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 . An assay method for identifying an agent which modulates the interaction of a hypoxia inducible factor (HIF) hydroxylase with a substrate of the hydroxylase, wherein said substrate is a HIF polypeptide,
 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   wherein the HIF hydroxylase is chosen from:   (a) the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, FLJ21620 (BAB15101) or C1orf12 (NP071334);   (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 (a) or (b) having HIF hydroxylase activity.   
     
     
         57 . An assay method according to  claim 56 , wherein the HIF associated activity is chosen from interaction with von Hippel-Lindau protein (VHL), HIF mediated transcription or expression of a reporter gene driven by a HIF regulated promoter. 
     
     
         58 . A polypeptide comprising:
 (a) the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, FLJ21620 (BAB15101) or C1orf12 (NP071334);   (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 (a) or (b) having HIF hydroxylase activity.   
     
     
         59 . A polynucleotide comprising a sequence which encodes a polypeptide as defined in  claim 58 . 
     
     
         60 . A polynucleotide according to  claim 59  which comprises
 (i) SEQ ID NO: 1, 3, 5 or 7 or a complementary sequence thereto; 
 (ii) a sequence which hybridizes 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. 
 
     
     
         61 . A polynucleotide according to  claim 59  which is DNA. 
     
     
         62 . An expression vector, comprising a polynucleotide as defined in  claim 59 . 
     
     
         63 . An antibody or a fragment thereof capable of specifically binding a polypeptide as defined in  claim 58 . 
     
     
         64 . A method of treating a condition associated with increased or reduced HIF activity or a condition in which it is desired to modulate HIF activity comprising administering a therapeutically effective amount of at least one substance chosen from a modulator of HIF hydroxylase activity, a polypeptide, a polynucleotide, a vector, and an antibody or fragment thereof to an individual in need thereof. 
     
     
         65 . A method according to  claim 64 , wherein said condition is chosen from ischaemia, wound healing, auto-, allo-, and xeno-transplantation, systemic high blood pressure, cancer, and inflammatory disorders. 
     
     
         66 . A method according to  claim 64 , 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. 
     
     
         67 . A method according to  claim 64 , 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. 
     
     
         68 . The method according to  claim 64 , wherein the polypeptide comprises:
 (a) the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, FLJ21620 (BAB15101) or C1orf12 (NP071334);   (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 (a) or (b) having HIF hydroxylase activity.   
     
     
         69 . The method according to  claim 64 , wherein the polynucleotide comprises a sequence which encodes 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 HIF hydroxylase activity.   
     
     
         70 . The method according to  claim 64 , wherein the expression vector comprises a polynucleotide sequence which encodes a polypeptide comprising:
 (a) the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, FLJ21620 (BAB15101) or C1orf12 (NP071334);   (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 (a) or (b) having HIF hydroxylase activity.   
     
     
         71 . The method according to  claim 64 , wherein the antibody or fragment thereof is capable of specifically binding a polypeptide comprising:
 (a) the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, FLJ21620 (BAB15101) or C1orf12 (NP071334);   (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 (a) or (b) having HIF hydroxylase activity.   
     
     
         73 . 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   wherein the HIF hydroxylase is chosen from:   (a) the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, FLJ21620 (BAB15101) or C1orf12 (NP071334);   (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 (a) or (b) having HIF hydroxylase activity.