ABSTRACT
BACKGROUND: Staphylococcus aureus is a common pathogenic microorganism in humans and animals. Type II NADH oxidoreductase (NDH-2) is the only NADH:quinone oxidoreductase present in this organism and represents a promising target for the development of anti-staphylococcal drugs. Recently, myricetin, a natural flavonoid from vegetables and fruits, was found to be a potential inhibitor of NDH-2 of S. aureus. The objective of this study was to evaluate the inhibitory properties of myricetin against NDH-2 and its impact on the growth and expression of virulence factors in S. aureus. RESULTS: A screening method was established to identify effective inhibitors of NDH-2, based on heterologously expressed S. aureus NDH-2. Myricetin was found to be an effective inhibitor of NDH-2 with a half maximal inhibitory concentration (IC50) of 2 µM. In silico predictions and enzyme inhibition kinetics further characterized myricetin as a competitive inhibitor of NDH-2 with respect to the substrate menadione (MK). The minimum inhibitory concentrations (MICs) of myricetin against S. aureus strains ranged from 64 to 128 µg/mL. Time-kill assays showed that myricetin was a bactericidal agent against S. aureus. In line with being a competitive inhibitor of the NDH-2 substrate MK, the anti-staphylococcal activity of myricetin was antagonized by MK-4. In addition, myricetin was found to inhibit the gene expression of enterotoxin SeA and reduce the hemolytic activity induced by S. aureus culture on rabbit erythrocytes in a dose-dependent manner. CONCLUSIONS: Myricetin was newly discovered to be a competitive inhibitor of S. aureus NDH-2 in relation to the substrate MK. This discovery offers a fresh perspective on the anti-staphylococcal activity of myricetin.
Subject(s)
Flavonoids , Microbial Sensitivity Tests , Staphylococcus aureus , Flavonoids/pharmacology , Flavonoids/chemistry , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , NADH Dehydrogenase/antagonists & inhibitors , NADH Dehydrogenase/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Animals , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/metabolism , Humans , Virulence Factors/antagonists & inhibitors , Virulence Factors/metabolismABSTRACT
Cancer cells are highly dependent on bioenergetic processes to support their growth and survival. Disruption of metabolic pathways, particularly by targeting the mitochondrial electron transport chain complexes (ETC-I to V) has become an attractive therapeutic strategy. As a result, the search for clinically effective new respiratory chain inhibitors with minimized adverse effects is a major goal. Here, we characterize a new OXPHOS inhibitor compound called MS-L6, which behaves as an inhibitor of ETC-I, combining inhibition of NADH oxidation and uncoupling effect. MS-L6 is effective on both intact and sub-mitochondrial particles, indicating that its efficacy does not depend on its accumulation within the mitochondria. MS-L6 reduces ATP synthesis and induces a metabolic shift with increased glucose consumption and lactate production in cancer cell lines. MS-L6 either dose-dependently inhibits cell proliferation or induces cell death in a variety of cancer cell lines, including B-cell and T-cell lymphomas as well as pediatric sarcoma. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI-1) partially restores the viability of B-lymphoma cells treated with MS-L6, demonstrating that the inhibition of NADH oxidation is functionally linked to its cytotoxic effect. Furthermore, MS-L6 administration induces robust inhibition of lymphoma tumor growth in two murine xenograft models without toxicity. Thus, our data present MS-L6 as an inhibitor of OXPHOS, with a dual mechanism of action on the respiratory chain and with potent antitumor properties in preclinical models, positioning it as the pioneering member of a promising drug class to be evaluated for cancer therapy. MS-L6 exerts dual mitochondrial effects: ETC-I inhibition and uncoupling of OXPHOS. In cancer cells, MS-L6 inhibited ETC-I at least 5 times more than in isolated rat hepatocytes. These mitochondrial effects lead to energy collapse in cancer cells, resulting in proliferation arrest and cell death. In contrast, hepatocytes which completely and rapidly inactivated this molecule, restored their energy status and survived exposure to MS-L6 without apparent toxicity.
