A Novel Small-Molecule Inhibitor of the Mycobacterium tuberculosis Demethylmenaquinone Methyltransferase MenG Is Bactericidal to Both Growing and Nutritionally Deprived Persister Cells

Abstract

Active tuberculosis (TB) and latent Mycobacterium tuberculosis infection both require lengthy treatments to achieve durable cures. This problem has partly been attributable to the existence of nonreplicating M. tuberculosis “persisters” that are difficult to kill using conventional anti-TB treatments. Compounds that target the respiratory pathway have the potential to kill both replicating and persistent M. tuberculosis and shorten TB treatment, as this pathway is essential in both metabolic states. We developed a novel respiratory pathway-specific whole-cell screen to identify new respiration inhibitors. This screen identified the biphenyl amide GSK1733953A (DG70) as a likely respiration inhibitor. DG70 inhibited both clinical drug-susceptible and drug-resistant M. tuberculosis strains. Whole-genome sequencing of DG70-resistant colonies identified mutations in menG (rv0558), which is responsible for the final step in menaquinone biosynthesis and required for respiration. Overexpression of menG from wild-type and DG70-resistant isolates increased the DG70 MIC by 4× and 8× to 30×, respectively. Radiolabeling and high-resolution mass spectrometry studies confirmed that DG70 inhibited the final step in menaquinone biosynthesis. DG70 also inhibited oxygen utilization and ATP biosynthesis, which was reversed by external menaquinone supplementation. DG70 was bactericidal in actively replicating cultures and in a nutritionally deprived persistence model. DG70 was synergistic with the first-line TB drugs isoniazid, rifampin, and the respiratory inhibitor bedaquiline. The combination of DG70 and isoniazid completely sterilized cultures in the persistence model by day 10. These results suggest that MenG is a good therapeutic target and that compounds targeting MenG along with standard TB therapy have the potential to shorten TB treatment duration.

Recent advances for identification of new scaffolds and drug targets for Mycobacterium tuberculosis

“…Ubiquinone methyl transferase (MenG) enzyme catalyzes methylation of dimethylmenaquinone into MK‐9 using S‐adenosyl methionine. Using respiratory pathway specific phenotypic screens, diphenyl amide has been identified as inhibitor for MenG enzyme from M. tuberculosis . This compound was bactericidal and showed synergy with INH, Rif, and bedaquiline in vitro .…”

“…Using respiratory pathway specific phenotypic screens, diphenyl amide has been identified as inhibitor for MenG enzyme from M. tuberculosis . This compound was bactericidal and showed synergy with INH, Rif, and bedaquiline in vitro . Furthermore, target‐based screening has led to identification of small molecule inhibitors against other enzymes involved in MK‐9 biosynthesis such as MenA, MenB, and MenE .…”

Demethylmenaquinone methyl transferase is a membrane domain-associated protein essential for menaquinone homeostasis in Mycobacterium smegmatis

“…as a surrogate electron carrier in M. tuberculosis in the presence of MenG inhibitor 516 (Sukheja et al, 2017). Second, MenG depletion in M. smegmatis did not lead to the 517 reduction in ATP production.…”

“…the lack of growth is due to the depletion of menaquinone. In M. tuberculosis, the growth 415 arrest by the MenG inhibitor was rescued by the addition of MK-4 as a surrogate 416 menaquinone (Sukheja et al, 2017). Therefore, we added MK-4 to see if exogenously 417 added menaquinone can rescue the growth of the mutant in the presence of ATC.…”

Allosteric Regulation of Vitamin K2 Biosynthesis in a Human Pathogen

“…MK levels are known to be tightly linked to the redox potential in bacteria [2,3,[7][8][9][10]; however, the molecular mechanisms that regulate this phenomenon are unclear. The first committed step in MK biosynthesis in Mtb is catalyzed by the thiamine-diphosphate (ThDP)-dependent enzyme MenD (2-succinyl-5-enolpyruvyl-6hydroxy-3-cyclohexadiene-1-carboxylate synthase, SEPHCHC synthase).…”