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.