Targeting the Hsp90 C-terminal domain to induce allosteric inhibition and selective client downregulation

•NSC145366 and bisphenol A-based symmetrical probes inhibit Hsp90 activity.•NSC145366 binds to the Hsp90 CTD, does not inhibit ATP binding to Hsp90 NTD or CTD.•NSC145366 selectively downregulates AR and BRCA1 client proteins in LNCaP cells.•Probes affect CTD oligomerization and allosterically inhibit NTD ATPase activity.•The symmetrical core of the probes dock at the dimer interface.

BackgroundInhibition of Hsp90 is desirable due to potential downregulation of oncogenic clients. Early generation inhibitors bind to the N-terminal domain (NTD) but C-terminal domain (CTD) inhibitors are a promising class because they do not induce a heat shock response. Here we present a new structural class of CTD binding molecules with a unique allosteric inhibition mechanism.MethodsA hit molecule, NSC145366, and structurally similar probes were assessed for inhibition of Hsp90 activities. A ligand-binding model was proposed indicating a novel Hsp90 CTD binding site. Client protein downregulation was also determined.ResultsNSC145366 interacts with the Hsp90 CTD and has anti-proliferative activity in tumor cell lines (GI50 = 0.2-1.9 μM). NSC145366 increases Hsp90 oligomerization resulting in allosteric inhibition of NTD ATPase activity (IC50 = 119 μM) but does not compete with NTD or CTD-ATP binding. Treatment of LNCaP prostate tumor cells resulted in selective client protein downregulation including AR and BRCA1 but without a heat shock response. Analogs had similar potencies in ATPase and chaperone activity assays and variable effects on oligomerization. In silico modeling predicted a binding site at the CTD dimer interface distinct from the nucleotide-binding site.ConclusionsA set of symmetrical scaffold molecules with bisphenol A cores induced allosteric inhibition of Hsp90. Experimental evidence and molecular modeling suggest that the binding site is independent of the CTD-ATP site and consistent with unique induction of allosteric effects.General significanceAllosteric inhibition of Hsp90 via a mechanism used by the NSC145366-based probes is a promising avenue for selective oncogenic client downregulation.

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