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Global infections by nontuberculous mycobacteria (NTM) are steadily rising. New drugs are needed to treat NTM infections, but the NTM drug pipeline remains poorly populated and focused on repurposing or reformulating approved antibiotics., Global infections by nontuberculous mycobacteria (NTM) are steadily rising. New drugs are needed to treat NTM infections, but the NTM drug pipeline remains poorly populated and focused on repurposing or reformulating approved antibiotics. We sought to accelerate de novo NTM drug discovery by testing advanced compounds with established activity against Mycobacterium tuberculosis. 3-Aminomethyl 4-halogen benzoxaboroles, a novel class of leucyl-tRNA synthetase inhibitors, were recently discovered as active against M. tuberculosis. Here, we report that the benzoxaborole EC/11770 is not only a potent antitubercular agent but is active against the M. abscessus and M. avium complexes. Focusing on M. abscessus, which causes the most-difficult-to-cure NTM disease, we show that EC/11770 retained potency against drug-tolerant biofilms in vitro and was effective in a mouse lung infection model. Resistant mutant selection experiments showed a low frequency of resistance and confirmed leucyl-tRNA synthetase as the target. This work establishes the benzoxaborole EC/11770 as a novel preclinical candidate for the treatment of NTM lung disease and tuberculosis and validates leucyl-tRNA synthetase as an attractive target for the development of broad-spectrum antimycobacterials.

We have targeted the Mycobacterium tuberculosis decaprenylphosphoryl-β-d-ribose oxidase (Mt-DprE1) for potential chemotherapeutic intervention of tuberculosis. A multicopy suppression strategy that overexpressed Mt-DprE1 in M. bovis BCG was used to profile the publically available GlaxoSmithKline antimycobacterial compound set, and one compound (GSK710) was identified that showed an 8-fold higher minimum inhibitory concentration relative to the control strain. Analogues of GSK710 show a clear relationship between whole cell potency and in vitro activity using an enzymatic assay employing recombinant Mt-DprE1, with binding affinity measured by fluorescence quenching of the flavin cofactor of the enzyme. M. bovis BCG spontaneous resistant mutants to GSK710 and a closely related analogue were isolated and sequencing of ten such mutants revealed a single point mutation at two sites, E221Q or G248S within DprE1, providing further evidence that DprE1 is the main target of these compounds. Finally, time-lapse microscopy experiments showed that exposure of M. tuberculosis to a compound of this series arrests bacterial growth rapidly followed by a slower cytolysis phase.

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis β-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related β-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis., Screens for bactericidal compounds have resulted in promising anti-tubercular hits. Here, the authors analyse in detail the target of an indazole sulfonamide (GSK3011724A), and find that it has a different mode of inhibition compared to other Kas inhibitors of fatty acid biosynthesis in bacteria.