RESUMO
Determining interacting cellular partners of drugs by chemical proteomic techniques is complex and tedious. Most approaches rely on activity-based probe profiling and compound-centric chemical proteomics. The anti-malarial artemisinin also exerts profound anti-cancer activity, but the mechanisms of action are incompletely understood. In the present investigation, we present a novel approach to identify artemisinin-interacting target proteins. Our approach overcomes usual problems in traditional fishing procedures, because the drug was attached to a surface without further chemical modification. The proteins identified effect among others, cell cycle arrest, apoptosis, inhibition of angiogenesis, disruption of cell migration, and modulation of nuclear receptor responsiveness. Furthermore, a bioinformatic approach confirmed experimentally identified proteins and suggested a large number of other interacting proteins. Theoretically predicted interaction partners may serve as a starting point to complete the whole set of proteins binding artemisinin.
Assuntos
Artemisininas/farmacologia , Biologia Computacional/métodos , Neoplasias Nasofaríngeas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteômica/métodos , Anticarcinógenos/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , HumanosRESUMO
Chagas disease, caused by Trypanosoma cruzi, is a widespread infection in Latin America. Currently, only 2 partially effective and highly toxic drugs, i.e., benznidazole and nifurtimox, are available for the treatment of this disease, and several efforts are underway in the search for better chemotherapeutic agents. Here, we have determined the trypanocidal activity of 2,3-diphenyl-1 ,4-naphthoquinone (DPNQ), a novel quinone derivative. In vitro, DPNQ was highly cytotoxic at a low, micromolar concentration (LD50 = 2.5 microM) against epimastigote, cell-derived trypomastigote, and intracellular amastigote forms of T. cruzi, but not against mammalian cells (LD50 = 130 microM). In vivo studies on the murine model of Chagas disease revealed that DPNQ-treated animals (3 doses of 10 mg/kg/day) showed a significant delay in parasitemia peak and higher (up to 60%) survival rate 70 days post-infection, when compared with the control group (infected, untreated). We also observed a 2-fold decrease in parasitemia between the control group (infected, untreated) and the treated group (infected, treated). No apparent drug toxicity effects were noticed in the control group (uninfected, treated). In addition, we determined that DPNQ is the first competitive inhibitor of T. cruzi lipoamide dehydrogenase (TcLipDH) thus far described. Our results indicate that DPNQ is a promising chemotherapeutic agent against T. cruzi.
Assuntos
Doença de Chagas/tratamento farmacológico , Naftoquinonas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Linhagem Celular , Doença de Chagas/parasitologia , Di-Hidrolipoamida Desidrogenase/antagonistas & inibidores , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos C3H , Naftoquinonas/química , Naftoquinonas/uso terapêutico , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Trypanothione reductase (TR) is both a valid and an attractive target for the design of new trypanocidal drugs. Starting from menadione, plumbagin, and juglone, three distinct series of 1,4-naphthoquinones (NQ) were synthesized as potential inhibitors of TR from Trypanosoma cruzi (TcTR). The three parent molecules were functionalized at carbons 2 and/or 3 by various polyamine chains. Optimization of TcTR inhibition and TcTR specificity versus human disulfide reductases was achieved with the 3,3'-[polyaminobis(carbonylalkyl)]bis(1,4-NQ) series 19-20, in which an optimum chain length was determined for inhibition of the trypanothione disulfide reduction. The most active derivatives against trypanosomes in cultures were also studied as subversive substrates of TcTR and lipoamide dehydrogenase (TcLipDH). The activities were measured by following NAD(P)H oxidation as well as coupling the reactions to the reduction of cytochrome c which permits the detection of one-electron transfer. For TcTR, 20(4-c) proved to be a potent subversive substrate and an effective uncompetitive inhibitor versus trypanothione disulfide and NADPH. Molecular modeling studies based on the known X-ray structures of TcTR and hGR were conducted in order to compare the structural features, dimensions, and accessibility of the cavity at the dimer interface of TcTR with that of hGR, as one of the putative NQ binding sites. TcLipDH reduced the plumbagin derivatives by an order of magnitude faster than the corresponding menadione derivatives. Such differences were not observed with the pig heart enzyme. The most efficient and specific subversive substrates of TcTR and TcLipDH exhibited potent antitrypanosomal activity in in vitro T. brucei and T. cruzi cultures. The results obtained here confirm that reduction of NQs by parasitic flavoenzymes is a promising strategy for the development of new trypanocidal drugs.
