Covid 19: RNA-dependent RNA Polymerase Assays for Biochemical Characterization and Antiviral Drug Discovery

Grants and Contracts Details

Description

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 month of April, four 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 two complementary and broadly applicable RdRp activity-based assays that will be used for antiviral drug discovery efforts. Our specific aims are to establish (1) a novel, real-time colorimetric-based assay and (2) a novel, end-point assay to probe RdRp activity using SARS-CoV-2 as the model polymerase system. It is expected that, by accomplishing these aims, the assays will enable a thorough biochemical characterize 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 by other groups. Finally, the activity-based assays 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
StatusActive
Effective start/end date7/20/216/30/24

Funding

  • National Institute of Allergy and Infectious Diseases: $417,474.00

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