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Scientific Reports

Scientific Reports

IF: 3.8
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Construction and validation of a cell based reporter assay for identifying inhibitors of SARS coronavirus 2 RNA dependent RNA polymerase activity

Published:26 May 2025 DOI: 10.1038/s41598-025-03813-y PMID: 40419748
Eunjeong Kang,?Haelim Yoon,?Junho Lee,?JinAh Lee,?Seungtaek Kim,?Inseong Jo,?Soo Bong Han,?Dae Gwin Jeong,?Sayeon Cho

Abstract

Targeting RNA-dependent RNA polymerase (RdRp), a highly conserved enzyme essential for SARS coronavirus 2 (SARS-CoV-2) replication and transcription, represents a promising antiviral strategy due to its lower mutation rate than structural proteins such as Spike. This study introduces a cell-based assay system for screening potential SARS-CoV-2 RdRp inhibitors, contributing to ongoing efforts to identify effective antiviral agents. The assay utilizes a reporter vector containing the 3' untranslated region (UTR), luciferase reporter gene, and 5' UTR gene, sequentially arranged in reverse under the control of the cytomegalovirus promoter in the pcDNA3.1 vector. Co-transfection with SARS-CoV-2 RdRp resulted an increase in luminescence-based quantification of RdRp activity, achieving a Z-factor of 0.605, indicative of high reproducibility and reliability for high-throughput screening. Established RdRp inhibitors, including remdesivir, molnupiravir, tenofovir, and sofosbuvir, significantly reduced reporter activity, with remdesivir exhibiting the strongest inhibition. A newly identified RdRp inhibitor was further validated through primer extension polymerase and NMPylation assays, along with virus-based experiments, confirming its inhibitory mechanism. These results highlight the utility of this screening system in identifying effective RdRp-targeting antivirals, reinforcing the strategic importance of RdRp inhibition in combating SARS-CoV-2 and emerging variants.

Substances (16)

Materials
Procduct Name CAS Molecular Formula Supplier Price
Tenofovir 147127-20-6 C9H14N5O4P 694 suppliers $5.00-$1710.00
Tenofovir 147127-20-6 C9H14N5O4P 694 suppliers $5.00-$1710.00
Tenofovir 147127-20-6 C9H14N5O4P 694 suppliers $5.00-$1710.00
Tenofovir 147127-20-6 C9H14N5O4P 694 suppliers $5.00-$1710.00
Sofosbuvir 1190307-88-0 C22H29FN3O9P 615 suppliers $16.00-$3990.00
Sofosbuvir 1190307-88-0 C22H29FN3O9P 615 suppliers $16.00-$3990.00
Sofosbuvir 1190307-88-0 C22H29FN3O9P 615 suppliers $16.00-$3990.00
Sofosbuvir 1190307-88-0 C22H29FN3O9P 615 suppliers $16.00-$3990.00
Molnupiravir 2349386-89-4 C13H19N3O7 314 suppliers $35.00-$46000.00
Molnupiravir 2349386-89-4 C13H19N3O7 314 suppliers $35.00-$46000.00

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