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Development of Novel Inhibitors of HSP90Pharmacological inhibition of the Hsp90 machinery is an exciting option for cancer therapy. Clinical efficacy of Hsp90 inhibitors is, however, less than expected. Binding of the co-chaperone p23 to Hsp90, and induced overexpression of anti-apoptotic proteins, Hsp70 and Hsp27, is thought to contribute to this undesired outcome. We therefore face an urgent need to develop much better inhibitors of the Hsp90 machinery that can effectively kill cancer cells with minimal side effects. The goal of this dissertation is to identify novel inhibitors of Hsp90 chaperoning machinery to efficiently kill cancer cells with minimal side effects on normal cell survival. First, we report that the natural product, gedunin, may provide a new alternative to inactivate the Hsp90 machine. We show that gedunin directly binds to the co-chaperone p23 and inactivates it, without inducing over-expression of Hsp27 and only a relatively modest induction of Hsp70. Using molecular docking and mutational analyses; we mapped the gedunin-binding site on p23. Functional analysis shows that gedunin inhibits p23 chaperoning activity, blocks its cellular interaction with Hsp90 and interferes with p23-mediated gene regulation. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the Cterminus. These results provide important insight into the molecular mechanism of action of this promising lead compound. Second, we report the development of a novel semi-high-throughput drugscreening assay to identify small molecule inhibitors of Hsp90 and its cochaperones. Our assay quantitatively measures the ability of Hsp90 and its cochaperones to refold the progesterone receptor (PR), a physiological client of Hsp90, in an in vitro assay performed in a 96-well plate format. We tested the NIH clinical collection drug library of 446 compounds and identified capsaicin as a “hit”. Our data show that capsaicin targets the Hsp70-Hsp90 chaperone complex in cells and alters Hsp70 multi-chaperone complexes. It induces cellular destabilization of Hsp90-Hsp70 client proteins and causes degradation of the Hsp70 (induced form) but not the Hsc70 (constitutive form) protein through lysosome-autophagy pathway. Cell survival assays showed that capsaicin selectively kills cancer cells by inducing mitophagy. Taken together, our data suggest that capsaicin could be used in combination with Hsp90 inhibitors for cancer treatment.