Assay development for identifying inhibitors of the mycobacterial FadD32 activity.
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.
Institut de Pharmacologie et de Biologie Structurale (IPBS), Centre National de la Recherche Scientifique (CNRS), Département "Mécanismes Moléculaires des Infections Mycobactériennes," Toulouse, France.
Ségolène
S
Galandrin
Valérie
V
Guillet
Rajendra S
RS
Rane
Mathieu
M
Léger
Radha
R
N
Nathalie
N
Eynard
Kaveri
K
Das
Tanjore S
TS
Balganesh
Lionel
L
Mourey
Mamadou
M
Daffé
Hedia
H
Marrakchi
D000995:Antitubercular Agents / Q000302:isolation & purification* / Q000494:pharmacology; D002855:Chromatography, Thin Layer / Q000379:methods; D055808:Drug Discovery / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D008025:Ligases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D020102:Mycobacterium smegmatis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D009171:Mycolic Acids / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D054928:Validation Studies as Topic
D000995:Antitubercular Agents; D009171:Mycolic Acids; D011994:Recombinant Proteins; D008025:Ligases
FAAL; assay adaptation; fatty acyl-AMP ligase; mycobacteria; screening of inhibitors