Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells

Jeffery D Eskew,Xiaoying Zhang,Pedro Morales,Jeffrey M Holzbeierlein,Alison Donnelly,Robert L Matts,Irene Dunwiddie,Stacey Hembruff,Brian Sj Blagg,Megan Swink,Alicia Duren,Roger A Rajewski,Jacob R Manjarrez,George A Vielhauer,Takrima Sadikot

doi:10.1186/1471-2407-11-468

Abstract

BackgroundThe molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells.MethodsPC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies.ResultsKU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model.ConclusionsOverall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.

Highlights

The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery
In the prostate cancer cell lines, PC-3 and DU145, KU174 was cytostatic at the single dose of 10 μM with values of 0.46 and 51.79, respectively
Based on previous publications in prostate cancer using an earlier analogue, F-4 [18], we chose to focus on the characterization of KU174 in the PC3-MM2 and LNCaP-LN3 cell-lines to further understand its mechanism of action and effects on Hsp90

Summary

Introduction

The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Like other N-terminal inhibitors, the efficacy of 17-AAG is hampered by the fact that Hsp inhibition itself initiates a heat shock response (HSR), resulting in the induction of Hsp and anti-apoptotic proteins such as Hsp and Hsp27 [9,10,11]. The largely cytostatic profile observed upon administration of 17-AAG across cancers is likely the result of the pro-survival Hsp induction. This is supported by studies showing that neutralizing Hsp and Hsp activity or their transcriptional inducer, HSF-1 augments the effect of 17-AAG and dramatically increases the extent of apoptosis [11,15,16]. Others have shown that combinatorial approaches consisting of 17-AAG and transcriptional inhibition of pro-survival Hsp’s improves the efficacy of 17-AAG [17]

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