AUTO-DECONVOLUTING COMBINATORIAL LIBRARIES

Abstract: The present invention relates to the field of apparatus and methods which provide the rapid generation of structure/activity relationships using auto-deconvoluting combinatorial libraries, which facilitate the invention of novel active compounds. The invention provides apparatus and methods which can be used for the rapid generation of structure/activity relationship (SAR) data, and, therefore, the characterisation of the active motif of any group of compounds. The invention provides libraries of compounds which interact with an active moiety, and apparatus and methods to identify such compounds. The active moieties may be (but are not limited to) enzymes, receptors, antibodies, etc. The interaction of the active moiety with the compounds of the library may be (but is not limited to) the interaction of a substrate or inhibitor with an enzyme, the interaction of a ligand with a receptor, the interaction of an antigen or antigenic epitope with an antibody, etc.

Description: Auto-Deconvolut nα Combinatorial Libraries The present invention relates to the field of apparatus and methods which provide the rapid generation of structure/activity relationships using auto-deconvolutmg combinatorial libraries, which facilitate the invention of novel active compounds. The invention provides apparatus and methods which can be used for the rapid generation of structure/activity relationship (SAR) data, and, therefore, the characterisation of the active motif of any group of compounds. The invention provides libraries of compounds which interact with an active moiety, and apparatus and methods to identify such compounds. The active moieties may be (but are not limited to) enzymes, receptors, antibodies, etc. The interaction of the active moiety with the compounds of the library may be (but is not limited to) the interaction of a substrate or inhibitor with an enzyme, the interaction of a ligand with a receptor, the interaction of an antigen or antigemc epitope with an antibody, etc The following list illustrates a number of enzyme classes and subclasses • Enzyme Classes 1. Oxidoreductase a) dehydrogenase b) oxidase c) peroxidase d) catalase 2. Hydrolase f) peptidase (proteolytic enzyme) 3. Transferase g) aminotransferase h) kinase I) glucosyltransferase 4. Lyase j ) decarboxylase k) dehydratease 5. Isomerase 1) racemase m) mutase 6. Ligase n) synthetase o) carboxylase Proteases of interest include (but are not limited to) : 1. Aspartyl proteases, such as renin, HIV, cathepsin D and cathespin E etc . 2. Metalloproteases, such as ECE, gelatinase A and B, collagenases , stromolysins etc. 3. Cysteinyl proteases, such as apopain, ICI, DerPI, cathepsin B, cathepsin K etc. SUBSTITUTE SHEET RULE 26) 4 Senne proteases, such as thrombm, factor Vila, factor Xa, elastase, trypsin. 5 Threonyl proteases, such as proteasome S. Proteases Many therapeutically useful drugs act as enzyme inhibitors In particular, proteolytic enzyme inhibitors have been the focus of much attention in the pharmaceutical industry, because they play a variety of roles m a multitude of biological systems Their proteolytic activities are related to processes ranging from cell invasion associated with metastatic cancer to evasion of an immune response, as seen in certain parasitic organisms, from nutrition to intracellular signalling to the site-specific proteolysis of viral proteases and eukaryotic hormone-processing enzymes However, the traditional random screening methods for the identification of lead molecules as inhibitors of proteolytic enzymes are often laborious and time- consuming Therefore new and efficient methods which can accelerate the drug discovery process are greatly in demand. We consider that proteases contain an active catalytic site which tends to become increasingly activated as the recognition pockets 1 { S 1 and S 2 etc) and (S,. 1 and S 2 ' etc) become better occupied. Therefore, it is important that those parts (P x and P 2 etc) ( P x ' and P 2 ' etc) of the inhibitor that best fit into these pockets are identified as quickly as possible m order to design novel protease inhibitors. Therefore, we have devised a combinatorial method for the rapid identification of these binding motifs which will greatly expedite the synthesis of inhibitors of a variety of proteolytic enzymes such as aspartyl proteases, seπne proteases, metallo proteases and cystemyl proteases. The use of a fluorescence resonance energy transfer (FRET) substrate for the analysis of proteolytic enzyme specificity was first published by Carmel. 2 Since then many different quenched fluorogenic substrates for measuring enzyme inhibition have been described m the literature " u These substrates contain a fluorophore, F, m a P position ( vide supra) , which is quenched by another group, Q, present in a P position ( vide supra) and separated from F by the scissile bond The advantage of the positioning of these residues, F and Q, is that cleavage of a peptide bond occurs between the two natural residues and, therefore, represents a more natural hydrolytic event rather than the cleavage and release of a C-terminal chromophore . For example, Bratovanova and Petkov 12 have synthesised fluorogenic substrates from peptide 4-nιtroanιlιdes . N- acylation of peptide 4-n troanιlιdes with the ammobenzoyl (ABz) group yielded substrates that are internally quenched by the presence of the 4-nιtroanιlιde moiety. Upon hydrolysis of the ammoacyl-4-nιtroanιlιde bond, the highly fluorescent N-ABz group is released attached either to an ammo acid or peptide Immobilised libraries, where substrates are attached to a polymer or biopolymer support, have also been used for mapping protease binding sites. 13 Singh et al . reported recently that enzymatic substrate activity of 38 selected octapeptides attached via a linker to controlled pore glass is predictive of the same activity of similar peptides in solution. However, these results are preliminary and only for a specific example Therefore, it is not clear whether immobilised substrates attached to polymers can reliably replace soluble substrates in mapping the hindered protease binding sites, especially since the hydrophilic or lipophilic nature of the polymer and the size of the interstices with the polymer are bound to influence the reaction between the enzyme and its substrates . Mixtures of internally quenched, fluorogenic substrates have also recently been described in which the quencher group, Q, is 2, 4-dιnιtrophenyl (Dnp) and is attached to the P side of the scissile bond, while the fluorogenic group, is N-methyl anthran lic acid (Nma) and is attached Examples of other Donor-Acceptor Chromophore Pairs that have been applied to Biological Systems are shown in Table 1. Table 1 . Donor-Acceptor Chromophore Pairs That Have Been Applied To Biological Samples Donor Acceptor Donor Acceptor Naphthalene Dansyl IANBD DDPM IAEDANS DDPM ANAI, 2-anthracene N-acetylimidazole; BPE, B-phycoerythrin; CF, carboxyfluorescein succinimidyl ester; CPM, 7-doethylamιno-3-(4'maletmιdylphenyl)-4-methylcoumann; CY5, carboxymethylindocyanine-N-hydroxysuccinimidyl ester; dιI-C 18 , l , l '-dιoctadecyl-3,3,3'3 '- tetramethyl-indocarbocyanme, DιO-C, 4 , 3,3'-dιtetradecyloxacarbocyanιne, DABM, 4- dιmethylanιnιphenylazo-phenyl-4'-maleιmιde; DACM, (7-(dιmethylamιno)coumann-4-yl)- acetyl, DANZ, dansylazindine, DDPM, N-(4-dιrnethylarruno-3-5-d_rιιtrophenyl)rrιaleιrnιde, DACM, dι-methylamιno-4-maleιmιdostιlbene; DMSM, N-(2,5-dιmethoxystιben-4-yl)- maleimide, DNP, 2,4-dιnιtrophenyl, e-A, l ,N 6 -ethenoadenosιne;EIA, 5-(ιodoacetetamιdo)eosιn, EITC, eosιn-5-ιsothιocyanate; ENAI, eosin N-acetyhmidazole, EM, eosin maleimide, ErlTC, erythrosιn-5 '-ιsothιocyanate, ETSC, eosin thiosemicarbazide, F 2 DPB, 1 ,5-dιfluoro- 2,4'dιnιtrobenzene; F 2 DPS, 4,4'-dιfluro-3,3 , dinιtropheylsulphone; FITC, fluorescein N- acety midazole; FTS, fluorescein thiosemicarbazide; IAANS, 2-((4'- ιodoacetamιdo)anιhno)naphthalene-6-sulphonιc acid, IAEDANS, 5-(2- ((ιodoacetyl)armno)ethylam_no)-naphthlene- l -sulphonιc acid, IAF, 5- lodoacetarmdofluorescein; IANBD, N-((2-(ιodoacetoxy)ethyl)-N-methyl)arruno-7-nιrrober__-2- oxal ,3,dιazole, IPM, 3(4-ιsothιocyanatophenyl)7-dιethyl-4-methylcoumann; ISA, 4- (ιodoacetamιdo)salιcylιc acιd,LRH, hssamιnerho-2,l,3-benzoxadιazol-4-yl, NCP, N- cyclohexyl-N'-( l-pyrenyl)carbodnmιde, ODR, octadecylrhodamine, PM, N-(l-pyrene)- maleimide, SRHsulphurhodamine, TMR, tetramethylrhodamine, TNP, tnnitrophenyl, TR, Texas red from Wu, P and Brand, L 1994 Anal Biochem 218, 1-13 The specificity of soluble peptide libraries have been determined. 15 16 Ber an et al . described 16 an HPLC mass spectrometry technique m which 6 mixtures of 128 peptides were synthesised which were N-termmally labelled with the Dnp group in order to allow UV monitoring on the HPLC. The disadvantage of this approach is that each assay mixture has to be individually analysed, because no fluorogenic substrate is revealed, and that the effective concentration of each separate component s limited by the size of the mixture because of overall solubility f ctors. Drev et al. 17 have suggested the use of individually synthesised fluorogenic substrates for the determination of enzyme activity using a chromophore which chelates lanthamde ions. Garmann and Phillips have suggested the use of FRET substrates m which the fluorogenic and quencher moieties are attached via thiol or ammo functional groups after the peptide has been synthesised, but this has the disadvantage that they are not in library form and that these functional ammo and thiol groups need to be selectively revealed after the peptide has been synthesised. Wang et al . have suggested the use of the EDA S and DABCYL fluorescor and quencher pairing for the individual synthesis of substrates for proteolytic enzymes . The above methods which have used FRET techniques for the mapping of the active site around a specific protease suffer from one or more of the following disadvantages- l because of general aqueous insolubility they do not produce mixtures of compounds a form suitable for high throughput screening in aqueous solution, ii the derivatised compounds cannot be prepared in combinatorial library form using solid phase techniques . in. the mixtures which have been used 8 9 were not self- decodmg, and needed time-consuming deconvolutive resynthesis for identification of the active molecules Kinases Protein kinases are intracellular enzymes that play key roles in cell growth, differentiation and mter-cell communication. Aberrant protein kinase activity has been implicated in many disease states including several forms of cancer and severe-combined immunodeficiency disease All seπne/threonine and tyros e protein kinases have a region of approximately 300 ammo acids known as the catalytic subunit which has evolved from a common ancestor kinase (Hunter et al, 1991) . Crystal structure determination of several kinases has shown that they all have a common bi-lobal structure The ammo-terminal part of the subunit encodes a small lobe responsible for the binding of ATP, whereas the carboxy-terminal residues encode a larger lobe important for protein substrate binding. In the tertiary structure of the active kinase, both the ATP and the protein substrate binding sites are brought together allowing transfer of the ATP γ-phosphate to the ammo acid acceptor on the protein substrate The prote /peptide binding groove stretches across the face of the large lobe between two α-helices and under the small lobe. This groove therefore contains the residues important for substrate specificity of the kmase. Protein kinases are arranged in kinase cascades withm the cell, providing the ability for signal amplification in