Selective in vivo and in vitro effects of a small molecule inhibitor of cyclin-dependent kinase 4

Background: Cyclin-dependent kinase 4 (Cdk4) represents a prime target for the treatment of cancer because most human cancers are characterized by ove rexpression of its activating partner cyclin D1, loss of the natural Cdk4-s pecific inhibitor p16, or mutation(s) in Cdk4's catalytic subunit, All of t hese can cause deregulated cell growth, resulting in tumor formation. We so ught to identify a small molecule that could inhibit the kinase activity of Cdk4 in vitro and to then ascertain the effects of that inhibitor on cell growth and tumor volume in vivo. Methods: A triaminopyrimidine derivative, CINK4 (a chemical inhibitor of Cdk4), was identified by screening for compo unds that could inhibit Cdk4 enzyme activity in vitro. Kinase assays were p erformed on diverse human Cdks and on other kinases that were expressed in and purified from insect cells to determine the specificity of CINK4. Cell cycle effects of CINK4 on tumor and normal cells were studied by flow cytom etry, and changes in phosphorylation of the retinoblastoma protein (pRb), a substrate of Cdk4, were determined by western blotting. The effect of the inhibitor on tumor growth in vivo was studied by use of tumors established through xenografts of HCT116 colon carcinoma cells in mice. Statistical tes ts were two-sided. Results: CINK4 specifically inhibited Cdk4/ cyclin D1 in vitro. It caused growth arrest in tumor cells and in normal cells and prev ented pRb phosphorylation. CINK4 treatment resulted in statistically signif icantly (P = .031) smaller mean tumor volumes in a mouse xenograft model. C onclusions: Like p16, the natural inhibitor of Cdk4, CINK4 inhibits Cdk4 ac tivity in vitro and slows tumor growth in vivo. The specificity of CINK4 fo r Cdk4 raises the possibility that this small molecule or one with a simila r structure could have therapeutic value.