Bc1 Complex Research Articles

Unusual semiquinone as an electron buffer in enzymatic quinone oxidation

Cytochromes bc1 and c b6f are part of respiratory or photosynthetic machinery. The main role of these enzymes is to build proton motive force across the bioenergetic membranes by coupling the proton translocations with electron transfer from the pool of membrane-soluble quinones to water-soluble redox proteins. Despite many years of research, the mechanism of quinol oxidation is not fully understood. It is assumed that unstable form of a partially oxidized quinol – semiquinone is an intermediate state of this process and that it is also a potential electron donor in the side reaction of superoxide generation. This semiquinone has remained experimentally elusive over years but recently a semiquinone interacting with the reduced iron-sulfur cluster was identified as a new state of the enzyme. The results indicate that semiquinone coupled to the iron-sulfur cluster is most probably an additional state that can prevent side reactions, including superoxide generation.

High-throughput virtual screening approach involving pharmacophore mapping, ADME filtering, molecular docking and MM-GBSA to identify new dual target inhibitors of PfDHODH and PfCytbc1 complex to combat drug resistant malaria

Emerging cases of drug resistance against Artemisinin combination therapies which are the current and the last line of defense against malaria makes the situation very alarming. Due to the liability of single-target drugs to be more prone to drug resistance, the trend of development of dual or multi-target inhibitors is emerging. Recently, a malaria box molecule, MMV007571 which is a well known new permeability pathways inhibitor was investigated to be also multi-targeting Plasmodium falciparum dihydroorotate dehydrogenase and cytochrome bc1 complex. The aspiration behind this study was to use the information of its pharmacophoric features essential for binding as two of its new targets. In this regard, high throughput virtual screening involving pharmacophore mapping, ADME filtering, molecular docking, and MM-GBSA calculations were carried out. This approach has lead to the identification of two new hits namely DT00V1902 and DT00V1922 which binds with -37.85 and -24.65 kcal/mol of more stable ΔG Bind energy at two targets than the lead molecule, MMV007571. The screened compounds are indicated to be carry improvement in binding potential and pharmacokinetic characters as per in silico studies. The authors propose that DT00V1902 and DT00V1922 can be forwarded for experimental validation and clinical studies for antimalarial chemotherapy. Communicated by Ramaswamy H. Sarma

ABC1K10a, an atypical kinase, functions in plant salt stress tolerance

BackgroundABC1K (Activity of BC1 complex Kinase) is an evolutionarily primitive atypical kinase family widely distributed among prokaryotes and eukaryotes. The ABC1K protein kinases in Arabidopsis are predicted to localize either to the mitochondria or chloroplasts, in which plastid-located ABC1K proteins are involved in the response against photo-oxidative stress and cadmium-induced oxidative stress.ResultsHere, we report that the mitochondria-localized ABC1K10a functions in plant salt stress tolerance by regulating reactive oxygen species (ROS). Our results show that the ABC1K10a expression is induced by salt stress, and the mutations in this gene result in overaccumulation of ROS and hypersensitivity to salt stress. Exogenous application of the ROS-scavenger GSH significantly represses ROS accumulation and rescues the salt hypersensitive phenotype of abc1k10a. ROS overaccumulation in abc1k10a mutants under salt stress is likely due to the defect in mitochondria electron transport chain. Furthermore, defects of several other mitochondria-localized ABC1K genes also result in salt hypersensitivity.ConclusionsTaken together, our results reveal that the mitochondria-located ABC1K10a regulates mitochondrial ROS production and is a positive regulator of salt tolerance in Arabidopsis.

Design and synthesis of potent inhibitors of bc1 complex based on natural product neopeltolide

Neopeltolide, a natural product isolated from deep-water sponge specimen of the family neopeltidae, has been proven to be a novel inhibitor of cytochrome bc1. In this study, a series of neopeltolide derivatives was designed by replacing the 14-membered macrolactone with indole ring and confirmed by 1H NMR, 13C NMR, and HRMS. Based on the binding mode of 12h with bc1 complex, the IC50 values of compounds 16a-f (ranging from 0.70 to 1.46 μM) were improved significantly than the ester derivatives 12a-u by replacing the ester with amide linker. Subsequently, the molecular docking results indicated that compound 16e could form a π-π interaction with Phe274 and two H-bonds with Glu271 and His161 and the latter H-bond was found to account for its high activity. The present work accelerates the discovery of novel bc1 complex inhibitors to deal with the resistance that the existing bc1 complex inhibitors are facing and provides a valuable idea for the design of new fungicides.