post-transduction pathways This amplification relies on the upstream kinase specifically activating its downstream partner. For this reason, protein kinases have developed remarkable substrate specificities which prevent unwanted crosstalk between different kmase cascades. We believe that such substrate specificity can be exploited in the design of selective protein kinase inhibitors. Existing protein kinase inhibitors Non-peptide inhibitors Most available non-peptide protein kinase inhibitors do not target the substrate binding region, but compete with ATP for enzyme binding. These inhibitors can be engineered using conventional medicinal chemistry to demonstrate specific enzyme selectivity. An example of this strategy was the modification of the fungal metabolite staurosporme, a general protein kinase inhibitor, to the bis-mdolyl-maleimides Ro 31-7549 and Ro 31-8425 (fig. 17) which are selective protein kmase C inhibitors (Muid et al , 1991) Unfortunately, the potency of ATP antagonist inhibitors can be dramatically reduced m certam intracellular compartments due to the high (millimolar) levels of ATP. The aromatic compound erbstat (fig. 18) produced by certain Streptomyces strains was identified in screens for reversal of protein tyrosine kinase-induced cell transformation. Erbstatm was subsequently shown to inhibit the EGFR kinase m vi tro with a k. of 5.58 μM and typical lineweaver-Burke competitive kinetics against a peptide substrate, while remaining non-competitive with ATP (Umezawa and Imoto, 1991) . The compound is readily inactivated in calf serum and so could not be considered as a serious candidate for therapeutic use. Attempts have been made to both increase the stability of erbstatm, by replacement of the N-formyl moiety, and bias the inhibition towards specific tyrosine kinases using a systematic modification of all parts of the molecule. These synthetic erbstatm derivatives are known as tyrphostms (tyrosine phosphorylation .inhibitors) and are mainly used as tools in enzyme/signal transduction research, but have had limited success m animal models of disease (see Levitzki and Gazit, 1995 for an extensive review) . Pseudosubstrate inhibitors Research into the structure and function of protein kinases uncovered a natural method of regulating catalytic activity known as pseudosubstrate inhibition. This method uses sequences, either on the same molecule as the catalytic site (protein kinase C) or on a regulatory subunit molecule (cyclic cAMP-dependent protein kmase) that bind into the active site and inhibit kmase activity. These sequences do not have a phospho-acceptor residue, but modification of a single specific residue to a phospho-acceptor results the formation of a good substrate sequence. It is for this reason that the sequences are known as pseudosubstrates . Peptides based on pseudosubstrate motifs have been used as tools for inhibition of specific proteins kinases such as cyclic cA P-dependent protein kinase (PKA) , protein kinase C (PKC) , myosin light chain kinase (MLCK) and calmodul - dependent protein kinase II (Kemp et al , 1994) . Pseudosubstrate peptides of less than 20 ammo acids have been shown to be potent low nanomolar inhibitors in vi tro with pseudosubstrate-based peptides of six ammo acids inhibiting protein kinases at less than one micromolar (Kemp et al , 1991) . It is clear that there is scope for a small molecular weight inhibitor of protein kinases that competes with substrate for binding to the active site A rational and peptide proven approach would be to base these inhibitors on substrate molecules which can specifically access the catalytic site. By focusing on pseudosubstrates, this approach would also have the advantage of "m-built" specificity, an element which is generally missing when leads are identified from random screening approaches. Protein kinase inhibitors in disease treatment Currently, there are no specific protein kinase inhibitors available for clinical use. Nevertheless, the potential for such compounds is clear with every major pharmaceutical company having projects for kmase inhibition m a number of therapeutic areas, most notably oncology, autoimmunity and transplant rejection. Prototypic kmase inhibitor compounds are widely available from many companies, and are used as tools for n vi tro signal transduction research Several have formed the basis for extensive medicinal chemistry programmes designed to engineer both selectivity and enhanced activity into these compounds. A number of examples have been reported which indicate that this strategy has had some success in producing compounds active both in vi tro withm cell assay systems, and m animal models of disease Staurosporme analogues (CGP41251 and UCN-01) have been used to show anti-tumour activity in vivo (Meyer et al , 1989, Akmagaka et al , 1991) and selective PKC inhibitors have been shown to work in animal models of acute inflammation (Bradshaw et al, 1993) Moreover, the BMN tyrphostm AG490 has been shown to inhibit ly phoblastic leukaemia growth in animal models, correlating with its ability to inhibit the non-receptor intracellular tyrosine kinase JAK-2 (Meydan et al , 1996) These promising preclmical studies have yet to be translated into the clinic, and it is possible that unforeseen toxicological problems, perhaps related to compound specificity, could be delaying their progress. During the past few years, there has been an explosion of knowledge relating to kinase structure, function and activity. New kinases have also been identified, some of which have a sufficiently restricted cellular distribution that one could envisage that a specific inhibitor would have selective effects e.g. a ZAP70 inhibitor would selectively inhibit activation of T cells and, to a lesser extent, NK cells. Together, these aspects offer the promise of new approaches for the development of kmase inhibitors to treat a range of conditions. Method of Protein Kinase Inhibitor Design Generation of initial substrate information A technique has recently been developed by L Cantley's laboratory to provide, consensus peptide kinase substrate information (Songyang et al, 1994) . Firstly, a degenerate peptide library of peptides with a central phospho- acceptor such as tyrosine or serme/threonme flanked by four unknown ammo acids on each side is synthesised The library is then phosphorylated by the protein kinase of interest and phosphorylated peptides isolated by DEAE- sephacel and ferric chelation chromatography. The phosphopeptide mixture is then sequenced and the frequency of each ammo acid at every position -4 to +4 assessed to give a preferred substrate sequence. These studies have yielded consensus substrate information, and do not allow a detailed analysis of particular preferences for neighbouring residue interactions. Similarly, the analysis was performed solely with natural ammo acids. Filamentous phage are now also being used to generate substrate information (Miami, Biotechnology Conference, 1996) . Substrate information can also be obtained from knowledge of the physiological substrates for the kinase in vi vo . Description of the Drawings Figures 1 to 14 inclusive exemplify component distributions m the plates of a library matrix; Figure 15 illustrates a reaction scheme for production of compound number 4: Boc-Val-Ala-Leu-H wherein l isobutylchloroformate, N-methylmorphol e, then N,0- dimethylhydroxylamme HCl, THF. ii. HCl, dioxan. m. isobutylchloroformate, N- methylmorphlme, then Boc-Ala-OH, THF. lv HCl, dioxan V. Boc-Val-Osuc, N-methylmorpholme, DMF Vi . LAH; Figure 16 illustrates a reaction scheme for production of active inhibitors of Der PI . Figure 17 shows the molecular structure of the Roche protein kinase C inhibitors. A; Ro 31-8425/002, 3-[8(RS)- (ammomethyl) -6,7,8, 9-tetrahydropryrιdol [1, 2-a] mdol-10- yl] -4- (l-methyl-3-mdolyl) -IH-pyrrole-2, 5-dιone hydrochloride. B; Ro 31-7549/001, 3- [1- (3-ammopropyl) -3- mdolyl] -4- (l-methyl-3-mdolyl) -lH-pyrrole-2, 5-dιone hydrochloride Figure 18 shows the molecular structure of the protein tyrosine kinase inhibitor erbstatm, isolated from Streptomyces MH435-hF3 Brief Description of the Invention The present mvention relates to the field of apparatus and methods which provide the rapid generation of structure-activity relationships using auto-deconvolutmg combinatorial libraries, which facilitate the mvention of novel active compounds. We describe herein apparatus and methods which can be used for the rapid generation of structure-activity relationship (SAR) data and, therefore, the characterisation of the active motif of any group of compounds. The invention provides two orthogonal sets of mixtures of compounds m solution providing two complementary combinatorial libraries indexed in two dimensions for autodeconvolution. These are referred to as primary and secondary libraries. The general concept of two orthogonal sets of mixtures indexed in two dimensions can be applied to various permutations of numbers of wells, plate layout, number of permutations per mixture etc. However, according to the mvention the numerical interrelationship is defined as indicated below for libraries containing compounds with four variable groups B,C,D and E. General Deconvolution Formulae -Bb-Cc-Dd-n(Ee) - (I) 1) Primary and Secondary plates preferably have the same number of compounds per well [X] : otherwise there are two values, having X p and X s respectively. 2) The primary library comprises [np] plates. If Rp.Cp=Rs.Cs, then the number of plates in the secondary library is also [np] . If not, the number of plates in the secondary library [ns] is: ns = Rp .Cp . np Rs.Cs eg. A primary library of np=4 , Rp=8, Cp=10 can be set out in an Rs=4, Cs=5 secondary library with the number of plates equal to: ns = 8 x 10 . np 4 x 5 = 16 plates. Number of compounds per well -Bb-Cc-Dd-np(Ee) - (1) Number of possible combinations [k] is given by: k = b.c.d.np.e (2) When number of wells on a plate = [N] , number of compounds per well = [X] and number of plates = [np] : k = X.N.np (3) However, number of wells [N] is also defined by the number of rows [Rp] and number of columns [Cp] : N = Rp. Cp (4) Combining (3) and (4) : k = X.Rp .Cp.np (5) Combining (2) and (5) : b.c .d.np.e = X.Rp.Cp.np (6) Cancelling [np] from both sides of the equation.