In vitro and in vivo inhibition of the HSP90 molecular chaperone by novel, synthetic, potent pyrazole/isoxazole resorcinol 5-amide analogs

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The molecular chaperone HSP90 is required for the ATP-dependent folding, conformation, activity and stability of several oncogenic client proteins. Hence, agents that inhibit the function of HSP90 hold significant promise for cancer therapy. The first-in-class HSP90 inhibitor to enter clinical trials is 17-allylamino-17-demethoxygeldanamycin (17-AAG). Given the potential limitations of 17-AAG, it is appropriate to develop alternative chemical series. X-ray crystallography studies of our previously reported high-throughput screening hit, the synthetic 3, 4 diaryl pyrazole resorcinol CCT018159 (Cheung KM et al. Bioorg Med Chem Lett 2005;15:3338-43), was used to design the 5-amide analogs. Here we report the biological and pharmacological properties of the pyrazole VER49009 and the isoxazole VER50589. These compounds exhibited up to 40-fold more potency than CCT018159 against HSP90 and human cancer cells. X-ray crystallography studies demonstrated a virtually identical mode of binding for these two inhibitors. The IC₅₀ values for VER49009 and VER50589 were 167 ± 9 nM and 143 ± 23 nM, respectively in an ATPase enzyme assay and 47 ± 9 nM and 21 ± 4 nM in a fluorescent binding assay. Mean cellular GI₅₀ values for VER49009 and VER50589 across a panel of human cancer cell lines were 644.1 and 71.9 nM, respectively. The increased potency observed for VER50589 may be attributed to greater cellular uptake. Unlike 17-AAG, the cellular sensitivity of the pyrazole and isoxazole resorcinol analogs was independent of NQO1/DT-diaphorase and P-glycoprotein expression. Both agents caused induction of HSP70 and HSP27, and downregulation of many oncogenic client proteins including C-RAF, B-RAF, CDK4 and a protein methyl transferase. Phospho-ERK1/2 and phospho-AKT levels were also reduced. Pharmacokinetic studies of VER50589 in HCT116 human colon cancer xenografts showed accumulation of compound at tumor levels above cellular GI₅₀ for 24 hr post dosing. Extent and duration of PD changes in an orthotopic ovarian carcinoma model confirmed the superiority of VER50589. Daily doses of 100 mg/kg VER50589 to athymic mice bearing HCT116 tumor xenografts gave ~30% growth inhibition with clear changes in PD markers, including induction of HSP70 and depletion of ERBB2. The pyrazole/isoxazole compounds compare favourably with 17-AAG and illustrate the value of structure-based design and optimisation.