FMS‐like tyrosine kinase 3 (FLT3) has been found to be mutated in ~ 30% of acute myeloid leukaemia patients. Small‐molecule inhibitors targeting FLT3 that are currently approved or still undergoing clinical trials are subject to drug resistance due to FLT3 mutations. How these mutations lead to drug resistance is hitherto poorly understood. Herein, we studied the molecular mechanism of the drug resistance mutations D835N, Y842S and M664I, which confer resistance against the most advanced inhibitors, quizartinib and PLX3397 (pexidartinib), using enzyme kinetics and computer simulations. In vitro kinase assays were performed to measure the comparative catalytic activity of the native protein and the mutants, using a bacterial expression system developed to this aim. Our results reveal that the differential drug sensitivity profiles can be rationalised by the dynamics of the protein–drug interactions and perturbation of the intraprotein contacts upon mutations. Drug binding induced a single conformation in the native protein, whereas multiple conformations were observed otherwise (in the mutants or in the absence of drugs). The end‐point kinetics measurements indicated that the three resistant mutants conferred catalytic activity that is at least as high as that of the reference without such mutations. Overall, our calculations and measurements suggest that the structural dynamics of the drug‐resistant mutants that affect the active state and the increased conformational freedom of the remaining inactive drug‐bound population are the two major factors that contribute to drug resistance in FLT3 harbouring cancer cells. Our results explain the mechanism of drug resistance mutations and can aid to the design of more effective tyrosine kinase inhibitors.

中文翻译：

---

阐明FLT3抗性突变的分子机制。

已发现约30％的急性髓细胞性白血病患者发生FMS类酪氨酸激酶3（FLT3）突变。目前已批准或仍在进行临床试验的靶向FLT3的小分子抑制剂由于FLT3突变而具有耐药性。迄今为止，对这些突变如何导致耐药性的了解还很少。在这里，我们使用酶动力学和计算机模拟研究了耐药突变D835N，Y842S和M664I的分子机制，这些突变赋予了对最先进的抑制剂quizartinib和PLX3397（pexidartinib）的耐药性。体外使用为此目的开发的细菌表达系统，进行激酶测定以测量天然蛋白和突变体的比较催化活性。我们的研究结果表明，可以通过蛋白质-药物相互作用的动力学以及突变后蛋白质内接触的扰动来合理化差异药物敏感性分布。药物结合在天然蛋白中诱导了一个构象，而在其他情况下则观察到了多个构象（在突变体中或在没有药物的情况下）。终点动力学测量表明，三个抗性突变体的催化活性至少与没有此类突变的参比的催化活性一样高。总体，我们的计算和测量结果表明，影响活性状态的耐药突变体的结构动力学以及剩余的非活性药物结合种群的构象自由度的增加是导致带有FLT3的癌细胞产生耐药性的两个主要因素。我们的结果解释了耐药性突变的机制，可以帮助设计更有效的酪氨酸激酶抑制剂。