Abstract

Swine acute diarrhea syndrome coronavirus (SADS-CoV) poses a significant zoonotic risk. The absence of the structure of SADS-CoV main protease (M pro ) severely impedes the development of effective antiviral therapeutics. Here, we present the high-resolution structures of SADS-CoV M pro and its complexes with inhibitors 27h and SY110 , respectively. These two compounds inhibit SADS-CoV M pro through a novel inhibition mechanism. Residues 40–53 of SADS-CoV M pro adopt a single-helix conformation, in contrast to a coiled coil formed by two consecutive alpha-helices observed in SARS-CoV-2 M pro . These structural differences contribute to the varying inhibitor potency between Alphacoronavirus (α-CoV) and Betacoronavirus (β-CoV) M pro s. We subsequently demonstrate that the absence of residue ‘51’ in α-CoV M pro s plays a key role in these conformational changes. Furthermore, 27h was proved to efficiently suppress SADS-CoV replication in both cell-based assays and porcine intestinal organoids—marking the first such assessment. Overall, these findings reveal that intrinsic M pro dynamics influence inhibitor potency and provide insights for designing broad-spectrum M pro inhibitors. Swine acute diarrhea syndrome coronavirus (SADS-CoV) causes severe disease and high mortality in piglets, resulting in major economic losses. Notably, SADS-CoV has the ability to cross species barriers, highlighting the importance of anti-SADS-CoV research in reducing potential risks to human health. In this study, we determined the structures of SADS-CoV main protease (M pro ), a key antiviral target, in complex with two inhibitors, 27h and SY110 . By comparing the M pro structures with those of SARS-CoV-2, we found that a flexible region of SADS-CoV M pro can modulate inhibitor efficiency. Furthermore, these structural differences are conserved between Alphacoronavirus (α-CoV) and Betacoronavirus (β-CoV) M pro s. We subsequently identified the absence of a single am