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Liposomal FRET Assay Identifies Potent Drug‐Like Inhibitors of the Ceramide Transport Protein (CERT)
Author(s) -
Samaha Doaa,
Hamdo Housam H.,
Cong Xiaojing,
Schumacher Fabian,
Banhart Sebastian,
Aglar Öznur,
Möller Heiko M.,
Heuer Dagmar,
Kleuser Burkhard,
Saied Essa M.,
Arenz Christoph
Publication year - 2020
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.202003283
Subject(s) - ceramide , förster resonance energy transfer , sphingomyelin , golgi apparatus , endoplasmic reticulum , chemistry , biochemistry , vesicle , fluorescence , microbiology and biotechnology , biology , membrane , apoptosis , physics , quantum mechanics
Ceramide transfer protein (CERT) mediates non‐vesicular transfer of ceramide from endoplasmic reticulum to Golgi apparatus and thus catalyzes the rate‐limiting step of sphingomyelin biosynthesis. Usually, CERT ligands are evaluated in tedious binding assays or non‐homogenous transfer assays using radiolabeled ceramides. Herein, a facile and sensitive assay for CERT, based on Förster resonance energy transfer (FRET), is presented. To this end, we mixed donor and acceptor vesicles, each containing a different fluorescent ceramide species. By CERT‐mediated transfer of fluorescent ceramide, a FRET system was established, which allows readout in 96‐well plate format, despite the high hydrophobicity of the components. Screening of a 2 000 compound library resulted in two new potent CERT inhibitors. One is approved for use in humans and one is approved for use in animals. Evaluation of cellular activity by quantitative mass spectrometry and confocal microscopy showed inhibition of ceramide trafficking and sphingomyelin biosynthesis.