Assuntos
Di-Hidrolipoamida Desidrogenase/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , NADH NADPH Oxirredutases/antagonistas & inibidores , Naftoquinonas/síntese química , Tripanossomicidas/síntese química , Trypanosoma cruzi/efeitos dos fármacos , Animais , Células Cultivadas , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Camundongos , Modelos Moleculares , Miocárdio/enzimologia , Naftoquinonas/química , Naftoquinonas/farmacologia , Oxirredução , Relação Estrutura-Atividade , Suínos , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/enzimologiaRESUMO
Trypanosoma brucei, the causative agent of African sleeping sickness, synthesizes deoxyribonucleotides via a classical eukaryotic class I ribonucleotide reductase. The unique thiol metabolism of trypanosomatids in which the nearly ubiquitous glutathione reductase is replaced by a trypanothione reductase prompted us to study the nature of thiols providing reducing equivalents for the parasite synthesis of DNA precursors. Here we show that the dithiol trypanothione (bis(glutathionyl)spermidine), in contrast to glutathione, is a direct reductant of T. brucei ribonucleotide reductase with a K(m) value of 2 mm. This is the first example of a natural low molecular mass thiol directly delivering reducing equivalents for ribonucleotide reduction. At submillimolar concentrations, the reaction is strongly accelerated by tryparedoxin, a 16-kDa parasite protein with a WCPPC active site motif. The K(m) value of T. brucei ribonucleotide reductase for T. brucei tryparedoxin is about 4 micrometer. The disulfide form of trypanothione is a powerful inhibitor of the tryparedoxin-mediated reaction that may represent a physiological regulation of deoxyribonucleotide synthesis by the redox state of the cell. The trypanothione/tryparedoxin system is a new system providing electrons for a class I ribonucleotide reductase, in addition to the well known thioredoxin and glutaredoxin systems described in other organisms.
Assuntos
DNA/metabolismo , Glutationa/metabolismo , Ribonucleotídeo Redutases/metabolismo , Espermidina/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Glutationa/análogos & derivados , Proteínas de Protozoários/metabolismo , Espermidina/análogos & derivados , Especificidade por SubstratoRESUMO
(2,2':6',2"-terpyridine)platinum(II) complexes possess pronounced cytostatic activities against trypanosomes and leishmania. As shown here, the complexes are irreversible inhibitors of trypanothione reductase (TR) from Trypanosoma cruzi, the causative agent of Chagas' disease. The most effective derivatives are the (4'-chloro-2, 2':6',2"-terpyridine)platinum(II) ammine and the (4-picoline)(4'-p-bromophenyl-2,2':6',2" -terpyridine)platinum(II) complexes which in the presence of NADPH inhibit TR with second-order rate constants of about 1.3 x 10(4) M(-1) s(-1). The modified enzyme species possess increased oxidase activities. The inhibition is not reversed upon dialysis or treatment with low-molecular-mass thiols. Kinetic and spectroscopic data suggest that Cys52 in the active site has been specifically altered. Inhibition of this key enzyme of parasite thiol metabolism probably contributes to the antitrypanosomal activity of the compounds. In contrast to the parasite enzyme, most (terpyridine)platinum complexes interact only reversibly with human glutathione reductase and an initial inhibition is completely abolished during the course of the assay.