Subject(s)
Antineoplastic Agents , Cell Proliferation , Electron Transport Complex I , Mitochondria , Saccharomyces cerevisiae Proteins , Animals , Humans , Electron Transport Complex I/metabolism , Electron Transport Complex I/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Mice , Cell Line, Tumor , Mitochondria/metabolism , Mitochondria/drug effects , Cell Proliferation/drug effects , Uncoupling Agents/pharmacology , Oxidative Phosphorylation/drug effects , Xenograft Model Antitumor Assays , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/drug effects , Rats , NADH Dehydrogenase/metabolism , NADH Dehydrogenase/antagonists & inhibitorsABSTRACT
In this article, two Keggin-type polyoxometalates [Co(L)2]3[PMo12O40] (1) and [Co(L)2]3[PW12O40] (2) (HL = 2-acetylpyrazine thiosemicarbazone) were prepared and fully characterized. The compounds are stable in aqueous solution with different pH values and show superior antibacterial activity against Escherichia coli (E. coli: minimal inhibitory concentration (MIC) = 0.00375, 0.12 µg/mL), Agrobacterium tumefaciens (A. tumefaciens: MIC = 0.06, 0.12 µg/mL), Bacillus subtilis (B. subtilis: MIC = 0.015, 0.06 µg/mL) and especially for Staphylococcus aureus (S. aureus: MIC = 0.00048, 0.015 µg/mL) for 1 and 2, respectively. The time kill studies showed the entire killing of specific bacteria during 4 to 8 h. In addition, the possible antibacterial mechanism of compound 1 was explored systematically. The experimental results proved that cell wall/membrane damage, leakage of protein, inhibition of respiratory chain dehydrogenases activity, enhancement of intracellular reactive oxygen species (ROS) and depletion of glutathione (GSH) were the potential causes of bacteria death.
Subject(s)
Anti-Bacterial Agents/pharmacology , Coordination Complexes/pharmacology , Molybdenum/chemistry , Tungsten Compounds/pharmacology , Anti-Bacterial Agents/chemical synthesis , Bacteria/drug effects , Cell Membrane/drug effects , Cell Survival/drug effects , Cobalt/chemistry , Coordination Complexes/chemical synthesis , Glutathione/metabolism , Microbial Sensitivity Tests , NADH Dehydrogenase/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Tungsten Compounds/chemical synthesisABSTRACT
Targeting energy metabolism in Mycobacterium tuberculosis (Mtb) is a new paradigm in the search for innovative anti-TB drugs. NADH:menaquinone oxidoreductase is a non-proton translocating type II NADH dehydrogenase (NDH-2) that is an essential enzyme in the respiratory chain of Mtb and is not found in mammalian mitochondria. Phenothiazines (PTZs) represent one of the most known class of NDH-2 inhibitors, but their use as anti-TB drugs is currently limited by the wide range of potentially serious off-target effects. In this work, we designed and synthesized a series of new PTZs by decorating the scaffold in an unconventional way, introducing various halogen atoms. By replacing the sulfur atom with selenium, a dibromophenoselenazine 20 was also synthesized. Among the synthesized poly-halogenated PTZs (HPTZs), dibromo and tetrachloro derivatives 9 and 11, along with the phenoselenazine 20, emerged with a better anti-TB profile than the therapeutic thioridazine (TZ). They targeted non-replicating Mtb, were bactericidal, and synergized with rifampin and bedaquiline. Moreover, their anti-TB activity was found to be related to the NDH-2 inhibition. Most important, they showed a markedly reduced affinity to dopaminergic and serotonergic receptors respect to the TZ. From this work emerged, for the first time, as the poly-halogenation of the PTZ core, while permitting to maintain good anti-TB profile could conceivably lead to fewer CNS side-effects risk, making more tangible the use of PTZs for this alternative therapeutic application.
