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

Lipid amidases of therapeutic relevance include acid ceramidase (AC), N-acylethanolaminehydrolyzing acid amidase, and fatty acid amide hydrolase (FAAH).Although fluorogenic substrates have been developed for the three enzymes and highthroughput 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 1deoxydihydroceramide RBM1-151, a 1-deoxy derivative and vinilog of RBM14-C12, as a novel substrate of amidases.This compound is hydrolyzed by AC ( app K m ¼ 7.0 μM; app V max ¼ 99.3 nM/min), N-acylethanolamine-hydrolyzing acid amidase ( app K m ¼ 0.73 μM; app V max ¼ 0.24 nM/min), and FAAH ( app K m ¼ 3.6 μM; app V max ¼ 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.