African swine fever (ASF) is a viral disease in swine that results in high mortality in domestic pigs and causes considerable economic losses. Currently, there is no effective vaccine or drugs available for treatment. Identification of new anti-ASFV drugs is urgently needed. Here, the pS273R protein of the African swine fever virus (ASFV) is a specific SUMO-1-like cysteine protease that plays an important role in its replication process. To inhibit virus replication and improve treatment options, a set of small-molecule compounds, targeted inhibitors against the ASFV pS273R protease, were obtained through molecular screening by homology modeling and molecular docking based on structural information of pS273R. Our results clearly demonstrated that the 14th carbon atom of the cysteinase inhibitor E-64 could form one CS covalent bond with the Cys 232 amino acid of the pS273R protease and seven additional hydrogen bonds to maintain a stable binding state. Simultaneously, cell viability, immunophenotyping, and in vitro enzyme activity inhibition assays were performed to comprehensively evaluate E-64 characteristics. Our findings demonstrated that 4 mmol/L E-64 could effectively inhibit the enzyme activity center of the pS273R protease by preventing pS273R protease from lysing pp62, while promoting the upregulation of immune-related cytokines at the transcription level. Moreover, cell viability results revealed that 4 mmol/L E-64 was not cytotoxic. Taken together, we identified a novel strategy to potentially prevent ASFV infection in pigs by blocking the activity of pS273R protease with a small-molecule inhibitor.

非洲猪瘟 (ASF) 是一种猪的病毒性疾病，可导致家猪的高死亡率并造成相当大的经济损失。目前，没有有效的疫苗或药物可用于治疗。迫切需要鉴定新的抗 ASFV 药物。在这里，非洲猪瘟病毒 (ASFV) 的 pS273R 蛋白是一种特定的 SUMO-1 样半胱氨酸蛋白酶，在其复制过程中起重要作用。为了抑制病毒复制和改善治疗方案，基于 pS273R 的结构信息，通过同源建模和分子对接的分子筛选，获得了一组小分子化合物，针对 ASFV pS273R 蛋白酶的靶向抑制剂。我们的结果清楚地表明，半胱氨酸酶抑制剂 E-64 的第 14 个碳原子可以形成一个 CS 与 pS273R 蛋白酶的 Cys 232 氨基酸和七个额外的氢键共价键，以保持稳定的结合状态。同时，进行细胞活力、免疫表型和体外酶活性抑制试验以综合评估 E-64 特性。我们的研究结果表明，4 mmol/L E-64 可以通过阻止 pS273R 蛋白酶裂解 pp62 来有效抑制 pS273R 蛋白酶的酶活性中心，同时在转录水平上促进免疫相关细胞因子的上调。此外，细胞活力结果显示 4 mmol/L E-64 没有细胞毒性。总之，我们确定了一种新策略，通过用小分子抑制剂阻断 pS273R 蛋白酶的活性来潜在地预防猪的 ASFV 感染。