Subject(s)
Antitubercular Agents/pharmacology , Organoselenium Compounds/pharmacology , Phenothiazines/pharmacology , Animals , Antitubercular Agents/chemical synthesis , Antitubercular Agents/metabolism , Antitubercular Agents/toxicity , Chlorocebus aethiops , Drug Synergism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/toxicity , HEK293 Cells , Humans , Microsomes, Liver/metabolism , Molecular Structure , Mycobacterium smegmatis/drug effects , Mycobacterium tuberculosis/drug effects , NADH Dehydrogenase/antagonists & inhibitors , Organoselenium Compounds/chemical synthesis , Organoselenium Compounds/metabolism , Organoselenium Compounds/toxicity , Parasitic Sensitivity Tests , Phenothiazines/chemical synthesis , Phenothiazines/metabolism , Phenothiazines/toxicity , Protein Binding , Receptors, Dopamine D2/metabolism , Receptors, Serotonin/metabolism , Structure-Activity Relationship , Vero CellsABSTRACT
Introduction. Rhein (4, 5-dihydroxyanthraquinone-2-carboxylic acid) has various properties, including anti-inflammatory, antioxidant and anticancer activities. However, the mechanism underlying the role of rhein in antimicrobial activity remains largely unknown.Aim. This study aims to identify potential natural compounds of rhein that are capable of inhibiting Cutibacterium acnes and elucidate the effects of rhein on NADH dehydrogenase-2 activity in C. acnes.Methodology. The anti-C. acnes activity of compounds was analysed using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), the paper disc diffusion test and the checkerboard dilution test. To check whether rhein was inhibitory, putative type II NADH dehydrogenase (NDH-2) of C. acnes was analysed, cloned and expressed in Escherichia coli, and then NDH-2 purification was assessed with Ni-NTA before rhein inhibition of NADH dehydrogenase-2 activity was checked with ferricyanide [K3Fe(CN)6] as a substrate.Results. The results showed that the MIC of rhein against C. acnes was 6.25 µg ml-1, while the MBC was 12.5 µg ml-1, and there was a 38 mm inhibition zone in the paper disc diffusion test. Rhein showed an additive two- to fourfold reduction of the MIC value with four antibiotics on the checkerboard dilution test. The purified NADH dehydrogenase gene product showed a size of approximately 51 kDa and had a V max of 23 µmol and a K m of 280 µm. The inhibitory effect of rhein against NADH dehydrogenase-2 activity was non-competitive with ferricyanide [K3Fe(CN)6] with a K i value of 3.5-4.5 µm.Conclusion. This study provided evidence of the inhibitory effects of rhein on the growth of C. acnes by blocking of NADH dehydrogenase-2 activity. This mechanism of inhibitory activity in the reduction of ROS formation and ATP productivity should be further tested in C. acnes and the question of whether rhein inhibits the natural growth of C. acnes should be investigated.
Subject(s)
Anthraquinones/pharmacology , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/pharmacology , Gram-Positive Bacterial Infections/microbiology , NADH Dehydrogenase/antagonists & inhibitors , Propionibacterium acnes/drug effects , Propionibacterium acnes/enzymology , Anthraquinones/therapeutic use , Anti-Bacterial Agents/therapeutic use , Antioxidants/pharmacology , Antioxidants/therapeutic use , Enzyme Activation/drug effects , Enzyme Inhibitors/therapeutic use , Gram-Positive Bacterial Infections/drug therapy , Humans , Kinetics , Microbial Sensitivity Tests , Microbial Viability/drug effects , Propionibacterium acnes/growth & development , Reactive Oxygen Species/metabolism , Recombinant ProteinsABSTRACT
Oxidative stress in cells caused by excessive production of reactive oxygen species (ROS) and decreased antioxidant defense is implicated in the cytotoxicity of xenobiotics including drugs and environmental chemicals. Endosulfan, a highly toxic organochlorine insecticide, causes cytotoxic cell death by inducing oxidative stress. We have investigated the biochemical basis of induction of oxidative stress, involving the role of NADH dehydrogenase and the possible role of Na+, K+-ATPase in endosulfan cytotoxicity and, whether the cytotoxicity could be attenuated by targeting ROS induction using the natural flavonoid antioxidant, quercetin, in Ehrlich ascites tumor (EAT) cells. Exposure of cells to endosulfan caused cytotoxic cell death (necrosis) which was associated with induction of ROS, lipid peroxidation as well as a reduction in glutathione levels, concomitant with loss of NADH dehydrogenase and Na+, K+-ATPase activity in a dose-dependent manner, indicating that oxidative stress and perturbation of membrane function are the major causes of endosulfan cytotoxicity. Our results showed that quercetin, protected against endosulfan-induced cytotoxicity and significantly abrogated oxidative stress, and ameliorated the inhibition of NADH dehydrogenase and Na+, K+-ATPase activity in EAT cells. Our study presents evidence that NADH dehydrogenase inhibition plays an important role in oxidative stress-mediated cytotoxicity, and perturbed membrane function as evident from inhibition of sodium-potassium pump is involved in cytotoxic cell death.
