The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.

Bacterial dormancy is a major impediment to the eradication of tuberculosis (TB), because currently used drugs primarily target actively replicating bacteria. Therefore, decoding of the critical survival pathways in dormant tubercle bacilli is a research priority to formulate new approaches for killing these bacteria. Employing a network-based gene expression analysis approach, we demonstrate that redox active vitamin C (vit C) triggers a multifaceted and robust adaptation response in Mycobacterium tuberculosis (Mtb) involving ~ 67% of the genome. Vit C-adapted bacteria display well-described features of dormancy, including growth stasis and progression to a viable but non-culturable (VBNC) state, loss of acid-fastness and reduction in length, dissipation of reductive stress through triglyceride (TAG) accumulation, protective response to oxidative stress, and tolerance to first line TB drugs. VBNC bacteria are reactivatable upon removal of vit C and they recover drug susceptibility properties. Vit C synergizes with pyrazinamide, a unique TB drug with sterilizing activity, to kill dormant and replicating bacteria, negating any tolerance to rifampicin and isoniazid in combination treatment in both in-vitro and intracellular infection models. Finally, the vit C multi-stress redox models described here also offer a unique opportunity for concurrent screening of compounds/combinations active against heterogeneous subpopulations of Mtb. These findings suggest a novel strategy of vit C adjunctive therapy by modulating bacterial physiology for enhanced efficacy of combination chemotherapy with existing drugs, and also possible synergies to guide new therapeutic combinations towards accelerating TB treatment.

DNA gyrase is a clinically validated target for developing drugs against Mycobacterium tuberculosis (Mtb). Despite the promise of fluoroquinolones (FQs) as anti-tuberculosis drugs, the prevalence of pre-existing resistance to FQs is likely to restrict their clinical value. We describe a novel class of N-linked aminopiperidinyl alkyl quinolones and naphthyridones that kills Mtb by inhibiting the DNA gyrase activity. The mechanism of inhibition of DNA gyrase was distinct from the fluoroquinolones, as shown by their ability to inhibit the growth of fluoroquinolone-resistant Mtb. Biochemical studies demonstrated this class to exert its action via single-strand cleavage rather than double-strand cleavage, as seen with fluoroquinolones. The compounds are highly bactericidal against extracellular as well as intracellular Mtb. Lead optimization resulted in the identification of potent compounds with improved oral bioavailability and reduced cardiac ion channel liability. Compounds from this series are efficacious in various murine models of tuberculosis.

A pharmacophore-based search led to the identification of thiazolopyridine ureas as a novel scaffold with antitubercular activity acting through inhibition of DNA Gyrase B (GyrB) ATPase. Evaluation of the binding mode of thiazolopyridines in a Mycobacterium tuberculosis (Mtb) GyrB homology model prompted exploration of the side chains at the thiazolopyridine ring C-5 position to access the ribose/solvent pocket. Potent compounds with GyrB IC50 ≤ 1 nM and Mtb MIC ≤ 0.1 μM were obtained with certain combinations of side chains at the C-5 position and heterocycles at the C-6 position of the thiazolopyridine core. Substitutions at C-5 also enabled optimization of the physicochemical properties. Representative compounds were cocrystallized with Streptococcus pneumoniae (Spn) ParE; these confirmed the binding modes predicted by the homology model. The target link to GyrB was confirmed by genetic mapping of the mutations conferring resistance to thiazolopyridine ureas. The compounds are bactericidal in vitro and efficacious in vivo in an acute murine model of tuberculosis.