with similar functionality to the peptide may be placed. Accordingly, in a second aspect the present invention provides a non-peptide structure which has a backbone conformation substantially identical to the backbone conformation of a cyclic pentapeptide wherein the bonds joining the α-carbon atoms of the five amino acids in the pentapeptide are such that (i) α-carbonl has (a) a phi angle of between -125.1 and - 49.3 and a psi angle of between -81.3 and -20.6, or (b) a phi angle of between 55.4 and 77.5 and a psi angle of between -85.0 and -65.8; (ii) α-carbon2 has a phi angle of between -144.8 and -72.1 and a psi angle of 22.6 and 117.0; (iii) α-carbon3 has (a) a phi angle of between -69.2 and - 32.0 and a psi angle of between 116.3 and 137.6, or (b) a phi angle of between 13.9 and 66.3 and a psi angle of between 80.0 and 110,1; (iv) α-carbon4 has (a) a phi angle of between -109.5 and - 47.9 and a psi angle of between 113.5 and 166.9, or (b) a phi angle of - 60.2 and a psi angle of -68.0; and (v) α-carbon5 has (a) a phi angle of between 78.0 and 120.5 and a psi angle of between 58.0 and 97.3, or (b) a phi angle of between 53.1 and 118.1 and a psi angle of between -146.0 and -67.0. or (c) a phi angle of -71.1 and a psi angle of -73.2. In a third apect the present invention also provides a method of generating a potential inhibitor of PLA 2 which method includes generating a compound which has a backbone conformation substantially identical to the backbone conformation of a cyclic pentapeptide wherein the bonds joining the α-carbon atoms of the five amino acids in the pentapeptide are such that (i) α-carbonl has (a) a phi angle of between -125.1 and - 49.3 and a psi angle of between -81.3 and -20.6, or (b) a phi angle of between 55.4 and 77.5 and a psi angle of between -85.0 and -65.8: (ii) α-carbon2 has a phi angle of between -144.8 and -72.1 and a psi angle of 22.6 and 117.0; (iii) α-carbon3 has (a) a phi angle of between -69.2 and - 32.0 and a psi angle of between 116.3 and 137.6, or (b) a phi angle of between 13.9 and 66.3 and a psi angle of between 80.0 and 110.1; (iv) α-carbon4 has (a) a phi angle of between -109.5 and - 47.9 and a psi angle of between 113.5 and 166,9, or (b) a phi angle of - 60.2 and a psi angle of -68.0; and (v) α-carbon5 has (a) a phi angle of between 78.0 and 120.5 and a psi angle of between 58.0 and 97.3, or (b) a phi angle of between 53.1 and 118.1 and a psi angle of between -146.0 and -67.0, or (c) a phi angle of -71.1 and a psi angle of -73.2 and testing the compound for inhibitory activity against PLA 2. The present inventors have also found that the FLSYK equivalent D-peptide, synthesised with the reverse sequence, inhibits the activity of Type II PLA Accordingly, in a fourth aspect the present invention provides a peptide which inhibits the enzymatic activity of Type II PLA 2 , wherein the peptide comprises D-amino acids and includes an amino acid sequence which corresponds to the reverse sequence of a region between residues 69 to 75 of the PLA 2 . In a preferred embodiment of this aspect of the invention the peptide has an amino acid sequence which corresponds to the reverse sequence of the region between residues 70 to 74 of PLA 2 . In a preferred embodiment of the present invention the PLA 2 is human PLA 2 . In a further preferred embodiment the peptide is a pentapeptide which consists of D-amino acids. In a further embodiment the peptide has the following formula: A1-A2-A3-A4-A5-A6-A7 in which Al is H or one or two D-amino acids A2 is k or r or h or D-citrulline A3 is y or q or D-2,naphthylalanine A4 is s or t or c or D-homoserine A5 is 1 or v or i or D-nor-leucine A6 is f or y or w or D-norleucine or D- 2 ,naphthylalanine A7 is OH or one or two D- amino acids. Throughout the description and claims of the present application, D-amino acids are distinguished from L-amino acids by representation by small case letters, wherein the letters are the standard single letter amino acid symbols. For example, "f ' represents D- phenylalanine whereas "F" represents L-phenylalanine. In another preferred embodiment of the present invention Ai is H and Λ7 is OH. In a further preferred embodiment the peptide is kyslf. It will be appreciated by those skilled in the art that a number of modifications or substitutions may be made to the D- peptides of the present without substantially decreasing the biological activity of the peptide. This may be achieved by various changes, such as insertions, deletions and substitutions, either conservative or non- conservative in the peptide sequence where such changes do not substantially decrease the biological activity of the peptide. By conservative substitutions of the side chains the intended combinations may embrace polarity (n,q,s,t,y;d,e;k,r,h), hydrophobicity (v.i,l,m,f.w,y,k) and aromaticity (f,y,w). It may also be possible to add various groups (which include organic molecules and peptidomimetics) to the peptide of the present invention to confer advantages such as increased potency without substantially decresing the biological activity. Further, the peptides may be cyclic peptides. Although the use of D-peptides in biological systems has been previously reported (9,10), the efficacy of D-peptides is critically dependent on the mode of interaction of the peptide with the enzyme. The present inventors have found that the side chains of the sequence kyslf effectively mimic those in FLSYK, although the direction of the chain is reversed, the backbone NH and C=0 of the amide links are exchanged and the charged carboxy and amino termini reside on different amino acids. The present inventors have therefore found that the sequence and side chain interactions of the amino acids are of major importance in the inhibition of PLA 2 by peptides from the region spanning residues 69-75. Further, the backbone hydrogen bonding and ionic interactions at the N- and C-termini appear to have little effect on inhibition of PLA 2 by these peptides. A person skilled in the art will therefore appreciate that other peptide and non-peptide compounds which provide substantially the same spatial geometry and polarity as the D-peptides of the present invention should also mimic the inhibitory effects of the D-peptides of the present invention. It is intended that such peptides and compounds are also included within the scope of the present invention. Accordingly, the present invention extends to a compound wherein the spatial geometry and polarity of the compound substantially corresponds to that of a peptide according to the third aspect of the present invention, wherein the compound is capable of inhibiting the enzymatic activity of Type II phospholipase A2. It will be appreciated that D-peptide inhibitors of the present invention provide advantages over the previously disclosed L- peptide inhibitors. Naturally occurring proteins consist almost exclusively of L-amino acids and their breakdown to the constituent amino acids is effected by enzymes that have evolved to cleave L-amino acids only. Consequently, inhibitory molecules consisting of D-amino acids would be expected to have much longer life time in biological systems. The present inventors have confirmed that the D- pentapeptide, kyslf. inhibits human synovial Type II PLA 2 to approximately the same degree as does the L-peptide FLSYK. The resistance of the D-peptide to proteolytic digestion should enhance its attraction as an inhibitor for in vivo use. In a fifth aspect the present invention provides a composition for use in treating a subject suffering from septic shock, rheumatoid arthritis and/or other inflammatory diseases, the composition including a therapeutically acceptable amount of a structure or peptide of the present invention and a pharmaceutically acceptable carrier. In a sixth aspect the present invention provides a method of treating septic shock and/or inflammatory disease in a subject which includes administering to the subject a structure or peptide of the present invention. It will be appreciated by those skilled in the art that the structure of the present invention provides a model structure which may be used to design or screen for compounds (either naturally occurring or synthetic) which have PLA 2 inhibitory activity. Accordingly, in a seventh aspect the present invention provides a method of screening an agent for potential PLA 2 inhibitory activity which method includes analysing the structure of the agent for similarities with the three dimensional structure of the peptide FLSYK. wherein the peptide FLSYK is in a conformation which allows the peptide to bind to (i) at least one amino acid in the N-terminal helix region of PLA 2 selected from Asnl, Val3, Asn4, His6, Arg7, LyslO and Leul2; and (ii)at least one amino acid in the region spanning amino acid residues 70-77 of PLA 2 . In an eighth apect the present invention provides a method of generating a potential inhibitor of PLA 2 which method includes generating a compound which has a conformation which is substantially identical to the three dimensional structure of the peptide FLSYK, wherein the peptide FLSYK is in a conformation which allows the peptide to bind to (i) at least one amino acid in the N-terminal helix region of PLA 2 selected from Asnl, Val3, Asn4, His6, Arg7, LyslO and Leul2; and (ii)at least one amino acid in the region spanning amino acid residues 70-77 of PLA 2 and testing the compound for inhibitory activity against PLA 2. 