Multiplexed, multimodal profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq.

Multiplexed assays of variant effect are powerful tools for assessing the impact of protein sequence variation, but are limited to measuring a single protein property and often rely on indirect readouts of intracellular protein function. Here, we developed LAbeling with Barcodes and Enrichment for biochemicaL analysis by sequencing (LABEL-seq), a platform for the multimodal profiling of thousands of protein variants in cultured human cells. Multimodal measurement of ~20,000 variant effects for ~1,600 BRaf variants using LABEL-seq revealed that variation at positions that are frequently mutated in cancer had minimal effects on folding and intracellular abundance but could dramatically alter activity, protein-protein interactions, and druggability. Integrative analysis of our multimodal measurements identified networks of positions with similar roles in regulating BRaf's signaling properties and enabled predictive modeling of variant effects on complex processes such as cell proliferation and small molecule-promoted degradation. LABEL-seq provides a scalable approach for the direct measurement of multiple biochemical effects of protein variants in their native cellular context, yielding insight into protein function, disease mechanisms, and druggability.

Profiling of drug resistance in Src kinase at scale uncovers a regulatory network coupling autoinhibition and catalytic domain dynamics.

Kinase inhibitors are effective cancer therapies, but resistance often limits clinical efficacy. Despite the cataloging of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively profiled the resistance of ∼3,500 Src tyrosine kinase mutants to four different ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Src's catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Src's phosphotransferase activity were prone to the development of resistance. Unexpectedly, we found that a resistance-prone cluster of residues located on the top face of the N-terminal lobe of Src's catalytic domain contributes to autoinhibition by reducing catalytic domain dynamics, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how drug resistance profiling can be used to define potential resistance pathways and uncover new mechanisms of kinase regulation.