Targeting Drug Resistance in EGFR with Covalent Inhibitors: A Structure-Based Design Approach

Receptor tyrosine kinases represent one of the prime targets in cancer therapy, as the dysregulation of these elementary transducers of extracellular signals, like the epidermal growth factor receptor (EGFR), contributes to the onset of cancer, such as non-small cell lung cancer (NSCLC). Strong efforts were directed to the development of irreversible inhibitors and led to compound CO-1686, which takes advantage of increased residence time at EGFR by alkylating Cys797 and thereby preventing toxic effects. Here, we present a structure-based approach, rationalized by subsequent computational analysis of conformational ligand ensembles in solution, to design novel and irreversible EGFR inhibitors based on a screening hit that was identified in a phenotype screen of 80 NSCLC cell lines against approximately 1500 compounds. Using protein X-ray crystallography, we deciphered the binding mode in engineered cSrc (T338M/S345C), a validated model system for EGFR-T790M, which constituted the basis for further rational design approaches. Chemical synthesis led to further compound collections that revealed increased biochemical potency and, in part, selectivity toward mutated (L858R and L858R/T790M) vs nonmutated EGFR. Further cell-based and kinetic studies were performed to substantiate our initial findings. Utilizing proteolytic digestion and nano-LC-MS/MS analysis, we confirmed the alkylation of Cys797.受体酪氨酸激酶（Receptor tyrosine kinases）是癌症治疗的核心靶点之一，这类细胞外信号的初级转导分子失调会诱发癌症，例如表皮生长因子受体（epidermal growth factor receptor, EGFR）失调可引发非小细胞肺癌（non-small cell lung cancer, NSCLC）。学界大力开展不可逆抑制剂的研发工作，最终得到了化合物CO-1686；该化合物通过烷基化Cys797位点延长其在EGFR上的驻留时间，从而规避毒副作用。本研究提出一种基于结构的设计策略，通过后续对溶液中构象性配体集合的计算分析进行优化，基于从80株非小细胞肺癌细胞系针对约1500种化合物的表型筛选中获得的命中化合物，设计新型不可逆EGFR抑制剂。我们利用蛋白质X射线晶体学解析了工程化改造的cSrc（T338M/S345C）的结合模式，该体系是EGFR-T790M的验证模型，为后续的合理设计策略奠定了基础。通过化学合成获得了更多化合物集合，这些化合物展现出更强的生化活性，且部分化合物对突变型（L858R及L858R/T790M）EGFR相较于野生型EGFR具有选择性。我们进一步开展了基于细胞的实验与动力学研究，以验证初始研究结果。我们采用蛋白水解酶切结合纳米液相色谱-串联质谱（nano-LC-MS/MS）分析，证实了Cys797位点的烷基化修饰。