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: The isolated peptide studied, showing the bonds that are allowed to rotate. Figure 2 (A and B): Energy profile of models. The calculated energy of human Type II PLA 2 and the docked pentapeptides FLSYK (2A) and TVSYT (2B) plotted against the identifying number of the conformation. Figure 3: Frequency analysis of number of contacts from FLSYK against residue number of human Type II PLA 2 . It shows only two small groups of amino acid residues in PLA 7 are involved. Figure 4 (A to C): Stereo diagrams of three representative generic conformations from docking calculations. A. Group I conformation number 173; B. Group II conformation number 242; C. Group II conformation number 435. Figure 5 (A and B): (A) Synthetic peptides FLSYK (0.4 mg/ml). and NLVNFHRMIKLTTG (0.6 mg/ml). were incubated in 20mM Tris HCl pH 8.5 at room temperature. Aliquots (10 μl) were taken at Ohrs, 22hrs and lOOhrs and analysed by HPLC as described in Materials and Methods. Retention times were 16.0 min and 19.0 min for the individual peptides FLSYK and residues 1-14 respectively. Asterisk indicates peak fraction collected for further analysis. (B) The peak fraction was subjected to N-terminal sequence analysis. Amino acid residues appearing in each cycle are identified by their amino acid. Repetitive yields for each amino acid derivative in the first four sequencing cycles are shown. Figure 6 (A and B): Association of FLSYK with Residues 67-75 of Human Type II PLA 2 . A. Shows the complex of the two peptides (asterisk) formed after 14 days at room temperature. B. Is the mixture at zero time. Figure 7: Conceptual presentation of inhibitor binding to PLA 2 (for explanation see text). Figure 8 (A and B) : (A) Energy conformations of docked cFLSYR (A) and (B) frequency analysis of number of contacts from cFLSYR against residue number of type II PLA 2 . Figure 9 (A to E) : Ramachandran Plots showing the phi-psi angles for each of the α-carbon 1 (A), α-carbon 2 (B), α-carbon 3 (C). α-carbon 4 (D) and α-carbon 5 (E) in cFLSYR. Figure 10(A to D): Inhibitory analogues of PLA 2 . (A) FLSYK( D ), TVSYT (O), LSYKF ( O ). (B) FLSYK( D ), TVSYT( ), FLTYK( O ). (C) FLSYK( D ), TVSYT (< ), FLS(2-Naphthylalanine)K ( O ), (2- Naphthylalanine)LSYK ( Δ ). (D) FLSYK( D ), TVSYT ( ). cyclic FLSYR ( O ). Figure 11: Amino acid sequence of the human synovial fluid PLA2. The amino acids positions coloured (•) represent gaps in the sequence and are used to standardise the numbering according to Renetseder et al (12.12a). Figure 12 (A and B): (A) Inhibition curves for D-peptides kyslf ( O) (residues 70-74 as the reverse sense) and (B) fkysl (residues 71-75 in the reverse sense) ( Δ ) are analysed. PLA2 L-FLSYK ( ϋ ) and TVSYT ( jare included as controls. Examples Materials and Methods Purification of human type II PLA 2 Human Type II PLA 2 was purified from conditioned medium generated from stably-transfected Chinese hamster ovary cell line (5A2) expressing 350 μg per litre cloned enzyme. Conditioned medium was centrifuged at low speed and the pellet, containing 90% of the sPLA 2 , was extracted with 10% (w/w) ammonium sulphate, dialysed into phosphate buffered saline solution and purified using polysulfoethyl aspartamide strong-cation exchange chromatography (4.6 x 200 mm column, PolyLC, 0.5M KCI gradient, buffer system 25mM KH 2 P0 4 , pH3.0. acetonitrile 7:3 v/v). The active PLA 2 fraction was further purified by reverse -phase HPLC (Aquapore RP300 column. 1 x 100mm, Perkin Elmer, 0 to 70% acetonitrile gradient in 0.1% TFA). The fraction was pure as judged by gel electrophoresis followed by silver staining (50). The identity of the enzyme was confirmed by Western blotting using antisera raised to synthetic peptides of human Type II PLA 2 (data not shown). The substrate specificity of the purified enzyme showed a preference for phosphatidylethyanolamine over phosphatidylcholine (3). Enzyme was stored at 4°C in 35% acetonitrile in 0.1% TFA and diluted immediately prior to use. The enzyme was initially quantified by amino acid analysis and the concentration was checked routinely using an ELISA developed in our laboratory (8). Snake venom PLA 2 from the Crotalus durissus and Crotalus atrox were purchased from Boehringer Mannheim and Sigma, respectively. They were stored according to the manufactures' instructions and used without further purification. Peptide synthesis Peptide synthesis was carried out in a peptide synthesiser (Applied Biosystems Model 430A) using t-butyloxycarbonyl chemistry and peptides were recovered by hydrogen fluoride cleavage (Auspep, Australia). Some peptides were supplied by Chiron Mimotopes (Australia). All peptides were purified using reverse phase HPLC. Peptide sequences and purity were confirmed by N-terminal sequence analysis and amino acid