Abstract
Enterovirus D68 (EV-D68) is a respiratory viral pathogen that primarily infects children under the age of 8. Although EV-D68 infection typically leads to moderate to severe respiratory illnesses, recent years have seen increasing cases of EV-D68 triggered neurological complications such as acute flaccid myelitis (AFM). There is currently no vaccine or antiviral available for EV-D68; we therefore aimed to develop potent and specific small molecule antivirals against EV-D68. In this study, we report our discovery of a viral capsid inhibitor R856932 that inhibits multiple contemporary EV-D68 strains with single-digit to submicromolar efficacy. Mechanistic studies have shown that the tetrazole compound R856932 binds to the hydrophobic pocket of viral capsid protein VP1, thereby preventing viral uncoating and release of viral genome in the infected cells. The mechanism of action of R856932 was confirmed by time-of-addition, Western blot, RT-qPCR, viral heat inactivation, serial viral passage, and reverse genetics experiments. A single mutation located at VP1, A129V, confers resistance against R856932. However, a recombination virus encoding VP1-A129V appeared to have compromised fitness of replication compared to the wild-type EV-D68 virus as shown by the competition growth assay. Overall, the hit compound identified in this study, R856932, represents a promising starting point with a confirmed mechanism of action that can be further developed into EV-D68 antivirals.