Identification and biochemical characterization of DC07090 as a novel potent small molecule inhibitor against human...

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.