Repurposing existing antiviral agents and discovery of novel antiviral target against emerging coronaviruses

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had caused over 700 million confirmed cases and six million deaths worldwide. Antiviral screening is an essential strategy for drug discovery for emerging viral outbreaks. Antiviral screening allows rapid repurposing and characterization of existing drugs as therapeutics, especially when specific treatments are lacking. In addition to drug repurposing, another crucial objective of antiviral screening is to identify new antiviral targets for novel therapeutic development. In this thesis, both drug repurposing and de novo discovery of novel antivirals against coronaviruses were achieved via screening of chemical libraries. In Chapter 2, 22 existing broad-spectrum antiviral agents were evaluated for their anti-SARS-CoV-2 activity in vitro. These antiviral agents were chosen because of their reported broad-spectrum activity on viruses including SARS-CoV and Middle Eastern respiratory Syndrome coronavirus (MERS-CoV). Utilizing viral load reduction and plaque reduction assays, betaferon (IFN-β1b) and remdesivir were validated to exhibit the strongest antiviral potency against SARS-CoV-2 among the tested drugs. Additionally, time-of-drug-addition assay was performed for two other anti-SARS-CoV-2 agents, namely, the lipogenesis modulators AM580 and 25-hydroxylcholesterol, which inhibited the post-entry steps of the SARS-CoV-2 replication cycle. In Chapter 3, 50,213 compounds from the SMARTTM library were examined for their cytopathic effects (CPE) inhibition in VeroE6 cells. In the primary screening, 168 compounds were identified with over 60% CPE inhibition. In the secondary screening, 40 compounds were shortlisted for their dose-dependent antiviral activities. Further validation using plaque reduction and MTT cell viability assays confirmed five compounds (compounds 16, 65, 77, 132, and 172) with anti-SARS-CoV-2 activity in vitro. Compound 172 was investigated further because of its highest selectivity index. Interestingly, compound 172 could inhibit several SARS-CoV-2 variants of concern (VOC) and multiple other human-pathogenic coronaviruses including SARS-CoV, MERS-CoV, and HCoV-229E. Compound 172 also exhibited antiviral activity in both the golden Syrian hamster and K18-human angiotensin-converting enzyme 2-transgenic mouse models. Escape mutant indicated that the antiviral activity of compound 172 was associated with 3-chymotrypsin-like protease (3CLpro) S301, which was validated by a 3CLpro S301P recombinant virus with antiviral resistance. Furthermore, compound 172 was confirmed to interact with 3CLpro by fluorescence-resonance-energy-transfer-based protease activity assay and surface plasmon resonance spectroscopy. Based on forward and reverse genetics, enzymatic assays, and circular dichroism spectrometry, compound 172 was shown to inhibit 3CLpro by binding to a previously uncharacterized allosteric site associated with S301 near the dimerization interface that destabilizes 3CLpro secondary structures. Molecular docking simulation also predicted the stable binding of compound 172 at the 3CLpro dimeric interface. Most importantly, compound 172 can achieve drug synergism with nirmatrelvir (active compound of paxlovid) at nanomolar concentration. These findings highlight the potential of combination therapy to reduce the likelihood of antiviral resistance, identify the presence of an alternative druggable target on coronavirus 3CLpro, and provide insights for the development of allosteric protease inhibitors. Overall, the findings in this thesis illustrate the significance of drug repurposing and de novo discovery in the development of antiviral therapeutics for emerging coronaviruses.