Enterovirus 2C Protein Drug Design, Assay Development, and Drug Resistance Studies

In this Account, we describe our efforts to establish enteroviral 2C as a tractable target for antiviral drug discovery by integrating medicinal chemistry, molecular pharmacology, resistance profiling, structural modeling, and assay development. Rather than relying solely on conventional phenotypic high-throughput screening, our initial studies focused on the rational optimization of reported 2C inhibitors identified from a drug-repurposing campaign. This strategy led to quinoline-based antivirals with substantially improved potency and selectivity against enterovirus D68 (EV-D68), providing early medicinal chemistry insights into ligand recognition by enteroviral 2C and supporting the feasibility of chemically targeting this protein. Building on these findings, we subsequently developed pyrazolopyridine-based inhibitors, which evolved into one of the most potent classes of enteroviral 2C inhibitors reported to date. Mechanistic studies combining differential scanning fluorimetry (DSF), serial viral passage, reverse genetics, and molecular modeling supported direct engagement of a conserved allosteric pocket corresponding to the previously defined (S)-fluoxetine binding site. Structure-guided optimization led to Jun6504, a rationally designed 2C inhibitor with improved metabolic stability, favorable pharmacokinetic properties, and robust in vivo efficacy in a neonatal mouse model of EV-D68-induced paralysis. Collectively, these studies provided strong pharmacological validation of 2C as a therapeutically actionable antiviral target. In parallel, we recognized that the absence of direct, scalable biochemical assays was a major impediment to 2C-targeted antiviral discovery. To address this bottleneck, we developed a fluorescence polarization-based binding assay that monitors ligand engagement of the conserved allosteric pocket in enteroviral 2C proteins. Using Jun14157, a fluorescent probe derived from the pyrazolopyridine inhibitor series, this platform enabled direct, quantitative, and high-throughput characterization of ligand-2C interactions across EV-D68, EV-A71, and CVB3. Coupling this assay with structure-based virtual screening further enabled the identification of new allosteric inhibitor scaffolds and established a generalizable discovery platform for enteroviral 2C inhibitors. Together, these studies define an integrated framework for enteroviral 2C antiviral discovery, spanning hit-to-lead optimization, mechanism of action studies, resistance mapping, pharmacological validation, and assay innovation. Continued advances in inhibitor-bound structural elucidation, broad-spectrum inhibitor design, and next-generation therapeutic strategies should further accelerate the development of clinically useful antivirals targeting enteroviral 2C proteins.