Cell-Based Covalent-Capture Deubiquitinase Assay for Inhibitor Discovery

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Ubiquitination is a crucial post-translational modification regulating cellular processes.
• Deubiquitinases (DUBs) counteract ubiquitination, and their dysregulation is linked to various diseases.
• Targeting DUBs offers therapeutic potential and aids in understanding ubiquitination pathways.

Purpose of the Study:

• To develop a robust, cell-based assay for high-throughput screening of deubiquitinase (DUB) inhibitors.
• To enable the discovery of novel, cell-permeable DUB inhibitors in a native cellular environment.
• To validate the assay's utility by screening against human USP15.

Main Methods:

• Development of a cell-based assay using a cell-permeable ubiquitin probe (Biotin-cR10-Ub-PA) for covalent DUB labeling.
• Quantification of DUB activity using Amplified Luminescent Proximity Homogeneous Assay (AlphaLISA).
• Proof-of-concept screening of human USP15 against a protease inhibitor library.

Main Results:

• The developed cell-based DUB assay is robust and suitable for high-throughput screening.
• The assay successfully identified known pan-DUB inhibitor PR-619 and other novel DUB inhibitors against USP15.
• Demonstrated the assay's adaptability for discovering inhibitors against various human DUBs.

Conclusions:

• The novel cell-based DUB assay facilitates the discovery of potent and cell-permeable DUB inhibitors.
• This assay is a valuable tool for advancing DUB-targeted therapeutics and fundamental research.
• The platform is readily adaptable for screening against a wide range of deubiquitinases.