Lung cancer is commonly caused by activating mutations in the epidermal growth factor receptor (EGFR). Allosteric kinase inhibitors are unaffected by common ATP-site resistance mutations and represent a promising therapeutic strategy for targeting drug-resistant EGFR variants. However, allosteric inhibitors are antagonized by kinase dimerization, and understanding this phenomenon has been limited to cellular experiments. To facilitate the study of allosteric inhibitor pharmacology, we designed and purified a constitutive EGFR kinase dimer harboring the clinically relevant L858R/T790M mutations. Kinetic characterization revealed that the EGFR kinase dimer is more active than monomeric EGFR(L858R/T790M) kinase and has the same K_m,ATP. Biochemical profiling of a large panel of ATP-competitive and allosteric EGFR inhibitors showed that allosteric inhibitor potency decreased by >500-fold in the kinase dimer compared with monomer, yielding IC₅₀ values that correlate well with Ba/F3 cellular potencies. Thus, this readily purifiable constitutive asymmetric EGFR kinase dimer represents an attractive tool for biochemical evaluation of EGFR inhibitor pharmacology, in particular for allosteric inhibitors.

中文翻译：

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用于研究抑制剂药理学的组成型 EGFR 激酶二聚体

肺癌通常是由表皮生长因子受体 (EGFR) 的激活突变引起的。变构激酶抑制剂不受常见 ATP 位点耐药突变的影响，是一种针对耐药 EGFR 变异体的有前途的治疗策略。然而，变构抑制剂会被激酶二聚化所拮抗，并且对这种现象的理解仅限于细胞实验。为了促进变构抑制剂药理学的研究，我们设计并纯化了含有临床相关 L858R/T790M 突变的组成型 EGFR 激酶二聚体。动力学表征表明，EGFR 激酶二聚体比单体 EGFR(L858R/T790M) 激酶活性更高，并且具有相同的 K _m,ATP。对大量 ATP 竞争性和变构 EGFR 抑制剂的生化分析表明，与单体相比，激酶二聚体中的变构抑制剂效力降低了 500 倍以上，产生的 IC ₅₀值与 Ba/F3 细胞效力密切相关。因此，这种易于纯化的组成型不对称 EGFR 激酶二聚体代表了一种用于 EGFR 抑制剂药理学生化评估的有吸引力的工具，特别是对于变构抑制剂。