Novobiocin defines an Asn28 allosteric pocket that governs SARS-CoV-2 main protease activity.

The repeated outbreaks of coronavirus show how hard it is to keep antiviral effectiveness when the virus mutates. Coronaviruses depend on tightly regulated proteolytic processing mediated by the 3-chymotrypsin-like protease (3CLpro), making it a key antiviral target. Most current inhibitors engage the catalytic site, an approach that remains vulnerable to resistance driven by active-site mutations. Here, we investigate an alternative strategy based on allosteric regulation of 3CLpro by targeting a pocket surrounding residue Asn28, previously shown to influence enzymatic activity and dimer stability. Structure-based virtual screening identified novobiocin as a candidate ligand for this region, which lies adjacent to but distinct from the catalytic center. Biophysical experiments showed direct binding of novobiocin to 3CLpro in solution, with sub-micromolar affinity (Kd ∼ 3 × 10-7 M). Protease thermal stability and dimeric assembly were lowered by ligand binding. Enzymatic assays revealed a pronounced reduction in catalytic turnover with minimal effects on substrate binding, consistent with an allosteric mechanism of inhibition, and yielded IC50 values of ∼0.5 μM across independent assays. Molecular docking and simulation analyses supported stable binding at the Asn28-associated pocket and revealed localized changes in conformational dynamics. These findings show that novobiocin allosterically inhibits 3CLpro and identify the Asn28-associated pocket as a relevant target for developing inhibitors with improved resistance to viral evolution.