MAPK Pathway Inhibition Reshapes Kinase Chemical Probe Reactivity Reflecting Cellular Activation States.

Despite the pivotal role of oncogenic kinases in cancer initiation, progression, and therapeutic resistance, functionally profiling their activity and conformational dynamics in live cells remains challenging. Existing methods often fail to capture inhibitor-bound structural states of kinases, particularly in clinically relevant contexts, such as treatment response and acquired resistance, where genomic data alone are insufficient. Here, we use activity-based protein profiling (ABPP) to monitor composite amino acid reactivity changes, across cysteine, lysine, and carboxylic acid residues, as a hypothesis-generating readout of kinase state in live cells. Using electrophilic probes, we show that treatment of BRAFV600E mutant melanoma cells with vemurafenib or trametinib decreases overall cysteine and lysine reactivity in BRAFV600E and MEK1/2, likely reflecting composite changes in amino acid accessibility across multiple reactive residues associated with inhibitor binding. Changing the order of probe addition and inhibitor treatment altered the labeling outcomes, consistent with competitive engagement and structural stabilization. Comparative analysis of ATP-competitive BRAFV600E inhibitors vemurafenib and dabrafenib indicated differences in aspartate and glutamate labeling patterns, consistent with the possibility that ABPP may detect inhibitor-associated variations in residue accessibility, which could reflect differences in inhibitor-bound conformations. In inhibitor-resistant melanoma models, ABPP detected differences in residue reactivity relative to parental cells, which aligned with known resistance-associated features, such as BRAF overexpression and the MEK2 Q60P activation mutation. Moreover, global proteome analyses of cysteine and lysine reactivity upon BRAFV600E inhibition revealed probe-accessible cysteine labeling changes on KSR2, suggesting a potential MAPK pathway remodeling. Together, these findings highlight ABPP as a valuable chemical biology approach for investigating inhibitor-dependent changes in kinase residue reactivity, offering a framework to investigate how kinase conformational dynamics and signaling pathway adaptation influence the therapeutic response and resistance in cancer.