Needles in a Haystack: Identification and Characterization of Inhibitors of Lassa Virus RNA-Dependent RNA Polymerase

• Author / Creator

  Gate, Gabrielle C

• The all-too-recent coronavirus disease 2019 (COVID-19) pandemic is a case study in what happens when suitable antiviral measures are not in place prior to an outbreak. Severe acute respiratory syndrome virus 2 (SARS-CoV-2) was the causative agent of the COVID-19 pandemic, and has claimed more than 6.5 million lives since its discovery in 20191. During the scramble to find effective treatments for COVID-19, our lab demonstrated the potency of the nucleotide analogue remdesivir as an inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp)2. To date, remdesivir remains one of only a handful of approved treatments for COVID-193. Unfortunately, it has also been shown that mutations in the RdRp can confer resistance to remdesivir4. As such, it is crucial to continue to expand the arsenal of antiviral agents available, not only for SARS-CoV-2, but for other viruses with high epidemic potential.

  The viral RdRp replicates the viral genome, and is essential for the viral life cycle of most RNA viruses, making it a logical target for drug development5,6. While nucleotide analogues like remdesivir have been successfully used to target RdRps in the past, allosteric inhibitors may also be used. Allosteric inhibitors are especially attractive in that they are less likely to be toxic in vivo than their substrate analogue counterparts7. Allosteric inhibitors can also be used in conjunction with nucleotide analogues, which can increase the efficacy of treatment and reduce the potential for resistance7. The goal of this project was to screen for novel allosteric compounds that inhibit the RdRp, or L-protein, of Lassa fever virus (LASV). This virus, like SARS-CoV-2, was identified by the World Health Organization as a priority pathogen back in 2017, noted for its pandemic potential8. Unlike SARS-CoV-2, there is currently no approved vaccine for LASV, although a vaccine is currently in clinical trials9. In addition, data supporting the antiviral drug ribavirin as an effective treatment for LASV is limited10.

  In the interest of pandemic preparedness, the goals of this project were to: 1) identify small molecule inhibitors of LASV RdRp, and 2) assess various properties of those inhibitors to identify which compounds warrant further investigation as candidates for drug development into new antiviral drugs to treat LASV and related viruses. Beginning with a library of 100,000 compounds, we used an in vitro polymerase activity assay to systematically narrow a pool of candidate compounds based on their potency (ie. half-maximal inhibitory concentration, IC50), selectivity, chemical stability, and mechanism of action. Ultimately, we identified five chemically stable compounds with high potency and appropriate selectivity. Of these, three compound appeared to be competitive, while the other two appeared to be allosteric inhibitors, although the possibility of covalent inhibition couldn’t be ruled out in this study. As such, while further investigation is still required, these five candidates provide a solid foundation for future LASV drug development efforts.

• Subjects / Keywords

  • viral polymerases
  • RdRp
  • in vitro polymerase assays
  • high-throughput drug screening
  • antiviral
  • non-nucleoside inhibitors
  • non-nucleotide inhibitors

• Graduation date

  Fall 2024

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-ym3k-tw48

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.