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 Restricted; Files Only

Lulkin, Nicole (Spring 2022)

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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

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