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 . An assay method for identifying an agent which modulates the interaction of a hypoxia inducible factor (HIF) hydroxylase with a substrate of the hydroxylase the method comprising: 
 contacting a HIF hydroxylase and a test substance in the presence of a substrate of the hydroxylase under conditions in which the hydroxylase interacts with the substrate in the absence of the test substance; and    determining the interaction, or lack of interaction of, the hydroxylase and the substrate.    
     
     
         2 . An assay method according to  claim 1 , wherein the interaction of the hydroxylase with the substrate in the presence of the test substance is determined by measuring the hydroxylase activity of the hydroxylase.  
     
     
         3 . An assay method according to  claim 2 , wherein the hydroxylase and the test substance are contacted in the presence of 2-oxoglutarate and the hydroxylase activity is determined by determining the turnover of said 2-oxoglutarate to succinate and CO 2 .  
     
     
         4 . An assay method according to  claim 2  or  3 , wherein the hydroxylase activity is determined by determining the hydroxylation of one or more proline residues in the substrate.  
     
     
         5 . An assay method according to any one of the preceding claims, wherein the substrate is a HIF polypeptide.  
     
     
         6 . An assay method according to  claim 5 , wherein the HIF polypeptide comprises HIF-1α, a fragment thereof comprising a proline equivalent to Pro 564 and/or Pro 402 of HIF-1α or a peptide analogue of HIF-1α or fragment thereof comprising a proline equivalent to Pro 564 and/or Pro 402 of HIF-1α, and wherein hydroxylation of Pro 564 and/or Pro 402 o or HIF-1α or of a said equivalent proline is determined.  
     
     
         7 . An assay method according to any one of the preceding claims, wherein the substrate comprises a polypeptide sequence comprising residues 556-574, such as 549 to 582 of the human HIF-1α sequence.  
     
     
         8 . An assay method according to any one of the preceding claims, wherein the HIF hydroxylase is a prolyl hydroxylase.  
     
     
         9 . An assay method according to any of the preceding claims, wherein the HIF hydroxylase comprises: 
 (a) the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8;    (b) a variant thereof having at least 60% identity to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 and having HIF hydroxylase activity; or    (c) a fragment of either thereof having HIF hydroxylase activity.    
     
     
         10 . An assay method according to any one of the preceding claims, wherein the HIF hydroxylase is selected from SEQ ID NO: 2, 4, 6 or 8, FLJ21620 (BAB105101) or Clorf12(NP071334).  
     
     
         11 . An assay method according to any one of  claims 5  to  10 , comprising providing a cell expressing a HIF polypeptide; expressing the HIF hydroxylase recombinantly in the cell; contacting the cell with a test substance and monitoring for HIF polypeptide abundance or stability or HIF-associated activity in the cell to determine the effect of the test substance on HIF hydroxylase activity.  
     
     
         12 . An assay method according to  claim 11 , wherein the HIP associated activity is selected from interaction with von Hippel-Lindau protein (VHL), HIF-mediated transcription or expression of a reporter gene driven by a HIF regulated promoter.  
     
     
         13 . An assay method according to any one of the preceding claims, wherein the HIF hydroxylase is a human HIF hydroxylase.  
     
     
         14 . An assay method according to any one of the preceding claims, wherein the HIF polypeptide is human HIF-1α or HIF-2α.  
     
     
         15 . A method for providing a pharmaceutical composition, which method comprises: 
 (a) identifying an agent by an assay method as defined in any one of  claims 1  to  14 ; and    (b) formulating the agent thus identified with a pharmaceutically acceptable excipient.    
     
     
         16 . An agent identified by an assay method according to any one of  claims 1  to  14 .  
     
     
         17 . A polypeptide comprising: 
 (a) the amino acid sequence of SEQ ID NO:  2 ,  4 ,  6  or  8 ;    (b) a variant thereof having at least 60% identity to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 and having HIF hydroxylase activity; or    (c) a fragment of either thereof having HIP hydroxylase activity.    
     
     
         18 . A polynucleotide comprising a sequence which encodes a polypeptide as defined in  claim 17 .  
     
     
         19 . A polynucleotide according to  claim 18  which comprises 
 (i) SEQ ID NO: 1, 3, 5 or 7 or a complementary sequence thereto;  
 (ii) a sequence which hybridises under stringent conditions to the sequence defined in (i);  
 (iii) a sequence which is degenerate as a result of the genetic code to a sequence as defined in (i) or (ii);  
 (iv) a sequence having at least 60% identity to a sequence as defined in (i); or  
 (v) a fragment of any of the sequences (i), (ii), (iii) or (iv), and which encodes a polypeptide having hydroxylase activity or capable of generating antibodies specific for a HIF hydroxylase.  
 
     
     
         20 . A polynucleotide according to  claim 18  or  19  which is DNA.  
     
     
         21 . An expression vector, comprising a polynucleotide as defined in any one of  claims 18  to  20 .  
     
