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
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• 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 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 .
• 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 ⁇ -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).
• influenza virus neuraminidase 34
• HIV protease for a selected sample see reference 35
• purine nucleoside phosphorylase for a selected sample see reference 35
• purine nucleoside phosphorylase for a selected sample see reference 35
• thymidylate synthase
• 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.
• 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
• 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.
• the PLA 2 is human PLA 2 .
• 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.
• 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
• 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
• Aromatic residue better than linear side chain e.g. naphthylalanine.
• Position 2 (a) Hydrophobic group, preferably Leu; and/or
• Position 3 (a) Polar group on side chain (for H-bonding); and/or
• Position 4 Hydrophobic group larger than Phe (e.g. 2- naphthylalanine); and/or
• 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:
• 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 ⁇ -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.
• 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 ⁇ -carbon atoms of the five amino acids in the pentapeptide are such that
• ⁇ -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;
• ⁇ -carbon2 has a phi angle of between -144.8 and -72.1 and a psi angle of 22.6 and 117.0;
• ⁇ -carbon3 has (a) a phi angle of between -69.2 and -
• ⁇ -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 -
• ⁇ -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
• 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 ⁇ -carbon atoms of the five amino acids in the pentapeptide are such that
• ⁇ -carbonl has (a) a phi angle of between -125.1 and -
• ⁇ -carbon2 has a phi angle of between -144.8 and -72.1 and a psi angle of 22.6 and 117.0;
• ⁇ -carbon3 has (a) a phi angle of between -69.2 and -
• ⁇ -carbon4 has (a) a phi angle of between -109.5 and -
• ⁇ -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
• 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 .
• the peptide has an amino acid sequence which corresponds to the reverse sequence of the region between residues 70 to 74 of PLA 2 .
• the PLA 2 is human PLA 2 .
• the peptide is a pentapeptide which consists of D-amino acids.
• the peptide has the following formula:
• 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
• A7 is OH or one or two D- amino acids.
• 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.
• Ai is H and ⁇ 7 is OH.
• the peptide is kyslf.
• 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.
• 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).
• peptides may be cyclic peptides.
• D-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 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.
• 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.
• 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.
• 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.
• 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.
• 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
• 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
• 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
• 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).
• NLVNFHRMIKLTTG (0.6 mg/ml). were incubated in 20mM Tris HCl pH 8.5 at room temperature. Aliquots (10 ⁇ l) 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.
• 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 ⁇ -carbon 1 (A), ⁇ -carbon 2 (B), ⁇ -carbon 3 (C). ⁇ -carbon 4
• FIG. 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)
• Human Type II PLA 2 was purified from conditioned medium generated from stably-transfected Chinese hamster ovary cell line (5A2) expressing 350 ⁇ g 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 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
• 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).
• PE substrate solution was prepared by dissolving freshly desiccated PE (5.5nmol) in 25 ⁇ l of 2% (w/v) sodium deoxycholate, then diluting to the final concentration of 0.22nmol PE/25 ⁇ l with 2 x assay buffer (lOOmM Tris-HCI, pH8.0, lOmM CaCl 2 . 300mM NaCl).
• the sample (25 ⁇ l) was prepared by mixing lO ⁇ l of PLA 2 at the appropriate concentration with 15 ⁇ l test peptide at the appropriate concentration, both diluted in lOmM Tris-HCI pH ⁇ .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 ⁇ l substrate solution (pre warmed at 37°C) to the sample. Final assay conditions were 50mM Tris-HCI. pH 8.0, 5mM CaCl 2 , 4.4 ⁇ M PE, ImM sodium deoxycholate in all cases. Reactions were incubated for 30 min at 37°C and terminated by the addition of lOOmM EDTA (lO ⁇ l).
• the reaction mixture (30 ⁇ l), 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 3.72 ⁇ mol/min/mg. Time course studies showed that the assay was linear over 60 minutes at all PLA 2 concentration used (data not shown).
• FIG. 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.
• 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 ⁇ helix, ⁇ pleated sheet). This map plots phi, the twist about the C ⁇ -N bond axis, against psi, the twist about the C ⁇ -C axis.
• the Ramachandran Plots were generated by the program ProCheck (J. Thornton).
• 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.
• 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.
• 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.
• 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
• 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).
• PE/ABI Perkin Elmer/Applied Biosystem
• Figure 7 is a pictorial representation of FLSYK in juxtaposition with its PLA 2 contact residues. Only C ⁇ 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 C ⁇ 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).
• FIG. 9 shows plots of phi-psi angles for each of the carbon ⁇ 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 carbon ⁇ atoms. These plots show the phi-psi angles cluster to defined regions in space.
• 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

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