- b.c.d.e = X.Rp.Cp (7) Two of the variables (e.g. b.c) on the left side of the equation must each be equal in number to the number of columns [Cp] , whilst a remaining variable (e.g. d) on the 16 - left side must be equal in number to the number of rows [Rp] . So: [Cp] 2 . Rp.e = X.Rp.Cp (8) Cancelling [Cp] and [Rp] from both sides of the equation: Cp.e = X (9) where [e] is the number of variants along a fixed row; and if Rp=Cp, then Rp.e = X. Example for a 10 x 10 x 8 x 8 format over 4 plates np.e e = 2 10 x 2 = X X = 20. From an understanding of the general deconvolution formulae shown above, those skilled in the art will readily appreciate that the advantageous results of self- deconvolution according to the invention are obtainable utilising a number of different arrangements of wells, plate layouts, mixtures etc and that such variants on the preferred embodiment illustrated herein are intended to be within the scope of the present invention. The molecules may be cyclic or alicyclic. They may be linear or cyclic on the same structure or BCDE can be on a central scaffold (linear or cyclic) or one of the residues can be the backbone Where the symbol -B-C-D-E- or -B-C-D-nE- is used herein, it is to be understood to include all of these linear and cyclic variants within its definition At least one but not all the bonds between B, C, D, E need to be scissile Non scissile bonds may include sulphonamide, urea, aminomethylene . Several non-limiting examples of "scaffold" molecules are shown in Table 2, in which substituents Rj . -R « correspond to possible variable groups B, C, D and E. In a first aspect the invention provides novel compounds represented by the formula A-B-C-D-nE-F [I] in which; A represents a fluorescor internally quenched by F; B, C, D, and E represent groups such that the scissile bond between any two of these groups is a suitable bond, F represents a quencher capable of internally quenching the fluorescor A; and n represents an integer between 1 and 4 inclusive. In some embodiments the suitable bond is an unsubstituted amide bond (see Example 1) ; m other embodiments the suitable bond is an ester bond (see Example 2) . In preferred embodiments B, C, D, E are ammo acids or hydroxy acids That is a molecule with an amine or hydroxy terminus and a carboxylic acid terminus. The amme/hydroxy group may be positioned on the same carbon atom or separated by a number of atoms and atom types. In a second aspect the invention provides a combinatorial library of FRET compounds comprising a mixture of compounds of formula [I] In a third aspect the invention provides for the use of such a combinatorial FRET library a method which provides rapid generation of structure-activity relationships (SAR) which comprises detection and measurement of proteolytic enzyme activity by carrying out an assay with a library of combinatorial FRET (fluorescence resonance energy transfer) molecules to find a substrate or substrates for the enzyme. According to this method an identified substrate can be synthesised and used m biological assay for proteolytic enzymes Novel substrates are included m the scope of the invention. In a forth aspect the invention provides for the use of such a combinatorial FRET library m a method for detection and measurement of proteolytic enzyme activity against compounds of the library. In a fifth aspect the invention provides a method which comprises the identification of an enzyme inhibitor or inhibitors wherein a FRET compound which has been TABLE 2 identified as a substrate is used in an inhibition assay with the enzyme separately against a panel of possible inhibitors. • In a sixth aspect the invention provides a set of compounds which comprises two complementary FRET compound libraries. Such a set will be referred to hereafter as "apparatus" because it allows for the screening or assay method for identifying substrates or inhibitors of proteolytic enzymes. This set of compounds constituting an apparatus is capable of providing an auto-deconvoluting combinatorial library as will be described below. In a seventh aspect the invention provides a method of identifying and synthesising an inhibitor of a proteolytic enzyme which comprises detection and measurement of proteolytic enzyme activity by carrying out an assay with a library of combinatorial FRET (fluorescence resonance energy transfer) molecules, deconvoluting the library to find a substrate or substrates for the enzyme and synthesis of an inhibitor based on the substrate or substrates. The direct product of this method is one or more novel proteolytic enzyme inhibitors. In an eighth aspect the invention provides an inhibition assay which uses a FRET molecule, which has been identified as a substrate for the enzyme, wherein the molecule is assayed with the enzyme separately against a panel of possible inhibitors . In a ninth aspect the invention provides a complementary pair of compound libraries LI and L2 which constitute a set containing compounds of formula: Aa-Bb-Cc-Dd-n(Ee) -Ff-Gg giving a x b x c x d x e x f x g = Mn compounds in each library, there being a predetermined number (PI, P2) of mixtures each consisting of a predetermined number (Ql, Q2) of individual identifiable compounds m each library, wherein both LI and L2 contam the same Mn compounds, but where any two compounds which are found together in one mixture of Ql compounds of LI are not found together in any one of the P2 mixtures of L2. In a tenth aspect the invention provides a method of screening for enzymic activity using the libraries LI, L2 described above m which the PI mixtures of LI and the P2 mixtures of L2 are each placed separately into individual wells of well plates, the well plates having wells arranged m a format adapted to allow deduction of a unique active compound formula from the presence of activity one well of LI and one well of L2. The apparatus of the invention preferably comprises two complementary compound libraries, LI and L2, each containing n x 1600 compounds of the invention, of the type A-B 1-10 -C l l0 -Dj a -nfEj 2 ) -F-G [II] , in which: A = a fluorescor internally quenched by F, preferably an unsubstituted or substituted anthranilic acid derivative, connected by an amide bond to B B, C, D, E, are natural or unnatural ammo acid residues connected together by suitable bonds, although B, C, D and E can be any set of groups, provided that the scissile bond between D-E is an unsubstituted bond F = a quencher capable of internally quenching the fluorescor A, preferably an unsubstituted or substituted 3-nιtrotyrosme derivative. G = optionally present and is a hydrophilic moiety, preferably an aspartyl amide moiety. If present, G advantageously ensures that all compounds in the library are imparted with aqueous solubility. Also, G should not be a substrate for any type of enzyme. n = any integer between 1 and 4 inclusive. In an alternative, the scissile bond could be between B-C or C-D. (Note that A and F herein correspond generally and respectively to moieties F and Q of the prior art referred to above) . The numbers represented in subscript following residues B, C, D and E refer to the number of possibilities from which those residues are selected. Thus, by way of illustrative example, A-B j 5 -C-D-E ! 2 -F-G represents a mixture of the following ten compounds: A-B 2 -C-D-E 1 -F-G A-B 4 -C-D-E 1 -F-G A-B α -C-D-E 2 -F-G A-B 3 -C-D-E 2 -F-G A-B 4 -C-D-E 2 -F-G A-B 5 -C-D-E 2 -F-G The general combinatorial formula for each library can be expressed as : providing I l0 x l0 x 8 x n x 2 lx l = 1600n compounds. Both compound libraries, LI and L2, of the above type are synthesized using solid phase techniques using the Multipin approach 24 such that each library contains 1600n compounds as 80n mixtures of 20 distinct, identifiable compounds. These 20 component mixtures are then placed separately into each of 80 wells of a 96 well plate (the other two lanes are used for control experiments) and then screened against a known quantity of the protease. Thus it is an important part of the invention that regardless of the number of compounds contained in the two libraries LI and L2 (e.g. in the preferred embodiment: 1600n, where n = any integer between 1 and 4) the libraries themselves are complementary and amenable to deconvolution without recourse to resynthesis. It is also an important part of the invention that the library matrix has been especially formatted so that the most important site pairings P 2 and P x for proteolytic enzymes can be identified immediately without recourse to resynthesis. Those compounds of the type A-B-C-D-E-F-G that are the better substrates for the protease will be cleaved, and can be readily identified because the fluorescor, A, will be cleaved from its nearby quencher F, a time dependent manner which can be easily quantified. The fluorescent quenching by F of A only occurs when the two are in nearby proximity, normally within 30 angstrom units Hence cleavage of a scissile bond (e.g. the scissile bond D-E) allows F to move further away from A and thus allow A to fluoresce when excited by light of the correct wavelength 1. In this manner the most active compound can be rapidly identified without the need for further resynthesis and deconvolution Moreover, the wells that show the most rapid development of fluorescence can also be analysed by mass spectrometry, since by comparison with the original mixture, the identity of the most efficient substrate can be found by its disappearance into its two component parts, e.g A-B- C-D and E-F-G. Hence the problem of library deconvolution can be overcome and the most active substrate for the enzyme can be rapidly identified. In addition, after the initial treatment of the proteolytic enzyme with the library mixtures, LI and L2, the residual enzymatic activity in each well can be quantified by the addition of the most potent fluorogenic substrate for the enzyme, SI, which is found in the 16xn compound library Because of the nature of the library design this can be quickly prepared and purified If there is no appearance of increased fluorescence with the known substrate, SI, then the presence of an enzyme inhibitor can be inferred, which again can be quickly identified without the need for resynthesis. The general description of the library layout will now be described with reference to figures 1 to 14. For example, when n=l and the library contains 1600 compounds, m the first column of the first row (Al) (Fig.l) m the first plate (PI) of the library LI, (hereinafter designated as location A1,P1,L1) there will be one C component, C 1# one D component, D 1( the ten B components and the two E components (E τ and E 2 ) (Fig 2) In the tenth column of the first row (A10) m the first plate (PI) of the library LI, (hereinafter designated as location A10,P1,L1) there will be one C component, C 10 , one D component, D l( the ten B components and the two E components (E t and E-) . In the tenth column of the eighth row (H10) m the first plate (PI) of the library LI, (hereinafter designated as location H10,P1,L1) there will be one C component, C 10 , one D component, D β , the ten B components and the two E components (E Υ and E 2 ) . Hence all 1600 components are present in the one plate, because the 80 wells each contain 20 components. A second complementary library is synthesised as follows (Fig. 3) . In the first column of the first row (Al) of the first plate (PI) of the library, L2, (hereinafter designated as location A1,P1,L2), there will be ten C components, two D components (Di and D 2 ) , one B component, Bj, and one E component, Ej . In the tenth column of the first row (A10) of the first plate (PI) of the library, L2 , (hereinafter designated as location A10,P1,L2) , there will be ten C components, two D components (D ! and D 2 ) , one B component, B 10 , and one E component, E^ . In the first column of the second row (Bl) of the first plate (PI) of the library, L2, (hereinafter designated as location B1,P1,L2) , there will be ten C components, two D components (Dj and D 2 ) , one B component, B 1( and one E component, E 2 . In the tenth column of the second row (B10) of the first plate (PI) of the library, L2, (B10,P1,L2) there will be ten C components, two D components (D, and D 2 ) , one B component, B 10 , and one E component, E 2 . Hence only the first two rows are used to accommodate 400 compounds in total. In the first column of the first row (Al) of the second plate (P2) of the library, L2, (hereinafter designated as location A1,P2,L2) , there will be ten C components, two D components (D 3 and D 4 ) , one B component, B x , and one E component, Ej . (Fig. 4) . In the tenth column of the first row (A10) of the second plate (P2) of the library, L2, (hereinafter designated as location A10,P2,L2), there will be ten C components, two D components (D 3 and D , one B component, B 10 , and one E component, E l r In the first column of the second row (Bl) of the second plate (P2) of the library, L2, (hereinafter designated as location B1,P2,L2) , there will be ten C components, two D components (D 3 and D 4 ) , one B component, B 1( and one E component, E 2 . In the tenth column of the second row (BIO) of the second plate (P2) of the library, L2, (B10,P2,L2) , there will be ten C components, two D components (D 3 and D.,) , one B component, B 10 , and one E component, E 2 . Hence only the first two rows are used to accommodate 400 compounds in total . In the first column of the first row (Al) of the third plate (P3) of the library, L2, (hereinafter designated as location A1,P3,L2) , there will be ten C components, two D components (D G and D 6 ) , one B component, B 1( and one E component, λ (Fig 5) . In the tenth column of the first row (A10) of the third plate (P3) of the library, L2, (hereinafter designated as location A10,P3,L2) , there will be ten C components, two D components (D s and D 6 ) , one B component, B l0 , and one E component, E x . In the first column of the second row (Bl) of the third plate (P3) of the library, L2, (hereinafter designated as location B1,P3,L2), there will be ten C components, two D components (D 5 and D 6 ) , one B component, B and one E component, E 2 . In the tenth column of the second row (BIO) of the third plate (P3) of the library, L2, (B10,P3,L2) , there will be ten C components, two D components (D 5 and D 6 ) , one B component, B 10 and one E component, E 2 . Hence only the first two rows are used to accommodate 400 compounds in total . In the first column of the first row (Al) of the fourth plate (P4) of the library, L2, (hereinafter designated as location A1,P4,L2), there will be ten C components, two D components (D 7 and D θ ) , one B component, B l t and one E component, E x (Fig. 6) . In the tenth column of the first row (A10) of the fourth plate (P4) of the library, L2, (hereinafter designated as location A10,P4,L2) , there will be ten C components, two D components (D 7 and D 8 ) , one B component, B 10 , and one E component, Ej . In the first column of the second row (Bl) of the fourth plate (P4) of the library, L2 , (hereinafter designated as location B1,P4,L2), there will be ten C components, two D components (D 7 and D 8 ) , one B component, B 1( and one E component, E 2 . In the tenth column of the second row (BIO) of the fourth plate (P4) of the library, L2, (B10,P4,L2) , there will be ten C components, two D components (D 7 and D a ) , one B component, B 10 , and one E component, E 2 . Hence only the first two rows are used to accommodate 400 compounds in total . In this fashion two complementary libraries, LI and L2 are prepared. In library, LI, each of the 80 of wells contains a mixture of 20 components providing 1600 compounds for screening. In library, L2, four plates are used in which only the first two rows are employed, providing 20 wells of 20 components per well per plate, and furnishing the same 1600 compounds as are present in library LI, but in a format in which no two compounds found together in library, LI, will be found together in library, L2.. Thus it is an important part of the invention that the compounds contained in the two libraries LI and L2 are themselves complementary, in that any two compounds which are found together in a 20 component mixture in the same location (e.g. A1P1L1) in library LI, are not found together in any of the 20 component mixtures in any location of the library L2. Thus, for example, with reference to the primary library PI LI of figure 2 and the secondary libraries PI L2 , P2 , L2 , P3 L2 and P4 L2 of figures 3-6 it is possible to deconvolute an exemplary sequence: -B,-C,-D,-E, If this sequence is a substrate fluorescence will occur in PI LI at C 3 D,. This gives the information that the substrate is -?-C 3 -D 4 -?- If fluorescence occurs in P2 L2 at B 2 Ej it indicates a substrate The confirmation of the substrate as should be provided by non-fluorescence of PI L2, P3 L2 and P4 L2 which all contain -B 2 -C 3 -X-E 3 - where X is not D„ . In practice it is likely that more than one sequence will result in a substrate. Information as to which positions B-C-D-E- are sensitive to change (i.e. require a specific group) and which are insensitive (i.e. can tolerate more than one choice of group) in the context of the whole sequence gives valuable SAR data which can be used to model and/or synthesise related compounds. In analogous examples, where separately n=2, 3 or 4, extra plates are constructed in library LI format to accommodate the component pairs E 3 and E 4 (n =2) , E 5 and E 6 (n = 3) , and E 7 and E 8 (n = 4) , respectively. For the respective deconvolution libraries of the type, L2, the respective rows in the plates PI, P2 , P3 , and P4, are increasingly filled with the paired components O Ύ and D 2 , D 3 and D„, and D 5 and D 6 , and D 7 and D 8 , respectively. For example, when n = 3, and the library contains 4800 compounds, in the first column of the first row (Al) in the first plate (PI) of the library LI, (hereinafter designated as location A1,P1,L1) there will be one C component, C x , one D component, D 1# the ten B components and the two E components ( E λ and E 2 ) . In the tenth column of the first row (A10) in the first plate (PI) of the library LI, (hereinafter designated as location A10,P1,L1) there will be one C component, C 10 , one D component, D x , the ten B components and the two E components ( E λ and E 2 ) . In the tenth column of the eighth row (H10) in the first plate (PI) of the library LI, (hereinafter designated as location H10,P1,L1) there will be one C component, C 10 , one D component, D β , the ten B components and the two E components (Ei and E 2 ) . Hence 1600 components are present in the one plate, because the 80 wells each contain 20 components . In the first column of the first row (Al) in the second plate (P2) of the library LI, (hereinafter designated as location A1,P2,L1) there will be one C component, C 1( one D component, D 1; the ten B components and the two E components (E 3 and E„) . In the tenth column of the first row (A10) in the second plate (P2) of the library LI, (hereinafter designated as location A10,P2,L1) there will be one C component, C ι0 , one D component, D α , the ten B components and the two E components (E 3 and E 4 ) . In the tenth column of the eighth row (H10) in the second plate (P2) of the library LI, (hereinafter designated as location H10,P1,L1) there will be one C component, C 10 , one D component, D β , the ten B components and the two E components (E 3 and E 4 ) . Hence 1600 components are present in the one plate, because the 80 wells each contain 20 components . In the first column of the first row (Al) in the third plate (P3) of the library LI, (hereinafter designated as location A1,P3,L1) there will be one C component, C 1# one D component, D x , the ten B components and the two E components (E 5 and E 6 ) . In the tenth column of the first row (A10) m the third plate (P3) of the library LI, (hereinafter designated as location A10,P3,L1) there will be one C component, C 10 , one D component, D : , the ten B components and the two E components (E 5 and E 6 ) . In the tenth column of the eighth row (H10) in the third plate (P3) of the library LI, (hereinafter designated as location H10,P3,L1) there will be one C component, C 10 , one C component, C 8 , the ten B components and the two E components (E 5 and E 6 ) . Hence 1600 components are present the one plate, because the 80 wells each contain 20 components. In total the three plate, PI, P2 and P3 , contain 1600 compounds/plate 4800 compounds m total For example, when n = 4, and the library contains 6400 compounds, in the first column of the first row (Al) in the first plate (PI) of the library LI, (hereinafter designated as location A1,P1,L1) there will be one C component, C lr one D component, D 1( the ten B components and the two E components (E 3 and E 2 ) (Fig. 7) . In the tenth column of the first row (A10) the first plate (PI) of the library LI, (hereinafter designated as location A10,P1,L1) there will be one C component, C 10 , one D component, D lf the ten B components and the two E components (E x and E 2 ) In the tenth column of the eighth row (H10) m the first plate (PI) of the library LI, (hereinafter designated as location H10,P1,L1) there will be one C component, C 10 , one D component, D 8 , the ten B components and the two E components (E L and E 2 ) . Hence all 1600 components are present in the one plate, because the 80 wells each contain 20 components. In the first column of the first row (Al) in the second plate (P2) of the library LI, (hereinafter designated as location A1,P2,L1) there will be one C component, C 1; one D component, Dj, the ten B components and the two E components (E 3 and E 4 ) (Fig. 8) . In the tenth column of the first row (A10) in the second plate (P2) of the library LI, (hereinafter designated as location A10,P2,L1) there will be one C component, C 10 , one D component, D,, the ten B components and the two E components (E 3 and E 4 ) . In the tenth column of the eighth row (H10) in the second plate (P2) of the library LI, (hereinafter designated as location H10,P2,L1) there will be one C component, C 10 , one D component, D β , the ten B components and the two E components (E 3 and E 4 ) . In the first column of the first row (Al) in the third plate (P3) of the library LI, (hereinafter designated as location A1,P3,L1) there will be one C component, C 1# one D component, D x , the ten B components and the two E components (E 5 and EJ (Fig. 9) . In the tenth column of the first row (A10) in the third plate (P3) of the library LI, (hereinafter designated as location A10,P3,L1) there will be one C component, C 10 one D component, D the ten B components and the two E components (E 5 and EJ . In the tenth column of the eighth row (H10) in the third plate (P3) of the library LI, (hereinafter designated as location H10,P3,L1) there will be one C component, C 10 , one D component, D β , the ten B components and the two E components (E s and EJ . In the first column of the first row (Al) in the fourth plate (P4) of the library LI, (hereinafter designated as location A1,P4,L1) there will be one C component, C 1( one D component, D,, the ten B components and the two E components (E 7 and EJ (Fig. 10) Likewise, in the tenth column of the first row (A10) in the fourth plate (P4) of the library LI, (hereinafter designated as location A10,P4,L1) there will be one C component, C 10 , one D component, D 1( the ten B components and the two E components (E 7 and EJ . In the tenth column of the eighth row (H10) in the fourth plate (P4) of the library LI, (hereinafter designated as location H10,P4,L1) there will be one C component, C 10 , one D component, D 8 , the ten B components and the two E components (E 7 and EJ . A second complementary library is synthesised as follows. In the first column of the first row (Al) of the first plate (PI) of the library, L2 , (hereinafter designated as location A1,P1,L2) , there will be ten C components, two D components (O λ and D 2 ) , one B component, B l; and one E component, Ej . (Fig. 11) . In the tenth column of the first row (A10) of the first plate (PI) of the library, L2, (hereinafter designated as location A10,P1,L2), there will be the ten C components, two D components (D x and D 2 ) , one B component, B 10 , and one E component, E 1 . In the first column of the eighth row (HI) of the first plate (PI) of the library, L2 , (hereinafter designated as location H1,P1,L2), there will be the ten C components, two D components (D x and D , one B component, B lf and one E component, E 8 . In the tenth column of the eighth row (HIO) of the first.plate (PI) of the library, L2, (H10,P1,L2) there will be the ten C components, two D components (Di and D 2 ) , one B component, B 10 , and one E component, E„. Hence the matrix containing all ten columns and all eight rows are used to accommodate 1600 compounds