The p16 status of tumor cell lines identifies small molecule inhibitors specific for cyclin-dependent kinase 4

Loss of p16 functional activity leading to disruption of the p16/cyclin-dep endent kinase (CDK) 4:cyclin D/retinoblastoma pathway is the most common ev ent in human tumorigenesis, suggesting that compounds with CDK4 kinase inhi bitory activity may be useful to regulate cancer cell growth. To identify s uch inhibitors, the 60 cancer cell lines of the National Cancer Institute d rug screen panel were examined for p16 alterations (biallelic deletion, int ragenic mutations, or absent p16 protein), and the growth-inhibitory activi ty of more than 50,000 compounds against these 60 cell lines was compared w ith their p16 status. One compound, 3-amino thioacridone (3-ATA; NSC 680434 ), whose growth-inhibitory activity correlated with the p16 status of the c ell lines had an IC50 of 3.1 mu M in a CDK4 kinase assay. In addition, four compounds structurally related to 3-ATA inhibited CDK4 kinase with IC(50)s ranging from 0.2-2.0 mu M. All five of these compounds were less potent in hibitors of cell division cycle 2 and CDK2 kinases, with IC(50)s 30- to 500 -fold higher than that for CDK4. ATP competition experiments demonstrated a noncompetitive mode of inhibition for 3-ATA (K-i = 5.5 mu M) and a linear mixed mode for benzothiadiazine (NSC 645787; K-i = 0.73 mu M). We have succ essfully demonstrated a novel approach to identify specific CDK4 kinase inh ibitors that may selectively induce growth inhibition of p16-altered tumors .