高通量筛选并鉴定抗堪萨斯分枝杆菌活性化合物的研究

doi:10.19982/j.issn.1000-6621.20240518

中国防痨杂志 ›› 2025, Vol. 47 ›› Issue (5): 597-604.doi: 10.19982/j.issn.1000-6621.20240518

高通量筛选并鉴定抗堪萨斯分枝杆菌活性化合物的研究

胡前芳¹, 钟如杰², 尚媛媛², 张旭霞²^,³, 李姗姗²(), 王伟²^,³()

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

收稿日期:2024-11-21 出版日期:2025-05-10 发布日期:2025-04-29
通信作者: 李姗姗,Email:lss9011@126.com;王伟,Email:wangwei010@aliyun.com
基金资助:
北京市科技新星计划(20230484295);科技部重点研发计划项目(2024YFC2311200)

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

Hu Qianfang¹, Zhong Rujie², Shang Yuanyuan², Zhang Xuxia²^,³, Li Shanshan²(), Wang Wei²^,³()

¹Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing 300016, China
²Bacterial 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
³National 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

Received:2024-11-21 Online:2025-05-10 Published:2025-04-29
Contact: Li Shanshan, Email: lss9011@126.com; Wang Wei, Email: wangwei010@aliyun.com
Supported by:
Beijing Science and Technology New Star Program(20230484295);National Key Research and Development Program of China(2024YFC2311200)

摘要/Abstract

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A₄₅₀=3.027,P-HCl A₄₅₀=1.984,t=4.183,P=0.014),而沃拉帕沙在4μg/ml(DMSO对照A₄₅₀=3.027,沃拉帕沙A₄₅₀=1.959,t=4.342,P=0.012)和8μg/ml(DMSO对照A₄₅₀=3.027,沃拉帕沙A₄₅₀=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24h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99.97%,沃拉帕沙处理后细胞存活率为84.97%,t=3.098,P=0.021)。结论: 构建了抗堪萨斯分枝杆菌活性化合物的高通量筛选平台,发现P-HCl和VP对堪萨斯分枝杆菌具有显著的抗菌活性和抗生物膜活性,为开发新型抗堪萨斯分枝杆菌药物提供了潜在候选化合物。

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

Abstract:

Objective: 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 1.6 μg/ml and 1.23 μg/ml, respectively, and both of them showed a MIC of 2 μg/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 8 μg/ml (A₄₅₀=3.027 vs. 1.984, t=4.183, P=0.014), whereas vorapaxar showed significant inhibitory effects at both 4 μg/ml (A₄₅₀=3.027 vs. 1.959, t=4.342, P=0.012) and 8 μg/ml (A₄₅₀=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 20 μmol/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 compounds for the development of new anti-M.kansasii drugs.

Key words: Mycobacterium kansasii, High-throughput screening, Promethazine, Vorapaxar, Biofilms

中图分类号:

R978.1

胡前芳, 钟如杰, 尚媛媛, 张旭霞, 李姗姗, 王伟. 高通量筛选并鉴定抗堪萨斯分枝杆菌活性化合物的研究[J]. 中国防痨杂志, 2025, 47(5): 597-604. doi: 10.19982/j.issn.1000-6621.20240518

Hu Qianfang, Zhong Rujie, Shang Yuanyuan, Zhang Xuxia, Li Shanshan, Wang Wei. High-throughput screening and identification of compounds with anti-Mycobacterium kansasii activity[J]. Chinese Journal of Antituberculosis, 2025, 47(5): 597-604. doi: 10.19982/j.issn.1000-6621.20240518

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化合物名称	临床状态	分子量	化学式	形态	MIC(μmol/L)	MIC(μg/ml)
沃拉帕沙	FDA批准	492.58	C₂₉H₃₃FN₂O₄	游离碱	2.50	1.23
盐酸异丙嗪	FDA批准	320.88	C₁₇H₂₀ClN₂S	盐酸盐	5.00	1.60
AM251	其他	555.24	C₂₂H₂₁Cl₂IN₄O	游离碱	10.00	5.55
甲磺酸溴隐亭	FDA批准	750.70	C₃₂H₄₀BrN₅O₅·CH₄O₃S	甲磺酸盐	10.00	7.51
马来酸PRX-08066	其他	517.96	C₂₃H₂₁ClFN₅O₄S	马来酸盐	10.00	5.18
氢溴酸沃替西汀	其他	379.36	C₁₈H₂₃BrN₂S	氢溴酸盐	10.00	3.79
匹莫范色林	其他	1005.20	C₅₄H₇₄F₂N₆O₁₀	酒石酸盐	10.00	10.05
JMS-17-2	其他	419.95	C₂₅H₂₆ClN₃O	游离碱	10.00	4.20
阿立哌唑	其他	448.39	C₂₃H₂₇Cl₂N₃O₂	游离碱	10.00	4.48
二甲酸丙氯拉嗪	其他	606.09	C₂₈H₃₂ClN₃O₈S	二甲基马来酸盐	10.00	6.06
马来酸环丙西平	其他	386.40	C₂₀H₂₂N₂O₆	马来酸盐	10.00	3.86
盐酸硫利达嗪	其他	407.04	C₂₁H₂₇ClN₂S₂	盐酸盐	20.00	8.14
盐酸阿莫地喹	FDA批准	428.78	C₂₀H₂₄Cl₃N₃O	二水合物	10.00	4.29
NIBR189	临床前阶段	429.31	C₂₁H₂₁BrN₂O₃	游离碱	10.00	4.29
二氢氯化二水合阿莫地喹	其他	464.81	C₂₀H₂₈Cl₃N₃O₃	二氢氯化物二水合物	10.00	4.65
JK184	其他	350.44	C₁₉H₁₈N₄OS	游离碱	20.00	7.01
APD597	临床试验阶段	479.55	C₂₁H₂₉N₅O₆S	游离碱	20.00	9.59

菌株号	利福平	克拉霉素	盐酸异丙嗪	沃拉帕沙
ATCC 12478	0.25	0.50	2.00	2.00
1	0.50	0.50	2.00	2.00
2	0.50	0.50	2.00	2.00
3	0.25	0.50	4.00	>4.00
4^a	>2.00	0.50	2.00	2.00
5^a	>2.00	0.50	2.00	2.00

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

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