# An intrinsic loop-mediated structural stability modulating inhibitor potency in the SADS-CoV and SARS-CoV-2 main proteases

## TL;DR

Researchers found structural differences in a key viral enzyme from SADS-CoV compared to SARS-CoV-2, which affect how well inhibitors work, and tested a new inhibitor that reduces virus replication in pig cells.

## Contribution

The study reveals a novel inhibition mechanism and structural dynamics in SADS-CoV Mpro that influence inhibitor potency and guide broad-spectrum antiviral design.

## Key findings

- SADS-CoV Mpro has a single-helix region instead of a coiled coil seen in SARS-CoV-2 Mpro, affecting inhibitor binding.
- Inhibitor 27h effectively suppresses SADS-CoV replication in cell-based assays and porcine intestinal organoids.
- The absence of residue 51 in α-CoV Mpros drives structural differences and modulates inhibitor efficiency.

## Abstract

Swine acute diarrhea syndrome coronavirus (SADS-CoV) poses a significant zoonotic risk. The absence of the structure of SADS-CoV main protease (Mpro) severely impedes the development of effective antiviral therapeutics. Here, we present the high-resolution structures of SADS-CoV Mpro and its complexes with inhibitors 27h and SY110, respectively. These two compounds inhibit SADS-CoV Mpro through a novel inhibition mechanism. Residues 40–53 of SADS-CoV Mpro adopt a single-helix conformation, in contrast to a coiled coil formed by two consecutive alpha-helices observed in SARS-CoV-2 Mpro. These structural differences contribute to the varying inhibitor potency between Alphacoronavirus (α-CoV) and Betacoronavirus (β-CoV) Mpros. We subsequently demonstrate that the absence of residue ‘51’ in α-CoV Mpros 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 Mpro dynamics influence inhibitor potency and provide insights for designing broad-spectrum Mpro 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 (Mpro), a key antiviral target, in complex with two inhibitors, 27h and SY110. By comparing the Mpro structures with those of SARS-CoV-2, we found that a flexible region of SADS-CoV Mpro can modulate inhibitor efficiency. Furthermore, these structural differences are conserved between Alphacoronavirus (α-CoV) and Betacoronavirus (β-CoV) Mpros. We subsequently identified the absence of a single amino acid in α-CoV Mpros as a key factor driving these structural changes. We further showed that 27h could reduce SADS-CoV replication not only in cultured cells but also in porcine intestinal organoids, providing the first evidence of anti-SADS-CoV Mpro activity in a physiologically relevant pig model. Together, our results highlight how natural Mpro flexibility shapes inhibitor effectiveness and offer new guidance for developing broad-spectrum coronavirus antivirals.