Trypanosoma Cruzi Trypanothione Reductase Research Articles

In silico identification and evaluation of new Trypanosoma cruzi trypanothione reductase (TcTR) inhibitors obtained from natural products database of the Bahia semi-arid region (NatProDB)

Trypanosoma cruzi Trypanothione Reductase (TcTR) is one of the therapeutic targets studied in the development of new drugs against Chagas' disease. Due to its biodiversity, Brazil has several compounds of natural origin that were not yet properly explored in drug discovery. Therefore, we employed the Virtual Screening against TcTR aiming to discover new inhibitors from the Natural Products Database of the Bahia Semi-Arid region (NatProDB). This database has a wide chemical diversity favoring the discovery of new chemical entities. Subsequently, we analyzed the best docking conformations using self-organizing maps (AuPosSOM) aiming to verify their interaction sites at TcTR. Finally, the Pred-hERG, the Aggregator Advisor, the FAF-DRUGS and the pkCSM results allowed us to evaluate, respectively, the cardiotoxicity, aggregation capacity, presence of false positives (PAINS) and its toxicity. Thus, we selected three molecules that could be tested in in vitro assays in the hope that the computational results reported here would favor the development of new anti-chagasic drugs.

Crassiflorone derivatives that inhibit Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH) and Trypanosoma cruzi trypanothione reductase (TcTR) and display trypanocidal activity

Crassiflorone is a natural product with anti-mycobacterial and anti-gonorrhoeal properties, isolated from the stem bark of the African ebony tree Diospyros crassiflora. We noticed that its pentacyclic core possesses structural resemblance to the quinone-coumarin hybrid 3, which we reported to exhibit a dual-targeted inhibitory profile towards Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH) and Trypanosoma cruzi trypanothione reductase (TcTR). Following this basic idea, we synthesized a small library of crassiflorone derivatives 15-23 and investigated their potential as anti-trypanosomatid agents. 19 is the only compound of the series showing a balanced dual profile at 10 μM (% inhibitionTbGAPDH = 64% and % inhibitionTcTR = 65%).In phenotypic assay, the most active compounds were 18 and 21, which at 5 μM inhibited Tb bloodstream-form growth by 29% and 38%, respectively. Notably, all the newly synthesized compounds at 10 μM did not affect viability and the status of mitochondria in human A549 and 786-O cell lines, respectively. However, further optimization that addresses metabolic liabilities including solubility, as well as cytochromes P450 (CYP1A2, CYP2C9, CYP2C19, and CYP2D6) inhibition, is required before this class of natural product-derived compounds can be further progressed.

Docking-based screening of natural product database in quest for dual site inhibitors of Trypanosoma cruzi trypanothione reductase (TcTR)

Chagas disease is caused by the protozoan Trypanosoma cruzi prevalent in the endemic regions of Latin American countries. With the existing drugs having severe toxic side effects, neither a vaccine nor an effective treatment is available for this chronic disease till date. In a pursuit to develop novel drug candidates against Chagas disease, we looked at the vast reservoir of mother nature and built an in-house library of 110 natural plant metabolites from medicinal plants having proven trypanocidal activities. This battery of compounds was examined against T. cruzi trypanothione reductase (1GXF) via computational methods using Autodock. 18-acetoxy-5,6-deoxy-5-withenolide (B.E. = −11.05 kcal/mol) and Sarachine (B.E. = −10.86 kcal/mol) turned out to be the top scorers in this study. Further pharmacophore detection of the high scorers resulted in a hybrid model considering the structural and spatial characteristics of the molecules. This can be further used to generate promising drug candidates aligned for clinical development in the drug design pipeline.

Novel aryl β-aminocarbonyl derivatives as inhibitors of Trypanosoma cruzi trypanothione reductase: binding mode revised by docking and GRIND2-based 3D-QSAR procedures

Trypanothione reductase has long been investigated as a promising target for chemotherapeutic intervention in Chagas disease, since it is an enzyme of a unique metabolic pathway that is exclusively present in the pathogen but not in the human host, which has the analog Glutathione reductase. In spite of the present data-set includes a small number of compounds, a combined use of flexible docking, pharmacophore perception, ligand binding site prediction, and Grid-Independent Descriptors GRIND2-based 3D-Quantitative Structure-Activity Relationships (QSAR) procedures allowed us to rationalize the different biological activities of a series of 11 aryl β-aminocarbonyl derivatives, which are inhibitors of Trypanosoma cruzi trypanothione reductase (TcTR). Three QSAR models were built and validated using different alignments, which are based on docking with the TcTR crystal structure, pharmacophore, and molecular interaction fields. The high statistical significance of the models thus obtained assures the robustness of this second generation of GRIND descriptors here used, which were able to detect the most important residues of such enzyme for binding the aryl β-aminocarbonyl derivatives, besides to rationalize distances among them. Finally, a revised binding mode has been proposed for our inhibitors and independently supported by the different methodologies here used, allowing further optimization of the lead compounds with such combined structure- and ligand-based approaches in the fight against the Chagas disease.

Synthesis and evaluation of substrate analogue inhibitors of trypanothione reductase

Trypanothione reductase (TR) is found in the trypanosomatid parasites, where it catalyses the NADPH-dependent reduction of the glutathione analogue, trypanothione, and is a key player in the parasite’s defenses against oxidative stress. TR is a promising target for the development of antitrypanosomal drugs; here, we report our synthesis and evaluation of compounds 3–5 as low micromolar Trypanosoma cruzi TR inhibitors. Although 4 and 5 were designed as potential irreversible inhibitors, these compounds, as well as 3, displayed reversible competitive inhibition. Compound 3 proved to be the most potent inhibitor, with a Ki = 2 µM.

In vitro activity of hypnophilin from Lentinus strigosus: a potential prototype for Chagas disease and leishmaniasis chemotherapy

Hypnophilin and panepoxydone, terpenoids isolated from Lentinus strigosus, have significant inhibitory activity on Trypanosoma cruzi trypanothione reductase (TR). Although they have similar TR inhibitory activity at 10 μg/mL (40.3 μM and 47.6 μM for hypnophilin and panepoxydone, respectively; ~100%), hypnophilin has a slightly greater inhibitory activity (~71%) on T. cruzi amastigote (AMA) growth in vitro as well as on in vitro phytohemagglutinin (PHA)-induced peripheral blood mononuclear (PBMC) proliferation (~70%) compared to panepoxydone (69% AMA inhibition and 91% PBMC inhibition). Hypnophilin and panepoxydone at 1.25 μg/mL had 67% inhibitory activity onLeishmania (Leishmania) amazonensis amastigote-like (AMA-like) growth in vitro. The panepoxydone activity was accompanied by a significant inhibitory effect on PHA-induced PBMC proliferation, suggesting a cytotoxic action. Moreover, incubation of human PBMC with panepoxydone reduced the percentage of CD16(+) and CD14(+) cells and down-regulated CD19(+), CD4(+) and CD8(+) cells, while hypnophilin did not alter any of the phenotypes analyzed. These data indicate that hypnophilin may be considered to be a prototype for the design of drugs for the chemotherapy of diseases caused by Trypanosomatidae.

Molecular docking of a series of peptidomimetics in the trypanothione binding site of T. cruzi Trypanothione Reductase

Chagas’ disease (CD) has been responsible for many deaths and disabilities mainly in South America. Currently, 40 million people are at risk of acquiring this disease and, existing therapies are still unsatisfactory, presenting harsh side effects. Therefore, the development of new chemical entities to reverse this state is critical. A series of peptidomimetics, developed by Mc Kie et al. (2001) [11], showed a reversible and competitive inhibition against Trypanosoma cruzi Trypanothione Reductase (TR). These inhibitors may be used as basis of lead compounds in the design of new drug candidates for the treatment of CD. In this work, we have docked this series of peptidomimetics into the TR binding site, using the FlexX algorithm as implemented in the Sybyl program, in order to access the binding mode of this class of compounds in the target enzyme.

