Abstract

The 90-kDa heat shock protein (Hsp90) assists in the proper folding of numerous mutated or overexpressed signal transduction proteins that are involved in cancer. Inhibiting Hsp90 consequently is an attractive strategy for cancer therapy as the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. Here we report a novel C-terminal Hsp90 inhibitor, designated KU675, which exhibits potent anti-proliferative and cytotoxic activity along with client protein degradation without induction of the heat shock response (HSR) in both androgen dependent and independent prostate cancer cell lines. In addition, KU675 demonstrates direct inhibition of Hsp90 complexes as measured by the inhibition of luciferase refolding in prostate cancer cells. In direct binding studies, the internal fluorescence signal of KU675 was utilized to determine the binding affinity of KU675 to recombinant Hsp90α, Hsp90β and Hsc70 proteins. The K_d for Hsp90α was determined to be 191 μM while the K_d for Hsp90β was 726 μM, demonstrating a preference for Hsp90α. Western blot experiments with prostate cancer cell lines treated with KU675 supported this selectivity, by inducing the degradation of Hsp90α-dependent client proteins. KU675 also displayed binding to Hsc70 with a K_d value at 76.3 μM, which was supported in cellular by lower levels of Hsc70 specific client proteins upon western blot analyses. Overall, these findings suggest that KU675 is an Hsp90 C- terminal inhibitor as well as dual inhibitor of Hsc70, and may have potential use for the treatment of cancer.