Bailey Fiona P, Andreev Veselin I, Eyers Patrick A
Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
Methods Enzymol. 2014;548:117-46. doi: 10.1016/B978-0-12-397918-6.00005-7.
Acquired resistance to targeted kinase inhibitors is a well-documented clinical problem that is potentially fatal for patients to whom a suitable back-up is not available. However, protein kinase alleles that promote resistance to inhibitors can be exploited experimentally as gold-standards for "on"- and "off"-target validation strategies and constitute a powerful resource for assessing the ability of new or combined therapies to override resistance. Clinical resistance to kinase inhibitors is an evident in all tyrosine kinase-driven malignancies, where high rates of mutation drive tumor evolution toward the insidious drug-resistant (DR) state through a variety of mechanisms. Unfortunately, this problem is likely to intensify in the future as the number of target kinases, approved inhibitors, and clinical indications increase. To empower the analysis of resistance in kinases, we have validated a bioinformatic, structural, and cellular workflow for designing and evaluating resistance at key mutational hotspots among kinome members. In this chapter, we discuss how mutation of amino acids in the gatekeeper and hinge-loop regions (collectively termed the "resistance tetrad") and the DFG motif represent an effective approach for generating panels of DR kinase alleles for chemical genetics and biological target validation.
获得性靶向激酶抑制剂耐药是一个有充分文献记载的临床问题，对于没有合适替代治疗方案的患者来说可能是致命的。然而，促进对抑制剂耐药的蛋白激酶等位基因可在实验中用作“靶向”和“脱靶”验证策略的金标准，并构成评估新疗法或联合疗法克服耐药能力的强大资源。激酶抑制剂的临床耐药在所有酪氨酸激酶驱动的恶性肿瘤中都很明显，在这些肿瘤中，高突变率通过多种机制推动肿瘤向隐匿的耐药（DR）状态演变。不幸的是，随着靶向激酶、获批抑制剂和临床适应症数量的增加，这个问题在未来可能会加剧。为了加强对激酶耐药性的分析，我们已经验证了一种生物信息学、结构和细胞工作流程，用于设计和评估激酶组成员中关键突变热点处的耐药性。在本章中，我们将讨论守门人和铰链环区域（统称为“耐药四联体”）以及DFG模体中的氨基酸突变如何代表一种有效的方法，用于生成用于化学遗传学和生物学靶点验证的DR激酶等位基因组。
