Hand, foot and mouth disease (HFMD) is a serious, highly contagious disease. HFMD caused by Enterovirus 71 (EV71), results in severe complications and even death. The pivotal role of EV71 3C^pro in the viral life cycle makes it an attractive target for drug discovery and development to treat HFMD. In this study, we identified novel EV71 3C^pro inhibitors by docking-based virtual screening. Totally 50 compounds were selected to test their inhibitory activity against EV71 3C^pro. The best inhibitor DC07090 exhibited the inhibition potency with an IC₅₀ value of 21.72 ± 0.95 μM without apparent toxicity (CC₅₀ > 200 μM). To explore structure-activity relationship of DC07090, 15 new derivatives were designed, synthesized and evaluated in vitro enzyme assay accordingly. Interestingly, four compounds showed inhibitory activities against EV71 3C^pro and only DC07090 inhibited EV71 replication with an EC₅₀ value of 22.09 ± 1.07 μM. Enzyme inhibition kinetic experiments showed that the compound was a reversible and competitive inhibitor. The Ki value was determined to be 23.29 ± 12.08 μM. Further molecular docking, MD simulation and mutagenesis studies confirmed the binding mode of DC07090 and EV71 3C^pro. Besides, DC07090 could also inhibit coxsackievirus A16 (CVA16) replication with an EC₅₀ value of 27.76 ± 0.88 μM. Therefore, DC07090 represents a new non-peptidyl small molecule inhibitor for further development of antiviral therapy against EV71 or other picornaviruses.

手足口病（HFMD）是一种严重的，高度传染性的疾病。肠道病毒71（EV71）引起的手足口病会导致严重的并发症甚至死亡。EV71 3C ^pro在病毒生命周期中的关键作用使其成为治疗HFMD的药物发现和开发的有吸引力的目标。在这项研究中，我们通过基于对接的虚拟筛选确定了新型EV71 3C ^pro抑制剂。总共选择了50种化合物来测试它们对EV71 3C ^pro的抑制作用。最好的抑制剂DC07090表现出抑制作用，IC ₅₀值为21.72± 0.95μM，无明显毒性（CC ₅₀ > 200μM）。为了探索DC07090的构效关系，设计了15种新的衍生物，进行了合成和体外酶评价。有趣的是，四种化合物显示出对EV71 3C ^pro的抑制活性，只有DC07090抑制了EV71复制，EC ₅₀值为22.09± 1.07μM 。酶抑制动力学实验表明该化合物是可逆的竞争性抑制剂。Ki值确定为23.29±12.08μM。进一步的分子对接，MD模拟和诱变研究证实了DC07090和EV71 3C ^pro的结合模式。此外，DC07090还可以通过EC ₅₀抑制柯萨奇病毒A16（CVA16）复制值为27.76±0.88μM。因此，DC07090代表了一种新的非肽基小分子抑制剂，可用于进一步开发针对EV71或其他小核糖核酸病毒的抗病毒治疗。