# RNA-dependent RNA Polymerase Assays for Biochemical Characterization and Antiviral Drug Discovery

> **NIH NIH R21** · UNIVERSITY OF KENTUCKY · 2021 · $417,474

## Abstract

SARS-CoV-2 is a positive-sense, single-stranded RNA [(+)ssRNA] virus that relies on its RNA-dependent RNA
polymerase (RdRp) for survival. Within the months of April and May, five groups have independently reported
the production of recombinant SAR-CoV-2 RdRp and a preliminary activity assessment of this essential enzyme.
Included in these studies was direct evidence that the triphosphate version of the nucleoside analogue
remdesivir, which has received Emergency Use Authorization from the Food and Drug Administration to treat
SARS-CoV-2, is incorporated in place of ATP into the growing RNA oligonucleotide, ultimately leading to chain
termination. Other nucleobase and nucleoside analogues including EIDD-1931 and favipiravir, the latter of which
has been approved in Japan to treat the (-)ssRNA influenza virus, have demonstrated promise as therapeutic
agents against SARS-CoV-2 by likely interfering with RNA metabolism via inhibition or processing as alternative
substrates for RdRp. This and other data suggest that RdRp is an excellent target for the discovery and
development of novel SARS-CoV-2 therapeutics. However, one of the major bottlenecks in exploiting RdRp as
a drug target is the relatively low throughput activity-based assays that are costly, prone to interference, and/or
lack flexibility in the experimental design. The primary objective of this proposal is to develop a broadly applicable
RdRp activity-based assay that will be used for antiviral drug discovery efforts. Our specific aim is to establish a
novel, real-time assay using a five-enzyme coupled system with a colorimetric readout. The new assay will be
directly compared to the traditional polyacrylamide gel electrophoresis and a liquid scintillation proximity end-
point assay, and further validated with high resolution mass spectrometry. It is expected that, by accomplishing
this aim, the assay will enable a thorough biochemical characterization of SARS-CoV-2 RdRp and, for the first
time, enable the testing of synthetic compound and natural product libraries to identify inhibitors, alternative
substrates, modulators, or effectors of SARS-CoV-2 RdRp activity in a high throughput screening format. Notably,
the strategy implemented herein is expected to complement on-going structural-based anti-SARS-CoV-2
discovery efforts. Finally, the activity-based assay can be readily adapted for RdRp from other (+)ssRNA and (-
)ssRNA viruses in an effort to identify therapeutics against a broad spectrum of pandemic-causing viruses.

## Key facts

- **NIH application ID:** 10164176
- **Project number:** 1R21AI157903-01
- **Recipient organization:** UNIVERSITY OF KENTUCKY
- **Principal Investigator:** Steven Gary Van Lanen
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $417,474
- **Award type:** 1
- **Project period:** 2021-07-20 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10164176

## Citation

> US National Institutes of Health, RePORTER application 10164176, RNA-dependent RNA Polymerase Assays for Biochemical Characterization and Antiviral Drug Discovery (1R21AI157903-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10164176. Licensed CC0.

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