Abstract
Enterovirus D68 (EV-D68) virus is a nonpolio enterovirus that typically causes respiratory illness and, in severe cases, can lead to paralysis and death in children. There is currently no vaccine or antiviral for EV-D68. We previously discovered the viral 2A protease (2A(pro)) as a viable antiviral drug target and identified telaprevir as a 2A(pro) inhibitor. 2A(pro) is a viral cysteine protease that cleaves the viral VP1-2A polyprotein junction. In this study, we report the X-ray crystal structures of EV-D68 2A(pro), wild-type, and the C107A mutant and the structure-based lead optimization of telaprevir. Guided by the X-ray crystal structure, we predicted the binding pose of telaprevir in 2A(pro) using molecular dynamics simulations. We then utilized this model to inform structure-based optimization of the telaprevir's reactive warhead and P1-P4 substitutions. These efforts led to the discovery of 2A(pro) inhibitors with improved antiviral activity than telaprevir. These compounds represent promising lead compounds for further development as EV-D68 antivirals.