Small-Molecule Inhibitors Allosterically Control Bruton’s Tyrosine Kinase Assembly on the Membrane

Abstract

Bruton’s tyrosine kinase (Btk) is targeted by small-molecule active-site inhibitors used to treat lymphomas and leukemias. Beyond its catalytic activity, Btk participates in signaling assemblies through scaffolding functions that can contribute to drug resistance. Here, we show that Btk forms micron-scale assemblies on membranes and that this behavior is allosterically regulated by ligand binding at the active site. Using supported lipid bilayers and total internal reflection fluorescence microscopy, we find that nucleotides and inhibitors including ibrutinib, acalabrutinib, and dasatinib promote assembly, whereas fenebrutinib and pirtobrutinib do not. Membrane assembly is independent of catalysis but correlates with ligand-dependent changes in Btk conformational flexibility measured by hydrogen-deuterium exchange. These results reveal a previously unrecognized connection between active-site ligand binding and mesoscale membrane organization, suggesting a mechanism by which kinase inhibitors may differentially affect Btk scaffolding functions implicated in drug resistance.