Charge polarization imposed by the binding site facilitates enzymatic redox reactions of quinone

Quinone reduction site (Qi) of cytochrome bc1 represents one of the canonical sites used to explore the enzymatic redox reactions involving semiquinone (SQ) states. However, the mechanism by which Qi allows the completion of quinone reduction during the sequential transfers of two electrons from the adjacent heme bH and two protons to C1- and C4-carbonyl remains unclear. Here we established that the SQ coupled to an oxidized heme bH is a dominant intermediate of catalytic forward reaction and, contrary to the long-standing assumption, represents a significant population of SQ detected across pH 5–9. The pH dependence of its redox midpoint potential implicated proton exchange with histidine. Complementary quantum mechanical calculations revealed that the SQ anion formed after the first electron transfer undergoes charge and spin polarization imposed by the electrostatic field generated by histidine and the aspartate/lysine pair interacting with the C4- and C1-carbonyl, respectively. This favors a barrierless proton exchange between histidine and the C4-carbonyl, which continues until the second electron reaches the SQi. Inversion of charge polarization facilitates the uptake of the second proton by the C1-carbonyl. Based on these findings we developed a comprehensive scheme for electron and proton transfers at Qi featuring the equilibration between the anionic and neutral states of SQi as means for a leak-proof stabilization of the radical intermediate. The key catalytic role of the initial charge/spin polarization of the SQ anion at the active site, inherent to the proposed mechanism, may also be applicable to the other quinone oxidoreductases.

Palmitoylome profiling indicates that androgens promote the palmitoylation of metabolism-related proteins in prostate cancer-derived LNCaP cells

To explore potential therapeutic targets other than androgen-deprivation treatment for prostate cancer by screening the proteins induced by androgen at palmitoylation modification level in LNCaP cells. The LNCaP cells were treated with androgen (Methyltrienolone, R1881, 5 nmol/L) or dimethyl sulfoxide (DMSO) for 24 h, and then labeled with alkynyl palmitic acid Alk-C16 (100 μmol/L). After that, the cells were collected, lysed, the total protein was extracted, agarose beads labeled with azide (1 mmol/L) were added, and the click-chemistry reaction was carried out at room temperature for 1 h. The covalent bond formed by click-chemistry reaction of azide and alkynyl group was used to enrich the palmitoylated proteins on agarose beads. Label-free quantitation (LFQ) was used to compare the protein palmitoylation level of R1881 treated and untreated cells to screen the proteins induced by androgen at palmitoylation modification level. In this experiment, 907 potential palmitoylated proteins (mascot score>2, P<0.05) were identified, among which 430 proteins had LFQ values not zero at least twice. Among the 430 proteins, the palmitoylation levels of 92 candidates were increased by androgen treatment, and their LFQ values were significantly upregulated (>1.5-fold, P<0.05) in ≥2 samples of androgen-treated vs. untreated LNCaP cells. We also used the software of cytoscape to classify the 92 proteins, and found that the known functional proteins of them could be divided into three categories: metabolism related, protein folding related and translation initiation related. Among them, metabolism related proteins included lipid metabolism (6), glucose metabolism (7) and respiratory electron transport chain (8), and a small amount of amino acid metabolism (2) and other metabolism related proteins (2). Notably, the ratio of LFQ of cytochrome b-c1 complex subunit 2 (UQCRC2) was significantly (>3-fold, P<0.05) higher in androgen-treated cells compared with untreated cells, indicating that the palmitoylation level of UQCRC2 was enhanced by androgen most significantly than that of others. The second was long-chain acyl CoA dehydrogenase (ACADVL) related to lipid metabolism and glucose 6-phosphate dehydrogenase (PGD) related to glucose metabolism, but the LFQ ratio of them was less than 3-fold. The research on palmitoylation mechanism of metabolism, especially the proteins related to respiratory electron transport chain, will provide a new guidance for the diagnosis and treatment of prostate cancer and the development of targeted drugs.

