Assays, methods and means

Abstract

A novel class of hydroxylases is described having the amino acid sequence of SEQ ID NO: 2, 4, 6 and 8, and variants and fragments thereof having HIF hydroxylation activity. The polypeptides of the invention have in particular prolyl hydroxylase activity. An assay method monitors the interaction of the HIF hydroxylase with a substrate. Modulators of HIF hydroxylase are provided for use in the treatment of a condition associated with increased or decreased HIF levels or activity or for the treatment of a condition where it is desirable to modulate HIF levels or activity.

1 - 55 . (canceled) 
     
     
         56 . A pharmaceutical composition comprising
 a substance that has been identified as inhibiting the activity of a HIF hydroxylase in mediating the hydroxylation of one or more proline residues of a HIF-α protein by means of an assay method in which
 a HIF prolyl hydroxylase and a substrate of the hydroxylase are contacted under conditions in which the hydroxylase interacts with the substrate, in the presence or absence of a test substance; and 
 the interaction, or lack of interaction of, the hydroxylase and the substrate is determined by measuring the hydroxylase activity of the hydroxylase; 
 wherein the HIF prolyl hydroxylase is a fragment of SEQ ID NO: 6 that
 contains the sequence HXD[X] n H, X being any amino acid and n being any number between 1 and 200, 
 contains a β-barrel jelly roll structure, 
 retains HIF prolyl hydroxylase activity, and 
 hydroxylates the proline residue of a motif LXXLXP contained in the substrate, where X is any amino acid; and 
 
   a pharmaceutically acceptable excipient.   
     
     
         57 . The pharmaceutical composition in  claim 56 , wherein the fragment of SEQ ID NO: 6 used in the assay method has the HXD portion of the motif HXD[X] n H on the second strand of the β-barrel jelly roll structure. 
     
     
         58 . The pharmaceutical composition in  claim 57 , wherein the fragment of SEQ ID NO: 6 used in the assay method has the remaining H of the motif LXXLXP on or close to the seventh strand thereof. 
     
     
         59 . The pharmaceutical composition of  claim 56 , wherein the fragment of SEQ ID NO: 6 used in the assay method hydroxylates the proline residue of a motif LXXLAP contained in the substrate, where X is any amino acid. 
     
     
         60 . The pharmaceutical composition of  claim 56 , wherein the fragment of SEQ ID NO: 6 used in the assay method contains the sequence M(X) 13 HXD(X) 4 D(X) 7 Y(X) 14 L(X) 14 P(X) 10 D(X) 4 HXV(X) 6 R where X is any amino acid residue. 
     
     
         61 . The pharmaceutical composition of  claim 56 , wherein the substance selectively inhibits the activity of HIF prolylhydroxylases. 
     
     
         62 . The pharmaceutical composition of  claim 61 , wherein the substance selectively inhibits the activity of HIF prolyl hydroxylases relative to that of other 2-oxoglutarate dependent oxygenases. 
     
     
         63 . The pharmaceutical composition of  claim 62 , wherein the other oxygenases are collagen prolyl hydroxylases (CPH). 
     
     
         64 . The pharmaceutical composition of  claim 56 , wherein the substance is an inhibitor of a collagen prolyl hydroxylase or a modification thereof. 
     
     
         65 . The pharmaceutical composition of  claim 56 , wherein the substance is a 2-oxoglutarate analogue. 
     
     
         66 . The pharmaceutical composition of  claim 56 , wherein the substance competes with HIF-α for binding to the HIF prolyl hydroxylase. 
     
     
         67 . The pharmaceutical composition of  claim 56 , wherein the substance has the following formula:
   R 1 -A*B*C*D(R 2 ) y      
       where the group R 1  is capable of forming an electrostatic interaction with the side chain of the arginine, A*B is a chain of two atoms which are, independently, carbon, oxygen, nitrogen or sulphur, C*D is a chain of two atoms which are, independently, carbon, oxygen, nitrogen, or sulphur, and y is 0 or 1, with A, B, C and D being linked to one another by single and/or double and/or triple bonds, such that when y is 0 or 1 at least one of the atoms of A, B, C or D is capable of chelating with a metal group, and when y is 1 said chain is attached to R 2  which is capable of chelating with a metal group. 
     
