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1.
Eur J Med Chem ; 182: 111632, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31499363

RESUMO

ß-amino acids and their analogues are gathering increased attention not only because of their antibacterial and antifungal activity, but also for their use in designing peptidomimetics with increased oral bioavailability and resistance to metabolic degradation. In this study, a series of α-phenyl substituted chalcones, α-phenyl, ß-amino substituted dihydrochalcones and ß-amino acid derivatives were synthesized and evaluated for their antileishmanial efficacy against experimental visceral leishmaniasis (VL). Among all synthesized derivatives, 10c showed promising antileishmanial efficacy against both extracellular promastigote and intracellular amastigote (IC50 8.2 µM and 20.5 µM respectively) of L. donovani with negligible cytotoxic effect towards J774 macrophages and Vero cells. 10c effectively reduced spleen and liver parasite burden (>90%) in both hamster and Balb/c model of VL without any hepatotoxicity. In vitro pharmacokinetic analysis showed that 10c was stable in gastric fluid and plasma of Balb/c mice at 10 µg/ml. Further analysis of the molecular mechanism revealed that 10c entered into the parasite by depolarizing the plasma membrane rather than forming nonspecific pores and induced molecular events like loss in mitochondrial membrane potential with a gradual decline in ATP production. This, in turn, did not induce programmed cell death of the parasite; rather 10c induced bioenergetic collapse of the parasite by decreasing ATP synthesis through specific inhibition of mitochondrial complex III activity. Altogether, our results allude to the therapeutic potential of ß-amino acid derivatives as novel antileishmanials, identifying them as lead compounds for further exploration in the design of potent candidates for the treatment of visceral leishmaniasis.


Assuntos
Aminoácidos/farmacologia , Antiprotozoários/farmacologia , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Aminoácidos/química , Animais , Antiprotozoários/química , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Relação Dose-Resposta a Droga , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Leishmania donovani/metabolismo , Leishmaniose Visceral/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Células Vero
2.
Molecules ; 24(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426298

RESUMO

Amongst the various approaches to contain aflatoxin contamination of feed and food commodities, the use of inhibitors of fungal growth and/or toxin biosynthesis is showing great promise for the implementation or the replacement of conventional pesticide-based strategies. Several inhibition mechanisms were found taking place at different levels in the biology of the aflatoxin-producing fungal species such as Aspergillus flavus: compounds that influence aflatoxin production may block the biosynthetic pathway through the direct control of genes belonging to the aflatoxin gene cluster, or interfere with one or more of the several steps involved in the aflatoxin metabolism upstream. Recent findings pointed to mitochondrial functionality as one of the potential targets of some aflatoxin inhibitors. Additionally, we have recently reported that the effect of a compound belonging to the class of thiosemicarbazones might be related to the energy generation/carbon flow and redox homeostasis control by the fungal cell. Here, we report our investigation about a putative molecular target of the 3-isopropylbenzaldehyde thiosemicarbazone (mHtcum), using the yeast Saccharomyces cerevisiae as model system, to demonstrate how the compound can actually interfere with the mitochondrial respiratory chain.


Assuntos
Aflatoxinas/antagonistas & inibidores , Antifúngicos/farmacologia , Regulação Fúngica da Expressão Gênica , Mitocôndrias/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Tiossemicarbazonas/farmacologia , Aflatoxinas/biossíntese , Antifúngicos/química , Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/enzimologia , Aspergillus flavus/genética , Sítios de Ligação , Transporte de Elétrons/efeitos dos fármacos , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Simulação de Acoplamento Molecular , Família Multigênica , Ligação Proteica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Tiossemicarbazonas/química
3.
PLoS One ; 14(4): e0214023, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30964863

