The cyclin-dependent protein kinases are key regulators of cell cycle progression. Aberrant expression or altered activity of distinct cyclin-dependent kinase (CDK) complexes results in escape of cells from cell cycle control, leading to unrestricted cell proliferation. CDK inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells, and identifying small-molecule CDK inhibitors has been a major focus in cancer research. Several CDK inhibitors are entering the clinic, the most recent being selective CDK2 and CDK4 inhibitors. We have identified a diaminopyrimidine compound, R547, which is a potent and selective ATP-competitive CDK inhibitor. In cell-free assays, R547 effectively inhibited CDK1/cyclin B, CDK2/cyclin E, and CDK4/cyclin D1 (K i = 1 -3 nmol/L) and was inactive (K i > 5,000 nmol/L) against a panel of >120 unrelated kinases. In vitro, R547 effectively inhibited the proliferation of tumor cell lines independent of multidrug resistant status, histologic type, retinoblastoma protein, or p53 status, with IC 50 s V 0.60 Mmol/L. The growth-inhibitory activity is characterized by a cell cycle block at G 1 and G 2 phases and induction of apoptosis. R547 reduced phosphorylation of the cellular retinoblastoma protein at specific CDK phosphorylation sites at the same concentrations that induced cell cycle arrest, suggesting a potential pharmacodynamic marker for clinical use. In vivo, R547 showed antitumor activity in all of the models tested to date, including six human tumor xenografts and an orthotopic syngeneic rat model. R547 was efficacious with daily oral dosing as well as with once weekly i.v. dosing in established human tumor models and at the targeted efficacious exposures inhibited phosphorylation of the retinoblastoma protein in the tumors. The selective kinase inhibition profile and the preclinical antitumor activity of R547 suggest that it may be promising for development for use in the treatment of solid tumors. R547 is currently being evaluated in phase I clinical trials.

The cyclin-dependent kinases (CDKs) and their cyclin partners are key regulators of the cell cycle. Since deregulation of CDKs is found with high frequency in many human cancer cells, pharmacological inhibition of CDKs with small molecules has the potential to provide an effective strategy for the treatment of cancer. The 2,4-diamino-5-ketopyrimidines 6 reported here represent a novel class of potent and ATP-competitive inhibitors that selectively target the cyclin-dependent kinase family. This diaminopyrimidine core with a substituted 4-piperidine moiety on the C2-amino position and 2-methoxybenzoyl at the C5 position has been identified as the critical structure responsible for the CDK inhibitory activity. Further optimization has led to a good number of analogues that show potent inhibitory activities against CDK1, CDK2, and CDK4 but are inactive against a large panel of serine/threonine and tyrosine kinases (K(i) > 10 microM). As one of these representative analogues, compound 39 (R547) has the best CDK inhibitory activities (K(i) = 0.001, 0.003, and 0.001 microM for CDK1, CDK2, and CDK4, respectively) and excellent in vitro cellular potency, inhibiting the growth of various human tumor cell lines including an HCT116 cell line (IC(50) = 0.08 microM). An X-ray crystal structure of 39 bound to CDK2 has been determined in this study, revealing a binding mode that is consistent with our SAR. Compound 39 demonstrates significant in vivo efficacy in the HCT116 human colorectal tumor xenograft model in nude mice with up to 95% tumor growth inhibition. On the basis of its superior overall profile, 39 was chosen for further evaluation and has progressed into Phase I clinical trial for the treatment of cancer.

Cyclin-dependent kinase 4 (CDK4)/cyclin D has a key role in regulating progression through late G 1 into S phase of the cell cycle. CDK4-cyclin D complexes then persist through the latter phases of the cell cycle, although little is known about their potential roles. We have developed small molecule inhibitors that are highly selective for CDK4 and have used these to define a role for CDK4-cyclin D in G 2 phase. The addition of the CDK4 inhibitor or small interfering RNA knockdown of cyclin D3, the cyclin D partner, delayed progression through G 2 phase and mitosis. The G 2 phase delay was independent of ATM/ATR and p38 MAPK but associated with elevated Wee1. The mitotic delay was because of failure of chromosomes to migrate to the metaphase plate. However, cells eventually exited mitosis, with a resultant increase in cells with multiple or micronuclei. Inhibiting CDK4 delayed the expression of the chromosomal passenger proteins survivin and borealin, although this was unlikely to account for the mitotic phenotype. These data provide evidence for a novel function for CDK4-cyclin D3 activity in S and G 2 phase that is critical for G 2 /M progression and the fidelity of mitosis. Cyclin-dependent kinase 4 (CDK4)3 binds cyclin D to provide a mechanistic link between extracellular growth signals and the initiation of entry into S phase from G 0 /G 1 . Its role in this pathway is to phosphorylate and inactive members of the retinoblastoma protein (Rb) family, which includes Rb, p107, and p130 (1, 2). This phosphorylation allows the release and activation of E2F transcription factors, which in turn up-regulates the genes required for S phase. CDK4 is regulated by cyclin D binding, phosphorylation, and association with inhibitory protein subunits of the INK4 family. Unlike other CDK-cyclin complexes, it is present at constant levels throughout the cell cycle in continuously proliferating cells, although its activity varies.Mutations in the CDK4-cyclin D/Rb pathway are commonly found in many types of cancer. A number of viral oncogenes directly target the inactivation of Rb, and overexpression of cyclin D is common in breast cancer. This is particularly relevant in melanoma, where germ line mutations of the cyclin-dependent kinase inhibitor p16 INK4A or CDK4 mutations that disrupt p16 binding are carried in melanoma-prone kindred (for review, see Ref.3). In addition, somatic p16 INK4A loss or mutation occurs in up to 30% of melanomas (3, 4), and a small proportion of melanomas carry somatic CDK4 mutations that disrupt p16INK4A binding (4, 5). This points to the importance of the cyclin D/CDK4/Rb pathway in controlling G 0 /G 1 cell cycle progression. However, the role of CDK4-cyclin D in the phosphorylation and inactivation of Rb can in some cases be effectively substituted by cyclin E-CDK2 (6), and knockout mouse models have shown redundancy of CDK4 and CDK6, whereas knock-out of both had only minimal effect on the cell cycle of cells (7,8). These data indicate that CDK4 is not essential for cell cycle progression, so ...

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