     
         68 . The pharmaceutical composition of  claim 67 , wherein at least one of A, B, C and D is not carbon in the substance formula. 
     
     
         69 . The pharmaceutical composition of  claim 67 , wherein the A*B*C*D chain in the substance formula is selected from the group consisting of C—N—C—C, C—C—C═O, and C—O—C—C. 
     
     
         70 . The pharmaceutical composition of  claim 67 , wherein A*B*C*D in the substance formula forms part of a ring. 
     
     
         71 . The pharmaceutical composition of  70 , wherein the ring is pyrolidine or an unsaturated derivative thereof. 
     
     
         72 . The pharmaceutical composition of  67 , wherein R 1  in the substance formula is an acid group. 
     
     
         73 . The pharmaceutical composition of  67 , wherein R 2  in the substance formula is selected from the group consisting of —SH, —OH, —CO 2 H, —SO 3 H, —B(OH) 2 , —PO 3 H 2 , —NHOH, —CONHR 3 , —CONHOR 3 , —CONHR 3 , and —CONR 3 OR 3  where R3 is a branched or straight chain alkyl group of 1 to 6 carbon atoms which can be functionalized. 
     
     
         74 . The pharmaceutical composition of  claim 56 , wherein the substance is a N-containing heterocyclic compound having one of the following formulae: 
       
         
           
           
               
               
           
         
       
       where R 1  to R 5  may be H, a branched or straight C 1  to C 6  alkyl chain such as Me, a 4 to 7 membered heterocyclic ring optionally containing 1 or more N, S, O or P atoms, or a 5 or 6 membered aromatic ring, optionally containing 1 or more N, O or S atoms, which can be fused to another ring, or a said alkyl chain substituted by a said aromatic group, A=substituted alkylene, B=CO 2 H, NHSO 2 CF 3 , tetrazolyl, imidazolyl or 3-hydroxyisoxazolyl, and m is 0 or 1, 
       
         
           
           
               
               
           
         
       
       where R i  to R iv  may independently be H, a branched or straight chain alkyl of from 1 to 6 C atoms, a halogen group (i.e. fluoro-, chloro-, bromo- or iodo-), a carboxylate group, a 4 to 7 membered heterocyclic ring optionally containing 1 or more N, S, O or P atoms, a 5 or 6 membered aromatic ring, optionally containing 1 or more N, O or S atoms which can be fused to another ring or a said alkyl chain substituted by a said aromatic ring, or a C(═O)XR group as defined below, X is O, NH, NR, where R is H, OH, a branched or straight chain alkyl of from 1 to 6 C atoms which can be functionalised, alkoxy containing a branched or straight chain alkyl of from 1 to 6 C atoms which can be functionalised, a 4 to 7 membered heterocyclic ring optionally containing 1 or 2 N, S, O or P atoms, a 5 or 6 membered aromatic ring, optionally containing 1 or 2 N, O or S atoms which can be fused to another ring, such that RX is typically straight or branched C 1  to C 6  alkoxy, and m is 0 or 1, 
       
         
           
           
               
               
           
         
       
       where X=O, Y N or CR 3 , m=O or 1, A=substituted alkylene, B=CO 2 H, NHSO 2 CF 3 , tetrazolyl, imidazolyl or 3-hydroxyisoxazolyl, R 1 , R 2  and R 3  may independently be H, OH, halo, cyano, CF 3 , NO 2 , CO 2 H, alkyl, cycloalkyl, cycloalkoxy, aryl, aralkynyl, alkynylcarbonyl, alkylcarbonyloxy, carbamoyl, alkynyloxyalkyl, alkenyloxy, alkoxyalkoxy, alkynyl, retinyloxycarbonyl, alkenyloxycarbonyloxy, where R 1  and R 2  or R 2  and R 3 =(CH 2 )O in which 1-2 CH 2  groups of the saturated or C:C unsaturated chain may be replaced by O, S, SO, SO 2  or imino, o=3-5, R4=H, and 
       
