Specificity of 005A, a small molecule inhibitor of p18. (A) Predicted 005A and p18 protein binding interactions at the amino acid level. (B) The structure of 005A. (C) Binding energy changes for p18 (i) and CDK6 (ii) residues in the presence and absence of ligand 005A. (D) Surface plasma resonance (SPR) results of the binding activity of p18 (i), p18^Mut (ii), and p18^Del (iii) at various concentrations of compound 005A. (E) Co-immunoprecipitation (Co-IP) assay using Flag taged CDK6 and Myc taged p18 and its mutants. HEK293T cells were treated with 005A for 24 hours after 48-hour transient transfection. (F) In vitro CDK6 kinase activity assays were performed in the indicated conditions, Rb phosphating levels indicate the CDK6 kinase activity in these assays (n = 3). All data represent the means ± SD. Compared with control unless specified: ***P < .001 by 2-tailed unpaired t test.