Activation of Hsp90 Enzymatic Activity and Conformational Dynamics through Rationally Designed Allosteric Ligands

Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current pharmacological approaches block the chaperone with ATP-competitive inhibitors. Herein, a general approach to perturb Hsp90 through design of new allosteric ligands aimed at modulating its functional dynamics is proposed. Based on the characterization of a first set of 2-phenylbenzofurans showing stimulatory effects on Hsp90 ATPase and conformational dynamics, new ligands were developed that activate Hsp90 by targeting an allosteric site, located 65 æ from the active site. Specifically, analysis of protein responses to first-generation activators was exploited to guide the design of novel derivatives with improved ability to stimulate ATP hydrolysis. The molecules’ effects on Hsp90 enzymatic, conformational, cochaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. These designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary

Targeting the Hsp40/Hsp70 chaperone axis as a novel strategy to treat castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) is characterized by reactivation of androgen receptor (AR) signaling, in part by elevated expression of AR splice variants (ARv) including ARv7, a constitutively active, ligand binding domain (LBD)-deficient variant whose expression has been correlated with therapeutic resistance and poor prognosis. In a screen to identify small-molecule dual inhibitors of both androgen-dependent and androgen-independent AR gene signatures, we identified the chalcone C86. Binding studies using purified proteins and CRPC cell lysates revealed C86 to interact with Hsp40. Pull-down studies using biotinylated-C86 found Hsp40 present in a multiprotein complex with full-length (FL-) AR, ARv7, and Hsp70 in CRPC cells. Treatment of CRPC cells with C86 or the allosteric Hsp70 inhibitor JG98 resulted in rapid protein destabilization of both FL-AR and ARv, including ARv7, concomitant with reduced FL-AR- and ARv7-mediated transcriptional activity. The glucocorticoid receptor, whose elevated expression in a subset of CRPC also leads to androgen-independent AR target gene transcription, was also destabilized by inhibition of Hsp40 or Hsp70. In vivo, Hsp40 or Hsp70 inhibition demonstrated single-agent and combinatorial activity in a 22Rv1 CRPC xenograft model. These data reveal that, in addition to recognized roles of Hsp40 and Hsp70 in FL-AR LBD remodeling, ARv lacking the LBD remain dependent on molecular chaperones for stability and function. Our findings highlight the feasibility and potential benefit of targeting the Hsp40/Hsp70 chaperone axis to treat prostate cancer that has become resistant to standard antiandrogen therapy.Significance: These findings highlight the feasibility of targeting the Hsp40/Hsp70 chaperone axis to treat CRPC that has become resistant to standard antiandrogen therapy. Cancer Res; 78(14); 4022-35. ©2018 AACR

Hsp90 and co-chaperones twist the functions of diverse client proteins

Hsp90 molecular chaperones are required for the stability and activity of a diverse range of client proteins that have critical roles in signal transduction, cellular trafficking, chromatin remodeling, cell growth, differentiation, and reproduction. Mammalian cells contain three types of Hsp90s: cytosolic Hsp90, mitochondrial Trap-1, and Grp94 of the endoplasmic reticulum. Each of the Hsp90s, as well as the bacterial homolog, HtpG, hydrolyze ATP and undergo similar conformational changes. Unlike the other forms of Hsp90, cytosolic Hsp90 function is dependent on a battery of co-chaperone proteins that regulate the ATPase activity of Hsp90 or direct Hsp90 to interact with specific client proteins. This review will summarize what is known about Hsp90's ability to mediate the folding and activation of diverse client proteins that contribute to human diseases, such as cancer and fungal and viral infections

Out with the old: Hsp90 finds amino acid residue more useful than co-chaperone protein

Redundant functions maintained from single to multi-cellular organisms have made Saccharomyces cere-visiae an important model for the analysis of con-served com-plex cellular processes. Yeast has been especially useful in understanding the regulation and function of the essential molecular chaperone, Heat Shock Protein 90 (Hsp90). Research focused on Hsp90 has determined that it is highly regulated by both co-chaperones and posttranslational modifications. A recent study per-formed by (Zuehlke et al., 2017) demonstrates that the function of one co-chaperone in yeast is replaced by posttranslational modification (PTM) of a single amino acid within Hsp90 in higher eukaryotes

Chaperoning the Chaperone: A Role for the Co-chaperone Cpr7 in Modulating Hsp90 Function in Saccharomyces cerevisiae

