Authors
Biswas, I., Wang, Q., McCann, J. T., Tchesnokov, E. P., Nguyen, L., Saini, M., Cantero, J., Revalde, J. L., Gotte, M., Renslo, A., Neitz, R. J., Arkin, M. R., Arnold, E., Ruiz, F. X.
Abstract
Enterovirus D68 (EV-D68) is a non-polio picornavirus that has caused increasing rates of severe respiratory illness and acute flaccid myelitis in children worldwide this century. There are no approved vaccines or antivirals for EV-D68. Thus, we conducted a crystallographic fragment screening (CFS) and a high-throughput screening (HTS) biochemical assay against the EV-D68 RNA-dependent RNA polymerase 3D (3Dpol) to identify ligandable sites and non-nucleoside compounds that can spearhead anti-enteroviral drug discovery. The CFS, involving 650 fragments, identified 68 hit compounds (~10% hit rate) distributed across 3Dpol, including the functionally relevant sites RNA template channel, Active site, and RNA primer channel, and the previously unknown "Thumb site II" and "Index-middle finger pocket". Inhibition assays confirmed that compounds binding to each site can inhibit EV-D68 3Dpol activity. The HTS, a fluorescence-based PicoGreen biochemical assay, permitted screening 50,000 compounds of the ChemBridge Premium Library (0.77% hit rate). After a second-round dose-response screening, we identified 5-aminoindazole as a promising scaffold that inhibits EV-D68 3Dpol, including hit-to-lead compound 727590, which displayed an IC50 value of 25 {micro}M and preliminary structure-activity relationships. These hits offer amenable starting points for discovery and development of non-nucleoside inhibitors and provide opportunities for structure-based drug design against enteroviruses.
Preprint server:
bioRxiv
The authors list and abstract were imported from bioRxiv on 12 Jul 2026.
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