Project Abstract: Certain viruses in the picornaviridae family, specifically enterovirus-D68 (EV68), have emerged as global health concerns over the last decade with severe symptomatic infections with EV68 able to result in long lasting neurological deficits and death. There are currently no US Food and Drug Administration approved drugs for any non-polio enterovirus, highlighting the need to develop strategies against these lethal enteroviral strains. One particularly attractive class of potential drugs are small molecules inhibitors, which can act as direct-acting antiviral (DAA) inhibitors towards the conserved active site of EV68 3C protease. This main viral protease is a cysteine protease conserved in the 3C family, responsible for cleaving eight sites along the viral polyprotein, which is essential for viral propagation. DAAs designed to target 3C proteases can potentially achieve robust inhibition across enterovirus species. However, as drug resistance in viruses can be prevalent, it is paramount to design inhibitors less susceptible to resistance mutations. It was demonstrated previously in the Schiffer Lab that when bound to protease, viral substrates occupy a conserved three-dimensional volume called the substrate envelope. It was also demonstrated that inhibitors that extend beyond the substrate envelope are more susceptible to drug resistance mutations. By utilizing the substrate envelope and cocrystal structures of the proteases, DAAs designed to fit within the three-dimensional consensus volume as naturally occurring substrates will interact primarily with functionally important residues and be less susceptible to drug resistance mutations. The central hypothesis of this proposal is that cocrystallization of EV68 3C protease with its natural substrates will enable the calculation of the substrate envelope to inform on substrate specificity, which will also aid in the design of robust pan-3C-protease inhibitors. In Aim 1, I will determine the cocrystal structures of EV68 3C protease bound to viral substrates. I will then use these structures to elucidate the molecular mechanism of substrate specificity for EV68 3C protease and calculate the substrate envelope. These data will aid in small- molecule design to create DAAs with improved resilience to mutations that can confer drug resistance. In Aim 2, I will design and test novel DAAs that target EV68 3C protease. I will first characterize previously designed inhibitors for other 3C and 3C-like proteases with the substrate envelope to establish a starting compound based on potency. Inhibitors based on the scaffold will be designed, synthesized, and tested in a FRET-based enzyme inhibition assay. Crystallization of novel potent compounds with EV68 3C and their characterization within the substrate envelope will assess inhibitors’ susceptibility to drug resistance mutations. Overall, this study aims to develop a robust, novel compounds with resistance-thwarting protease inhibition against the emerging pathogen that is EV68.