Abstact

Viral macrodomains, which hydrolyze mono-ADP-ribosylated proteins to evade host immunity, represent emerging antiviral targets, yet their druggability remains underexplored. GS-441524, the active metabolite of remdesivir, has been identified as an inhibitor of the SARS-CoV-2 (severe acute respiratory syndrome coronavirus) macrodomain (Nsp3b). Herein, the structure-activity relationship governing macrodomain recognition by the ribosylated moiety using a panel of nucleoside analogs, revealing that phosphate configuration and nucleobase identity critically modulate binding affinity. GS-441524 derivatives exhibit up to 200-fold higher affinity compared to adenosine-based ligands. A novel sulfamoyl derivative demonstrates superior inhibitory potency, attributable to its occupation of the phosphate subsite and formation of a stabilizing hydrogen-bond network. These findings provide molecular insights into Nsp3b-ligand interactions and establish a rational framework for the development of high-affinity, structure-guided inhibitors targeting viral macrodomains.