Assuntos
2,2'-Dipiridil/síntese química , Inibidores Enzimáticos/síntese química , Glutationa Redutase/antagonistas & inibidores , NADH NADPH Oxirredutases/antagonistas & inibidores , Compostos Organoplatínicos/síntese química , Tripanossomicidas/síntese química , Trypanosoma cruzi/química , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Animais , Diálise , Inibidores Enzimáticos/química , Glutationa Redutase/química , Humanos , Cinética , Ligantes , NADH NADPH Oxirredutases/química , Compostos Organoplatínicos/química , Oxirredução , Espectrofotometria , Compostos de Sulfidrila/química , Tripanossomicidas/químicaRESUMO
Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi, the causative agent of Chagas' disease, was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. LADH lipoamide reductase and diaphorase activities decreased as a function of incubation time and composition of the MPO/H2O2/halide system, a transient increase preceding the loss of diaphorase activity. Iodide, bromide, thiocyanide and chloride were effective components of MPO/H2O2 or MPO/NADH systems. Catalase prevented LADH inactivation by the MPO/NADH/halide systems in agreement with H2O2 production by NADH-supplemented LADH. Thiol compounds (L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine) and Captopril prevented LADH inactivation by the MPO/H2O2/NaCl system and by NaOCl, thus supporting HOCl as agent of the MPO/H2O2/NaCl system. MPO/H2O2/NaNO2 and MPO/NADH/NaNO2 inactivated LADH, the reaction being prevented by MPO inhibitors and thiol compounds. T. cruzi LADH was affected by MPO-dependent systems like myocardial LADH, allowance being made for the variation of the diaphorase activity and the greater sensitivity of the T. cruzi enzyme to MPO/H2O2/halide systems.
Assuntos
Di-Hidrolipoamida Desidrogenase/antagonistas & inibidores , Ácido Hipocloroso/farmacologia , Neutrófilos/fisiologia , Nitritos/farmacologia , Peroxidase/fisiologia , Proteínas de Protozoários/antagonistas & inibidores , Explosão Respiratória , Trypanosoma cruzi/enzimologia , Acetilcisteína/farmacologia , Animais , Brometos/farmacologia , Captopril/farmacologia , Catalase/farmacologia , Cisteína/farmacologia , Citotoxicidade Imunológica , Glutationa/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Cinética , Miocárdio/enzimologia , NAD/metabolismo , Neutrófilos/enzimologia , Oxirredução , Penicilamina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Cloreto de Sódio/farmacologia , Compostos de Sódio/farmacologia , Compostos de Sulfidrila/farmacologia , Ácido Tióctico/análogos & derivados , Ácido Tióctico/metabolismo , Triptofano/farmacologia , Tirosina/farmacologiaRESUMO
Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi, the causative agent of Chagas' disease, was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. LADH lipoamide reductase and diaphorase activities decreased as a function of incubation time and composition of the MPO/H2O2/halide system, a transient increase preceding the loss of diaphorase activity. Iodide, bromide, thiocyanide and chloride were effective components of MPO/H2O2 or MPO/NADH systems. Catalase prevented LADH inactivation by the MPO/NADH/halide systems in agreement with H2O2 production by NADH-supplemented LADH. Thiol compounds (L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine) and Captopril prevented LADH inactivation by the MPO/H2O2/NaCl system and by NaOCl, thus supporting HOCl as agent of the MPO/H2O2/NaCl system. MPO/H2O2/NaNO2 and MPO/NADH/NaNO2 inactivated LADH, the reaction being prevented by MPO inhibitors and thiol compounds. T. cruzi LADH was affected by MPO-dependent systems like myocardial LADH, allowance being made for the variation of the diaphorase activity and the greater sensitivity of the T. cruzi enzyme to MPO/H2O2/halide systems.
