Date of Award

9-14-2022

Publication Type

Thesis

Degree Name

M.Sc.

Department

Chemistry and Biochemistry

Keywords

COVID-19;Enzyme Kinetics;Inhibitor;RdRp;RNA dependent RNA polymerase;SARS CoV-2

Supervisor

Kenneth Ng

Abstract

The recent pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) highlights the importance of developing more effective approaches to the diseases caused by RNA virus pathogens. The RNA-dependent RNA polymerase (RdRp) is essential for the replication of RNA viruses, which makes it an attractive target for antiviral drug development. The main goal of this thesis is to develop a platform to explore the molecular mechanisms of catalysis and inhibition for the SARS CoV-2 RdRp, thus providing new approaches for developing effective inhibitors to combat viral infections. An efficient expression system and purification protocol was developed for the multi-subunit SARS CoV-2 RdRp using Trichoplusia ni and a baculovirus expression system for the nsp12 subunit, and E. coli and a T7 bacteriophage promoter expression system for the nsp7 and nsp8 subunits. With a reliable source of the enzyme in hand, an in vitro fluorescence-based RNA synthesis assay was developed and validated to provide a sensitive and efficient tool to explore mechanisms of catalysis and inhibition. Several different putative inhibitors were tested under initial rate, steady-state conditions to provide novel insights relating to kinetic effects and the molecular mechanisms of inhibition. By establishing a platform for exploring the activity of the SARS CoV-2 RdRp in vitro, my work has helped to create a novel and powerful tool to assist with discovering novel antiviral compounds. Future work will focus on further refining and extending this platform, as well as applying the platform to discover and characterize a broad range of putative inhibitors. This work is expected to help develop more effective antiviral compounds, which will help prepare for future outbreaks and pandemics caused by emerging viral pathogens.

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