A fluorogenic substrate for the detection of lipid amidases in intact cells.

Journal of lipid research 2024 Mar

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Lipid amidases of therapeutic relevance include acid ceramidase (AC), N-acylethanolamine-hydrolyzing acid amidase, and fatty acid amide hydrolase (FAAH). Although fluorogenic substrates have been developed for the three enzymes and high-throughput methods for screening have been reported, a platform for the specific detection of these enzyme activities in intact cells is lacking. In this article, we report on the coumarinic 1-deoxydihydroceramide RBM1-151, a 1-deoxy derivative and vinilog of RBM14-C12, as a novel substrate of amidases. This compound is hydrolyzed by AC (appKm = 7.0 μM; appVmax = 99.3 nM/min), N-acylethanolamine-hydrolyzing acid amidase (appKm = 0.73 μM; appVmax = 0.24 nM/min), and FAAH (appKm = 3.6 μM; appVmax = 7.6 nM/min) but not by other ceramidases. We provide proof of concept that the use of RBM1-151 in combination with reported irreversible inhibitors of AC and FAAH allows the determination in parallel of the three amidase activities in single experiments in intact cells. Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.

Citation

Mireia Casasampere, Johnson Ung, Alejandro Iñáñez, Carine Dufau, Kazuhito Tsuboi, Josefina Casas, Su-Fern Tan, David J Feith, Nathalie Andrieu-Abadie, Bruno Segui, Thomas P Loughran, José Luis Abad, Gemma Fabrias. A fluorogenic substrate for the detection of lipid amidases in intact cells. Journal of lipid research. 2024 Mar;65(3):100520