RESUMO

The battle against malaria has been substantially impeded by the recurrence of drug resistance in Plasmodium falciparum, the deadliest human malaria parasite. To counter the problem, novel antimalarial drugs are urgently needed, especially those that target unique pathways of the parasite, since they are less likely to have side effects. The mitochondrial type II NADH dehydrogenase (NDH2) of P. falciparum, PfNDH2 (PF3D7_0915000), has been considered a good prospective antimalarial drug target for over a decade, since malaria parasites lack the conventional multi-subunit NADH dehydrogenase, or Complex I, present in the mammalian mitochondrial electron transport chain (mtETC). Instead, Plasmodium parasites contain a single subunit NDH2, which lacks proton pumping activity and is absent in humans. A significant amount of effort has been expended to develop PfNDH2 specific inhibitors, yet the essentiality of PfNDH2 has not been convincingly verified. Herein, we knocked out PfNDH2 in P. falciparum via a CRISPR/Cas9 mediated approach. Deletion of PfNDH2 does not alter the parasite's susceptibility to multiple mtETC inhibitors, including atovaquone and ELQ-300. We also show that the antimalarial activity of the fungal NDH2 inhibitor HDQ and its new derivative CK-2-68 is due to inhibition of the parasite cytochrome bc1 complex rather than PfNDH2. These compounds directly inhibit the ubiquinol-cytochrome c reductase activity of the malarial bc1 complex. Our results suggest that PfNDH2 is not likely a good antimalarial drug target.


Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos/genética , NADH Desidrogenase/genética , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Animais , Antimaláricos/uso terapêutico , Sistemas CRISPR-Cas , Células Cultivadas , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Eritrócitos/parasitologia , Técnicas de Inativação de Genes , Humanos , Malária Falciparum/sangue , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , NADH Desidrogenase/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Quinolonas/farmacologia , Quinolonas/uso terapêutico
4.
Int Urol Nephrol ; 51(7): 1207-1218, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31020626

RESUMO

OBJECTIVES: The dysfunction of mitochondrial respiratory chain induced by cisplatin results in overproduction of reactive oxygen species (ROS) which contributes to kidney injury. The current study aimed to evaluate the effect of a mitochondrial electron transport inhibitors of rotenone (mitochondrial complex I inhibitor) and azoxystrobin (mitochondrial complex III inhibitor), in cisplatin-induced kidney injury. METHODS: In vivo, cisplatin was administered to male C57BL/6J mice by a single intraperitoneal (i.p.) injection (20 mg/kg). Then the mice were treated with or without 200 ppm rotenone in food. Mice were sacrificed after cisplatin administration for 72 h. The serum and the kidney tissues were collected for further analysis. In vitro, mouse proximal tubular cells (mPTCs) were treated with cisplatin (5 µg/mL) and rotenone/azoxystrobin for 24 h. Flow cytometry, Western blotting, and TUNEL staining were used to evaluate the cell injury. RESULTS: In vivo, rotenone treatment obviously ameliorated cisplatin-induced renal tubular injury evidenced by the improved histology and blocked NGAL upregulation. Meanwhile, cisplatin-induced renal dysfunction shown by the increased levels of serum creatinine (Scr), blood urea nitrogen (BUN), and cystatin C were significantly reduced by rotenone treatment. Moreover, the increments of cleaved caspase-3 and transferase dUTP nick-end labeling (TUNEL)-positive cells were markedly decreased in line with the attenuated mitochondrial dysfunction and oxidative stress after rotenone administration. In vitro, rotenone and azoxystrobin protected against mitochondrial dysfunction, oxidative stress, and renal tubular cell apoptosis induced by cisplatin. CONCLUSIONS: Our results demonstrated that inhibition of mitochondrial activity significantly attenuated cisplatin nephrotoxicity possibly by inhibiting mitochondrial oxidative stress.


Assuntos
Lesão Renal Aguda , Cisplatino/farmacologia , Mitocôndrias , Pirimidinas/farmacologia , Rotenona/farmacologia , Estrobilurinas/farmacologia , Lesão Renal Aguda/induzido quimicamente , Lesão Renal Aguda/metabolismo , Lesão Renal Aguda/patologia , Lesão Renal Aguda/prevenção & controle , Animais , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Rim/metabolismo , Rim/patologia , Testes de Função Renal/métodos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Resultado do Tratamento , Desacopladores/farmacologia
5.
FEBS Lett ; 593(5): 499-503, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30734287

RESUMO

Methylene blue (MB) is a promising prodrug to treat mitochondrial dysfunctions that is currently being used in clinical trials for Alzheimer's disease. MB can penetrate the blood brain barrier, accumulating in brain mitochondria where it acts as a redox mediator in the electron transfer chain (ETC). Mitochondrial flavins are thought to reduce MB, which is then oxidized by cytochrome c, thereby bypassing inhibited Complex I of ETC. We found that in mouse brain mitochondria, MB fails to restore the membrane potential and respiration inhibited by antimycin. Furthermore, antimycin inhibits MB-induced H2 O2 generation. Our data suggest that the acceptor of electrons from MB is a Qo ubiquinol-binding site of Complex III; thus, MB-based drugs might not be helpful in mitochondrial dysfunctions involving Complex III inhibition.