Abstract Heat-shock protein 90 (Hsp90) of Saccharomyces cerevisiae is an abundant essential eukaryotic molecular chaperone involved in the activation and stabilization of client proteins, including several transcription factors and oncogenic kinases. Hsp90 undergoes a complex series of conformational changes and interacts with partner co-chaperones such as Sba1, Cpr6, Cpr7, and Cns1 as it binds and hydrolyzes ATP. In the absence of nucleotide, Hsp90 is dimerized only at the carboxy-terminus. In the presence of ATP, Hsp90 also dimerizes at the amino-terminus, creating a binding site for Sba1. Truncation of a charged linker region of yeast Hsp90 (Hsp82Δlinker) was known to disrupt the ability of Hsp82 to undergo amino-terminal dimerization and bind Sba1. We found that yeast expressing Hsp82Δlinker constructs exhibited a specific synthetic lethal phenotype in cells lacking CPR7. The isolated tetratricopeptide repeat domain of Cpr7 was both necessary and sufficient for growth in those strains. Cpr6 and Cpr7 stably bound the carboxy-terminus of wild-type Hsp82 only in the presence of nonhydrolyzable ATP and formed an Hsp82–Cpr6–Cpr7 ternary complex. However, in cells expressing Hsp82Δlinker or lacking CPR7, Cpr6 was able to bind Hsp82 in the presence or absence of nucleotide. Overexpression of CNS1, but not of other co-chaperones, in cpr7 cells restored nucleotide-dependent Hsp82–Cpr6 interaction. Together, our results suggest that the in vivo functions of Cpr7 include modulating Hsp90 conformational changes, mediating proper signaling of the nucleotide-bound state to the carboxy-terminus of Hsp82, or regulating Hsp82–Cpr6 interaction

Interaction of Heat Shock Protein 90 and the Co-chaperone Cpr6 with Ura2, a Bifunctional Enzyme Required for Pyrimidine Biosynthesis

Background: Hsp90 and co-chaperones are critical for the folding and activation of client proteins. Results: Hsp90 and Cpr6, but not other tetratricopeptide repeat (TPR)-containing co-chaperones, interact with Ura2. Conclusion: The TPR domain of Cpr6 has unique functions, including interaction with Ura2, an enzyme required for pyrimidine biosynthesis. Significance: Identification of co-chaperone-specific interactions is crucial to understanding how to selectively target Hsp90 functions. The molecular chaperone heat shock protein 90 (Hsp90) is an essential protein required for the activity and stability of multiple proteins termed clients. Hsp90 cooperates with a set of co-chaperone proteins that modulate Hsp90 activity and/or target clients to Hsp90 for folding. Many of the Hsp90 co-chaperones, including Cpr6 and Cpr7, contain tetratricopeptide repeat (TPR) domains that bind a common acceptor site at the carboxyl terminus of Hsp90. We found that Cpr6 and Hsp90 interacted with Ura2, a protein critical for pyrimidine biosynthesis. Mutation or inhibition of Hsp90 resulted in decreased accumulation of Ura2, indicating it is an Hsp90 client. Cpr6 interacted with Ura2 in the absence of stable Cpr6-Hsp90 interaction, suggesting a direct interaction. However, loss of Cpr6 did not alter the Ura2-Hsp90 interaction or Ura2 accumulation. The TPR domain of Cpr6 was required for Ura2 interaction, but other TPR containing co-chaperones, including Cpr7, failed to interact with Ura2 or rescue CPR6 -dependent growth defects. Further analysis suggests that the carboxyl-terminal 100 amino acids of Cpr6 and Cpr7 are critical for specifying their unique functions, providing new information about this important class of Hsp90 co-chaperones

The Hsp90 cochaperones Cpr6, Cpr7, and Cns1 interact with the intact ribosome

The abundant molecular chaperone Hsp90 is essential for the folding and stabilization of hundreds of distinct client proteins. Hsp90 is assisted by multiple cochaperones that modulate Hsp90's ATPase activity and/or promote client interaction, but the in vivo functions of many of these cochaperones are largely unknown. We found that Cpr6, Cpr7, and Cns1 interact with the intact ribosome and that Saccharomyces cerevisiae lacking CPR7 or containing mutations in CNS1 exhibited sensitivity to the translation inhibitor hygromycin. Cpr6 contains a peptidyl-prolyl isomerase (PPIase) domain and a tetratricopeptide repeat (TPR) domain flanked by charged regions. Truncation or alteration of basic residues near the carboxy terminus of Cpr6 disrupted ribosome interaction. Cns1 contains an amino-terminal TPR domain and a poorly characterized carboxy-terminal domain. The isolated carboxy-terminal domain was able to interact with the ribosome. Although loss of CPR6 does not cause noticeable growth defects, overexpression of CPR6 results in enhanced growth defects in cells expressing the temperature-sensitive cns1-G90D mutation (the G-to-D change at position 90 encoded by cns1). Cpr6 mutants that exhibit reduced ribosome interaction failed to cause growth defects, indicating that ribosome interaction is required for in vivo functions of Cpr6. Together, these results represent a novel link between the Hsp90 molecular-chaperone machine and protein synthesis