The Art of Unwinding: Discovery of SARS-CoV-2 Helicase (nsp13) Inhibitors and Mechanistic Investigation of the Replication-Transcription Complex Restricted; Files Only
Castro, Jose Miguel (Summer 2026)
Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has caused a global pandemic resulting in >779 million cases and over 7 million deaths. Although the current therapeutics have significantly reduced diseases burden, resistance-associated mutations have highlighted the limitations of current therapeutics resulting in a need for new viral targets. The SARS-CoV-2 nonstructural protein 13 (nsp13) helicase is a highly conserved protein among betacoronaviruses and a critical component of the replication-transcription complex (RTC). This helicase unwinding activity is essential for viral propagation making it an attractive target for future pan-coronavirus antiviral development. This dissertation investigates SARS-CoV-2 nsp13 as a novel antiviral target. First, an ultra-high throughout nucleic acid unwinding assay was used to screen an in-house library of approved FDA-drugs and bioactive compounds against nsp13 activity. The screen identified forty compounds with inhibitory activity against nsp13 with IC50 ranging from 1.4-10 μM. Ten hits were selected for further characterization. Several compounds bound directly to nsp13 and did not disrupt the protein ATPase activity, suggesting an alternative mechanism of inhibition. Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) identified the Zinc binding domain (ZBD) and the RecA-like domains as major regions of differential exchange upon ligand binding. Furthermore, the selected compounds demonstrated antiviral activity against infectious SARS-Cov-2 in multiple cell lines. To further understand the mechanism of action and enable future structural studies, this work established the required experimental framework to investigate nsp13 within the context of the RTC. Electrophoretic mobility shift assays, mass photometry, and primer extension assays confirmed the incorporation of nsp13 into an active RTC complex. Preliminary negative stain electron microscopy informed future optimization strategies for future cryogenic electron microscopy (cryo-EM) studies. Collectively, this dissertation establishes nsp13 as a potential antiviral target and provides the foundational tools for future mechanistic, and structural experiments aimed at pan-coronavirus antiviral development.
Table of Contents
Chapter 1: Introduction
1. Viruses as human pathogens 1
1.1 Positive-sense ssRNA viruses 2
1.2 Coronaviruses 4
1.2.1 Historical overview 6
1.2.2 Impacts and Pandemic Preparedness for Emerging Coronaviruses 11
1.2.3 The importance of developing antivirals against newer coronaviruses 13
1.3 SARS-CoV-2 genome organization, Life cycle and methods to study 14
1.3.1 SARS-CoV-2 replication cycle 18
1.3.2 Variants of Concern 26
1.3.3 Research tools 29
1.4 Treatments and Therapeutic Targets 34
1.4.1 Vaccines 35
1.4.2 Antibody-based treatments 40
1.4.3 Antivirals 42
1.5 Viral Helicases 49
1.5.1 Nsp13 Helicase 52
1.6 Research goals 57
1.7 Chapter 1- References 60
Chapter 2: Dimming the Corona: Studying SARS-Coronavirus-2 at Reduced Biocontainment Level Using Replicons and Virus-Like Particles
2. Introduction 90
2.1 SARS-CoV-2 Life Cycle 92
2.2 What are Replicons? 95
2.3 What are Virus-Like Particles (VLPs)? 101
2.4 Future Perspectives 106
2.5 Chapter 2 - Tables 108
2.6 Chapter 2 - Figures 112
2.7 Chapter 2 - References 116
Chapter 3: Stalling the Enemy: Targeting Nsp13 for Next-Generation SARS-CoV-2 Antivirals
3. Introduction 135
3.1 Results 136
3.1.1 FRET-Based Unwinding Assay to Measure SARS-CoV-2 nsp13 Activity 136
3.1.2 Screening for Small Molecule Inhibitors of nsp13 137
3.1.3 Validation of Selected Hits from the FRET Screening Assay 137
3.1.4 Effect of Inhibitors on ATPase Activity of nsp13 138
3.1.5 Biophysical Characterization of Small-Molecule Inhibitors of nsp13 138
3.1.5.1 Testing for Binding of Inhibitors to nsp13 138
3.1.5.2 Testing for Binding of Inhibitors to dsRNA 139
3.1.6 Using HDX-MS to Investigate Conformational Flexibility of nsp13 upon Ligand Binding 140
3.1.7 Antiviral Efficacy of nsp13 Inhibitors in Reporter-Replicon Assays and Stably Expressing Reporter-Replicon Cell Line 141
3.1.8 Antiviral Efficacy of Selected Inhibitors of nsp13 Against SARS-CoV-2 Infection 142
3.2 Discussion 143
3.3 Materials and Methods 148
3.4 Chapter 3 - Table 158
3.5 Chapter 3 - Figures 159
3.6 Chapter 3 - Supplemental Material 166
3.7 Chapter 3 - References 177
Chapter 4: Structural insight into the SARS-CoV-2 replication-transcription complex (RTC) in the presence of helicase (nsp13)
4. Introduction 197
4.1 Results 198
4.1.1 Purification of nsp13 and RTC components 198
4.1.2 Assessing activity of holo-RTC 200
4.1.3 Holo-RTC and nsp13-RTC assembly determination 200
4.1.4 Evaluation of candidate inhibitors of SARS-CoV-2 RTC 201
4.1.5 Preliminary structural characterization of the nsp13-RTC by negative-stain EM 203
4.2 Discussion 204
4.3 Methods 206
4.4 Chapter 4 - Figures 213
4.5 Chapter 4 - References 225
Chapter 5: General Discussion and Conclusions
5. Summary of key findings 227
5.1 State of the Field 231
5.1.1 Coronavirus knowledge enabling nsp13 antiviral research 231
5.1.2 Different chemical spaces explored in nsp13 inhibitor discovery 232
5.2 Future Directions 235
5.3 Chapter 5 - References 238
About this Dissertation
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File download under embargo until 20 August 2032 | 2026-06-30 15:34:38 -0400 | File download under embargo until 20 August 2032 |
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