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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. IMPORTANCE This study shows that MenG, which is responsible for the last enzymatic step in menaquinone biosynthesis, may be a good drug target for improving TB treatments. We describe the first small-molecule inhibitor (DG70) of Mycobacterium tuberculosis MenG and show that DG70 has characteristics that are highly desirable for a new antitubercular agent, including bactericidality against both actively growing and nonreplicating mycobacteria and synergy with several first-line drugs that are currently used to treat TB.

In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was < or = 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria.

Volume 60, no. 6, p. [3497–3508][1], 2016. Page 3507, left column, lines 2 and 3: “nonnucleoside reverse transcriptase inhibitors (e.g., zidovudine)” should read “nucleoside reverse transcriptase inhibitors (e.g., zidovudine).” [1]: /lookup/doi/10.1128/AAC.03049-15