SMaSh: A Streptavidin Mass Shift Assay for Rapidly Quantifying Target Occupancy by Irreversible Inhibitors

The streptavidin mass shift (SMaSh) assay is a robust and fast approach for quantifying target protein occupancy by a covalent inhibitor or ligand. It exploits the biotin-streptavidin bond using the Simple Western platform. One measurement on a single sample determines both total and occupied target protein simultaneously and is, therefore, self-normalizing. The approach works in diverse and complex biological matrices and, with no need for matched vehicle-treated controls, readily applies to tissues from animal pharmacology models. Assessing occupancy is critical in the development of targeted covalent drugs. We demonstrate its use by characterizing and validating a variety of chemical probes for Bruton's tyrosine kinase (BTK, UniprotKB Q10607) and mitogen-activated protein kinase (ERK1/2/MAPK1/2, UniprotKB P28482 and P27361) and determining target engagement of covalent inhibitors for both targets and off-target engagement for ERK. We demonstrated that it works in cell lysates, tissues, and human peripheral blood mononuclear cells. The SMaSh assay is superior to traditional methods and broadly useful as a tool in assessing covalent biological probes or targeted covalent inhibitors.