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 - 14 . (canceled) 
     
     
         15 . A method of treating or preventing a hypoxic or ischemic related disorder in a subject, comprising administering to a subject in need thereof a compound which decreases prolyl hydroxylase expression or activity, such that said hypoxic or ischemic related disorder is treated, wherein the compound is identified by an assay comprising the steps of:
 a) bringing into contact a prolyl hydroxylase, a HIF1α polypeptide moiety consisting of amino acids 555-575 of wild type HIFα, numbered in accordance with wild type HIFα, and a putative modulator compound under conditions where prolyl hydroxylase, in the absence of modulator, is capable of acting on said HIF1α polypeptide moiety; and   b) measuring the degree of prolyl hydroxylase inhibition caused by said modulator compound.   
     
     
         16 - 21 . (canceled) 
     
     
         22 . A method of increasing angiogenesis or vascularization in a subject, comprising administering to a subject in need thereof a compound which decreases prolyl hydroxylase expression or activity identified by an assay comprising the steps of:
 a) bringing into contact a prolyl hydroxylase, a HIF1α polypeptide moiety consisting of amino acids 555-575 of wild type HIFα, numbered in accordance with wild type HIFα, and a putative modulator compound under conditions where prolyl hydroxylase, in the absence of modulator, is capable of acting on said HIF1α polypeptide moiety; and   b) measuring the degree of prolyl hydroxylase inhibition caused by said modulator compound.   
     
     
         23 - 54 . (canceled) 
     
     
         55 . The method of  claim 15 , wherein said assay further comprises pVHL. 
     
     
         56 . The method of  claim 15 , wherein the hypoxic or ischemic related disorder is tissue ischemia. 
     
     
         57 . The method of  claim 22 , wherein said assay further comprises pVHL.