激酶抑制剂是有效的癌症治疗方法，但抗药性常常限制了临床疗效。尽管已经鉴定了许多抗药性突变，但对激酶抑制剂抗药性的理解仍然不完善。在这里，我们全面地分析了约3,500个Src酪氨酸激酶突变体对四种不同的竞争ATP抑制剂的抗药性。我们发现，竞争ATP抑制剂抗药性突变分布在Src的催化结构域内。除了抑制剂接触位点以外，参与调节Src磷酸转移酶活性的残基也容易发展抗药性。出乎意料的是，我们发现位于Src催化结构域N-末端瓣的顶面的一个易发展抗药性的残基簇通过减少催化结构域动力学来促进自抑制，并且该簇中的突变通过降低抑制剂亲和力和促进激酶过度活化来产生抗药性。综上所述，我们的研究证明了如何利用药物抗药性分析来界定潜在的抗药性途径，并揭示了激酶调控的新机制。版权所有 © 2023 Elsevier Ltd. 保留所有权利。

Kinase inhibitors are effective cancer therapies, but resistance often limits clinical efficacy. Despite the cataloging of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively profiled the resistance of ∼3,500 Src tyrosine kinase mutants to four different 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, we found that a resistance-prone cluster of residues located on the top face of the N-terminal lobe of Src's catalytic domain contributes to autoinhibition by reducing catalytic domain dynamics, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how drug resistance profiling can be used to define potential resistance pathways and uncover new mechanisms of kinase regulation.Copyright © 2023 Elsevier Ltd. All rights reserved.