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With current treatments addressing only a fraction of pathogens and new viral threats constantly evolving, there is a critical need to expand our existing therapeutic arsenal. To speed the rate of discovery and better prepare against future threats, we establish a high-throughput platform capable of screening compounds against 40 diverse viral proteases simultaneously. This multiplex approach is enabled by using cellular biosensors of viral protease activity combined with DNA-barcoding technology, as well as several design innovations that increase assay sensitivity and correct for plate-to-plate variation. Among >100,000 compound-target interactions explored within our initial screen, a series of broad-acting inhibitors against coronavirus proteases were uncovered and validated through orthogonal assays. A medicinal chemistry campaign was performed to improve one of the inhibitor's potency while maintaining its broad activity. This work highlights the power of multiplex screening to efficiently explore chemical space at a fraction of the time and costs of previous approaches., Competing Interests: Disclosure and competing interests statement. AC, SJH, and SJR have submitted a patent related to the design and implementation of multiplex small molecule screens. BRS is an inventor on patents and patent applications involving small molecules, holds equity in and serves as a consultant to Exarta Therapeutics and ProJenX Inc., holds equity in Sonata Therapeutics, and serves as a consultant to Weatherwax Biotechnologies Corporation and Akin Gump Strauss Hauer & Feld LLP. DDH is a co-founder of TaiMed Biologics and RenBio, consultant to WuXi Biologics, Brii Biosciences, Apexigen, and Veru Inc., and board director for Vicarious Surgical., (© 2025. The Author(s).)

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 1 . The rapid development of highly effective vaccines 2,3 against SARS-CoV-2 has altered the trajectory of the pandemic, and antiviral therapeutics 4 have further reduced the number of COVID-19 hospitalizations and deaths. Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses that encode various structural and non-structural proteins, including those critical for viral RNA replication and evasion from innate immunity 5 . Here we report the discovery and development of a first-in-class non-covalent small-molecule inhibitor of the viral guanine-N7 methyltransferase (MTase) NSP14. High-throughput screening identified RU-0415529, which inhibited SARS-CoV-2 NSP14 by forming a unique ternary S-adenosylhomocysteine (SAH)-bound complex. Hit-to-lead optimization of RU-0415529 resulted in TDI-015051 with a dissociation constant (K d ) of 61 pM and a half-maximal effective concentration (EC 50 ) of 11 nM, inhibiting virus infection in a cell-based system. TDI-015051 also inhibited viral replication in primary small airway epithelial cells and in a transgenic mouse model of SARS CoV-2 infection with an efficacy comparable with the FDA-approved reversible covalent protease inhibitor nirmatrelvir 6 . The inhibition of viral cap methylases as an antiviral strategy is also adaptable to other pandemic viruses., Competing Interests: Competing interests: The authors declare no competing interests. The Rockefeller University filed patent application PCT/US2024/012695 titled ‘Sulfonamide-1H-pyrrole-2-carboxamide inhibitors of SARS-CoV-2 NSP14 methyltransferase and derivatives thereof’., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)