Assuntos
Animais , Humanos , Ácido Hipocloroso/farmacologia , Di-Hidrolipoamida Desidrogenase , Neutrófilos/fisiologia , Nitritos , Peroxidase , Proteínas de Protozoários/antagonistas & inibidores , Explosão Respiratória , Trypanosoma cruzi , Acetilcisteína/farmacologia , Ácido Tióctico/análogos & derivados , Ácido Tióctico/metabolismo , Brometos , Captopril , Catalase , Cisteína/farmacologia , Cloreto de Sódio/farmacologia , Compostos de Sódio/farmacologia , Citotoxicidade Imunológica , Espécies Reativas de Oxigênio/metabolismo , Glutationa , Glicina , Cinética , Miocárdio , NAD , Neutrófilos/enzimologia , Oxirredução , Penicilamina , Peróxido de Hidrogênio/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Compostos de Sulfidrila , Triptofano , TirosinaRESUMO
We have examined the occurrence of the R1 and R2 subunits of ribonucleotide reductase during the life cycle of Trypanosoma brucei. Whereas the R1 protein is present throughout the life cycle, the R2 protein is not found in cell cycle-arrested short stumpy trypanosomes. RT-PCR/hybridization analysis revealed almost equal amounts of the R1 and R2 mRNAs in all life cycle stages of the parasite. The data indicate that ribonucleotide reductase of African trypanosomes is developmentally controlled by post-transcriptional regulation of the R2 subunit.
Assuntos
Estágios do Ciclo de Vida/genética , Ribonucleotídeo Redutases/genética , Trypanosoma brucei brucei/enzimologia , Animais , Western Blotting , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Camundongos , Camundongos Endogâmicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleotídeo Redutases/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. With MPO/H2O2/NaCl, LADH lipoamide reductase and diaphorase activities significantly decreased as a function of incubation time. Iodide, bromide, thiocyanide and chloride effectively supplemented the MPO/H2O2 system, KI and NaCl being the most and the least effective supplements, respectively. LADH inactivation by MPO/H2O2/NaCl and by NaOCl was similarly prevented by thiol compounds such as GSH, L-cysteine, N-acetylcysteine, penicillamine and N-(2-mercaptopropionyl-glycine) in agreement with the role of HOCI in LADH inactivation by MPO/H2O2/NaCl. LADH was also inactivated by MPO/NADH/halide, MPO/H2O2/NaNO2 and MPO/NADH/NaNO2 systems. Catalase prevented the action of the NADH-dependent systems, thus supporting H2O2 production by NADH-supplemented LADH. MPO inhibitors (4-aminobenzoic acid hydrazide, and isoniazid), GSH, L-cysteine, L-methionine and L-tryptophan prevented LADH inactivation by MPO/H2O2/NaNO2. Other MPO systems inactivating LADH were (a) MPO/H2O2/chlorpromazine; (b) MPO/H2O2/monophenolic systems, including L-tyrosine, serotonin and acetaminophen and (c) MPO/H2O2/di- and polyphenolic systems, including norepinephrine, catechol, nordihydroguaiaretic acid, caffeic acid, quercetin and catechin. Comparison of the above effects and those previously reported with pig myocardial LADH indicates that both enzymes were similarly affected by the MPO-dependent systems, allowance being made for T. cruzi LADH diaphorase inactivation and the greater sensitivity of its LADH lipoamide reductase activity towards the MPO/H2O2/NaCl system and NaOCl.
Assuntos
Di-Hidrolipoamida Desidrogenase/antagonistas & inibidores , Peroxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Técnicas In Vitro , Peroxidase/farmacologia , Fenol/metabolismo , Fenol/farmacologia , Fenotiazinas/metabolismo , Fenotiazinas/farmacologia , Sais/metabolismo , Sais/farmacologia , Nitrito de Sódio/metabolismo , Nitrito de Sódio/farmacologiaRESUMO
Trypanosomes and Leishmania, the causative agents of several tropical diseases, lack the glutathione/glutathione reductase system but have trypanothione/trypanothione reductase instead. The uniqueness of this thiol metabolism and the failure to detect thioredoxin reductases in these parasites have led to the suggestion that these protozoa lack a thioredoxin system. As presented here, this is not the case. A gene encoding thioredoxin has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The single copy gene, which encodes a protein of 107 amino acid residues, is expressed in all developmental stages of the parasite. The deduced protein sequence is 56% identical with a putative thioredoxin revealed by the genome project of Leishmania major. The relationship to other thioredoxins is low. T. brucei thioredoxin is unusual in having a calculated pI value of 8.5. The gene has been overexpressed in Escherichia coli. The recombinant protein is a substrate of human thioredoxin reductase with a K(m) value of 6 microM but is not reduced by trypanothione reductase. T. brucei thioredoxin catalyzes the reduction of insulin by dithioerythritol, and functions as an electron donor for T. brucei ribonucleotide reductase. The parasite protein is the first classical thioredoxin of the order Kinetoplastida characterized so far.