Assuntos
Antimicina A/análogos & derivados , Encéfalo/efeitos dos fármacos , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Azul de Metileno/farmacocinética , Mitocôndrias/efeitos dos fármacos , Animais , Antimicina A/farmacologia , Barreira Hematoencefálica , Encéfalo/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Feminino , Peróxido de Hidrogênio/metabolismo , Masculino , Azul de Metileno/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo
6.
J Agric Food Chem ; 67(10): 2774-2781, 2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30794394

RESUMO

The marine natural product neopeltolide was isolated from a deep-water sponge specimen of the family Neopeltidae. Neopeltolide has been proven to be a new type of inhibitor of the cytochrome bc1 complex in the mitochondrial respiration chain. However, its detailed inhibition mechanism has remained unknown. In addition, neopeltolide is difficult to synthesize because of its very complex chemical structure. In the present work, the binding mode of neopeltolide was determined for the first time by integrating molecular docking, molecular dynamics simulations, and molecular mechanics Poisson-Boltzmann surface area calculations, which showed that neopeltolide is a Qo site inhibitor of the bc1 complex. Then, according to guidance via inhibitor-protein interaction analysis, structural modification was carried out with the aim to simplify the chemical structure of neopeltolide, leading to the synthesis of a series of new neopeltolide derivatives with much simpler chemical structures. The calculated binding energies (Δ Gcal) of the newly synthesized analogues correlated very well ( R2 = 0.90) with their experimental binding free energies (Δ Gexp), which confirmed that the computational protocol was reliable. Compound 45, bearing a diphenyl ether fragment, was successfully designed and synthesized as the most potent candidate (IC50 = 12 nM) against porcine succinate cytochrome c reductase. The molecular modeling results indicate that compound 45 formed a π-π interaction with Phe274 and two hydrogen bonds with Glu271 and His161. The present work provides a new starting point for future fungicide discovery to overcome the resistance that the existing bc1 complex inhibitors are facing.


Assuntos
Produtos Biológicos/química , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Inibidores Enzimáticos/química , Macrolídeos/química , Animais , Sítios de Ligação , Complexo III da Cadeia de Transporte de Elétrons/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Poríferos/química
7.
Cell Mol Life Sci ; 76(7): 1381-1396, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30666338

RESUMO

Ubiquinol cytochrome c reductase core protein I (UQCRC1) is a component of the complex III in the respiratory chain. Its biological functions are unknown. Here, we showed that knockout of UQCRC1 led to embryonic lethality. Disrupting one UQCRC1 allele in mice (heterozygous mice) of both sexes did not affect their growth but reduced UQCRC1 mRNA and protein in the brain. These mice had decreased complex III formation, complex III activity and ATP content in the brain at baseline. They developed worsened neurological outcome after brain ischemia/hypoxia or focal brain ischemia compared with wild-type mice. The ischemic cerebral cortex of the heterozygous mice had decreased mitochondrial membrane potential and ATP content as well as increased free radicals. Also, the heterozygous mice performed poorly in the Barnes maze and novel object recognition tests. Finally, UQCRC1 was expressed abundantly in neurons and astrocytes. These results suggest a critical role of UQCRC1 in embryo survival. UQCRC1 may also be important by forming the complex III to maintain normal brain ischemic tolerance, learning and memory.