     
         22 . An antibody or a fragment thereof capable of specifically binding a polypeptide as defined in  claim 17 .  
     
     
         23 . An agent according to  claim 16 , a polypeptide according to  claim 17 , a polynucleotide according to any one of  claims 18  to  20 , a vector according to  claim 21  or an antibody according to  claim 22  for use in a method of treatment of the human or animal body by therapy.  
     
     
         24 . A pharmaceutical composition comprising an agent according to  claim 16 , a polypeptide according to  claim 17 , a polynucleotide according to any one of  claims 18  to  20 , a vector according to  claim 21  or an antibody according to  claim 22  and a pharmaceutically acceptable excipient.  
     
     
         25 . Use of a modulator of HIF hydroxylase activity, an agent according to  claim 16 , a polypeptide according to  claim 17 , a polynucleotide according to any one of  claims 18  to  20 , a vector according to  claim 21  or an antibody according to  claim 22  in the manufacture of a medicament for the treatment of a condition associated with increased or decreased HIF levels or activity or the treatment of a condition where it is desirable to modulate HIF levels or activity.  
     
     
         26 . Use according to  claim 25 , wherein said condition is selected from the group consisting of ischaemia, wound healing, auto-, allo-, and xeno-transplantation, systemic high blood pressure, cancer, and inflammatory disorders.  
     
     
         27 . Use according to  claim 25  or  26 , wherein the modulator of HIF hydroxylase activity is a collagen prolyl hydroxylase (CPH) inhibitor which inhibits the biological activity of a HIF hydroxylase and wherein the condition is one associated with reduced HIF levels or activity.  
     
     
         28 . Use according to  claim 27 , wherein said inhibitor has the following formula:  
       R 1 -A*B*C*D(R 2 ) y    (A)  
       where the group R 1  is capable of forming an electrostatic interaction with the sidechain of the arginine which 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, which chain can be functionalised; A, B, C and D being linked to one another by single and/or double and/or triple bonds, y is 0 or 1; and C*D is a chain of two atoms which are, independently, carbon, oxygen, nitrogen, or sulphur, which chain C-D can be functionalised, such that when y is 0 or 1 at least one of the atoms of said chain C-D is capable of chelating with a metal group and when y is 1 said chain C-D is attached to R 2  which is capable of chelating with a metal group.  
     
     
         29 . Use according to  claim 28 , wherein R 1  is —CO 2 H, —SO 3 H, —B(OH) 2  or —PO 3 H 2 .  
     
     
         30 . Use according to  claim 28  or  29 , wherein y is 0 and C and/or D is attached to ═S, ═O, —SH or —OH.  
     
     
         31 . Use according to  claim 28  or  29 , wherein y is 1 and R 2  is —SH, —OH, —CO 2 H, —SO 3 H, —B(OH) 2 , —PO 3 H 2 , —NHOH, —CONHR 3 , —CONHOR 3 , —CONR 3 OH or CONR 3 OR 3  where in R 3  is a branched or straight chain alkyl group of 1 to 6 carbon atoms which can be functionalised.  
     
     
         32 . Use according to any one of  claims 28  to  31 , wherein said inhibitor has the following formula:  
       
         
           
           
               
               
           
         
       
       where R 1  and R may independently be H, a branched or straight C 1  to C 6  alkyl chain which can be functionalised, any natural amino acid side chain, 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 can be fused to another ring; and X is NH, NR″ where R″ is OH, a branched or straight C 1  to C 6  alkyl chain which can be functionalised or O; and Y is O or S.  
     
     
         33 . Use according to  claim 32 , wherein said inhibitor is dimethyloxalylglycine.  
     
     
         34 . Use according to any one of  claims 28  to  31 , wherein said inhibitor has the following formula:  
       
         
           
           
               
               
           
         
       
       where R 1  and R may independently be H, a branched or straight C 1  to C 6  alkyl chain, any natural amino acid side chain, 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 can be fused to another ring; and Y is O or S.  
     
     
         35 . Use according to any one of  claims 28  to  31 , wherein said inhibitor has the following formula:  
       
         
           
           
               
               
           
         
       
       where R I  to R V  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 C(═O)XR group in which: 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 containing i 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 O, and mis 0or 1.  
     
     
         36 . Use according to any one of  claims 28  to  31 , wherein said inhibitor has the following formula:  
       
         
           
           
               
               
           
         
       
       where R III  to R V  may independently be H, a branched or straight C 1  to C 6  alkyl chain, optionally with 1, 2, 3, 4 or 5 halo substitutions, 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 can be fused to another ring.  
     
     
         37 . Use according to anyone of  claims 28  to  31 , wherein said inhibitor has the following formula:  
       
         
           
           
               
               
           
         
       
       where R 1 , R 2 , R 3  and R 4  may independently be H, a branched or straight C 1  to C 6  alkyl chain, OH, 0-alkyl having a branched or straight C 1  to C 6  alkyl chain optionally containing 1 or 2 N, O or S atoms, COOH, a branched or straight C 1  to C 6  alkyl ester, 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 more N, O or S atoms, which can be fused to another ring.  
     
