title: Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway.
abstract: Hypoxia-inducible factor 1 (HIF-1) is a master regulator of the transcriptional response to oxygen deprivation. HIF-1 has been implicated in the regulation of genes involved in angiogenesis [e.g., vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase] and anaerobic metabolism (e.g., glycolytic enzymes). HIF-1 is essential for angiogenesis and is associated with tumor progression. In addition, overexpression of HIF-1 alpha has been demonstrated in many common human cancers. Therefore, HIF-1 is an attractive molecular target for development of novel cancer therapeutics. We have developed a cell-based high-throughput screen for the identification of small molecule inhibitors of the HIF-1 pathway. We have genetically engineered U251 human glioma cells to stably express a recombinant vector in which the luciferase reporter gene is under control of three copies of a canonical hypoxia-responsive element (U251-HRE). U251-HRE cells consistently expressed luciferase in a hypoxia- and HIF-1-dependent fashion. We now report the results of a pilot screen of the National Cancer Institute "Diversity Set," a collection of approximately 2000 compounds selected to represent the greater chemical diversity of the National Cancer Institute chemical repository. We found four compounds that specifically inhibited HIF-1-dependent induction of luciferase but not luciferase expression driven by a constitutive promoter. In addition, these compounds inhibited hypoxic induction of VEGF mRNA and protein expression in U251 cells. Interestingly, three compounds are closely related camptothecin analogues and topoisomerase (Topo)-I inhibitors. We show that concomitant with HIF-1 and VEGF inhibition, the activity of the Topo-I inhibitors tested is associated with induction of cyclooxygenase 2 mRNA expression. The luciferase-based high-throughput screen is a feasible tool for the identification of small molecule inhibitors of HIF-1 transcriptional activation. In addition, our results suggest that altered Topo-I function may be associated with repression of HIF-1-dependent induction of gene expression.
authors.affiliation: Developmental Therapeutics Program-Tumor Hypoxia Laboratory, Science Applications International Corporation-Frederick, Inc. and National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702-1201, USA.
authors.forename: Annamaria
authors.initials: A
authors.lastname: Rapisarda
authors.forename: Badarch
authors.initials: B
authors.lastname: Uranchimeg
authors.forename: Dominic A
authors.initials: DA
authors.lastname: Scudiero
authors.forename: Mike
authors.initials: M
authors.lastname: Selby
authors.forename: Edward A
authors.initials: EA
authors.lastname: Sausville
authors.forename: Robert H
authors.initials: RH
authors.lastname: Shoemaker
authors.forename: Giovanni
authors.initials: G
authors.lastname: Melillo
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002166:Camptothecin / Q000031:analogs & derivatives; D004273:DNA, Neoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D004354:Drug Screening Assays, Antitumor; D016228:Endothelial Growth Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005910:Glioma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D008222:Lymphokines / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020218:Response Elements / Q000187:drug effects / Q000235:genetics; D019772:Topotecan / Q000494:pharmacology; D014157:Transcription Factors; D015533:Transcriptional Activation / Q000187:drug effects* / Q000502:physiology; D014162:Transfection; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
chemical_list: D000970:Antineoplastic Agents; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D016228:Endothelial Growth Factors; D004791:Enzyme Inhibitors; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008222:Lymphokines; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D019772:Topotecan; D008156:Luciferases; D002166:Camptothecin