Assay Development for Identifying Inhibitors of the Mycobacterial FadD32 Activity
| Autor: | N Radha, Ségolène Galandrin, Hedia Marrakchi, Mathieu Léger, Lionel Mourey, Tanjore Balganesh, Rajendra S. Rane, Nathalie Eynard, Kaveri Das, Mamadou Daffé, Valérie Guillet |
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| Přispěvatelé: | Institut de pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées |
| Rok vydání: | 2013 |
| Předmět: |
[SDV]Life Sciences [q-bio]
Mycobacterium smegmatis Antitubercular Agents Validation Studies as Topic Models Biological 01 natural sciences Biochemistry Substrate Specificity Analytical Chemistry Microbiology Ligases Mycobacterium tuberculosis 03 medical and health sciences chemistry.chemical_compound Biosynthesis Drug Discovery Protein purification ComputingMilieux_MISCELLANEOUS 030304 developmental biology chemistry.chemical_classification 0303 health sciences DNA ligase biology 010405 organic chemistry Fatty acid biology.organism_classification Recombinant Proteins High-Throughput Screening Assays 3. Good health 0104 chemical sciences Enzyme Mycolic Acids chemistry Molecular Medicine Chromatography Thin Layer Adenosine triphosphate Protein Binding Biotechnology |
| Zdroj: | Journal of Biomolecular Screening Journal of Biomolecular Screening, SAGE Publications, 2013, 18 (5), pp.576-587. ⟨10.1177/1087057112474691⟩ |
| ISSN: | 2472-5552 1087-0571 |
| Popis: | FadD32, a fatty acyl-AMP ligase (FAAL32) involved in the biosynthesis of mycolic acids, major and specific lipid components of the mycobacterial cell envelope, is essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate and is conserved in all the mycobacterial species sequenced so far, thus representing a promising target for the development of novel antituberculous drugs. Here, we describe the optimization of the protein purification procedure and the development of a high-throughput screening assay for FadD32 activity. This spectrophotometric assay measuring the release of inorganic phosphate was optimized using the Mycobacterium smegmatis FadD32 as a surrogate enzyme. We describe the use of T m (melting temperature) shift assay, which measures the modulation of FadD32 thermal stability, as a tool for the identification of potential ligands and for validation of compounds as inhibitors. Screening of a selected library of compounds led to the identification of five novel classes of inhibitors. |
| Databáze: | OpenAIRE |
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