Antimycobacterial drug discovery using Mycobacteria-infected amoebae identifies anti-infectives and new molecular targets

Abstract

Tuberculosis remains a serious threat to human health world-wide, and improved efficiency of medical treatment requires a better understanding of the pathogenesis and the discovery of new drugs. In the present study, we performed a whole-cell based screen in order to complete the characterization of 168 compounds from the GlaxoSmithKline TB-set. We have established and utilized novel previously unexplored host-model systems to characterize the GSK compounds, i.e. the amoeboid organisms D. discoideum and A. castellanii, as well as a microglial phagocytic cell line, BV2. We infected these host cells with Mycobacterium marinum to monitor and characterize the anti-infective activity of the compounds with quantitative fluorescence measurements and high-content microscopy. In summary, 88.1% of the compounds were confirmed as antibiotics against M. marinum, 11.3% and 4.8% displayed strong anti-infective activity in, respectively, the mammalian and protozoan infection models. Additionally, in the two systems, 13–14% of the compounds displayed pro-infective activity. Our studies underline the relevance of using evolutionarily distant pathogen and host models in order to reveal conserved mechanisms of virulence and defence, respectively, which are potential “universal” targets for intervention. Subsequent mechanism of action studies based on generation of over-expresser M. bovis BCG strains, generation of spontaneous resistant mutants and whole genome sequencing revealed four new molecular targets, including FbpA, MurC, MmpL3 and GlpK.

Crystal Structures of Membrane Transporter MmpL3, an Anti-TB Drug Target

“…The diamines and indolcarboxamides have already entered clinical trials or preclinical studies (Rao et al, 2013;World Health Organization, 2018). The current literature suggest they all target MmpL3 since the resistant mutations have been specifically mapped to the mmpL3 gene Grzegorzewicz et al, 2012;Ioerger et al, 2013;Kozikowski et al, 2017;La Rosa et al, 2012;Lun et al, 2013;McNeil et al, 2017;Rao et al, 2013;Rayasam, 2014;Stanley et al, 2012;Tahlan et al, 2012;Trofimov et al, 2018;Zheng et al, 2018). Since our structural data are only for four MmpL3 inhibitors, we investigated if molecular docking could indicate how six other putative MmpL3 inhibitors might bind to this protein.…”

Identification of novel anti-bacterial compounds from a chemically highly diverse pathways-based library using phenotypic screens in amoebae host models

“…On the other hand, identification of infection enhancers in the set of compounds was quite surprising, although the same observation was already reported in the phenotypic screen of the GlaxoSmithKlyne TB-set of anti-mycobacterial antibiotics in the A. castellanii-M. marinum model of infection (Trofimov et al 2018). Notably here, five compounds were identified that increased at least 2-fold the intracellular M. marinum bacterial load (Fig.…”

“…To evaluate the effect of hit compounds on host fitness, we used D. discoideum cells expressing GFP-ABD to measure toxic and growth inhibitory activities of compounds (Trofimov et al 2018). In parallel, a cell viability test using Alamar blue was performed using A. castellanii 4 hours after hit compound addition at a 30μM concentration.…”

“…marinum-Amoebae system and the more standard Mtb-macrophages models. This study also underlined the relevance of using evolutionary distant pathogen and host models to reveal conserved mechanisms of virulence and defence (Trofimov et al 2018).…”

“…Alternative in cellulo screening systems have also been established to take advantage of previously underexplored amoeba host-models (Kicka et al 2014; Harrison et al 2013). For example, D. discoideum and A. castellanii were used to characterize compounds of the GSK TB-set of anti-mycobacterial compounds (Trofimov et al 2018). This study showed that most compounds previously selected for their antibiotic activity against Mtb and M. smegmatis (Ballell et al 2013) were active against the closely related M. marinum , but showed little or no activity in the intracellular context of an infection.…”