Department of Hematology, Dongguan Institute of Clinical Cancer Research, Affiliated Dongguan Hospital, Southern Medical University, Dongguan City, 523050, Guangdong Province, China; Dongguan Institute of Clinical Cancer Research, Dongguan Key Laboratory of Precision Diagnosis and Treatment for Tumors, Affiliated Dongguan Hospital, Southern Medical University, Dongguan City, 523050, Guangdong Province, China.
Department of Intensive Care Unit, The First Dongguan Affiliated Hospital, Guangdong Medical University, Dongguan City, 523710, Guangdong Province, China.
J Mol Graph Model. 2024 Jan;126:108623. doi: 10.1016/j.jmgm.2023.108623. Epub 2023 Sep 12.
Drug resistance to Bruton's Tyrosine Kinase (BTK) inhibitors presents a challenge in treating B-cell malignancies, and the mechanism behind drug resistance remains unclear. In this study, we focused on the BTK L528W mutation and investigated the underlying mechanisms of resistance to ibrutinib (including prototype and its active metabolite from, PCI-45227) using a combination of bioinformatics analysis, and molecular dynamics (MD) simulations. Protein stability of wild type (WT) BTK and L528W mutant was predicted using DUET, PoPMuSiC, and I-Mutant2.0. We performed MD simulations of six systems, apo-WT, metabolite-WT, prototype-WT and their mutants, to analyze the significant conformational and BTK-inhibitor binding affinity changes induced by the L528W mutation. Results show that the L528W mutation reduces the conformational stability of BTK compared to the WT. Principal component analysis (PCA) based free energy landscape (FEL) analysis shows that the L528W mutant ensemble tends to form more conformation clusters and exhibit higher levels of local minima than the WT counterpart. The interaction analysis reveal that the L528W mutation disrupts the strong hydrogen bond between Cys481 and inhibitors and reduces the number of hydrogen bonds between inhibitors and BTK in the L528W mutant complex structures compared to the WT. Porcupine plot analysis in association with cross-correlation analysis show the high-intensity flexible motion exhibited by the P-loop region. MM/GBSA calculations show that the L528W mutation in metabolite-BTK and prototype-BTK complexes increases binding free energy compared to the WT, with a reduction in binding affinity confirmed by per-residue energy decomposition. Specifically, the binding free energy increases from -57.86 kcal/mol to -48.26 kcal/mol for the metabolite-BTK complex and from -62.04 kcal/mol to -50.55 kcal/mol for the prototype-BTK complex. Overall, our study finds that the L528W mutation reduces BTK stability, decreases binding affinity, and leads to drug resistance and potential disease recurrence.
布鲁顿酪氨酸激酶（BTK）抑制剂的耐药性在治疗 B 细胞恶性肿瘤方面带来了挑战，而耐药的机制仍不清楚。在这项研究中，我们专注于 BTK L528W 突变，并使用生物信息学分析和分子动力学（MD）模拟相结合的方法，研究了对伊布替尼（包括原型及其来自 PCI-45227 的活性代谢物）耐药的潜在机制。使用 DUET、PoPMuSiC 和 I-Mutant2.0 预测野生型（WT）BTK 和 L528W 突变体的蛋白稳定性。我们对六个系统（apo-WT、metabolite-WT、prototype-WT 及其突变体）进行了 MD 模拟，以分析 L528W 突变引起的显著构象和 BTK-抑制剂结合亲和力变化。结果表明，与 WT 相比，L528W 突变降低了 BTK 的构象稳定性。基于主成分分析（PCA）的自由能景观（FEL）分析表明，L528W 突变体集合倾向于形成更多的构象簇，并表现出比 WT 对应物更高的局部最小值水平。相互作用分析表明，L528W 突变破坏了 Cys481 和抑制剂之间的强氢键，并减少了 L528W 突变体复合物结构中抑制剂和 BTK 之间氢键的数量。与互相关分析结合的刺猬图分析表明 P 环区域表现出高强度的柔性运动。MM/GBSA 计算表明，与 WT 相比，在代谢物-BTK 和原型-BTK 复合物中，L528W 突变增加了结合自由能，通过残基能量分解证实了结合亲和力的降低。具体来说，代谢物-BTK 复合物的结合自由能从-57.86 kcal/mol 增加到-48.26 kcal/mol，原型-BTK 复合物的结合自由能从-62.04 kcal/mol 增加到-50.55 kcal/mol。总的来说，我们的研究发现，L528W 突变降低了 BTK 的稳定性，降低了结合亲和力，并导致耐药性和潜在的疾病复发。
