title: Echinomycin, a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity.
abstract: 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.
authors.affiliation: Science Applications International Corporation-Frederick, Inc., Frederick, Maryland, USA.
authors.forename: Dehe
authors.initials: D
authors.lastname: Kong
authors.forename: Eun Jung
authors.initials: EJ
authors.lastname: Park
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Stephen
authors.forename: Maura
authors.initials: M
authors.lastname: Calvani
authors.forename: John H
authors.initials: JH
authors.lastname: Cardellina
authors.forename: Anne
authors.initials: A
authors.lastname: Monks
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Fisher
authors.forename: Robert H
authors.initials: RH
authors.lastname: Shoemaker
authors.forename: Giovanni
authors.initials: G
authors.lastname: Melillo
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000737:chemistry / Q000378:metabolism*; D004448:Echinomycin / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005910:Glioma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000903:Antibiotics, Antineoplastic; D004273:DNA, Neoplasm; D051793:Hypoxia-Inducible Factor 1; D012333:RNA, Messenger; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D004448:Echinomycin; D008156:Luciferases