Discovery of SARS-CoV-2 Antivirals from Zoonotic Peptides and Repurposed Drugs Restricted; Files Only
von Beck, Troy (Fall 2023)
Abstract
SARS-CoV-2, the causative viral agent behind the COVID-19 pandemic and a disease whose notoriety is on par with the black plague and the Spanish flu, has driven unprecedented research and discovery from the medical and scientific fields. Since its emergence in late 2019, global efforts to identify novel antivirals and vaccines for this devastating disease have yielded innovative solutions in the development of mRNA vaccines, coronavirus protease inhibitors, and broad-spectrum inhibitors of RNA-dependent RNA polymerases. These medical advances not only exist to limit the morbidity and mortality of SARS-CoV-2 but exist as safeguards against the future zoonotic spillover of other coronavirus and closely related arterivirus species. Development of successful and lasting SARS-CoV-2 antivirals is critically dependent on the targeting of highly conserved viral structures. This definition inherently excludes the spike protein which readily evolves with each new variant, instead shifting the focus towards other viral determinants necessary for viral replication and constrained by interactions with invariant nucleic acid, protein, and lipid substrates.
Here, we employ in silico, in vitro, and in vivo techniques to explore and characterize latent anti-SARS-CoV-2 activity in zoonotic cathelicidins and existing therapeutics which can serendipitously inhibit the viral endoribonuclease encoded by nsp15. In our first experimental aims, we identified a boar cathelicidin derivative termed “Yongshi,” which exhibits consistent, albeit modest, inhibition of SARS-CoV-2 and its many variants of concern in cell culture. Through careful time of addition and protein dynamics studies, we posit a theoretical inhibitory mechanism requiring direct interaction between Yongshi and SARS-CoV-2 and potentially involving specific interactions with the conserved heptad repeat domains. In our second set of studies, we employ newly developed in silico screening techniques to first identify human-approved drugs with probable binding to the SARS-CoV-2 nsp15 protein, and then validate them via enzymatic assays with recombinant nsp15. This yielded two previously unknown inhibitors of nsp15, pibrentasvir and atovaquone which were active against both SARS-CoV-2 and the related HCoV-OC43 virus. These findings expand our understanding of cathelicidins in antiviral immunity and identify the first SARS-CoV-2 nsp15 inhibitor active at human bioavailable concentrations in cell culture.
Table of Contents
Chapter 1: Introduction. 1
Origins of the COVID-19 Pandemic. 1
First appearance and description of SARS-CoV-2. 1
Pandemic spread and public health measures. 2
The development and deployment of SARS-CoV-2 therapeutics and vaccines. 3
Viral evolution and appearance of major variants of concern. 5
Lifecycle of SARS-CoV-2 in the host. 6
Cellular Entry. 6
Replication. 9
Evasion of immune defenses. 11
Shedding of progeny. 13
Small molecule inhibitors of Nsp15. 16
Nsp15 structure and function. 16
Nsp15 and its role in the viral lifecycle. 19
Current state of Nsp15 inhibitor development. 22
Antimicrobial Peptides. 23
Cathelicidins as a source of highly diverse AMPs. 24
Mechanisms and Specificity of Cathelicidin Activity. 26
Summary. 29
Figures. 31
Figure 1. Overview of the SARS-CoV-2 Life cycle. 31
Figure 2. SARS-CoV-2 mechanism of innate immunity suppression by nsp15. 32
Figure 3. Mechanisms of virus recognition and neutralization by cathelicidins. 33
Chapter 2: A wild boar cathelicidin peptide derivative inhibits severe acute respiratory syndrome coronavirus-2 and its drifted variants. 34
Summary. 34
Introduction. 35
Methods. 38
Results. 43
Discussion. 51
Figures. 55
Figure 1. A wild boar cathelicidin PMAP-36R inhibits SARS-CoV-2 infection of Vero hACE2 cells. 57
Figure 2. The PMAP-36R cathelicidin derivative pSer possesses reduced cytotoxicity. 59
Figure 3. The PMAP-36R derivative Yongshi (pSer) mediates SARS-CoV-2 inhibition via acting on both virions and cells. 60
Figure 4. The D-enantiomer of Yongshi loses SARS-CoV-2 specificity. 61
Figure 5. Yongshi retains inhibitory activity against emergent SARS-CoV-2 variants alpha, beta, gamma, kappa, and delta. 62
Figure 6. Deep-learning sequence alignment algorithm and computational modeling predict stable binding interactions between Yongshi and HR1. 63
Figure 7. Phylogenetic tree of HR1 segments in disparate coronaviruses. 64
Figure 8. Yongshi binds to HR1 peptide with higher affinity than HR2. 65
Figure 9. Yongshi remains active against the SARS-CoV-2 Omicron Variant. 66
Tables. 67
Table 1. Calculated IC50 and TD50 values for PMAP-36R derivatives and control LL-37 or OVA peptides. 67
Table 2. Calculated IC50 values for Yongshi against drifted SARS-CoV-2 variants. 68
Supplemental Information. 69
Supplemental Table 1. Sequence of peptides used in this study. 72
Supplemental Figure 1. Representative IHC Staining of Vero E6 hACE2 cells infected with SARS-CoV-2 and treated with pSer. 73
Supplemental Figure 2. Dose-response curves of other cathelicidin peptides with greater than 50% SARS-CoV-2 inhibition at 50µM. 75
Chapter 3: Atovaquone and pibrentasvir inhibit the SARS-CoV-2 endoribonuclease and restrict infection in vitro but not in vivo. 76
Summary. 76
Introduction. 77
Methods. 79
Results. 86
Discussion. 91
Figures. 95
Figure 1. Binding pockets of 11 drugs on monomeric nsp15 predicted by in silico screening with FRAGSITE2 on monomeric nsp15. 95
Figure 2. In vitro screening results of predicted inhibitors against nsp15 nuclease activity. 96
Figure 3. Inhibition of SARS-CoV-2 infection by in silico predicted inhibitors. 97
Figure 4. Inhibition of HCoV-OC43 infection by atovaquone and pibrentasvir. 99
Figure 5. Efficacy of prophylactic atovaquone and pibrentasvir treatment in a mouse model of SARS-CoV-2 infection. 101
Tables. 101
Table 1. Ranking of FDA-approved drugs predicted by FRAGsite to bind nsp15 from SARS-CoV-2. Compounds are grouped by binding pocket 1 (orange) or binding pocket 2 (blue). 101
Table 2. Additional compounds predicted by other in silico studies to bind nsp15 from SARS-CoV2. 102
Supplemental Information. 103
Supplemental Figure 1. Western blot analysis of HEK293T cells expressing strep-tagged nsp15 of SARS-CoV-2. 103
Supplemental Figure 2. Binding pockets of 11 drugs on hexameric nsp15 predicted by an in silico screening with FRAGSITE2. 105
Supplemental Figure 3. Evaluation of drug-induced nsp15 aggregation. 107
Supplemental Figure 4. MTS formazan formation viability assay. 108
Supplemental Figure 5. Flow cytometric quantification of HCoV-OC43 infected cells with and without nsp15 inhibitor treatment. 110
Chapter 4: Discussion. 112
Repurposed drugs and biologics as novel antiviral compounds. 112
Targeting conserved structures produces pandemic ready solutions. 116
The future of Yongshi and nsp15 inhibitors. 118
Figures. 121
Figure 1. Characterization of a self-amplifying mRNA construct for therapeutic delivery of Yongshi. 122
Appendix A: Methods for Yongshi self-amplifying mRNA construct experiments. 123
Works Cited 127
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