Identification of small molecule compounds that inhibit the HIF-1 signaling pathway.

Methods Development


BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) is the major hypoxia-regulated transcription factor that regulates cellular responses to low oxygen environments. HIF-1 is composed of two subunits: hypoxia-inducible HIF-1alpha and constitutively-expressed HIF-1beta. During hypoxic conditions, HIF-1alpha heterodimerizes with HIF-1beta and translocates to the nucleus where the HIF-1 complex binds to the hypoxia-response element (HRE) and activates expression of target genes implicated in cell growth and survival. HIF-1alpha protein expression is elevated in many solid tumors, including those of the cervix and brain, where cells that are the greatest distance from blood vessels, and therefore the most hypoxic, express the highest levels of HIF-1alpha. Therapeutic blockade of the HIF-1 signaling pathway in cancer cells therefore provides an attractive strategy for development of anticancer drugs. To identify small molecule inhibitors of the HIF-1 pathway, we have developed a cell-based reporter gene assay and screened a large compound library by using a quantitative high-throughput screening (qHTS) approach. RESULTS: The assay is based upon a beta-lactamase reporter under the control of a HRE. We have screened approximate 73,000 compounds by qHTS, with each compound tested over a range of seven to fifteen concentrations. After qHTS we have rapidly identified three novel structural series of HIF-1 pathway Inhibitors. Selected compounds in these series were also confirmed as inhibitors in a HRE beta-lactamase reporter gene assay induced by low oxygen and in a VEGF secretion assay. Three of the four selected compounds tested showed significant inhibition of hypoxia-induced HIF-1alpha accumulation by western blot analysis. CONCLUSION: The use of beta-lactamase reporter gene assays, in combination with qHTS, enabled the rapid identification and prioritization of inhibitors specific to the hypoxia induced signaling pathway.


Xia, Menghang; Bi, Kun; Huang, Ruili; Cho, Ming-Hsuang; Sakamuru, Srilatha; Miller, Susanne C; Li, Hua; Sun, Yi; Printen, John; Austin, Christopher; Inglese, James;


  • Cell Line, Tumor
  • Genes, Reporter
  • Humans
  • Hypoxia-Inducible Factor 1/ antagonists & inhibitors
  • Hypoxia-Inducible Factor 1/ metabolism
  • Oxygen/ metabolism
  • RNA Interference
  • Signal Transduction/ drug effects
  • beta-Lactamases/ genetics

External Links