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

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 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 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 (K-app(m) = 7.0 mu M; V-app(max) = 99.3 nM/min), N-acyle-thanolamine-hydrolyzing acid amidase (K-app(m) = 0.73 mu M; V-app(max) = 0.24 nM/min), and FAAH (K-app(m) = 3.6 mu M; V-app(max) = 7.6 nM/min) but not by other ceramidases.(jlr) 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.