Cytochrome Bc1 Inhibitors Research Articles

Lansoprazole interferes with fungal respiration and acts synergistically with amphotericin B against multidrug-resistant Candida auris

ABSTRACT Candida auris has emerged as a problematic fungal pathogen associated with high morbidity and mortality. Amphotericin B (AmB) is the most effective antifungal used to treat invasive fungal candidiasis, with resistance rarely observed among clinical isolates. However, C. auris possesses extraordinary resistant profiles against all available antifungal drugs, including AmB. In our pursuit of potential solutions, we screened a panel of 727 FDA-approved drugs. We identified the proton pump inhibitor lansoprazole (LNP) as a potent enhancer of AmB’s activity against C. auris. LNP also potentiates the antifungal activity of AmB against other medically important species of Candida and Cryptococcus. Our investigations into the mechanism of action unveiled that LNP metabolite(s) interact with a crucial target in the mitochondrial respiratory chain (complex III, known as cytochrome bc1 ). This interaction increases oxidative stress within fungal cells. Our results demonstrated the critical role of an active respiratory function in the antifungal activity of LNP. Most importantly, LNP restored the efficacy of AmB in an immunocompromised mouse model, resulting in a 1.7-log (∼98%) CFU reduction in the burden of C. auris in the kidneys. Our findings strongly advocate for a comprehensive evaluation of LNP as a cytochrome bc1 inhibitor for combating drug-resistant C. auris infections.

Characterization of Two Novel Inhibitors of the Mycobacterium tuberculosis Cytochrome bc1 Complex.

Drug-resistant tuberculosis is a global health care threat calling for novel effective treatment options. Here, we report on two novel cytochrome bc1 inhibitors (MJ-22 and B6) targeting the Mycobacterium tuberculosis respiratory chain with excellent intracellular activities in human macrophages. Both hit compounds revealed very low mutation frequencies and distinct cross-resistance patterns with other advanced cytochrome bc1 inhibitors.

Nitric Oxide-Dependent Electron Transport Chain Inhibition by the Cytochrome bc1 Inhibitor and Pretomanid Combination Kills Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of human tuberculosis, harbors a branched electron transport chain, preventing the bactericidal action of cytochrome bc1 inhibitors (e.g., TB47). Here, we investigated, using luminescent mycobacterial strains, the in vitro combination activity of cytochrome bc1 inhibitors and nitric oxide (NO) donors including pretomanid (PMD) and explored the mechanisms of combination activity. The TB47 and PMD combination quickly abolished the light emission of luminescent bacilli, as was the case for the combination of TB47 and aurachin D, a putative cytochrome bd inhibitor. The TB47 and PMD combination inhibited M. tuberculosis oxygen consumption, decreased ATP levels, and had a delayed bactericidal effect. The NO scavenger carboxy-PTIO prevented the bactericidal activity of the drug combination, suggesting the requirement for NO. In addition, cytochrome bc1 inhibitors were largely bactericidal when administered with DETA NONOate, another NO donor. Proteomic analysis revealed that the cotreated bacilli had a compromised expression of the dormancy regulon proteins, PE/PPE proteins, and proteins required for the biosynthesis of several cofactors, including mycofactocin. Some of these proteomic changes, e.g., the impaired dormancy regulon induction, were attributed to PMD. In conclusion, combination of cytochrome bc1 inhibitors with PMD inhibited M. tuberculosis respiration and killed the bacilli. The activity of cytochrome bc1 inhibitors can be greatly enhanced by NO donors. Monitoring of luminescence may be further exploited to screen cytochrome bd inhibitors.

Natural Compound-derived Cytochrome bc1 Complex Inhibitors as Antifungal Agents.

The high incidence of fungal pathogens has become a global issue for crop protection. A promising strategy to control fungal plant infections is based on the use of nature-inspired compounds. The cytochrome bc1 complex is an essential component of the cellular respiratory chain and is one of the most important fungicidal targets. Natural products have played a crucial role in the discovery of cytochrome bc1 inhibitors, as proven by the development of strobilurins, one of the most important classes of crop-protection agents, over the past two decades. In this review, we summarize advances in the exploration of natural product scaffolds for the design and development of new bc1 complex inhibitors. Particular emphasis is given to molecular modeling-based approaches and structure–activity relationship (SAR) studies performed to improve the stability and increase the potency of natural precursors. The collected results highlight the versatility of natural compounds and provide an insight into the potential development of nature-inspired derivatives as antifungal agents.

