In vitro Biological Characterization of a Novel, Synthetic Diaryl Pyrazole Resorcinol Class of Heat Shock Protein 90 Inhibitors

Abstract

The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target. Derivatives of the natural product geldanamycin, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), were the first HSP90 ATPase inhibitors to enter clinical trial. Synthetic smallmolecule HSP90 inhibitors have potential advantages. Here, we describe the biological properties of the lead compound of a new class of 3,4-diaryl pyrazole resorcinol HSP90 inhibitor (CCT018159), which we identified by high-throughput screening. CCT018159 inhibited human HSP90B with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH 2 -terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI 50 value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 Mmol/L. Unlike 17-AAG, the in vitro antitumor activity of the pyrazole resorcinol analogues is independent of NQO1/ DT-diaphorase and P-glycoprotein expression. The molecular signature of HSP90 inhibition, comprising increased expression of HSP72 protein and depletion of ERBB2, CDK4, C-RAF, and mutant B-RAF, was shown by Western blotting and quantified by time-resolved fluorescent-Cellisa in human cancer cell lines treated with CCT018159. CCT018159 caused cell cytostasis associated with a G 1 arrest and induced apoptosis. CCT018159 also inhibited key endothelial and tumor cell functions implicated in invasion and angiogenesis. Overall, we have shown that diaryl pyrazole resorcinols exhibited similar cellular properties to 17-AAG with potential advantages (e.g., aqueous solubility, independence from NQO1 and P-glycoprotein). These compounds form the basis for further structure-based optimization to identify more potent inhibitors suitable for clinical development. [Cancer Res 2007;67(5):2206-16]

Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues

“…Compounds VER-49009 and VER-50589 were synthesized as described (29,30 Cell Culture Unless otherwise stated, cell lines were from the American Type Culture Collection (LGC Promochem, Middlesex, United Kingdom) and cultured as described (27). Penicillin (100 units/mL) and streptomycin (100 Ag/mL; Invitrogen, Paisley, United Kingdom) were added to the medium for human breast cancer cells.…”

“…Penicillin (100 units/mL) and streptomycin (100 Ag/mL; Invitrogen, Paisley, United Kingdom) were added to the medium for human breast cancer cells. Human umbilical vein endothelial cells (HUVEC) and the nontumorigenic cell lines MCF10a (human mammary gland) and PNT2 (human prostate epithelial cells immortalized with SV40) were from the European Collection of Animal Cell Cultures (Wiltshire, United Kingdom) and cultured as described (27). All lines were screened routinely for Mycoplasma (Venor GeM kit, Minerva Biolabs, Berlin, Germany).…”

“…Protein Production and X-ray Crystallography Protein production, crystallization, and X-ray crystallography were done as described (27,29,31).…”

“…Cell Proliferation Assay Antiproliferative effects were measured using the sulforhodamine B assay (15,27). HUVEC sensitivity was determined by an alkaline phosphatase method (35).…”

“…As seen from their chemical structures, VER-49009 and VER-50589 differ only in the pyrazole versus isoxazole heterocycle. The pyrazole to isoxazole switch does not affect the critical hydrogen bonding network exhibited by the pyrazole resorcinol unit that anchors this class of inhibitors to the HSP90 NH 2 -terminal ATP site (26,27,29), while also avoiding steric changes that would not be tolerated. We show here that the isoxazole has greater binding affinity than the corresponding pyrazole and also exhibits enhanced cellular uptake, leading to more potent HSP90 inhibition and antiproliferative Structural differences between the pyrazole VER-49009 (blue ) and the corresponding isoxazole VER-50589 (red ) are highlighted.…”