# Kinetic and structural basis for SARS-CoV-2 RNA-dependent RNA polymerase specificity and inhibition

> **NIH NIH R01** · UNIVERSITY OF TEXAS AT AUSTIN · 2022 · $577,840

## Abstract

Project Summary/Abstract
Although there is much hope for an effective vaccine to combat COVID-19, a pressing need remains to develop
direct acting antivirals in the event that vaccines fail to provide protective immunity, for the treatment of acute
infections, and for future coronavirus strains that might evade existing vaccines. The SARS coronavirus (CoV-
2) RNA-dependent RNA polymerase (RdRp) is an attractive target because inhibitors of viral RNA-dependent
polymerases form the cornerstone of antiviral drug combination therapy for successful treatment of HIV and
hepatitis C virus infections. Remdesivir, a nucleotide analog developed by Gilead, is already showing promise
in clinical trials. The long-term goal of this research is to facilitate the development of more effective, less toxic
drugs directed against the SARS CoV-2 RdRp. The rationale for this research is based on prior experience
demonstrating that accurate measurements of the kinetics of nucleotide incorporation and excision by the viral
polymerase/exonuclease translates directly to understanding viral RNA replication and can guide the design of
robust assays to find effective inhibitors. Kinetic analysis will be based on single turnover rapid-kinetic
measurements of polymerization to provide definitive results to define the mechanistic basis for nucleotide
selectivity. Our working hypothesis is that an effective nucleotide analog can be identified and its therapeutic
potential quantified based on analysis of the kinetics of incorporation relative to the kinetics of excision by the
proofreading exonuclease. Specifically, the aims of this research are to quantify the kinetics of nucleotide
incorporation using single turnover kinetic analysis in order to establish the mechanism and overall fidelity of the
RNA replication. Parallel studies will establish the kinetic and mechanistic basis for inhibition for nucleotide
analogs. We will also include extensive characterization of the kinetics of the proofreading exonuclease to define
the rules governing removal of mismatched base pairs and nucleotide analogs. We will also us cryoEM with
samples based on our biochemical knowledge to obtain structures of the polymerase with Remdesivir
incorporated and of the RdRp with the exonuclease. These studies are innovative in that they take advantage of
the most advanced methods of single turnover kinetic analysis and global data fitting developed by the PI to
establish the kinetic and thermodynamic basis for polymerase specificity to reveal the basis for discrimination
against nucleotide analogs. No other lab is applying such standards to this important problem. Moreover, this
quantitative analysis provides an accurate vector pointing toward more effective inhibitors in structure/activity
relationship studies. The work is soundly based the the PI's prior work and on preliminary data explaining the
kinetic basis for the effectiveness of Remdesivir in competing with ATP. The proposed research will signi...

## Key facts

- **NIH application ID:** 10452645
- **Project number:** 5R01AI163336-02
- **Recipient organization:** UNIVERSITY OF TEXAS AT AUSTIN
- **Principal Investigator:** KENNETH ALLEN JOHNSON
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $577,840
- **Award type:** 5
- **Project period:** 2021-07-16 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10452645

## Citation

> US National Institutes of Health, RePORTER application 10452645, Kinetic and structural basis for SARS-CoV-2 RNA-dependent RNA polymerase specificity and inhibition (5R01AI163336-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10452645. Licensed CC0.

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