Assessment of Clofazimine and TB47 Combination Activity against Mycobacterium abscessus Using a Bioluminescent Approach.

Mycobacterium abscessus is intrinsically resistant to most antimicrobial agents. The emerging infections caused by M. abscessus and the lack of effective treatment call for rapid attention. Here, we intended to construct a selectable marker-free autoluminescent M. abscessus strain (designated UAlMab) as a real-time reporter strain to facilitate the discovery of effective drugs and regimens for treating M. abscessus The UAlMab strain was constructed using the dif/Xer recombinase system. In vitro and in vivo activities of several drugs, including clofazimine and TB47, a recently reported cytochrome bc1 inhibitor, were assessed using UAlMab. Furthermore, the efficacy of multiple drug combinations, including the clofazimine and TB47 combination, were tested against 20 clinical M. abscessus isolates. The UAlMab strain enabled us to evaluate drug efficacy both in vitro and in live BALB/c mice in a real-time, noninvasive fashion. Importantly, although TB47 showed marginal activity either alone or in combination with clarithromycin, amikacin, or roxithromycin, the drug markedly potentiated the activity of clofazimine, both in vitro and in vivo This study demonstrates that the use of the UAlMab strain can significantly facilitate rapid evaluation of new drugs and regimens. The clofazimine and TB47 combination is effective against M. abscessus, and dual/triple electron transport chain (ETC) targeting can be an effective therapeutic approach for treating mycobacterial infections.

Cyclization-blocked proguanil as a strategy to improve the antimalarial activity of atovaquone

Atovaquone-proguanil (Malarone®) is used for malaria prophylaxis and treatment. While the cytochrome bc1-inhibitor atovaquone has potent activity, proguanil’s action is attributed to its cyclization-metabolite, cycloguanil. Evidence suggests that proguanil has limited intrinsic activity, associated with mitochondrial-function. Here we demonstrate that proguanil, and cyclization-blocked analogue tBuPG, have potent, but slow-acting, in vitro anti-plasmodial activity. Activity is folate-metabolism and isoprenoid biosynthesis-independent. In yeast dihydroorotate dehydrogenase-expressing parasites, proguanil and tBuPG slow-action remains, while bc1-inhibitor activity switches from comparatively fast to slow-acting. Like proguanil, tBuPG has activity against P. berghei liver-stage parasites. Both analogues act synergistically with bc1-inhibitors against blood-stages in vitro, however cycloguanil antagonizes activity. Together, these data suggest that proguanil is a potent slow-acting anti-plasmodial agent, that bc1 is essential to parasite survival independent of dihydroorotate dehydrogenase-activity, that Malarone® is a triple-drug combination that includes antagonistic partners and that a cyclization-blocked proguanil may be a superior combination partner for bc1-inhibitors in vivo.

Targeted Structure-Activity Analysis of Endochin-like Quinolones Reveals Potent Qi and Qo Site Inhibitors of Toxoplasma gondii and Plasmodium falciparum Cytochrome bc1 and Identifies ELQ-400 as a Remarkably Effective Compound against Acute Experimental Toxoplasmosis.

Cytochrome bc1 inhibitors have been broadly studied as human and veterinary medicines and agricultural fungicides. For the most part, cytochrome bc1 inhibitors compete with ubiquinol at the ubiquinol oxidation (Qo) site or with ubiquinone at the quinone reduction (Qi) site. 4(1 H)-Quinolones with 3-position substituents may inhibit either site based on quinolone ring substituents. 4(1 H)-Quinolones that inhibit the Qi site are highly effective against toxoplasmosis, malaria, and babesiosis and do not inhibit human cytochrome bc1. We tested a series of 4(1 H)-Quinolones against wild-type and drug resistant strains of Toxoplasma gondii and Plasmodium falciparum. These experiments identified very potent compounds that inhibit T. gondii proliferation at picomolar concentrations. The most potent compounds target the Qo site, and for these compounds, an alkyl side chain confers potency against T. gondii greater than that of bulkier side chains. Our experiments also show that substituents on the quinolone ring influenced selectivity between T. gondii and P. falciparum and between Qo and Qi site-mediated activity. Comparison of the parasite cytochrome b sequences identified amino acids that are associated with drug resistance in P. falciparum that exist naturally in wild-type T. gondii. These underlying differences may influence drug susceptibility. Finally, a Qo site active 4(1 H)-quinolone-3-diarylether tested in a murine model of toxoplasmosis was superior to atovaquone, resulting in survival from Type I strain T. gondii infection. These experiments identify highly effective compounds for toxoplasmosis and provide valuable insight into the structure-activity relationship of cytochrome bc1 inhibitors.