Subject(s)
Endosulfan/toxicity , Insecticides/toxicity , NADH Dehydrogenase/antagonists & inhibitors , Oxidative Stress/drug effects , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Animals , Antioxidants/pharmacology , Carcinoma, Ehrlich Tumor , Cell Death , Glutathione/metabolism , Lipid Peroxidation , Mice , Quercetin/pharmacology , Reactive Oxygen Species/metabolismABSTRACT
High-throughput drug discovery is highly dependent on the targets available to accelerate the process of candidates screening. Traditional chemical proteomics approaches for the screening of drug targets usually require the immobilization/modification of the drug molecules to pull down the interacting proteins. Recently, energetics-based proteomics methods provide an alternative way to study drug-protein interaction by using complex cell lysate directly without any modification of the drugs. In this study, we developed a novel energetics-based proteomics strategy, the solvent-induced protein precipitation (SIP) approach, to profile the interaction of drugs with their target proteins by using quantitative proteomics. The method is easy to use for any laboratory with the common chemical reagents of acetone, ethanol, and acetic acid. The SIP approach was able to identify the well-known protein targets of methotrexate, SNS-032, and a pan-kinase inhibitor of staurosporine in cell lysate. We further applied this approach to discover the off-targets of geldanamycin. Three known protein targets of the HSP90 family were successfully identified, and several potential off-targets including NADH dehydrogenase subunits NDUFV1 and NDUFAB1 were identified for the first time, and the NDUFV1 was validated by using Western blotting. In addition, this approach was capable of evaluating the affinity of the drug-target interaction. The data collectively proved that our approach provides a powerful platform for drug target discovery.
Subject(s)
HSP90 Heat-Shock Proteins/antagonists & inhibitors , Methotrexate/pharmacology , NADH Dehydrogenase/antagonists & inhibitors , Oxazoles/pharmacology , Proteomics , Staurosporine/pharmacology , Thiazoles/pharmacology , Acetic Acid/chemistry , Acetone/chemistry , Cells, Cultured , Drug Discovery , Drug Evaluation, Preclinical , Ethanol/chemistry , HEK293 Cells , HSP90 Heat-Shock Proteins/chemistry , HeLa Cells , High-Throughput Screening Assays , Humans , Methotrexate/chemistry , NADH Dehydrogenase/chemistry , NADH Dehydrogenase/metabolism , Oxazoles/chemistry , Solvents/chemistry , Staurosporine/chemistry , Thiazoles/chemistryABSTRACT
The viability of Mycobacterium tuberculosis (Mtb) depends on energy generated by its respiratory chain. Cytochrome bc1-aa3 oxidase and type-2 NADH dehydrogenase (NDH-2) are respiratory chain components predicted to be essential, and are currently targeted for drug development. Here we demonstrate that an Mtb cytochrome bc1-aa3 oxidase deletion mutant is viable and only partially attenuated in mice. Moreover, treatment of Mtb-infected marmosets with a cytochrome bc1-aa3 oxidase inhibitor controls disease progression and reduces lesion-associated inflammation, but most lesions become cavitary. Deletion of both NDH-2 encoding genes (Δndh-2 mutant) reveals that the essentiality of NDH-2 as shown in standard growth media is due to the presence of fatty acids. The Δndh-2 mutant is only mildly attenuated in mice and not differently susceptible to clofazimine, a drug in clinical use proposed to engage NDH-2. These results demonstrate the intrinsic plasticity of Mtb's respiratory chain, and highlight the challenges associated with targeting the pathogen's respiratory enzymes for tuberculosis drug development.
Subject(s)
Antitubercular Agents/therapeutic use , Drug Development , Electron Transport Complex III/genetics , Electron Transport Complex IV/genetics , Mycobacterium tuberculosis/genetics , NADH Dehydrogenase/genetics , Tuberculosis/drug therapy , Adaptation, Physiological/genetics , Animals , Callithrix , Electron Transport , Electron Transport Complex III/antagonists & inhibitors , Electron Transport Complex IV/antagonists & inhibitors , Gene Knockdown Techniques , Imidazoles/pharmacology , In Vitro Techniques , Lung/drug effects , Lung/pathology , Mice , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , NADH Dehydrogenase/antagonists & inhibitors , Piperidines/pharmacology , Pyridines/pharmacology , Tuberculosis, Pulmonary/drug therapy , Tuberculosis, Pulmonary/pathologyABSTRACT
With the emergence of drug-resistant Plasmodium falciparum, the treatment of malaria has become a significant challenge; therefore, the development of antimalarial drugs acting on new targets is extremely urgent. In Plasmodium falciparum, type II nicotinamide adenine dinucleotide (NADH) dehydrogenase (NDH-2) is responsible for catalyzing the transfer of two electrons from NADH to flavin adenine dinucleotide (FAD), which in turn transfers the electrons to coenzyme Q (CoQ). As an entry enzyme for oxidative phosphorylation, NDH-2 has become one of the popular targets for the development of new antimalarial drugs. In this study, reliable motion trajectories of the NDH-2 complex with its co-factors (NADH and FAD) and inhibitor, RYL-552, were obtained by comparative molecular dynamics simulations. The influence of cofactor binding on the global motion of NDH-2 was explored through conformational clustering, principal component analysis and free energy landscape. The molecular interactions of NDH-2 before and after its binding with the inhibitor RYL-552 were analyzed, and the key residues and important hydrogen bonds were also determined. The results show that the association of RYL-552 results in the weakening of intramolecular hydrogen bonds and large allosterism of NDH-2. There was a significant positive correlation between the angular change of the key pocket residues in the NADH-FAD-pockets that represents the global functional motion and the change in distance between NADH-C4 and FAD-N5 that represents the electron transfer efficiency. Finally, the possible non-competitive inhibitory mechanism of RYL-552 was proposed. Specifically, the association of inhibitors with NDH-2 significantly affects the global motion mode of NDH-2, leading to widening of the distance between NADH and FAD through cooperative motion induction; this reduces the electron transfer efficiency of the mitochondrial respiratory chain. The simulation results provide useful theoretical guidance for subsequent antimalarial drug design based on the NDH-2 structure and the respiratory chain electron transfer mechanism.