in total. In the first column of the first row (Al) of the second plate (P2) of the library, L2, (hereinafter designated as location A1,P2,L2), there will be ten C components, two D components (D 3 and DJ , one B component, B 1( and one E component, E 3 (Fig. 12) . In the tenth column of the first row (A10) of the second plate (P2) of the library, L2, (hereinafter designated as location A10,P2,L2) , there will be ten C components, two D components (D 3 and DJ , one B component, B 10 , and one E component, E 3 . In the first column of the second row {Bl) of the second plate (P2) of the library, L2, (hereinafter designated as location B1,P2,L2) , there will be ten C components, two D components (D 3 and DJ , one B component, B lf and one E component, E 2 . In the tenth column of the eighth row (HIO) of the second plate (P2) of the library, L2, (H10,P2,L2), there will be ten C components, two D components (D 3 and DJ , one B component, B 1D , and one E component, E 8 . In the first column of the first row (Al) of the third plate (P3) of the library, L2, (hereinafter designated as location A1,P3,L2), there will be ten C components, two D components (D 5 and DJ , one B component, B 1( and one E component, E τ (Fig. 13) . In the tenth column of the first row (A10) of the third plate (P3) of the library, L2, (hereinafter designated as location A10,P3,L2) , there will be ten C components, two D components (D s and DJ , one B component, B 10 , and one E component, E 1 . In the first column of the second row (Bl) of the third plate (P3) of the library, L2, (hereinafter designated as location B1,P3,L2) , there will be ten C components, two D components (D 5 and DJ , one B component, B lr and one E component, E 2 . In the tenth column of the eighth row (HIO) of the third plate (P3) of the library, L2, (H10,P3,L2) , there will be ten C components, two D components (D s and DJ , one B component, B 10 , and one E component, E 8 . In the first column of the first row (Al) of the fourth plate (P4) of the library, L2, (hereinafter designated as location A1,P4,L2) , there will be ten C components, two D components (D 7 and DJ , one B component, B 1( and one E component, E Ύ (Fig.14) . In the tenth column of the first row (A10) of the fourth plate (P4) of the library, L2 , (hereinafter designated as location A10,P4,L2) , there will be ten C components, two D components (D 7 and DJ , one B component, B 10 , and one E component, E x . In the first column of the second row (Bl) of the fourth plate (P4) of the library, L2, (hereinafter designated as location B1,P4,L2) , there will be ten C components, two D components (D 7 and DJ , one B component, B_, and one E component, E 2 . In the tenth column of the eighth row (HIO) of the fourth plate (P4) of the library, L2, (H10,P4,L2) , there will be ten C components, two D components (D 7 and DJ , one B component, B 10 , and one E component, E s . The FRET strategy is based on the synthesis of two orthogonal sets of mixtures in solution. These solutions are each indexed in two dimensions. Thus the data from, for example, a protease scan identifies the most active compounds without the need for decoding or resynthesis. The positional preferences of sub-units (in this case amino acids) are optimised with respect to all other variant positions simultaneously. The synergistic relationship between all four positions is realised and both positive, beneficial and negative, deactivating data are generated. This leads to families (sub-populations) of substrates and their sub-unit preferences. The data can be fed into molecular modelling programs to generate pharmacophoric descriptors that encompass both the desirable features (from the positive data) and indicate undesirable interactions (from the negative data sets) . Note that a one dimensional scan only indicates one position at a time as 'most active' and does not explore the synergistic relationship between positions. The general methodology exemplified above with regard to the use of complementary combinatorial FRET libraries for the identification of proteolytic enzyme substrates, is equally applicable for identification of compounds from a library which interact with another active moiety. Combinatorial libraries of compounds containing four variable groups B, C, D and E can be produced an interactions with active moieties detected using suitable reporters or markers . For example, the active moiety may be a protein kinase. Protein kinases include (but are not limited to) the following: ZAP-70, Syk, p56 lck , p59 (yn , Yes, Hck, Src, Btk, Blk, Lyn, Raf, EGF kinase, Insulin receptor kinase, cyclin dependent kinases, Bcl-Abl, lkB-kinase C-terminal kinases, lkB N- terminal kinases, Jak kinases, MAP kinase kinases, MAP kinase kinase kinases, MAP kinases (Erks 1-3, p38 and Jun kinases) , STAT family C-terminal serine/threonine kinases, protein kinase A, protein kinase B, all protein kinase C isoforms . The active interaction of a protein kinase with a compound in a library may be detected, for example, by radioactively labelled phosphorylation of the substrate in the library. Thus it is possible to identify peptide substrates of protein tyrosine kinase by phosphorylation of exogenous peptide substrates present in a library. The assay uses the transfer of 33 P from radiolabelled ATP to lysine-tagged tyrosine-peptides substrate as a measure of kinase activity. The peptide binds to negatively charged membrane and is washed free of unincorporated [γ-"P] -ATP. The detection of β-energy emission using a fluor- containing scintillant on a β-counter instrument provides the measurement of peptide phosphorylation. In another example the active moiety could be a glycosidase and the interaction would be with enzyme substrates the library. In such a case a scissile bond between at least one of the groups B, C, D and E would be an acetal or glycosidic bond The groups B, C, D and E could be selected from furanosides and pyranosides In another example the active moiety could be a nuclease In such a case the scissile bond would be a phosphodiester bond and the groups B, C, D and E would be nucleotides The general methodology of the present invention can also be applied to the detection of an interaction between an active moiety such as a receptor and a library containing ligands for the receptor Examples of receptor/ligand interactions are shown by way of non-limitmg illustration below Chemokme receptor/chemok e l e. CKR3/Eotaxm SH3 domam/prolme ammo acid sequence i.e. Grb2/S0S peptide WW domam/prolme ammo acid sequence i.e. Yap (Yes- associated protein) /RSV Gag protein SH2 domam/phosphopeptide i.e Grb2/LNK phosphopeptide Lect doma /ligand l e. CD72/CD5. Further more, combinatorial libraries of possible antigens or antigenic epitopes could be tested for interaction with an active antibody moiety The reporter or marker for detection of a reaction in receptor/ligand and antibody/antigen libraries could be similar. Suitable reporters are, for example, (but not limited to) : 1. Biotin tag on the ligand using streptavidm-HRP for detection, 2. Alkaline phosphatase tag, 3. Radioisotope tag. The mvention will now be described by reference to the following examples. Example 1: Cysteine Protease Inhibitors and Substrates In this Example the proteolytic enzyme of interest is Der PI, which is found in house dust mite faeces. The example illustrates the synthesis of a number of FRET compounds in which the suitable bond is an unsubstituted amide bond, their use as a library for screening for potential substrates of Der PI, and subsequent identification and synthesis of active inhibitors of the enzyme. Purification of Der pi . Crude mite extract (-lOOmg, SmithKlme-Beecham, U.K) was dissolved in 5mL Phosphate Buffered Saline (PBS; 50 mM potassium phosphate; pH 7.4 containing 150 mM NaCl Der pi was purified by affinity column chromatography using 4C1 antibody (indoor Biotechnology, Deeside, U.K.) The crude preparation was mixed with -2 mL of affinity resm for 2 h at 4°C and then washed with 2-3 volumes of PBS. Elution of bound protein was carried out using 5mM glycme containing 50%(v/v) ethylene glycol. Fractions (2.2 mL) were collected and neutralised with 0.8 mL of 0.2 M sodium phosphate buffer, pH 7.0. The fractions were pooled and dialysed overnight against 4 L PBS followed by a second dialysis against 2 L PBS for 2-3h. The total protein was concentrated as required by ultrafiltration (MacroSep; Flowgen, U.K.) Synthesis of compounds The compounds were synthesised using the Multipm approach 25 26 using Fmoc-Rmk amide Macro crowns (Chiron Mimotypes Pty , Ltd., 11 Duerdin Street, Clayton Victoria 3168, Australia) with a loading of 7 μMoles. The ammo acid residues of each of the compounds were linked using amide bonds in a suitable form. The coupling chemistry employed is similar to that reported m the literature 27 for fluorenylmethoxycarbonyl protected am o acids and activated pentafluorophenyl esters, in which the side-chains are protected using acid labile protecting groups known to those skilled in the art, such as Boc- (for the -NH 2 of Lysine, -NH 2 of anthranilic acid and guanid o of argmine) , tBu- (for the -OH groups of ser e, threon e and tyrosine) , t-Bu for the -COOH group of Aspartic acid and Glutamic acid, Trityl- (for the Amide of Asparagine and Glutamine, and the amme functionality of the Histidme ring. The N-α-fluorenylmethoxycarbonyl protecting group of the coupled residues were cleaved using 20% piperid e m dimethylformamide (DMF) for 30 minutes at 20° C. The coupling reactions for the free acids such as Boc-ABz-OH (Boc-2-ammobenzoιc acid), and Fmoc- (3-nitro) tyrosme-OH were accomplished using 10 equivalents of a mixture of the free acid (1 eq.) :TBTU (0.98 eq ) . HOBt (0.98 eq.) : N- methylmorpholine (1.96 eq.) in dimethylformamide (500 μL) as solvent for 5 hours at 20° C. The other ammo acids were coupled as their pentafluorophenyl esters 26 for 2-6 hours. Hence, in order to couple approximately equal ratios of each component in the mixture of the derivatised amino acids as their pentafluorophenyl esters, a solution of a total of 0.98 equivalents (relative to the ammo group loading on the crown) of the mixture of amino acid pentafluorophenyl esters • HOBT (1 eq.) DMF (500 μL) were coupled for 16 hours at 20° C. The pins were then washed well with DMF and then recoupled using the same mixture under the same conditions. A third coupling of 10 equivalents (relative to the ammo group loading of the crown) for 2 hours m DMF was performed using this coupling protocol with equimolar mixtures of the derivatised pentafluorophenyl esters of the ammo acids m slightly less than 1 equivalent, it is possible to obtain approximately equal amounts of the coupled products to the crown. In this fashion the libraries are constructed with 20 compounds present on each crown. The compounds were cleaved from the crowns directly into the 80 designated wells of the desired 96 well plate. In the cleavage protocol each crown was treated with a mixture (600 μL) containing trifluoroacetic acid (95%) , triethylsilane (5%) for 2 hours at 20° C. The crowns were then washed with trifluoroacetic acid (500 μL) and this was then combined with the cleavage solution. The Fmoc-Rink amide Macro crowns (Chiron Mimotypes Pty., Ltd , 11 Duerdin Street, Clayton Victoria 3168, Australia) at 7 μMol loading per crown, were coupled with a 10 fold excess of a mixture containing L-Fmoc-As (O-t-Bu) -OH (leq) using TBTU (0 98 eq) and N-methylmorpholme (1.96 eq.) m the presence of HOBt (0.98 eq.) in DMF at 0.14M concentration After deblocking of the Fmoc group with 20% pipeπdme in DMF for 30 minutes and subsequent washing with DMF and then methanol, coupling of the Fmoc- (3- nitro) tyrosme-OH was