         
           
           
               
               
           
         
       
       where A=(substituted alkylene), B=(modified) carboxy, tetrazolyl, imidazolyl, 3-hydroxyisoxazolyl, R4=H, OH, halo, cyano, CF 3 , NO 2 , CO 2 H, alkyl (e.g. branched or straight chain C 1 -C 6  alkyl), cycloalkyl, cycloalkylalkyl, cycloalkylalkoxy, cycloalkoxyalkyl, aryl, aralkyl, aralkoxy, hydroxyalkyl, alkenyl, alkynyl, alkynyloxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, arylcarbonyloxy, cinnamoyl, alkenylcarbonyl, arylcarbamoyl or aralkoxycarbonyloxy. 
     
     
         75 . The pharmaceutical composition of  claim 56 , wherein the substance has one of the following formulae: 
       
         
           
           
               
               
           
         
       
       where R, R i  to R vi  may independently be H, a branched or straight C 1  to C6 alkyl chain, a 4 to 7 membered heterocyclic ring optionally containing 1 or 2 N, S, O or P atoms, a 5 or 6 membered aromatic ring, optionally containing 1 or 2 N, O or S atoms, which can be fused to another ring, or a said alkyl chain substituted by a said aromatic ring, preferably H or methyl, R 2 O is hydrogen or acyl typically aromatic acyl such as benzoyl, X is NH, NR″, where R″ is OH, Me, alkyl, OMe, Oalkyl with a C 1  to C 6  alkyl chain, and Y is O or S. 
     
     
         76 . The pharmaceutical composition of  claim 56 , wherein the substance has one of the following formulae: 
       
         
           
           
               
               
           
         
       
       where R 1  is H, a branched or straight C 1  to C 6  alkyl chain which can be functionalised, any natural amino acid side chain for example of glutamic acid, a 4 to 7 membered heterocyclic ring optionally containing 1 or 2 N, S, O or P atoms or a 5 or 6 membered aromatic ring, optionally containing 1 or 2 N, O or S atoms which may be fused to another ring or a said alkyl chain substituted by a said aromatic ring and each of R 2  to R 6 , which may be the same or different, is as defined for R1 or is NH2 or OR 7  where R 7  is as defined for R 1  and E represents a monocyclic ring system such as thiophene or pyran and E′ is absent or forms with E a bicyclic ring system such as naphthalene or indole, E′ typically being benzene. 
     
     
         77 . The pharmaceutical composition of  claim 56 , wherein the substrate is a HIF-α protein or fragment thereof having one or more prolyl residues that is/are hydroxylated in conditions in which the hydroxylase interacts with the substrate and the hydroxylase activity is determined by measuring the hydroxylation of one or more proline residues of the substrate. 
     
     
         78 . The pharmaceutical composition of  claim 56 , wherein the conditions under which the assay method is carried out include the presence of 2-oxoglutarate. 
     
     
         79 . The pharmaceutical composition of  claim 78 , wherein the hydroxylase activity is determined in the assay method by measuring the turnover of the 2-oxoglutarate to succinate and carbon dioxide. 
     
     
         80 . The pharmaceutical composition of  claim 56 , wherein the conditions under which the assay method is carried out include the presence of dioxygen. 
     
     
         81 . The pharmaceutical composition of  claim 56 , wherein the conditions under which the assay method is carried out include the presence of ascorbate. 
     
     
         82 . The pharmaceutical composition of  claim 56 , wherein the conditions under which the assay method is carried out include the presence of ferrous iron. 
     
     
         83 . The pharmaceutical composition of  claim 56 , wherein the assay method is carried out in the presence of a reducing agent.