Structural basis for substrate-assisted catalysis and small-molecule inhibition of alphavirus macrodomains

Abstract Sporadic alphavirus infections are pandemic threats that evade innate immunity via macrodomains which remove host mono-ADP-ribose (ADPr). Through high-resolution crystallography of apo and ADPr-bound forms, we here define a conserved MacroD-like fold and a shared hydrogen-bonding network for macrodomains from Eastern equine encephalitis (EEEV), Venezuelan equine encephalitis (VEEV), and Chikungunya (CHIKV) alphaviruses. Our detailed active site structures support a substrate-assisted hydrolysis mechanism where the ADPr α-phosphate activates the catalytic water for nucleophilic attack. Furthermore, we identify the suramin trisulfonic moiety as a potent inhibitor. Crystal structures with solution-state SAXS show suramin occupies the ADPr-binding pocket and induces reversible, inhibitor-mediated oligomerization. Biophysical and enzymatic assays confirm low-micromolar inhibition of EEEV glycohydrolase activity, while cell-based trans -replication assays demonstrate broad-spectrum suppression of EEEV, VEEV, and CHIKV replication. Collectively, our findings define the structural basis for alphaviral macrodomain catalysis, key alphaviral macrodomain druggable features, and a promising scaffold for targeted broad-spectrum antiviral therapeutics.