accomplished using 10 equivalents of a mixture of the Fmoc- (3-nitro) tyrosine-OH (1 eq.) :TBTU (0.98 eq.) : HOBt (0.98 eq.) : N-methylmorpholme (1.96 eq.) in dimethylformamide as solvent at 0.14 M concentration for 5 hours at 20° C. Removal of the Fmoc group { vide infra) was followed by coupling of the mixtures of ammo acids in the ratios outlined and under the conditions described ( vide infra) . In a particular example the ammo acids comprising group B include Ala, Val, Leu, Ser, Asn, Gin, Glu, Lys, Phe, Pro. The ammo acids comprising group C include Ala, Val, Leu, Ser, Asn, Gin, Glu, Lys, Phe, Pro The ammo acids comprising group D include Ala, Val, lie, Leu, Nle, Ser, Glu, Phe. For n=4, the ammo acids comprising group E include Ala, Val, lie, Leu, Nle, Ser, Glu, Phe. Otherwise any selection from the ammo acids can be made for n=l, 2, or 3. The plates containing the combined cleavage solutions and were then evaporated to dryness to yield the component mixtures using a rotary centrifuge ("SPEEDVAC", Savant Instruments Inc., Farmmgdale, NY) at 800 rpm for 1 hour at 20° C under a reduced pressure of 10 2 mmHg. Each component was then transferred to the final mother plate using a (50%: 45%: 5%) mixture of acetonitrile: water: acetic acid. The plates were then lyophilised to dryness using at 20° C under a reduced pressure of 10 2 mmHg, and then stored at -20° C. In this fashion libraries of the type shown in Figures 2-14 were prepared. In further detail, the Multipm approach which was employed is described below: Multipin Synthesis Of Potential Substrates of Der pi The 'Chiron' multipm kit consists of a standard 8 x 12 pin holder containing 96 'pin stems' to which are reversibly attached 'crowns' . The 'crowns' provide a reactive polymer surface upon which a growing peptide is anchored during solid phase peptide synthesis. Each crown (the equivalent of the peptide-res m standard solid phase synthesis) can be considered to be an independent reactor by performing simultaneous synthesis in individual ImL wells of industry standard 96 well plates. Each well, and thus each crown, can be charged with a unique set of reagents providing unique sequences to each crown. Common steps such as washing or removal of N protection can be performed concomitantly. Synthesis is based upon the use of Nα- fluorenylmethyloxycarbonyl (Fmoc) protected amino acids. Side-chains of tri-functional amino acids are protected with acid labile groups such as trityl or tert-butyl. The addition of amino-acid residues to the growing peptide chain, a process termed 'coupling' proceeds through the utilisation of pre-formed pentafluorophenyl (pfp) esters or activation of the free acid, using the reagents HBTU or BOP in the presence of tertiary base (NMM) and HOBt as catalyst . The experimental techniques used are fully documented (Maeji, N. J. Bray, A. M. Valerio, R. M. and Wang, W., Peptide Research, 8(1), 33-38, 1995 and Valerio, R. M. Bray, A. M. and Maeji, N. J. Int . J. Pept . Prot . Res, 44, 158-165, 1994) and the mam steps are briefly as follows. General Methods Preparation of Multipin Assembly Whilst wearing standard plastic gloves, Fmoc-Rink Amide derivitized macrocrowns are assembled (simply clipped) onto stems and slotted into the 8 x 12 stem holder in the desired pattern for synthesis. Removal of N -F oc Protection A 250mL solvent resistant bath is charged with 200 ml of a 20% piperidine/DMF solution. The multipin assembly is added and deprotection allowed to proceed for 30 minutes. The assembly is then removed and excess solvent removed by brief shaking. The assembly is then washed consecutively with (200mL each) , DMF (5mins) and MeOH (Smins, 2mms, 2mins) and left to air dry for 15m ns. Quantitative UV Measurement of Fmoc Chromophore Release A lcm path length UV cell is charged with 1.2mL of a 20% piperidine/DMF solution and used to zero the absorbance of the UV spectrometer at a wavelength of 290nm. A UV standard is then prepared consisting of 5.Omg Fmoc- Asp(OBut) -Pepsyn KA (0.08mmol/g) in 3.2mL of a 20% piperidine/DMF solution. This standard gives Abs 290 = O.55- 0.65 (at RT) . An aliquot of the multipin deprotection solution is then diluted as appropriate to give a theoretical Abs 290 = 0.6, and this value compared with the actual experimentally measured absorbance showing the efficiency of previous coupling reaction. Coupling of amino-acid residues Whilst the multipm assembly is drying, the appropriate Nce-Fmoc ammo acid pfp esters (10 equivalents calculated from the loading of each crown) and HOBt (10 equivalents) required for the particular round of coupling are accurately weighed into suitable containers. Alternatively, the appropriate Nα-Fmoc ammo acids (10 equivalents calculated from the loading of each crown) , desired coupling agent e.g. HBTU (9.9 equivalents calculated from the loading of each crown) and activation eg HOBt (9.9 equivalents calculated from the loading of each crown), NMM (19.9 equivalents calculated from the loading of each crown) are accurately weighed into suitable containers. The protected and activated Fmoc ammo acid derivatives are then dissolved in DMF (500μl for each macrocrown, e.g. for 20 acrocrowns, 20 x lOeq x 7mmoles of derivative would be dissolved in 10 OOOμL DMF) The appropriate derivatives are then dispensed to the appropriate wells ready for commencement of the 'coupling cycle' As a standard, coupling reactions are allowed to proceed for 2- 6 hours (depending upon nature of coupling e.g. Ala to Ala 2 hours Val to Leu 6 hours . When coupling Fmoc ammo-acid pentafluorophenyl esters, lOeq of derivative in DMF (400μl) with bromophenol blue stock solution (lOOμl) is used for each macrocrown. This allows monitoring of the progress of the acylation reaction through the disappearance of the deep blue coloration of bromophenol blue in the presence of unreacted amme to a pale yellow upon completion of acylation. Preparation of Bromophenol Blue Stock Solution Bromophenol blue (20mg) is dissolved in DMF (50mL) and HOBt (lOmg) added. Washing Following Coupling If a 20% piperidine/DMF deprotection is to immediately follow the coupling cycle, then the multipm assembly is briefly shaken to remove excess solvent washed consecutively with (200mL each) , MeOH (5mms) and DMF (5mms) and deprotected (see above) . If the multipm assembly is to be stored, then a full washing cycle consisting brief shaking then consecutive washes with (200mL each) , DMF (5mms) and MeOH (5mms, 2mms, 2mms) is performed. Acidolytic Mediated Cleavage of Peptide-Pin Assembly Acid mediated cleavage protocols are strictly performed in a fume hood. A polystyrene 96 well plate (ImL / well) is labelled, then the tare weight measured to the nearest mg Appropriate wells are then charged with a trifluoroacetic acid / triethylsilane (95:5, v/v, 600μl) cleavage solution, in a pattern corresponding to that of the multipm assembly to be cleaved. The multipm assembly is added, the entire construct covered in tin foil and left for 2hrs . The multip assembly in then added to another polystyrene 96 well plate (ImL /well) containing trifluoroacetic acid / triethylsilane (95:5, v/v, 600μl) (as above) for 5 m s . The cleaved assembly is washed with DMF (200μL, 5ττuns) , MeOH (200μL, 5mιns) , the spent crowns removed and discarded, the ste s removed and washed by sonication methanol (lhr, RT) Work up of Cleaved Peptides The primary polystyrene cleavage plate (2hr cleavage) and the secondary polystyrene plate (5mm wash) (see above) are then placed m the SpeedVac and the solvents removed (minimum drying rate) for 90mms. The contents of the secondary polystyrene plate (see above) are transferred to their corresponding wells on the primary plate using an acetonitrile / water / acetic acid (50:45:5, v/v/v) solution (3 x 150μl) and the spent secondary plate discarded. Analysis of Products l.Oμl of each well (see above) is diluted to 400μl with 0.1%aq TFA and analysed by HPLC-MS. Column Vydac C4 (214TP52, narrowbore, 21 x 250mm) . Eluents :- Solvent A = 0.1%aq trifluoroacetic acid, Solvent B = acetonitrile / 10%A. Gradient : - 10-90% B m A over 27mms, 250ml / mm, 215nm UV detection. The individual substrates described below were prepared by the above methods and shown by HPLC-MS to be >95% with the correct mass. Final Lyophilisation of Peptides The primary polystyrene plate (plus the washings from the secondary plate) is covered with tin foil, held to the plate with an elastic band A pin prick is placed m the foil directly above each well and the plate placed at -80°C for 30mms. The plate is then lyophilised on the 'Heto freeze drier' overnight. Where appropriate individual peptides were then weighed and dissolved to lOmM stock solutions n DMSO prior to biological screening. Alternatively the 20 component mixture is weighed and the peptιde/20 component ratio is calculated Further coupling of ammo acid residues was carried out according to the multipm approach described above. Whilst the multip assembly was drying, the appropriate Nα-Fmoc amino acids (10 equivalents calculated from the loading of each crown) , HATU coupling agent (9.9 equivalents calculated from the loading of each crown) , HOAt catalyst (9.9 equivalents calculated from the loading of each crown) and DIPEA (19.9 equivalents calculated from the loading of each crown) were accurately weighed into suitable containers. The protected Nα-Fmoc ammo acids and coupling agents were then dissolved in DMF (500 μl for each macrocrown) and activated by the addition of DIPEA. The appropriate derivatives were then dipensed to their appropriate wells and as standard coupling to each macro crown was allowed to proceed for 2 hours . When coupling particularly hindered ammo acid residues such as N- ethyl, Cα-Methyl or unusual amino acids (whose coupling efficiency is unknown) the coupling reaction was repeated, as standard, for a further 2 hours Substrates for Der pi Using the general techniques described above, the following compounds were prepared and assayed as potential substrates against Der pi purified as described above. Peptide [SEQ ID Nos. 1-76] Measured K_, (μM) Abz-Val-Ala-Nle-Ser-Tyr(NOJ -Asp-NH 2 12 H-Val-Ala-Nle-Ser-TyrN0 2 -Asp-NH 2 NS H-Ala-Nle-Ser-Tyr(N0J -Asp-NH 2 NS Ac-Val-Ala-Nle-Ser-Tyr(NOJ -Asp-NH 2 NS H-Val-Ala-Nle-Ser-Tyr(NOJ -NH 2 NS H-Ala-Nle-Ser-Tyr(NOJ -Asp-NH 2 NS Ac-Val-Ala-Nle-Ser-Tyr(NOJ -NH 2 NS Abz-Val-Ala-Nle-Ser-Tyr (NOJ -NH 2 NM Abz-Val-Ala-Nle-Ser-NH 2 NM Abz-Val-Ala-Nle-Ser-Phe-Asp-NH 2 NM Abz-Val-Ala-Nle-Ser-Tyr-Asp-NH 2 NM Abz-Val-Ala-Nle-Ser-Ala-Asp-NH 2 NM Abz-Val-Ala-Nle-Ser-Lys-Asp-NH 2 NM Abz-Val-Ala-Nle-Ser-eAHA-Asp-NH 2 NM Abz-Ala-Nle-Ser-Tyr (N0 2 ) -Asp-NH 2 NM Abz-Nle-Ser-Tyr(NOJ -Asp-NH 2 NS Bz-Val-Ala-Nle-Ser-Tyr (NOJ -NH 2 NM a Bz (2-carboxy) -Val-Ala-Nle-Ser-Tyr (NOJ -NH 2 NM a Chex-Val-Ala-Nle-Ser-Tyr(N0 2 ) -NH 2 NM a n-Bu-Val-Ala-Nle-Ser-Tyr (N0 2 ) -NH 2 NM a Piv-Val-Ala-Nle-Ser-Tyr (N0 2 ) -NH 2 NM a Bz-Val-Ala-Nle-Ser-Tyr (NOJ -NH 2 NM a Abz-Val-Ala-Lys-Ser-Tyr (NOJ -Asp-NH 2 14 Abz-Val-Ala-Gln-Ser-Tyr (N0 2 ) -Asp-NH 2 6 Abz-Val-Ala-Thr-Ser-Tyr (N0 2 ) -Asp-NH 2 6 Abz-Val-Ala-hLeu-Ser-Tyr(NO ; ) -Asp-NH 2 4 Abz-Val-Ala-Cha-Ser-Tyr(N0 2 ) -Asp-NH 2 5 Abz-Val-Ala-His-Ser-Tyr (NOJ -Asp-NH, > 20 Abz-Val-Ala-ACH-Ser-Tyr(NOJ -Asp-NH 2 NS Abz-Val-Ala-DNle-Ser-Tyr (NOJ -Asp-NH 2 NS Abz-Val-Ala-3pyr-Ser-Tyr (N0 2 ) -Asp-NH 2 10 Abz-Val-Ala-Hyp-Ser-Tyr (NOJ -Asp-NH 2 NS Abz-Val-Ala-ACP-Ser-Tyr(N0 2 ) -Asp-NH 2 NS Abz-Val-Lys-hLeu-Ser-Tyr(N0 2 ) -Asp-NH 2 35 Abz-Val-DAla-hLeu-Ser-Tyr (NOJ -Asp-NH 2 NS Abz-Val-Tic-hLeu-Ser-Tyr(NOJ -Asp-NH 2 NS Abz-Val-ACH-hLeu-Ser-Tyr(N0 2 ) -Asp-NH 2 NS Abz-Val-Met (0) -hLeu-Ser-Tyr(N0 2 ) -Asp-NH 2 35 Abz-Val-2Nal-hLeu-Ser-Tyr(N0 2 ) -Asp-NH 2 NS Abz-Val-ACP-hLeu-Ser-Tyr(N0 2 ) -Asp-NH 2 NS Abz-Val-DLys-hLeu-Ser-Tyr(NOJ -Asp-NH 2 NS Abz-Val-DGln-hLeu-Ser-Tyr(NOJ -Asp-NH 2 NS Abz-Val-3pyr-hLeu-Ser-Tyr (N0 2 ) -Asp-NH 2 NS Abz-Val-Cha-hLeu-Ser-Tyr (NOJ -Asp-NH 2 NS Abz-DVal-Ala-hLeu-Ser-Tyr(N0 2 ) -Asp-NH 2 NS Abz-Gln-Ala-hLeu-Ser-Tyr (NOJ -Asp-NH 2 12 Abz-Lys-Ala-hLeu-Ser-Tyr(NOJ -Asp-NH 2 >15 Abz-Tic-Ala-hLeu-Ser-Tyr (N0 2 ) -Asp-NH 2 NS Abz-ACH-Ala-hLeu-Ser-Tyr(N0 2 ) -Asp-NH 2 NS Abz-Met (0) -Ala-hLeu-Ser-Tyr(NOJ -Asp-NH 2 20 Abz-3pyr-Ala-hLeu-Ser-Tyr(N0 2 ) -Asp-NH 2 >10 Abz-2Nal-Ala-hLeu-Ser-Tyr(N0 2 ) -Asp-NH 2 15 Abz-Leu-Ala-hLeu-Ser-Tyr (N0 2 ) -Asp-NH 2 18 Abz-Cha-Ala-hLeu-Ser-Tyr(N0 2 ) -Asp-NH 2 9 Abz-Bip-Ala-hLeu-Ser-Tyr(N0 2 ) -Asp-NH 2 2.5 Abz-Bip-Ala-hLeu-Tyr-Tyr (NOJ -Asp-NH 2 3 Abz-Bip-Ala-hLeu-Leu-Tyr(N0 2 ) -Asp-NH 2 3.7 Abz-Bip-Ala-hLeu-Lys-Tyr(N0 2 ) -Asp-NH 2 2 Abz-Bip-Ala-hLeu-Asp-Tyr(N0 2 ) -Asp-NH 2 5.0 Abz-Bip-Ala-hLeu-Abu-Tyr (N0 2 ) -Asp-NH 2 1 . 