Journal
CHEMISTRY-A EUROPEAN JOURNAL
Volume 26, Issue 70, Pages 16616-16621Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/chem.202003283
Keywords
enzyme assays; Fö rster resonance energy transfer (FRET); liposomes; sphingolipids; transport proteins
Categories
Funding
- Deutsche Forschungsgemeinschaft [AR 376/10-1]
- Government of Egypt
- EXIST program of the BMBF
- School of Analytical Sciences Adlershof (SALSA) - DFG
- Projekt DEAL
Ask authors/readers for more resources
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 Forster 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available