Figure 1.

Screening for membrane permeabilizing agents identifies BT-08 as a hit compound with in vivo and ex vivo anti-mycobacterial activity.

(A) Chemical structure of BTP15 and ethoxzolamide served as a starting point to generate a benzothiazole compound library, from which BT-08 was the most promising hit. WT M. marinum cultures were grown (7H9 medium with albumin–dextrose–saline [ADS] and tyloxapol) in the presence of test compounds (10 μM) before the EtBr uptake assay was performed. A WT M. marinum strain expressing the porin mspA served as a positive control (+mspA). After the addition of EtBr, the fluorescence intensity (arbitrary fluorescence units) was measured for 3 h. Data are presented as the mean of triplicates ± range. (B) Dose-dependent activity of BT-08 on M. marinum during the ethidium bromide uptake assay (7H9 medium with ADS and tyloxapol). Data are presented as the mean of triplicates ± range. (C) Dose-dependent activity of BT-08 on M. marinum during the resazurin uptake assay (7H9 medium with ADS and tyloxapol). Data are presented as the mean of triplicates ± range. (D) Dose-dependent activity of BT-08 in the zebrafish embryo infection model. Embryos were yolk-infected with M. marinum expressing tdTomato, and treatment was performed by immersion. Each data point represents the integrated red fluorescence intensity of a single zebrafish embryo, and the signal of each group is expressed as the mean ± SD of the mean. Statistical significance was determined by one-way ANOVA, following Dunnett’s multiple comparison test by comparing the signal from the DMSO-treated control sample with each treatment group (****P ≤ 0.0001). CTL represents the non-infected group. (E) Effect of BT-08 on bacterial growth of M. marinum M^USA and M. tuberculosis H37Rv was assessed using the resazurin reduction microtiter plate assay. DMSO-treated sample represents 100% bacterial growth. Data are presented as the mean of duplicates ± range. (F) Intracellular activity of BT-08 in THP-1 macrophages infected with M. tuberculosis expressing gfp. The expression of gfp was induced by the addition of ATc. To detect macrophages (gray bars), the nuclei were stained with Hoechst dye. The GFP signal within each macrophage was quantified, representing the amount of viable bacteria (green bars). DMSO- and rifampicin (10 μM)-treated samples served as a negative and positive control, respectively. Data are presented as the mean of duplicates ± range.