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 . A method of identifying a compound that modulates proline hydroxylation of the alpha subunit of hypoxia inducible factor (HIFα), the method comprising: 
 a) bringing into contact a compound and a non-hydroxylated HIFα polypeptide comprising one or more hydoxylatable proline residues under conditions wherein the HIFα polypeptide, in the absence of compound, is hydroxylated on at least one proline residue; and    b) determining the ability of the compound to modulate proline hydroxylation of HIFα.    
     
     
         2 . The method of  claim 1 , wherein the bringing into contact step further comprises pVHL.  
     
     
         3 . The method of  claim 1 , wherein said HIFα polypeptide comprises the amino acid sequence Y-X 1 -Leu-X 2 -Pro-X 3 -X 4 -X 5 -X 6 -Y′, wherein 
 a) X 1 , X 2 , X 4 , X 5 , and X 6  are independently Gly, Ala, Val, Leu, Ile, Pro, Met, Phe, or Trp;    b) X 3  is Ser, Thr, or Tyr; and    c) Y and Y′ are independently present or absent and, if present, independently comprise a peptide having from 1 to 600 amino acids.    
     
     
         4 . The method of  claim 1 , wherein said HIFα polypeptide moiety comprises the amino acid sequence corresponding to the N-terminal residues 1-600 of HIF1α, numbered in accordance with wild type HIF1α.  
     
     
         5 . The method of  claim 1 , wherein said HIFα polypeptide moiety comprises a 4 to 12 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α, numbered in accordance with wild type HIF1α.  
     
     
         6 . The method of  claim 1 , wherein said HIFα polypeptide moiety comprises a 12 to 14 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α, numbered in accordance with wild type HIF1α.  
     
     
         7 . The method of  claim 1 , wherein said HIFα polypeptide moiety comprises a 20 to 30 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α, numbered in accordance with wild type HIF1α.  
     
     
         8 . The method of  claim 1  wherein said HIFα polypeptide moiety comprises a 80 to 120 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α, numbered in accordance with wild type HIF1α.  
     
     
         9 . The method of  claim 1 , wherein said HIFα polypeptide moiety comprises amino acids 555-575 of human HIF1α, numbered in accordance with wild type HIFα.  
     
     
         10 . The method of  claim 1 , wherein modulation is an increase in HIFα proline hydroxylation.  
     
     
         11 . The method of  claim 1 , wherein modulation is a decrease in HIFα proline hydroxylation.  
     
     
         12 . The method of  claim 11 , wherein the decrease in HIFα proline hydroxylation is due to inhibition of a prolyl hydroxylase.  
     
     
         13 . A polypeptide comprising amino acids 555-575 of wild type HIF1α, numbered in accordance with wild type HIF1α, provided the polypeptide is not a naturally-occurring HIF1α.  
     
     
         14 . The method of  claim 1 , wherein the HIFα polypeptide is selected from the group consisting of HIF1α, HIF2α, and HIF3α 
     
     
         15 . A method of treating or preventing tissue damage due to hypoxia or ischemia in a subject, the method comprising administering to the subject a compound identified by the method of  claim 1 , thereby treating or preventing tissue damage in the subject.  
     
     
         16 . The method of  claim 15 , wherein the hypoxia or ischemia is due to a condition selected from the group consisting of myocardial infarction, stroke, cancer, and diabetes.  
     
     
         17 . The method of  claim 15 , wherein the hypoxia or ischemia is due to a condition selected from the group consisting of deep vein thrombosis, pulmonary embolus, and renal failure.  
     
     
         18 . The method of  claim 15 , wherein the half-life of HIFα in the subject is increased compared to a subject not exposed to the compound.  
     
     
         19 . A method of increasing angiogenesis or vascularization in a subject, the method comprising administering to the subject a compound identified by the method of  claim 1 , thereby increasing angiogenesis or vascularization in the subject.  
     
     
         20 . A method of treating cancer in a subject, the method comprising administering to the subject a compound identified by the method of  claim 1 , thereby treating cancer in the subject.  
     
     
         21 . A method for treating or preventing tissue damage due to hypoxia or ischemia in a subject, the method comprising administering to the subject a compound which decreases prolyl hydroxylation of HIFα, such that said tissue damage is treated or prevented.  
     
     
         22 . The method of  claim 21  wherein the hypoxia or ischemia is due to a condition selected from the group consisting of myocardial infarction, stroke, cancer, diabetes, deep vein thrombosis, pulmonary embolus, and renal failure.