Substrate recognition and cleavage mechanism of the monkeypox virus core protease

Area of Science:

• Virology
• Structural Biology
• Medicinal Chemistry

Background:

• Poxviruses, including smallpox and mpox, are significant human health threats.
• The poxvirus core protease (Core^Pro) is crucial for viral maturation and a conserved antiviral target.
• The three-dimensional structure of Core^Pro was previously unknown, hindering drug development.

Purpose of the Study:

• To determine the structure of mpox virus Core^Pro in its apo form and in complex with an inhibitor.
• To characterize the catalytic mechanism and substrate-binding mode of Core^Pro.
• To design and develop novel peptidomimetic inhibitors targeting Core^Pro for broad-spectrum anti-poxvirus activity.

Main Methods:

• X-ray crystallography was used to determine the structures of apo-mpox virus Core^Pro and its complex with aloxistatin.
• Characterization of the catalytic intermediate state using an aldehyde derivative of a natural substrate (I-G18).
• Structure-based drug design of peptidomimetic inhibitors with a nitrile warhead targeting the catalytic cysteine (Cys328).

Main Results:

• The structure revealed Core^Pro forms a homodimer with a unique "dancing-couple" fold.
• Substrate binding induces a conformational shift in the active site from closed to open.
• Designed peptidomimetic inhibitors demonstrated potent inhibition (IC₅₀ 44.9-100.3 nM) and broad anti-poxvirus activity.

Conclusions:

• The determined structures provide critical insights into the Core^Pro mechanism and structure-activity relationships.
• Novel peptidomimetic inhibitors show promise for developing wide-spectrum antiviral therapies against poxvirus infections.
• This work lays the foundation for future antiviral drug discovery targeting conserved poxvirus proteases.