Rcf2 revealed in cryo-EM structures of hypoxic isoforms of mature mitochondrial III-IV supercomplexes

The organization of the mitochondrial electron transport chain proteins into supercomplexes (SCs) is now undisputed; however, their assembly process, or the role of differential expression isoforms, remain to be determined. In Saccharomyces cerevisiae, cytochrome c oxidase (CIV) forms SCs of varying stoichiometry with cytochrome bc1 (CIII). Recent studies have revealed, in normoxic growth conditions, an interface made exclusively by Cox5A, the only yeast respiratory protein that exists as one of two isoforms depending on oxygen levels. Here we present the cryo-EM structures of the III2-IV1 and III2-IV2 SCs containing the hypoxic isoform Cox5B solved at 3.4 and 2.8 Å, respectively. We show that the change of isoform does not affect SC formation or activity, and that SC stoichiometry is dictated by the level of CIII/CIV biosynthesis. Comparison of the CIV5B- and CIV5A-containing SC structures highlighted few differences, found mainly in the region of Cox5. Additional density was revealed in all SCs, independent of the CIV isoform, in a pocket formed by Cox1, Cox3, Cox12, and Cox13, away from the CIII-CIV interface. In the CIV5B-containing hypoxic SCs, this could be confidently assigned to the hypoxia-induced gene 1 (Hig1) type 2 protein Rcf2. With conserved residues in mammalian Hig1 proteins and Cox3/Cox12/Cox13 orthologs, we propose that Hig1 type 2 proteins are stoichiometric subunits of CIV, at least when within a III-IV SC.

Assembly the [2Fe‐2S] Cluster in the Cytochrome bc1 Complex from Saccharomyces cerevisiae

ObjectiveParticipation of the Fe‐S‐IBG subsystem in the assembly of the [2Fe‐2S] cluster into the bc1 complex.MethodsMutants of S. cerevisiae: Δiba57, Δisa1, Δgrx5, and Δrip1 were grown in YPD medium at 30°C. Mitochondria were isolated by differential centrifugation according to Auchére et al. (2008). Respiratory complex and aconitase activities were evaluated according to Gomez et al. (2014). Mitochondria were treated with digitonin to evaluate the formation of respiratory supercomplexes by electrophoresis in Polyacrylamide Blue Native Gels (BN‐PAGE), and after 2‐dimensional SDS‐PAGE analysis according to Schagger et al. (2000). Detection of the Rip1p subunit was performed by Western blot using the anti‐Rip1p monoclonal antibody at 1:20,000 dilution and developed with anti‐mouse IgG HRP‐conjugate.AbstractThe iron‐sulfur clusters [Fe‐S] are inorganic cofactors contained in enzymes involved in multiple cellular processes. In eukaryotes, [Fe‐S] synthesis mainly occurs in the mitochondrial matrix by the biogenesis of Iron Sulfur Cluster system (ISC). This system involves two steps: the novo assembly of the [Fe–S] clusters on scaffold proteins, and the transference of the [Fe–S] cluster to target apo‐proteins. These steps involve the participation of several proteins, which perform specific reactions. The ISC subsystem Fe‐S‐IBG conformed by the Grx5p, Isa1p, Isa2p, and Iba57p proteins is essential for the synthesis of the [4Fe‐4S] clusters. Yeast mutants in the Fe‐S‐IBG subsystem showed deficiency in respiration and excessive ROS generation was found, similarly to the mutant Δrip1, which gene encodes for the Rieske subunit of cytochrome bc1. In this work, the activity of the cytochrome bc1 in mitochondria from the Δiba57,Δisa1, Δgrx5, and Δrip1 mutants strains were determined. Results showed that the cytochrome bc1 complex in mitochondria from the Δiba57,Δisa1, and Δrip1 mutants was dysfunctional; whereas in the Δgrx5, the cytochrome bc1 activity was partially decreased in comparison with the WT strain. BN‐PAGE analysis shows that the respiratory complex lost the capacity to form supercomplexes in mitochondria from the Δiba57 and Δisa1 mutants, Immunodetection assays showed that Rip1p was absent in the iba57Δ and Δisa1 mutants; whereas the Δgrx5 mutant shows an unaltered pattern profile of the supercomplexes bands in BN‐PAGE, without the loss of Rieske protein. In conclusion, results indicate that the Isa1p and Iba57p proteins are involved in the insertion of the [2Fe‐2S] cluster on the Rip1p apoprotein, suggesting that the Fe‐S‐IBG subsystem is not exclusive for the maturation/assembly of proteins with [4Fe‐4S] clusters, but also is involved in [2Fe‐2S] clusters assembly such as occurred in the Rieske subunit.

