Galandrin Ségolène, Guillet Valérie, Rane Rajendra S, Léger Mathieu, N Radha, Eynard Nathalie, Das Kaveri, Balganesh Tanjore S, Mourey Lionel, Daffé Mamadou, Marrakchi Hedia
Institut de Pharmacologie et de Biologie Structurale (IPBS), Centre National de la Recherche Scientifique (CNRS), Département "Mécanismes Moléculaires des Infections Mycobactériennes," Toulouse, France.
J Biomol Screen. 2013 Jun;18(5):576-87. doi: 10.1177/1087057112474691. Epub 2013 Jan 30.
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.
FadD32是一种参与分枝菌酸生物合成的脂肪酰-AMP连接酶（FAAL32），分枝菌酸是结核分枝杆菌细胞壁主要且特有的脂质成分，对于结核病病原体结核分枝杆菌的存活至关重要。该蛋白在三磷酸腺苷存在的情况下催化脂肪酸转化为酰基腺苷酸（酰基-AMP），并且在迄今为止测序的所有分枝杆菌物种中都保守，因此是开发新型抗结核药物的一个有前景的靶点。在此，我们描述了蛋白质纯化程序的优化以及针对FadD32活性的高通量筛选测定法的开发。这种测量无机磷酸盐释放的分光光度测定法使用耻垢分枝杆菌FadD32作为替代酶进行了优化。我们描述了使用测量FadD32热稳定性调节的Tm（熔解温度）位移测定法，作为鉴定潜在配体和验证化合物作为抑制剂的工具。对选定化合物库的筛选导致鉴定出五类新型抑制剂。
