The Qi Site of Cytochrome b is a Promiscuous Drug Target in Trypanosoma cruzi and Leishmania donovani.

Available treatments for Chagas' disease and visceral leishmaniasis are inadequate, and there is a pressing need for new therapeutics. Drug discovery efforts for both diseases principally rely upon phenotypic screening. However, the optimization of phenotypically active compounds is hindered by a lack of information regarding their molecular target(s). To combat this issue we initiate target deconvolution studies at an early stage. Here, we describe comprehensive genetic and biochemical studies to determine the targets of three unrelated phenotypically active compounds. All three structurally diverse compounds target the Qi active-site of cytochrome b, part of the cytochrome bc1 complex of the electron transport chain. Our studies go on to identify the Qi site as a promiscuous drug target in Leishmania donovani and Trypanosoma cruzi with a propensity to rapidly mutate. Strategies to rapidly identify compounds acting via this mechanism are discussed to ensure that drug discovery portfolios are not overwhelmed with inhibitors of a single target.

Dual-active antifungal agents containing strobilurin and SDHI-based pharmacophores.

Crop disease management often implies repeated application of fungicides. However, the increasing emergence of fungicide-resistant pathogens requires their rotation or combined use. Tank-mix combinations using fungicides with different modes of action are often hard to manage by farmers. An alternative and unexploited strategy are bifunctional fungicides, i.e. compounds resulting from conjugation of the pharmacophores of fungicides with different mechanisms of action. In this paper we describe a new approach to antifungal treatments based on the synthesis of dual agents, obtained by merging the strobilurin and succinate dehydrogenase inhibitor pharmacophores into a new entity. The compounds were tested against important fungal plant pathogens and showed good inhibition of Pyricularia oryzae and Sclerotinia sclerotiorum with activity comparable to commercial fungicides. The inhibition of the cytochrome bc1 and the succinate dehydrogenase enzyme activity confirmed that the new molecules are endowed with a dual mechanism of action. These results were further supported by molecular modelling which showed that selected compounds form stable complexes with both cytochrome b subunit and succinate dehydrogenase enzyme. This work can be considered an important first step towards the development of novel dual-action agents with optimized structure and improved interaction with the targets.

Exposing Anopheles mosquitoes to antimalarials blocks Plasmodium parasite transmission.

Bites of Anopheles mosquitoes transmit Plasmodium falciparum parasites that cause malaria, which kills hundreds of thousands of people every year. Since the turn of this century, efforts to prevent the transmission of these parasites via the mass distribution of insecticide-treated bed nets have been extremely successful, and have led to an unprecedented reduction in deaths from malaria1. However, resistance to insecticides has become widespread in Anopheles populations2-4, which has led to the threat of a global resurgence of malaria and makes the generation of effective tools for controlling this disease an urgent public health priority. Here we show that the development of P. falciparum can be rapidly and completely blocked when female Anopheles gambiae mosquitoes take up low concentrations of specific antimalarials from treated surfaces-conditions that simulate contact with a bed net. Mosquito exposure to atovaquone before, or shortly after, P. falciparum infection causes full parasite arrest in the midgut, and prevents transmission of infection. Similar transmission-blocking effects are achieved using other cytochrome b inhibitors, which demonstrates that parasite mitochondrial function is a suitable target for killing parasites. Incorporating these effects into a model of malaria transmission dynamics predicts that impregnating mosquito nets with Plasmodium inhibitors would substantially mitigate the global health effects of insecticide resistance. This study identifies a powerful strategy for blocking Plasmodium transmission by female Anopheles mosquitoes, which has promising implications for efforts to eradicate malaria.

[Peculiarities of the Structural-Functional State of the Cytochrome Part of Liver Mitochondrial Respiratory Chain under Conditions of Acetaminophen-induced Hepatitis against the Background of Alimentary Deprivation of Protein].

Activity of the key enzyme of the cytochrome part of the respiratory chain--cytochrome oxidase, quantitative redistribution of mitochondrial cytochromes b, c1, c and aa3, activity of the key enzymes of cytochromes' heme metabolism--delta-aminolevulinate synthase and heme oxygenase under conditions of acetaminophen-induced hepatitis against the background of alimentary deprivation of protein were studied. It was found out, that under conditions of acetaminophen-induced hepatitis against the background of alimentary deprivation of protein, an inhibition of cytochrome oxidase activity and a decrease in the quantitative content of mitochondrial cytochromes against the background of the increase in the delta-aminolevulinate synthase and heme oxygenase activity are observed. In animals with toxic liver injury, maintained under conditions of alimentary deprivation of protein, a progressive decrease in the quantitative content of mitochondrial cytochromes b, c1, c and aa3 against the background. of the increase in heme oxygenase activity and preservation of delta-aminolevulinate synthase activity on the control level is identified. The conclusion was made, that alimentary deprivation of protein is a critical factor for the development of the disturbances of structural-functional integrity of the cytochromic part of the respiratory chain. The identified changes may be considered as one of the possible mechanisms of energy biotransformation system disturbances under conditions of alimentary deprivation of protein.

