Department of Chemistry and Biochemistry, University of California , Los Angeles, California 90095, United States.
Celgene Avilomics Research , Bedford, Massachusetts 01730, United States.
J Chem Theory Comput. 2016 Apr 12;12(4):2066-78. doi: 10.1021/acs.jctc.5b01221. Epub 2016 Apr 4.
Epidermal growth factor receptor (EGFR) inhibitors interrupt EGFR-dependent cellular signaling pathways that lead to accelerated tumor growth and proliferation. Mutation of a threonine in the inhibitor binding pocket, known as the "gatekeeper", to methionine (T790M) confers acquired resistance to several EGFR-selective inhibitors. We studied interactions between EGFR inhibitors and the gatekeeper residues of the target protein. Thermodynamic integration (TI) with Amber14 indicates that the binding energies of gefitinib and AEE788 to the active state of the T790M mutant EGFR is 3 kcal/mol higher than to the wild type (WT), whereas ATP binding energy to the mutant is similar to the WT. Using metadynamics MD simulations with NAMD v2.9, the conformational equilibrium between the inactive resting state and the catalytically competent activate state was determined for the WT EGFR. When combined with the results obtained by Sutto and Gervasio, our simulations showed that the T790M point mutation lowers the free energy of the active state by 5 kcal/mol relative to the inactive state of the enzyme. Relative to the WT, the T790M mutant binds gefitinib more strongly. The T790M mutation is nevertheless resistant due to its increased binding of ATP. By contrast, the binding of AEE788 to the active state causes a conformational change in the αC-helix adjacent to the inhibitor binding pocket, that results in a 2 kcal/mol energy penalty. The energy penalty explains why the binding of AEE788 to the T790M mutant is unfavorable relative to binding to WT EGFR. These results establish the role of the gatekeeper mutation on inhibitor selectivity. Additional molecular dynamics (MD) simulations, TI, and metadynamics MD simulations reveal the origins of the changes in binding energy of WT and mutants.
表皮生长因子受体（EGFR）抑制剂会阻断 EGFR 依赖的细胞信号通路，从而导致肿瘤生长和增殖加速。抑制剂结合口袋中的一个苏氨酸突变为蛋氨酸（T790M），称为“守门员”，这会导致对几种 EGFR 选择性抑制剂产生获得性耐药。我们研究了 EGFR 抑制剂与靶蛋白的“守门员”残基之间的相互作用。Amber14 的热力学积分（TI）表明，吉非替尼和 AEE788 与 T790M 突变型 EGFR 的活性状态的结合能比野生型（WT）高 3 千卡/摩尔，而突变型的 ATP 结合能与 WT 相似。使用 NAMD v2.9 的元动力学 MD 模拟，确定了 WT EGFR 从非活性静息状态到催化活性激活状态的构象平衡。将我们的模拟结果与 Sutto 和 Gervasio 的结果相结合，表明 T790M 点突变使酶的活性状态的自由能相对于非活性状态降低了 5 千卡/摩尔。与 WT 相比，T790M 突变体与吉非替尼的结合更强。然而，由于其与 ATP 的结合增加，T790M 突变体仍然具有耐药性。相比之下，AEE788 与活性状态的结合会导致抑制剂结合口袋附近的αC-螺旋发生构象变化，从而导致 2 千卡/摩尔的能量罚分。能量罚分解释了为什么 AEE788 与 T790M 突变体的结合不如与 WT EGFR 的结合有利。这些结果确定了“守门员”突变对抑制剂选择性的作用。额外的分子动力学（MD）模拟、TI 和元动力学 MD 模拟揭示了 WT 和突变体结合能变化的起源。