Elucidating the Effects of Distal Charges on the Reduction Potential of the Rieske Protein

The Rieske protein plays a key role in both cellular respiration and photosynthesis, transferring electrons through the respective cytochrome bc1 and b6f complexes of each process. It is characterized by a [2Fe‐2S] cluster and is unique in that one iron is ligated by nitrogens from two solvent‐exposed histidine residues while the other is ligated by the sulfurs of two cysteines. In the electron transport chain (ETC), the Rieske protein oxidizes ubiquinol and reduces cytochrome c, transferring electrons between complexes II and IV.Previous work has demonstrated how hydrogen bonds, solvent accessibility of the ligands, and the presence of nearby charged residues can impact the reduction potential of the protein. However, the full extent of this third element, how far from the cluster these charged residues can reside and still impact the reduction potential, has yet to be explored. Two types of mutants have been created to probe the effects of these distal charges. First, mutations removing positively charged residues were predicted to decrease reduction potential. Likewise, mutations removing negatively charged residues were expected to increase reduction potential. Each mutation either resides a unique distance from the cluster or produces a different change in overall charge, demonstrating the impact of each factor both individually and additively. Lastly, the pKa values of the histidine ligands were also expected to be inversely related to reduction potential.Using pH‐dependent UV‐Visible spectrophotometry, the pKa values of these mutants were determined and correlated to these relationships. While the overall trend of pKa values increased as expected for the negative‐charge mutants, confounding elements also appeared, such as a third pKa sigmoid. Furthermore, E146R, a positive‐charge mutant, also demonstrated increased pKa values, opposite to the hypothesized trend. New mutants that implement a neutral to positive substitutions have been developed to probe the effect of making the protein more positive.Support or Funding InformationMurchison Summer Undergraduate Research Fellowship, Welch Foundation

Timing of dimerization of the bc1 complex during mitochondrial respiratory chain assembly

The mitochondrial bc1 complex plays an important role in mitochondrial respiration. It transfers electrons from ubiquinol to the soluble electron shuttle cytochrome c and thereby contributes to the proton motive force across the inner mitochondrial membrane. In the yeast Saccharomyces cerevisiae, each monomer consists of three catalytic and seven accessory subunits. The bc1 complex is an obligate homo-dimer in all systems. It is currently not known when exactly during the assembly dimerization occurs. In this study, we determined that the dimer formation is an early event. Specifically, dimerization is mediated by the interaction of a stable tetramer formed by the two Cor subunits, Cor1 and Cor2, that joins assembly intermediate II, containing the fully hemylated cytochrome b and the two small accessory proteins, Qcr7 and Qcr8. Addition of cytochrome c1 and Qcr6 can either occur concomitantly or independently of dimerization. These results reveal a strict order in assembly, where dimerization occurs after stabilization of co-factor acquisition by cytochrome b. Finally, assembly is completed by addition of the remaining subunits.

Design, synthesis, and evaluation of new 2-(quinoline-4-yloxy)acetamide-based antituberculosis agents

Using a classical molecular simplification approach, a series of 36 quinolines were synthesized and evaluated as in vitro inhibitors of Mycobacterium tuberculosis (M. tuberculosis) growth. Structure–activity relationship (SAR) studies leaded to potent antitubercular agents, with minimum inhibitory concentration (MIC) values as low as 0.3 μM against M. tuberculosis H37Rv reference strain. Furthermore, the lead compounds were active against multidrug-resistant strains, without cross-resistance with some first- and second-line drugs. Testing the molecules against a spontaneous mutant strain containing a single mutation in the qcrB gene (T313A) indicated that the synthesized quinolines targeted the cytochrome bc1 complex. In addition, leading compounds were devoid of apparent toxicity to HepG2 and Vero cells and showed moderate elimination rates in human liver S9 fractions. Finally, the selected structures inhibited M. tuberculosis growth in a macrophage model of tuberculosis infection. Taken together, these data indicate that this class of compounds may furnish candidates for the future development of antituberculosis drugs.

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.

Probing the Open Global Health Chemical Diversity Library for Multistage-Active Starting Points for Next-Generation Antimalarials.

