title: Biochemical and cellular characterization of VRX0466617, a novel and selective inhibitor for the checkpoint kinase Chk2.
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 micromol/L. These doses of VRX0466617 inhibited, to some extent, the phosphorylation of Chk2 Ser(19) and Ser(33-35), but not of Chk2 Thr(68), which is phosphorylated by the upstream ataxia-telangiectasia mutated (ATM) kinase. Interestingly, VRX0466617 induced the phosphorylation of Chk2 Thr(68) 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.
authors.affiliation: Department of Experimental Oncology, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milan, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Carlessi
authors.forename: Giacomo
authors.initials: G
authors.lastname: Buscemi
authors.forename: Gary
authors.initials: G
authors.lastname: Larson
authors.forename: Zhi
authors.initials: Z
authors.lastname: Hong
authors.forename: Jim Zhen
authors.initials: JZ
authors.lastname: Wu
authors.forename: Domenico
authors.initials: D
authors.lastname: Delia
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000528:radiation effects; D064007:Ataxia Telangiectasia Mutated Proteins; D001562:Benzimidazoles / Q000737:chemistry / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D049109:Cell Proliferation / Q000187:drug effects* / Q000528:radiation effects; D002478:Cells, Cultured / Q000187:drug effects / Q000528:radiation effects; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D015394:Molecular Structure; D017239:Paclitaxel / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011839:Radiation, Ionizing; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology*; D013912:Threonine / Q000737:chemistry; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D001562:Benzimidazoles; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013844:Thiazoles; D025521:Tumor Suppressor Proteins; C520468:VRX0466617; D013912:Threonine; D012694:Serine; D004317:Doxorubicin; C031000:benzimidazole; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; C000605869:Chek1 protein, mouse; C578633:Chek2 protein, mouse; D017346:Protein Serine-Threonine Kinases; D017239:Paclitaxel; D002945:Cisplatin