Subject(s)
Antimalarials/chemistry , Ketones/chemistry , NADH Dehydrogenase/antagonists & inhibitors , Plasmodium falciparum/enzymology , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/chemistry , Quinolines/chemistry , Electron Transport , Flavin-Adenine Dinucleotide/chemistry , Hydrogen Bonding , Models, Molecular , Molecular Structure , NAD/chemistry , NADH Dehydrogenase/chemistry , Oxidation-Reduction , Protein Binding , Structure-Activity Relationship , ThermodynamicsABSTRACT
Due to its pivotal role in NADH oxidation and ATP synthesis, mitochondrial complex I (CI) emerged as a crucial regulator of cellular metabolism. A functional CI relies on the sequential assembly of nuclear- and mtDNA-encoded subunits; however, whether CI assembly status is involved in the metabolic adaptations in CI deficiency still remains largely unknown. Here, we investigated the relationship between CI functions, its structure and the cellular metabolism in 29 patient fibroblasts representative of most CI mitochondrial diseases. Our results show that, contrary to the generally accepted view, a complex I deficiency does not necessarily lead to a glycolytic switch, i.e. the so-called Warburg effect, but that this particular metabolic adaptation is a feature of CI assembly defect. By contrast, a CI functional defect without disassembly induces a higher catabolism to sustain the oxidative metabolism. Mechanistically, we demonstrate that reactive oxygen species overproduction by CI assembly intermediates and subsequent AMPK-dependent Pyruvate Dehydrogenase inactivation are key players of this metabolic reprogramming. Thus, this study provides a two-way-model of metabolic responses to CI deficiencies that are central not only in defining therapeutic strategies for mitochondrial diseases, but also in all pathophysiological conditions involving a CI deficiency.
Subject(s)
Electron Transport Complex I/metabolism , Mitochondria/metabolism , Citric Acid Cycle , Fibroblasts/cytology , Fibroblasts/metabolism , Glycolysis , Humans , Metabolic Engineering , Mitochondrial Diseases/metabolism , Mitochondrial Diseases/pathology , NADH Dehydrogenase/antagonists & inhibitors , NADH Dehydrogenase/genetics , NADH Dehydrogenase/metabolism , Principal Component Analysis , RNA Interference , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolismABSTRACT
BACKGROUND: Araucaria angustifolia extract (AAE) is a polyphenol-rich extract that has gained interest as a natural anticancer agent. Recent work suggests that AAE induces oxidative damage and apoptosis through its action on decreasing complex I activity of the mitochondrial Electron Transport Chain (ETC). AIMS AND METHODS: In the present study, we aimed to further examine the specific targets by which AAE exerts proapoptotic effects in HEp-2 cancer cells. Specifically, the effect of AAE on the: 1) levels of pyruvate dehydrogenase was assessed by ELISA assay; 2) levels of mitochondrial ETC complexes, focusing on complex I at the gene transcript and protein level relevant to ROS generation was evaluated by multiplex ELISA followed by qRT-PCR and immunoblotting; 3) mitochondrial network distribution analysis was assessed by MitoTracker Red CMXRos; and 4) chemical variations on DNA was evaluated by dot-blotting in HEp-2 cells. RESULTS: Results demonstrated that AAE increased protein levels of PDH, switching energy metabolism to oxidative metabolism. Protein expression levels of complex I and III were found decreased in AAE-treated HEp-2 cells. Analyzing the subunits of complex I, changes in protein and gene transcript levels of NDUFS7 and NDUFV2 were found. Mitochondria staining after AAE incubation revealed changes in the mitochondrial network distribution. AAE was able to induce DNA hypomethylation and decreased DNA (cytosine-5)-methyltransferase 1 activity. CONCLUSION: Our data demonstrate for the first time that AAE alters expression of NDUFS7 and NDUFV2 mitochondrial subunits and induce epigenetic changes in HEp-2 cancer cells leading to a possible suppression of oncogenes.