Abstract
To identify novel inhibitors of Mycobacterium tuberculosis cell envelope biosynthesis, we employed a two-step approach. First, we screened the diverse synthetic small molecule 71,544-compound Enamine library for growth inhibitors using the non-pathogenic surrogate Mycobacterium bovis BCG as screening strain and turbidity as readout. Second, 16 confirmed hits were tested for their ability to induce the cell envelope stress responsive promoter piniBAC controlling expression of red fluorescent protein in an M. bovis BCG reporter strain. Using a fluorescence readout, the acetamide E11 was identified. Resistant mutant selection and whole genome sequencing revealed the mycolic acid transporter Mmpl3 as a candidate target of E11. Biochemical analysis using mycobacterial spheroplasts and various membrane assays suggest that E11 indirectly inhibits MmpL3-facilitated translocation of trehalose monomycolates by proton motive force disruption. E11 showed potent bactericidal activity against growing and non-growing M. tuberculosis, low cytotoxic, and hemolytic activity and a dynamic structure activity relationship. In addition to activity against M. tuberculosis, E11 was active against the non-tuberculous mycobacterium M. abscessus, an emerging opportunistic pathogen. In conclusion, we identified a novel bactericidal anti-mycobacterial lead compound targeting MmpL3 providing an attractive starting point for optimization.
Highlights
Mycobacterium tuberculosis (Mtb), the world’s most successful pathogen, is responsible for an estimated 1.3 million deaths and 10.4 million new tuberculosis (TB) infections every year (WHO, 2017)
Various topographies have been proposed for the cell envelope of mycobacteria with the most widely accepted model introducing a schematic division into three subdomains, the outer capsule, the tripartite cell wall consisting of the outer membrane (OM) bound to arabinogalactan-peptidoglycan complex, and the inner membrane (IM) (Daffé and Marrakchi, 2017)
A robust single-point M. bovis BCG screening assay in 384-well format and simple turbidity measurement as readout was used to screen the Enamine library of 71,544 synthetic small molecule compounds for mycobacterial growth inhibitors in duplicates at a concentration of 12.5 μM
Summary
Mycobacterium tuberculosis (Mtb), the world’s most successful pathogen, is responsible for an estimated 1.3 million deaths and 10.4 million new tuberculosis (TB) infections every year (WHO, 2017). The cell envelope of mycobacteria is a unique composition of carbohydrates and complex lipids which contribute to pathogenicity and set the genus Mycobacterium apart from other prokaryotes (Alderwick et al, 2015). It plays a critical role during infection by protecting intracellular (pathogenic) mycobacteria from the harsh environment of the phagosomal compartment (Gengenbacher and Kaufmann, 2012) and it acts as a permeability barrier for antibiotics in non-replicating nutrient-starved Mtb (Sarathy et al, 2013). INH and EMB have been part of the TB standard-of-care chemotherapy for more than 50 years demonstrating that mycobacterial cell envelope biosynthesis is a clinically validated intervention level and attractive for current drug discovery (Bhat et al, 2017)
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