Activity-Based Probe for N-Acylethanolamine Acid Amidase

Area of Science:

• Biochemistry
• Pharmacology
• Chemical Biology

Background:

• N-Acylethanolamine acid amidase (NAAA) is a lysosomal enzyme crucial for lipid signaling molecule metabolism.
• NAAA inhibitors, like α-amino-β-lactone derivatives, show potential in modulating inflammatory responses.
• Previous studies identified potent NAAA inhibitors ARN077 and ARN726 with distinct in vivo applications.

Purpose of the Study:

• To design and validate the first activity-based protein profiling (ABPP) probe for in vivo NAAA detection.
• To utilize the probe for identifying catalytically active NAAA in cellular and tissue models.
• To confirm the inhibitory mechanisms of ARN077 and ARN726 using competitive ABPP.

Main Methods:

• Synthesis and validation of a novel click-chemistry activity-based probe (ABP) derived from ARN726.
• In vitro and in vivo application of the ABP in NAAA detection.
• Competitive ABPP assays to assess the inhibitory activity of ARN077 and ARN726 against NAAA.

Main Results:

• The synthesized compound 1 functions as an effective in vitro and in vivo click-chemistry ABP for NAAA.
• Compound 1 successfully captured catalytically active NAAA in HEK293 cells and rat lung tissue.
• Competitive ABPP confirmed that ARN726 and ARN077 are potent NAAA inhibitors in vitro and in vivo.

Conclusions:

• Compound 1 represents a valuable new tool for detecting activated NAAA in biological systems.
• This ABP facilitates the investigation of NAAA's physiological and pathological roles.
• The findings validate the utility of ABPP for studying enzyme activity and inhibitor efficacy in complex biological contexts.