analysis. Analytical Methods Amino acid analyses were carried out after pre-column derivatisation using phenylisothiocyanate chemistry (48,49). N- terminal sequence analysis was performed on a pulsed-liquid amino acid sequencer (Applied Biosystems, Model 477A) coupled to a HPLC system (Applied Biosystems, Model 120A). Synthetic peptide mixtures derived from non-covalent association experiments were analysed by chromatography on a 1090M Hewlett-Packard HPLC (100 mm x 2.1mm i.d. Hypersil ODS column, 28 ϋ C, 0.1% (v/v) TFA acid, linear 25 min gradient from 0 to 100%>, acetonitrile/water (90/10 v/v) containing 0.09% TFA). Enzyme Assays PLA 2 enzyme activity was primarily measured in a mixed- micelle assay using l-acyl-2-[l- 14 C] arachidonyl-L-3- phosphatidylethanolamine (PE) and sodium deoxycholate (50). The PE substrate solution was prepared by dissolving freshly desiccated PE (5.5nmol) in 25μl of 2% (w/v) sodium deoxycholate, then diluting to the final concentration of 0.22nmol PE/25μl with 2 x assay buffer (lOOmM Tris-HCI, pH8.0, lOmM CaCl 2 . 300mM NaCl). The sample (25μl) was prepared by mixing lOμl of PLA 2 at the appropriate concentration with 15 μl test peptide at the appropriate concentration, both diluted in lOmM Tris-HCI pHδ.O. Peptides were pre-incubated with PLA 2 for 30 minutes at 37°C prior to addition of substrate. The reaction was started by the addition of 25 μl substrate solution (pre warmed at 37°C) to the sample. Final assay conditions were 50mM Tris-HCI. pH 8.0, 5mM CaCl 2 , 4.4μM PE, ImM sodium deoxycholate in all cases. Reactions were incubated for 30 min at 37°C and terminated by the addition of lOOmM EDTA (lOμl). The reaction mixture (30μl), was separated by thin layer chromatography (kieselgel 60F 2r , 4 , Merck, chloroform:methanol:acetic acid (90:10:1) and spots identified by overnight exposure with Kodak X-OMAT AR film. Radioactivity at the origin and at the liberated arachidonic acid front was counted by liquid scintillation and PLA 2 activity expressed as pmol PE hydrolysed/min. The specific activity of the enzyme was 3.72μmol/min/mg. Time course studies showed that the assay was linear over 60 minutes at all PLA 2 concentration used (data not shown). Some experiments were performed in triplicate using the 3 H-labelled Escerichia coli membrane assay (51). Briefly, 96-well microtitre plates were blocked with 1% BSA in PBS for 16 hours at 4°C. Plates were washed twice with 0.05% Tween 20 in PBS. Enzyme and peptides at varying concentrations were incubated in 50μl assay buffer (250mM Tris-HCI pH 9.0, lOmM CaCl z . ImM β-mercaptoethanol) at 37°C for 30 minutes. Fifty μl substrate (autoclaved E.coli suspension [5, 6, 8, 9, 11, 14, 15 - 3 H(N)] arachiodonate labelled, DuPont- NENjsuspended in assay buffer was then added and incubated at 37°C for 30 minutes. Final reaction conditions were 0.2nM PLA 2 12.6μM Pi. The reaction was stopped by addition of 25 μl 4M HCl and 25 μl fatty- acid-free BSA (40mg/ml). A 50μl aliquot of each reaction was removed to determine total counts in the assay. The remaining sample was centrifuged at 1300xg for 15 minutes and 50μl of supernatant was removed to measure arachidonic acid release. Radioactivity in both samples was determined by liquid scintillation counting (Top count. Packard). Enzyme activity was expressed as % hydrolysis. Modelling Calculations Docking calculations on the human PLA 2 have been performed in order to ascertain possible binding locations for the pentapeptide. Figure 1 shows the mainchain bonds that are free to rotate in the docking calculation FLSYK. The docking method is Monte-Carlo (52). The FLSYK peptide is docked onto the PLA 2 molecule [accession code 1POD from the Brookhaven Protein Data Bank (27,53)]. The PLA 2 molecule includes 2 integral internal water molecules. 24 torsion angles (as shown in Figure 1) were included in the Monte Carlo algorithm along with 6 global translational and rotational parameters. The starting position of the FLSYK was the same conformation found in the protein but positioned adjacent to the sequence in the human type II PLA2. This position has no other significance. The initial FLSYK conformer and side-chains identified in the vicinity of the active site were energy minimised by 300 steps of conjugate energy minimisation using the Consistent Valence Force Field (cvff). Residues included in the calculation were Asn 1-Thr 13, Ala 18, Ala 19, Tyr 22-Gly 32, Cys 45, His 48. Asp 49. Tyr 52, Lys 53. Glu 56, Thr 68-Phe 75, He 82, Leu 95. Asp 99, Lys 100. Ala 102, Ala 103, Phe 106, Tyr 120-Asn 122. Each subsequent docked conformation was only saved if it was within 10 kcal of the last saved conformation and the root mean square deviation of the FLSYK conformer had to be 0.5 A or greater than a previously found conformation. 