Pirtobrutinib structure, binding mode, and effects on BTK stability and activity. (A) Chemical structure of pirtobrutinib. (B) Superposition of the crystal structures of BTK and BTK C481S in complex with pirtobrutinib (magenta and light gray cartoon/stick representation, respectively; selected water molecules are shown as red spheres, and hydrogen bond interactions are illustrated as dashed lines). (C) BTK in complex with pirtobrutinib (magenta cartoon/stick representation) overlayed with BTK covalently bound to ibrutinib (cyan cartoon/stick representation; PDB ID 5P9J). (D) Dose-response curves showing inhibition of BTK and BTK C481S kinase activity by pirtobrutinib (n = 10) (mean and SD are graphed). (E) Tm graphs showing pirtobrutinib- and cBTKi-induced increases in full-length and SH3-SH2-KD BTK Tm. Light and dark blue bars represent the first and second pirtobrutinib-induced melting events, respectively; ∗P < .05 (compared with Apo Tm); #P < .05 (compared with each cBTKi’s Tm). (F) Dose-response curves showing inhibition of cellular Y223 BTK phosphorylation in HEK293 cells stably expressing BTK by pirtobrutinib, ibrutinib, zanubrutinib, and acalabrutinib (n = 3) (mean and SD are graphed). FL, full-length; SD, standard deviation.