Substrates and inhibitors of proteolytic enzymes

Abstract

The present invention relates to the field of compounds which are substrates or inhibitors of proteolytic enzymes and to apparatus and methods for identifying substrates or inhibitors for proteolytic enzymes. We have devised a combinatorial method for the rapid identification of binding motifs which will greatly expedite the synthesis of inhibitors of a variety of proteolytic enzymes such as aspartyl proteases, serine proteases, metallo proteases and cysteinyl proteases.

1 . A complementary pair of compound libraries L 1  and L 2  which constitute a set containing combinatorial FRET compounds of formula:  
       Aa-Bb-Cc-Dd-n(Ee)-Ff-Gg  
       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 wherein all compounds are in aqueous solution; and  
 n represents an interger between 1 and 4 inclusive;  
 giving a×b×c×d×e×f×g=Mn compounds in each library, there being a predetermined number (P 1 , P 2 ) of mixtures each consisting of a predetermined number (Q 1 , Q 2 ) of individual identifiable compounds in each library, wherein both L 1  and L 2  contain the same Mn compounds, but wherein any two compounds which are found together in one mixture of Q 1  compounds of L 1  are not found together in any one of the P 2  mixtures of L 2 .  
 
     
     
         2 . A method of screening for enzymic activity, wherein the activity is the interaction of proteolytic enzyme applied to a well with one or more compounds of a mixture in the well, using the libraries L 1 , L 2  according to  claim 1 , in which the P 1  mixtures of L 1  and the P 2  mixtures of L 2  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 L 1  and one well of L 2 .  
     
     
         3 . A method according to  claim 2  wherein the format complies with general deconvolution formulae in which:  
                   (   i   )                   n                 s     =         Rp   ·   Cp   ·       Rs   ·   Cs          N                 p                   (ii) k=b.c.d.np.e (iii) k=x.N.np (iv) N=Rp.Cp (v) K=X.Rp.Cp.np (vi) b.c.d.e=X.Rp.Cp (vii) Cp.e=X (viii) Rp.e=X, if Rp=Cp  
       and wherein 
 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.  
 
     
     
         4 . A method according to  claim 3  wherein 
 n= 4   
 ns=16  
 Rp=8  
 Rs=4  
 Cp=10  
 Cs=5  
 K=6400  
 N=80  
 x=20  
 
     
     
         5 . A library pair according to  claim 1  wherein the scissile bond is between D and E.  
     
     
         6 . A library pair according to  claim 1  wherein A represents an unsubstituted or substituted anthranilic acid derivative.  
     
     
         7 . A library pair according to  claim 1  wherein B, C, D and E independently represent natural or unnatural amino acid residues.  
     
     
         8 . A library pair according to  claim 1  wherein F represents an unsubstituted or substituted 3-nitrotyrosine derivative.  
     
     
         9 . A library pair according to  claim 1  wherein the scissile bond is a suitable bond selected from the group consisting of an unsubstituted amide bond, and an ester bond.  
     
     
         10 . A library pair according to  claim 1  wherein the formula further comprises G which ensures the compound is imparted with aqueous solubility.  
     
     
         11 . A library pair according to  claim 10  which is represented by the formula A-B-C-D-n(E)-F-G in which A, B, C, D, E, F and n are as defined in  claim 1;  and G represents a hydrophilic moiety which is not an enzyme substrate.  
     
     
         12 . A library pair according to  claim 10  or  11  of general formula:  
       Abz-B-C-D-E-Tyr(NO2)-Asp-NH2  
       Where 
 B=Valine>Alanine, Glutamine, Leucine, Phenylalanine  
 C=Alanine>>Glutamine, or Lysine.  
 D=Leucine, Norleucine or Alanine>Serine  
 E=Serine  
 
     
     
         13 . Apparatus which comprises an auto-deconvoluting set of compounds according to  claim 1  that facilitates the invention of novel inhibitors of proteolytic enzymes and the rapid generation of structure-activity relationships (SAR) by the detection and measurement of proteolytic enzyme activity.  
     
     
         14 . Apparatus according to claims  13  wherein each library comprises a mixture of compounds represented by the formula:  
       A-B 1-10 -C 1-10 -D 1-8 -n (E 1-2 )-F.  
     
     
         15 . Apparatus according to  claim 14  wherein the formula further comprises G which ensures that compounds in the library are imparted with aqueous solubility.  
     
     
         16 . Apparatus according to  claim 15  wherein each library comprises a mixture of compounds represented by the formula:  
       A-B 1-10 -C 1-10 -D 1-8 -n (E 1-2 )-F-G  
       in which; 
 G represents a hydrophilic moiety which is not an enzyme substrate.  
 
     
     
         17 . Apparatus according to  claim 15  or  16  in which G represents an aspartyl amide moiety.  
     
     
         18 . Apparatus according to any one of  claims 13  to  17  wherein the library comprises 1600n compounds as 80n mixtures of 20 distinct, identifiable compounds.  
     
     
         19 . Apparatus according to  claim 18  wherein the mixtures of 20 distinct, identifiable compounds are placed separately into each of 80 wells of an microtitre plate.  
     
     
         20 . Use of a combinatorial FRET library pair according to  claim 1  in 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 pair of combinatorial FRET (fluorescence resonance energy transfer) molecules to find a substrate or substrates for the enzyme.  
     
     
         21 . Use of a combinatorial FRET library pair according to  claim 1  in a method for detection and measurement of proteolytic enzyme activity against compounds of the library.  
     
     
         22 . 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 pair of combinatorial FRET (fluorescence resonance energy transfer) molecules according to  claim 1 , deconvoluting the library to find a substrate or substrates for the enzyme and synthesis of an inhibitor based on the substrate or substrates.  
     
     
         23 . An inhibition assay which uses a FRET molecule, which has been identified as a substrate for the enzyme by the method of  claim 2 , wherein the molecule is assayed with the enzyme separately against a panel of possible inhibitors.  
     
     
         24 . A method which provides the rapid generation of structure-activity relationships (SAR) which comprises detection and measurement of proteolytic enzyme activity by carrying out an assay with a library pair of combinatorial FRET (fluorescence resonance energy transfer) molecules according to  claim 1  to find a substrate or substrates for the enzyme.  
     
     
         25 . A method which comprises the identification of an enzyme inhibitor or inhibitors wherein a FRET compound which has been identified as a substrate by the method of  claim 2  is used in an inhibition assay with the enzyme separately against a panel of possible inhibitors.  
     
     
         26 . A method according to  claim 25  wherein the assay is performed by using the apparatus according to any of  claims 13  to  19 .  
     
     
         27 . A method according to any preceding method claim wherein the compounds of the library pair are synthesized using a solid phase technique.  
     
     
         28 . A library pair, apparatus, method or assay substantially as described herein with reference to example 1.