Loss of p16 functional activity leading to disruption of the p16/cyclin-dependent kinase (CDK) 4:cyclin D/retinoblastoma pathway is the most common event in human tumorigenesis, suggesting that compounds with CDK4 kinase inhibitory activity may be useful to regulate cancer cell growth. To identify such inhibitors, the 60 cancer cell lines of the National Cancer Institute drug screen panel were examined for p16 alterations (biallelic deletion, intragenic mutations, or absent p16 protein), and the growth-inhibitory activity of more than 50,000 compounds against these 60 cell lines was compared with their p16 status. One compound, 3-amino thioacridone (3-ATA; NSC 680434), whose growth-inhibitory activity correlated with the p16 status of the cell lines had an IC50 of 3.1 microM in a CDK4 kinase assay. In addition, four compounds structurally related to 3-ATA inhibited CDK4 kinase with IC50s ranging from 0.2-2.0 microM. All five of these compounds were less potent inhibitors of cell division cycle 2 and CDK2 kinases, with IC50s 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 microM) and a linear mixed mode for benzothiadiazine (NSC 645787; K(i) = 0.73 microM). We have successfully demonstrated a novel approach to identify specific CDK4 kinase inhibitors that may selectively induce growth inhibition of p16-altered tumors.