A high-throughput target-based screening approach for the identification and assessment of Mycobacterium tuberculosis mycothione reductase inhibitors.

The World Health Organization's goal to combat tuberculosis (TB) is hindered by the emergence of anti-microbial resistance, therefore necessitating the exploration of new drug targets. Multidrug regimens are indispensable in TB therapy as they provide synergetic bactericidal effects, shorten treatment duration, and reduce the risk of resistance development. The research within our European RespiriTB consortium explores Mycobacterium tuberculosis energy metabolism to identify new drug candidates that synergize with bedaquiline, with the aim of discovering more efficient combination drug regimens. In this study, we describe the development and validation of a luminescence-coupled, target-based assay for the identification of novel compounds inhibiting Mycobacterium tuberculosis mycothione reductase (MtrMtb), an enzyme with a role in the protection against oxidative stress. Recombinant MtrMtb was employed for the development of a highly sensitive, robust high-throughput screening (HTS) assay by coupling enzyme activity to a bioluminescent readout. Its application in a semi-automated setting resulted in the screening of a diverse library of ~130,000 compounds, from which 19 hits were retained after an assessment of their potency, selectivity, and specificity. The selected hits formed two clusters and four fragment molecules, which were further evaluated in whole-cell and intracellular infection assays. The established HTS discovery pipeline offers an opportunity to deliver novel MtrMtb inhibitors and lays the foundation for future efforts in developing robust biochemical assays for the identification and triaging of inhibitors from high-throughput library screens. IMPORTANCE: The growing anti-microbial resistance poses a global public health threat, impeding progress toward eradicating tuberculosis. Despite decades of active research, there is still a dire need for the discovery of drugs with novel modes of action and exploration of combination drug regimens. Within the European RespiriTB consortium, we explore Mycobacterium tuberculosis energy metabolism to identify new drug candidates that synergize with bedaquiline, with the aim of discovering more efficient combination drug regimens. In this study, we present the development of a high-throughput screening pipeline that led to the identification of M. tuberculosis mycothione reductase inhibitors.

Effect of HIV on mortality among hospitalised patients in South Africa.

Background: HIV and AIDS continues to impose substantial healthcare challenges in sub-Saharan Africa, but there are limited local data comparing inpatient outcomes between people with HIV (PLWH) and those uninfected. Objectives: To compare cause-specific mortality among hospitalised adolescents and adults, stratified by HIV-serostatus. Method: A cross-sectional analysis was performed, analysing cause-specific inpatient mortality data and total admissions, from 01 January 2017 to 30 June 2020, at Tshepong Hospital, North West province, South Africa. Results: The overall inpatient mortality rate decreased from 14.5% (95% confidence interval [CI]: 13.4-16.0) in 2017, to 11.3% (95% CI: 10.6-11.9) in 2020; P < 0.001. People living with HIV accounted for 53.9% (n = 2342) of inpatient deaths, 22.6% (n = 984) were HIV-seronegative patients and 23.5% (n = 1020) patients with unknown HIV-serostatus. People with HIV died at younger ages (median: 44 years, interquartile range [IQR]: 35.8-54.2) compared to HIV-seronegative inpatients (median: 64.4 years, IQR: 55.5-73.9); P < 0.001. Leading causes of death were pneumonia (19.9%, n = 863), then pulmonary and extrapulmonary tuberculosis (15.0%, n = 654). People with HIV who had CD4+ counts < 350 cells/mL or viral load ≥ 1000 copies/mL had increased risk of death from tuberculosis compared to virally suppressed patients (adjusted relative risk: 2.10 [95% CI: 1.44-3.04, P < 0.009] and 1.56 [95% CI: 1.22-2.00, P < 0.001]). Conclusion: Our study, conducted in a regional hospital in South Africa, showed PLWH had higher mortality rates and died at younger ages compared to HIV-seronegative patients.

Mycobacterium tuberculosis H2S Functions as a Sink to Modulate Central Metabolism, Bioenergetics, and Drug Susceptibility.

