Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a respiratory infection with a global distribution. TB chemotherapy is faced with many challenges, including the continual evolution of drug-resistant Mtb and the treatment failures resulting from the inactivity of many drugs against latent TB. These challenges highlight the need to develop more effective TB drugs. With the approval of pretomanid and delamanid for TB treatment, nitro-containing compounds have remerged as promising antimycobacterial compounds that can be developed into TB drugs.In this dissertation, I describe the mechanisms-of-action of 10 new nitro-containing compounds that have potent antitubercular activities. These compounds were discovered from our previous high throughput screen of the Molecular Libraries Small Molecule Repository (MLSMR). Using a forward genetic selection approach, I showed that three of these compounds, the nitrofuranyl piperazines (HC2209, HC2210, HC2211), depend on the cofactor F420 (deazaflavin) activation machinery for their antimycobacterial activity. This is a well-characterized activation system that is used by pretomanid and delamanid for their reductive activation into toxic metabolites. Unlike pretomanid that completely loses its activity against Mtb in the absence of the deazaflavin-dependent nitroreductase (Ddn), I showed that these three compounds partially depend on Ddn and possibly, a secondary unknown F420-dependent nitroreductase. Therefore, these nitrofurans have the possibility of being used in the treatment of pretomanid-resistant TB cases that are caused by ddn mutations. Additionally, these three nitrofurans differ from pretomanid in their activity against M. abscessus (Mab), a mycobacterial species with high intrinsic drug resistance. While pretomanid is inactive against Mab, the nitrofurans maintain their inhibitory activities against the pathogen. Additionally, I used a transcriptional profiling approach to demonstrate that HC2210 has differing effects on both Mab and Mtb. While HC2210 is bactericidal in Mtb and impacts different genes, including those involved in respiration; in Mab, HC2210 is bacteriostatic and does not affect the expression of respiratory genes. Interestingly, the genetic selection of HC2210-resistant mutants in Mab identified glycerol kinase (GlpK) as a resistance factor in Mab. Much is known about the role of this gene in driving antibiotic resistance in Mtb, but little is known about it in Mab. The works presented in this dissertation remains one of the few reports of these protein as a resistance driver in Mab. In addition to the nitrofuranyl piperazines, I genetically characterized the mechanism-of-action of four dinitrobenzamides (HC2217, HC2226, HC2238, and HC2239) and showed that they lose their activity against dprE1 mutants in both Mtb and M. smegmatis. This is predictable since many dinitrobenzamide-based compounds have been biochemically characterized as DprE1 inhibitors. Interestingly, HC2250, a nitrofuranyl hydrazide, also loses its activity against the mutant, suggesting a DprE1-dependent mechanism. Transcriptional profiling of HC2250-treated and HC2238-treated cultures supports the possibility that HC2250 is a DprE1 inhibitor. This is the first report of a nitrofuran scaffold as a putative DprE1 inhibitor. However, HC2250 differs from the dinitrobenzamides in its bactericidal activity against dormant Mtb under hypoxic conditions, with this activity occurring in a DprE1-independent manner. I have also demonstrated that HC2250 and HC2210 have in vivo efficacy in a murine model of TB, indicating their promising potential for development as TB drugs. Lastly, a targeted mutant screening approach and cheminformatics was used to provide an early assessment of the mechanisms-of-action of some growth inhibitors from the MLSMR screen. Surprisingly, this approach identified isoniazid analogs that partially retain their antimycobacterial activity against a Tn:katG mutant. Additionally, I identified many nitro-containing clusters in the MLSMR dataset, including the nitrofuranyl benzothiazoles that show enhanced activity against a mmpL3 mutant pool and a Tn:katG mutant. This is a classic example of collateral sensitivity. Overall, this dissertation used chemical-genetic approaches to characterize the mechanisms-of-action of new nitro-containing compounds and provides proof-of-concept for their potential development as TB drugs.
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