High Content Screening Identifies Decaprenyl-Phosphoribose 2′ Epimerase as a Target for Intracellular Antimycobacterial Inhibitors

Abstract

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2′ epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.

Fighting Against Resistant Strains: The Case of Benzothiazinones and Dinitrobenzamides

“…Recently, two new promising antitubercular drugs were developed (Makarov et al, 2009;Christophe et al, 2009). Both these drugs have the same cellular target, the DprE1 of M. tuberculosis (Manina et al, 2010b).…”

“…The DprE1 protein contains a FAD-binding N-terminal and a C-terminal D-arabinono-1,4-lactone oxidase-like enzyme domains (Wolucka, 2008). What is evident now, however, is that the decaprenylphosphoribose 2'-epimerase enzyme is a very important and authentic validated target for antitubercular drugs, being the target for at least three different classes of antitubercular drugs, namely benzothiazinones, dinitrobenzamides, and benzoquinoxalines whose lead compound is VI-9376, a molecule structurally related to benzothiazinones (Makarov et al, 2009;Christophe et al, 2009;Magnet et al, 2010).…”

“…In summary, DprE1, as well as its orthologs, are not only optimal targets for benzothiazinones, dinitrobenzamides and the lastly described benzoquinoxalines (Makarov et al, 2009;Christophe et al, 2009;Magnet et al, 2010), but also promising targets for the discovery of new molecules in treating infections caused by M. tuberculosis, Mycobacterium leprae, Nocardia spp., Rhodococcus spp., and Corynebacterium spp.…”

Tuberculosis: Still a Way to go ahead for a Lead- A Review

“…BTZ043 undergoes nitro-reduction in physiological environment and inhibits DprE1 enzyme covalently through a cysteine residue of the active site. Advantage of this molecule is it doesn't have shown any antagonist action to the other drugs of the anti-TB regimen and as well with some molecules under the trials like, PA-824, SQ109 [43][44][45][46][47][48].…”

Antitubercular In Vitro Drug Discovery: Tools for Begin the Search

“…Measurement of luminescence has shown to provide a rapid alternative to the counting of colonies as a means of evaluate mycobacterial viability (Protopopova et al, 2006). Profiting from mycobacteria expressing GFP, a vast array of recent technologies, based on fluorescence such as confocal microscopy or flow cytometry are now being applied to test new anti-TB drugs intracellular activity (Christophe et al, 2009). …”