Li Qin, Luo Jiayi, Fu Chenggong, Kang Wenqingqing, Wang Lingling, Tong Henry, Lun Zhaorong, Zhang Qianqian, Lai Dehua, Liu Huanxiang
Faculty of Applied Sciences, Macao Polytechnic University, Macao SAR, China.
MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China.
Pharmaceuticals (Basel). 2025 May 23;18(6):778. doi: 10.3390/ph18060778.
Human African trypanosomiasis (HAT), caused by , is a neglected tropical disease with limited treatments, highlighting the pressing need for new drugs. Cell division cycle-2-related kinase 12 (CRK12), a pivotal protein involved in the cell cycle regulation of , has emerged as a promising therapeutic target for HAT, yet effective CRK12 inhibitors remain lacking. An integrated strategy combining computational modeling, virtual screening, molecular dynamics (MD) simulations, and experimental validation was adopted to discover potential inhibitors against CRK12. By using the predicted and refined 3D structure of CRK12 from AlphaFold2 and MD simulation, over 1.5 million compounds were screened based on multiple-scale molecular docking, and 26 compounds were selected for evaluation of biological activity based on anti- bioassays. Dose-response curves were generated for the most potent inhibitors, and the interaction mechanism between the top four compounds and CRK12 was explored by MD simulations and MM/GBSA binding free energy analysis. Of the 26 compounds, six compounds demonstrated sub-micromolar to low-micromolar IC values (0.85-3.50 µM). The top four hits, F733-0072, F733-0407, L368-0556, and L439-0038, exhibited IC values of 1.11, 1.97, 0.85, and 1.66 µM, respectively. Binding free energy and energy decomposition analyses identified ILE335, VAL343, PHE430, ALA433, and LEU482 as hotspot residues for compound binding. Hydrogen bonding analysis demonstrated that these compounds can form stable hydrogen bonds with the hinge residue ALA433, ensuring their stable binding within the active site. This study establishes a robust and cost-effective pipeline for CRK12 inhibitor discovery, identifying several novel inhibitors demonstrating promising anti-HAT activity. The newly discovered scaffolds exhibit structural diversity distinct from known CRK12 inhibitors, providing valuable lead compounds for anti-trypanosomal drug development.
人类非洲锥虫病（HAT）由[具体病原体未给出]引起，是一种治疗方法有限的被忽视热带病，凸显了对新药的迫切需求。细胞分裂周期2相关激酶12（CRK12）是参与[相关生物的名称未给出]细胞周期调控的关键蛋白，已成为HAT有前景的治疗靶点，但仍缺乏有效的CRK12抑制剂。采用了一种结合计算建模、虚拟筛选、分子动力学（MD）模拟和实验验证的综合策略来发现针对CRK12的潜在抑制剂。通过使用来自AlphaFold2的预测和优化后的CRK12三维结构以及MD模拟，基于多尺度分子对接筛选了超过150万种化合物，并基于抗[相关生物的名称未给出]生物测定选择了26种化合物进行生物活性评估。为最有效的抑制剂生成了剂量反应曲线，并通过MD模拟和MM/GBSA结合自由能分析探索了前四种化合物与CRK12之间的相互作用机制。在这26种化合物中，有六种化合物表现出亚微摩尔至低微摩尔的IC值（0.85 - 3.50µM）。前四个命中化合物F733 - 0072、F733 - 0407、L368 - 0556和L439 - 0038的IC值分别为1.11、1.97、0.85和1.66µM。结合自由能和能量分解分析确定ILE335、VAL343、PHE430、ALA433和LEU482为化合物结合的热点残基。氢键分析表明这些化合物可与铰链残基ALA433形成稳定的氢键，确保它们在活性位点内的稳定结合。本研究建立了一个强大且经济高效的CRK12抑制剂发现流程，鉴定出几种具有有前景的抗HAT活性的新型抑制剂。新发现的支架展现出与已知CRK12抑制剂不同的结构多样性，为抗锥虫药物开发提供了有价值的先导化合物。