H2S is a potent gasotransmitter in eukaryotes and bacteria. Host-derived H2S has been shown to profoundly alter M. tuberculosis (Mtb) energy metabolism and growth. However, compelling evidence for endogenous production of H2S and its role in Mtb physiology is lacking. We show that multidrug-resistant and drug-susceptible clinical Mtb strains produce H2S, whereas H2S production in non-pathogenic M. smegmatis is barely detectable. We identified Rv3684 (Cds1) as an H2S-producing enzyme in Mtb and show that cds1 disruption reduces, but does not eliminate, H2S production, suggesting the involvement of multiple genes in H2S production. We identified endogenous H2S to be an effector molecule that maintains bioenergetic homeostasis by stimulating respiration primarily via cytochrome bd. Importantly, H2S plays a key role in central metabolism by modulating the balance between oxidative phosphorylation and glycolysis, and it functions as a sink to recycle sulfur atoms back to cysteine to maintain sulfur homeostasis. Lastly, Mtb-generated H2S regulates redox homeostasis and susceptibility to anti-TB drugs clofazimine and rifampicin. These findings reveal previously unknown facets of Mtb physiology and have implications for routine laboratory culturing, understanding drug susceptibility, and improved diagnostics.

Bedaquiline reprograms central metabolism to reveal glycolytic vulnerability in Mycobacterium tuberculosis.

The approval of bedaquiline (BDQ) for the treatment of tuberculosis has generated substantial interest in inhibiting energy metabolism as a therapeutic paradigm. However, it is not known precisely how BDQ triggers cell death in Mycobacterium tuberculosis (Mtb). Using 13C isotopomer analysis, we show that BDQ-treated Mtb redirects central carbon metabolism to induce a metabolically vulnerable state susceptible to genetic disruption of glycolysis and gluconeogenesis. Metabolic flux profiles indicate that BDQ-treated Mtb is dependent on glycolysis for ATP production, operates a bifurcated TCA cycle by increasing flux through the glyoxylate shunt, and requires enzymes of the anaplerotic node and methylcitrate cycle. Targeting oxidative phosphorylation (OXPHOS) with BDQ and simultaneously inhibiting substrate level phosphorylation via genetic disruption of glycolysis leads to rapid sterilization. Our findings provide insight into the metabolic mechanism of BDQ-induced cell death and establish a paradigm for the development of combination therapies that target OXPHOS and glycolysis.

Hydrogen sulfide stimulates Mycobacterium tuberculosis respiration, growth and pathogenesis.

Hydrogen sulfide (H2S) is involved in numerous pathophysiological processes and shares overlapping functions with CO and •NO. However, the importance of host-derived H2S in microbial pathogenesis is unknown. Here we show that Mtb-infected mice deficient in the H2S-producing enzyme cystathionine ß-synthase (CBS) survive longer with reduced organ burden, and that pharmacological inhibition of CBS reduces Mtb bacillary load in mice. High-resolution respirometry, transcriptomics and mass spectrometry establish that H2S stimulates Mtb respiration and bioenergetics predominantly via cytochrome bd oxidase, and that H2S reverses •NO-mediated inhibition of Mtb respiration. Further, exposure of Mtb to H2S regulates genes involved in sulfur and copper metabolism and the Dos regulon. Our results indicate that Mtb exploits host-derived H2S to promote growth and disease, and suggest that host-directed therapies targeting H2S production may be potentially useful for the management of tuberculosis and other microbial infections.

New 2-Ethylthio-4-methylaminoquinazoline derivatives inhibiting two subunits of cytochrome bc1 in Mycobacterium tuberculosis.

The emergence of multi-drug (MDR-TB) and extensively-drug resistant tuberculosis (XDR-TB) is a major threat to the global management of tuberculosis (TB) worldwide. New chemical entities are of need to treat drug-resistant TB. In this study, the mode of action of new, potent quinazoline derivatives was investigated against Mycobacterium tuberculosis (M. tb). Four derivatives 11626141, 11626142, 11626252 and 11726148 showed good activity (MIC ranging from 0.02-0.09 µg/mL) and low toxicity (TD50 ≥ 5µg/mL) in vitro against M. tb strain H37Rv and HepG2 cells, respectively. 11626252 was the most selective compound from this series. Quinazoline derivatives were found to target cytochrome bc1 by whole-genome sequencing of mutants selected with 11626142. Two resistant mutants harboured the transversion T943G (Trp312Gly) and the transition G523A (Gly175Ser) in the cytochrome bc1 complex cytochrome b subunit (QcrB). Interestingly, a third mutant QuinR-M1 contained a mutation in the Rieske iron-sulphur protein (QcrA) leading to resistance to quinazoline and other QcrB inhibitors, the first report of cross-resistance involving QcrA. Modelling of both QcrA and QcrB revealed that all three resistance mutations are located in the stigmatellin pocket, as previously observed for other QcrB inhibitors such as Q203, AX-35, and lansoprazole sulfide (LPZs). Further analysis of the mode of action in vitro revealed that 11626252 exposure leads to ATP depletion, a decrease in the oxygen consumption rate and also overexpression of the cytochrome bd oxidase in M. tb. Our findings suggest that quinazoline-derived compounds are a new and attractive chemical entity for M. tb drug development targeting two separate subunits of the cytochrome bc1 complex.

