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.

尽管致癌激酶（oncogenic kinases）在癌症发生、进展及治疗耐药中发挥关键作用，但在活细胞中对其活性与构象动态开展功能谱分析仍颇具挑战。现有方法往往难以捕获激酶结合抑制剂的结构状态，尤其是在临床相关场景中，比如治疗响应与获得性耐药，此时仅依靠基因组数据往往不足以提供充分信息。本研究采用基于活性的蛋白质谱分析（activity-based protein profiling, ABPP），以半胱氨酸、赖氨酸与羧酸残基的综合氨基酸反应性变化作为假设生成性读数，用以表征活细胞内的激酶状态。通过亲电探针，我们发现向BRAFV600E突变黑色素瘤细胞施加维莫非尼（vemurafenib）或曲美替尼（trametinib），可降低BRAFV600E与MEK1/2的整体半胱氨酸及赖氨酸反应性，这大概率反映了与抑制剂结合相关的多个反应性残基的氨基酸可及性综合变化。调整探针添加与抑制剂处理的先后顺序会改变标记结果，这与竞争性结合与结构稳定的现象相符。对ATP竞争性BRAFV600E抑制剂维莫非尼与达拉非尼（dabrafenib）的比较分析显示，二者在天冬氨酸与谷氨酸标记模式上存在差异，这符合ABPP可检测到抑制剂相关的残基可及性变化的可能性，而这类变化或可反映抑制剂结合构象的差异。在抑制剂耐药的黑色素瘤模型中，ABPP检测到与亲本细胞相比的残基反应性差异，这与已知的耐药相关特征（如BRAF过表达与MEK2 Q60P激活突变）相符。此外，对BRAFV600E抑制后半胱氨酸与赖氨酸反应性的全局蛋白质组分析显示，KSR2上存在探针可及的半胱氨酸标记变化，这提示MAPK通路存在潜在重塑。综上，这些研究结果表明ABPP是一种极具价值的化学生物学方法，可用于探究激酶残基反应性的抑制剂依赖性变化，为研究激酶构象动态与信号通路适应如何影响癌症治疗响应与耐药提供了研究框架。