Figure 2.

Inhibitor Binding to CDK1 and CDK2

(A and B) Isothermal titration calorimetry (ITC) thermograms to assess AZD5438 binding to CDK1-cyclin B and CDK2-cyclin A (A) and cyclin-free CDK1 and CDK2 (B). For each sample, CDK1 and CDK2 were phosphorylated (on T161 or T160, respectively). AZD5438 shows reduced binding to cyclin-free CDK1 compared with CDK1-cyclin (B).

(C) Surface plasmon resonance (SPR) studies to determine the binding of AZD5438 to CDK1 and CDK2. Unphosphorylated CDK1 and CDK2 as GST fusions were immobilized on the SPR chip via anti-GST antibody coupling. Accompanying sets of ITC thermograms and SPR traces that evaluate Dinaciclib, SU9516, Alvocidib, and CGP74514A binding are presented in Figure S2.

(D) Bar chart to compare the fold difference in binding affinity between cyclin-free CDK1 and CDK2 and their cyclin-associated forms. CDK1:CDK1-cyclin B and CDK2:CDK2-cyclin A ratios are shown in black and gray, respectively. ITC experiments conducted in the presence of Cks2 are presented in Figure S3.

(E) ITC-derived energetic experimental data (ΔH, -TΔS, and ΔG) for the binding of AZD5438 to CDK1 and CDK2 and their respective cognate cyclins.