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Research Articles: Therapeutics, Targets, and Development

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

¹ Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, United Kingdom; ² Section of Structural Biology, The Institute of Cancer Research, Chester Beatty Laboratories, London, United Kingdom; and ³ Vernalis Ltd., Granta Park, Great Abington, Cambridge, United Kingdom

Requests for reprints: Paul Workman, Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Belmont, Sutton, Surrey, SM2 5NG, United Kingdom. Phone: 44-20-8722-4301; Fax: 44-20-8722-4324. E-mail: Paul.Workman{at}icr.ac.uk

Abstract

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 IC₅₀ 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 GI₅₀ 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 GI₅₀ for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as HSP90 inhibitors. [Mol Cancer Ther 2007;6(4):1198–211]

Grant support: Cancer Research UK [CUK] programme grants C309/A2187 and C309/A8274, Wellcome Trust, and Vernalis Ltd.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: P. Workman is a Cancer Research UK Life Fellow. J. Holmes is funded by a studentship from Cancer Research UK.

⁴ http://www.picard.ch

⁵ Supplementary materials for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

⁶ Unpublished data.

Received 3/ 5/07; revised 3/13/07; accepted 3/14/07.

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