Nucleophilic amino acids make important contributions to protein function, including performing key roles in catalysis and serving as sites for post-translational modification. Electrophilic groups that target amino-acid nucleophiles have been used to create covalent ligands and drugs, but have, so far, been mainly limited to cysteine and serine. Here we report a chemical proteomic platform for the global and quantitative analysis of lysine residues in native biological systems. We quantified, in total, more than 9000 lysines in human cell proteomes and identified several hundred residues with heightened reactivity that are enriched at protein functional sites and can frequently be targeted by electrophilic small molecules. We discovered lysine-reactive fragment electrophiles that inhibit enzymes by active site and allosteric mechanisms, as well as disrupt protein-protein interactions in transcriptional regulatory complexes, emphasizing the broad potential and diverse functional consequences of liganding lysine residues throughout the human proteome.

Highlights d Chemical proteomics identifies cysteine reactivity changes in activated T cells d Chemical proteomics maps ligandable cysteines in diverse immune-relevant proteins d Cysteine-directed electrophilic compounds suppress T cells by distinct mechanisms d Electrophile-cysteine interactions promote the degradation of immune proteins

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Summary Patients with non-small cell lung cancer (NSCLC) that have kinase-activating epidermal growth factor receptor (EGFR) mutations are highly responsive to first- and second-generation EGFR inhibitors. However, these patients often relapse due to a secondary, drug-resistant mutation in EGFR where the gatekeeper threonine is converted to methionine (T790M). Several third-generation EGFR inhibitors have been developed that irreversibly inactivate T790M-EGFR while sparing wild-type EGFR, thus reducing epithelium-based toxicities. Using chemical proteomics, we show here that individual T790M-EGFR inhibitors exhibit strikingly distinct off-target profiles in human cells. The FDA-approved drug osimertinib (AZD9291), in particular, was found to covalently modify cathepsins in cell and animal models, which correlated with lysosomal accumulation of the drug. Our findings thus show how chemical proteomics can be used to differentiate covalent kinase inhibitors based on global selectivity profiles in living systems and identify specific off-targets of these inhibitors that may impact drug activity and safety.

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