3.0 A was the maximum move distance for the translational components of the move and energy minimization was not performed if the structure resulting from the random move was more than 1000 kcal higher in energy than the last minimised structure. The calculations have been performed with the Molecular Simulations Software (54). A Ramachandran Plot is a conformational map of a polypeptide chain, describing the ranges of bond angles permissible and the main types of structure (eg α helix, β pleated sheet). This map plots phi, the twist about the Cα-N bond axis, against psi, the twist about the Cα-C axis. The Ramachandran Plots were generated by the program ProCheck (J. Thornton). Results Low energy interactions of FLSYK with just two segments of PLA 2 . Some 426 conformations have been found within 33.1 kcal of the minimum energy conformation. In a control calculation using TVSYT as the pentapeptide no conformation was found of energy comparable with that in the FLSYK calculation. See Figure 2. The region of binding has been determined on the basis of the contacts between the docked FLSYK and the human Type II PLA2 in this calculation. It is identified as Asn 1, Val 3, Asn 4. His 6, Arg 7, Lys 10, Leu 12, Ser 72, Tyr 73, Lys 74, Phe 75, Ser 76, Asn 77. Figure 3 shows the contacting residues with a graph of residue number versus number of contact made with FLSYK in the calculations. Three groups of conformations have been identified. These groups are broadly defined structures respresented by conformations 83-209 (Group I), conformations 210-278 (Group II) and conformations 279-500 (Group III), as shown in Figure 4. All the conformations exhibit relatively closed conformations in which the terminii are in proximity. Conformations describing the features of these are shown in Figure 4. In the first group represented by conformation 173 (Figure 4A) the aromatic ring of tyrosine of FLSYK and is stacked over and is against the ring of Phe 75 and alongside the main chain of Lys 74 of the human Type II PLA 2 . The hydroxyl of the tyrosine is also in proximity to the main chain of Phe 75. Leucine of FLSYK makes hydrophobic contact at Arg 7. The serine of FLSYK is in contact with Asn 4 and its lysine contacts Ser 72. In the second group represented by conformation 242 (Figure 4B) the phenylalanine of FLSYK contacts Arg 7, Tyr 73 and Lys 74. The tyrosine of FLSYK contacts Val 3 and its lysine contacts Ser 72. In the third group represented by conformation 435 (Figure 4C) the backbone at tyrosine of FLSYK contacts Asn 4, phenylalanine of FLSYK contacts Ser 72 and Tyr 73. Leucine and serine of FLSYK contact the Arg 7, as does its phenylalanine. Lysine is in contact with Val 3. Analogues Peptide Synthesis Peptides were synthesized on the Perkin Elmer/Applied Biosystem (PE/ABI). T-boc (t-butyloxycarbonyl) chemistry was used. Peptide synthesis was carried out in solid phase on PAM-resin (PAM- phenylacetamidomethyl) or MBHA-resin (MBHA-pp- methylbenzhydrylamino) supplied by PE/ABI. Peptide bonds were formed either via symmetric anhydride coupling or via HOBt (p- hydroxybenzotriazole) ester activation (55). Protected amino acids used in the peptide synthesis were purchased from Auspep (Melbourne, Australia) and protected semi-organic amino acids were purchased from Syn the tech (Oregon. USA). Hydrogen fluoride (HF) cleavage of the fully protected peptide from the solid support was performed by Auspep Pty Ltd, Melbourne. The resin was treated for 60 minutes in 10ml HF, with 1.3g of phenol as a scavenger. Peptides were extracted into an aqueous phase (30% acetonitrile/water, v/v) and scavengers were washed out with ether. The aqueous extracts were then lyophihzed to yield the crude product. Side chain protection groups chosen for each amino acid were removed during the cleavage process. A number of peptides were also purchased from Chiron Mimotopes (Aus.) and Macromolecular Resources (Colorado, US). Crude synthetic products were subjected to HPLC (both ion exchange and reverse phase chromatography), and a major peak representing the desired peptide was observed. To confirm peptide purity and identity, purified peptides were then subjected to both sequence and mass spectroscopy analysis for sequence and molecular weight confirmation. Biophysical evidence for interaction of residues 70-74 with the NH2- terminal amino acids of human sPLA 2 To confirm that these two peptides did not fortuitously co-elute under these conditions, purified synthetic peptides (FLSYK and NLVNFHR) in Tris-HCI buffer (pH 8.5) were mixed in approximately equal molar ratios and chromatographed. The peptides eluted as two separate peaks (data not shown). On standing for 24 hours, a third peak, only partially resolved