Protein kinases are one of the most important drug targets in the past 10 years. Understanding the inhibitor association processes will profoundly impact new binder designs with preferred binding kinetics. However, after more than a decade of effort, a complete atomistic-level study of kinase inhibitor binding pathways is still lacking. As all kinases share a similar scaffold, we used p38 kinase as a model system to investigate the conformational dynamics and free energy transition of inhibitor binding toward kinases. Two major kinase conformations, Asp-Phe-Gly (DFG)-in and DFG-out, and three types of inhibitors, type I, II, and III, were thoroughly investigated in this work. We performed Brownian dynamics simulations and up to 340 μs Gaussian-accelerated molecular dynamics simulations to capture the inhibitor binding paths and a series of conformational transitions of the p38 kinase from its apo to inhibitor-bound form. Eighteen successful binding trajectories, including all types of inhibitors, are reported herein. Our simulations suggest a mechanism of inhibitor recruitment, a faster ligand association step to a pre-existing DFG-in/DFG-out p38 protein, followed by a slower molecular rearrangement step to adjust the protein-ligand conformation followed by a shift in the energy landscape to reach the final bound state. The ligand association processes also reflect the energetic favor of type I and type II/III inhibitor binding through ATP and allosteric channels, respectively. These different binding routes are directly responsible for the fast (type I binders) and slow (type II/III binders) kinetics of different types of p38 inhibitors. Our findings also echo the recent study of p38 inhibitor dissociation, implying that ligand unbinding could undergo a reverse path of binding, and both processes share similar metastates. This study deepens the understanding of molecular and energetic features of kinase inhibitor-binding processes and will inspire future drug development from a kinetic point of view.

蛋白激酶是过去10年最重要的药物靶点之一。了解抑制剂缔合过程将深刻影响具有优选结合动力学的新粘合剂设计。然而，经过十多年的努力，仍然缺乏对激酶抑制剂结合途径的完整原子水平研究。由于所有激酶共享相似的支架，我们使用 p38 激酶作为模型系统来研究抑制剂与激酶结合的构象动力学和自由能转变。这项工作对两种主要激酶构象 Asp-Phe-Gly (DFG)-in 和 DFG-out 以及三种类型的抑制剂（I 型、II 型和 III 型）进行了彻底研究。我们进行了布朗动力学模拟和高达 340 μs高斯加速分子动力学模拟，以捕获抑制剂结合路径以及 p38 激酶从 apo 到抑制剂结合形式的一系列构象转变。本文报道了十八种成功的结合轨迹，包括所有类型的抑制剂。我们的模拟表明了一种抑制剂募集机制，即与预先存在的 DFG-in/DFG-out p38 蛋白的更快的配体关联步骤，随后是较慢的分子重排步骤以调整蛋白-配体构象，随后发生能量变化景观达到最终的束缚状态。配体缔合过程也反映了 I 型和 II/III 型抑制剂分别通过 ATP 和变构通道结合的能量偏好。这些不同的结合途径直接影响不同类型 p38 抑制剂的快速（I 型结合物）和慢速（II/III 型结合物）动力学。 我们的研究结果也呼应了最近对 p38 抑制剂解离的研究，这意味着配体解离可能经历相反的结合路径，并且这两个过程具有相似的转移。这项研究加深了对激酶抑制剂结合过程的分子和能量特征的理解，并将从动力学的角度启发未来的药物开发。