Monkeypox is a viral illness caused by the monkeypox virus (MPXV), a species of the genus Orthopoxvirus. MPXV has posed a significant global health threat since 2022. The I7L protease, essential for viral maturation, has emerged as a key target for antiviral drug development. However, effort to develop effective inhibitors has been hampered by limited understanding of substrate recognition, the absence of structural data, and the lack of reliable enzymatic assays.

In a study published in Advanced Science, a research team led by Prof. XU Yechun from the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences (CAS) and Prof. ZHANG Leike from the Wuhan Institute of Virology of CAS reported the first high-resolution crystal structure of the MPXV I7L protease, and identified unique conformational changes of this protease, enabling the design of nanomolar-level covalent inhibitors as promising leads for broad-spectrum antiviral development.

Researchers first solved three high-resolution crystal structures of the MPXV I7L protease, and revealed that the protease forms a stable dimer. They then identified a flexible “cap region” near the active site, which exhibited both open and closed conformations, indicating its role in dynamically regulating substrate access. Structural analysis followed by molecular dynamics (MD) simulations confirmed that this cap functions as a conformational switch during substrate binding.

Using AlphaFold3-based modeling of protease-substrate structures and Quantum Mechanics/Molecular Mechanics (QM/MM) calculations, researchers explored detailed molecular mechanisms of substrate recognition and hydrolysis by the I7L protease. They revealed key roles of S1–S5 subsites in the substrate binding and a rate-limiting step (deacylation) in the hydrolysis reaction profile.

Moreover, researchers designed peptidomimetic covalent inhibitors featuring a nitrile warhead. To evaluate inhibitory activity of compounds, they developed a high-throughput Fluorescence Resonance Energy Transfer (FRET)-based assay, which led to the discovery of several potent hits. Among them, compound 11 showed the most potent inhibitory activity against the I7L protease, with an IC₅₀ value of 69 nM. In cell-based assays, it effectively inhibited vaccinia virus replication with an EC₅₀ value of 6.0 μM and showed no significant cytotoxicity at concentrations up to 400 μM (CC₅₀>400 μM).

Researchers performed multiple sequence alignment across orthopoxvirus species and monkeypox clades (Clade I, IIa, IIb). They revealed that the I7L protease is highly conserved, and observed no mutations at key inhibitor-binding sites, suggesting that these inhibitors could offer broad-spectrum activity against various orthopoxviruses and their variants. 

This work provides a comprehensive understanding of the MPXV I7L protease’s structure, dynamics, and function, and presented a successful example of structure-based design of covalent peptidomimetic inhibitors. It also provides valuable lead compounds for the development of antiviral drugs against MPXV and other orthopoxviruses.