Year: 2026
Type: article
Abstract: 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–nsp6 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.
Source: Biosensors
DOI: 10.3390/bios16050253
Institutions: Altai State University, State Research Center of Virology and Biotechnology VECTOR, Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Novosibirsk Institute of Organic Chemistry, Boreskov Institute of Catalysis, Siberian Branch of the Russian Academy of Sciences
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,654
Funder: Russian Science Foundation
Award: G7341920508
OpenAlex ID: W7160025291