7 Abz -Bip-Ala-hLeu-Cha-Tyr (NOJ -Asp-NH 2 2 . 5 Abz-Bip-Ala-hLeu-Met (O) -Tyr (NOJ -Asp-NH 2 5 Abz-Bip-Ala-hLeu-Thr-Tyr(NOJ -Asp-NH 2 2.5 Abz-Bip-Ala-hLeu-3pyr-Tyr(N0 2 ) -Asp-NH 2 4 Abz-Bip-Ala-hLeu-Bu'Gly-Tyr (NOJ -Asp-NH 2 4 Abz-Bip-Ala-hLeu-Hyp-Tyr (N0 2 ) -Asp-NH 2 4 Abz-Phe-Val-Ala-Nle-Ser-Tyr (NOJ -Asp-NH 2 NM Abz-3.Pyr-Val-Ala-Nle-Ser-Tyr(N0 2 ) -Asp-NH 2 NM Abz-l.Naph-Val-Ala-Nle-Ser-Tyr(N0 2 ) -Asp-NH 2 17 Abz-2.Naph-Val-Ala-Nle-Ser-Tyr (N0 2 ) -Asp-NH 2 NM Abz-Tyr-Val-Ala-Nle-Ser-Tyr (N0 2 ) -Asp-NH 2 NM Abz-Bip-Val-Ala-Nle-Ser-Tyr (N0 2 ) -Asp-NH 2 10 Abz-Lys-Val-Ala-Nle-Ser-Tyr(NOJ -Asp-NH 2 15 Abz-Glu-Val-Ala-Nle-Ser-Tyr(N0 2 ) -Asp-NH 2 20 Abz-Leu-Val-Ala-Nle-Ser-Tyr(NOJ -Asp-NH 2 NM Abz-Hyp-Val-Ala-Nle-Ser-Tyr (NOJ -Asp-NH 2 NS NS indicates that the peptide was not hydrolysed by Der pi . NM indicates that the peptide was a substrate for Der pi , but the K_, was not measured. NM a indicates that the peptide was a substrate for Der pi , and its cleavage was followed by HPLC-MS showing hydrolysis to occur between-Nle-Ser- . Ranked in order of cleavage rate : -Bz > n-But > Piv > Bz (2-carboxy) > Abz . Coupling of ammo acid residues was carried out according to the multipm approach described above. Whilst the multipm assembly was drying, the appropriate Nα-Fmoc amino acids (10 equivalents calculated from the loading of each crown) , HATU coupling agent (9.9 equivalents calculated from the loading of each crown) , HOAt catalyst (9 9 equivalents calculated from the loading of each crown) and DIPEA (19.9 equivalents calculated from the loading of each crown) were accurately weighed into suitable containers. The protected Nα-Fmoc ammo acids and coupling agents were then dissolved DMF (500 μl for each macrocrown) and activated by the addition of DIPEA. The appropriate derivatives were then dispensed to their appropriate wells and standard coupling to each macrocrown was allowed to proceed for 2 hours . When coupling particularly hindered ammo acid residues such as N-Methyl, Cα-Methyl or unusual ammo acids (whose coupling efficiency is unknown) the coupling reaction was repeated, as standard, for a further 2 hours. The following sequences were synthesised m this way Peptide Sequence [SEQ ID Nos. 77-85] Measured Km(μM) Abz-Val-Ala- (NMe)Nle-Ser-Tyr(NOJ -Asp-NH 2 NS Abz-Val- (NMe)Ala-Nle-Ser-Tyr(NOJ -Asp-NH 2 NS Abz-Val-Ala-Aib-Ser-Tyr (NOJ -Asp-NH 2 NS Abz-Val-Aib-Nle-Ser-Tyr (N0 2 ) -Asp-NH 2 NS Abz-Deg-Ala-Nle-Ser-Tyr (N0 2 ) -Asp-NH 2 NS nBu-D.Ser-D.Nle-D.Ala-D.Val-p.Aba-NH 2 NS Bz-Val-Ala-Statme-Ser-eAha-NH 2 NS Abz-p.Aba-Nle-Ser-Tyr (N0 2 ) -Asp-NH 2 NS Abz-Cmpi-Nle-Ser-Tyr (NOJ -Asp-NH 2 NS NS-Not Substrate: Not hydrolysed by Der pi NM-Substrate but not measured: Substrate for Der pi but not measured. Assay procedure Each mixture of 20 compounds in the libraries of the apparatus described herein was screened at a concentration of 1.0 μM per compound an assay against the cystemyl protease Der pi . The most active wells were identified by the rate of emission of fluorescence at 420 nm when the samples were irradiated at 320 nm. An analysis of the two complementary libraries showed that the best substrates for the enzyme were: Abz-B-C-D-E-Tyr(N02) -Asp-NH2 Where B= Valme>Alanme , Glutamme , Leuc e , Phenylalanme C= Alanme>>Glutamme , or Lysme . D= Leucme , Norleucme or Alanme>Serme E=Serme The best substrate was : [SEQ ID : 1] Abz -Val -Ala-Nle-Ser-Tyr (N0 2 ) -Asp-NH 2 This compound was then resynthesised as a single component using the peptide synthesis methodology described herein. The k cac /K-, value for the pure substrate the Der pi assay was measured as 3.5x10^ l s l , and was considered to be suitably high for use m a high throughput assay for the general screening of inhibitors of Der pi . High throughput assay development Plate assays were carried out m 96 well plate format, using 0.1 ug of Der pi per 100 μL assay volume in each well and using 20 μM of the substrate. All assays were performed in Assay Buffer (AB; 50 mM potassium phosphate, pH 8.25 containing ImM ethylenediammetetraacetic acid (EDTA) and ImM dithiothreitol (DTT) The Der pi enzyme is pre-activated by addition of DTT and this in incubated at room temperature for 5 mm. prior to initiation of the assay. As an example for the screening methodology, each well contains a 5 μL of a 20 μM solution of the test compound in DMSO, 10 μL of a 200 μM aqueous solution of the substrate, 2., and 85 μL of Der pi m AB is added to initiate the reaction. Enzyme activity s monitored by fluorescence using 320 nm for excitation and 420nm for the emission wavelengths using a Labsystems Fluroskan Ascent machine. Kinetic measurements were carried out using a Hitachi F-4500 Fluorescence Spectrophotometer. Synthesis of inhibitors of Der pi The best substrate described above was shown by HPLC-mass spectroscopic analysis of the enzyme/ assay solution, to be cleaved between the Norleucme-Serme amide bond. Replacement of the terminal Abz group by a series of derivatives (e.g. Boc-, Pivaloyl, Benzoyl, and 2- carboxy- Benzoyl) affected substrate activity and specificity for the Der pi enzyme. W th this knowledge of the P x -PJ cleavage site and for the P 4 -P 3 -P 2 -P ! motif, the compound Boc-Val-Ala-Leu-H, 4_, was synthesised as shown Scheme la, figure 15. Attachment of a suitable Michael acceptor such as CH=CH- C02Et, and -CH=CH-S0 2 Ph to the motif (Scheme 2, figure 16) , provided active inhibitors of the enzyme with apparent IC S0 values of 50nM, 1000 nM and lOOnM respectively. Based on the sequence 4, a series of acyloxymethylketone compounds having active Der PI inhibitor activity was prepared. Details of the preparation and activity of this novel group of cysteine protease inhibitor compounds are discussed in co-pending International Application Publication Number WO97/04004. Example 2: Viral Protease Inhibitors and Substrates Design of Depsipeptides Another suitable bond in a compound of general formula (I) , (II) or (III) according to the mvention is an ester bond to form a depsipeptide The incorporation of depsipeptide substrates aided the identification of substrates for low reactivity viral proteases, such as viral seπne proteases . For example, substrates of the general formula n [Abz-Bj . i o -Ci m -Di β y [COO] -E x 8 -Tyr (NOJ -Asp-NHJ were produced However, a significant proportion of viral proteases only recognise substrate sequences larger than those represented by the general structure above. It is well acknowledged that by the very nature of action of a viral protease (function is to cleave immature viral proteins into the mature viral package) one automatically receives data concerning the natural substrate sites Thus, the general structure above can be extended by introducing extra fixed ammo-acids at appropriate sites. A logical extension would be to introduce the known Pl-Pl' cleavage site as the depsipeptide bond, then subsequently introduce the four variant positions following the standard format hus; n [Abz -Bi i- - Ci i- -Dj . g - Ej . 8 - Pl y [COO] - PI ' -Tyr ( NOJ -Asp -NHJ Furthermore, if these substrates again proved to be too small, one may use the known substrate sequences to introduce additional fixed positions. For instance, with Hepatitis NS3 protease it is known that the natural P6 position is a conserved acidic residue (aspartic or glutamic acid) Thus one could extend the above structure as detailed below. n [Abz-Pβ-Bi i o -Ci lo -Dj . ,,-Ei β-Pl y [COO] -PI' -Tyr (NOJ -Asp-NHJ The novel methodology described herein greatly faciltiates the mvention of therapeutically useful proteolytic enzyme inhibitors and is commercially exploitable This is because the best substrate motif for the proteolytic enzyme can be rapidly identified, and, since there exist m the literature a variety of ways for attaching motifs which react with the active site of a proteolytic enzyme, especially for aspartyl, metallo, ser e and cystemyl proteases, an enzyme inhibitor can be readily synthesised. Moreover, amide bond replacements or transition state mimetics can be incorporated into the molecule, which would be especially useful for the inhibition of aspartyl or metallo proteases. The method described also facilitates the rapid development of a screening assay for novel protease inhibitors. The most potent fluorogenic substrate discovered by library screening can subsequently be used for the detection of inhibitors of the particular proteolytic enzyme under scrutiny. The presence of an inhibitor withm the compound libraries described is readily detected by retreatment of the assay mixture with the most active fluorogenic substrate, which will allow the immediate measurement of the remaining proteolytic enzyme activity The invention provides self-decoding, combinatorial fluorogenic libraries, and it will greatly facilitate the design and invention of novel protease inhibitors because l. The peptides of the library may have increased aqueous solubility m comparison to peptides containing similar and other fluorogenic and quencher groups. ii . The peptides are stable to contaminating exopeptidases . in The self deconvolution method described, coupled with the continuous analysis of the rate of substrate cleavage data, allows the immediate identification of the most active binding motif contained withm the substrate library. lv. The method allows for the rapid assessment of the enzyme assay mixture for any compounds m the library that are acting as enzyme inhibitors Example 3: Kinase Inhibitors and Substrates As previously indicated, the present invention is a broadly applicable methodology and is not limited to the use of FRET libraries to detect protease inhibitor/substrate compounds. An example of another use of the invention is the detection of the interaction of a kinase with a substrate/inhibitor by monitoring radioactively labelled phosphorylation of compounds of complementary combinatorial libraries. The Identification of a ZAP-70 Tyrosme Kmase Inhibitor ZAP-70 is a intracellular kinase essential for T-cell signal transduction from the T-cell antigen receptor. Mutations resulting n non-functional ZAP-70 kinase have been observed in patients suffering from a severe combined immunodeficiency disease. Drug down-modulation of ZAP-70 kmase activity has therefore been seen by the ma]or pharmaceutical companies as a potential method of down- regulatmg the immune system in an antigen-independent manner. Such a drug could be used both transplant treatment and as a therapy m autoimmune diseases. Generation of ZAP-70 kinase catalytic domain The full length protein ZAP-70 tyros e kinase possesses amino-terminal tandem SH2 domains which allow enzyme recruitment to phosphorylated tyrosine residues on the T cell antigen receptor, but are unnecessary for protein kinase catalytic activity. Removal of the SH2 domains results m a constitutively active, stable ZAP-70 kinase. Specific oligonucleotide