The human FGF receptors (FGFRs) play critical roles in various human cancers, and several FGFR inhibitors are currently under clinical investigation. Resistance usually results from selection for mutant kinases that are impervious to the action of the drug or from up-regulation of compensatory signaling pathways. Preclinical studies have demonstrated that resistance to FGFR inhibitors can be acquired through mutations in the FGFR gatekeeper residue, as clinically observed for FGFR4 in embryonal rhabdomyosarcoma and neuroendocrine breast carcinomas. Here we report on the use of a structure-based drug design to develop two selective, next-generation covalent FGFR inhibitors, the FGFR irreversible inhibitors 2 (FIIN-2) and 3 (FIIN-3). To our knowledge, FIIN-2 and FIIN-3 are the first inhibitors that can potently inhibit the proliferation of cells dependent upon the gatekeeper mutants of FGFR1 or FGFR2, which confer resistance to first-generation clinical FGFR inhibitors such as NVP-BGJ398 and AZD4547. Because of the conformational flexibility of the reactive acrylamide substituent, FIIN-3 has the unprecedented ability to inhibit both the EGF receptor (EGFR) and FGFR covalently by targeting two distinct cysteine residues. We report the cocrystal structure of FGFR4 with FIIN-2, which unexpectedly exhibits a "DFG-out" covalent binding mode. The structural basis for dual FGFR and EGFR targeting by FIIN3 also is illustrated by crystal structures of FIIN-3 bound with FGFR4 V550L and EGFR L858R. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance and provide the first example, to our knowledge, of a kinase inhibitor that covalently targets cysteines located in different positions within the ATP-binding pocket.

中文翻译：

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开发可克服对第一代 FGFR 激酶抑制剂耐药性的共价抑制剂。

人类 FGF 受体 (FGFR) 在各种人类癌症中发挥关键作用，目前有几种 FGFR 抑制剂正在进行临床研究。耐药性通常是由于选择了不受药物作用影响的突变激酶或补偿性信号通路的上调。临床前研究表明，对 FGFR 抑制剂的抗性可以通过 FGFR 看门人残基的突变获得，正如临床观察到的胚胎横纹肌肉瘤和神经内分泌乳腺癌中的 FGFR4。在这里，我们报告使用基于结构的药物设计来开发两种选择性的下一代共价 FGFR 抑制剂，即 FGFR 不可逆抑制剂 2 (FIIN-2) 和 3 (FIIN-3)。据我们所知，FIIN-2 和 FIIN-3 是第一种能够有效抑制依赖于 FGFR1 或 FGFR2 的看门突变体的细胞增殖的抑制剂，这些突变体赋予对第一代临床 FGFR 抑制剂（如 NVP-BGJ398 和 AZD4547）的抗性。由于反应性丙烯酰胺取代基的构象灵活性，FIIN-3 具有通过靶向两个不同的半胱氨酸残基共价抑制 EGF 受体 (EGFR) 和 FGFR 的前所未有的能力。我们报告了 FGFR4 与 FIIN-2 的共晶结构，它出人意料地表现出“DFG-out”共价结合模式。FIIN3 对 FGFR 和 EGFR 双重靶向的结构基础也通过与 FGFR4 V550L 和 EGFR L858R 结合的 FIIN-3 的晶体结构来说明。