A6(ShanghaiTech University)

Poxviruses cause severe diseases, including smallpox and mpox, that pose major threats to human health. The poxvirus core protease (Core^Pro) is essential for viral maturation and is highly conserved in poxviruses, making it an attractive antiviral target¹. However, the structure of Core^Pro remains unknown, hampering antiviral development. Here we determined the apo structure of monkeypox virus (MPXV) Core^Pro and the structure of Core^Pro in a complex with the inhibitor aloxistatin, a drug candidate for muscular dystrophy². These structures show that Core^Pro forms a homodimer that features a unique 'dancing couple' fold. The catalytic intermediate state of Core^Pro was characterized by an aldehyde derivative from a natural substrate (I-G18). This derivative binds covalently to the catalytic Cys328, shifting the active site of the viral protease from a closed conformation in the apo form to a favourable open conformation upon substrate binding. On the basis of the Core^Pro-I-G18 complex, we designed a series of peptidomimetic inhibitors with a nitrile warhead, which could covalently anchor with the catalytic Cys328. These compounds inhibit Core^Pro with half-maximal inhibitory concentrations of 44.9-100.3 nM, and exhibit potent and broad anti-poxvirus activity. Our studies provide a basis for designing wide-spectrum inhibitors against poxvirus infections.