Galandrin, Segolene and Guillet, Valerie and Rane, Rajendra S and Leger, Mathieu and Radha, N and Eynard, Nathalie and Das, Kaveri and Balganesh, Tanjore S and Mourey, Lionel and Daffe, Mamadou and Marrakchi, Hedia (2013) Assay development for identifying inhibitors of the mycobacterial FadD32 activity. In: Journal of Biomolecular Screening, 18 (5). pp. 576-587.
Full text not available from this repository. (Request a copy)Abstract
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 Tm (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.
Item Type: | Journal Article |
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Publication: | Journal of Biomolecular Screening |
Publisher: | Sage Publications Inc |
Additional Information: | Copyright of this article belongs to Sage Publications Inc. |
Keywords: | Mycobacteria; Fatty Acyl-AMP Ligase; FAAL; Assay Adaptation; Screening of Inhibitors |
Department/Centre: | Autonomous Societies / Centres > Society for Innovation and Development |
Date Deposited: | 11 Jul 2013 05:49 |
Last Modified: | 04 Feb 2019 05:26 |
URI: | http://eprints.iisc.ac.in/id/eprint/46811 |
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