Figure 6. The gatekeeper residue in Wee1 contributes to inhibitor specificity. (A) Ribbon diagrams of published structures of Chk1 (PDB code 2HY0) and Wee1 (PDB code 3BI6) bound with the inhibitors bosutinib or Bos-I which are superimposed (main). Insets show enlarged view of the pocket bound to their inhibitors (gray backbone). The gatekeeper residues are shown in pink (black arrow) for both kinases. (B) In vitro kinase assays were used to assess the ability of bosutinib and Bos-I (0.5–10 µM) to inhibit recombinant Wee1 WT and Wee1 gatekeeper mutants. Phosphorylation of purified cdc2 (p-Cdc2 Y15) was used as a read-out for Wee1 activity. Kinase reactions were performed at least twice. A representative western blot is shown. (C) Ribbon diagram of the Wee1 (PDB code 3BI6) gatekeeper mutant N376F (shown in pink, highlighted with arrow). Insets show enlarged view of bosutinib and Bos-I and the positioning of the gatekeeper residue.