from NLVNFHR. appeared. This peak contained FLSYK in addition to NLVNFHR (data not shown). Further experiments were carried out to confirm this observation using FLSYK and residues 1-14 (NLVNFHRMIKLTTG) of the human Type II PLA 2 . The results (Figures 5 and 6) showed that the third peak originated from a non-covalent association between the two chromatographically distinct peptides which occurred on standing. In contrast, when the experiment was repeated using a synthetic peptide (WDIYR) derived from the 70-74 region of the C.durissus Type II PLA 2 in place of FLSYK, no new peak was seen (data not shown). Computer graphics predict that low energy interactions occur with residues 72 (Ser), 73 (Tyr), 74 (Lys) and 75 (Phe) as well as with the N- terminal residues 1 (Asn), 3 (Val), 4 (Asn), and 7 (Arg). These interactions provide a plausible explanation for the essential nature of all five residues of the pentapeptide inhibitor. Conceptual model of FLSYK binding to defined regions of PLA 2 . Figure 7 is a pictorial representation of FLSYK in juxtaposition with its PLA 2 contact residues. Only Cα positions and relevant side-chains are shown but it can be appreciated that backbone interactions (which are clear from other models) could also play a part. A feature of the model is that FLSYK (in the centre of the model with Cα residues labelled 1 to 5) is configured as a hair pin, the loop being centred on the serine at residue 3. It presumably also has a planar character by virtue of a pseudo-cyclic structure caused by interaction of the N- and C- terminal amino and carboxyl groups of the pentapeptide, that is, linking 1 to 5. Both the N-terminal helix and the 70-77 stretch of chain are on the surface of the molecule and involved in interfacial reactions with their external environment. Binding of the lysine side chain of peptide residue 5 could occur via PLA 2 residues 1 (Asn), 4 (Asn) and 72 (Ser); peptide residues 4 and 3 (Tyr and Ser) interact with PLA 2 residue 3 (Val); peptide residues 3 and 2 (Ser and Leu) have interactions with arginine at residue 7 of PLA 2 and peptide residues 2 and 1 (Leu and Phe) are close to PLA 2 residues 72. 73, 74 and 75 (Ser, Tyr, Lys and Phe respectively). At the very least these interactions would be expected to restrict the ability of the enzyme to adopt an active conformation in the presence of substrate. It is of interest that the OH of Tyrosine, residue 4 of FLSYK, is not essential and can be replaced by a second aromatic ring (that is Y becomes a 2-naphthylalanine residue). It is tempting to speculate from the diagram that this side chain could project into the hydrophobic channel and foul the ingress of a substrate molecule. These modelling studies predict that a cyclic structure in which a peptide bond is formed between the N- and C- terminus of a linear compound would act as a specific inhibitor. As shown in Table 2 and Figure 10D the cyclic compound FLSYR does inhibit human sPLA 2 in the E.coli assay. When this compound is used in modelling experiments using the same methodology as described in Figure 2A and 2B, the energy profile shown in Figure 8A is generated. Further analysis of the location of the cyclic compounds contact residues on PLA , show that this compound docks to the same relative region of sPLA 2 as the linear compound, Figure 8B. Specifically, the contact residues are Asn-1. Val-3, Asn-4. Arg-7, Leu-12. Ser- 72. Tyr-73, Lys-74, Phe-75, Ser-76 and Asp-77. This cyclic modelling experiment can be used to identify specific conformations on the ring structure which could be used as scaffolds to synthesise peptidomimetic and organic based inhibitors of PLA 2 . This information is summarised in Figure 9 and Table 1. Figure 9 shows plots of phi-psi angles for each of the carbonα atoms identified in the cyclic structure (numbered 1-5 in Figure 7). for each of the 460 low energy (E_< 322 kcal) configurations generated by the modelling experiments. Table 1 summarises the phi-psi angles for each of the carbonα atoms. These plots show the phi-psi angles cluster to defined regions in space. The limits of phi-psi angles in these regions define specific structures which the cyclic peptide backbone may assume when bound to PLA 2 . These constraints could be generated synthetically by one skilled in the art using both peptidomimetic and organic synthetic approaches. These scaffolds could then be used to provide reactive centres identified as important from our linear peptide analogue experiments (for example, a hydroxyl group or some equivalent chemical entity on the Cα3 carbon of the ring). Table 1 cFLSYR 460 conformations with E < 322 kcal Phi range 1 Psi range' min max min max α-Carbon 1 (i) -125.1 -49.3 -81.3 -20.6 Phe (ii) 55.4 77.5 -85.0 -65.8 α-Carbon 