Background:The anti-TB prodrugs isoxyl (ISO) and thiacetazone (TAC) inhibit mycolic acid biosynthesis. Results: We show that ISO and TAC both target the dehydration step of the FAS-II elongation system. Conclusion: ISO and TAC share the same mode of action. Significance: ISO and TAC are the first antibiotics reported to target the FAS-II dehydratase(s) of Mycobacterium tuberculosis.

The treatment of tuberculosis is becoming more difficult due to the ever increasing prevalence of drug resistance. Thus, it is imperative that novel anti-tuberculosis agents, with unique mechanisms of action, be discovered and developed. The direct anti-tubercular testing of a small compound library led to discovery of adamantyl urea hit compound 1. In this study, the hit was followed up through the synthesis of an optimization library. This library was generated by systematically replacing each section of the molecule with a similar moiety until a clear structure activity relationship was obtained with respect to anti-tubercular activity. The best compounds in this series contained a 1-adamantyl-3-phenyl urea core and had potent activity against Mycobacterium tuberculosis plus an acceptable therapeutic index. It was noted that the compounds identified and the pharmacophore developed is consistent with inhibitors of epoxide hydrolase family of enzymes. Consequently, the compounds were tested for inhibition of representative epoxide hydrolases: M. tuberculosis EphB and EphE; and human soluble epoxide hydrolase. Many of the optimized inhibitors showed both potent EphB and EphE inhibition suggesting the antitubercular activity is through inhibition of multiple epoxide hydrolyase enzymes. The inhibitors also showed potent inhibition of humans soluble expoxide hydrolyase, but limited cytotoxicity suggesting that future studies must be towards increasing the selectivity of epoxide hydrolyase inhibition towards the M. tuberculosis enzymes.

Background: Molecular programs employed by Mycobacterium tuberculosis (Mtb) for the establishment of non-replicating persistence (NRP) are poorly understood. In order to investigate mechanisms regulating entry into NRP, we asked how cell cycle regulation is linked to downstream adaptations that ultimately result in NRP. Based on previous reports and our recent studies, we reason that, in order to establish NRP, cells are halted in the cell cycle at the point of septum formation by coupled regulatory mechanisms. Results: Using bioinformatic consensus modeling, we identified an alternative cell cycle regulatory element, Soj Mtb encoded by rv1708. Soj Mtb coordinates a regulatory mechanism involving cell cycle control at the point of septum formation and elicits the induction of the MazF6 toxin. MazF6 functions as an mRNA interferase leading to bacteriostasis that can be prevented by interaction with its cognate antitoxin, MazE6. Further, MazEF6 acts independently of other Maz family toxin:antitoxin pairs. Notably, soj Mtb and mazEF6 transcripts where identified at 20, 40 and 100 days post-infection in increasing abundance indicating a role in adaption during chronic infection. Conclusions: Here we present the first evidence of a coupled regulatory system in which cell cycle regulation via Soj Mtb is linked to downstream adaptations that are facilitated through the activity of the MazEF6 TA pair.