The 1,5-diarylpyrrole derivative BM212 was previously shown to be active against multidrug-resistant clinical isolates and Mycobacterium tuberculosis residing within macrophages as well as against Mycobacterium avium and other atypical mycobacteria. To determine its mechanism of action, we identified the cellular target. Spontaneous Mycobacterium smegmatis, Mycobacterium bovis BCG, and M. tuberculosis H37Rv mutants that were resistant to BM212 were isolated. By the screening of genomic libraries and by whole-genome sequencing, we found that all the characterized mutants showed mutations in the mmpL3 gene, allowing us to conclude that resistance to BM212 maps to the MmpL3 protein, a member of the MmpL (mycobacterial membrane protein, large) family. Susceptibility was unaffected by the efflux pump inhibitors reserpine, carbonylcyanide m-chlorophenylhydrazone, and verapamil. Uptake/efflux experiments with [ 14 C]BM212 demonstrated that resistance is not driven by the efflux of BM212. Together, these data strongly suggest that the MmpL3 protein is the cellular target of BM212.T he rise of multidrug-resistant (MDR) and extensively drugresistant (XDR) Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), makes the validation of new antitubercular agents a major global priority. Since tubercular drug resistance is chromosomally encoded (17), chemotherapeutic agents directed against new cellular targets are likely to be effective against both drug-sensitive and drug-resistant M. tuberculosis strains (5,12,13,18). Target identification and validation are usually achieved by either genetic or chemical approaches. The former has the advantage of identifying a likely cellular target a priori but yields no information with regard to the druggability of the target and the access of the drug to the target (a particular problem in mycobacteria [23]). It is therefore not surprising that no current antitubercular agents have been identified through rational drug design (23). Alternatively, the identification of a cellular target candidate through chemical screening has the advantage of knowing that the compound can bind and affect the cellular target in vivo. The identification of the target for an active compound allows the rational modification of lead candidates through medicinal chemistry while ensuring that the compound retains activity against its primary target. However, finding which proteins are inhibited by a compound can be quite challenging.We randomly screened a library of compounds to identify structures of interest for further development. Several azole compounds containing imidazole, pyrrole, toluidine, or methanamine groups were tested for antimycobacterial activity. Among them, 1-{[1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl]methyl}-4-methylpiperazine (BM212) (Fig. 1) proved to be active against multidrug-resistant clinical isolates, against M. tuberculosis residing within macrophages, and against Mycobacterium avium as well as other nontuberculous mycobacteria (7). The identification of BM21...

Mononuclear phagocytes such as monocytes, tissue-specific macrophages and dendritic cells are primary actors in both innate and adaptive immunity. These professional phagocytes can be parasitized by intracellular bacteria, turning them from housekeepers to hiding places and favoring chronic and/or disseminated infection. One of the most infamous is the bacteria that cause tuberculosis (TB), which is the most pandemic and one of the deadliest diseases with one third of the world's population infected, and an average of 1.8 million deaths/year worldwide.Here we demonstrate the effective targeting and intracellular delivery of antibiotics to infected macrophages both in vitro and in vivo, using pH sensitive nanoscopic polymersomes made of PMPC-PDPA block copolymer. Polymersomes showed the ability to significantly enhance the efficacy of the antibiotics killing Mycobacterium bovis, Mycobacterium tuberculosis and another established intracellular pathogen the Staphylococcus aureus. Moreover, they demonstrated to easily access TB-like granuloma tissues -one of the harshest environments to penetrate -in zebrafish models. We thus successfully exploited this targeting for the effective eradication of several intracellular bacteria, including the M. tuberculosis -the etiological agent of human TB.

Objectives To develop a robust phenotypic antimicrobial susceptibility testing (AST) method with a correctly set breakpoint for pretomanid (Pa), the most recently approved anti-tuberculosis drug. Methods The Becton Dickinson Mycobacterial Growth Indicator Tube™ (MGIT) system was used at six laboratories to determine the MICs of a phylogenetically diverse collection of 356 Mycobacterium tuberculosis complex (MTBC) strains to establish the epidemiological cut-off value for pretomanid. MICs were correlated with WGS data to study the genetic basis of differences in the susceptibility to pretomanid. Results We observed ancient differences in the susceptibility to pretomanid among various members of MTBC. Most notably, lineage 1 of M. tuberculosis, which is estimated to account for 28% of tuberculosis cases globally, was less susceptible than lineages 2, 3, 4 and 7 of M. tuberculosis, resulting in a 99th percentile of 2 mg/L for lineage 1 compared with 0.5 mg/L for the remaining M. tuberculosis lineages. Moreover, we observed that higher MICs (≥8 mg/L), which probably confer resistance, had recently evolved independently in six different M. tuberculosis strains. Unlike the aforementioned ancient differences in susceptibility, these recent differences were likely caused by mutations in the known pretomanid resistance genes. Conclusions In light of these findings, the provisional critical concentration of 1 mg/L for MGIT set by EMA must be re-evaluated. More broadly, these findings underline the importance of considering the global diversity of MTBC during clinical development of drugs and when defining breakpoints for AST.