Most phenotypic screens aiming to discover new antimalarial chemotypes begin with low cost, high-throughput tests against the asexual blood stage (ABS) of the malaria parasite life cycle. Compounds active against the ABS are then sequentially tested in more difficult assays that predict whether a compound has other beneficial attributes. Although applying this strategy to new chemical libraries may yield new leads, repeated iterations may lead to diminishing returns and the rediscovery of chemotypes hitting well-known targets. Here, we adopted a different strategy to find starting points, testing ∼70,000 open source small molecules from the Global Health Chemical Diversity Library for activity against the liver stage, mature sexual stage, and asexual blood stage malaria parasites in parallel. In addition, instead of using an asexual assay that measures accumulated parasite DNA in the presence of compound (SYBR green), a real time luciferase-dependent parasite viability assay was used that distinguishes slow-acting (delayed death) from fast-acting compounds. Among 382 scaffolds with the activity confirmed by dose response (<10 μM), we discovered 68 novel delayed-death, 84 liver stage, and 68 stage V gametocyte inhibitors as well. Although 89% of the evaluated compounds had activity in only a single life cycle stage, we discovered six potent (half-maximal inhibitory concentration of <1 μM) multistage scaffolds, including a novel cytochrome bc1 chemotype. Our data further show the luciferase-based assays have higher sensitivity. Chemoinformatic analysis of positive and negative compounds identified scaffold families with a strong enrichment for activity against specific or multiple stages.

Mitochondria respiratory chain studied by electron paramagnetic resonance spectroscopy

Aerobic respiration is the indispensable physiology of higher organism living on earth, since the required energy for biological activities is supplied mainly by this metabolism. It is well established that mitochondria plays an important role in converting the stable chemical energy stored in foods into the active and utilizable energy supplying to life activities. Upon the process, the energy conversion is carried out by the respiratory electron transport chain, which consists of five distinct and coupled protein complexes (termed complex I−V) embedded in the mitochondria intima. Among them, complex I (NADH-ubiquinone oxidoreductase), III (cytochrome bc 1) and IV (cytochrome c oxidase) are the electron-coupled proton-pumping enzymes. Except for the complex V (i.e., ATP synthase), each complex has several redox cofactors to promote the electron transfer within themselves, and the transfer from complex I and II to complex III is shuttled by membrane-embedded ubiquinones, from complex III to IV by the soluble cytochrome c . When the reductive electron is donated by the initial donors NADH or FADH2 to the final acceptor molecular oxygen O2, a transmembrane proton motive force is formed simultaneously. The latter is the driving force for the ATP synthesis via ATP synthase with a ratio of three protons per ATP. All the aforementioned processes are also known as oxidative phosphorylation, and the arrangement of the electron carriers therein is called as respiratory electron transport chain. In the stepwise electron relay, these carriers undergo the redox changes, giving rise to the respective paramagnetic and diamagnetic spin states. These single-electron relays are prone to the electron paramagnetic resonance spectroscopy (EPR), which is a powerful and noninvasive technique to monitor the unpaired electron in situ . As shown in the overwhelming literatures, EPR technique has been adopted to reveal the electron transfer pathway in different biological activities. Herein, the application of EPR to study the dynamic electron transfer in mitochondria is reviewed briefly. However, this conventional application required rather high concentration of the active center at a level of 1–10 µmol/L. Actually, most of the intermediates of the electron carriers are very active and short-life. In the human body, only a few tissues can accumulate such required concentration of paramagnetic substances, e.g., in blood, heart and liver. For the past decade, a more sensitive single-molecule magnetic resonance technique based on the nitrogen-vacancy (NV) center of diamond has been developing dramatically. In principle, this advanced EPR technique requires the less sample or concentration, even down to the single biomolecule, and provides the time-resolved dynamic information. Lately, the studies of single-biomolecule EPR at room temperature or in aqueous solution have been reported consecutively in single protein (MAD2/mitotic arrest deficient-2) and single DNA (tethered DNA duplexes). Finally, a perspective of single-biomolecule magnetic resonance technique is given for the further study on mitochondrion and relative physiological activities. The single molecule EPR technique is also helpful to unveil the other specific information of biological processes, for example, drug screening, personalized medicine and other major applications.

