Multiplexed Gel-based ABPP Strategy for Target-agnostic Serine Hydrolase Inhibitor Screening.

Area of Science:

• Biochemistry
• Chemical Biology
• Enzymology

Background:

• Serine hydrolases (SHs) are crucial enzymes in human physiology, but many lack selective inhibitors for functional studies.
• Approximately 240 human SHs exist, presenting a significant target class for drug discovery and biological research.

Purpose of the Study:

• To develop a scalable screening platform for identifying selective chemical inhibitors of human serine hydrolases.
• To discover novel inhibitors for underexplored SHs and facilitate their functional validation.

Main Methods:

• Development of a medium-throughput screening platform using multiplexed gel-based activity-based protein profiling (ABPP).
• Target-agnostic screening of 1,664 covalent compounds against mouse brain proteome.
• Chemical proteomics and overexpression studies for target identification and validation.

Main Results:

• Identification of three micromolar-potent fatty acid amide hydrolase (FAAH) inhibitors with urea warheads.
• Discovery of one epoxide-based inhibitor targeting an unannotated protein.
• Identification and validation of an interaction with alpha/beta-hydrolase domain-containing protein 2 (ABHD2).

Conclusions:

• The developed ABPP platform provides a scalable workflow for discovering selective inhibitors of SHs.
• This approach aids in the functional validation and mechanism-of-action studies of previously uncharacterized proteins.
• The study highlights the potential for identifying inhibitors against metabolic SHs and other enzyme classes.