Ibrutinib binds to the ATP-binding pocket of HCK and blocks ATP binding. (A) Docking model of the ibrutinib (purple stick) aligned with the co-crystal structure of HCK (green ribbon)-A419259 (yellow stick). The hydrogen bonds (depicted as red dashed lines) between the aminopyrimidine moiety of ibrutinib and the hinge region of HCK are indicated. Docking studies showed that ibrutinib bound to the ATP-binding pocket of HCK with calculated affinity energy (ΔG) of −10.5 kcal/mol. (B) Results from pull-down experiments using SVB, IB-BTN, and CC-292 (CC-BTN) to detect BTK and HCK binding in MYD88-mutated BCWM.1, MWCL-1, and TMD-8 cells. (C) Results from kinase active-site inhibition assays utilizing an ATP-BTN probe that was used to pull down active kinases in the presence of ibrutinib, CC-292, or A419259 in lysates from BCWM.1 WM cells. ATP-BTN, ATP-biotin; IB-BTN, biotinylated ibrutinib; SVB, streptavidin beads.