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Carcinoma, Squamous Cell/drug therapy , Epigenesis, Genetic/drug effects , Laryngeal Neoplasms/drug therapy , Mitochondria/drug effects , Plant Extracts/pharmacology , Polyphenols/pharmacology , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Proliferation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Epigenesis, Genetic/genetics , Humans , Laryngeal Neoplasms/metabolism , Laryngeal Neoplasms/pathology , Mitochondria/metabolism , NADH Dehydrogenase/antagonists & inhibitors , NADH Dehydrogenase/genetics , NADH Dehydrogenase/metabolism , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Polyphenols/chemistry , Polyphenols/isolation & purification , Structure-Activity Relationship , Tracheophyta/chemistryABSTRACT
Tumor hypoxia reduces the effectiveness of radiation therapy by limiting the biologically effective dose. An acute increase in tumor oxygenation before radiation treatment should therefore significantly improve the tumor cell kill after radiation. Efforts to increase oxygen delivery to the tumor have not shown positive clinical results. Here we show that targeting mitochondrial respiration results in a significant reduction of the tumor cells' demand for oxygen, leading to increased tumor oxygenation and radiation response. We identified an activity of the FDA-approved drug papaverine as an inhibitor of mitochondrial complex I. We also provide genetic evidence that papaverine's complex I inhibition is directly responsible for increased oxygenation and enhanced radiation response. Furthermore, we describe derivatives of papaverine that have the potential to become clinical radiosensitizers with potentially fewer side effects. Importantly, this radiosensitizing strategy will not sensitize well-oxygenated normal tissue, thereby increasing the therapeutic index of radiotherapy.
Subject(s)
Cell Hypoxia/drug effects , Lung Neoplasms/radiotherapy , Mitochondria/drug effects , NADH Dehydrogenase/antagonists & inhibitors , Oxygen/metabolism , Papaverine/pharmacology , Radiation-Sensitizing Agents/pharmacology , Animals , CRISPR-Cas Systems , Cell Hypoxia/radiation effects , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Electron Transport Complex I , Female , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Mitochondria/radiation effects , NADH Dehydrogenase/genetics , Phosphodiesterase Inhibitors/pharmacology , Radiation Tolerance , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Energy generation is a promising area of drug discovery for both bacterial pathogens and parasites. Type II NADH dehydrogenase (NDH-2), a vital respiratory membrane protein, has attracted attention as a target for the development of new antitubercular and antimalarial agents. To date, however, no potent, specific inhibitors have been identified. Here, we performed a site-directed screening technique, tethering-fragment based drug discovery, against wild-type and mutant forms of NDH-2 containing engineered active-site cysteines. Inhibitory fragments displayed IC50 values between 3 and 110⯵M against NDH-2 mutants. Possible binding poses were investigated by in silico modelling, providing a basis for optimisation of fragment binding and improved potency against NDH-2.