Assuntos
Isquemia Encefálica/patologia , Cognição/fisiologia , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/metabolismo , Isquemia Encefálica/metabolismo , Resistência à Doença , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/genética , Embrião de Mamíferos/metabolismo , Feminino , Masculino , Aprendizagem em Labirinto , Potencial da Membrana Mitocondrial , Memória/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Espécies Reativas de Oxigênio/metabolismo
8.
Pest Manag Sci ; 75(7): 1831-1846, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30636031

RESUMO

BACKGROUND: UK-2A is an antifungal antibiotic produced by Streptomyces sp. 517-02. Derivatization of its picolinamide OH to form the isobutyryl acetal led to the discovery of fenpicoxamid (InatreqTM active), which is currently under development as a fungicide by Dow AgroSciences LLC. This paper documents efforts to achieve additional efficacy enhancements through semi-synthetic modification of the benzyl substituent of the UK-2A macrocycle. RESULTS: Of 34 analogs prepared, the most active had mitochondrial electron transport IC50 values 1.5- to 3.7-fold higher than UK-2A (IC50 0.86 nM). The cyclohexyl analog (38, IC50 1.23 nM) was the most intrinsically active derivative, and inhibited in vitro growth of Zymoseptoria tritici (EC50 2.8 ppb) and Leptosphaeria nodorum (EC50 6.2 ppb) more strongly than UK-2A (EC50 5.3 and 11.3 ppb for Z. tritici and L. nodorum, respectively). Heterocyclic ring systems and polar linker functionalities resulted in substantial activity loss. Several analogs (20, 22, 23, 24, 36 and 38) translated Z. tritici in vitro growth inhibition activity to in planta disease control more effectively than did UK-2A, with log D being a key factor in this regard. CONCLUSIONS: UK-2A is amenable to further modification at the benzyl position on the macrocycle, which provides opportunities for manipulation of physical properties while retaining strong intrinsic and antifungal activity. © 2019 Society of Chemical Industry.


Assuntos
Ascomicetos/efeitos dos fármacos , Fungicidas Industriais/síntese química , Ustilago/efeitos dos fármacos , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Fungicidas Industriais/química , Fungicidas Industriais/farmacologia , Concentração Inibidora 50 , Lactonas/síntese química , Lactonas/química , Lactonas/farmacologia , Mitocôndrias , Piridinas/síntese química , Piridinas/química , Piridinas/farmacologia , Relação Estrutura-Atividade , Triticum/microbiologia
9.
Physiol Plant ; 167(2): 188-204, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30467859

RESUMO

Research has begun to elucidate the signal transduction pathway(s) that control cellular responses to changes in mitochondrial status. Important tools in such studies are chemical inhibitors used to initiate mitochondrial dysfunction. This study compares the effect of different inhibitors and treatment conditions on the transcript amount of nuclear genes specifically responsive to mitochondrial dysfunction in leaf of Nicotiana tabacum L. cv. Petit Havana. The Complex III inhibitors antimycin A (AA) and myxothiazol (MYXO), and the Complex V inhibitor oligomycin (OLIGO), each increased the transcript amount of the mitochondrial dysfunction genes. Transcript responses to OLIGO were greater during treatment in the dark than in the light, and the dark treatment resulted in cell death. In the dark, transcript responses to AA and MYXO were similar to one another, despite MYXO leading to cell death. In the light, transcript responses to AA and MYXO diverged, despite cell viability remaining high with either inhibitor. This divergent response may be due to differential signaling from the chloroplast because only AA also inhibited cyclic electron transport, resulting in a strong acceptor-side limitation in photosystem I. In the light, chemical inhibition of chloroplast electron transport reduced transcript responses to AA, while having no effect on the response to MYXO, and increasing the response to OLIGO. Hence, when studying mitochondrial dysfunction signaling, different inhibitor and treatment combinations differentially affect linked processes (e.g. chloroplast function and cell fate) that then contribute to measured responses. Therefore, inhibitor and treatment conditions should be chosen to align with specific study goals.


Assuntos
Cloroplastos/metabolismo , Mitocôndrias/metabolismo , Transdução de Sinais , Tabaco/genética , Antimicina A/farmacologia , Cloroplastos/efeitos da radiação , Transporte de Elétrons/efeitos dos fármacos , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Luz , Metacrilatos/farmacologia , Mitocôndrias/efeitos da radiação , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , Oligomicinas/farmacologia , Complexo de Proteína do Fotossistema I/efeitos dos fármacos , Complexo de Proteína do Fotossistema I/genética , Complexo de Proteína do Fotossistema I/metabolismo , Folhas de Planta/genética , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Tiazóis/farmacologia , Tabaco/fisiologia , Tabaco/efeitos da radiação
10.
Pest Manag Sci ; 75(8): 2107-2114, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30426681

