Abnormal proliferation mediated by disruption of the normal cell cycle mechanisms is a hallmark of virtually all cancer cells. Compounds targeting complexes between cyclin-dependent kinases (CDK) and cyclins, such as CDK2/cyclin A and CDK2/cyclin E, and inhibiting their kinase activity are regarded as promising antitumor agents to complement the existing therapies. From a high-throughput screening effort, we identified a new class of CDK2/cyclin A/E inhibitors. The hit-to-lead expansion of this class is described. X-ray crystallographic data of early compounds in this series, as well as in vitro testing funneled for rapidly achieving in vivo efficacy, led to a nanomolar inhibitor of CDK2/cyclin A (N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(2-naphthyl)acetamide (41), PNU-292137, IC50 = 37 nM) with in vivo antitumor activity (TGI > 50%) in a mouse xenograft model at a dose devoid of toxic effects.

cdk2⅐cyclin E and cdk5⅐p25 are two members of the cyclin-dependent kinase family that are potential therapeutic targets for oncology and Alzheimer's disease, respectively. In this study we have investigated the mechanism for these enzymes. Kinases catalyze the transfer of phosphate from ATP to a protein acceptor, thus utilizing two substrates, ATP and the target protein. For a two-substrate reaction, possible kinetic mechanisms include: ping-pong, sequential random, or sequential ordered. To determine the kinetic mechanism of cdk2⅐GST-cyclin E and cdk5⅐GST-p25, kinase activity was measured in experiments in which concentrations of peptide and ATP substrates were varied in the presence of dead-end inhibitors. A peptide identical to the peptide substrate, but with a substitution of valine for the phosphoacceptor threonine, competed with substrate with a K i value of 0.6 mM. An aminopyrimidine, PNU 112455A, was identified in a screen for inhibitors of cdk2. Nonlinear least squares and Lineweaver-Burk analyses demonstrated that the inhibitor PNU 112455A was competitive with ATP with a K i value of 2 M. In addition, a co-crystal of PNU 112455A with cdk2 showed that the inhibitor binds in the ATP binding pocket of the enzyme. Analysis of the inhibitor data demonstrated that both kinases use a sequential random mechanism, in which either ATP or peptide may bind first to the enzyme active site. For both kinases, the binding of the second substrate was shown to be anticooperative, in that the binding of the first substrate decreases the affinity of the second substrate. For cdk2⅐GST-cyclin E the kinetic parameters were determined to be K m, ATP ‫؍‬ 3.6 ؎ 1.0 M, K m, peptide ‫؍‬ 4.6 ؎ 1.4 M, and the anticooperativity factor, ␣ ‫؍‬ 130 ؎ 44. For cdk5⅐GST-p25, the K m, ATP ‫؍‬ 3.2 ؎ 0.7 M, K m, peptide ‫؍‬ 1.6 ؎ 0.3 M, and ␣ ‫؍‬ 7.2 ؎ 1.8.

Vasopressin-activated Ca2+-mobilizing (VACM)-1 gene product is a 780-amino acid membrane protein that shares sequence homology with cullins, a family of genes involved in the regulation of cell cycle. However, when expressed in vitro, VACM-1 attenuates basal and vasopressin- and forskolin-induced cAMP production. Mutating the PKA-dependent phosphorylation site in the VACM-1 sequence (S730AVACM-1) prevents this inhibitory effect. To further examine the biological role of VACM-1, we studied the effect of VACM-1 and S730AVACM-1 proteins on cellular proliferation and gene expression in Chinese hamster ovary and COS-1 cells. Cellular proliferation of VACM-1-expressing cell lines was significantly lower compared with that of the vector-transfected cells, whereas it was significantly increased in S730AVACM-1-derived cell lines. Furthermore, expression of VACM-1 but not S730AVACM-1 protein retarded cytokinesis and prevented MAPK phosphorylation. Screening with the Human PathwayFinder-1 GEArray system and subsequent Western blot analysis demonstrated that VACM-1 induces p53 mRNA and protein expression. In summary, VACM-1 inhibits cellular growth by a mechanism that involves cAMP, MAPK phosphorylation, and p53 expression.

The vasopressin-activated calcium-mobilizing (VACM-1) protein is a unique arginine vasopressin (AVP) receptor which shares sequence homology with the cullins, genes involved in the regulation of cell cycle transitions. Unlike either cullins or AVP receptors, however, VACM-1 is expressed exclusively in the vascular endothelial cells and in the renal collecting tubule cells. In order to test the hypothesis that the expression of VACM-1 might be correlated with the cell cycle, and to establish an endothelial cell model for the VACM-1 receptor, we examined VACM-1 expression in rat adrenal medulla endothelial cells (RAMEC). Northern and Western blot analyses of mRNA and protein from RAMEC identified presence of 6.4 kb mRNA and a Mr 81 kDa protein, respectively. Immunostaining of RAMEC with anti-VACM-1 antibodies and Western blot analyses indicated that in RAMEC, VACM-1 protein expression is dependent on the cell cycle. VACM-1 protein virtually disappears during the S phase and localizes to the cytosol during cell division and to the cell membrane at the completion of cytokinesis. Furthermore, pretreatment of RAMEC with anti-VACM-1 specific antibodies increased basal levels of Ca2+and attenuated the AVP-dependent increase in cytosolic Ca2+. In summary, these results indicate that VACM-1 protein expression in RAMEC membrane is linked to the cell cycle, and consequently, VACM-1 may be involved in the regulation of cell division.

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