Synthesis of a Novel Ceramide Analogue and its Use in a High-Throughput Fluorogenic Assay for Ceramidases

Autor: Virginie Garcia, Gemma Fabriàs, Thierry Levade, Josefina Casas, Carmen Bedia
Přispěvatelé: Simon, Marie Francoise, Research Unit on BioActive Molecules, Departamento de Química Orgánica Biológica, Instituto de Investigaciones Quimicas y Ambientales de Barcelona, Institut de médecine moléculaire de Rangueil (I2MR), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées- Institut Fédératif de Recherche Bio-médicale Institution (IFR150)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Rok vydání: 2007
Předmět:
MESH: Amidohydrolases
Male
MESH: Microsomes
Liver
Acid Ceramidase
Ceramidases
MESH: Drug Design
Biochemistry
Substrate Specificity
chemistry.chemical_compound
Microtiter plate
MESH: Animals
Enzyme Inhibitors
Cells
Cultured
chemistry.chemical_classification
biology
Hydrolysis
MESH: Indicators and Reagents
Ceramidase
MESH: Enzyme Inhibitors
Microsomes
Liver
MESH: Ceramidases
Molecular Medicine
Biological Assay
MESH: Hydrolysis
MESH: Cells
Cultured
Ceramide
MESH: Rats
MESH: Acid Ceramidase
MESH: Biological Assay
Ceramides
Amidohydrolases
Cell Line
Animals
Humans
Rats
Wistar
Molecular Biology
MESH: Humans
Organic Chemistry
MESH: Rats
Wistar
MESH: Ceramides
Molecular biology
MESH: Male
Enzyme assay
In vitro
MESH: Cell Line
Rats
Enzyme
chemistry
Drug Design
biology.protein
MESH: Substrate Specificity
Indicators and Reagents
Lysosomes
MESH: Lysosomes
Zdroj: ChemBioChem
ChemBioChem, 2007, 8 (6), pp.642-8. ⟨10.1002/cbic.200600533⟩
ISSN: 1439-7633
1439-4227
DOI: 10.1002/cbic.200600533
Popis: International audience; Several investigations have shown that acid ceramidase inhibitors are potential antiproliferative and cytostatic drugs for cancer chemotherapy. The combinatorial chemistry approach for the discovery of acid ceramidase inhibitors requires the availability of a high-throughput enzyme assay. The synthesis of a novel fluorogenic ceramidase substrate, and its processing both in vitro and in cultured cells in a microtiter plate layout, are reported in this article. This coumarinic substrate was hydrolyzed in vitro (rat liver lysosomes) with Km and Vmax values of 113 microM and 3.6 pmol min-1 mg-1, respectively. Similarly, hydrolysis occurred in intact cultured cells that overexpressed acidic ceramidase. The assay was validated for the identification and characterization of acidic ceramidase inhibitors by using several alpha-ketoamide ceramide analogues, whose inhibitory activity had been previously described.
Databáze: OpenAIRE
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