Assuntos
Genes de Protozoários , Tiorredoxinas/genética , Trypanosoma brucei brucei/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Ditiotreitol/metabolismo , Dosagem de Genes , Humanos , Insulina/metabolismo , Dados de Sequência Molecular , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Proteínas Recombinantes , Ribonucleotídeo Redutases/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
In Kinetoplastida, trypanothione and trypanothione reductase (TRYR) provide an intracellular reducing environment, substituting for the glutathione-glutathione reductase system found in most other organisms. To investigate the physiological role of TRYR in Trypanosoma brucei, we generated cells containing just one trypanothione reductase gene, TRYR, which was under the control of a tetracycline-inducible promoter. This enabled us to regulate TRYR activity in the cells from less than 1% to 400% of wild-type levels by adjusting the concentration of added tetracycline. In normal growth medium (which contains reducing agents), trypanosomes containing less than 10% of wild-type enzyme activity were unable to grow, although the levels of reduced trypanothione and total thiols remained constant. In media lacking reducing agents, hypersensitivity towards hydrogen peroxide (EC50 = 3.5 microM) was observed compared with the wild type (EC50 = 223 microM). The depletion of TRYR had no effect on susceptibility to melarsen oxide. The infectivity and virulence of the parasites in mice was dependent upon tetracycline-regulated TRYR activity: if the trypanosomes were injected into mice in the absence of tetracycline, no infection was detectable; and when tetracycline was withdrawn from previously infected animals, the parasitaemia was suppressed.
Assuntos
NADH NADPH Oxirredutases/metabolismo , Estresse Oxidativo , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/patogenicidade , Animais , Antibacterianos/farmacologia , Antiprotozoários/farmacologia , Arsenicais/farmacologia , Ciclo Celular/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Doxiciclina/farmacologia , Inibidores Enzimáticos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , NADH NADPH Oxirredutases/efeitos dos fármacos , NADH NADPH Oxirredutases/genética , Regiões Promotoras Genéticas , Compostos de Sulfidrila/metabolismo , Tetraciclina/farmacologia , Tripanossomíase Africana/tratamento farmacológico , VirulênciaRESUMO
Lipoamide dehydrogenase (LipDH), trypanothione reductase (TR), and glutathione reductase (GR) catalyze the NAD(P)H-dependent reduction of disulfide substrates. TR occurs exclusively in trypanosomatids which lack a GR. Besides their physiological reactions, the flavoenzymes catalyze the single-electron reduction of nitrofurans with the concomitant generation of superoxide anions. Here, we report on the interaction of clinically used antimicrobial nitrofurans with LipDH and TR from Trypanosoma cruzi, the causative agent of Chagas' disease (South American trypanosomiasis), in comparison to mammalian LipDH and GR. The compounds were studied as inhibitors and as subversive substrates of the enzymes. None of the nitrofurans inhibited LipDH, although they did interfere with the disulfide reduction of TR and GR. When the compounds were studied as substrates, T. cruzi LipDH showed a high rate of nitrofuran reduction and was even more efficient than its mammalian counterpart. Several derivatives were also effective subversive substrates of TR, but the respective reaction with human GR was negligible. Nifuroxazide, nifuroxime, and nifurprazine proved to be the most promising derivatives since they were redox-cycled by both T. cruzi LipDH and TR and had pronounced antiparasitic effects in cultures of T. cruzi and Trypanosoma brucei. The results suggest that those nitrofuran derivatives which interact with both parasite flavoenzymes should be revisited as trypanocidal drugs.
