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 method of treatment of a hypoxic condition by administering a substance that inhibits the hydroxylation of one or more prolyl residues of a human HIF-α protein mediated by a HIF prolyl hydroxylase, wherein that substance has been identified as inhibiting such hydroxylation by means of an assay comprising
 contacting a HIF prolyl hydroxylase and a substrate of the hydroxylase under a condition in which the hydroxylase interacts with the substrate, in the presence or absence of a test substance; and 
 determining the interaction, or lack of interaction of, the hydroxylase and the substrate by measuring the hydroxylase activity of the hydroxylase;
 wherein the HIF prolyl hydroxylase is chosen from (1) the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8; and (2) a fragment thereof containing a β-barrel jelly roll structure and having HIF hydroxylase activity. 
 
 
     
     
         57 . The method of  claim 56 , wherein the HIF prolyl hydroxylase used in the assay contains the motif HXD[X] n H, where X is any amino acid and n is any number between 1 and 200, and where the HXD portion of the HXD[X] n H motif is on the second strand of the β-barrel jelly roll structure. 
     
     
         58 . The method of  claim 57 , wherein the HIF prolyl hydroxylase used in the assay 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. 
     
     
         59 . The method of  claim 57 , wherein the HIF prolyl hydroxylase used in the assay has the remaining H of the HXD[X] n H motif on the seventh strand thereof. 
     
     
         60 . The method of  claim 56 , wherein the HIF prolyl hydroxylase used in the assay hydroxylates the prolyl residue of a motif LXXLXP contained in the substrate, where X is any amino acid. 
     
     
         61 . The method of  claim 56 , wherein the HIF prolyl hydroxylase used in the assay has the amino acid sequence of SEQ ID NO: 4. 
     
     
         62 . The method of  claim 56 , wherein the HIF prolyl hydroxylase used in the assay is a fragment of SEQ ID NO: 4 containing a β-barrel jelly roll structure and having HIF hydroxylase activity. 
     
     
         63 . The method of  claim 56 , wherein the HIF prolyl hydroxylase used in the assay has the amino acid sequence SEQ ID NO: 6. 
     
     
         64 . The method of  claim 56 , wherein the HIF prolyl hydroxylase used in the assay is a fragment of SEQ ID NO: 6 containing a β-barrel jelly roll structure and having HIF hydroxylase activity. 
     
     
         65 . The method of  claim 56 , wherein the hypoxic condition is anaemic, histotoxic, stagnant or generalized hypoxia. 
     
     
         66 . The method of  claim 65 , wherein the hypoxic condition is ischaemic hypoxia. 
     
     
         67 . The method of  claim 66 , wherein the treatment is applied following tissue damage. 
     
     
         68 . The method of  claim 66 , wherein the treatment is prophylactic. 
     
     
         69 . The method of  claim 65 , wherein the hypoxic condition is anaemic hypoxia. 
     
     
         70 . The method of  claim 56 , wherein the substance is a 2-oxoglutarate analogue. 
     
     
         71 . The method 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 which together with other residues binds the 5-carboxylate of 2-oxoglutarate during catalysis, 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. 
     
     
         72 . The method of  claim 71 , wherein at least one of A, B, C and D is not carbon in the substance formula. 
     
     
         73 . The method of  claim 71 , wherein the A*B*C*D chain in the substance formular is selected from the group consisting of C—N—C—C, C—C—C═O, and C—O—C—C. 
     
     
         74 . The method of  claim 71 , wherein A*B*C*D in the substance formula forms part of a ring. 
     
     
         75 . The method of  claim 74 , wherein the ring is pyrolidine or an unsaturated derivative thereof. 
     
     
         76 . The method of  claim 71 , wherein R 1  in the substance formula is an acid group. 
     
     
         77 . The method of 71, 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. 
     
     
         78 . The method 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. 
     
     
         79 . The method of  claim 56 , wherein the substance has one of the following formulae: 
       
         
           
           
               
               
           
         
       
       where R I  to R vi  may independently be H, a branched or straight C 1  to C 6  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. 
     
     
         80 . The method 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. 
     
     
         81 . The method of  claim 56 , wherein the substrate used in the assay is a HIF-α protein or fragment thereof having one or more prolyl residues that is/are hydroxylated in a condition 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. 
     
     
         82 . The method of  claim 56 , wherein the condition under which the assay is carried out include the presence of 2-oxoglutarate and wherein the hydroxylase activity is determined by measuring the turnover of the 2-oxoglutarate to succinate and carbon dioxide. 
     
     
         83 . The method of  claim 56 , wherein the condition under which the assay is carried out include the presence of dioxygen. 
     
     
         84 . The method of  claim 56 , wherein the condition under which the assay is carried out include the presence of ascorbate and ferrous iron. 
     
     
         85 . The method of  claim 56 , wherein the assay is carried out in the presence of a reducing agent.