     
         38 . Use according to any one of  claims 28  to  31 , wherein said inhibitor has the following formula;  
       
         
           
           
               
               
           
         
       
       where R I  to R V  may independently be H, a branched or straight C 1  to C 6  alkyl chain, optionally with 1, 2, 3, 4 or 5 halo substitutions, 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 can be fused to another ring.  
     
     
         39 . Use according to any one of  claims 28  to  31 , wherein said inhibitor has the formula:  
       
         
           
           
               
               
           
         
       
       where A*B*C*D are as defined in  claim 28  and R 21  is hydrogen or acyl.  
     
     
         40 . Use according to  claim 25 , wherein the modulator of HIF hydroxylase is a selective inhibitor of the HIF hydroxylase polypeptide and the condition is one associated with reduced HIF levels or activity.  
     
     
         41 . A method of treating a condition associated with increased or reduced HIF activity or a condition in which is desired to modulate HIF activity comprising administering a therapeutically effective amount of an agent according to  claim 16 , a polypeptide according to  claim 17 , a modulator of the activity of a polypeptide according to  claim 17 , a poly,nucleotide according to any one of  claims 18  to  20 , a vector according to  claim 21  or an antibody according to  claim 22  to an individual in need thereof.  
     
     
         42 . A method of modulating the amount of HIF polypeptide in a cell comprising contacting the cell with a substance which inhibits the prolyl hydroxylase activity of a HIF hydroxylase.  
     
     
         43 . A method according to  claim 41  or  42 , wherein the modulator is an agent according to  claim 16 .  
     
     
         44 . A method according to any one of  claims 41  to  43 , wherein the modulator inhibits the biological activity of a HIF hydroxylase but does not inhibit biological activity of a collagen prolyl hydroxylase.  
     
     
         45 . A method of making a pharmaceutical composition comprising admixing an agent according to  claim 16 , a polypeptide according to  claim 17 , a modulator of the activity of a polypeptide according to  claim 17 , a polynucleotide according to any one of  claims 18  to  20 , a vector according to  claim 21  or an antibody according to  claim 22  with a pharmaceutically acceptable excipient.  
     
     
         46 . A method of producing a HIF hydroxylase comprising: 
 (a) causing expression from nucleic acid which encodes a HIF hydroxylase in a suitable expression system to produce the polypeptide recombinantly; and    (b) determining the hydroxylation of a HIF polypeptide by said recombinantly produced polypeptide.    
     
     
         47 . An assay method for identifying a HIF hydroxylase comprising, 
 (a) providing a test polypeptide,    (b) bringing into contact an HIF polypeptide and the test polypeptide under conditions in which the HIF polypeptide is hydroxylated by a HIF hydroxylase; and    (c) determining hydroxylation of the HIF polypeptide.    
     
     
         48 . Use of an polypeptide according to  claim 17  for the prolyl hydroxylation of a HIF polypeptide.  
     
     
         49 . An isolated polypeptide of SEQ ID NO: 9. 
 DLDLEMLAP*YIPMDDDFQL (SEQ ID NO: 9.)    wherein P* is 4-hydroxy proline.    
     
     
         50 . An isolated polypeptide which is a variant of SEQ ID NO: 9, wherein such variants comprises from 1 to 4 amino acid substitutions of any amino acid apart from P*, said variant retaining the ability to antagonise the interaction of a HIF-α subunit with VHL.  
     
     
         51 . An assay for a modulator of the interaction between VHL and HIF-α comprising contacting a polypeptide according to  claim 49  or  claim 50  with a HIF-1α binding domain of VHL in the presence of a test substance and determining the interaction of the polypeptides.  
     
     
         52 . An assay for an inhibitor of VHL-mediated HIF-α destruction, which comprises providing a HIF-α, or fragment thereof which includes a VHL-binding portion, together with its cognate prolyl-hydroxylase under conditions suitable for the hydroxylation of a proline residue in the HIF-α VHL-binding domain; 
 providing a putative modulator of hydroxylation; and  
 determining whether the amount of hydroxylation of said proline residue has been modulated by said putative modulator.  
 
     
     
         53 . An assay for a promoter of hydroxylation of a proline residue in HIF-α, which comprises: 
 providing HIF-α, or fragment thereof which includes a VHL-binding portion, under hypoxic conditions, said HIF-α or fragment thereof containing a proline residue in the VHL-binding domain;  
 providing a putative hydroxylation promoting agent; and  
 determining whether said agent provides for hydroxylation of said proline.  
 
     
     
         54 . A method of identifying a substrate of a polypeptide as defined in  claim 17 , comprising contacting a test polypeptide substrate with a HIF hydroxylase according to  claim 17 , and monitoring for hydroxylation of said test polypeptide substrate.  
     
     
         55 . A polynucleotide comprising: 
 (a) an antisense polynucleotide to the polynucleotide of  claim 19  or    (b) an RNAi to the polynucleotide of  claim 19.