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

Inhibition 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.

A 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.

NSC145366 interacts with the Hsp90 CTD and has anti-proliferative activity in tumor cell lines (GI₅₀=0.2-1.9μM). NSC145366 increases Hsp90 oligomerization resulting in allosteric inhibition of NTD ATPase activity (IC₅₀=119μM) 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.

A 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.

Allosteric inhibition of Hsp90 via a mechanism used by the NSC145366-based probes is a promising avenue for selective oncogenic client downregulation.