Echinomycin, a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity

Cancer Res. 2005 Oct 1;65(19):9047-55. doi: 10.1158/0008-5472.CAN-05-1235.


The identification of small molecules that inhibit the sequence-specific binding of transcription factors to DNA is an attractive approach for regulation of gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that controls genes involved in glycolysis, angiogenesis, migration, and invasion, all of which are important for tumor progression and metastasis. To identify inhibitors of HIF-1 DNA-binding activity, we expressed truncated HIF-1alpha and HIF-1beta proteins containing the basic-helix-loop-helix and PAS domains. Expressed recombinant HIF-1alpha and HIF-1beta proteins induced a specific DNA-binding activity to a double-stranded oligonucleotide containing a canonical hypoxia-responsive element (HRE). One hundred twenty-eight compounds previously identified in a HIF-1-targeted cell-based high-throughput screen of the National Cancer Institute 140,000 small-molecule library were tested in a 96-well plate ELISA for inhibition of HIF-1 DNA-binding activity. One of the most potent compounds identified, echinomycin (NSC-13502), a small-molecule known to bind DNA in a sequence-specific fashion, was further investigated. Electrophoretic mobility shift assay experiments showed that NSC-13502 inhibited binding of HIF-1alpha and HIF-1beta proteins to a HRE sequence but not binding of the corresponding proteins to activator protein-1 (AP-1) or nuclear factor-kappaB (NF-kappaB) consensus sequences. Interestingly, chromatin immunoprecipitation experiments showed that NSC-13502 specifically inhibited binding of HIF-1 to the HRE sequence contained in the vascular endothelial growth factor (VEGF) promoter but not binding of AP-1 or NF-kappaB to promoter regions of corresponding target genes. Accordingly, NSC-13502 inhibited hypoxic induction of luciferase in U251-HRE cells and VEGF mRNA expression in U251 cells. Our results indicate that it is possible to identify small molecules that inhibit HIF-1 DNA binding to endogenous promoters.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antibiotics, Antineoplastic / pharmacology*
  • Breast Neoplasms / drug therapy
  • Breast Neoplasms / genetics
  • Breast Neoplasms / metabolism
  • Cell Line, Tumor
  • DNA, Neoplasm / chemistry
  • DNA, Neoplasm / metabolism*
  • Echinomycin / pharmacology*
  • Enzyme-Linked Immunosorbent Assay
  • Glioma / drug therapy
  • Glioma / genetics
  • Glioma / metabolism
  • Humans
  • Hypoxia-Inducible Factor 1 / antagonists & inhibitors*
  • Hypoxia-Inducible Factor 1 / biosynthesis
  • Hypoxia-Inducible Factor 1 / genetics
  • Hypoxia-Inducible Factor 1 / metabolism
  • Luciferases / antagonists & inhibitors
  • Luciferases / biosynthesis
  • Luciferases / genetics
  • Promoter Regions, Genetic
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / genetics
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Vascular Endothelial Growth Factor A / biosynthesis
  • Vascular Endothelial Growth Factor A / genetics


  • Antibiotics, Antineoplastic
  • DNA, Neoplasm
  • Hypoxia-Inducible Factor 1
  • RNA, Messenger
  • Recombinant Proteins
  • Vascular Endothelial Growth Factor A
  • Echinomycin
  • Luciferases