title: Identification of novel small molecule inhibitors of hypoxia-inducible factor-1 that differentially block hypoxia-inducible factor-1 activity and hypoxia-inducible factor-1alpha induction in response to hypoxic stress and growth factors.
abstract: Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that is activated in response to hypoxia and growth factors. HIF-1 plays a central role in tumor progression, invasion, and metastasis. Overexpression of the HIF-1alpha subunit has been observed in many human cancers and is associated with a poor prognostic outcome with conventional treatments. Targeting HIF-1 using novel small molecule inhibitors is, therefore, an attractive strategy for therapeutic development. We have generated U2OS human osteosarcoma cells stably expressing a luciferase reporter construct under the control of a hypoxia response element (U2OS-HRE-luc). The U2OS-HRE-luc cells were robustly and reproducibly sensitive to hypoxic stress in a HIF-1-dependent manner. We developed an automated U2OS-HRE-luc cell-based assay that was used in a high-throughput screen to identify compounds that inhibited HIF-1 activity induced by treatment with the hypoxia mimetic, deferoxamine mesylate. We performed a pilot screen of the National Cancer Institute Diversity Set of 2,000 compounds. We identified eight hit compounds, six of these were also identified by Rapisarda et al. in an independent hypoxia screen. However, there were two novel hit compounds, NSC-134754 and NSC-643735, that did not significantly inhibit constitutive luciferase activity in U2OS cells (U2OS-luc). We showed that both NSC-134754 and NSC-643735 significantly inhibited HIF-1 activity and HIF-1alpha protein induced by deferoxamine mesylate. Interestingly, NSC-134754 but not NCS-643735 inhibited HIF-1 activity and HIF-1alpha protein induced by hypoxia and significantly inhibited Glut-1 expression. Finally, we showed that both NCS-134754 and NCS-643735 inhibited HIF-1alpha protein induced by insulin-like growth factor-1. Our cell-based assay approach has successfully identified novel compounds that differentially target hypoxia and/or growth factor-mediated induction of HIF-1alpha.
authors.affiliation: Cell Growth Regulation and Angiogenesis Team, Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey, United Kingdom.
authors.forename: Noan-Minh
authors.initials: NM
authors.lastname: Chau
authors.forename: Paul
authors.initials: P
authors.lastname: Rogers
authors.forename: Wynne
authors.initials: W
authors.lastname: Aherne
authors.forename: Veronica
authors.initials: V
authors.lastname: Carroll
authors.forename: Ian
authors.initials: I
authors.lastname: Collins
authors.forename: Edward
authors.initials: E
authors.lastname: McDonald
authors.forename: Paul
authors.initials: P
authors.lastname: Workman
authors.forename: Margaret
authors.initials: M
authors.lastname: Ashcroft
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology*; D008156:Luciferases / Q000378:metabolism; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D010078:Oxazines / Q000494:pharmacology*; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*
chemical_list: D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007546:Isoquinolines; C502338:NSC-134754; C502337:NSC-643735; D009687:Nuclear Proteins; D010078:Oxazines; D014157:Transcription Factors; C091971:DX 52-1; D007334:Insulin-Like Growth Factor I; D008156:Luciferases