Assuntos
Di-Hidrolipoamida Desidrogenase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , NADH NADPH Oxirredutases/antagonistas & inibidores , Nitrofuranos/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Feminino , Humanos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Naftoquinonas/química , Naftoquinonas/farmacologia , Nitrofuranos/química , Oxirredução , Oxirredutases/antagonistas & inibidores , Relação Estrutura-Atividade , Especificidade por Substrato , Trypanosoma cruzi/enzimologiaRESUMO
The potential for chemotherapeutic exploitation of thiol metabolism in parasitic protozoa is reviewed here by Luise Krauth-Siegel and Graham Coombs. The review is based largely on discussions held at a meeting of the COST B9 Action entitled 'Chemotherapy of Protozoal Infections'*. The major questions posed were: which enzymes are the best to target; what further information is required to allow their use for rational drug development; and how can this be achieved most efficiently? Not surprisingly, only partial answers could be obtained in many cases, but the interactive discussion between the multidisciplinary group of participants provided thought-provoking ideas and will help direct future research.
Assuntos
Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Leishmania/enzimologia , NADH NADPH Oxirredutases/antagonistas & inibidores , Plasmodium/enzimologia , Compostos de Sulfidrila/metabolismo , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Animais , Avaliação Pré-Clínica de Medicamentos , Glutationa/análogos & derivados , Glutationa/biossíntese , Glutationa/metabolismo , Glutationa/fisiologia , Leishmania/efeitos dos fármacos , Leishmania/metabolismo , NADH NADPH Oxirredutases/metabolismo , Plasmodium/efeitos dos fármacos , Plasmodium/metabolismo , Estudos Prospectivos , Espermidina/análogos & derivados , Espermidina/biossíntese , Espermidina/metabolismo , Espermidina/fisiologia , Tiorredoxina Dissulfeto Redutase/metabolismoRESUMO
The nitroimidazole derivative Megazol is a highly active compound used against several strains of Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanomiasis). With the aim of gaining an insight into the probable mode of action, the interaction of Megazol with different redox enzymes was studied in comparison to that of Nifurtimox and Metronidazole. The three nitroaromatic compounds are reduced by L-lactate cytochrome c-reductase, adrenodoxin reductase, and NADPH:cytochrome P-450 reductase (EC 1.6.2.4), the efficiencies of the enzymatic reductions being roughly related to the reduction potentials of these pseudo-substrates. As the enzyme responsible for the reduction of Megazol within the parasite has not yet been identified, the nitroimidazole was assayed with T. cruzi lipoamide dehydrogenase and trypanothione reductase. Megazol did not inhibit the physiological reactions but proved to be a weak substrate of both flavoenzymes. The single electron reduction of the compound by NADPH:cytochrome P-450 reductase, by rat liver as well as by trypanosome microsomes was confirmed by ESR experiments. As shown here, Megazol interferes with the oxygen metabolism of the parasite, but its extra activity when compared to Nifurtimox may be related to other features not yet identified.
Assuntos
Ferredoxina-NADP Redutase/metabolismo , Metronidazol/farmacocinética , NADH Desidrogenase/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Nifurtimox/farmacocinética , Nitroimidazóis/farmacocinética , Tiadiazóis/farmacocinética , Animais , Biotransformação , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Espectroscopia de Ressonância de Spin Eletrônica , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase (Citocromo) , Estrutura Molecular , Oxirredução , Ratos , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), a garlic-derived natural compound, is a covalent inhibitor as well as a substrate of human glutathione reductase (GR) and Trypanosoma cruzi trypanothione reductase (TR). The 2.1-A resolution crystal structure of GR inhibited by (E)-ajoene revealed a mixed disulfide between the active site Cys58 and the CH2=CH-CH2-SO-CH2-CH=CH-S moiety of ajoene. The modified enzyme has a markedly increased oxidase activity when compared to free GR. GR reduces (Z)-ajoene with a kcat/Km of 6.8 x 10(3) M-1 s-1 yielding 4,5,9-trithiadodeca-1, 6,11-triene (deoxyajoene) and 4,8,9,13-tetrathiahexadeca-1,6,10, 15-tetraene as stable reaction products. The reaction leads also to the formation of single-electron reduced products and concomitantly superoxide anion radicals as shown by coupling the reaction to the reduction of cytochrome c. The interactions between the flavoenzymes and ajoene are expected to increase the oxidative stress of the respective cell. The antiparasitic and cytostatic actions of ajoene may at least in part be due to the multiple effects on key enzymes of the antioxidant thiol metabolism.
