Can’t Stop the Cleaving: Effects of Mutations in SARS-CoV-2 Mpro on Activity and Drug Efficacy 公开

Lee, Rachel (Fall 2024)

Permanent URL: https://etd.library.emory.edu/concern/etds/pn89d800s?locale=zh
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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the Coronavirus Disease 2019 (COVID-19) pandemic, which has costed millions of lives globally. The main protease (Mpro) of SARS-CoV-2 is a potential drug target for COVID-19 treatments due to its critical role in the SARS-CoV-2 infectious cycle and dissimilarity to human proteases, and it is the target of Paxlovid, a Pfizer antiviral that includes the Mpro inhibitor nirmatrelvir (NIR). The E166V mutation in Mpro was found in patients with repeated and prolonged treatment with Paxlovid. E166V results in high NIR resistance but comes with a fitness cost due to distortion of the active site and loss of dimerization, which is necessary for proteolytic activity. The L141T mutation was identified in SARS-CoV to stabilize the active site of the enzyme and increase activity. I hypothesize that introduction of the L141T mutation would increase dimerization and thus the enzymatic activity of the Omicron (BA.1) E166V Mpro without decreasing NIR resistance. Using a FRET-based activity assay, I determined that the L141T/E166V shows slightly decreased activity and NIR resistance compared to E166V. Using SEC-MALS, I demonstrated that the L141T mutation rescues dimerization in solution from the E166V, indicating that increasing dimerization does not automatically improve activity. In addition, we observed substrate inhibition kinetics in the BA.1 E166V Mpro, which has not been previously characterized.

Table of Contents

Introduction………………………………………………………………………………………………………….………1

Materials and Methods

             Protein Expression & Purification………………………………………………………..…………….5

             Kinetics Assay……………………………………………………………………………………………………6

             IC50 Assay……………………………………………………………………………………………..…………7

             SEC-MALS………………………………………………………………………………………………..……….9

Results…………………………………………………………………………………………………………….………….12

Discussion…………………………………………………………………………………………………………..……….17

References……………………………………………………………………………………………………….………….19

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