Small-molecule inhibitors of non-structural protein 15 (NSP15) for the treatment of SARS-CoV-2

• Author / Creator

  Chen, Jerry

• Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is
  responsible for the coronavirus disease 2019 (COVID-19) pandemic. As of 2022, the viral spread is still ongoing and has claimed the lives of at least 6.24 million globally. Despite being more than two years into the pandemic, the need to find effective treatments for SARS-CoV-2 remains. This project aims to screen for small molecule inhibitors of SARS-CoV-2 non-structural protein 15 (NSP15). NSP15 is a relatively conserved RNA uracil endonuclease implicated in the evasion of host interferon and immune defenses. NSP15 was found to be essential for coronavirus replication in-vivo and represents a promising therapeutic target for treating SARS-CoV-2 infections. Currently, only a limited number of NSP15 inhibitors have been identified and these have only limited therapeutic potential. As a result, this project aims to further expand the repertoire of potential coronavirus treatments by finding novel SARS-CoV-2 NSP15 inhibitors through high-throughput screening.
  Methods: To screen for NSP15 small-molecule compounds, a fluorescent resonance energy transfer (FRET)-based NSP15 activity assay was designed and optimized. Using this assay, a high-throughput screen of over 108,000+ compounds for NSP15 inhibition, and a secondary screen of the top 1280 hits, was performed. Top compounds were further validated by orthogonal assays such as for dose-dependency using the FRET-based assay, the Amplex Red assay for redox cycling, and an RNA gel cleavage assay. Furthermore, compound inhibition mechanisms were determined through Michaelis-Menten titration experiments. Finally, compound cytotoxicity tests were
  ii
  performed and the efficacy of the compounds against SARS-CoV-2 viral infection was measured in the Vero cell line using plaque assay.
  Results: I successfully validated and optimized a FRET-based NSP15 activity assay, including the validation of NSP15 characteristics and enzyme kinetics. From the high- throughput screen, I validated six novel compounds for NSP15 inhibition using the various orthogonal assays. These compounds inhibited NSP15 in in-vitro biochemical assays at ranges from 5-95 μM with competitive, mixed, and non-competitive mechanisms. Surprisingly, three compounds, CID5675221, Hexachlorophene, and IPA3, showed strong efficacy in Vero cells against SARS-CoV-2 viral replication and was determined to have positive selectivity index 50 (SI50) ratios.
  Conclusions: From the high-throughput screen of SARS-CoV-2 NSP15 inhibitors, I validated six novel NSP15 inhibitors using in-vitro enzymatic assays. Of the six compounds, three compounds showed favorable selectivity index 50 (SI50) ratios in Vero cell culture. As a result, these compounds could bode well for a downstream hit-to- lead pipeline. Future experiments will further elucidate the binding affinity and structural mechanism by which these compounds bind to the NSP15 protein, and to further evaluate their potential as therapeutics for the treatment of COVID-19.

• Subjects / Keywords

  • SARS-CoV-2
  • NSP15
  • small molecule screen

• Graduation date

  Fall 2022

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-m21r-n443

• 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.