Development of a Novel FRET Probe for the Real‐Time Determination of Ceramidase Activity

Abstract

Fretful novelty: We developed two novel doubly labelled fluorescent ceramide analogues that exhibit significant FRET and undergo hydrolysis by ceramidases. We present a fluorescent sphingolipid FRET probe that allows homogeneous ratiometric determination of enzyme activity in real-time.

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

“…In contrast, fl uorogenic substrates are susceptible for high-throughput confi gurations. Reported ceramidase fl uorogenic substrates include FRETbased dually labeled ceramides ( 12,26 ) in which donor and microsomes from tetracycline-treated ACER2-TET-ON cells had signifi cantly higher levels of free bases than controls ( Fig. 5D ).…”

“…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 a previous article ( 10 ), we reported on the use of a coumarinic analog of ceramide, namely RBM14C16 ( Fig.…”

Inhibitors of Ceramidases

“…In these assays the ceramidase activity can be determined by monitoring the release of the fluorescent molecule from the substrate upon hydrolysis by fluorimetry. Over the last two decades, a variety of singly-labelled fluorescent ceramide substrates have been designed, with 7-nitro-2-1,3-benzoxadiazol (NBD) Tani et al, 1999Tani et al, , 1998, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) (He et al, 1999), Nile red (Bhabak et al, , 2013b or diphenylhexatriene (DPH) (Antes et al, 1992), as a fluorophore tag, attached to either the end of the fatty acid or sphingosine of ceramide. Several studies revealed that these substrates have preference toward a particular enzyme sub-type/s (Fig.…”

Small Molecule Inhibitors of Ceramidases

“…The NR-Cer-C 12 -NBD , was found to be efficiently hydrolyzed by recombinant acid and neutral ceramidases. However, in cultured cells the probe was rapidly enriching to Golgi apparatus and no cleavage was observed [113]. This limitation may be overcome in future by the development of a liposomal transporter.…”