Assuntos
Dissulfetos/farmacologia , Inibidores Enzimáticos/farmacologia , Glutationa Redutase/antagonistas & inibidores , NADH NADPH Oxirredutases/antagonistas & inibidores , Extratos Vegetais/farmacologia , Trypanosoma cruzi/enzimologia , Animais , Sítios de Ligação , Catálise , Cristalografia por Raios X , Dissulfetos/química , Inibidores Enzimáticos/química , Glutationa Redutase/metabolismo , Humanos , Cinética , Espectroscopia de Ressonância Magnética , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Extratos Vegetais/química , SulfóxidosRESUMO
Series of 9-amino and 9-thioacridines have been synthesized and studied as inhibitors of trypanothione reductase (TR) from Trypanosoma cruzi, the causative agent of Chagas' disease. The compounds are structural analogues of the acridine drug mepacrine (quinacrine), which is a competitive inhibitor of the parasite enzyme, but not of human glutathione reductase, the closest related host enzyme. The 9-aminoacridines yielded apparent K(i) values for competitive inhibition between 5 and 43 microM. The most effective inhibitors were those with the methoxy and chlorine substituents of mepacrine and NH(2) or NHCH(CH(3))(CH(2))(4)N(Et)(2) at C9. Detailed kinetic analyses revealed that in the case of 9-aminoacridines more than one inhibitor molecule can bind to the enzyme. In contrast, the 9-thioacridine derivatives inhibit TR with mixed-type kinetics. The kinetic data are discussed in light of the three-dimensional structure of the TR-mepacrine complex. The conclusion that structurally very similar acridine compounds can give rise to completely different inhibition patterns renders modelling studies and quantitative structure-activity relationships difficult.
Assuntos
Acridinas/farmacologia , Inibidores Enzimáticos/farmacologia , NADH NADPH Oxirredutases/antagonistas & inibidores , Trypanosoma cruzi/efeitos dos fármacos , Acridinas/química , Animais , Inibidores Enzimáticos/química , Cinética , Espectroscopia de Ressonância Magnética , Relação Estrutura-Atividade , Trypanosoma cruzi/enzimologiaRESUMO
Tyr114 and Tyr197 are highly conserved residues in the active site of human glutathione reductase, Tyr114 in the glutathione disulfide (GSSG) binding site and Tyr197 in the NADPH site. Mutation of either residue has profound effects on catalysis. Y197S and Y114L have 17% and 14% the activity of the wild-type enzyme, respectively. Mutation of Tyr197, in the NADPH site, leads to a decrease in Km for GSSG, and mutation of Tyr114, in the GSSG site, leads to a decrease in Km for NADPH. This behavior is predicted for enzymes operating by a ping-pong mechanism where both half-reactions partially limit turnover. Titration of the wild-type enzyme or Y114L with NADPH proceeds in two phases, Eox to EH2 and EH2 to EH2-NADPH. In contrast, Y197S reacts monophasically, showing that excess NADPH fails to enhance the absorbance of the thiolate-FAD charge-transfer complex, the predominant EH2 form of glutathione reductase. The reductive half-reactions of the wild-type enzyme and of Y114L are similar; FAD reduction is fast (approximately 500 s-1 at 4 degreesC) and thiolate-FAD charge-transfer complex formation has a rate of 100 s-1. In Y197S, these rates are only 78 and 5 s-1, respectively. The oxidative half-reaction, the rate of reoxidation of EH2 by GSSG, of the wild-type enzyme is approximately 4-fold faster than that of Y114L. These results are consistent with Tyr197 serving as a gate in the binding of NADPH, and they indicate that Tyr114 assists the acid catalyst His467'.
