高通量筛选并鉴定抗堪萨斯分枝杆菌活性化合物的研究  

High-throughput screening and identification of compounds with anti-Mycobacterium kansasii activity

作　　者：胡前芳 钟如杰 尚媛媛[2] 张旭霞[2,3] 李姗姗 王伟[2,3] Hu Qianfang;Zhong Rujie;Shang Yuanyuan;Zhang Xuxia;Li Shanshan;Wang Wei(Department of Respiratory and Critical Care Medicine,The First Affiliated Hospital of Chongqing Medical University,Chongqing 300016,China;Bacterial Immunology Laboratory/Beijing Key Laboratory for Drug-Resistant Tuberculosis Research,Beijing Chest Hospital,Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing 101149,China;National Clinical Laboratory on Tuberculosis/Beijing Key Laboratory for Drug-Resistant Tuberculosis Research,Beijing Chest Hospital,Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing 101149,China)

机构地区：[1]重庆医科大学附属第一医院呼吸与危重症医学科,重庆300016 [2]首都医科大学附属北京胸科医院/北京市结核病胸部肿瘤研究所,细菌免疫学实验室/耐药结核病研究北京市重点实验室,北京101149 [3]首都医科大学附属北京胸科医院/北京市结核病胸部肿瘤研究所,国家结核病临床实验室/耐药结核病研究北京市重点实验室,北京101149

出　　处：《中国防痨杂志》2025年第5期597-604,共8页Chinese Journal of Antituberculosis

基　　金：北京市科技新星计划(20230484295);科技部重点研发计划项目(2024YFC2311200)。

摘　　要：目的:堪萨斯分枝杆菌感染的治疗面临耐药性增加和药物选择有限的挑战。本研究旨在构建高通量筛选抗堪萨斯分枝杆菌活性化合物平台,寻找新型抗堪萨斯分枝杆菌活性的化合物,以应对这一临床需求。方法:构建基于96孔板的高通量筛选体系,对G蛋白偶联受体(G protein-coupled receptor,GPCR)化合物库进行初筛,以寻找具有抗堪萨斯分枝杆菌活性的候选化合物。初筛后,选择活性最显著的候选化合物进行进一步评估,包括抗临床菌株活性实验、时间-杀菌曲线实验、生物膜形成抑制实验及细胞毒性测试实验。实验数据采用t检验和方差分析进行统计学分析,以P<0.05表示差异有统计学意义。结果:通过高通量筛选,从GPCR化合物库中初步筛选出17种对堪萨斯分枝杆菌标准菌株ATCC 12478具有抑菌活性的候选化合物。其中,盐酸异丙嗪(promethazine HCl,P-HCl)和沃拉帕沙(vorapaxar,VP)抑菌活性最为显著,对ATCC 12478菌株的最低抑菌浓度(minor inhibitory concentration,MIC)分别为1.6μg/ml及1.23μg/ml,作为候选化合物进行后续实验。抗临床菌株活性实验显示,两种化合物对两株利福平耐药临床分离株的MIC均为2μg/ml。时间-杀菌曲线实验提示,两种化合物对ATCC 12478菌株的抑菌活性呈浓度依赖性。生物膜形成抑制实验显示,两种化合物对ATCC 12478菌株的抗生物膜效应呈浓度依赖性,其中,盐酸异丙嗪在8μg/ml时显示出抑制作用(DMSO对照A_(450)=3.027,P-HCl A_(450)=1.984,t=4.183,P=0.014),而沃拉帕沙在4μg/ml(DMSO对照A_(450)=3.027,沃拉帕沙A_(450)=1.959,t=4.342,P=0.012)和8μg/ml(DMSO对照A_(450)=3.027,沃拉帕沙A_(450)=2.024,t=4.493,P=0.019)时显示出抑制作用。在细胞毒性测试中,P-HCl与THP-1细胞共培养24 h后,未观察到明显的细胞存活率下降,与对照组相比差异无统计学意义;沃拉帕沙仅在20μmol/L浓度下导致细胞存活率轻微降低(DMSO对照细胞存活率为99.97Objective:To establish a high-throughput screening(HTS)platform for compounds actively against Mycobacterium kansasii and identify novel candidates.Methods:A 96-well plate-based HTS system was developed to screen a G protein-coupled receptor(GPCR)compound library for candidates with anti-M.kansasii activity.Selected candidates were evaluated for antibacterial activity against clinical strains,time-kill kinetics,biofilm inhibition,and cytotoxicity.Data were statistically analyzed using t-tests and analysis of variance(ANOVA)at P<0.05.Results:Seventeen candidate compounds with anti-M.kansasii activity were identified.Promethazine hydrochloride and vorapaxar exhibited best performance against the M.kansasii standard strain(ATCC 12478),with minimum inhibitory concentrations(MIC)of 1.6μg/ml and 1.23μg/ml,respectively,and both of them showed a MIC of 2μg/ml against rifampin-resistant clinical isolates.Time-kill curve experiments and Biofilm inhibition assays demonstrated their anti-ATCC 12478 activity and anti-biofilm effect were both concentration-dependent.Promethazine hydrochloride exhibited an inhibitory effect only at 8μg/ml(A_(450)=3.027 vs.1.984,t=4.183,P=0.014),whereas vorapaxar showed significant inhibitory effects at both 4μg/ml(A_(450)=3.027 vs.1.959,t=4.342,P=0.012)and 8μg/ml(A_(450)=3.027 vs.2.024,t=4.493,P=0.019),with all differences being statistically significant.There was no statistically significant difference in cell viability compared to the control group after cell being cultured with Promethazine hydrochloride for 24 hours.Vorapaxar caused a slight reduction in cell viability only at a concentration of 20μmol/L compared with control group(survival rate=84.97%vs.99.97%,t=3.098,P=0.021).Conclusion:A high-throughput screening platform for compounds with anti-M.kansasii activity was successfully established,and both promethazine hydrochloride and vorapaxar exhibited significant anti-M.kansasii activity as well as concentration-dependent antibiofilm activity,providing potential candidate compoun

关 键 词：分枝杆菌 堪萨斯 高通量筛选 异丙嗪 沃拉帕沙 生物膜 

分 类 号：R978.1[医药卫生—药品]