An amiloride derivative is active against the F1Fo-ATP synthase and cytochrome bd oxidase of Mycobacterium tuberculosis.

Increasing antimicrobial resistance compels the search for next-generation inhibitors with differing or multiple molecular targets. In this regard, energy conservation in Mycobacterium tuberculosis has been clinically validated as a promising new drug target for combatting drug-resistant strains of M. tuberculosis. Here, we show that HM2-16F, a 6-substituted derivative of the FDA-approved drug amiloride, is an anti-tubercular inhibitor with bactericidal properties comparable to the FDA-approved drug bedaquiline (BDQ; Sirturo®) and inhibits the growth of bedaquiline-resistant mutants. We show that HM2-16F weakly inhibits the F1Fo-ATP synthase, depletes ATP, and affects the entry of acetyl-CoA into the Krebs cycle. HM2-16F synergizes with the cytochrome bcc-aa3 oxidase inhibitor Q203 (Telacebec) and co-administration with Q203 sterilizes in vitro cultures in 14 days. Synergy with Q203 occurs via direct inhibition of the cytochrome bd oxidase by HM2-16F. This study shows that amiloride derivatives represent a promising discovery platform for targeting energy generation in drug-resistant tuberculosis.

Microtiter Screening Reveals Oxygen-Dependent Antimicrobial Activity of Natural Products Against Mastitis-Causing Bacteria.

In this study we investigated the influence of oxygen availability on a phenotypic microtiter screen to identify new, natural product inhibitors of growth for the bovine mastitis-causing microorganisms; Streptococcus uberis, Staphylococcus aureus, and Escherichia coli. Mastitis is a common disease in dairy cattle worldwide and is a major cause of reduced milk yield and antibiotic usage in dairy herds. Prevention of bovine mastitis commonly relies on the application of teat disinfectants that contain either iodine or chlorhexidine. These compounds are used extensively in human clinical settings and increased tolerance to chlorhexidine has been reported in both Gram-positive and Gram-negative microorganisms. As such new, non-human use alternatives are required for the agricultural industry. Our screening was conducted under normoxic (20% oxygen) and hypoxic (<1% oxygen) conditions to mimic the conditions on teat skin and within the mammary gland respectively, against two natural compound libraries. No compounds inhibited E. coli under either oxygen condition. Against the Gram-positive microorganisms, 12 inhibitory compounds were identified under normoxic conditions, and 10 under hypoxic conditions. Data revealed a clear oxygen-dependency amongst compounds inhibiting growth, with only partial overlap between oxygen conditions. The oxygen-dependent inhibitory activity of a naturally occurring quinone, ß-lapachone, against S. uberis was subsequently investigated and we demonstrated that this compound is only active under normoxic conditions with a minimum inhibitory concentration and minimum bactericidal concentration of 32 µM and kills via a reactive oxygen species-dependent mechanism as has been demonstrated in other microorganisms. These results demonstrate the importance of considering oxygen-availability in high-throughput inhibitor discovery.

The synthesis and evaluation of quinolinequinones as anti-mycobacterial agents.

A library of thirty-two quinolinequinones (QQs) with various amine substituents at the 6- and 7-positions were synthesised efficiently and in good yields for evaluation as potential anti-tuberculosis agents. Mycobacterium tuberculosis growth inhibition assays demonstrated that QQs bearing moderate length alkyl chains (i.e. heptylphenylamino- and octylamino-QQs), and aryl groups (i.e. phenylethylamino- and benzylamino-QQs) exhibited encouraging inhibitory activity, while QQ analogue 7-chloro-6-propargylamino-quinoline-5,8-dione (16b) had excellent inhibitory activity (MIC = 8 µM). The cLogP values and redox activities of the QQs were determined, and neither readout correlated with the anti-mycobacterial activities of the compounds. Notwithstanding, mode of action studies of 16b revealed that treatment of M. tuberculosis with this compound led to activation of NADH-dependent oxygen consumption suggesting a redox cycling mechanism. To this end, the promising anti-mycobacterial activity of several QQs and their ability to perturb oxygen management leading to an uncontrolled respiratory burst, as identified in this work and by others, demonstrates the merit of further optimising the anti-mycobacterial activity of this readily synthesised class of compound.

Activation of type II NADH dehydrogenase by quinolinequinones mediates antitubercular cell death.

OBJECTIVES: Quinolinequinones (QQ) have been shown to inhibit the growth of mycobacterial species, but their mode(s) of action and molecular target(s) remain unknown. To facilitate further development of QQ as antimycobacterial drugs, we investigated the molecular mechanism and target of QQ in mycobacteria. METHODS: Cell viability of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin was determined in the presence of QQ8c, a representative QQ compound, and isoniazid, a frontline antitubercular drug. The effect of QQ8c on mycobacterial energetics was studied using inverted membrane vesicles. NADH oxidation and formation of reactive oxygen species (ROS) were measured in the presence and absence of KCN. Generation of ROS was measured via oxygen consumption in an oxygen electrode. The effects of QQ8c were compared with the antimycobacterial drug clofazimine in side-by-side experiments. RESULTS: QQ8c challenge resulted in complete sterilization of cultures with no refractory resistant population observed. QQ8c stimulated NADH oxidation by type II NADH dehydrogenase (NDH-2) and oxygen consumption in inverted membrane vesicles. Large quantities of ROS were produced in the presence of QQ8. Even when oxygen consumption was blocked with KCN, activation of NDH-2 by QQ8c occurred suggesting QQ8c was redox cycling. CONCLUSIONS: QQ8c targets NDH-2 of the mycobacterial respiratory chain leading to activation of NADH oxidation and generating bactericidal levels of ROS in a manner similar to, but more effectively than, the antimycobacterial drug clofazimine. Our results validate respiratory-generated ROS as an avenue for antimycobacterial drug development.

Synthesis and activity of a diselenide bond mimetic of the antimicrobial protein caenopore-5.

Antimicrobial proteins are a rich source of new lead compounds for the development of new drugs that will tackle global resistance towards existing antibiotics. Caenopore-5 (Cp-5) is an antimicrobial protein (AMP) expressed in the intestine of the nematode Caenorhabditis elegans and is a member of the lipid binding saposin-like-protein family, composed of 5 α-helices and 3 disulfide bonds. Substitution of the 7Cys and 81Cys by two selenocysteine 7U and 81U afforded a selenocysteine analogue [7Sec-81Sec]-Cp-5, which displayed a higher stability (using thermal circular dichroism) compared to the native protein Cp-5. [7Sec-81Sec]-Cp-5 and an N-terminal truncated peptide exhibited cell permeability similar to the wild type Cp-5.