Identification of novel small-molecule inhibitors of hypoxia-inducible factor-1 transactivation and DNA binding.

Hypoxia-inducible factor-alpha (Hif-alpha) plays an important role in tumor growth by increasing resistance to apoptosis and the production of angiogenic factors, such as vascular endothelial growth factor (VEGF). Therefore, Hif-alpha is an attractive target for development of novel cancer therapeutics. We have generated Chinese hamster ovary cells, which stably express luciferase reporter construct under the control of a hypoxia response element to screen 15,000 compounds. We identified 40 compounds that inhibited hypoxic up-regulation of luciferase, and the top 30 compounds were further screened in a secondary assay using MDA-468 breast cancer cell line. Eight compounds were shown to inhibit VEGF expression in hypoxic cells at subtoxic concentrations. Three top putative Hif inhibitors, DJ12, DJ15, and DJ30, were chosen for further analysis. Transient transfection of cells with hypoxia-regulated luciferase reporter plasmids further validated that these compounds inhibit hypoxia up-regulated genes. All three compounds failed to inhibit Hif-1alpha protein levels but they did inhibit induction of downstream targets of Hif-alpha under hypoxia. Two of the three compounds were cell type specific, whereas compound DJ12 inhibited VEGF at subtoxic levels in breast cancer cell lines MDA-468 and ZR-75, melanoma cell line MDA-435, and pVHL mutant renal cancer cell lines RCC4 and 786-0. Compound DJ12 down-regulated mRNA of downstream targets of Hif-alpha, and significantly inhibited Hif-1alpha transactivation activity by blocking Hif-1alpha hypoxia response element-DNA binding. Our cell-based approach and deconvolution of the inhibitory effect of DJ12 has identified a novel compound that targets the hypoxia pathway by inhibiting Hif-alpha-inducible transcription.