Enterovirus D68 (EV-D68), a viral pathogen associated with moderate to severe respiratory illness in children, also infects the nervous system in rare cases and causes neurological complications such as polio-like acute flaccid myelitis (AFM). Despite a significant disease burden, no approved antiviral drugs or vaccines are available for EV-D68. Due to its medical importance, EV-D68 is currently listed as a priority pathogen by the National Institute of Allergy and Infectious Diseases. Because the occurrence of the next EV-D68 outbreak cannot be predicted, the need for a potent, broadly acting, and safe EV-D68 inhibitor is urgent. The objectives of this proposal are to explore novel viral proteins as antiviral drug targets and to develop corresponding chemical probes as tools for target validation. This proposal is built upon our recent discovery that the EV-D68 2A protein exerts cysteine protease activity that is specific for the viral polyprotein VP1-2A junction and its protease activity can be inhibited by an FDA-approved oral drug, telaprevir. We further showed that telaprevir had submicromolar to low micromolar potency against several contemporary human EV-D68 strains from clades A and B in different human cell lines, including human neuronal cells. The antiviral potency of telaprevir (IC50) against EV-D68 was similar to that of HCV, indicating clinical relevance of repurposing telaprevir as an EV-D68 antiviral. The mechanism underlying the inhibition of the EV-D68 virus by telaprevir was independently elucidated using a serial viral passage experiment, leading to the selection of an N84T mutation near the active site of EV-D68 2Apro protein that reduced the potency of telaprevir in enzymatic and cellular antiviral assays. To validate the hypothesis that EV-D68 2Apro is essential for viral replication both in vitro and in vivo, our goals are to develop 2Apro inhibitors with favorable pharmacokinetic properties and to use them as chemical probes to validate EV-D68 2Apro as a viable antiviral drug target in cell culture as well as in a neonatal mouse model of EV-D68-induced paralytic disease. Specifically, we propose to design more potent and selective EV- D68 2Apro inhibitors through structure-activity relationship studies of telaprevir. All designed compounds will be systematically tested in in vitro enzymatic and cellular assays and profiled for pharmacokinetic properties. Prioritized lead compounds will be selected to test in EV-D68 mouse models. The expected outcomes of the proposed research are to validate EV-D68 2Apro as a viable antiviral drug target and demonstrate the in vivo antiviral efficacy of EV-D68 2Apro inhibitors.