A Fungal-Selective Cytochrome bc1 Inhibitor Impairs Virulence and Prevents the Evolution of Drug Resistance

To cause disease, a microbial pathogen must adapt to the challenges of its host environment. The leading fungal pathogen Candida albicans colonizes nutrient-poor bodily niches, withstands attack from the immune system, and tolerates treatment with azole antifungals, often evolving resistance. To discover agents that block these adaptive strategies, we screened 300,000 compounds for inhibition of azole tolerance in a drug-resistant Candida isolate. We identified a novel indazole derivative that converts azoles from fungistatic to fungicidal drugs by selective inhibition of mitochondrial cytochrome bc1. Wesynthesized 103 analogs to optimize potency (half maximal inhibitory concentration 0.4?M) and fungal selectivity (28-fold over human). In additionto reducing azole resistance, targeting cytochromebc1 prevents C.albicans from adapting to the nutrient-deprived macrophage phagosome and greatly curtails its virulence in mice. Inhibiting mitochondrial respiration and restricting metabolic flexibility with this synthetically tractable chemotype provides an attractive therapeutic strategy to limit both fungal virulence and drug resistance.

Alterations of cellular redox homeostasis in cultured fibroblast-like renal cells upon exposure to low doses of cytochrome bc1 complex inhibitor kresoxim-methyl

In this paper we elucidate the effects of the cytochrome bc1 inhibitor, strobilurin fungicide kresoxim-methyl, on the redox balance of a mammalian renal cell line. To explore whether mammalian exposure to sub-nephrotoxic concentrations of kresoxim-methyl induces cellular and biochemical mechanisms of toxicity, its effects on cellular viability and, in particular, several parameters related to oxidative stress, mitochondrial respiratory function and apoptosis were examined in fibroblast-like renal Vero cells. Elevation of mitochondrial superoxide generation, together with a concomitant decrease in mitochondrial transmembrane potential was indicative of mitochondrial dysfunction. Losses on antioxidant enzyme activities and GSH, along with increased H2O2 and nitrite release were associated with oxidative stress and induced impaired cellular migration. Raise of intracellular calcium was also observed, while no experimental evidence of apoptosis was found. Our findings suggest that sub-nephrotoxic concentrations of kresoxim-methyl cause perturbation of multiple pathways in renal mammalian cellular redox homeostasis.

Rational Design of Highly Potent and Slow-Binding Cytochrome bc1 Inhibitor as Fungicide by Computational Substitution Optimization

Hit to lead (H2L) optimization is a key step for drug and agrochemical discovery. A critical challenge for H2L optimization is the low efficiency due to the lack of predictive method with high accuracy. We described a new computational method called Computational Substitution Optimization (CSO) that has allowed us to rapidly identify compounds with cytochrome bc1 complex inhibitory activity in the nanomolar and subnanomolar range. The comprehensively optimized candidate has proved to be a slow binding inhibitor of bc1 complex, ~73-fold more potent (Ki = 4.1 nM) than the best commercial fungicide azoxystrobin (AZ; Ki = 297.6 nM) and shows excellent in vivo fungicidal activity against downy mildew and powdery mildew disease. The excellent correlation between experimental and calculated binding free-energy shifts together with further crystallographic analysis confirmed the prediction accuracy of CSO method. To the best of our knowledge, CSO is a new computational approach to substitution-scanning mutagenesis of ligand and could be used as a general strategy of H2L optimisation in drug and agrochemical design.

