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Research Articles: Therapeutics, Targets, and Development

Biochemical and cellular characterization of VRX0466617, a novel and selective inhibitor for the checkpoint kinase Chk2

¹ Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy and ² Drug Discovery, Valeant Pharmaceuticals International, Costa Mesa, California

Requests for reprints: Domenico Delia, Department of Experimental Oncology, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milan, Italy. Phone: 39-2-2390-2641; Fax: 39-2-2390-2764. E-mail: domenico.delia{at}istitutotumori.mi.it

Abstract

VRX0466617 is a novel selective small-molecule inhibitor for Chk2 discovered through a protein kinase screening program. In this study, we provide a detailed biochemical and cellular characterization of VRX0466617. We show that VRX0466617 blocks the enzymatic activity of recombinant Chk2, as well as the ionizing radiation (IR)–induced activation of Chk2 from cells pretreated with the compound, at doses between 5 and 10 µmol/L. These doses of VRX0466617 inhibited, to some extent, the phosphorylation of Chk2 Ser¹⁹ and Ser^33–35, but not of Chk2 Thr⁶⁸, which is phosphorylated by the upstream ataxia-telangiectasia mutated (ATM) kinase. Interestingly, VRX0466617 induced the phosphorylation of Chk2 Thr⁶⁸ even in the absence of DNA damage, arising from the block of its enzymatic activity. VRX0466617 prevented the IR-induced Chk2-dependent degradation of Hdmx, concordant with the in vivo inhibition of Chk2. Analysis of ATM/ATM and Rad3-related substrates Smc1, p53, and Chk1 excluded a cross-inhibition of these kinases. VRX0466617 did not modify the cell cycle phase distribution, although it caused an increase in multinucleated cells. Whereas VRX0466617 attenuated IR-induced apoptosis, in short-term assays it did not affect the cytotoxicity by the anticancer drugs doxorubicin, Taxol, and cisplatin. These results underscore the specificity of VRX0466617 for Chk2, both in vitro and in vivo, and support the use of this compound as a biological probe to study the Chk2-dependent pathways. [Mol Cancer Ther 2007;6(3):935–44]

Grant support: Italian Association for Cancer Research, Italian Ministry of Health (Ricerca Finalizzata), Consiglio Nazionale Ricerche, and Telethon grant GGP05226.

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.

Received 9/13/06; revised 12/ 5/06; accepted 1/30/07.

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