Diversity-oriented synthesis probe targets Plasmodium falciparum cytochrome b ubiquinone reduction site and synergizes with oxidation site inhibitors.

BACKGROUND: The emergence and spread of drug resistance to current antimalarial therapies remains a pressing concern, escalating the need for compounds that demonstrate novel modes of action. Diversity-Oriented Synthesis (DOS) libraries bridge the gap between conventional small molecule and natural product libraries, allowing the interrogation of more diverse chemical space in efforts to identify probes of novel parasite pathways. METHODS: We screened and optimized a probe from a DOS library using whole-cell phenotypic assays. Resistance selection and whole-genome sequencing approaches were employed to identify the cellular target of the compounds. RESULTS: We identified a novel macrocyclic inhibitor of Plasmodium falciparum with nanomolar potency and identified the reduction site of cytochrome b as its cellular target. Combination experiments with reduction and oxidation site inhibitors showed synergistic inhibition of the parasite. CONCLUSIONS: The cytochrome b oxidation center is a validated antimalarial target. We show that the reduction site of cytochrome b is also a druggable target. Our results demonstrating a synergistic relationship between oxidation and reduction site inhibitors suggests a future strategy for new combination therapies in the treatment of malaria.

Genetic analysis and molecular characterisation of laboratory and field mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to QoI fungicides.

BACKGROUND: QoI fungicides, inhibitors of mitochondrial respiration, are considered to be at high risk of resistance development. In several phytopathogenic fungi, resistance is caused by mutations (most frequently G143A) in the mitochondrial cytochrome b (cytb) gene. The genetic and molecular basis of QoI resistance were investigated in laboratory and field mutants of Botryotinia fuckeliana (de Bary) Whetz. exhibiting in vitro reduced sensitivity to trifloxystrobin. RESULTS: B. fuckeliana mutants highly resistant to trifloxystrobin were obtained in the laboratory by spontaneous mutations in wild-type strains, or from naturally infected plants on a medium amended with 1-3 mg L(-1) trifloxystrobin and 2 mM salicylhydroxamic acid, an inhibitor of alternative oxidase. No point mutations were detected, either in the complete nucleotide sequences of the cytb gene or in those of the aox and Rieske protein genes of laboratory mutants, whereas all field mutants carried the G143A mutation in the mitochondrial cytb gene. QoI resistance was always maternally inherited in ascospore progeny of sexual crosses of field mutants with sensitive reference strains. CONCLUSIONS: The G143A mutation in cytb gene is confirmed to be responsible for field resistance to QoIs in B. fuckeliana. Maternal inheritance of resistance to QoIs in progeny of sexual crosses confirmed that it is caused by extranuclear genetic determinants. In laboratory mutants the heteroplasmic state of mutated mitochondria could likely hamper the G143A detection, otherwise other gene(s) underlying different mechanisms of resistance could be involved.

A chemical genomic analysis of decoquinate, a Plasmodium falciparum cytochrome b inhibitor.

Decoquinate has single-digit nanomolar activity against in vitro blood stage Plasmodium falciparum parasites, the causative agent of human malaria. In vitro evolution of decoquinate-resistant parasites and subsequent comparative genomic analysis to the drug-sensitive parental strain revealed resistance was conferred by two nonsynonymous single nucleotide polymorphisms in the gene encoding cytochrome b. The resultant amino acid mutations, A122T and Y126C, reside within helix C in the ubiquinol-binding pocket of cytochrome b, an essential subunit of the cytochrome bc(1) complex. As with other cytochrome bc(1) inhibitors, such as atovaquone, decoquinate has low nanomolar activity against in vitro liver stage P. yoelii and provides partial prophylaxis protection when administered to infected mice at 50 mg kg(-1). In addition, transgenic parasites expressing yeast dihydroorotate dehydrogenase are >200-fold less sensitive to decoquinate, which provides additional evidence that this drug inhibits the parasite's mitochondrial electron transport chain. Importantly, decoquinate exhibits limited cross-resistance to a panel of atovaquone-resistant parasites evolved to harbor various mutations in cytochrome b. The basis for this difference was revealed by molecular docking studies, in which both of these inhibitors were shown to have distinctly different modes of binding within the ubiquinol-binding site of cytochrome b.

Rapid method for detecting resistance to a QoI fungicide in Plasmopara viticola populations.