Determination of the Binding Interaction between Mitochondrial Electron Transport Chain Proteins Cytochrome C and Cytochrome C Oxidase

The electron transport chain (ETC) is a collection of proteins that are key to the mitochondria's ability to perform cellular respiration, which is responsible for creating the majority of the ATP that cells rely on for energy. Cytochrome c (Cc) is one of the electron transport chain proteins that is responsible for electron transfer from cytochrome bc1 to cytochrome c oxidase (CcO). This Cc interaction determines the rate of electron transfer for the entire mitochondrial respiratory chain. During ischemia events, these ETC proteins are dephosphorylated in an attempt to combat the drop in ATP levels. Once blood flow is resumed, the dephosphorylated ETC proteins replenish the ATP supply but also create superoxide species that initiate the tissue damage attributed to reperfusion injury. It has been shown that the serine 47 (S47) of Cc is a site of phosphoryl regulation. Human Cc mutant S47E-Cc (serine to glutamate) will be prepared to imitate this phosphorylation. The glutamate residue has the ability to mimic the presence of a negatively charged phosphoryl group. This is useful since intact phosphorylated Cc is difficult to purify by traditional methods. The binding affinity that the mutant Cc has for wild-type CcO and the electron transfer rates between the mutant Cc and wild-type CcO will be determined. Ultimately, this project will provide insight that is needed to settle the current debate over the binding model for Cc and CcO. There are currently two main models proposed; however, they each vary in the role of Cc's S47 residue during Cc and CcO binding. Supported by NIH grants GM20488 and 8P30GM103450.

Structure of the Bcs1 AAA-ATPase suggests an airlock-like translocation mechanism for folded proteins.

Some proteins require completion of folding before translocation across a membrane into another cellular compartment. Yet the permeability barrier of the membrane should not be compromised and mechanisms have remained mostly elusive. Here, we present the structure of Saccharomyces cerevisiae Bcs1, an AAA-ATPase of the inner mitochondrial membrane. Bcs1 facilitates the translocation of the Rieske protein, Rip1, which requires folding and incorporation of a 2Fe-2S cluster before translocation and subsequent integration into the bc1 complex. Surprisingly, Bcs1 assembles into exclusively heptameric homo-oligomers, with each protomer consisting of an amphipathic transmembrane helix, a middle domain and an ATPase domain. Together they form two aqueous vestibules, the first being accessible from the mitochondrial matrix and the second positioned in the inner membrane, with both separated by the seal-forming middle domain. On the basis of this unique architecture, we propose an airlock-like translocation mechanism for folded Rip1.

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.

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.

Naphthoquinones isolated from Eleutherine plicata herb: in vitro antimalarial activity and molecular modeling to investigate their binding modes

Plasmodium falciparum is the cause of malaria and has become resistant to the drugs used to treat the disease. Therefore, the development for novel compounds with antimalarial activity has become urgent. Plant species, such as naphthoquinone-rich Eleutherine plicata, may provide novel substances that exhibit antimalarial activity and serve as an alternative for the treatment of this disease. From this plant species, ethanol extracts were obtained, fractionated, and the isolated substances eleutherin, and isoleutherin were characterized by nuclear magnetic resonance. There in vitro activity against Plasmodium falciparum was examined using the traditional Microtest method using the extract, fractions, and isolated molecules. Eleutherin and isoleutherin showed the best activity toward the parasite with IC50 values of 10.45 and 8.70 µg/mL, respectively. Characterization of the binding mode of the compounds with a target enzyme and identification of the molecular interactions were revealed via molecular docking results. Eleutherin and isoeleutherin interacted with highly conserved residues from the binding cavity of the cytochrome bc1 complex, a protein found in mitochondria. Therefore, the eleutherin and isoeleutherin naphthoquinones showed antiplasmodial activity with a similar mechanism to that of atovaquone were able to interact with the cytochrome bc1 complex, and showed promise for antimalarial treatments.

A spontaneous mitonuclear epistasis converging on Rieske Fe-S protein exacerbates complex III deficiency in mice

We previously observed an unexpected fivefold (35 vs. 200 days) difference in the survival of respiratory chain complex III (CIII) deficient Bcs1lp.S78G mice between two congenic backgrounds. Here, we identify a spontaneous homoplasmic mtDNA variant (m.G14904A, mt-Cybp.D254N), affecting the CIII subunit cytochrome b (MT-CYB), in the background with short survival. We utilize maternal inheritance of mtDNA to confirm this as the causative variant and show that it further decreases the low CIII activity in Bcs1lp.S78G tissues to below survival threshold by 35 days of age. Molecular dynamics simulations predict D254N to restrict the flexibility of MT-CYB ef loop, potentially affecting RISP dynamics. In Rhodobacter cytochrome bc1 complex the equivalent substitution causes a kinetics defect with longer occupancy of RISP head domain towards the quinol oxidation site. These findings represent a unique case of spontaneous mitonuclear epistasis and highlight the role of mtDNA variation as modifier of mitochondrial disease phenotypes.