Compromised Metabolic Reprogramming Is an Early Indicator of CD8+ T Cell Dysfunction during Chronic Mycobacterium tuberculosis Infection.

The immunometabolic mechanisms underlying suboptimal T cell immunity in tuberculosis remain undefined. Here, we examine how chronic Mycobacterium tuberculosis (Mtb) and M. bovis BCG infections rewire metabolic circuits and alter effector functions in lung CD8+ T cells. As Mtb infection progresses, mitochondrial metabolism deteriorates in CD8+ T cells, resulting in an increased dependency on glycolysis that potentiates inflammatory cytokine production. Over time, these cells develop bioenergetic deficiencies that reflect metabolic "quiescence." This bioenergetic signature coincides with increased mitochondrial dysfunction and inhibitory receptor expression and was not observed in BCG infection. Remarkably, the Mtb-triggered decline in T cell bioenergetics can be reinvigorated by metformin, giving rise to an Mtb-specific CD8+ T cell population with improved metabolism. These findings provide insights into Mtb pathogenesis whereby glycolytic reprogramming and compromised mitochondrial function contribute to the breakdown of CD8+ T cell immunity during chronic disease, highlighting opportunities to reinvigorate immunity with metabolically targeted pharmacologic agents.

Arylvinylpiperazine Amides, a New Class of Potent Inhibitors Targeting QcrB of Mycobacterium tuberculosis.

New drugs are needed to control the current tuberculosis (TB) pandemic caused by infection with Mycobacterium tuberculosis We report here on our work with AX-35, an arylvinylpiperazine amide, and four related analogs, which are potent antitubercular agents in vitro All five compounds showed good activity against M. tuberculosisin vitro and in infected THP-1 macrophages, while displaying only mild cytotoxicity. Isolation and characterization of M. tuberculosis-resistant mutants to the arylvinylpiperazine amide derivative AX-35 revealed mutations in the qcrB gene encoding a subunit of cytochrome bc1 oxidase, one of two terminal oxidases of the electron transport chain. Cross-resistance studies, allelic exchange, transcriptomic analyses, and bioenergetic flux assays provided conclusive evidence that the cytochrome bc1-aa3 is the target of AX-35, although the compound appears to interact differently with the quinol binding pocket compared to previous QcrB inhibitors. The transcriptomic and bioenergetic profiles of M. tuberculosis treated with AX-35 were similar to those generated by other cytochrome bc1 oxidase inhibitors, including the compensatory role of the alternate terminal oxidase cytochrome bd in respiratory adaptation. In the absence of cytochrome bd oxidase, AX-35 was bactericidal against M. tuberculosis Finally, AX-35 and its analogs were active in an acute mouse model of TB infection, with two analogs displaying improved activity over the parent compound. Our findings will guide future lead optimization to produce a drug candidate for the treatment of TB and other mycobacterial diseases, including Buruli ulcer and leprosy.IMPORTANCE New drugs against Mycobacterium tuberculosis are urgently needed to deal with the current global TB pandemic. We report here on the discovery of a series of arylvinylpiperazine amides (AX-35 to AX-39) that represent a promising new family of compounds with potent in vitro and in vivo activities against M. tuberculosis AX compounds target the QcrB subunit of the cytochrome bc1 terminal oxidase with a different mode of interaction compared to those of known QcrB inhibitors. This study provides the first multifaceted validation of QcrB inhibition by recombineering-mediated allelic exchange, gene expression profiling, and bioenergetic flux studies. It also provides further evidence for the compensatory role of cytochrome bd oxidase upon QcrB inhibition. In the absence of cytochrome bd oxidase, AX compounds are bactericidal, an encouraging property for future antimycobacterial drug development.

