Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs

Abstract

Mycobacterium tuberculosis (Mtb) is an obligate aerobe that is capable of long-term persistence under conditions of low oxygen tension. Analysis of the Mtb genome predicts the existence of a branched aerobic respiratory chain terminating in a cytochrome bd system and a cytochrome aa3 system. Both chains can be initiated with type II NADH:menaquinone oxidoreductase. We present a detailed biochemical characterization of the aerobic respiratory chains from Mtb and show that phenothiazine analogs specifically inhibit NADH:menaquinone oxidoreductase activity. The emergence of drug-resistant strains of Mtb has prompted a search for antimycobacterial agents. Several phenothiazines analogs are highly tuberculocidal in vitro, suppress Mtb growth in a mouse model of acute infection, and represent lead compounds that may give rise to a class of selective antibiotics. Mycobacterium tuberculosis ͉ respiratory chainT he World Health Organization estimates that two billion people are infected with Mycobacterium tuberculosis (Mtb), and two million people die of the disease each year (1). Most individuals infected with the organism are latent carriers who have a 2-23% lifetime risk of developing reactivation tuberculosis (TB). The risk dramatically increases if the carrier's immune system is suppressed. Also, drug resistance is a serious concern; the isoniazid (INH)-resistance rate is Ϸ10%, and the rifampicin (RIF) resistance rate is Ϸ1%, with lower numbers in countries with effective TB programs and higher numbers in countries with deficient TB programs. The World Health Organization declared TB infections to be a global public health emergency (1), and the need to identify targets for antimicrobial therapy remains urgent.Mtb is capable of establishing persistent infection in the host by using a complex interplay between the host immune system and bacterial survival mechanisms. In the persistent infection, Mtb adapt to depletion of available oxygen and nutrients and enter a stage of nonreplicating persistence (NRP) in granulomatous or necrotic lesions. NRP Mtb are resistant to INH, ethambutol, and RIF, but they become sensitive to metronidazole in vitro (2). Given the critical role of oxygen in the generation of cellular energy and bacterial long-term survival, there is surprisingly little information on oxidative phosphorylation in Mtb. Clearly, oxidative phosphorylation is a central component in the production of ATP and the subsequent growth and pathogenesis of Mtb. Here, we characterize the aerobic respiratory pathway and show that NADH:menaquinone oxidoreductase (Ndh) is a key target for TB agents. Materials and MethodsMedia and Strains. Mtb H 37 R v was a gift from C. Imperatrice (Clinical Infectious Diseases, Hospital of the University of Pennsylvania) and Mycobacterium smegmatis Mc 2 155 was obtained from V. Mizrahi (National Health Laboratory Service, Johannesburg). Bacteria were cultured in 7H9 broth supplemented with 10% oleic acid-albumin-dextrose catalase͞0.5% glycerol͞0.05% Tween 80. Solid agar (15 g͞liter) was ad...

Nutrient-starved, non-replicating Mycobacterium tuberculosis requires respiration, ATP synthase and isocitrate lyase for maintenance of ATP homeostasis and viability

“…Maintaining a Energy metabolism of nutrient-starved M. tuberculosis constant ATP level was demonstrated to be critical for the survival of hypoxic M. tuberculosis, and the respiratory enzyme Ndh-2, but not Ndh-1, was shown to be required for the maintenance of ATP homeostasis (Rao et al, 2008). Various groups have reported that tubercle bacilli are susceptible to phenothiazines, a class of known Ndh-2 inhibitors (Amaral et al, 1996;Teh et al, 2007;Weinstein et al, 2005;Yano et al, 2006). The Rubin lab demonstrated the bactericidal activity of phenothiazine compounds on nutrient-starved M. tuberculosis (Xie et al, 2005).…”

Quinolinyl Pyrimidines: Potent Inhibitors of NDH-2 as a Novel Class of Anti-TB Agents

“…17 It is worth mentioning that this enzyme is not found in the mammalian mitochondria. The fundamental role of NDH-2 in Mtb respiration is supported by a wide range of biochemical 18 and transcriptional studies. 19 The Mtb genome contains two copies of ndh genes (ndh and ndhA).…”

“…17,21 The effect of ndh deletion mutant lacking NDH-2 on bacterial growth under various culture conditions and on animal infection has been reported. 18 Phenothiazines (a class of antipsychotic drugs) are reported to be inhibitors of NDH-2. 17 This class of compounds is known to inhibit the growth of bacteria in vitro as well as bacteria inside macrophages.…”

A Self‐Assembled Respiratory Chain that Catalyzes NADH Oxidation by Ubiquinone‐10 Cycling between Complex I and the Alternative Oxidase

“…Historically, they were used to demonstrate the construction of respiratory chains from individual enzymes and Q 10 , but their capabilities have not (with very few exceptions31) been exploited for detailed mechanistic studies. Furthermore, many quinone antagonists are already used as pharmaceuticals or pesticides,32, 33 and bioenergetic enzymes specific to parasites and phytopathogenic fungi (including the alternative NADH dehydrogenase and AOX) are being investigated as drug targets 34. 35 Our self‐assembled Q 10 PL system enables candidate drugs and inhibitors to be competed against physiologically relevant quinone substrates to provide properly defined and robust inhibitor dissociation constants for translational research.…”