The Janus kinases (JAK) are crucial targets in drug development for several diseases. However, accounting for the impact of possible structural rearrangements on the binding of different kinase inhibitors is complicated by the extensive conformational variability of their catalytic kinase domain (KD). The dynamic KD contains mainly four prominent mobile structural motifs: the phosphate-binding loop (P-loop), the αC-helix within the N-lobe, the Asp-Phe-Gly (DFG) motif, and the activation loop (A-loop) within the C-lobe. These distinct structural orientations imply a complex signal transmission path for regulating the A-loop’s flexibility and conformational preference for optimal JAK function. Nevertheless, the precise dynamical features of the JAK induced by different types of inhibitors still remain elusive. We performed comparative, microsecond-long, Gaussian accelerated molecular dynamics simulations in triplicate of three phosphorylated JAK2 systems: the KD alone, type-I ATP-competitive inhibitor (CI) bound KD in the catalytically active DFG-in conformation, and the type-II inhibitor (AI) bound KD in the catalytically inactive DFG-out conformation. Our results indicate significant conformational variations observed in the A-loop and αC helix motions upon inhibitor binding. Our studies also reveal that the DFG-out inactive conformation is characterized by the closed A-loop rearrangement, open catalytic cleft of N and C-lobe, the outward movement of the αC helix, and open P-loop states. Moreover, the outward positioning of the αC helix impacts the hallmark salt bridge formation between Lys882 and Glu898 in an inactive conformation. Finally, we compared their ligand binding poses and free energy by the MM/PBSA approach. The free energy calculations suggested that the AI’s binding affinity is higher than CI against JAK2 due to an increased favorable contribution from the total non-polar interactions and the involvement of the αC helix. Overall, our study provides the structural and energetic insights crucial for developing more promising type I/II JAK2 inhibitors for treating JAK-related diseases.

Janus 激酶 (JAK) 是多种疾病药物开发的关键靶点。然而，由于不同激酶抑制剂的催化激酶结构域 (KD) 的广泛构象变异性，考虑到可能的结构重排对不同激酶抑制剂的结合的影响变得复杂。动态 KD 主要包含四个突出的可移动结构基序：磷酸盐结合环（P 环）、N 叶内的 αC 螺旋、Asp-Phe-Gly (DFG) 基序和激活环（A-环）在C叶内。这些不同的结构方向意味着复杂的信号传输路径，用于调节 A 环的灵活性和构象偏好，以实现最佳的 JAK 功能。然而，不同类型抑制剂诱导的 JAK 的精确动力学特征仍然难以捉摸。我们对三个磷酸化 JAK2 系统进行了比较性、微秒长、高斯加速分子动力学模拟，一式三份：单独的 KD、催化活性 DFG 构象中的 I 型 ATP 竞争性抑制剂 (CI) 结合 KD ，以及类型 - II 抑制剂 (AI) 将 KD 结合在催化失活的 DFG- out构象中。我们的结果表明在抑制剂结合后 A 环和 αC 螺旋运动中观察到显着的构象变化。我们的研究还表明，DFG- out失活构象的特征是闭合的 A 环重排、N 和 C 叶开放的催化裂隙、αC 螺旋的向外运动和开放的 P 环状态。此外，αC 螺旋的向外定位会影响非活性构象中 Lys882 和 Glu898 之间标志性盐桥的形成。最后，我们通过 MM/PBSA 方法比较了它们的配体结合姿势和自由能。自由能计算表明，由于总非极性相互作用和αC螺旋的参与增加了有利贡献，AI对JAK2的结合亲和力高于CI。总的来说，我们的研究提供了结构和能量方面的见解，这对于开发更有前景的 I/II 型 JAK2 抑制剂来治疗 JAK 相关疾病至关重要。