Profiling of the drug resistance of thousands of Src tyrosine kinase mutants uncovers a regulatory network that couples autoinhibition to the dynamics of the catalytic domain

Protein kinase inhibitors are effective cancer therapies, but acquired resistance often limits clinical efficacy. Despite the cataloguing of numerous resistance mutations with model studies and in the clinic, we still lack a comprehensive understanding of kinase inhibitor resistance. Here, we measured the resistance of thousands of Src tyrosine kinase mutants to a panel of 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, a resistance-prone cluster of residues that are on the top face of the N-terminal lobe of the catalytic domain contributes to Src autoinhibition by reducing the dynamics of the catalytic domain, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how comprehensive profiling of drug resistance can be used to understand potential resistance pathways and uncover new mechanisms of kinase regulation.蛋白激酶抑制剂（Protein kinase inhibitors）是有效的癌症治疗手段，但获得性耐药往往会限制其临床疗效。尽管已有模型研究与临床实践对大量耐药突变完成编目，但我们对激酶抑制剂耐药性仍缺乏全面的认知。本研究检测了数千个Src酪氨酸激酶突变体（Src tyrosine kinase mutants）对一组ATP竞争性抑制剂（ATP-competitive inhibitors）的耐药性。我们发现，ATP竞争性抑制剂耐药突变遍布Src的催化结构域（catalytic domain）。除与抑制剂直接接触的残基外，参与调控Src磷酸转移酶活性的残基也易产生耐药性。出乎意料的是，位于催化结构域N端叶（N-terminal lobe）顶面的一组易耐药残基簇，可通过降低催化结构域的动态性介导Src的自抑制；该区域的突变则通过降低抑制剂亲和力、促进激酶过度激活而引发耐药。综上，本研究阐明了全面的耐药谱分析可用于解析潜在耐药通路，并揭示激酶调控的全新机制。