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Substrate and target selectivity of 4′-fluoroadenosine against viral and host polymerases
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DOI：10.1016/j.jbc.2026.113316.png)
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Abstract
En 中文
Developing safe and effective treatments against emerging RNA viruses is an important goal in pandemic preparedness efforts. 4′-fluorouridine (4′-FlU) is a broad-spectrum antiviral that was shown to inhibit viral RNA-dependent RNA polymerases (RdRps). Given its notable range of antiviral activity, this class of nucleoside analogs warrants further investigation. Here, we studied the antiviral activity and underlying mechanism of inhibition of 4′-fluoroadenosine (4′-FlA). Like 4′-FlU, 4′-FlA demonstrates a broad-spectrum of antiviral activity against eight prototypic viruses representing diverse families. Enzyme kinetics show that the triphosphate (4′-FlA-TP) is efficiently incorporated by viral RdRps. A cryo-EM structure of the RdRp of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in complex with double-stranded RNA and the incorporated monophosphate (4′-FlA-MP) characterizes interactions at the active site. The incorporated analog elicits heterogeneous inhibition patterns in primer extension reactions. In contrast, templates with embedded 4′-FlA-MP inhibit incorporation of complementary UTP across the viral RdRps. However, incorporation of 4′-FIA-TP is not limited to viral polymerases and likewise includes human mitochondrial RNA polymerase. These results demonstrate the general potential for 4′-fluorinated nucleotides as antiviral drugs and guide the development of more selective derivatives for medical use in appropriate settings.
Keywords:
RNA viruses
antiviral drugs
nucleotide analogs
enzymology
mechanism of action
RNA-dependent RNA polymerase
pandemic preparedness
EFdA
4′-ethynyl-2-fluoro-2′-deoxyadenosine
4′-FlA
4′-Fluoroadenosine
4′-FlU
4′-Fluorouridine
NHC
β-D-N4-hydroxycytidine
BHK
Baby hamster kidney cells
BSA
Bovine serum albumin
CCS
Cell culture supernatants
COVID-19
Coronavirus disease 2019
CCHFV
Crimean-Congo hemorrhagic fever
Cryo-EM
Cryogenic electron microscopy
DENV
Dengue virus
ddATP
Dideoxyadenosine
ddI
Dideoxyinosine
DMSO
Dimethyl sulfoxide
DP
Diphosphate
DMEM
Dulbecco’s Modified Eagle Medium
KF
E.coli DNA polymerase I Klenow Fragment
EBOV
Ebola virus
EMDB
Electron Microscopy Data Bank
FBS
Fetal bovine serum
CC50
Half-maximal cytotoxic concentration
EC50
Half-maximum effective concentration
hpi
Hours post-infection
Pol γ
Human DNA polymerase gamma
h-mtRNAP
Human mitochondrial RNA polymerase
HRV
Human rhinovirus
FluA
Influenza A virus
FluB
Influenza B virus
MP
Monophosphate
MOI
Multiplicity of infection
DDM
n-dodecyl β-D-maltoside
Ni-NTA
Nickel-nitrilotriacetic acid
NP
Nucleoprotein
NA
Nucleoside or nucleotide analog
OSF
Open Science Framework
P2A
Porcine teschovirus-1 2A peptide linker sequence
PDB
Protein Data Bank
RDV
Remdesivir
RSV
Respiratory syncytial virus
RNR
Ribonucleotide reductase
RdRp
RNA-dependent RNA polymerase
SI
Selectivity index
SARS-CoV-2
Severe acute respiratory syndrome coronavirus 2
ssRNA
single-stranded RNA
SDS-PAGE
Sodium dodecyl-sulfate polyacrylamide gel electrophoresis
SOF
Sofosbuvir
SD
Standard deviation
TFV
Tenofovir
TEV
Tobacco Etch Virus
TP
Triphosphate
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