Thierry Christophe, Hee Kyoung Jeon, Sae Yeon Lee, Denis Philippe Cedric Fenistein, Ulf Nehrbass, Priscille Brodin, Giovanna Riccardi, Ha Pham, Roland Brosch, Stewart T. Cole, Marie Kempf, Henrieta Škovierová, Jean-Philippe Carralot, Taegwon Oh, Monica Contreras-Dominguez, Eunjung Park, Jaeseung Kim, Laurent Marsollier, Won Kyung Shin, Zaesung No, Ji Youn Nam, Sunhee Kang, Min Jung Seo, Fanny Anne Ewann, Marie-Laure Joly-Guillou, Auguste Genovesio, Mary Jackson, Eun Hye Kim, Institut Pasteur Korea - Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP), Colorado State University [Fort Collins] (CSU), Università degli Studi di Pavia, Groupe d'Étude des Interactions Hôte-Pathogène (GEIHP), Université d'Angers (UA), International Tuberculosis Research Center [Masan, Korea] (ITRC), Pathogénomique mycobactérienne intégrée, Institut Pasteur [Paris], Ecole Polytechnique Fédérale de Lausanne (EPFL), This work was supported by Institut Pasteur Korea, by INSERM Avenir Grant (to PB), by the National Institute of Allergy and Infectious Diseases/National Institutes of Health grant AI064798 (to MJ), by the European Commission (LHSP-CT-2005-018923), and by the Association Française Raoul Follereau (to LM). MCD is a fellow of an INSERM-Poste Vert fellowship., European Project: LSHP-CT-2005-018923,NM4TB, Università degli Studi di Pavia = University of Pavia (UNIPV), and Institut Pasteur [Paris] (IP)
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., Author Summary Tuberculosis is still a major threat to global health. The disease in humans is caused by a bacterium, Mycobacterium tuberculosis, and treatment of an infected individual requires more than six months of chemotherapy. Because such a long course of treatment is required, compliance is low, which can result in the development of multidrug resistant strains (MDR-TB) and even extremely resistant strains (XDR-TB). Identifying new drug targets and potential lead therapeutic compounds are needed to combat MDR-XDR-TB. We developed a new type of assay based on the visualization of mycobacterium replication within host cells and applied it for the search of compounds that are able to chase the pathogen from its hideout. As a result, we found 20 new series of drug candidates that are effective against the bacilli in its hiding place, potentially addressing a crucial aspect in the resilience of the disease. We also showed that one series of compounds acts by inhibiting a key enzyme required for the synthesis of an essential component from the mycobacterial cell wall that is not targeted by any of the commercially available antituberculosis drugs. Altogether, our results pave the way for development of the next generation of antibacterial agents.