Lansoprazole is an antituberculous prodrug targeting cytochrome bc1.

Better antibiotics capable of killing multi-drug-resistant Mycobacterium tuberculosis are urgently needed. Despite extensive drug discovery efforts, only a few promising candidates are on the horizon and alternative screening protocols are required. Here, by testing a panel of FDA-approved drugs in a host cell-based assay, we show that the blockbuster drug lansoprazole (Prevacid), a gastric proton-pump inhibitor, has intracellular activity against M. tuberculosis. Ex vivo pharmacokinetics and target identification studies reveal that lansoprazole kills M. tuberculosis by targeting its cytochrome bc1 complex through intracellular sulfoxide reduction to lansoprazole sulfide. This novel class of cytochrome bc1 inhibitors is highly active against drug-resistant clinical isolates and spares the human H+K+-ATPase thus providing excellent opportunities for targeting the major pathogen M. tuberculosis. Our finding provides proof of concept for hit expansion by metabolic activation, a powerful tool for antibiotic screens.

Purification and characterisation of recombinant DNA encoding the alternative oxidase from Sauromatum guttatum

The alternative oxidase (AOX) is a non-protonmotive ubiquinol oxidase that is found in mitochondria of all higher plants studied to date. Structural and functional characterisation of this important but enigmatic plant diiron protein has been hampered by an inability to obtain sufficient native protein from plant sources. In the present study recombinant SgAOX (rSgAOX), overexpressed in a ΔhemA-deficient Escherichia coli strain (FN102), was solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. The kinetics of ubiquinol-1 oxidation by purified rSgAOX showed typical Michaelis–Menten kinetics (Km of 332μM and Vmax of 30μmol−1min−1mg−1), a turnover number 20μmols−1 and a remarkable stability. The enzyme was potently inhibited not only by conventional inhibitors such as SHAM and n-propyl gallate but also by the potent TAO inhibitors ascofuranone, an ascofuranone-derivative colletochlorin B and the cytochrome bc1 inhibitor ascochlorin. Circular dichroism studies revealed that AOX was approximately 50% α-helical and furthermore such studies revealed that rSgAOX and rTAO partially retained the helical absorbance signal even at 90°C (58% and 64% respectively) indicating a high conformational stability. It is anticipated that highly purified and active AOX and its mutants will facilitate investigations into the structure and reaction mechanisms of AOXs through the provision of large amounts of purified protein for crystallography and contribute to further progress of the study on this important plant terminal oxidase.

2D, 3D-QSAR and molecular docking of 4(1H)-quinolones analogues with antimalarial activities

Cytochrome bc1 has become a major focus as a molecular target in malaria parasites, which are the most important vector-borne infectious disease in the world. The inhibition of cytochrome bc1 blocks the mitochondrial respiratory chain and the consequent arrest of pyrimidine biosynthesis, which is essential for parasite development. The authors developed a theoretical study of two-dimensional, three-dimensional quantitative structure–activity relationships and a docking analysis of a series of 4(1H)-quinolones acting as cytochrome bc1 inhibitors. The predictive ability of the quantitative structure–activity relationship models was assessed using internal (leave-one-out cross-validation) and external (test set with 8 compounds) validation. From the two-dimensional quantitative structure–activity relationship models, the authors emphasized the following descriptors: GCUT_SLOGP_0, SLogP_VSA_5, Kier molecular flexibility index, electrophilicity index, the partition coefficient and the charge of atom 5 of the quinolone ring as the most important to explain the antimalarial activity of the compounds studied. Three-dimensional quantitative structure–activity relationship models showed that the substituents R1 and R4 in 4(1H)-quinolones analogues are key modulators to enhance the antimalarial activity. The appropriate binding conformations and orientations of these compounds interacting with cytochrome bc1 were also revealed by molecular docking. Based on the established models, 8 new compounds with highly predicted antimalarial activity have been theoretically designed and presented as a reference for synthesis and antimalarial evaluation.

