Pharmaceuticals and methods for treating hypoxia and screening methods therefor

Abstract

Light-generating fusion proteins having a ligand binding site and a light-generating polypeptide moiety and their use as diagnostics, in drug screening and discovery, and as therapeutics, are disclosed. The light-generating fusion protein has a feature where the bioluminescence of the polypeptide moiety changes upon binding of a ligand at the ligand binding site. The ligand may be, for example, an enzyme present in an environment only under certain conditions, e.g., ubiquitin ligase in a hypoxic state, such that the light-generating fusion protein is “turned on” only under such conditions.

1 - 54 . (canceled) 
     
     
         55 . An assay for a modulator of prolyl hydroxylation of HIF1α, comprising
 a) bringing into contact a source of HIF prolyl hydroxylase, a HIF1α polypeptide moiety and a putative modulator compound under conditions where HIF prolyl hydroxylase, in the absence of said putative modulator, is capable of acting on said HIF1α polypeptide moiety, wherein said HIF1α polypeptide moiety comprises a 20 amino acid sequence corresponding to the residues surrounding residue 564, inclusive, of HIF1α, numbered in accordance with wild-type HIF1α (SEQ ID NO:23), and further wherein residue 564 is a proline; and   b) measuring the degree of prolyl hydroxylation of HIF1α to determine the inhibition of prolyl hydroxylation of HIF1α caused by said modulator compound, wherein a putative modulator compound that causes inhibition of prolyl hydroxylation of HIF1α is determined to be a modulator of prolyl hydroxylation of HIF1α.   
     
     
         56 . A method of identifying a modulator of HIF prolyl hydroxylation, comprising the steps of:
 a) forming a test sample by bringing into contact a source of HIF prolyl hydroxylase, a putative prolyl hydroxylase modulator and a light-generating fusion protein comprising a HIFα polypeptide moiety capable of being hydroxylated by HIF prolyl hydroxylase and a light-generating polypeptide moiety, wherein the light generation of said light-generating polypeptide moiety changes as a consequence of hydroxylation of a proline of said HIFα polypeptide moiety by HIF prolyl hydroxylase, under conditions favorable for prolyl hydroxylation of HIFα; and   b) determining the ability of said putative prolyl hydroxylase modulator to modulate HIF prolyl hydroxylation by measuring the light generated in said test sample, whereby a putative prolyl hydroxylase modulator that causes modulation of light generation in said sample is identified as a modulator of HIF prolyl hydroxylation.