{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T16:35:09Z","timestamp":1785429309422,"version":"3.56.0"},"reference-count":36,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T00:00:00Z","timestamp":1777507200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100006769","name":"Russian Science Foundation","doi-asserted-by":"publisher","award":["24-75-00117"],"award-info":[{"award-number":["24-75-00117"]}],"id":[{"id":"10.13039\/501100006769","id-type":"DOI","asserted-by":"publisher"}]},{"award":["24-75-00117"],"award-info":[{"award-number":["24-75-00117"]}],"id":[{"id":"https:\/\/ror.org\/03y2gwe85","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biosensors"],"abstract":"<jats:p>A cell-based screening system for viral protease inhibitors was developed using firefly luciferase fragment complementation and validated on the SARS-CoV-2 3CLpro model. The optimal luciferase variant incorporating the VLQSGF proteolytic site (Luc III) retained 88% of its native activity. A critical requirement for system performance was the use of an extended nsp4\u2013nsp6 fragment of the viral polyprotein rather than the mature protease, underscoring the importance of the native context for 3CLpro activity. The bicistronic construct pCAG-Luc-III-IRES-nsp4-6 enables coordinated expression of the reporter and protease, thereby increasing assay reproducibility. IC50 values obtained in this system for nirmatrelvir and GC376 correlated with live-virus assay data but differed significantly from those of a cell-free FRET assay, reflecting the impact of cellular barriers. This approach combines simplicity, a standard substrate, and high reproducibility, making it promising for high-throughput screening in basic laboratory settings and adaptable to other viral proteases.<\/jats:p>","DOI":"10.3390\/bios16050253","type":"journal-article","created":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T10:47:28Z","timestamp":1777546048000},"page":"253","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Cell-Based Luciferase Assay for Testing SARS-CoV-2 3CL Protease Inhibitors"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8023-4453","authenticated-orcid":false,"given":"Dmitry N.","family":"Shcherbakov","sequence":"first","affiliation":[{"name":"State Research Center of Virology and Biotechnology VECTOR, Rospotrebnadzor, 630559 Novosibirsk, Russia"},{"name":"Research Institute of Biological Medicine Center for Recombinant Technologies, Altay State University, 656049 Barnaul, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ekaterina D.","family":"Mordvinova","sequence":"additional","affiliation":[{"name":"State Research Center of Virology and Biotechnology VECTOR, Rospotrebnadzor, 630559 Novosibirsk, Russia"},{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, Academician Lavrent\u2019ev Ave. 9, 630090 Novosibirsk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vadim O.","family":"Trufanov","sequence":"additional","affiliation":[{"name":"State Research Center of Virology and Biotechnology VECTOR, Rospotrebnadzor, 630559 Novosibirsk, Russia"},{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, Academician Lavrent\u2019ev Ave. 9, 630090 Novosibirsk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5010-9424","authenticated-orcid":false,"given":"Natalia V.","family":"Volkova","sequence":"additional","affiliation":[{"name":"State Research Center of Virology and Biotechnology VECTOR, Rospotrebnadzor, 630559 Novosibirsk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5935-2540","authenticated-orcid":false,"given":"Yulia V.","family":"Meshkova","sequence":"additional","affiliation":[{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, Academician Lavrent\u2019ev Ave. 9, 630090 Novosibirsk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9500-1713","authenticated-orcid":false,"given":"Maria K.","family":"Marenina","sequence":"additional","affiliation":[{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, Academician Lavrent\u2019ev Ave. 9, 630090 Novosibirsk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna V.","family":"Zaykovskaya","sequence":"additional","affiliation":[{"name":"State Research Center of Virology and Biotechnology VECTOR, Rospotrebnadzor, 630559 Novosibirsk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ekaterina A.","family":"Volosnikova","sequence":"additional","affiliation":[{"name":"State Research Center of Virology and Biotechnology VECTOR, Rospotrebnadzor, 630559 Novosibirsk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8481-0470","authenticated-orcid":false,"given":"Sophia S.","family":"Borisevich","sequence":"additional","affiliation":[{"name":"Synchrotron Radiation Facility, Siberian Circular Photon Source \u201cSKlF\u201d Boreskov Institute Catalysis, Siberian Branch of the Russian Academy of Sciences, Nikolskiy pr-t, 1, Koltsovo, 630559 Novosibirsk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3883-6335","authenticated-orcid":false,"given":"Svetlana V.","family":"Belenkaya","sequence":"additional","affiliation":[{"name":"State Research Center of Virology and Biotechnology VECTOR, Rospotrebnadzor, 630559 Novosibirsk, Russia"},{"name":"N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, Academician Lavrent\u2019ev Ave. 9, 630090 Novosibirsk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"4609","DOI":"10.1021\/cr010184f","article-title":"Viral Proteases","volume":"102","author":"Tong","year":"2002","journal-title":"Chem. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Swanstrom, R., Anderson, J., Schiffer, C., and Lee, S.K. (2009). Viral Protease Inhibitors. Antiviral Strategies, Springer. 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