Structure-Based Virtual Screening and Molecular Dynamic Simulation Studies to Identify Novel Cytochrome bc1 Inhibitors as Antimalarial Agents

Cytochrome bc1 (EC 1.10.2.2, bc1) is an essential component of the cellular respiratory chain, which catalyzes electron transfer from quinol to cytochrome c and concomitantly the translocation of protons across the membrane. It has been identified as a promising target in malaria parasites. The structure-based pharmacophore modelling and molecular dynamic simulation approach have been employed to identify novel inhibitors of cytochrome bc1. The best structure-based pharmacophore hypothesis (Hypo1) consists of one hydrogen bond acceptor (HBA), one general hydrophobic (HY), and two hydrophobic aromatic features (HYAr). Further, hydrogen interactions and hydrophobic interactions of known potent inhibitors with cytochrome bc1 were compared with Hypo1, which showed that the Hypo1 has good predictive ability. The validated Hypo1 was used to screen the chemical databases. The hits obtained were subsequently subjected to the molecular docking analysis to identify false-positive hits. Moreover, the molecular docking results were further validated by molecular dynamics simulations. Binding-free energy analysis using MM-GBSA method reveals that the van der Waals interactions and the electrostatic energy provide the basis for favorable absolute free energy of the complex. The five virtual hits were identified as possible candidates for the designing of potent cytochrome bc1 inhibitors.

FcStuA from Fusarium culmorum Controls Wheat Foot and Root Rot in a Toxin Dispensable Manner

Fusarium culmorum is one of the most harmful pathogens of durum wheat and is the causal agent of foot and root rot (FRR) disease. F. culmorum produces the mycotoxin deoxynivalenol (DON) that is involved in the pathogenic process. The role of the gene FcStuA, a StuA ortholog protein with an APSES domain sharing 98.5% homology to the FgStuA protein (FGSG10129), was determined by functional characterisation of deletion mutants obtained from two F. culmorum wild-type strains, FcUk99 (a highly pathogenic DON producer) and Fc233B (unable to produce toxin and with a mild pathogenic behavior). The ΔFcStuA mutants originating from both strains showed common phenotypic characters including stunted vegetative growth, loss of hydrophobicity of the mycelium, altered pigmentation, decreased activity of polygalacturonic enzymes and catalases, altered and reduced conidiation, delayed conidial germination patterns and complete loss of pathogenicity towards wheat stem base/root tissue. Glycolytic process efficiency [measured as growth on glucose as sole carbon (C) source] was strongly impaired and growth was partially restored on glutamic acid. Growth on pectin-like sources ranked in between glucose and glutamic acid with the following order (the lowest to the highest growth): beechwood xylan, sugarbeet arabinan, polygalacturonic acid, citrus pectin, apple pectin, potato azogalactan. DON production in the mutants originating from FcUK99 strain was significantly decreased (−95%) in vitro. Moreover, both sets of mutants were unable to colonise non-cereal plant tissues, i.e. apple and tomato fruits and potato tubers. No differences between mutants, ectopic and wild-type strains were observed concerning the level of resistance towards four fungicides belonging to three classes, the demethylase inhibitors epoxiconazole and tebuconzole, the succinate dehydrogenase inhibitor isopyrazam and the cytochrome bc1 inhibitor trifloxystrobin. StuA, given its multiple functions in cell regulation and pathogenicity control, is proposed as a potential target for novel disease management strategies.

Identification and Validation of Tetracyclic Benzothiazepines as Plasmodium falciparum Cytochrome bc1 Inhibitors

Here we report the discovery of tetracyclic benzothiazepines (BTZs) as highly potent and selective antimalarials along with the identification of the Plasmodium falciparum cytochrome bc(1) complex as the primary functional target of this novel compound class. Investigation of the structure activity relationship within this previously unexplored chemical scaffold has yielded inhibitors with low nanomolar activity. A combined approach employing genetically modified parasites, biochemical profiling, and resistance selection validated inhibition of cytochrome bc(1) activity, an essential component of the parasite respiratory chain and target of the widely used antimalarial drug atovaquone, as the mode of action of this novel compound class. Resistance to atovaquone is eroding the efficacy of this widely used antimalarial drug. Intriguingly, BTZ-based inhibitors retain activity against atovaquone resistant parasites, suggesting this chemical class may provide an alternative to atovaquone in combination therapy.