Comparative activity of the potent selective CDK2 inhibitor NU6102 in CDK2 wild-type and knock-out mouse embryo fibroblasts

4409 Cyclin-dependent kinases (CDK) regulate cell cycle progression, and CDK deregulation due to cyclin over-expression, mutation or tumor suppressor gene malfunction is a common feature of human cancer. CDKs are being actively investigated as targets for cancer therapy, and CDK inhibitors have entered clinical trials, e.g. flavopiridol, R-roscovitine and BMS387032. CDK2, in association with cyclins E and A, is involved in G1/S and S phase progression, and molecular pathology studies have implicated CDK2 as a target for drug therapy. However, recent molecular genetic studies in tumor cell lines and in knock-out mouse models have questioned the validity of CDK2 as a target (Tetsu and McCormick Cancer Cell 2003 3:233; Ortega et al., Nature Genetics 2003 35:25). Specifically, mice with homozygous deletion of CDK2 grow normally and give rise to embryonic fibroblasts (MEFs) with equivalent replicative capacity to their WT counterparts. We have recently described the design and synthesis of NU6102 (6-cyclohexylmethoxy-2-(4’-sulfamoylanilino) purine), a potent and selective CDK2 inhibitor (Davies at al., Nature Structural Biology 2002, 9:745). The current study describes the growth inhibitory activity of NU6102 in CDK2 WT and KO MEFs in comparison to the standard compound R-roscovitine. The CDK inhibitory activities of NU6102 and R-roscovitine were defined using a panel of purified human CDKs, ATP concentrations of 12.5 microM (CDK1,2 and 4) or 100 microM (CDK5 and 7), and appropriate substrates. Concentrations (microM) required to inhibit CDKs (CDK1/B; CDK2/A3; CDK4/D; CDK5/p25; CDK7/H) by 50% were: 0.25+/−0.05; 0.005+/−0.001; 1.5+/−0.7; 0.48+/−0.07; 4.4+/−0.8 to NU6102; However, R-roscovitine against these CDKs were 6; 0.41+/−0.05; 5.5+/−1.1; 0.87+/−0.14; 0.84+/−0.04, respectively. Thus NU6102 shows 50-fold selectivity for CDK2, whereas R-roscovitine is less selective. In a 5-day sulphorhodamine B growth inhibition assay, NU6102 had a GI50 value of 9+/−2 microM for CDK2 WT MEFs, whereas there was no growth inhibition up to and including 25 microM in the CDK KO MEFs. At concentrations where NU6102 showed differential activity against the CDK2 WT MEFs (