RESUMO

BACKGROUND: Complex III inhibitors targeting the Qi -site have been known for decades; some are used or being developed as antimicrobial compounds. Target site resistance mutations have been reported in laboratory-selected mutants and in field isolates. Here, we present a brief overview of mutations found in laboratory-selected resistant mutants. We also provide a study of mutations observed in field isolates of Plasmopara viticola, in particular the ametoctradin resistance substitution, S34L that we analysed in the yeast model. RESULTS: A survey of laboratory mutants showed that resistance could be caused by a large number of substitutions in the Qi -site. Four residues seemed key in term of resistance: N31, G37, L198 and K228. Using yeast, we analysed the effect of the ametoctradin resistance substitution S34L reported in field isolates of P. viticola. We showed that S34L caused a high level of resistance combined with a loss of complex III activity and growth competence. CONCLUSION: Use of single site Qi -site inhibitors is expected to result in the selection of resistant mutants. However, if the substitution is associated with a fitness penalty, as may be the case with S34L, resistance development might not be an insuperable obstacle, although careful monitoring is required. © 2018 Society of Chemical Industry.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/genética , Proteínas Mitocondriais/genética , Mutação , Oomicetos/genética , Seleção Genética , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/genética , Proteínas Mitocondriais/antagonistas & inibidores , Oomicetos/enzimologia
11.
ACS Infect Dis ; 5(2): 239-249, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30485737

RESUMO

Respiration is a promising target for the development of new antimycobacterial agents, with a growing number of compounds in clinical development entering this target space. However, more candidate inhibitors are needed to expand the therapeutic options available for drug-resistant Mycobacterium tuberculosis infection. Here, we characterize a putative respiratory complex III (QcrB) inhibitor, TB47: a pyrazolo[1,5- a]pyridine-3-carboxamide. TB47 is active (MIC between 0.016 and 0.500 µg/mL) against a panel of 56 M. tuberculosis clinical isolates, including 37 multi-drug-resistant and two extensively drug-resistant strains. Pharmacokinetic and toxicity studies showed promising profiles, including negligible CYP450 interactions, cytotoxicity, and hERG channel inhibition. Consistent with other reported QcrB inhibitors, TB47 inhibits oxygen consumption only when the alternative oxidase, cytochrome bd, is deleted. A point mutation in the qcrB cd2-loop (H190Y, M. smegmatis numbering) rescues the inhibitory effects of TB47. Metabolomic profiling of TB47-treated M. tuberculosis H37Rv cultures revealed accumulation of steps in the TCA cycle and pentose phosphate pathway that are linked to reducing equivalents, suggesting that TB47 causes metabolic redox stress. In mouse infection models, a TB47 monotherapy was not bactericidal. However, TB47 was strongly synergistic with pyrazinamide and rifampicin, suggesting a promising role in combination therapies. We propose that TB47 is an effective lead compound for the development of novel tuberculosis chemotherapies.


Assuntos
Antituberculosos/farmacologia , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Animais , Antituberculosos/farmacocinética , Feminino , Metabolômica , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Piridinas/farmacologia
12.
FEMS Yeast Res ; 19(2)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30500899

RESUMO

Scheffersomyces stipitis shows a high capacity to ferment xylose, with a strong oxygen dependence to allow NAD+ regeneration. However, without oxygen regeneration of NADH occurs by other metabolic pathways like alcoholic fermentation. There are few reports about inhibitors of mitochondrial respiration and their effects on growth and fermentation. This work aimed to explore the effect of cytochrome bc1 complex inhibition by antimycin A (AA), on growth and fermentation of S. stipitis using glucose, xylose and arabinose as carbon sources, at three agitation levels (0, 125 and 250 rpm). It was possible to discriminate between respiratory and fermentative metabolism in these different conditions using xylose or arabinose. Despite the inhibition of mitochondrial respiration, the glycolytic flux was active because S. stipitis metabolized glucose or xylose to produce ATP; on 0.5 M glucose the cells yielded 17-33 g L-1 ethanol. However, more complex results were obtained on xylose, which depended upon agitation conditions where ethanol production without agitation increased up to 11 g L-1. Inhibition of respiratory chain in S. stipitis could therefore be a good strategy to improve ethanol yields.


