2. Academic Validation
  3. A fluorogenic substrate for the detection of lipid amidases in intact cells

A fluorogenic substrate for the detection of lipid amidases in intact cells

  • J Lipid Res. 2024 Mar;65(3):100520. doi: 10.1016/j.jlr.2024.100520.
Mireia Casasampere 1,  Johnson Ung 2,  Alejandro Iñáñez 1,  Carine Dufau 3,  Kazuhito Tsuboi 4,  Josefina Casas 5,  Su-Fern Tan 6,  David J Feith 6,  Nathalie Andrieu-Abadie 3,  Bruno Segui 7,  Thomas P Loughran Jr 6,  José Luis Abad 8,  Gemma Fabrias 9
Affiliations

Affiliations

  • 1 Department of Biological Chemistry, Research Unit on BioActive Molecules, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain.
  • 2 Division of Hematology and Oncology, Department of Medicine, University of Virginia School of Medicine, Charlottesville, VA, USA; Department of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA, USA.
  • 3 INSERM UMR 1037, Cancer Research Center of Toulouse (CRCT), Toulouse, France; Equipe Labellisée Fondation ARC pour la recherche sur le cancer, Toulouse, France.
  • 4 Department of Pharmacology, Kawasaki Medical School, Kurashiki, Okayama, Japan.
  • 5 Department of Biological Chemistry, Research Unit on BioActive Molecules, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain; CIBEREHD, Madrid, Spain.
  • 6 Division of Hematology and Oncology, Department of Medicine, University of Virginia School of Medicine, Charlottesville, VA, USA; University of Virginia Cancer Center, University of Virginia School of Medicine, Charlottesville, VA, USA.
  • 7 INSERM UMR 1037, Cancer Research Center of Toulouse (CRCT), Toulouse, France; Equipe Labellisée Fondation ARC pour la recherche sur le cancer, Toulouse, France; Université Toulouse III - Paul Sabatier, Toulouse, France.
  • 8 Department of Biological Chemistry, Research Unit on BioActive Molecules, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain. Electronic address: joseluis.abad@iqac.csic.es.
  • 9 Department of Biological Chemistry, Research Unit on BioActive Molecules, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain; CIBEREHD, Madrid, Spain; Spanish National Research Council (CSIC)'s Cancer Hub, Madrid, Spain. Electronic address: gemma.fabrias@iqac.csic.es.
PMID: 38369184 DOI: 10.1016/j.jlr.2024.100520
Abstract

Lipid amidases of therapeutic relevance include acid Ceramidase (AC), N-acylethanolamine-hydrolyzing acid amidase, and fatty acid amide hydrolase (FAAH). Although fluorogenic substrates have been developed for the three Enzymes and high-throughput methods for screening have been reported, a platform for the specific detection of these enzyme activities in intact cells is lacking. In this article, we report on the coumarinic 1-deoxydihydroceramide RBM1-151, a 1-deoxy derivative and vinilog of RBM14-C12, as a novel substrate of amidases. This compound is hydrolyzed by AC (appKm = 7.0 μM; appVmax = 99.3 nM/min), N-acylethanolamine-hydrolyzing acid amidase (appKm = 0.73 μM; appVmax = 0.24 nM/min), and FAAH (appKm = 3.6 μM; appVmax = 7.6 nM/min) but not by Other ceramidases. We provide proof of concept that the use of RBM1-151 in combination with reported irreversible inhibitors of AC and FAAH allows the determination in parallel of the three amidase activities in single experiments in intact cells.

Keywords

N-acylethanolamine acid amidase; N-palmitoylethanolamine; anandamide; ceramidases; ceramides; chemical synthesis; enzymology; fatty acid amide hydrolase; lipids; sphingolipids.

Figures
Products