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Liposomal FRET Assay Identifies Potent Drug-Like Inhibitors of the Ceramide Transport Protein (CERT) |
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| Authors: | Doaa Samaha Dr Housam H Hamdo Dr Xiaojing Cong Dr Fabian Schumacher Dr Sebastian Banhart Öznur Aglar Prof Dr Heiko M Möller Dr Dagmar Heuer Prof Dr Burkhard Kleuser Dr Essa M Saied Prof Dr Christoph Arenz |
| Institution: | 1. Insitute for Chemistry, Humboldt Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany
Department of Pharmaceutical Chemistry, College of Pharmacy, Helwan University, Cairo, 11795 Egypt;2. Insitute for Chemistry, Humboldt Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany;3. CNRS, Institut de Chimie de Nice, Université Côte d'Azur, 06108 Nice, France;4. Department of Toxicology, Institute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114–116, 14558 Nuthetal, Germany Department of Molecular Biology, University of Duisburg-Essen, Hufelandstrasse 55, 45147 Essen, Germany;5. Unit ‘Sexually Transmitted Bacterial Infections', Department of Infectious Diseases, Robert Koch Institute, 13353 Berlin, Germany;6. Universität Potsdam, Institut für Chemie, Karl- Liebknecht- Strasse 24–25, Haus 25, 14476 Golm, Germany;7. Department of Toxicology, Institute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114–116, 14558 Nuthetal, Germany |
| Abstract: | 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. |
| Keywords: | enzyme assays Förster resonance energy transfer (FRET) liposomes sphingolipids transport proteins |