Assuntos
Glutationa Redutase/metabolismo , Tirosina/metabolismo , Substituição de Aminoácidos/genética , Sítios de Ligação , Catálise , Transporte de Elétrons , Ativação Enzimática/genética , Polarização de Fluorescência , Glutationa/análogos & derivados , Glutationa/farmacologia , Glutationa Redutase/antagonistas & inibidores , Glutationa Redutase/genética , Humanos , Leucina/genética , NADP/metabolismo , Oxirredução , Serina/genética , Espectrometria de Fluorescência , Tirosina/genéticaRESUMO
A gene has been cloned from Trypanosoma brucei which encodes a protein of 144 amino acid residues containing the thioredoxin-like motif WCPPCR. Overexpression of the gene in E. coli resulted in 4 mg pure protein from 100 ml bacterial cell culture. Recombinant T. brucei tryparedoxin acts as a thiol-disulfide oxidoreductase. It is spontaneously reduced by trypanothione. This dithiol, exclusively found in parasitic protozoa, also reduces E. coli glutaredoxin but not thioredoxin. The trypanothione/tryparedoxin couple is an effective reductant of T. brucei ribonucleotide reductase. Like thioredoxins it has a poor GSH:disulfide transhydrogenase activity. The catalytic properties of tryparedoxin are intermediate between those of classical thioredoxins and glutaredoxins which indicates that these parasite proteins may form a new class of thiol-disulfide oxidoreductases.
Assuntos
Tiorredoxinas/metabolismo , Trypanosoma brucei brucei/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Catálise , Clonagem Molecular , DNA Complementar , Escherichia coli/genética , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMO
The gene of an NADP+-specific glutamate dehydrogenase was cloned from Plasmodium falciparum, the causative agent of tropical malaria. Southern-blot analysis indicates a single-copy gene. The gene encodes a protein with 470 residues which has 50% of all residues identical with those of the glutamate dehydrogenases from other low eukaryotes and eubacteria. In contrast, the sequence identity with the human enzyme is marginal, which underlines the long evolutionary distance between parasite and host. The gene was overexpressed in Escherichia coli. The kinetic properties of the recombinant enzyme are in good agreement with those of the authentic enzyme. The parasite enzyme is inhibited by D-glutamate and glutarate, but not by chloroquine. Like other coenzyme-specific glutamate dehydrogenases, but in contrast to the dual-specific mammalian enzymes, the P. falciparum enzyme is not affected by GTP and ADP. The physical and chemical properties of the protein are in accordance with the cytosol being the major localization. The gene does not encode a cleavable mitochondrial presequence and the Mr of the recombinant protein and the protein isolated from the parasite are indistinguishable on SDS/PAGE. Western-blot analysis of stage-specific parasites shows that glutamate dehydrogenase is present in all intraerythrocytic stages. The signal increased continuously from rings, early trophozoites to late trophozoites and decreased slightly in the segmenter stage. Glutamate dehydrogenase, suggested to be the major source of NADPH in the parasite, is an attractive target molecule for the rational development of new antimalarial drugs.
Assuntos
Biomarcadores/química , Regulação Enzimológica da Expressão Gênica/genética , Glutamato Desidrogenase/genética , Plasmodium falciparum/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cloroquina/farmacologia , Clonagem Molecular , Inibidores Enzimáticos/farmacologia , Eritrócitos/microbiologia , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Ribonucleotide reductase (RR) is an attractive drug target molecule. The gene of the R2 protein of Trypanosoma brucei RR (nrd B) has been cloned. It encodes a protein of 337 residues which shows about 60% identity with other eukaryotic R2 proteins. All residues which bind the iron center, the tyrosyl radical or are supposed to participate in the radical transfer are conserved in the trypanosomal protein sequence. Overexpression of the gene in E. coli resulted in 2-5 mg pure R2 protein from 100 ml bacterial cell culture. Northern blot analysis revealed a transcript of 1.85 kb in bloodstream and procyclic forms of the parasite.