Synthesis of a Novel Ceramide Analogue and its Use in a High‐Throughput Fluorogenic Assay for Ceramidases

Abstract

Several investigations have shown that acid ceramidase inhibitors are potential antiproliferative and cytostatic drugs for cancer chemotherapy. The combinatorial chemistry approach for the discovery of acid ceramidase inhibitors requires the availability of a high-throughput enzyme assay. The synthesis of a novel fluorogenic ceramidase substrate, and its processing both in vitro and in cultured cells in a microtiter plate layout, are reported in this article. This coumarinic substrate was hydrolyzed in vitro (rat liver lysosomes) with Km and Vmax values of 113 microM and 3.6 pmol min-1 mg-1, respectively. Similarly, hydrolysis occurred in intact cultured cells that overexpressed acidic ceramidase. The assay was validated for the identification and characterization of acidic ceramidase inhibitors by using several alpha-ketoamide ceramide analogues, whose inhibitory activity had been previously described.

Fluorescence Assays for Biotransformations

“…The oxidative cleavage of 1,2 -aminoalcohol for designing selective substrates for amidases such as the phenacetylamide 42 suitable for screening penicillin G amidase has been exploited (Scheme 1.12 ) [32,43] . A related fl uorogenic substrate for ceramidase was also recently reported by others [50] . The phenacetyl derivative 43 also serves a fl uorogenic substrate for amidases [37] .…”

Activity of neutral and alkaline ceramidases on fluorogenic N-acylated coumarin-containing aminodiols

“…Most ceramidase inhibitors have been discovered after either rational design or screening of a small series of compounds. Although a number of procedures for the determination of ceramidase activities have been reported ( 9 ), massive screening relies on the availability of high-throughput methods, only a few of which have been described (10)(11)(12). These include a fl uorescent sphingolipid fl uorescence resonance energy transfer (FRET) probe that allows homogeneous ratiometric determination of enzyme activity in real-time ( 12 ).…”

“…In all cases, reactions were stopped with 25 l/well of methanol and then 100 l/well of NaIO 4 [2.5 mg/ml in 100 mM glycine-NaOH buffer (pH 10.6)] was added . After incubation at 37°C for 1 h in the dark, 100 l/well of 100 mM glycine-NaOH buffer (pH 10.6) was added and fl uorescence was measured spectrophotometrically at excitation and emission by guest, on May 10, 2018 www.jlr.org Downloaded from substrates, the assay was optimized with this enzyme. Because an aldehyde is produced in the oxidation step of the development phase of the procedure ( 1 ), the presence of primary amines in the solution must be avoided.…”

A simple fluorogenic method for determination of acid ceramidase activity and diagnosis of Farber disease

“…Incubation of the substrate in the presence of different amounts of FD1 AcCer 10× protein extracts, behind this assay has already been reported by our group ( 25 ). Briefl y, it is based on the use of a fl uorogenic substrate having the same stereochemistry as that of natural ceramide and bearing a coumarin group in the sphingoid base moiety.…”

“…All these disadvantages make both the aCDase assay and the diagnosis of FD restricted to very few expert laboratories. Recently, our group published a versatile fl uorimetric procedure to determine the activity of ceramidases by using a synthetic ceramide analog carrying a 2-oxo-2H-chromen-7-yloxy moiety in the CH 3 -terminal part of the sphingoid chain ( 25 ). This structure enables the release of umbelliferone after hydrolysis by ceramidase, periodate oxidation of the resulting aminodiol, and further ␤ -elimination of the aldehyde oxidation product.…”