polymerase chain reaction (PCR) primers were used to amplify amino acids 308 to 619 and add a carboxy- erminal hexahistidine sequence using human Jurkat cDNA as a template. This amplicon was cloned into the baculoviral transfer vector pACUW51 (Pharmmgen) and recombmant baculovirus produced by homologous recombination Sf9 insect cells. CatZAP(H6) Purification Low titre stocks of mixed CatZAP-70 baculovirus were used to infect sub-confluent monolayers of Sf9 cells. Cells were harvested 3 days post-mfection, washed in PBS and osmotic lysis performed immediately. Isolation of CatZAP(Hj protein was carried out using cobalt column chelation chromatography, fractions eluted using 100 mM imidazole and kinase activity determined by 3 P transfer. High throughput protein kinase screening assay The interaction of the active protein kinase moiety with compounds of a combinatorial peptide library can be determined. A rapid and very sensitive assay was developed for measuring the amount of peptide substrate phosphorylation produced by CatZAP(Hj . The peptide library was synthesised with a positively charged tetra-lysme tag. After peptide incubation with the protein kinase and ATP, containing trace amounts of radiolabelled ["P-γ] -ATP, the reaction mixture is passed through a negatively charged phosphocellulose membrane using a 96 well plate vacuum manifold system (Millipore) . Positively charged peptide is bound by the membrane, whereas unincorporated [33P-γ] - ATP is washed through. Scmtillant is then applied to the membrane and the amount of phosphorylated peptide calculated from the level of radioactivity on the membrane, measured using a well plate scintillation counter This type of assay can be readily applied to other protein kinase systems. The result of this screening assay is the identification of one or more protein kinase substrate motifs, which appear to have the common features of a negatively charged N-terminus and a conserved Pro at the C-termmus. For other protein tyrosme kinases, the existing libraries may provide some information, although it is envisaged that additional libraries will require to be built. For Ser/Thr kmases, the technology may be adapted by the use of a central Ser or Thr residue as the phosphoacceptor moiety. In addition, technology is being developed withm the chemistry department which will allow for the generation of cyclic peptide libraries. Incorporation of a central phosphoacceptor residue in these libraries will provide a source of constrained molecules for use in kinase assays Abbreviations Abbreviations used herein are as follows: Abbreviations for ammo acids and nomenclature of peptide structures follow the recommendations given m. IUPAC-IUB Commission on Biochemical Nomenclature, (J. Biol . Chem, 247, 997, 1971) . All chiral ammo acids are of the L conf guration unless otherwise stated. Other abbreviations used are : -Abu , b-amino butyric acid, : Abz, 2-amino benzoyl : ACH, 1-amιno-l-carboxy-cyclohexane : ACP, 1-amino-1-carboxy- cyclopropane : Bip, Biphenylalanme : n-Bu, n- butoxycarbonyl : Bz, Benzoyl : Bz (2-carboxy) , 2- carboxybenzoyl : Bu'Oly, tert-Butylglycyl • BOP, benzotπazoyl-oxy-tπs- (dimethylamino) -phosphonium hexafluorophosphate : Cha, cyclohexylalanme . Chex, 1- carboxycyclohexyl : eAHA, gamma ammohexanoyl : HBTU, 0- benzotπazoyl-N,N,N' ,N' -tetramethyluromum hexafluorophosphate : HOBt, 1-hydroxybenzotrιazole : Hyp, trans-4-hydroxyprolmyl : hLeu, homoleucyl : 2Nal, 2- napthylalanme : NMM, N-methylmorpholme : Piv, pivoyl : 3pyr, 3-pyridylalanine : Tic, 2-carboxytetrahydroqumolyl : Tyr (NOJ , 3-nιtrotyrosme. DMF, dimethylformamide; Fmoc, fluorenylmethoxycarbonyl; HPLC, high performance liquid chromatography; Pfp, pentafluorophenyl, tBoc, tert-butoxycarbonyl,- tBu, tert- butyl; TFA, trifluoroacetic acid; Pmc, pentamethyl chroman, Pbf, pentamethylbenzofuran, TBTU, 2-(lH- Benztrotrιazole-1-yl) -1,1,1,3, 3-tetramethyluronium tetrafluoroborate; Tr , Trityl. p.Aba, 4-ammobenzoyl; Aib, Ammoisobutyπc acid; Bip, Biphenylalanme; nBu, n-Butyl; Bz, Benzoyl; Cmpi, Carboxymethylpiperazme; Deg, Diethylglycme; DIPEA, N,N- Diisopropyl-ethyla me; HATU, 0- (7-azabezotrιazol-l-yl) - 1 , 1, 3 , 3-tetramethyluronium hexafluorophosphate; HOAT, 1- hydroxy-7-azabenzotπazole, Naph, Naphthylalanme; 3. Pyr, 3-pyridiylalanme; Tyr(N0 2 ) , 3-nitro- yrosine References 1. I. Schlechter and A. Berger, Biochem. Biophys. Res. Commun., 1967, 22, 157-162) 2. A. Carmel et al . , FEBS Lett., 1973, 10, 11- 3 M. M. Meldal and I. Svendsen, J. Chem. Soc. Perkin Trans.1, 1995, 1591-1596, 4. M. Meldal and K. Breddam, Anal. Biochem., 1991, 195. 141-147, 5. T. Forster, Ann. Phys . , 1948, 6 , 55. 6. A. Yaron, A. Carmel, and E. Katchalski-Katzir, Anal. Biochem. 1979, 9_5, 228 and references therein. 7. S.A. Latt et al . , Anal. Biochem., 1972, 0, 56. 8. A. Persson et al . , Anal. Biochem., 1977, 81, 2,96. 9. I . Yu Filppova et al . , Bioorg. Khim. , 1986, 12., 1172. 10. J. Pohl et al . , Anal. Biochem., 1987, 165. 96. 11. S.J. Pollack et al., J. Am. Chem. Soc, 1989, 111. 5961. 12. E. K. Bratovanova and D.D. Petkov, Analytical Biochem., 1987, 162. 213. 13. J. Singh et al . , J. Med. Chem., 1995, 3_8, 217-219 and references therein. 14. M. Green et al . in ~Innovation and Perspectives m Solid Phase Synthesis' (R. Epton Ed.) Mayflower Worldwide Ltd., Birmingham, UK. 1994, 239-244. 15. J. R. Petithory et al . , Proc. Natl. Acad. Sci. USA, 1991, 8 11510-11514 16. J. Berman et al . , J. Biol. Chem., 1992, 267. 1434- 1437. 17. H. Drevm, A, -T. Martin, J. Carlsson, S. Oscarsson, T. Lovgren, I. Hemmila and M. Kwiatkowski, WO89/10975, (5-5-1988) 18. A. J. Garman and N. G. Phillips, WO 94/28166 (27-5- 1993) 19. G.T. Wang and E.D. Matayoshi , E.P. 428000 (3-11- 1989) 20. G. A. Krafft, G. T. Wang and E.D. Matayoshi, EP 428000, (3-11-1989) . 21. G. R. Marshall and M. V. Toth, USP 5,164,300, (11- 12-1990) . 22. G. R. Marshall and M. V. Toth, USP 5,011,910, (28- 12-1989) . 23. K. T. Chapman, N. A. Thornberry, M. Maccoss, J. R. Weidner, R. A. Mumford, W. K. Hagmann, EP 528487A (16-8-1991) 24. R. P. Haugland, WO 93/04077, (23-8-1991) 25. R. M. Valerio, A. M. Bray N. J. Maeji, Int. J. Peptide Protein Res. , 1994, 4_4, 158-165 26. M. Bastos, N. J. Maeji and R. H. Abeles, Proc. natl. Acad. Sci. , 1995, 92., 6738-6742. 27. ^ Solid Phase Peptide Synthesis' , E. Atherton and R. C. Sheppard, IRL Press 1989.

Claims: Claims 1. Libraries of compounds which interact with an active moiety, the libraries comprising two orthogonal sets of mixtures of compounds in solution providing two complementary combinatorial libraries (referred to as primary and secondary libraries) indexed in two dimensions for autodeconvolution, thereby allowing characterisation of an active motif any group of compounds 2 A complementary pair of compound libraries LI and L2 according to claim 1 which constitute a set containing compounds having four variable groups B, C, D, E of formula -Bb-Cc-Dd-n(Ee) - giving b x c x d x e = Mn compounds in each library, there being a predetermined number (PI, P2) of mixtures each consisting of a predetermined number (Ql, Q2) of individual identifiable compounds in each library, wherein both LI and L2 contain the same Mn compounds, but wherem any two compounds which are found together in one mixture of Ql compounds of LI are not found together in any one of the P2 mixtures of L2. 3. A method of screening for interaction of an active moiety applied to a well with one or more compounds of a mixture in the well, using the libraries LI, L2 according to claim 2, in which the PI mixtures of LI and the P2 mixtures of L2 are each placed separately into individual wells of well plates, the well plates having wells arranged in a format adapted to allow deduction of a unique active compound formula from the presence of activity in one well of LI and one well of L2. 4. A method according to claim 3 wherem the format complies with general deconvolution formulae m which: (l) ns = Rp.Cp. Np Rs . Cs (ii) k = b.c.d.np.e (m) k = x.N.np (IV) N = Rp.Cp (v) K = X.Rp.Cp.np (vi) b.c.d.e = X.Rp.Cp (vn) Cp.e = X and wherem np = number of primary plates ns = number of secondary plates Rp = number of primary rows Rs = number of secondary rows Cp number of primary columns Cs = number of secondary columns K = number of combinations of compounds N = number of wells on a plate, and X = number of compounds per well. 5. A method according to claim 4 wherein 6. A method according to claim 3 wherein the active moiety is selected from the group consisting of enzymes, receptors and antibodies. 7. A method according to claim 6 wherem interaction of the active moiety with the compounds of the library is selected from the group consisting of the interaction of a substrate or inhibitor with an enzyme, the interaction of a ligand with a receptor, and the interaction of an antigen or antigenic epitope with an antibody. 8. A method according to claim 6 wherein the active moiety is selected from the group consisting of the following enzyme classes: 1. Ox doreductase a ) dehydrogenase b) oxidase c ) peroxidase d) catalase 2 . Hydrolase f ) peptidase (proteolytic enzyme ) 3 . Transf erase g ) am otrans f erase h) kmase l ) glucosyl transf erase 4 . Lyase j ) decarboxylase k) dehydratease 5. Isomerase 1) racemase m) mutase 6. Ligase s n) synthetase o) carboxylase 9. A method according to claim 8 wherem the active moiety is a protease selected from the group consisting of the following: 0 1. Aspartyl proteases, such as renin, HIV, cathepsin D and cathespin E. 2. Metalloproteases, such as ECE, gelatinase A and B, collagenases, stromolysins . 3. Cystemyl proteases, such as apopam, ICI, DerPI, s cathepsin B, cathepsin K. 4. Serine proteases, such as thrombm, factor Vila, factor Xa, elastase, trypsin. 5. Threonyl proteases, such as proteasome S. 10. A method according to claim 3 wherem the active moiety is a kinase and the interaction is with a kinase substrate or inhibitor. 11. A method according to claim 3 wherein the active moiety is a receptor and the interaction is with a ligand. 12 A method according to claim 3 wherem the active moiety is an antibody and the interaction is with an antigen or antigenic epitope. 13. A method according to claim 3 wherem the mixture of compounds in all of the wells of primary library LI contain all variations of a first two variable groups and each well contains a unique pair of the two other variable groups, and wherein the mixture of compounds in all of the wells of secondary library L2 contain all variations of the two other variable groups and each well contains a unique pair of the first two variable groups 14. A method according to claim 13 wherem the primary library contains all variations of variable groups B and E, and unique pairs of variable groups C and D; and the secondary library contains all variations of variable groups C and D, and unique pairs of variable groups B and E. 15. A method according to claim 3 or 4 which comprises the steps of forming libraries according to claim 1 or 2, placing the libraries into the wells of well plates m a format according to claim 3 or 4, applying an active moiety to the wells, screening for an interaction of the active moiety with one or more compounds of a mixture in a well of primary library LI and secondary library L2 and deducing from the respective LI and L2 well positions a unique active compound formula.

Inventors: HART TERANCE, JOHNSON TONY, QUIBELL MARTIN, HART, TERANCE, JOHNSON, TONY, QUIBELL, MARTIN
Applicants: HART TERANCE, JOHNSON TONY, PEPTIDE THERAPEUTICS LTD, QUIBELL MARTIN, HART, TERANCE, JOHNSON, TONY, PEPTIDE THERAPEUTICS LIMITED, QUIBELL, MARTIN
Application year: 1997
Main classification: C07K1
IPC codes: B01J19, C07K1, C07K5, C07K7, C12N9, C40B30, C40B60, G01N33