Mechanistic insights into coronavirus inhibition by a distinct base-modified adenosine analog targeting the viral polymerase
- 1Molecular, Structural and Translational Virology, Rega Institute, Department of Microbiology, Immunology and Transplantation, KU Leuven, Belgium
Abstract
Nucleoside analog (NA) inhibitors of viral polymerases are a chemically diverse and prominent class of antivirals, with most agents bearing sugar modifications to induce chain termination. Compounds with a modified base yet natural ribose are comparatively rare and typically incorporated to act as virus mutagens, raising significant safety concerns. How nucleobase modifications interfere with polymerization and the molecular basis of this effect remain poorly understood. We here define the mechanism of 6-ethyl-modified 7-deazaadenosine (EDA), a recently identified coronavirus (CoV) inhibitor that does not induce virus mutagenesis. Its strong activity spans across multiple CoVs and cell models and is dictated by the 6-ethyl substituent, with poor tolerance for smaller, larger or heteroatom-containing substituents. At the level of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), EDA-triphosphate incorporates as a mimic of ATP but not other NTPs, inducing immediate stalling but also excision by the viral 3'-to-5' exonuclease. While EDA incorporation prevents extension with a second EDA residue, the stalling of natural NTP incorporation is transient and still allows the formation of longer products. A similar phenomenon is observed with template-embedded EDA. Cryo-EM analysis with SARS-CoV-2 RdRp revealed that 3'-terminal EDA is accommodated without distortion of the template:primer duplex, but its 6-ethyl group induces rearrangements in the NTP binding pocket that prevent stable binding of the incoming nucleotide. The mechanistic findings were validated through antiviral studies with SARS-CoV-2 mutants bearing core substitutions in the RdRp or exonuclease. Together, these findings position EDA as a mechanistically distinct base-modified NA prototype that has relevance beyond coronaviruses and underscores the ever-expanding versatility of NA-based polymerase inhibitors.
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BibTeX
@misc{bakirtzoglou2026mechanistic,
title = {{Mechanistic insights into coronavirus inhibition by a distinct base-modified adenosine analog targeting the viral polymerase}},
author = {N. BAKIRTZOGLOU and B. Selisko and Y. Huang and P. Nie and B. Ribeiro de Castro Ramos and R. Van Berwaer and L. Schurmans and V. Fattorini and Y. Zhou and Y. Pan and X. An and J. Vandeput and B. Van Loy and B. Vanmechelen and P. Maes and D. Liu and P. Herdewijn and B. Canard and Z. Lou and E. Groaz and L. Naesens and A. Stevaert},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.28.761875},
url = {https://www.biorxiv.org/content/10.64898/2026.10.28.761875v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Mechanistic insights into coronavirus inhibition by a distinct base-modified adenosine analog targeting the viral polymerase
AU - N. BAKIRTZOGLOU
AU - B. Selisko
AU - Y. Huang
AU - P. Nie
AU - B. Ribeiro de Castro Ramos
AU - R. Van Berwaer
AU - L. Schurmans
AU - V. Fattorini
AU - Y. Zhou
AU - Y. Pan
AU - X. An
AU - J. Vandeput
AU - B. Van Loy
AU - B. Vanmechelen
AU - P. Maes
AU - D. Liu
AU - P. Herdewijn
AU - B. Canard
AU - Z. Lou
AU - E. Groaz
AU - L. Naesens
AU - A. Stevaert
PY - 2026
DA - 2026/10/28
PB - bioRxiv
DO - 10.64898/2026.10.28.761875
UR - https://www.biorxiv.org/content/10.64898/2026.10.28.761875v1
AB - Nucleoside analog (NA) inhibitors of viral polymerases are a chemically diverse and prominent class of antivirals, with most agents bearing sugar modifications to induce chain termination. Compounds with a modified base yet natural ribose are comparatively rare and typically incorporated to act as virus mutagens, raising significant safety concerns. How nucleobase modifications interfere with polymerization and the molecular basis of this effect remain poorly understood. We here define the mechanism of 6-ethyl-modified 7-deazaadenosine (EDA), a recently identified coronavirus (CoV) inhibitor that does not induce virus mutagenesis. Its strong activity spans across multiple CoVs and cell models and is dictated by the 6-ethyl substituent, with poor tolerance for smaller, larger or heteroatom-containing substituents. At the level of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), EDA-triphosphate incorporates as a mimic of ATP but not other NTPs, inducing immediate stalling but also excision by the viral 3'-to-5' exonuclease. While EDA incorporation prevents extension with a second EDA residue, the stalling of natural NTP incorporation is transient and still allows the formation of longer products. A similar phenomenon is observed with template-embedded EDA. Cryo-EM analysis with SARS-CoV-2 RdRp revealed that 3'-terminal EDA is accommodated without distortion of the template:primer duplex, but its 6-ethyl group induces rearrangements in the NTP binding pocket that prevent stable binding of the incoming nucleotide. The mechanistic findings were validated through antiviral studies with SARS-CoV-2 mutants bearing core substitutions in the RdRp or exonuclease. Together, these findings position EDA as a mechanistically distinct base-modified NA prototype that has relevance beyond coronaviruses and underscores the ever-expanding versatility of NA-based polymerase inhibitors.
ER -