Mycobacterium tuberculosis is one of the leading causes of death due to a single infectious pathogen. The evolution and spread of drug resistant strains requires new antibiotics to control the TB pandemic. Over the last decade, the lipid flippase MmpL3 has been identified as a potential drug candidate based on its essential nature for cell viability and repeated identification as the lead target of small molecule inhibitors of Mtb growth. Using a combined untargeted and targeted whole cell phenotypic screen I identified novel inhibitors of this valued target. A combination of lipid profiling and an innovative competitive binding assay supported MmpL3 as the target of these inhibitors. Cross resistance profiling of MmpL3 inhibitors against twenty-four unique mmpL3 Mtb mutants demonstrated that the level of resistance is associated with the proximity of resistant mutants to essential residues for protein function. Further, these resistance profiles suggested that MmpL3 inhibitors fall into separate clades depending on their chemical scaffolds. The results of this screen led to the development of novel potent analogs for one of the identified MmpL3 inhibitors, HC2099. These analogs were active against clinically relevant drug resistant Mtb strains that cause treatment failure in patients. Active analogs were able to kill Mtb inside of infected macrophages, an infectious niche of Mtb, without inducing cytotoxicity against these important immune cells. One of these analogs, MSU-43085, was orally bioavailable and successfully inhibited Mtb growth in infected mice, supporting further development and highlighting the therapeutic potential of this series. High throughput screens are often used to identify new inhibitors of Mtb growth. However, prioritized hits form these screens often identify similar targets such as MmpL3, lipid synthesis enzymes, redox cyclers, as well as inhibitors of the electron transport chain. Follow up studies of these inhibitors are often time consuming, costly and result in the rediscovery of previously identified targets. While this is not necessarily detrimental to Mtb drug discovery, as these reoccurring targets have therapeutic potential. The continued prioritization of inhibitors for these common metabolic pathways potentially limits the identification of inhibitors for novel targets. Therefore, additional steps that identify inhibitors of these common pathways could reshape how high throughput screen hits are prioritized. By applying the targeted mutant screen used to identify MmpL3 inhibitors to a non-prioritized library of hits from a high throughput screen, we identified more than fifty new potential MmpL3 inhibitors. Using an iterative strategy of applying additional mutants of commonly identified targets, this strategy promises to lead to parallel follow-up studies of inhibitors with known and unknown mechanisms of action. The ability of Mtb to enter into quiescent states in response to host stresses is one of the leading causes for the extended time to cure and evolution of drug resistance. These states can be induced by several environmental stresses including acidic pH, hypoxia, and others. In an effort to study this adaptation in the rapidly growing mycobacterial species M. smegmatis, we identified a lethal sodium citrate phenotype. Transcriptional profiling and genetic screening of mutants tolerant to sodium citrate indicated that this phenotype was due to the combined action of both chelation and osmotic stresses. Cell viability could be reduced from sodium citrate killing by cation and osmoprotectant supplementation. From these experiments we propose a model that can be applied to study carbon source uptake and probe the role of genes identified from the forward genetic screen with unknown function
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