Synthesis and Validation of the First Cell-Impermeable Hsp90α-Selective Inhibitors.

Hsp90α is an isoform of the heat shock protein 90 (Hsp90) family of molecular chaperones and mediates the folding and activation of ∼400 client proteins. However, inhibition of intracellular Hsp90α has caused detrimental side effects and significantly hindered the clinical development of Hsp90 inhibitors. As an alternative strategy, 14 Hsp90α-selective inhibitors were synthesized to introduce permanently charged moieties onto the solvent-exposed portion of the Hsp90α binding site to produce cell-impermeable extracellular Hsp90α-selective inhibitors. The resulting lead compounds were cell-permeable dimethylamine 14 (NDNA3), with an affinity of 0.51 µM for Hsp90α and >196-fold selectivity over the other Hsp90 isoforms, and cell-impermeable quaternary ammonium 17 (NDNA4), with an affinity of 0.34 µM for Hsp90α and >294-fold selectivity. The permanently charged analogs were determined to have low membrane permeability, to be nontoxic against Ovcar-8 and MCF-10A cells, to avoid disruption of hERG channel maturation, and not to induce the heat shock response or Hsp90α-dependent client degradation.

Elucidation of novel TRAP1-Selective inhibitors that regulate mitochondrial processes.

Hsp90 isoform-selective inhibitors represent a new paradigm for novel anti-cancer drugs as each of the four isoforms have specific cellular localization, function, and client proteins. The mitochondrial isoform, TRAP1, is the least understood member of the Hsp90 family due to the lack of small molecule tools to study its biological function. Herein, we report novel TRAP1-selective inhibitors used to interrogate TRAP1's biological function along with co-crystal structures of such compounds bound to the N-terminus of TRAP1. Solution of the co-crystal structure allowed for a structure-based approach that resulted in compound 36, which is a 40 nM inhibitor with >250-fold TRAP1 selectivity over Grp94, the isoform with the highest structural similarity to TRAP1 within the N-terminal ATP binding site. Lead compounds 35 and 36 were found to selectively induce TRAP1 client protein degradation without inducing the heat shock response or disrupting Hsp90-cytosolic clients. They were also shown to inhibit OXPHOS, alter cellular metabolism towards glycolysis, disrupt TRAP1 tetramer stability, and disrupt the mitochondrial membrane potential.

Structure-Activity Relationship Study of Tertiary Alcohol Hsp90α-Selective Inhibitors with Novel Binding Mode.

The heat shock protein 90 (Hsp90) family of molecular chaperones mediates the folding and activation of client proteins associated with all 10 hallmarks of cancer. Herein, the design, synthesis, and biological validation of Hsp90α-selective inhibitors that contain a tertiary alcohol are reported. Forty-one analogues were synthesized to modulate hydrogen-bonding interactions and to probe for steric and hydrophobic interactions within the Hsp90α binding site. Cocrystal structures of lead compound 23d (IC50 = 0.25 µM, 15-fold selective vs Hsp90ß) and a 5-fluoroisoindoline derivative (KUNA-111) revealed a novel binding mode that induced conformational changes within Hsp90α's N-terminal domain. The lead Hsp90α-selective inhibitors did not manifest significant antiproliferative activity, but they did result in selective and dose-dependent degradation of Hsp90α clients in the cellular environment. Additional studies will be sought to determine the effects of the novel conformational change induced by 23d.