Subject(s)
Bacterial Proteins/metabolism , Drug Design , Enzyme Inhibitors/metabolism , Membrane Proteins/metabolism , NADH Dehydrogenase/metabolism , Bacillaceae/enzymology , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Cysteine/chemistry , Cysteine/genetics , Enzyme Inhibitors/chemistry , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/chemistry , Membrane Proteins/genetics , Molecular Docking Simulation , Mutagenesis, Site-Directed , Mutation , NADH Dehydrogenase/antagonists & inhibitors , NADH Dehydrogenase/chemistry , NADH Dehydrogenase/genetics , Protein BindingABSTRACT
Malaria control is threatened by a limited pipeline of effective pharmaceuticals against drug-resistant strains of Plasmodium falciparum Components of the mitochondrial electron transport chain (ETC) are attractive targets for drug development, owing to exploitable differences between the parasite and human ETC. Disruption of ETC function interferes with metabolic processes including de novo pyrimidine synthesis, essential for nucleic acid replication. We investigated the effects of ETC inhibitor selection on two distinct P. falciparum clones, Dd2 and 106/1. Compounds CK-2-68 and RYL-552, substituted quinolones reported to block P. falciparum NADH dehydrogenase 2 (PfNDH2; a type II NADH:quinone oxidoreductase), unexpectedly selected mutations at the quinol oxidation (Qo) pocket of P. falciparum cytochrome B (PfCytB). Selection experiments with atovaquone (ATQ) on 106/1 parasites yielded highly resistant PfCytB Y268S mutants seen in clinical infections that fail ATQ-proguanil treatment. In contrast, ATQ pressure on Dd2 yielded moderately resistant parasites carrying a PfCytB M133I or K272R mutation. Strikingly, all ATQ-selected mutants demonstrated little change or slight increase of sensitivity to CK-2-68 or RYL-552. Molecular docking studies demonstrated binding of all three ETC inhibitors to the Qo pocket of PfCytB, where Y268 forms strong van der Waals interactions with the hydroxynaphthoquinone ring of ATQ but not the quinolone ring of CK-2-68 or RYL-552. Our results suggest that combinations of suitable ETC inhibitors may be able to subvert or delay the development of P. falciparum drug resistance.
Subject(s)
Cytochromes b/genetics , NADH Dehydrogenase/antagonists & inhibitors , Plasmodium falciparum/genetics , Antimalarials/pharmacology , Drug Resistance/drug effects , Electron Transport Chain Complex Proteins/genetics , Malaria, Falciparum/drug therapy , Malaria, Falciparum/genetics , Malaria, Falciparum/parasitology , Molecular Docking Simulation/methods , Mutation/genetics , Plasmodium falciparum/drug effects , Quinolones/pharmacologyABSTRACT
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.
Subject(s)
Mycobacterium tuberculosis/enzymology , NADH Dehydrogenase/chemistry , Quinazolinones/chemistry , Molecular Structure , NADH Dehydrogenase/antagonists & inhibitors , Quinazolinones/chemical synthesis , Quinazolinones/pharmacology , Structure-Activity RelationshipABSTRACT
We identified a series of novel 7-phenyl benzoxaborole compounds with activity against Mycobacterium tuberculosis. Compounds had a range of activity with inhibitory concentrations (IC90) as low as 5.1 µM and no cytotoxicity against eukaryotic cells (IC50 > 50 µM). Compounds were active against intracellular mycobacteria cultured in THP-1 macrophages. We isolated and characterized resistant mutants with mutations in NADH dehydrogenase (Ndh) or the regulatory protein Mce3R. Mutations suggest that Ndh may be the target of this series.
Subject(s)
Boron Compounds/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Mycobacterium tuberculosis/drug effects , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Boron Compounds/chemistry , Boron Compounds/toxicity , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/toxicity , Dose-Response Relationship, Drug , Drug Resistance, Bacterial , Humans , Microbial Sensitivity Tests , Mutation , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , NADH Dehydrogenase/antagonists & inhibitors , NADH Dehydrogenase/genetics , NADH Dehydrogenase/metabolism , THP-1 CellsABSTRACT
The generation of ATP through oxidative phosphorylation is an essential metabolic function for Mycobaterium tuberculosis (Mtb), regardless of the growth environment. The typeâ II NADH dehydrogenase (Ndh-2) is the conduit for electrons into the pathway, and is absent in the mammalian genome, thus making it a potential drug target. Herein, we report the identification of two types of small molecules as selective inhibitors for Ndh-2 through a multicomponent high-throughput screen. Both compounds block ATP synthesis, lead to effects consistent with loss of NADH turnover, and importantly, exert bactericidal activity against Mtb. Extensive medicinal chemistry optimization afforded the best analogue with an MIC of 90â nm against Mtb. Moreover, the two scaffolds have differential inhibitory activities against the two homologous Ndh-2 enzymes in Mtb, which will allow precise control over Ndh-2 function in Mtb to facilitate the assessment of this anti-TB drug target.