2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and Mycobacterium tuberculosis: Structure-Activity Relationships, Mechanism of Action and Absorption, Distribution, Metabolism, and Excretion Characterization.

Mycobacterium tuberculosis ( MTb) possesses two nonproton pumping type II NADH dehydrogenase (NDH-2) enzymes which are predicted to be jointly essential for respiratory metabolism. Furthermore, the structure of a closely related bacterial NDH-2 has been reported recently, allowing for the structure-based design of small-molecule inhibitors. Herein, we disclose MTb whole-cell structure-activity relationships (SARs) for a series of 2-mercapto-quinazolinones which target the ndh encoded NDH-2 with nanomolar potencies. The compounds were inactivated by glutathione-dependent adduct formation as well as quinazolinone oxidation in microsomes. Pharmacokinetic studies demonstrated modest bioavailability and compound exposures. Resistance to the compounds in MTb was conferred by promoter mutations in the alternative nonessential NDH-2 encoded by ndhA in MTb. Bioenergetic analyses revealed a decrease in oxygen consumption rates in response to inhibitor in cells in which membrane potential was uncoupled from ATP production, while inverted membrane vesicles showed mercapto-quinazolinone-dependent inhibition of ATP production when NADH was the electron donor to the respiratory chain. Enzyme kinetic studies further demonstrated noncompetitive inhibition, suggesting binding of this scaffold to an allosteric site. In summary, while the initial MTb SAR showed limited improvement in potency, these results, combined with structural information on the bacterial protein, will aid in the future discovery of new and improved NDH-2 inhibitors.

Susceptibility of Mycobacterium tuberculosis Cytochrome bd Oxidase Mutants to Compounds Targeting the Terminal Respiratory Oxidase, Cytochrome c.

We deleted subunits I (cydA) and II (cydB) of the Mycobacterium tuberculosis cytochrome bd menaquinol oxidase. The resulting ΔcydA and ΔcydAB mutants were hypersusceptible to compounds targeting the mycobacterial bc1 menaquinol-cytochrome c oxidoreductase and exhibited bioenergetic profiles indistinguishable from strains deficient in the ABC-type transporter, CydDC, predicted to be essential for cytochrome bd assembly. These results confirm CydAB and CydDC as potential targets for drugs aimed at inhibiting a terminal respiratory oxidase implicated in pathogenesis.

Correction: Mycobacterium tuberculosis arrests host cycle at the G1/S transition to establish long term infection.

[This corrects the article DOI: 10.1371/journal.ppat.1006389.].

Mycobacterium tuberculosis arrests host cycle at the G1/S transition to establish long term infection.

Signals modulating the production of Mycobacterium tuberculosis (Mtb) virulence factors essential for establishing long-term persistent infection are unknown. The WhiB3 redox regulator is known to regulate the production of Mtb virulence factors, however the mechanisms of this modulation are unknown. To advance our understanding of the mechanisms involved in WhiB3 regulation, we performed Mtb in vitro, intraphagosomal and infected host expression analyses. Our Mtb expression analyses in conjunction with extracellular flux analyses demonstrated that WhiB3 maintains bioenergetic homeostasis in response to available carbon sources found in vivo to establish Mtb infection. Our infected host expression analysis indicated that WhiB3 is involved in regulation of the host cell cycle. Detailed cell-cycle analysis revealed that Mtb infection inhibited the macrophage G1/S transition, and polyketides under WhiB3 control arrested the macrophages in the G0-G1 phase. Notably, infection with the Mtb whiB3 mutant or polyketide mutants had little effect on the macrophage cell cycle and emulated the uninfected cells. This suggests that polyketides regulated by Mtb WhiB3 are responsible for the cell cycle arrest observed in macrophages infected with the wild type Mtb. Thus, our findings demonstrate that Mtb WhiB3 maintains bioenergetic homeostasis to produce polyketide and lipid cyclomodulins that target the host cell cycle. This is a new mechanism whereby Mtb modulates the immune system by altering the host cell cycle to promote long-term persistence. This new knowledge could serve as the foundation for new host-directed therapeutic discovery efforts that target the host cell cycle.

Turning the respiratory flexibility of Mycobacterium tuberculosis against itself.

