Biochemical Studies on the Mechanism of Action of Remdesivir and Other Nucleotide Analog Polymerase Inhibitors during the COVID-19 Pandemic

• Author / Creator

  Gordon, Calvin J

• Several disease-causing RNA virus families, capable of infecting humans, remain a significant global health threat due to the lack of effective medical countermeasures. This was perhaps best exemplified early in the COVID-19 pandemic when spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) went unperturbed. To counteract future outbreaks, epidemics, and pandemics, effective antiviral drugs available for immediate use are needed. However, pharmaceutical intervention strategies are challenged by the diverse nature of the RNA viruses. Therefore, a proactive approach is required, directing research efforts toward prototypic RNA viruses representing different viral families. Placing emphasis on broadly acting antiviral agents can help amplify preparedness efforts. Required for viral genome replication, the viral RNA-dependent RNA polymerase (RdRp), specifically the highly conserved catalytic active site, represents a logical therapeutic target for broad-spectrum nucleoside analogs. To this end, remdesivir (RDV), β-D-N4-hydroxycytidine (NHC), and GS-7682, have demonstrated antiviral activity against several different RNA virus families. This thesis details the biochemical characterization of the active 5′-triphosphate (TP) form of the aforementioned nucleoside analogs against various viral RdRp.
  Aligning with the prototypic framework, chapter 3 presents the investigation of RDV-TP against Middle East respiratory syndrome coronavirus (MERS-CoV) RdRp initiated before the COVID-19 pandemic. Here, I help illustrate the first mechanism of action of RDV-TP against a model coronavirus RdRp. I found that RDV-TP is incorporated more efficiently than its natural counterpart ATP and inhibits RNA synthesis via delayed chain termination. Importantly, these studies enabled the timely transition into pandemic rapid response efforts and the investigation of SARS-CoV-2 when vaccines and antivirals were not available. Chapters 4 and 5 present a more complete mechanism of action for RDV-TP against SARS-CoV-2 RdRp, identifying two different modes of inhibition during virus replication. Exemplifying the utility of our biochemical techniques, I also investigated other clinically relevant broad-spectrum nucleotide analogs, providing an early indication of their efficacy that preclinical and clinical studies would later confirm.
  The intravenous administration of RDV inherently restricts treatment regimens to hospitalized patients. Considering this, molnupiravir, the prodrug of NHC, represented an orally available treatment for COVID-19. In chapter 6, I show the first biochemical mechanism for molnupiravir against SARS-CoV-2, explaining the mutagenic effect observed in previous studies. Crucial to this investigation was the ability to monitor base pairing tendencies during RNA virus transcription and replication that facilitate error catastrophe. Together, my investigation of RDV and molnupiravir supported their approval and authorization for the treatment of COVID-19 by the Food and Drug Administration, respectively.
  RDV contains a 1′-cyano modification and stands as the preferred treatment for coronavirus infection. Conversely, GS-7682 is a novel 4′-cyano modified nucleoside prodrug that targets other high priority RNA viruses, namely picorna- and pneumoviruses. In chapters 7 and 8, RDV-TP and GS-646939, the active metabolite of GS-7682, are investigated against a panel of viral RdRp. We show that efficient incorporation is required to elicit an antiviral effect, RDV-TP universally inhibits RNA synthesis when embedded in the template-strand, and GS-646939 acts predominately as an immediate chain terminator. The observed mechanistic differences between RDV-TP and GS-646939 offer assurance that 1′- and 4′-cyano modified nucleotides are viable candidates to target a spectrum of RNA viruses. These insights will guide the development of nucleotide analogs as effective therapeutics available for the next pandemic.

• Subjects / Keywords

  • RNA-dependent RNA polymerase
  • Nucleotide Analog
  • Remdesivir
  • Molnupiravir
  • GS-7682

• Graduation date

  Fall 2024

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-ptgr-fg03

• 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.