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
  • 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: [email protected].
  • 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: [email protected].
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.
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