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 overexpression of its activating partner cyclin D1, loss of the natural Cdk4-specific inhibitor p16, or mutation(s) in Cdk4's catalytic subunit. All of these can cause deregulated cell growth, resulting in tumor formation. We sought 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 compounds that could inhibit Cdk4 enzyme activity in vitro. Kinase assays were performed 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 cytometry, 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 tests were two-sided.


RESULTS
CINK4 specifically inhibited Cdk4/cyclin D1 in vitro. It caused growth arrest in tumor cells and in normal cells and prevented pRb phosphorylation. CINK4 treatment resulted in statistically significantly (P: =.031) smaller mean tumor volumes in a mouse xenograft model.


CONCLUSIONS
Like p16, the natural inhibitor of Cdk4, CINK4 inhibits Cdk4 activity in vitro and slows tumor growth in vivo. The specificity of CINK4 for Cdk4 raises the possibility that this small molecule or one with a similar structure could have therapeutic value.
Oncology Research, Novartis Pharma AG, Basel, Switzerland.
R
R
Soni
T
T
O'Reilly
P
P
Furet
L
L
Muller
C
C
Stephan
S
S
Zumstein-Mecker
H
H
Fretz
D
D
Fabbro
B
B
Chaudhuri
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000201:enzymology; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016161:Genes, Retinoblastoma / Q000187:drug effects*; D006801:Humans; D051379:Mice; D012516:Osteosarcoma / Q000188:drug therapy* / Q000201:enzymology; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D011743:Pyrimidines / Q000494:pharmacology*; D016196:S Phase / Q000187:drug effects; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; C423962:CINK4; D004791:Enzyme Inhibitors; D011743:Pyrimidines; C012070:2,4,6-triaminopyrimidine; D018844:Cyclin-Dependent Kinases