Assay Development for Identifying Inhibitors of the Mycobacterial FadD32 Activity

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 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.

Diarylcoumarins inhibit mycolic acid biosynthesis and kill Mycobacterium tuberculosis by targeting FadD32

“…In addition, the validation of FadD32 as a "druggable" target should spur research into novel compound series that also target this important enzyme. A recent report of the development of a high-throughput screening assay against Mycobacterium smegmatis FadD32 suggests the possibility that additional inhibitors that target M. tuberculosis FadD32 could be identified using an enzymatic assay (36). Early testing against intact M. tuberculosis is important given known difficulties of translating hits from enzymatic assays into leads with activity against whole cells (37).…”

The Mycobacterial Cell Envelope--Lipids

“…4). High-throughput screening assays were reported for FadD32 (Galandrin et al 2013) and the antigens 85 Elamin et al 2009;Sanki et al 2009;Favrot et al 2013). The latter assays led to the identification of a number of inhibitors of antigens 85, one of them, known as ebselen, also shows activity against Mtb in culture (MIC of 20 mg/mL).…”

Insight into Structure-Function Relationships and Inhibition of the Fatty Acyl-AMP Ligase (FadD32) Orthologs from Mycobacteria

“…Plasmids-The cloning of the fadD32 genes from M. tuberculosis, M. smegmatis, and Corynebacterium glutamicum has been described elsewhere (13,17). The M. marinum fadD32 gene was cloned according to published procedures, by PCR amplification from MYCM53 total DNA with the following primers: MmfadD32, 5Ј CTCGCATATGATGGCGTACCAC-AACCCGTTC; MmfadD32, 3Ј AGCTCATATGCTTGT-TGGCTTGCGCGTCCTC.…”

“…The M. tuberculosis genome encodes more than 60 AFEs involved in numerous essential biochemical processes, which therefore constitute attractive targets for the development of new antituberculous drugs (15). FadD32 has been identified as an important susceptible (16) and potentially druggable (13,17,18) target. We report here the full biochemical and biophysical characterization of four mycobacterial FadD32 enzymes.…”