Structural basis for the bi-specificity of USP25 and USP28 inhibitors

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Cancer therapeutics face challenges in directly targeting oncogenes.
• Deubiquitylases (DUBs) that stabilize oncogenes are promising therapeutic targets.
• USP28 and USP25 are key DUBs implicated in cancer, with existing non-selective inhibitors.

Purpose of the Study:

• To structurally and functionally characterize USP28 inhibition by small-molecule inhibitors.
• To understand the mechanism of inhibition for USP28 and USP25.
• To identify key residues and binding pockets for developing selective inhibitors.

Main Methods:

• Structural characterization of USP28 with inhibitors (AZ1, Vismodegib, FT206).
• Functional assays to assess enzyme activity and inhibition.
• Site-directed mutagenesis to investigate the role of key residues.

Main Results:

• Inhibitors bind to a common pocket in USP28, explaining similar potency against USP28 and USP25.
• A conserved glutamate residue (E366/E373) is critical for pocket stability, inhibitor binding, and enzyme activity.
• Mutating this glutamate residue impacts inhibitor binding and enzyme activity.

Conclusions:

• The identified common binding pocket and the crucial glutamate residue provide insights into USP28/USP25 inhibition.
• Targeting this pocket or mutating the glutamate could facilitate the development of selective USP28 or USP25 inhibitors.
• This research advances strategies for developing novel cancer therapeutics by targeting specific deubiquitylases.