Liposomal FRET Assay Identifies Potent Drug-Like Inhibitors of the Ceramide Transport Protein (CERT).
Förster resonance energy transfer (FRET)
enzyme assays
liposomes
sphingolipids
transport proteins
Journal
Chemistry (Weinheim an der Bergstrasse, Germany)
ISSN: 1521-3765
Titre abrégé: Chemistry
Pays: Germany
ID NLM: 9513783
Informations de publication
Date de publication:
15 Dec 2020
15 Dec 2020
Historique:
received:
13
07
2020
revised:
01
10
2020
pubmed:
14
10
2020
medline:
20
2
2021
entrez:
13
10
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 33047409
doi: 10.1002/chem.202003283
pmc: PMC7756341
doi:
Substances chimiques
Carrier Proteins
0
Ceramides
0
Pharmaceutical Preparations
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
16616-16621Subventions
Organisme : Deutsche Forschungsgemeinschaft
ID : AR 376/10-1
Informations de copyright
© 2020 The Authors. Published by Wiley-VCH GmbH.
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