Investigating Novel SARS-CoV-2 Inhibitors: Identification of Potent SARS-CoV-2 Antiviral Compounds and Development of a Luciferase-Based Reporter Vector for Papain-Like Protease Inhibition Public

Abstract

The rapid spread of SARS-CoV-2 across the world has taken a tremendous toll on all sectors of society and led to high mortality rates. While vaccines remain the most effective way to decrease hospitalizations and deaths due to SARS-CoV-2, they are challenging to distribute globally, demonstrate decreased efficacy against emerging SARS-CoV-2 variants, and have lower benefits in populations such as younger children and immunocompromised individuals. Therefore, antiviral drugs are needed alongside vaccines as a treatment for SARS-CoV-2.

I screened twenty-one novel antiviral compounds for inhibition of SARS-CoV-2 viral replication. Four of these novel compounds were nucleoside analogues targeting SARS-CoV-2 RNA-dependent RNA polymerase (non-structural protein 12) and seventeen targeting the SARS-CoV-2 papain-like protease (PLpro). To safely screen the potential antivirals in BSL2 biocontainment facilities, I primarily used a SARS-CoV-2 replicon system in transient and stable cell-line formats. These screening systems allowed me to study the effects of the SARS-CoV-2 exonuclease nsp14 and remdesivir (RDV) resistance-associated nsp12 mutations on nucleoside analogue potency. I also developed a PLPro reporter vector to facilitate future PLpro antiviral compound validation and in-depth mechanistic examinations.

Collectively, our study revealed two small-molecule PLpro-inhibitors with sub-micromolar EC50 values and a PLpro Proteolysis Targeting Chimera (PROTAC) with a <1.5 μM EC50 value. We also determined that despite their predicted benefits, the PROTAC moieties we screen by more than 14-fold. Lastly, we found that the sensitivity of the PLpro cell-based luciferase assay at this stage did not allow for dose-response analysis of antiviral compounds. With antiviral drugs continuing to be developed against SARS-CoV-2, the inhibitory compounds identified in this study and analysis of various compound screening methods can inform the ongoing search for COVID-19 therapeutics.

Table of Contents

1. Introduction: 10

1.1: SARS-CoV-2 epidemiology and prevalence: 10

1.2: SARS-CoV-2 phylogeny and disease characteristics: 11

1.3 SARS-CoV-2 genome: 11

1.4: SARS-CoV-2 mechanism of infection and replication process: 13

1.5: SARS-CoV-2 Viral Proteases: 14

1.6: SARS-CoV-2 N-terminal 3’-to-5’ Exoribonuclease (ExoN): 15

1.7: SARS-CoV-2 Small Molecule Inhibitors: 17

1.8: SARS-CoV-2 Proteolysis Targeting Chimeras (PROTACS): 19

1.9: SARS-CoV-2 replicon system: 20

1.10: SARS-CoV-2 cell-based luciferase complementation assay: 21

2. Material and Methods: 22

2.1: Plasmids and replicons: 22

2.2: Cell lines: 23

2.3: Compounds: 24

2.4: RNA Dependent Reverse Polymerase Targeting Dose-Response Antiviral Activity Assay (EC50): 25

2.5: Novel PLpro Targeting Antiviral Efficacy of Compounds: 27

2.6: Novel PLpro Targeting Experimental Antiviral Activity Assay (EC50): 30

2.7: PLpro Reporter Plasmid Construction and Screening: 30

2.8: Statistical Analysis: 34

3. Results: 34

3.1: RdRp Dose-Response Antiviral Activity Assay (EC50): 34

3.2: Initial Screening of PLpro-Targeting Antiviral Compounds: 37

3.3: PLpro-Targeting Dose-Response Antiviral Activity Assay (EC50): 40

3.4: Validation and Testing of Novel PLPro Reporter Assay: 43

4. Discussion: 45

5. References: 51

About this Honors Thesis

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Biology, Virology Biology, Microbiology
Mot-clé	RNA polymerases Papain-like proteases Viral infection Antiviral agents SARS-CoV-2 Drug discovery Pandemic Luciferases
Committee Chair / Thesis Advisor	Stefan Sarafianos, Emory University
Committee Members	Dieter Jaeger, Emory University Philip Tedbury, Emory University

Dernière modification

Primary PDF

Thumbnail	Title	Date Uploaded	Actions
	Investigating Novel SARS-CoV-2 Inhibitors: Identification of Potent SARS-CoV-2 Antiviral Compounds and Development of a Luciferase-Based Reporter Vector for Papain-Like Protease Inhibition ()	2022-04-12 17:54:02 -0400	Press to Select an action Download

Supplemental Files

Thumbnail	Title	Date Uploaded	Actions