TY  - JOUR
A1  - Samaha, Doaa
A1  - Hamdo, Housam H.
A1  - Cong, Xiaojing
A1  - Schumacher, Fabian
A1  - Banhart, Sebastian
A1  - Aglar, Öznur
A1  - Möller, Heiko Michael
A1  - Heuer, Dagmar
A1  - Kleuser, Burkhard
A1  - Saied, Essa M.
A1  - Arenz, Christoph
T1  - Liposomal FRET assay identifies potent drug-like inhibitors of the Ceramide Transport Protein (CERT)
JF  - Chemistry - a European journal
N2  - 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.
KW  - enzyme assays
KW  - Forster resonance energy transfer (FRET)
KW  - liposomes
KW  - sphingolipids
KW  - transport proteins
Y1  - 2020
U6  - https://doi.org/10.1002/chem.202003283
SN  - 0947-6539
SN  - 1521-3765
VL  - 26
IS  - 70
SP  - 16616
EP  - 16621
PB  - Wiley-VCH
CY  - Weinheim
ER  -