BACKGROUND: The increasing occurrence of Qo inhibitor (QoI)-fungicide-resistant Plasmopara viticola (Berk. & MA Curtis) Berl. & DeToni populations is becoming a serious problem in the control of grapevine downy mildew worldwide. RESULTS: The authors have developed a rapid method for detecting resistance to a QoI fungicide, azoxystrobin, in P. viticola populations using the nested PCR-RFLP method. With this method, a glycine-to-alanine substitution was discovered at codon 143 in the cytochrome b gene of P. viticola populations found in Japan. CONCLUSION: It is proposed that the nested PCR-RFLP method is a high-speed, sensitive and reliable tool for detecting azoxystrobin-resistant P. viticola populations.

Combining Inhibitor Resistance-conferring Mutations in Cytochrome b Creates Conditional Synthetic Lethality in Saccharomyces cerevisiae.

The mitochondrial cytochrome bc(1) complex is an essential respiratory enzyme in oxygen-utilizing eukaryotic cells. Its core subunit, cytochrome b, contains two sites, center P and center N, that participate in the electron transfer activity of the bc(1) complex and that can be blocked by specific inhibitors. In yeast, there are various point mutations that confer inhibitor resistance at center P or center N. However, there are no yeast strains in which the bc(1) complex is resistant to both center P and center N inhibitors. We attempted to create such strains by crossing yeast strains with inhibitor-resistant mutations at center P with yeast strains with inhibitor-resistant mutations at center N. Characterization of yeast colonies emerging from the cross revealed that there were multiple colonies resistant against either inhibitor alone but that the mutational changes were ineffective when combined and when the yeast were grown in the presence of both inhibitors. Inhibitor titrations of bc(1) complex activities in mitochondrial membranes from the various yeast mutants showed that a mutation that confers resistance to an inhibitor at center P, when combined with a mutation that confers resistance to an inhibitor at center N, eliminates or markedly decreases the resistance conferred by the center N mutation. These results indicate that there is a pathway for structural communication between the two active sites of cytochrome b and open new possibilities for the utilization of center N as a potential drug target.

Cytochrome b gene structure and consequences for resistance to Qo inhibitor fungicides in plant pathogens.

The cytochrome b (cyt b) gene structure was characterized for different agronomically important plant pathogens, such as Puccinia recondita f sp tritici (Erikss) CO Johnston, P graminis f sp tritici Erikss and Hennings, P striiformis f sp tritici Erikss, P coronata f sp avenae P Syd & Syd, P hordei GH Otth, P recondita f sp secalis Roberge, P sorghi Schwein, P horiana Henn, Uromyces appendiculatus (Pers) Unger, Phakopsora pachyrhizi Syd & P Syd, Hemileia vastatrix Berk & Broome, Alternaria solani Sorauer, A alternata (Fr) Keissl and Plasmopara viticola (Berk & Curt) Berlese & de Toni. The sequenced fragment included the two hot spot regions in which mutations conferring resistance to QoI fungicides may occur. The cyt b gene structure of these pathogens was compared with that of other species from public databases, including the strobilurin-producing fungus Mycena galopoda (Pers) P Kumm, Saccharomyces cerevisiae Meyer ex Hansen, Venturia inaequalis (Cooke) Winter and Mycosphaerella fijiensis Morelet. In all rust species, as well as in A solani, resistance to QoI fungicides caused by the mutation G143A has never been reported. A type I intron was observed directly after the codon for glycine at position 143 in these species. This intron was absent in pathogens such as A alternata, Blumeria graminis (DC) Speer, Pyricularia grisea Sacc, Mycosphaerella graminicola (Fuckel) J Schröt, M fijiensis, V inaequalis and P viticola, in which resistance to QoI fungicides has occurred and the glycine is replaced by alanine at position 143 in the resistant genotype. The present authors predict that a nucleotide substitution in codon 143 would prevent splicing of the intron, leading to a deficient cytochrome b, which is lethal. As a consequence, the evolution of resistance to QoI fungicides based on G143A is not likely to evolve in pathogens carrying an intron directly after this codon.

Malarone treatment failure and in vitro confirmation of resistance of Plasmodium falciparum isolate from Lagos, Nigeria.

We report the first in vitro and genetic confirmation of Malarone (GlaxoSmithKline; atovaquone and proguanil hydrochloride) resistance in Plasmodium falciparum acquired in Africa. On presenting with malaria two weeks after returning from a 4-week visit to Lagos, Nigeria without prophylaxis, a male patient was given a standard 3-day treatment course of Malarone. Twenty-eight days later the parasitaemia recrudesced. Parasites were cultured from the blood and the isolate (NGATV01) was shown to be resistant to atovaquone and the antifolate pyrimethamine. The cytochrome b gene of isolate NGATV01 showed a single mutation, Tyr268Asn which has not been seen previously.