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.
