{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T07:41:39Z","timestamp":1792741299209,"version":"4.5.0"},"reference-count":29,"publisher":"Portland Press Ltd.","issue":"11","license":[{"start":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T00:00:00Z","timestamp":1791417600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/2.0\/"}],"funder":[{"DOI":"10.13039\/501100001412","name":"Council of Scientific and Industrial Research, India","doi-asserted-by":"publisher","award":["HCP0041"],"award-info":[{"award-number":["HCP0041"]}],"id":[{"id":"10.13039\/501100001412","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004507","name":"CSIR | Central Drug Research Institute","doi-asserted-by":"publisher","award":["MLP007"],"award-info":[{"award-number":["MLP007"]}],"id":[{"id":"10.13039\/501100004507","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["portlandpress.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2026,11,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The SARS-CoV-2 nonstructural protein 15 (NSP15) is a uridine-specific endoribonuclease involved in viral RNA processing and immune evasion, thereby contributing to viral pathogenesis. Although NSP15 has been structurally characterized in several studies, which report its endoribonuclease activity and regulatory features, important functional and regulatory gaps remain unaddressed, including quantitative stability, metal ion regulation, and inhibitor-binding kinetics. To address these gaps, we used an integrated approach to investigate NSP15 regulation and inhibition. Thermochemical stability studies indicated moderate NSP15 stability, with cooperative unfolding characterized by a Cm of 1.68 M urea and a Tm of \u223c70\u00b0C. Urea\u2013PAGE activity measurements revealed a strong metal ion dependence, with Mn2+ as the optimal activator and marked stimulation compared with Ca2+, Mg2+, and Zn2+. Biophysical experiments using SPR and fluorescence quenching confirmed a direct protein\u2013ligand interaction with dissociation constants of \u223c1\u20134\u00a0\u03bcM. Consistent with these results, the compounds inhibited endoribonuclease activity with IC50 values of \u223c8\u201330\u00a0\u03bcM. Alanine substitution of catalytic residues H235A and H250A completely abolished cleavage, while S294A within the B1 pocket, a uridine-specificity determinant, reduced activity by 51%\u201367% (10\u201350\u00a0nM), markedly weakened inhibitor binding, and increased inhibition susceptibility. Importantly, docking and simulation studies revealed stabilized active-site dynamics driven by strong polar interactions, as evidenced by solvent-accessible surface area, hydrogen-bonding, RMSD, RMSF, and radius of gyration (Rg) analyses, underscoring the coordinated contribution of polar contacts and hydrophobic interactions in the inhibitor-bound active-site conformation. Collectively, the present work provides quantitative, mutational, and functional characterization, forming a foundation for designing NSP15-specific antivirals.<\/jats:p>","DOI":"10.1042\/bcj20260313","type":"journal-article","created":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T13:43:27Z","timestamp":1791467007000},"page":"2163-2182","update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Functional and mechanistic characterization of SARS-CoV-2 NSP15 reveals metal-dependent regulation and inhibition by direct-binding small molecules"],"prefix":"10.1042","volume":"483","author":[{"given":"Hira\u00a0Singh","family":"Gariya","sequence":"first","affiliation":[{"name":"1Prof. G.N. Ramachandran Structural Biology Laboratory, Division of Biochemistry and Structural Biology, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sanjay","family":"Kumar","sequence":"additional","affiliation":[{"name":"1Prof. G.N. Ramachandran Structural Biology Laboratory, Division of Biochemistry and Structural Biology, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India"},{"name":"2Academy of Scientific and Innovative Research, Ghaziabad 201002, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anurag","family":"Singh","sequence":"additional","affiliation":[{"name":"1Prof. G.N. Ramachandran Structural Biology Laboratory, Division of Biochemistry and Structural Biology, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raja\u00a0Mani","family":"Tripathi","sequence":"additional","affiliation":[{"name":"1Prof. G.N. Ramachandran Structural Biology Laboratory, Division of Biochemistry and Structural Biology, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India"},{"name":"2Academy of Scientific and Innovative Research, Ghaziabad 201002, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Imran","family":"Ansari","sequence":"additional","affiliation":[{"name":"1Prof. G.N. 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Ramachandran Structural Biology Laboratory, Division of Biochemistry and Structural Biology, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India"},{"name":"2Academy of Scientific and Innovative Research, Ghaziabad 201002, India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"288","published-online":{"date-parts":[[2026,10,23]]},"reference":[{"key":"2026102303370745500_B1","doi-asserted-by":"publisher","first-page":"157","DOI":"10.1016\/j.virol.2017.12.024","article-title":"An \u201cOld\u201d protein with a new story: coronavirus endoribonuclease is important for evading host antiviral defenses","volume":"517","author":"Deng","year":"2018","journal-title":"Virology"},{"key":"2026102303370745500_B2","doi-asserted-by":"publisher","first-page":"8094","DOI":"10.1073\/pnas.1921485117","article-title":"Coronavirus endoribonuclease targets viral polyuridine sequences to evade activating host sensors","volume":"117","author":"Hackbart","year":"2020","journal-title":"Proc. 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