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 inhibiting the activity of a human HIF prolyl hydroxylase in mediating the hydroxylation of one or more proline residues of a human HIF-α protein, comprising administering a substance to a subject, wherein the HIF prolyl hydroxylase is human EGLN-1, and wherein the substance is a 2-oxoglutarate analogue that binds to the active site metal iron ion of human EGLN-1 and to Arg383 of human EGLN-1. 
     
     
         57 . The method of  claim 56 , wherein the oxoglutarate analogue has been identified as inhibiting such hydroxylation by means of assays comprising:
 contacting a HIF prolyl hydroxylase and a substrate of the HIF prolyl hydroxylase under conditions in which the HIF prolyl hydroxylase interacts with the substrate, in the presence or absence of a test substance; and   determining the interaction, or lack of interaction of, the HIF prolyl hydroxylase and the substrate and/or by measuring the hydroxylase activity of the HIF prolyl hydroxylase.   
     
     
         58 . The method of  claim 56 , wherein angiogenesis is promoted by the treatment. 
     
     
         59 . The method of  claim 56 , wherein erythropoiesis is promoted by the treatment. 
     
     
         60 . The method of  claim 56 , wherein the condition is a hypoxic condition. 
     
     
         61 . The method of  claim 60 , wherein the hypoxic condition is anaemic, histotoxic, stagnant or generalized hypoxia. 
     
     
         62 . The method of  claim 61 , wherein the hypoxic condition is ischaemic hypoxia. 
     
     
         63 . The method of  claim 62 , wherein the treatment is applied following tissue damage. 
     
     
         64 . The method of  claim 62 , wherein the treatment is prophylactic. 
     
     
         65 . The method of  claim 61 , wherein the hypoxic condition is anaemic hypoxia. 
     
     
         66 . The method of  claim 56 , wherein the condition is associated with an inflammatory disorder. 
     
     
         67 . The method of  claim 56 , wherein the condition arises in transplantation. 
     
     
         68 . The method of  claim 56 , wherein the condition is a neurodegenerative disease. 
     
     
         69 . The method of  claim 56 , wherein the condition is a wound. 
     
     
         70 . The method of  claim 56 , wherein the condition is associated with systemic high blood pressure. 
     
     
         71 . The method of  claim 56 , wherein the substance selectively inhibits the activity of the HIF hydroxylase. 
     
     
         72 . The method of  claim 71 , wherein the substance selectively inhibits the activity of the HIF hydroxylase relative to that of other 2-oxoglutarate dependent oxygenases. 
     
     
         73 . The method of  claim 72 , wherein the other oxygenases are collagen prolyl hydroxylases (CPH). 
     
     
         74 . A method of inhibiting the activity of a human HIF prolyl hydroxylase in mediating the hydroxylation of one or more proline residues of a human HIF-α protein, comprising administering a substance to a subject, wherein the HIF prolyl hydroxylase is human EGLN-1 and contains a β-barrel jelly roll structure, and wherein the substance is a 2-oxoglutarate analogue that binds to the active site metal iron ion of human EGLN-1 and to the conserved arginine residue located between the seventh strand and the eight strand of the β-barrel jelly roll structure of human EGLN-1. 
     
     
         75 . A method of inhibiting the activity of a human HIF prolyl hydroxylase in mediating the hydroxylation of one or more proline residues of a human HIF-α protein, comprising administering a substance to a subject, wherein the HIF prolyl hydroxylase is human EGLN-1, and wherein the substance binds to the active site metal iron ion of human EGLN-1 and to Arg383 of human EGLN-1 and competes for binding with 2-oxoglutarate. 
     
     
         76 . A method of inhibiting the activity of a human HIF prolyl hydroxylase in mediating the hydroxylation of one or more proline residues of a human HIF-α protein, comprising administering a substance to a subject, wherein the HIF prolyl hydroxylase is human EGLN-1 and contains a β-barrel jelly roll structure, and wherein the substance binds to the active site metal iron ion of human EGLN-1 and to the conserved arginine residue located between the seventh strand and the eight strand of the β-barrel jelly roll structure of human EGLN-1 and competes for binding with 2-oxoglutarate.