2 (i) -144.8 -72.1 22.6 117.0 Leu α-Carbon 3 (i) -69.2 -32.0 116.3 137.6 Ser (ii) 13.9 66.3 80.0 110.1 α-Carbon 4 (i) -109.5 -47.9 113.5 166.9 Tyr (ii) -60.2 -60.2 -68.0 -68.0 α-Carbon 5 (i) 78.0 120.5 58.0 97.3 Arg (ii) 53.1 118.1 -146.0 -67.0 (iii) -71.1 -71.1 -73.2 -73.2 Assay of peptide analogues Table 2 lists a range of analogues which inhibited PLA 2 . This Table is a qualitative inhibition scale for the analogues such that + + + is equivalent to FLSYK inhibition as seen in the E.coli assay. + + is an analogue inhibition of PLA 2 that is less than FLSYK and + is an indication of small but detectable inhibition. The inhibition curves are depicted in Figure 10 for selected analogues. They range from zero inhibition for peptides outside the region 70-74 (Figure 10A). partial inhibition ( + +) for conservative amino acid replacements (Figure 10B), to inhibition equivalent to FLSYK (Figure 10C. + + +) or better (Figure 10D, + + + + ). Inhibition of human sPLA2 by kyslf To determine if the D-peptide kyslf (residues 74-70) was capable of inhibiting PLA 2 activity, different amounts were incubated with sPLA 2 . The peptide fkysl (residues 75-71) was also analysed and peptides FLSYK and TVSYT were included in the assays as controls. The peptide fyksl failed to inhibit human Type II PLA 2 (Figure 12B), while kyslf dose-dependently inhibited the enzyme activity over a wide concentration range (Figure 12A). 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. 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Claims: Claims: 1. A non-peptide structure which has a conformation and polarity such that the structure binds to (i) at least one amino acid in the N-terminal helix region of PLA 2 ; and/or (ii) at least one amino acid in the region spanning amino acid residues 70-77 of PLA 2 ; wherein binding of the structure to regions (i) and/or (ii) inhibits the enzymatic activity of PLA 2 . 2. A non-peptide structure according to claim 1 wherein the structure binds to (i) at least one amino acid in the N-terminal helix region of PLA 2 ; and (ii) at least one amino acid in the region spanning amino acid residues 70-77 of PLA 2 ; 3. A non-peptide structure according to claim 1 or claim 2 wherein the PLA 2 is human PLA 2 . 4. A non-peptide structure according to any one of claims 1 to 3 wherein the structure binds to at least one amino acid, more preferably at least two amino acids, in the N-terminal helix region selected from the group consisting of Asnl, Val3, Asn4, His6, Arg7, LyslO and Leul2. 5. A non-peptide structure according to any one of claims 1 to 4 wherein the structure has substantially the same spatial geometry and polarity as the peptide FLSYK wherein the peptide FLSYK is in a conformation which allows the peptide to bind to (i) at least one amino acid in the N-terminal helix region of PLA 2 selected from Asnl, Val3, Asn4, His6, Arg , LyslO and Leul2; and (ii) at least one amino acid in the region spanning amino acid residues 70-77 of PLA 2 . 6. A non-peptide structure according to claim 5 wherein the spatial geometry is delineated by the hydrogen bonding network provided by the backbone of the residues in FLSYK to residues 1.3,4,7,72,73,74 and 75 of PLA 2 . 7. A non-peptide structure according to claim 5 or claim 6 wherein the configuration of FLSYK is substantially as depicted in any one of Figures 4(A), 4(B) or 4(C). 8. A non-peptide structure which has a backbone conformation substantially identical to the general scaffold generated by cyclic pentapeptide wherein the bonds joining the α-carbon atoms of the five amino acids in the pentapeptide are such that (i) α-carbonl has (a) a phi angle of between -125.1 and -49.3 and a psi angle of between -81.3 and -20.6, or (b) a phi angle of between 55.4 and 77.5 and a psi angle of between -85.0 and -65.8; (ii) α-carbon2 has a phi angle of between -144.8 and -72.1 and a psi angle of 22.6 and 117.0; (iii) α-carbon3 has (a) a phi angle of between -69.2 and - 32.0 and a psi angle of between 116.3 and 137.6, or (b) a phi angle of between 13.9 and 66.3 and a psi angle of between 80.0 and 110.1; (iv) α-carbon4 has (a) a phi angle of between -109.5 and - 47.9 and a psi angle of between 113.5 and 166.9, or (b) a phi angle of - 60.2 and a psi angle of -68.0; and (v) α-carbon5 has (a) a phi angle of between 78.0 and 120.5 and a psi angle of between 58.0 and 97.3, or (b) a phi angle of between 53.1 and 118.1 and a psi angle of between -146.0 and -67.0, or (c) a phi angle of -71.1 and a psi angle of -73.2. 9. A composition for use in treating a subject suffering from septic shock, rheumatoid arthritis and/or other inflammatory diseases, the composition including a therapeutically acceptable amount of a structure according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier. 10. A method of treating septic shock and/or inflammatory disease in a subject which includes administering to the subject a non- peptide structure according to any one of claims 1 to 8. 