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-V54A 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 R523062 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 R523062 with a confirmed mechanism of action by targeting the viral 2C protein.