Institute of Biomechanics, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
Guangdong Provincial Engineering and Technology Research Center of Biopharmaceuticals, South China University of Technology, Guangzhou 510006, China.
Molecules. 2022 Nov 2;27(21):7451. doi: 10.3390/molecules27217451.
Bruton's tyrosine kinase (BTK) is a key protein from the TEC family and is involved in B-cell lymphoma occurrence and development. Targeting BTK is therefore an effective strategy for B-cell lymphoma treatment. Since previous studies on BTK have been limited to structure-function analyses of static protein structures, the dynamics of conformational change of BTK upon inhibitor binding remain unclear. Here, molecular dynamics simulations were conducted to investigate the molecular mechanisms of association and dissociation of a reversible (ARQ531) and irreversible (ibrutinib) small-molecule inhibitor to/from BTK. The results indicated that the BTK kinase domain was found to be locked in an inactive state through local conformational changes in the DFG motif, and P-, A-, and gatekeeper loops. The binding of the inhibitors drove the outward rotation of the C-helix, resulting in the upfolded state of Trp395 and the formation of the salt bridge of Glu445-Arg544, which maintained the inactive conformation state. Met477 and Glu475 in the hinge region were found to be the key residues for inhibitor binding. These findings can be used to evaluate the inhibitory activity of the pharmacophore and applied to the design of effective BTK inhibitors. In addition, the drug resistance to the irreversible inhibitor Ibrutinib was mainly from the strong interaction of Cys481, which was evidenced by the mutational experiment, and further confirmed by the measurement of rupture force and rupture times from steered molecular dynamics simulation. Our results provide mechanistic insights into resistance against BTK-targeting drugs and the key interaction sites for the development of high-quality BTK inhibitors. The steered dynamics simulation also offers a means to rapidly assess the binding capacity of newly designed inhibitors.
布鲁顿酪氨酸激酶（BTK）是 Tec 家族中的关键蛋白，参与 B 细胞淋巴瘤的发生和发展。因此，靶向 BTK 是治疗 B 细胞淋巴瘤的有效策略。由于之前对 BTK 的研究仅限于静态蛋白质结构的结构-功能分析，因此 BTK 与抑制剂结合时构象变化的动力学仍不清楚。在这里，进行了分子动力学模拟，以研究可逆（ARQ531）和不可逆（伊布替尼）小分子抑制剂与 BTK 结合和解离的分子机制。结果表明，BTK 激酶结构域通过 DFG 基序、P-、A-和守门员环中的局部构象变化被锁定在非活性状态。抑制剂的结合驱动 C-螺旋向外旋转，导致 Trp395 向上折叠和 Glu445-Arg544 形成盐桥，从而保持非活性构象状态。发现铰链区的 Met477 和 Glu475 是抑制剂结合的关键残基。这些发现可用于评估药效团的抑制活性，并应用于设计有效的 BTK 抑制剂。此外，不可逆抑制剂伊布替尼的耐药性主要来自 Cys481 的强相互作用，这一点通过突变实验得到了证明，并通过从定向分子动力学模拟测量断裂力和断裂时间进一步得到了证实。我们的研究结果为 BTK 靶向药物的耐药性提供了机制见解，并为开发高质量 BTK 抑制剂提供了关键的相互作用位点。定向动力学模拟也为快速评估新设计抑制剂的结合能力提供了一种手段。
