Structural basis for substrate-assisted catalysis and small-molecule inhibition of alphavirus macrodomains

Structural basis for substrate-assisted catalysis and small-molecule inhibition of alphavirus macrodomains | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Structural basis for substrate-assisted catalysis and small-molecule inhibition of alphavirus macrodomains Andrzej Joachimiak, Youngchang Kim, Nazar Gora, Changsoo Chang, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8652402/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Sporadic alphavirus infections are pandemic threats that evade innate immunity via macrodomains which remove host mono-ADP-ribose (ADPr). Through high-resolution crystallography of apo and ADPr-bound forms, we here define a conserved MacroD-like fold and a shared hydrogen-bonding network for macrodomains from Eastern equine encephalitis (EEEV), Venezuelan equine encephalitis (VEEV), and Chikungunya (CHIKV) alphaviruses. Our detailed active site structures support a substrate-assisted hydrolysis mechanism where the ADPr α-phosphate activates the catalytic water for nucleophilic attack. Furthermore, we identify the suramin trisulfonic moiety as a potent inhibitor. Crystal structures with solution-state SAXS show suramin occupies the ADPr-binding pocket and induces reversible, inhibitor-mediated oligomerization. Biophysical and enzymatic assays confirm low-micromolar inhibition of EEEV glycohydrolase activity, while cell-based trans-replication assays demonstrate broad-spectrum suppression of EEEV, VEEV, and CHIKV replication. Collectively, our findings define the structural basis for alphaviral macrodomain catalysis, key alphaviral macrodomain druggable features, and a promising scaffold for targeted broad-spectrum antiviral therapeutics. Health sciences/Molecular medicine Biological sciences/Drug discovery/Medicinal chemistry/Structure-based drug design alphaviruses macrodomain crystal structure solution studies ligand binding Full Text Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8652402","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":581943903,"identity":"7e85b7fe-fa66-4916-97a8-8a6f4b7de0bd","order_by":0,"name":"Andrzej 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