一种吡唑并吡啶酮类衍生物对新型冠状病毒SARS-CoV-2的抑制作用研究  

Study of the inhibitory effect of a pyrazolopyridone derivative against SARS-CoV-2 infection

作　　者：李银燕 姜琳瑞 陈至烜 梁锦龙 何晓雪 杨洁 LI Yinyan;JIANG Linrui;CHEN Zhixuan;LIANG Jinlong;HE Xiaoxue;YANG Jie(School of Pharmaceutical Sciences,Southern Medical University,Guangzhou 510515,China;Wuhan Institute of Virology,Chinese Academy of Sciences,Wuhan 430071,China)

机构地区：[1]南方医科大学药学院,广东广州510515 [2]中国科学院武汉病毒所,湖北武汉430071

出　　处：《沈阳药科大学学报》2024年第7期914-921,共8页Journal of Shenyang Pharmaceutical University

基　　金：国家自然科学基金资助项目(82073897,82373915)。

摘　　要：目的探究3-(4-氯苯基)-1,4-二苯基-1,4,5,7-四氢-6H-吡唑并[3,4-b]吡啶-6-酮(以下简称为J1)体外抑制新型冠状病毒(SARS-CoV-2)的作用及潜在作用机制。方法采用MTT法评价化合物J1对Vero-E6、ACE2/293T和HRT18细胞的毒性。利用感染性SARS-CoV-2和人冠状病毒OC43(HCoV-OC43)检测化合物J1的抗冠状病毒活性。采用SARS-CoV-2假病毒体外细胞感染模型和时间点加药实验检测J1抑制SARS-CoV-2病毒感染靶细胞的作用阶段。通过S蛋白介导的细胞融合抑制实验检测J1是否通过阻止病毒膜融合而抑制SARS-CoV-2的进入。采用SPR实验和分子对接技术阐明J1与SARS-CoV-2 S蛋白的作用。结果J1对Vero-E6、ACE2/293T和HRT18细胞的毒性较小,半数细胞毒性浓度CC50均大于100μmol·L^(-1)。J1能显著抑制SARS-CoV-2原始株和Delta变异株的活性,半数有效浓度EC50分别为607μmol·L^(-1)和221μmol·L^(-1)。此外,J1可抑制HCoV-OC43病毒的感染,显著减少NP蛋白和mRNA的表达。分子对接结果显示,J1通过氢键作用和疏水作用力与SARS-CoV-2 S蛋白活性位点稳定结合。机制研究结果表明,J1可抑制SARS-CoV-2病毒进入靶细胞,半数抑制浓度IC50为157μmol·L^(-1)。J1浓度依赖性抑制SARS-CoV-2 S蛋白介导的细胞-细胞膜融合,SPR实验证实J1与S蛋白具有较强结合能力。结论吡唑并吡啶酮类衍生物J1通过靶向S蛋白抑制SARS-CoV-2进入靶细胞。Objective To investigate the inhibitory effect and mechanism of 3-(4-chlorophenyl)-1,4-diphenyl-1,4,5,7-tetrahydro-6H-pyrazolo[3,4-b]pyridin-6-ketone(named J1)against SARS-CoV-2 infection in vitro.Methods The cytotoxicities of J1 on Vero-E6,ACE2/293T and HRT18 cells were evaluated using MTT assay.Subsequently,the anti-coronavirus activity of J1 was assessed using qRT-PCR and western blotting assay.Pseudovirus-based entry inhibition and time-of-addition assays were conducted to explore the antiviral mechanism of J1.The S protein-mediated cell-cell fusion assay determined whether J1 could block SARS-CoV-2 entry by inhibiting viral fusion.The interaction between the S protein and J1 was further explored through molecular docking and SPR assay.Results The compound had low cytotoxicity against Vero-E6 cells,ACE2/293T cells and HRT18 cells with a concentration of cytotoxicity 50%(CC50)up to 100μmol·L^(-1).Both the SARS-CoV-2 wild-type strain and the delta variant were significantly inhibited by J1,and the concentration for 50%of maximal effect(EC50)were 6.07μmol·L^(-1) and 2.21μmol·L^(-1),respectively.Furthermore,it was found that J1 inhibited HCoV-OC43 infection and reduced the expressions of NP protein and mRNA.Molecular docking results showed that J1 bound stably to the active site of the SARS-CoV-2 S protein through hydrogen bonding and hydrophobic forces interactions.Moreover,J1 inhibited the entry of SARS-CoV-2 PsV into the ACE2/293T cells with half maximal inhibitory concentration(IC50)of 1.57μmol·L^(-1) and exhibited a dose-dependent inhibition of SARS-CoV-2 S protein-mediated cell-cell fusion.SPR assay further confirmed that J1 exhibited a relatively strong binding affinity to SARS-CoV-2 S protein.Conclusion The pyrazolopyridone derivative J1 can effectively inhibit the entry of SARS-CoV-2 into cells by targeting S proteins.

关 键 词：SARS-CoV-2 S 蛋白 抗冠状病毒药物 小分子化合物 进入抑制剂 

分 类 号：R96[医药卫生—药理学]