Absolute binding energy calculation supported with metadynamic simulation and molecular dynamics improves inhibitors identification against GSK3-β for glaucoma treatment

The Wnt/β-catenin pathway is over-expressed in the trabecular meshwork and aqueous humour of glaucoma patients, causing increased intraocular pressure (IOP) and resistance to aqueous flow. Inhibiting GSK3-β, a key regulator of this pathway, can help manage IOP and aqueous outflow resistance, protecting photoreceptor cells and retinal ganglion cells (RGCs). In-silico screening of 4 million compounds using HTVS, SP and XP docking, MM-GBSA, and Molecular Dynamics (MD) simulation identified hit molecules. To improve its properties, analogs were designed. Final hit analogs were selected based on MD simulation results. Validation of the best analogs was done using Free Energy Landscape (FEL), well-tempered Metadynamics (wt-MetaD), and Absolute Binding Free Energy Perturbation (ABFEP) calculation. ZINC1250228067 was identified as the hit molecule, and its 22 analogs were designed. Initial MM-GBSA screening revealed 18 analogs with energies below −100 kcal/mol. Amongst all, analog 30 had the lowest post-MD MM-GBSA energy of −133.55 kcal/mol, maintained a hydrogen bond with VAL135, and a salt bridge with GLN185. FEL and wt-MetaD confirmed their stability, and ABFEP showed a superior binding energy of −8.45 ± 1.45 kcal/mol. The study identified a hit molecule and demonstrated that wt-MetaD and ABFEP effectively differentiate molecules with similar properties.

Wnt/β-catenin通路（Wnt/β-catenin pathway）在青光眼患者的小梁网（trabecular meshwork）与房水（aqueous humour）中呈过度表达状态，可引发眼压（intraocular pressure, IOP）升高与房水流出阻力增加。抑制该通路的关键调节因子糖原合成激酶3β（GSK3-β），能够辅助调控眼压与房水流出阻力，同时起到保护感光细胞（photoreceptor cells）及视网膜神经节细胞（retinal ganglion cells, RGCs）的作用。本研究通过高通量虚拟筛选（HTVS）、标准精度（SP）与精确精度（XP）分子对接、分子力学-广义玻恩表面积法（MM-GBSA）以及分子动力学（MD, Molecular Dynamics）模拟，对400万种化合物开展虚拟筛选，最终获得命中化合物。为优化候选化合物的性质，研究人员设计了一系列类似物，并基于分子动力学模拟结果筛选得到最终命中类似物。针对最优类似物的验证工作采用了自由能景观（FEL, Free Energy Landscape）、恒温元动力学（wt-MetaD, well-tempered Metadynamics）以及绝对结合自由能微扰（ABFEP, Absolute Binding Free Energy Perturbation）计算方法。研究鉴定出命中分子ZINC1250228067，并设计了其22种类似物；初始MM-GBSA筛选显示，其中18种类似物的结合能低于-100 kcal/mol。在所有类似物中，类似物30在分子动力学模拟后的MM-GBSA结合能最低，为-133.55 kcal/mol，且始终与缬氨酸135（VAL135）保持氢键相互作用，与谷氨酰胺185（GLN185）维持盐桥相互作用。自由能景观与恒温元动力学分析证实了该类似物的结构稳定性，绝对结合自由能微扰计算则显示其结合能优异，为-8.45±1.45 kcal/mol。本研究成功鉴定出一款青光眼治疗潜在命中分子，并证实恒温元动力学与绝对结合自由能微扰可有效区分性质相近的化合物。