Assuntos
Arabinose/metabolismo , Carbono/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Glucose/metabolismo , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Xilose/metabolismo , Antimicina A/metabolismo , Inibidores Enzimáticos/metabolismo , Etanol/metabolismo , Fermentação/efeitos dos fármacos , Glicólise , Análise do Fluxo Metabólico , Oxirredução , Saccharomycetales/efeitos dos fármacos
14.
Nat Commun ; 9(1): 5370, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30560872

RESUMO

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a neglected tropical skin disease that is most commonly found in children from West and Central Africa. Despite the severity of the infection, therapeutic options are limited to antibiotics with severe side effects. Here, we show that M. ulcerans is susceptible to the anti-tubercular drug Q203 and related compounds targeting the respiratory cytochrome bc1:aa3. While the cytochrome bc1:aa3 is the primary terminal oxidase in Mycobacterium tuberculosis, the presence of an alternate bd-type terminal oxidase limits the bactericidal and sterilizing potency of Q203 against this bacterium. M. ulcerans strains found in Buruli ulcer patients from Africa and Australia lost all alternate terminal electron acceptors and rely exclusively on the cytochrome bc1:aa3 to respire. As a result, Q203 is bactericidal at low dose against M. ulcerans replicating in vitro and in mice, making the drug a promising candidate for Buruli ulcer treatment.


Assuntos
Antibióticos Antituberculose/farmacologia , Úlcera de Buruli/tratamento farmacológico , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Mycobacterium ulcerans/efeitos dos fármacos , Doenças Negligenciadas/tratamento farmacológico , África , Animais , Antibióticos Antituberculose/uso terapêutico , Austrália , Úlcera de Buruli/microbiologia , Modelos Animais de Doenças , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium ulcerans/metabolismo , Doenças Negligenciadas/microbiologia , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Rifampina/farmacologia , Rifampina/uso terapêutico , Resultado do Tratamento
15.
Sci Adv ; 4(7): eaar5319, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30116775

RESUMO

Beige adipocytes can be induced from white adipocytes and precursors upon stimulation by cold temperatures and act like brown adipocytes to increase energy expenditure. Most in vivo studies examining the mechanisms for the induction of beige adipocytes have focused on subcutaneous white adipose tissue (sWAT; benign fat) in the mouse. How intra-abdominal WAT (aWAT; malignant fat) develops into beige adipocytes remains obscure, largely because there is a lack of a good animal model for the induction of beige adipocytes from aWAT. To better understand the development of beige adipocytes from mammalian WATs, especially aWAT, we induced beige adipocytes from bat aWAT and mouse sWAT by exposure to cold temperatures and analyzed their molecular signatures. RNA sequencing followed by whole genome-wide expression analysis shows that beige adipocytes induced from bat aWAT, rather than sWAT, have molecular signatures resembling those of mouse sWAT-induced beige adipocytes and exhibit dynamic profiles similar to those of classical brown adipocytes. In addition, we identified molecular markers that were highly enriched in beige adipocytes and conserved between bat aWAT and mouse sWAT, a set that included the genes Uqcrc1 and Letm1. Furthermore, knockdown of Uqcrc1 and Letm1 expression shows that they are required not only for beige adipocyte differentiation but also for preadipocyte maturation. This study presents a new model for research into the induction of beige adipocytes from aWAT in vivo, which, when combined with models where beige adipocytes are induced from sWAT, provides insight into therapeutic approaches for combating obesity-related diseases in humans.


Assuntos
Adipócitos Bege/metabolismo , Gordura Intra-Abdominal/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Adipócitos Bege/citologia , Adipogenia/efeitos dos fármacos , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Animais , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Diferenciação Celular/efeitos dos fármacos , Temperatura Baixa , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Genoma , Insulina/farmacologia , Gordura Intra-Abdominal/citologia , Camundongos , Modelos Animais , RNA/química , RNA/isolamento & purificação , RNA/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transcriptoma
16.
ACS Infect Dis ; 4(11): 1574-1584, 2018 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-30117728

RESUMO

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.


