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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