The Mycobacterium tuberculosis (Mtb) electron transport chain (ETC) has received significant attention as a drug target, however its vulnerability may be affected by its flexibility in response to disruption. Here we determine the effect of the ETC inhibitors bedaquiline, Q203 and clofazimine on the Mtb ETC, and the value of the ETC as a drug target, by measuring Mtb's respiration using extracellular flux technology. We find that Mtb's ETC rapidly reroutes around inhibition by these drugs and increases total respiration to maintain ATP levels. Rerouting is possible because Mtb rapidly switches between terminal oxidases, and, unlike eukaryotes, is not susceptible to back pressure. Increased ETC activity potentiates clofazimine's production of reactive oxygen species, causing rapid killing in vitro and in a macrophage model. Our results indicate that combination therapy targeting the ETC can be exploited to enhance killing of Mtb.

Ergothioneine Maintains Redox and Bioenergetic Homeostasis Essential for Drug Susceptibility and Virulence of Mycobacterium tuberculosis.

The mechanisms by which Mycobacterium tuberculosis (Mtb) maintains metabolic equilibrium to survive during infection and upon exposure to antimycobacterial drugs are poorly characterized. Ergothioneine (EGT) and mycothiol (MSH) are the major redox buffers present in Mtb, but the contribution of EGT to Mtb redox homeostasis and virulence remains unknown. We report that Mtb WhiB3, a 4Fe-4S redox sensor protein, regulates EGT production and maintains bioenergetic homeostasis. We show that central carbon metabolism and lipid precursors regulate EGT production and that EGT modulates drug sensitivity. Notably, EGT and MSH are both essential for redox and bioenergetic homeostasis. Transcriptomic analyses of EGT and MSH mutants indicate overlapping but distinct functions of EGT and MSH. Last, we show that EGT is critical for Mtb survival in both macrophages and mice. This study has uncovered a dynamic balance between Mtb redox and bioenergetic homeostasis, which critically influences Mtb drug susceptibility and pathogenicity.

The Physiology and Genetics of Oxidative Stress in Mycobacteria.

During infection, Mycobacterium tuberculosis is exposed to a diverse array of microenvironments in the human host, each with its own unique set of redox conditions. Imbalances in the redox environment of the bacillus or the host environment serve as stimuli, which could regulate virulence. The ability of M. tuberculosis to evade the host immune response and cause disease is largely owing to the capacity of the mycobacterium to sense changes in its environment, such as host-generated gases, carbon sources, and pathological conditions, and alter its metabolism and redox balance accordingly for survival. In this article we discuss the redox sensors that are, to date, known to be present in M. tuberculosis, such as the Dos dormancy regulon, WhiB family, anti-σ factors, and MosR, in addition to the strategies present in the bacillus to neutralize free radicals, such as superoxide dismutases, catalase-peroxidase, thioredoxins, and methionine sulfoxide reductases, among others. M. tuberculosis is peculiar in that it appears to have a hierarchy of redox buffers, namely, mycothiol and ergothioneine. We discuss the current knowledge of their biosynthesis, function, and regulation. Ergothioneine is still an enigma, although it appears to have distinct and overlapping functions with mycothiol, which enable it to protect against a wide range of toxic metabolites and free radicals generated by the host. Developing approaches to quantify the intracellular redox status of the mycobacterium will enable us to determine how the redox balance is altered in response to signals and environments that mimic those encountered in the host.

An enzyme-initiated Smiles rearrangement enables the development of an assay of MshB, the GlcNAc-Ins deacetylase of mycothiol biosynthesis.

MshB is the N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-D-glucopyranoside (GlcNAc-Ins) deacetylase active as one of the enzymes involved in the biosynthesis of mycothiol (MSH), a protective low molecular weight thiol present only in Mycobacterium tuberculosis and other actinomycetes. In this study, structural analogues of GlcNAc-Ins in which the inosityl moiety is replaced by a chromophore were synthesized and evaluated as alternate substrates of MshB, with the goal of identifying a compound that would be useful in high-throughput assays of the enzyme. In an unexpected and surprising finding one of the GlcNAc-Ins analogues is shown to undergo a Smiles rearrangement upon MshB-mediated deacetylation, uncovering a free thiol group. We demonstrate that this chemistry can be exploited for the development of the first continuous assay of MshB activity based on the detection of thiol formation by DTNB (Ellman's reagent); such an assay should be ideally suited for the identification of MshB inhibitors by means of high-throughput screens in microplates.