Assuntos
Antiprotozoários/farmacologia , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Éteres Fenílicos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Quinolonas/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasmose/tratamento farmacológico , Animais , Células Cultivadas , Descoberta de Drogas , Resistência a Medicamentos , Humanos , Malária Falciparum/tratamento farmacológico , Camundongos , Estrutura Molecular , Relação Estrutura-Atividade
17.
Cell Physiol Biochem ; 48(4): 1755-1770, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30078018

RESUMO

BACKGROUND/AIMS: CDH18 (cadherin 18) is specifically expressed in the central nervous system and associated with various neuropsychiatric disorders. In this study, the role of CDH18 in glioma carcinogenesis and progression was investigated. METHODS: The expression of CDH18 and its prognostic value in patients with gliomas were analyzed in public database and validated by real-time PCR/immunohistochemical staining (IHC) in our cohort. CCK-8 assay, transwell migration assay, wound healing assay, clonogenic assay and tumorigenicity assay were used to compare the proliferation, invasion and migration ability of glioma cells with different expressions of CDH18. iTRAQ-based quantitative proteomic analysis were used to reveal the downstream target of CDH18. Rescue experiments were conducted to further validate the relationship between UQCRC2 and CDH18. RESULTS: The expression of CDH18 was depressed in a ladder-like pattern from normal tissues to WHO IV gliomas, and was an independent prognostic factor in TCGA (The Cancer Genome Atlas), CGGA (the Chinese glioma genome-atlas) and our glioma cohorts (n=453). Functional experiments in vitro and in vivo demonstrated that CDH18 inhibited invasion/migration, enhanced chemoresistance and suppressed tumorigenicity of glioma cells. UQCRC2 was identified as the downstream target of CDH18 by proteomic analysis. The expression of UQCRC2 was gradually absent as the WHO grades of gliomas escalated and was positively correlated with the expression of CDH18. Furthermore, in vitro assays demonstrated that down-regulation of UQCRC2 partly reversed the inhibition of invasion/migration ability and chemoresistance in CDH18 overexpressed glioma cell lines. Survival analysis demonstrated that combined CDH18/UQCRC2 biomarkers significantly influenced the prognosis of glioma patients. CONCLUSIONS: The present research demonstrated that CDH18 exerted its tumor-suppressor role via UQCRC2 in glioma cells and CDH18 might serve as a therapeutic target for treating gliomas.


Assuntos
Neoplasias Encefálicas/patologia , Caderinas/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Glioma/patologia , Idoso , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Caderinas/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/genética , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Humanos , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Temozolomida
18.
J Biochem Mol Toxicol ; 32(9): e22195, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30004155

RESUMO

Targeting mitochondria respiration is an effective therapeutic strategy in renal cell carcinoma (RCC). Atovaquone is a FDA-approved antibiotic but is also known as a mitochondrial inhibitor. We found that atovaquone inhibited proliferation and induced apoptosis of RCC cells. Mechanistically, atovaquone inhibits mitochondrial respiration in a concentration-dependent and time-dependent manner, via targeting mitochondrial respiratory complex III. Although increased glycolysis was observed in atovaquone-treated cells, atovaquone decreased ATP levels. As a consequence of mitochondrial respiration inhibition, reactive oxygen species levels were increased by atovaquone. The complete rescue of atovaquone's effects by an antioxidant suggests the important role of oxidative stress in the action of atovaquone in RCC. Importantly, atovaquone enhanced the in vitro and in vivo efficacy of 5-fluorouracil (5-FU) and interferon-α (IFN-α). Our preclinical findings suggest that atovaquone is a useful addition for RCC treatment. Our work also further demonstrates that RCC is more dependent on mitochondrial respiration than glycolysis.


Assuntos
Antineoplásicos/farmacologia , Atovaquona/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Neoplasias Renais/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Animais , Anti-Helmínticos/química , Anti-Helmínticos/farmacologia , Antimetabólitos Antineoplásicos/química , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Atovaquona/antagonistas & inibidores , Atovaquona/uso terapêutico , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Humanos , Interferon-alfa/agonistas , Interferon-alfa/farmacologia , Interferon-alfa/uso terapêutico , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Camundongos SCID , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Distribuição Aleatória , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
J Biol Chem ; 293(26): 10363-10380, 2018 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-29739855

