Targeting the PAS-A domain of HIF-1α for development of small molecule inhibitors of HIF-1

Hypoxia inducible factor-1 (HIF-1) is a master regulator of cellular adaptation to oxygen deprivation and activates transcription of genes involved in tumor metabolism, angiogenesis, invasion and metastasis, all of which are implicated in cancer progression. Several domains of HIF-1 a mediate protein-protein interaction, which is essential for the formation of the active heterodimer with HIF-1 β. Targeting specific domains of HIF-1 a might lead to the identification of more selective inhibitors. HIF-1 a and HIF-1 β contain two Per-Arnt-Sim (PAS) domains, A and B, both of which appear to be important for heterodimer formation. In an attempt to identify small molecule inhibitors of the PAS-A domain of HIF-1 we expressed proteins containing amino acids 86-165 of HIF-1 a and amino acids 159-240 of HIF-1P fused to a His or FLAG tag, respectively. Expressed proteins retained functional activity as indicated by in vitro immunoprecipitation experiments and activation of luciferase expression in a mammalian two-hybrid system. Interestingly, over-expression of HIF-1 α-PAS-A domain was sufficient to abrogate hypoxic induction of HIF-1-dependent luciferase expression, supporting its potential role as drug target. An ELISA based on the interaction between FLAG-HIF-1β-PAS-A and HIF-1α-PAS-A-His was developed and used to screen libraries of synthetic compounds. NSC 50352 specifically inhibited PAS-A-dependent interaction between HIF-1α and HIF-1β, but not the interaction mediated by unrelated domains. However, NSC 50352 was devoid of activity in cell-based assays. Our results provide proof-of-principle that the PAS-A domain of HIF-1 a is a valid target for development of small molecule inhibitors.