title: Stimulating retinal blood vessel protection with hypoxia-inducible factor stabilization: identification of novel small-molecule hydrazones to inhibit hypoxia-inducible factor prolyl hydroxylase (an American Ophthalmological Society thesis).
abstract: OBJECTIVE
  To discover novel small molecules that inhibit hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD), a key enzyme that regulates the posttranslational stability and hence activity of HIF.
  
  
  METHODS
  NIH3T3 cell line stably transfected with firefly luciferase under a HIF-1-inducible promoter was used to screen a Chembridge library of 34,000 small molecules of molecular weight 250 to 550 Da. Positive hits were considered at 4.5-fold higher luminescence than control. Selected compounds were validated in vitro. The most effective dose was then used to treat mice expressing firefly luciferase fused to the oxygen-dependent degradation domain (lucODD) in order to determine the location of the receptor for systemic treatment with small-molecule HIF PHD inhibitors.
  
  
  RESULTS
  Twenty-three novel small molecules were discovered, the majority of which were hydrazones and hydrazines. Of the 23 compounds, each had different selectivity for expression of erythropoietin or vascular endothelial growth factor, two angiogenic, HIF-regulated gene products. In addition, each showed different selectivity for hepatocytes or kidney, or both or neither, when injected intraperitoneally in an in vivo reporter gene assay.
  
  
  CONCLUSIONS
  The discovery of multiple small molecules that inhibit HIF PHD identifies new reagents to develop strategies to prevent the degradation of HIF by its selective PHD. These molecules are novel hypoxia mimetics that may provide new strategies to protect retinovasculature from hyperoxia.
authors.affiliation: Department of Cell Biology, Lerner Research Institute (Dr Sears), and Ophthalmic Research, Cole Eye Institute (Dr Sears, Dr Hoppe), Cleveland Clinic, Cleveland, Ohio.
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Sears
authors.affiliation: Department of Cell Biology, Lerner Research Institute (Dr Sears), and Ophthalmic Research, Cole Eye Institute (Dr Sears, Dr Hoppe), Cleveland Clinic, Cleveland, Ohio.
authors.forename: George
authors.initials: G
authors.lastname: Hoppe
mesh_terms: D000818:Animals; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D006835:Hydrazones / Q000494:pharmacology*; D000860:Hypoxia / Q000517:prevention & control; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases / Q000037:antagonists & inhibitors*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D015427:Reperfusion Injury / Q000201:enzymology / Q000517:prevention & control*; D012164:Retinal Diseases / Q000201:enzymology / Q000517:prevention & control*; D012171:Retinal Vessels / Q000187:drug effects*; D014162:Transfection
chemical_list: D004791:Enzyme Inhibitors; C497443:Hif1a protein, mouse; D006835:Hydrazones; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008156:Luciferases; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases