Advancing Enterovirus Antiviral Discovery: A New Assay and Potent 2C Inhibitor Show Promise Against Paralysis
- Ray Sullivan
- Aug 29
- 4 min read
Enteroviruses, including EV-D68, EV-A71, and Coxsackievirus B3 (CVB3), are a significant global health threat, causing a spectrum of diseases from mild respiratory illness to severe neurological complications like acute flaccid myelitis. Despite their impact, there are currently no FDA-approved antiviral treatments for enterovirus infections. This pressing need has driven researchers to seek novel therapeutic targets, with the highly conserved 2C protein emerging as a prime candidate due to its essential role in viral replication and minimal similarity to host proteins. Recent work from Li, Demssie & Wang at Rutgers School of Pharmacy, addresses bottlenecks in enterovirus drug discovery by developing a robust fluorescence polarization (FP) assay for 2C inhibitors and identifying Jun6504, a rationally designed 2C inhibitor, with demonstrated in vivo efficacy against EV-D68-induced paralysis in a mouse model.
One major challenge in targeting the 2C protein has been the lack of suitable, high-throughput assays for inhibitor screening and characterization. Previous methods, such as the ATPase assay, suffered from intrinsically low activity with truncated 2C proteins, leading to a lack of observable inhibition for many reported compounds. Differential scanning fluorimetry or thermal shift assays lacked the sensitivity needed for quantitative binding affinity measurements.
To overcome these limitations, Li, Demssie, and Wang designed and validated a novel FP assay using a rationally developed probe, Jun14157. This probe was based on Jun1377, a potent pyrazolopyridine-based 2C allosteric inhibitor. The FP assay operates on a straightforward principle: an unbound Jun14157 probe rotates freely, exhibiting low polarization, but upon binding to the 2C allosteric site, its motion is restricted, leading to a high polarization signal. A competing inhibitor then displaces Jun14157, resulting in a decrease in the polarization signal.
The assay was meticulously optimized, demonstrating high reproducibility, excellent DMSO tolerance (up to 30%), and the ability to quantify binding affinities across various enteroviral 2C proteins (EV-D68, EV-A71, and CVB3). Its specificity was confirmed by its reduced binding affinity to drug-resistant 2C mutants (D183V and F190L), which are known to affect inhibitor binding at the allosteric pocket. The FP assay exhibited a strong correlation between inhibition constant (Ki) values and cellular antiviral EC50 values, the concentration of a drug which induces a response halfway between the baseline and maximum effect after a specified exposure time. This predictive capability significantly enhances its value for early-stage hit validation. Furthermore, the assay’s suitability for high-throughput screening was affirmed by a Z' factor of 0.69, well within the benchmark for robust screening assays. The Z′ factor is a statistical parameter used to evaluate and validate the quality and suitability of high-throughput screening assays. It provides a measure of the dynamic range, data variation, and overall robustness of an assay, making it a benchmark in drug discovery and other biological screening efforts. This platform led to the discovery and validation of two novel "Y"-shaped symmetric 2C inhibitors, Jun15716 and Jun15799, from a virtual screening campaign.
Building on the understanding of 2C inhibition, a separate study by Li, Rudy, Hu, Tan, et al., reported the rational design and characterization of Jun6504, a 2C inhibitor that demonstrates in vivo antiviral efficacy. While several 2C inhibitors had shown in vitro activity, few had translated to in vivo success in animal models.
Jun6504 emerged from lead optimization efforts aimed at improving the mouse liver microsomal stability of an earlier pyrazolopyridine compound, Jun571, while retaining the critical ionic hydrogen bonding interactions with key aspartate residues in the 2C allosteric pocket. Jun6504 exhibited potent, broad-spectrum antiviral activity against a wide array of enteroviruses, including multiple strains of EV-D68, EV-A71, CVB3, CVA6, CVA16, and poliovirus, with EC50 values typically in the low micromolar range. Its favorable in vitro and in vivo pharmacokinetic properties, including rapid absorption and suitable half-life, made it an ideal candidate for animal model studies.
The mechanism of action of Jun6504 was thoroughly elucidated through molecular dynamics simulations, thermal shift assays, and serial viral passage experiments for drug resistance. These studies confirmed that Jun6504 binds to the same allosteric pocket in the 2C protein as (S)-fluoxetine. Key mutations like D183V and F190L were identified as conferring drug resistance, highlighting their importance in the binding site.
Jun6504 demonstrated remarkable in vivo efficacy in a neonatal mouse model of EV-D68 infection. Treated mice showed significantly lower paralysis scores and increased body weight gain, even when treatment was initiated 24 hours post-infection. Mechanistically, Jun6504 treatment led to a significant reduction in viral titers in both the spinal cord and the infected quadriceps muscle. The study further confirmed that Jun6504 can cross the blood-brain barrier, reaching the spinal cord where it can exert its antiviral effects. Immunohistochemical analysis revealed that Jun6504 treatment preserved choline acetyltransferase-positive motor neurons and neuronal nuclei-positive neurons in the lumbar spinal cord, thereby mitigating neuronal damage and preserving motor function. This neuroprotective effect was linked to the drug markedly slowing the anterior progression of infection within the spinal cord.
Together, these two studies represent significant advancements in enterovirus antiviral drug discovery. The development of the FP assay provides a much-needed, high-sensitivity, and broadly applicable tool for quantifying binding affinities of 2C allosteric inhibitors, accelerating the screening and characterization of new compounds. Concurrently, the identification and in vivo validation of Jun6504 offer a promising candidate for further development as an EV-D68 antiviral, demonstrating that targeting the viral 2C protein can lead to effective treatments that combat both viral replication and disease progression, including paralysis. This integrated approach of assay development and rational inhibitor design sets a strong foundation for future enterovirus drug discovery efforts.
Li K, Demssie HA, Wang J. Design of a Fluorescence Polarization Probe for Enterovirus 2C Proteins. J Med Chem. 2025 Jul 10;68(13):14041-14053. doi: 10.1021/acs.jmedchem.5c01219. Epub 2025 Jun 21. PMID: 40542722; PMCID: PMC12333363.
Li K, Rudy MJ, Hu Y, Tan H, Lambrinidis G, Wu X, Georgiou K, Tan B, Frost J, Wilson C, Clarke P, Kolocouris A, Zhang QY, Tyler KL, Wang J. A rationally designed 2C inhibitor prevents enterovirus D68-infected mice from developing paralysis. Nat Commun. 2025 Jul 1;16(1):5987. doi: 10.1038/s41467-025-61083-8. PMID: 40593720; PMCID: PMC12216181.