Subject(s)
Anti-Bacterial Agents/pharmacology , Indazoles/pharmacology , Mycobacterium tuberculosis/enzymology , NADH Dehydrogenase/antagonists & inhibitors , Quinazolines/pharmacology , Drug Evaluation, Preclinical , Microbial Viability/drug effectsABSTRACT
Evidence continues to accumulate that pesticides are the leading candidates of environmental toxins that may contribute to the pathogenesis of Parkinson's disease. The mechanisms, however, remain largely unclear. According to epidemiological studies, we selected nine representative pesticides (paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan, fenpyroximate, tebufenpyrad, trichlorphon and carbaryl) which are commonly used in China and detected the effects of the pesticides on mitochondria and ubiquitin-proteasome system (UPS) function. Our results reveal that all the nine studied pesticides induce morphological changes of mitochondria at low concentrations. Paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan, fenpyroximate and tebufenpyrad induced mitochondria fragmentation. Furthermore, some of them (paraquat, rotenone, chlorpyrifos, fenpyroximate and tebufenpyrad) caused a significant dose-dependent decrease of intracellular ATP. Interestingly, these pesticides which induce mitochondria dysfunction also inhibit 26S and 20S proteasome activity. However, two out of the nine pesticides, namely trichlorphon and carbaryl, were found not to cause mitochondrial fragmentation or functional damage, nor inhibit the activity of the proteasome, which provides significant guidance for selection of pesticides in China. Moreover, our results demonstrate a potential link between inhibition of mitochondria and the UPS, and pesticide-induced Parkinsonism.
Subject(s)
Mitochondria/drug effects , Mitochondria/metabolism , Parkinson Disease/etiology , Parkinson Disease/metabolism , Pesticides/toxicity , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolism , Adenosine Triphosphate/metabolism , Apoptosis/drug effects , China/epidemiology , Dose-Response Relationship, Drug , Humans , Intracellular Space/metabolism , NADH Dehydrogenase/antagonists & inhibitors , Parkinson Disease/epidemiology , Unfolded Protein Response/drug effectsABSTRACT
INTRODUCTION: Tuberculosis (TB) is highly dangerous due to the development of resistance to first-line drugs. Moreover, Mycobacterium tuberculosis (Mtb) has also developed resistance to newly approved antitubercular drug bedaquiline. This necessitates the search for drugs acting on newer molecular targets. The energy metabolism of mycobacteria is the prime focus for the discovery of novel antitubercular drugs. Targeting type-2 NADH dehydrogenase (NDH-2) involved in the production of respiratory ATP could, therefore, be effective in treating the disease. Areas covered: This review describes the energetics of mycobacteria and the role of NDH-2 in ATP synthesis. Special attention has been given for genetic and chemical validations of NDH-2 as a molecular target. The reported kinetics and crystal structures of NDH-2 have been given in detail for better understanding of the enzyme. Expert opinion: NDH-2 is an essential enzyme for ATP synthesis and has a potential role in dormancy and persistence of Mtb. The human counterpart lacks this enzyme and hence NDH-2 inhibitors could have more clinical importance. Phenothiazines are potent inhibitor of NDH-2 and are effective against both drug-susceptible and drug-resistant Mtb. Thus, it is highly desirable to optimize phenothiazine class of compounds for the development of next generation anti-TB drugs.
Subject(s)
Antitubercular Agents/pharmacology , NADH Dehydrogenase/antagonists & inhibitors , Tuberculosis/drug therapy , Animals , Anti-Bacterial Agents/pharmacology , Drug Design , Drug Discovery , Drug Resistance, Neoplasm , Humans , Molecular Targeted Therapy , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , NADH Dehydrogenase/metabolism , Phenothiazines/pharmacology , Tuberculosis/microbiologyABSTRACT
Insects pollinate 75% of crops used for human consumption. Over the last decade, a substantial reduction in the abundance of pollinating insects has been recorded and recognized as a severe matter for food supply security. Many of the important food crops destined for human consumption are grown in greenhouses. A unique feature of greenhouse agriculture is the extensive use of fungicides to curb multiple fungal infections. The most widely used pollinating insects in greenhouses are commercially reared bumblebees. However, there is no data regarding the toxicity of fungicides to bumblebee mitochondria. To fill this gap in knowledge, we examined the effects of 16 widely used fungicides on the energetics of the flight muscles mitochondria of Bombus terrestris. We found that diniconazole and fludioxonil uncoupled the respiration of mitochondria; dithianon and difenoconazole inhibited it. By analyzing the action of these inhibitors on mitochondrial respiration and generation of reactive oxygen species, we concluded that difenoconazole inhibited electron transport at the level of Complex I and glycerol-3-phosphate dehydrogenase. Dithianon strongly inhibited succinate dehydrogenase and glycerol-3-phosphate dehydrogenase. It also strongly inhibited mitochondrial oxidation of NAD-linked substrates or glycerol 3-phosphate, but it had no effect on the enzymatic activity of Complex I. It may be suggested that dithianon inhibits electron transport downstream of Complex I, likely at multiply sites.