Pure and Applied Chemistry

Abstract: The hypoxia-inducible factor (HIF) takes part in transcriptional activation of hypoxia-responsive genes such as vascular endothelial growth factor (VEGF), insulin-like growth factor, and inducible nitric oxide synthase. Since VEGF plays an important role in pathological angiogenesis such as tumor growth and ischemic diseases, the inhibition of VEGF inducer HIF is an attractive approach for the inhibition of pathological angiogenesis. Recently, we have reported that the introduction of boronic acid and a carborane moiety into phenoxyacetanilide induced a potent inhibitory effect on HIF-1α activation under hypoxic conditions. In the present study, to clarify the mechanism of action of carboranylphenoxyacetanilide GN26361 against HIF inhibition, we designed and synthesized molecular probes of GN26361 substituted with benzophenone to induce covalent binding with the target protein by UV (photoaffinity labeling) and an acetylenic moiety to conjugate with the green-fluorescent Alexa Fluor 488-azide by click reaction. In-gel fluorescent imaging of target protein bound with the probe was identified as heat shock protein 60 (HSP60). Moreover, direct binding in gel fluorescent imaging was observed by photoaffinity labeling and click reaction of the probe with recombinant HSP60. These results indicate that HSP60 is the target protein of GN26361 and might be a new molecular target for HIF inhibition.