Mechanistic insights into coronavirus inhibition by a distinct base-modified adenosine analog targeting the viral polymerase
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.