Multi-tiered chemical proteomic maps of tryptoline acrylamide–protein interactions in cancer cells

Covalent chemistry is a versatile approach for expanding the ligandability of the human proteome. Activity-based protein profiling (ABPP) can infer the specific residues modified by electrophilic compounds through competition with broadly reactive probes. However, the extent to which such residue-directed platforms fully assess the protein targets of electrophilic compounds in cells remains unclear. Here we evaluate a complementary protein-directed ABPP method that identifies proteins showing stereoselective reactivity with alkynylated, chiral electrophilic compounds—termed stereoprobes. Integration of protein- and cysteine-directed data from cancer cells treated with tryptoline acrylamide stereoprobes revealed generally well-correlated ligandability maps and highlighted features, such as protein size and the proteotypicity of cysteine-containing peptides, that explain gaps in each ABPP platform. In total, we identified stereoprobe binding events for >300 structurally and functionally diverse proteins, including compounds that stereoselectively and site-specifically disrupt MAD2L1BP interactions with the spindle assembly checkpoint complex leading to delayed mitotic exit in cancer cells. The ligandability of the human proteome can be expanded using covalent chemistry. A multi-tiered chemical proteomic strategy now provides in-depth maps of tryptoline acrylamide–protein interactions in cancer cells. This platform afforded the discovery of stereoselective covalent ligands for hundreds of human proteins, including compounds that disrupt protein–protein interactions regulating the cell cycle.