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

Background: 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. 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.Methods: PC3-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.Results: KU174 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.Conclusions: Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.Peer reviewedBiochemistry and Molecular Biolog

Novobiocin–ferrocene conjugates possessing anticancer and antiplasmodial activity independent of HSP90 inhibition.

A series of tailored novobiocin–ferrocene conjugates was prepared in moderate yields and investigated for in vitro anticancer and antiplasmodial activity against the MDA-MB-231 breast cancer line and Plasmodium falciparum 3D7 strain, respectively. While the target compounds displayed moderate anticancer activity against the breast cancer cell line with IC50 values in the mid-micromolar range, compounds 10a–c displayed promising antiplasmodial activity as low as 0.889 µM. Furthermore, the most promising compounds were tested for inhibitory effects against a postulated target, heat shock protein 90 (Hsp90)

Probing Molecular Mechanisms of the Hsp90 Chaperone: Biophysical Modeling Identifies Key Regulators of Functional Dynamics

Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based “conformational selection” of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected residue clusters may be a rather general functional requirement encoded across molecular chaperones. The obtained insights may be useful in guiding discovery of allosteric Hsp90 inhibitors targeting protein interfaces with co-chaperones and protein binding clients

Activation of Hsp70 reduces neurotoxicity by promoting polyglutamine protein degradation

We sought novel strategies to reduce levels of the polyglutamine androgen receptor (polyQ AR) and achieve therapeutic benefits in models of spinobulbar muscular atrophy (SBMA), a protein aggregation neurodegenerative disorder. Proteostasis of the polyQ AR is controlled by the Hsp90/Hsp70-based chaperone machinery, but mechanisms regulating the protein’s turnover are incompletely understood. We demonstrate that overexpression of Hip, a co-chaperone that enhances binding of Hsp70 to its substrates, promotes client protein ubiquitination and polyQ AR clearance. Furthermore, we identify a small molecule that acts similarly to Hip by allosterically promoting Hsp70 binding to unfolded substrates. Like Hip, this synthetic co-chaperone enhances client protein ubiquitination and polyQ AR degradation. Both genetic and pharmacologic approaches targeting Hsp70 alleviate toxicity in a Drosophila model of SBMA. These findings highlight the therapeutic potential of allosteric regulators of Hsp70, and provide new insights into the role of the chaperone machinery in protein quality control

BPTF is required for c-MYC transcriptional activity and in vivo tumorigenesis

c-MYC oncogene is deregulated in most human tumours. Histone marks associated with transcriptionally active genes define high-affinity c-MYC targets. The mechanisms involved in their recognition by c-MYC are unknown. Here we report that c-MYC interacts with BPTF, a core subunit of the NURF chromatin-remodelling complex. BPTF is required for the activation of the full c-MYC transcriptional programme in fibroblasts. BPTF knockdown leads to decreased c-MYC recruitment to DNA and changes in chromatin accessibility. In Bptf-null MEFs, BPTF is necessary for c-MYC-driven proliferation, G1-S progression and replication stress, but not for c-MYC-driven apoptosis. Bioinformatics analyses unveil that BPTF levels correlate positively with c-MYC-driven transcriptional signatures. In vivo, Bptf inactivation in pre-neoplastic pancreatic acinar cells significantly delays tumour development and extends survival. Our findings uncover BPTF as a crucial c-MYC co-factor required for its biological activity and suggest that the BPTF-c-MYC axis is a potential therapeutic target in cancer.This work was supported, in part, by grants from Ministerio de Economía y Competitividad, Madrid, Spain (SAF2007–60860, SAF2011–29530 and ONCOBIO Consolider), Instituto de Salud Carlos III, Madrid, Spain (RTICC RD12/0036/0034), European Union Seventh Framework Programme (grant 256974), and an EIN/Roche-CNIO collaborative grant to F.X.R.; and grants from Instituto de Salud Carlos III (INTRASALUD PI12/00425 and RTICC RD12/0036/0037) to J.C.