高通量筛选靶向革兰阴性菌BamA蛋白的小分子药物初步研究

Preliminary study on high throughput screening small molecules targeting Gram-negative bacilli outer membrane protein BamA

目的通过高通量筛选获得针对革兰阴性杆菌BamA外膜蛋白的小分子抗菌药物。方法6株鲍曼不动杆菌临床菌株于2022年1—12月分离自中南大学湘雅三医院住院患者。基于分子对接试验,利用sybyl-X2.1软件对Chemdiv化合物数据库中的小分子化合物进行高通量虚拟筛选;对高通量筛选打分排名靠前的150个小分子进行体外生物学试验筛选,选取具有最佳抗菌活性的前4个小分子进行深入的分子对接分析,并选取对接分数最高的小分子8308-0401进行后续试验;通过棋盘稀释试验检测8308-0401与利福平的联用抗菌效果;最后,通过等离子表面共振试验检测8308-0401与靶蛋白BamA的亲合力。结果通过高通量虚拟筛选,发现了排名靠前的150个小分子的对接打分平均为5.63;通过体外生物学试验,发现了小分子8308-0401、8365-1335、C066-2507和L582-0346具有较强的抗菌活性;其中,8308-0401具有最高的分子对接分数,且与利福平联用对鲍曼不动杆菌标准菌株和临床菌株均具有协同抗菌活性;8308-0401与靶蛋白BamA具有较强的亲合力,其结合常数为182μmol/L。结论小分子8308-0401可靶向革兰阴性杆菌BamA外膜蛋白而发挥抗菌活性。

Objective High-throughput screening to obtain small molecular compounds against Gram-negative bacilli by targeting BamA outer membrane protein.Methods The sybyl-X2.1 software was used to perform high-throughput virtual screening of small molecular compounds in Chemdiv compound library based on the molecular docking.The top 150 hits by high-throughput screening were re-screened through in vitro biological experiments.The top 4 small molecules with obvious antibacterial activity were selected for in-depth molecular docking analysis,and the small molecule 8308-0401 with the highest docking score was selected for further experiments.The antibacterial effect of 8308-0401 combined with rifampicin was tested by checkerboard assay.Finally,the affinity between 8308-0401 and BamA was tested by plasma surface resonance assay.Results The docking score of the top 150 hits calculated by high-throughput virtual screening had a mean value of 5.63.In vitro biological experiments showed that small molecules 8308-0401,8365-1335,C066-2507 and L582-0346 exhibited strong antibacterial activity.Among those molecules,8308-0401 showed the highest molecular docking score,and synergistic antibacterial activity against both types of strains and clinical isolates when combined with rifampicin.8308-0401 has a strong affinity to BamA with binding a constant of 182μmol/L.Conclusion The small molecule 8308-0401 exerts antibacterial activity against Gram negative bacilli by targeting the outer membrane protein BamA.