Finding new anti-tuberculosis compounds with convincing in vivo activity is an ongoing global challenge to fight the emergence of multi-drug resistant Mycobacterium tuberculosis isolates. In this work, we exploited the medium-throughput capabilities of the zebrafish embryo infection model with Mycobacterium marinum as a surrogate for M. tuberculosis. Using a representative set of clinically established drugs, we demonstrate that this model could be predictive and selective for antibiotics that can be administered orally. We further used the zebrafish-infection model to screen 240 compounds from an anti-TB hit library for their in vivo activity and identified 14 highly active compounds. One of the most active compounds was the tetracyclic compound TBA161, which was studied in more detail. Analysis of resistant mutants revealed point mutations in aspS (rv2572c), encoding an aspartyl-tRNA synthetase. The target was genetically confirmed, and molecular docking studies propose possible binding of TBA161 in a pocket adjacent to the catalytic site. This study showed that the zebrafish-infection model is suitable to rapidly identify promising scaffolds with in vivo activity.
Screening strategies for anti-tuberculosis compounds using Mycobacterium tuberculosis are time-consuming and require BSL-3 facilities, which makes the development of high-throughput assays difficult and expensive. Mycobacterium marinum, a close genetic relative of M. tuberculosis, possesses several advantages as a suitable model for tuberculosis drug screening. However, despite the high genetic similarity, there are some obvious differences in susceptibility to some tuberculosis drugs between these two species, especially for the pro-drugs ethionamide and isoniazid. In this study, we aimed to improve M. marinum as a model for anti-tuberculosis drugs identification by heterologous expression of two common drug activators, EthA and KatG. These two activators were overexpressed in M. marinum and the strains were tested against ethionamide, isoniazid and a library of established antimycobacterial compounds from TbAlliance to compare drug susceptibility. Both in vitro and in vivo using zebrafish larvae, these genetically-modified M. marinum strains showed significantly higher susceptibility against ethionamide and isoniazid, which require activation by EthA and KatG. More importantly, a strain overexpressing both ethA and katG was potentially more susceptible to approximately 20% of the anti-tuberculosis hit compounds from the TB Alliance library. Most of these compounds were activated by EthA in M. marinum. Four of these compounds were selected for further analysis and three of them showed obvious EthA-dependent activity against M. tuberculosis. Overall, our developed M. marinum strains are valuable tools for high-throughput discovery of potential novel anti-tuberculosis pro-drugs.
The ESX-5 secretion system is essential for the viability and virulence of slow-growing pathogenic mycobacterial species. In this study, we identified a 1,2,4-oxadiazole derivative as a putative effector of the ESX-5 secretion system. We confirmed that this 1,2,4-oxadiazole and several newly synthesized derivatives inhibited the ESX-5-dependent secretion of active lipase LipY by Mycobacterium marinum (M. marinum). Despite reduced lipase activity, we did not observe a defect in LipY secretion itself. Moreover, we found that several other ESX-5 substrates, especially the high molecular-weight PE_PGRS MMAR_5294, were even more abundantly secreted by M. marinum treated with several 1,2,4-oxadiazoles. Analysis of M. marinum grown in the presence of different oxadiazole derivatives revealed that the secretion of LipY and the induction of PE_PGRS secretion were, in fact, two independent phenotypes, as we were able to identify structural features in the compounds that specifically induced only one of these phenotypes. Whereas the three most potent 1,2,4-oxadiazoles displayed only a mild effect on the growth of M. marinum or M. tuberculosis in culture, these compounds significantly reduced bacterial burden in M. marinum-infected zebrafish models. In conclusion, we report a 1,2,4-oxadiazole scaffold that dysregulates ESX-5 protein secretion.
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