RESUMO

Reactive oxygen and nitrogen species (ROS/RNS) such as superoxide (O2̇̄), hydrogen peroxide, lipid hydroperoxides, peroxynitrite, and hypochlorous and hypobromous acids play a key role in many pathophysiological processes. Recent studies have focused on mitochondrial ROS as redox signaling species responsible for promoting cell division, modulating and regulating kinases and phosphatases, and activating transcription factors. Many ROS also stimulate cell death and senescence. The extent to which these processes occur is attributed to ROS levels (low or high) in cells. However, the exact nature of ROS remains unknown. Investigators have used redox-active probes that, upon oxidation by ROS, yield products exhibiting fluorescence, chemiluminescence, or bioluminescence. Mitochondria-targeted probes can be used to detect ROS generated in mitochondria. However, because most of these redox-active probes (untargeted and mitochondria-targeted) are oxidized by several ROS species, attributing redox probe oxidation to specific ROS species is difficult. It is conceivable that redox-active probes are oxidized in common one-electron oxidation pathways, resulting in a radical intermediate that either reacts with another oxidant (including oxygen to produce O2̇̄) and forms a stable fluorescent product or reacts with O2̇̄ to form a fluorescent marker product. Here, we propose the use of multiple probes and complementary techniques (HPLC, LC-MS, redox blotting, and EPR) and the measurement of intracellular probe uptake and specific marker products to identify specific ROS generated in cells. The low-temperature EPR technique developed to investigate cellular/mitochondrial oxidants can easily be extended to animal and human tissues.


Assuntos
Mitocôndrias/metabolismo , Técnicas de Sonda Molecular , Espécies Reativas de Oxigênio/metabolismo , Aconitato Hidratase/metabolismo , Linhagem Celular , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Metabolismo Energético/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Mitocôndrias/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Superóxidos/metabolismo
20.
Respir Res ; 19(1): 69, 2018 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-29685148

RESUMO

BACKGROUND: Pulmonary arterial hypertension (PAH) is a fatal disease; however, the mechanisms directly involved in triggering and the progression of PAH are not clear. Based on previous studies that demonstrated a possible role of mitochondrial dysfunction in the pathogenesis of PAH, we investigated the effects of chronic inhibition of mitochondrial function in vivo in healthy rodents. METHODS: Right ventricle systolic pressure (RVSP) was measured in female rats at baseline and up to 24 days after inhibition of mitochondrial respiratory Complex III, induced by Antimycin A (AA, 0.35 mg/kg, given three times starting at baseline and then days 3 and 6 as a bolus injection into the right atrial chamber). RESULTS: Rodents exposed to AA demonstrated sustained increases in RVSP from days 6 through 24. AA-exposed rodents also possessed a progressive increase in RV end-diastolic pressure but not RV hypertrophy, which may be attributed to either early stages of PAH development or to reduced RV contractility due to inhibition of myocardial respiration. Protein nitration levels in plasma were positively correlated with PAH development in AA-treated rats. This finding was strongly supported by results obtained from PAH humans where plasma protein nitration levels were correlated with markers of PAH severity in female but not male PAH patients. Based on previously reported associations between increased nitric oxide production levels with female gender, we speculate that in females with PAH mitochondrial dysfunction may represent a more deleterious form, in part, due to an increased nitrosative stress development. Indeed, the histological analysis of AA treated rats revealed a strong perivascular edema, a marker of pulmonary endothelial damage. Finally, AA treatment was accompanied by a severe metabolic shift toward glycolysis, a hallmark of PAH pathology. CONCLUSIONS: Chronic mitochondrial dysfunction induces the combination of vascular damage and metabolic reprogramming that may be responsible for PAH development. This mechanism may be especially important in females, perhaps due to an increased NO production and nitrosative stress development.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Glicólise/fisiologia , Hipertensão Pulmonar/metabolismo , Pulmão/metabolismo , Mitocôndrias/metabolismo , Vasoconstrição/fisiologia , Animais , Antimicina A/toxicidade , Feminino , Glicólise/efeitos dos fármacos , Humanos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/fisiopatologia , Pulmão/efeitos dos fármacos , Pulmão/fisiopatologia , Masculino , Mitocôndrias/efeitos dos fármacos , Monocrotalina/toxicidade , Ratos , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacos , Disfunção Ventricular Direita/induzido quimicamente , Disfunção Ventricular Direita/metabolismo , Disfunção Ventricular Direita/fisiopatologia
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