Assays for modulators of asparaginyl hydroxylase

Abstract

The present invention is directed to methods of identifying an agent which modulates hydroxylation of hypoxia inducible factor (HIF), comprising contacting a HIF asparagine hydroxylase and a test substance in the presence of a substrate of the hydroxylase under conditions in which asparagine in the substrate is hydroxylated in the absence of the test substance; and determining hydroxylation of the substrate.

1 . A method of identifying an agent which modulates hydroxylation of hypoxia inducible factor (HIF), the method comprising:
 (a) contacting a HIF asparagine hydroxylase and a test substance in the presence of a substrate of the hydroxylase under conditions in which asparagine in the substrate is hydroxylated in the absence of the test substance; and   (b) determining hydroxylation of the substrate.   
     
     
         2 . The method according to  claim 1 , wherein the substrate is a HIF polypeptide. 
     
     
         3 . The method according to  claim 2 , wherein the HIF polypeptide comprises HIF-1α, a fragment thereof comprising Asn 803 of HIF-1α or a peptide analogue of HIF-1α or fragment thereof comprising an asparagine equivalent to Asn 803 of HIF-1α and wherein hydroxylation of Asn 803 or of a said equivalent asparagine is determined. 
     
     
         4 . The method according to  claim 3 , wherein the substrate comprises the CAD domain of the human HIF-1α sequence. 
     
     
         5 . The method according to  claim 1 , wherein the HIF asparagine hydroxylase comprises:
 (a) the amino acid sequence of SEQ ID NO: 2;   (b) a variant thereof having at least 60% identity to the amino acid sequence of SEQ ID NO: 2 and having HIF asparagine hydroxylase activity; or   (c) a fragment of either thereof having HIF asparagine hydroxylase activity.   
     
     
         6 . The method according to  claim 3 , wherein hydroxylation of the substrate is determined by monitoring the interaction of said HIF polypeptide with CH1 domain of p300. 
     
     
         7 . The method according  claim 1 , wherein the hydroxylation of the substrate is determined by monitoring for a HIF associated activity selected from interaction with CH1 domain of p300, HIF mediated transcription or expression of a reporter gene driven by a HIF regulated promoter. 
     
     
         8 . The method according to  claim 1 , further comprising formulating an agent identified as a modulator of HIF asparagine hydroxylase with a pharmaceutically acceptable recipient. 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . A method of treating a condition associated with increased or decreased HIF levels or activity or a condition where it is desired to modulate HIF activity, comprising administering a therapeutically effective amount of a modulator of HIF asparagine hydroxylase activity to an individual in need thereof. 
     
     
         12 . The method according to  claim 11 , wherein said condition is selected from the group consisting of ischemia, wound healing, auto-, allo-, and xeno-transplantation, systemic high blood pressure, cancer, inflammatory disorders and diabetes. 
     
     
         13 . The method according to  claim 11 , wherein the modulator of HIF asparagine hydroxylase is a selective inhibitor of the HIF asparagine hydroxylase polypeptide and the condition is one associated with reduced HIF levels or activity. 
     
     
         14 . 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 an agent according to  claim 9  to an individual in need thereof. 
     
     
         15 . A method of modulating HIF mediated activity in a cell comprising contacting the cell with a substance which inhibits the asparagine hydroxylase activity of a HIF hydroxylase. 
     
     
         16 . An assay for a promoter of hydroxylation of an asparagine residue in HIF-α which comprises:
 (a) providing HIF-α or fragment thereof which includes a CAD binding portion under hypoxic conditions, said HIF-α or fragment thereof containing an asparagine residue in the CAD domain;   (b) providing a putative hydroxylation promoting agent; and   (c) determining whether said agent provides for hydroxylation of said asparagine.