11. A method of screening an agent for potential PLA 2 inhibitory activity which method includes analysing the structure of the agent for similarities with the three dimensional structure of the peptide FLSYK, wherein the peptide FLSYK is in a conformation which allows the peptide to bind to (i) at least one amino acid in the N-terminal helix region of PLA 2 selected from Asnl, Val3, Asn4, His6, Arg7, LyslO and Leul2; and (ii)at least one amino acid in the region spanning amino acid residues 70-77 of PLA 2 and testing the compound for inhibitory activity against PLA 2 . 12. A method of generating a potential inhibitor of PLA 2 which method includes generating a compound which has a conformation which is substantially identical to the three dimensional structure of the peptide FLSYK, wherein the peptide FLSYK is in a conformation which allows the peptide to bind to (i) at least one amino acid in the N-terminal helix region of PLA 2 selected from Asnl, Val3, Asn4, His6, Arg7, LyslO and Leul2; and (ii)at least one amino acid in the region spanning amino acid residues 70-77 of PLA 2 and testing the compound for inhibitory activity against PLA 2 13. A method of generating a potential inhibitor of PLA 2 which method includes generating a compound which has a backbone conformation substantially identical to general scaffold generated by a cyclic pentapeptide wherein the bonds joining the α-carbon atoms of the five amino acids in the pentapeptide are such that (i) α-carbonl has (a) a phi angle of between -125.1 and -49.3 and a psi angle of between -81.3 and -20.6, or (b) a phi angle of between 55.4 and 77.5 and a psi angle of between -85.0 and -65.8; (ii) α-carbon2 has a phi angle of between -144.8 and -72.1 and a psi angle of 22.6 and 117.0; (iii) α-carbon3 has (a) a phi angle of between -69.2 and - 32.0 and a psi angle of between 116.3 and 137.6, or (b) a phi angle of between 13.9 and 66.3 and a psi angle of between 80.0 and 110.1; (iv) α-carbon4 has (a) a phi angle of between -109.5 and - 47.9 and a psi angle of between 113.5 and 166.9, or (b) a phi angle of - 60.2 and a psi angle of -68.0; and (v) α-carbon5 has (a) a phi angle of between 78.0 and 120.5 and a psi angle of between 58.0 and 97.3, or (b) a phi angle of between 53.1 and 118.1 and a psi angle of between -146.0 and -67.0, or (c) a phi angle of -71.1 and a psi angle of -73.2 and testing the compound for inhibitory activity against PLA 2 14. A peptide which inhibits the enzymatic activity of Type II PLA 2 , wherein the peptide comprises D-amino acids and includes an amino acid sequence which corresponds to the reverse sequence of a region between residues 69 to 75 of PLA 2 . 15. A peptide according to claim 14 wherein the peptide has an amino acid sequence which corresponds to the reverse sequence of the region between residues 70 to 74 of PLA 2 . 16. A peptide according to claim 14 or claim 15 wherein the PLA 2 is human PLA 2 . 17. A peptide according to any one of claims 14 to 16 wherein the peptide is a pentapeptide which consists of D-amino acids. 18. A peptide has the following formula: A1-A2-A3-A4-A5-A6-A7 in which Al is H or one or two D-amino acids A2 is k or r or h or D-citrulline A3 is y or q or D-2,naphthylalanine A4 is s or t or c or D-homoserine A5 is 1 or v or i or D-norleucine A6 is f or y or w or D-norleucine or D- 2.naphthylalanine A7 is OH or one or two D- amino acids. 19. A peptide according to claim 18 wherein Al is H and A7 is OH. 20. A peptide consisting of D-amino acids of the sequence kyslf. 21. A peptide according to any one of claims 14 to 20 which is a cyclic peptide. 22. A composition for use in treating a subject suffering from septic shock, rheumatoid arthritis and/or other inflammatory diseases, the composition including a therapeutically acceptable amount of a peptide according to any one of claims 14 to 21 and a pharmaceutically acceptable carrier. 23. A method of treating septic shock and/or inflammatory disease in a subject which includes administering to the subject a peptide according to any one of claims 14 to 21.

Inventors: BRYANT KATHERINE, CHURCH W BRET, INGLIS ADAM, SCOTT KIERAN, TSENG ALBERT, BRYANT, KATHERINE, CHURCH, W., BRET, INGLIS, ADAM, SCOTT, KIERAN, TSENG, ALBERT
Applicants: BRYANT KATHERINE, CHURCH W BRET, GARVAN INST MED RES, INGLIS ADAM, SCOTT KIERAN, TSENG ALBERT, BRYANT, KATHERINE, CHURCH, W., BRET, GARVAN INSTITUTE OF MEDICAL RESEARCH, INGLIS, ADAM, SCOTT, KIERAN, TSENG, ALBERT
Application year: 1997
Main classification: C07K7
IPC codes: A61K38, A61P19, A61P29, A61P31, C07K7, C12N9