Although the
heat shock protein 90 (HSP90) inhibitor
17-allylamino-17-demethoxygeldanamycin (17-AAG) shows clinical promise, potential limitations encourage development of alternative chemotypes. We discovered the 3,4-diarylpyrazole
resorcinol CCT018159 by high-throughput screening and used structure-based design to generate more potent
pyrazole amide analogues, exemplified by
VER-49009. Here, we describe the detailed
biological properties of
VER-49009 and the corresponding
isoxazole VER-50589. X-ray crystallography showed a virtually identical HSP90 binding mode. However, the dissociation constant (K(d)) of
VER-50589 was 4.5 +/- 2.2 nmol/L compared with 78.0 +/- 10.4 nmol/L for
VER-49009, attributable to higher enthalpy for
VER-50589 binding. A competitive binding assay gave a lower IC(50) of 21 +/- 4 nmol/L for
VER-50589 compared with 47 +/- 9 nmol/L for
VER-49009. Cellular uptake of
VER-50589 was 4-fold greater than for
VER-49009. Mean cellular antiproliferative GI(50) values for
VER-50589 and
VER-49009 for a human
cancer cell line panel were 78 +/- 15 and 685 +/- 119 nmol/L, respectively, showing a 9-fold potency gain for the
isoxazole. Unlike
17-AAG, but as with
CCT018159, cellular potency of these analogues was independent of
NAD(P)H:
quinone oxidoreductase 1/
DT-diaphorase and
P-glycoprotein expression. Consistent with HSP90 inhibition,
VER-50589 and
VER-49009 caused induction of HSP72 and HSP27 alongside depletion of client
proteins, including C-RAF, B-RAF, and
survivin, and the
protein arginine methyltransferase PRMT5. Both caused cell cycle arrest and apoptosis. Extent and duration of pharmacodynamic changes in an orthotopic human ovarian
carcinoma model confirmed the superiority of
VER-50589 over
VER-49009.
VER-50589 accumulated in HCT116 human
colon cancer xenografts at levels above the cellular GI(50) for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic
pyrazole/
isoxazole amide analogues as HSP90 inhibitors.