Simon Walker, University of Alberta

Simon M. Walker1, Arlo J. Loutan1, Egor P. Tchesnokov1, Dana Kocincova1, Calvin J. Gordon1, Ruby A. Escobedo2, Nathanial Jackson2, Luis Martinez-Sobrido2, Olivia A. Vogel3, Christopher F. Basler3, Kim Morsheimer4, Robert Davey4, Karen Wolff5, Anil Gupta5, Chenguang Yu5, Sumit Chanda5, Arnab Chatterjee5, and Matthias Götte1 1 Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada 2 Texas Biomedical Research Institute, San Antonio, TX, USA 3 Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA 4 Department of Virology, Microbiology and Immunology, Boston University, Boston, MA, USA 5 Calibr-Skaggs Institute for Innovative Medicines, a Division of Scripps Research, La Jolla, CA 92037, USA

Fighting & Responding

Developing safe and effective broad-spectrum antivirals targeting emerging RNA viruses, particularly priority pathogens such as influenza viruses, is key to pandemic preparedness efforts. 4′-fluorouridine (4′-FlU) has demonstrated antiviral activity against several positive and negative-sense RNA viruses. The active nucleoside triphosphate (NTP) targets the viral RNA-dependent RNA polymerase (RdRp). Given the notable range of antiviral activity, we asked whether 4′-fluorinated nucleotides exhibit a common mechanism of action (MOA) against prototypic viruses and their distinct RdRp complexes. We studied the antiviral activity of 4′-fluoroadenosine (4′-FlA) and determined substrate and target selectivity in biochemical assays. Similar to 4′-FlU, 4′-FlA demonstrates broad-spectrum activity, including potent inhibition of influenza viruses, as well as coronaviruses, flaviviruses, picornaviruses, pneumoviruses, arenaviruses, and nairoviruses. Enzyme kinetics show that 4′-FlA-TP is efficiently incorporated by each viral RdRp tested and elicits heterogeneous inhibition patterns in primer-extension reactions. Inhibition is not absolute and can be overcome to varying degrees, yielding modified templates. Incorporation of UTP opposite the template-embedded analog is inhibited, providing a unifying MOA across all RdRps tested in this study. We also demonstrate efficient incorporation of 4′-FIA-TP by human polymerases. Although enzyme inhibition is not evident here, the lack of selectivity raises concerns. These results demonstrate the general potential of 4′-fluorinated nucleotides as antivirals and highlight the need to design derivatives with improved selectivity towards viral polymerase targets.