Pharmacological Characterization of the Mechanism of Action of R523062, a Promising Antiviral for Enterovirus D68

Enterovirus D68 (EV-D68) is a re-emerging virus that causes moderate to severe respiratory diseases in children. In severe cases, EV-D68 infection can lead to neurological complications called acute flaccid myelitis (AFM). There is currently no antiviral or vaccine available for EV-D68. The goal of this study is to delineate the mechanism of action of a promising antiviral drug candidate R523062 that was identified through a phenotypic cytopathic effect (CPE)-based high-throughput screening. R523062 inhibits multiple contemporary EV-D68 strains with single-digit micromolar EC50 values and is less effective against the enterovirus A71 strains. Resistant mutants identified through serial viral passage experiments were mapped to four viral proteins including VP1-G178S, 2A-V112I, 2C-I227L/Q322R, and 3A-V54A. The involvements of VP1-G178S, 2A-V112I, and 3A-VS4A mutants in drug resistance were ruled out by the drug time-of-addition experiment, protease enzymatic assay, and the plaque assay with recombinant virus, respectively. In contrast, recombinant virus encoding the 2C-I227L/Q322R double mutants confers significant drug resistance, which is consistent with the result from serial passage experiments. The thermal shift binding assay showed R.523062 binds to the wild-type EV-D68 2C and 2C-Q322R but not 2C-I227L or 2C-I227L/Q322R, confirming 2C as the direct drug target of R523062 and 2C-I227L alone confers drug resistance. The 2C inhibitor R523062 also showed additive antiviral activity with the viral 2A protease inhibitor telaprevir as well as the viral capsid VP1 inhibitor R856932. Collectively, this study identified a promising EV-D68 antiviral drug candidate 8523062 with a confirmed mechanism of action by targeting the viral 2C protein.