Identification of PEDV inhibitors targeting 3CL protease

Ang Tian; Shutong Shi; Siying Zou; Shuaiyin Guan; Hao Wu; Zhen Li; Huanchun Chen; Yunfeng Song

doi:10.1016/j.virs.2025.06.002

August 2025

. doi: 10.1016/j.virs.2025.06.002

Citation: Ang Tian, Shutong Shi, Siying Zou, Shuaiyin Guan, Hao Wu, Zhen Li, Huanchun Chen, Yunfeng Song. Identification of PEDV inhibitors targeting 3CL protease .VIROLOGICA SINICA, 2025, 40(4) : 624-635. http://dx.doi.org/10.1016/j.virs.2025.06.002

以3CL蛋白酶为靶标鉴定PEDV抑制剂

田昂 ^a,b ,
石姝彤 ^a,b ,
邹思颖 ^a,b ,
关帅印 ^a,b ,
吴皓 ^a,b ,
李振 ^a,b ,
陈焕春 ^a,b ,
宋云峰 ^a,b,,

a 华中农业大学动物医学院, 武汉, 430070, 中国;
b 华中农业大学农业微生物资源发掘与利用全国重点实验室, 武汉, 430070, 中国

通讯作者： 宋云峰, syf@mail.hzau.edu.cn
收稿日期： 2025-02-18
录用日期： 2025-06-16

摘要

猪流行性腹泻（PED）由猪流行性腹泻病毒（PEDV）引起，是一种高度传染性的胃肠道疾病，其特征为呕吐、腹泻和脱水，对哺乳仔猪的致死率可接近100%。PEDV的3C样蛋白酶（3CLpro）对病毒复制至关重要，被视为开发抗病毒抑制剂的关键靶点。本研究旨在通过靶向3CLpro来筛选PEDV的小分子抑制剂。通过对ChemDiv化合物库中160万种化合物进行虚拟筛选，我们鉴定出四种潜在候选分子。分子动力学模拟（包括均方根偏差/RMSD、均方根涨落/RMSF、回转半径/Rg）分析表明，与单体酶相比，化合物-蛋白酶复合物的结构稳定性有所增强。所有化合物在Vero细胞中均表现出低细胞毒性（半数细胞毒性浓度/CC50 > 200 μM）。基于荧光共振能量转移（FRET）的活性测定显示，这些化合物对3CLpro具有剂量依赖性的抑制活性。在候选化合物中，F366-0161的抑制活性最弱，其半抑制浓度（IC50）为151.5 μM。其两个类似物3238-0395（IC50 = 121.4 μM）和L878-0493（IC50 = 123.6 μM）表现出中等程度的活性提升。Y041-1672被证实为最有效的抑制剂，其IC50为86.48 μM。在病毒复制抑制实验中，Y041-1672表现出抑制PEDV复制的能力，其半数有效浓度（EC50）达到17.97 μM，选择性指数（SI = CC50/EC50）为15.5。该化合物的抗病毒效果经RT-qPCR、空斑试验、免疫荧光和蛋白质印迹（Western blot）等多种方法验证。体外实验确认Y041-1672为最优抗病毒候选分子，加药时间点实验表明其抑制作用主要发生在病毒复制阶段。本研究成功筛选出可用于PEDV抗病毒药物开发的骨架分子，为猪流行性腹泻的治疗提供了策略性见解。
- 猪流行性腹泻病毒（PEDV）
- , 3CL蛋白酶
- , 虚拟筛选
- , 抗病毒

Identification of PEDV inhibitors targeting 3CL protease

Ang Tian ^a,b ,
Shutong Shi ^a,b ,
Siying Zou ^a,b ,
Shuaiyin Guan ^a,b ,
Hao Wu ^a,b ,
Zhen Li ^a,b ,
Huanchun Chen ^a,b ,
Yunfeng Song ^a,b,,

a. College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China;
b. National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, China

Corresponding author: Yunfeng Song, syf@mail.hzau.edu.cn
Received Date: 18 February 2025
Accepted Date: 16 June 2025

Abstract

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is a highly contagious gastrointestinal disease characterized by vomiting, diarrhea, and dehydration, with mortality rates approaching 100% among suckling piglets. The PEDV 3C-like protease (3CLpro) is essential for viral replication and regarded as a critical target for antiviral inhibitor development. In this study, we aimed to identify small-molecule inhibitors of PEDV by targeting 3CLpro. Virtual screening of 1.6 million compounds from the ChemDiv library identified four potential candidates. Molecular dynamics simulations, specifically analyzing RMSD, RMSF, and Rg, demonstrated increased structural stability of the compound-protease complexes compared to the monomeric enzyme. All compounds had low cytotoxicity in Vero cells (CC₅₀ > 200 μM). Fluorescence resonance energy transfer-based assays demonstrated dose-dependent inhibitory activity of the compounds against 3CLpro. Among the candidates, compound F366-0161 exhibited the weakest inhibition, with an IC₅₀ value of 151.5 μM. Two analogues, 3238-0395 (IC₅₀ of 121.4 μM) and L878-0493 (IC₅₀ of 123.6 μM), exhibited moderately enhanced activity. Y041-1672 was identified as the most effective inhibitor, with an IC₅₀ of 86.48 μM. In viral replication inhibition assays, Y041-1672 reduced PEDV replication, with an EC₅₀ of 17.97 μM and a selectivity index (SI) of 15.5 (CC₅₀/EC₅₀). These results were validated by RT-qPCR, plaque assays, immunofluorescence, and Western blot analyses. In vitro validation confirmed Y041-1672 as the optimal antiviral candidate, and time-of-addition experiments indicated that inhibition primarily occurred during viral replication. This study identifies scaffold molecules for PEDV antiviral drug development, providing strategic insights for PED treatment.
- Porcine epidemic diarrhea virus (PEDV)
- , 3CL protease
- , Virtual screening
- , Antiviral

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