Chemotherapy of chronic indeterminate Chagas disease: a novel approach to treatment

Treatment of Chagas disease is a controversial issue because the available drugs are highly toxic. Clomipramine is a tricyclic antidepressant drug that inhibits Trypanosoma cruzi's trypanothione reductase, provoking the death of the parasite and preventing the cardiac damage when used for the treatment of acutely infected mice. Here, we studied the effectiveness of clomipramine (5 mg/kg/day for one month) as chemotherapy for T. cruzi-infected mice in the chronic indeterminate stage of the infection. The animals were analyzed in the cardiac chronic phase. Survival of treated animals was 84% while for the untreated ones was 40%; most of the animals presented electrocardiographic abnormalities. Affinity and density of cardiac beta receptors from infected and treated mice were similar to those in the indeterminate phase, showing that clomipramine treatment stopped the increment of functional alterations provoked by the infection, while untreated mice presented affinity and density significantly diminished. Hearts from infected and untreated mice in the chronic stage presented mononuclear cells, necrosis and fiber dissolution while hearts from treated animals showed only isolated inflammatory infiltrates. Present results demonstrate that clomipramine used in the chronic indeterminate phase of the T. cruzi infection modified the natural evolution of the chagasic cardiopathy.

A potent trypanocidal component from the fungus Lentinus strigosus inhibits trypanothione reductase and modulates PBMC proliferation

The fungus Lentinus strigosus (Pegler 1983) (Polyporaceae, basidiomycete) was selected in a screen for inhibitory activity on Trypanosoma cruzi trypanothione reductase (TR). The crude extract of L. strigosus was able to completely inhibit TR at 20 microg/ml. Two triquinane sesquiterpenoids (dihydrohypnophilin and hypnophilin), in addition to two panepoxydol derivatives (neopanepoxydol and panepoxydone), were isolated using a bioassay-guided fractionation protocol. Hypnophilin and panepoxydone displayed IC50 values of 0.8 and 38.9 microM in the TR assay, respectively, while the other two compounds were inactive. The activity of hypnophilin was confirmed in a secondary assay with the intracellular amastigote forms of T. cruzi, in which it presented an IC50 value of 2.5 micro M. Quantitative flow cytometry experiments demonstrated that hypnophilin at 4 microM also reduced the proliferation of human peripheral blood monocluear cells (PBMC) stimulated with phytohemaglutinin, without any apparent interference on the viability of lymphocytes and monocytes. As the host immune response plays a pivotal role in the adverse events triggered by antigen release during treatment with trypanocidal drugs, the ability of hypnophilin to kill the intracellular forms of T. cruzi while modulating human PBMC proliferation suggests that this terpenoid may be a promising prototype for the development of new chemotherapeutical agents for Chagas disease.

Cytotoxic interactions of methylene blue with trypanosomatid-specific disulfide reductases and their dithiol products

Methylene blue (MB) is known to have trypanocidal activity. We tested the interactions of MB with a number of trypanosomatid-specific molecules of the antioxidant metabolism. At pH 7, trypanothione and other (di)thiols were oxidized to disulfides by the phenothiazine drug. MB inhibited Trypanosoma cruzi trypanothione reductase (TR) ( K i = 1.9 μM), and served as a significant subversive substrate of this enzyme ( K M = 30 μM, k cat = 4.9 s −1). With lipoamide dehydrogenase, the second thiol-generating flavoenzyme of T. cruzi, the catalytic efficiency for MB reduction was found to be almost 10 6 M −1 s −1. When the system MB-enzyme-molecular oxygen acts as a NAD(P)H-driven redox cycler, a reactive oxygen species, H 2O 2 or superoxide, is produced in each cycle. Since MB is an affordable, available, and accessible drug it might be tested – alone or in drug combinations – against trypanosomatid-caused diseases of animal and man.

Glutathionylation of Trypanosomal Thiol Redox Proteins

Trypanosomatids, the causative agents of several tropical diseases, lack glutathione reductase and thioredoxin reductase but have a trypanothione reductase instead. The main low molecular weight thiols are trypanothione (N(1),N(8)-bis-(glutathionyl)spermidine) and glutathionyl-spermidine, but the parasites also contain free glutathione. To elucidate whether trypanosomes employ S-thiolation for regulatory or protection purposes, six recombinant parasite thiol redox proteins were studied by ESI-MS and MALDI-TOF-MS for their ability to form mixed disulfides with glutathione or glutathionylspermidine. Trypanosoma brucei mono-Cys-glutaredoxin 1 is specifically thiolated at Cys(181). Thiolation of this residue induced formation of an intramolecular disulfide bridge with the putative active site Cys(104). This contrasts with mono-Cys-glutaredoxins from other sources that have been reported to be glutathionylated at the active site cysteine. Both disulfide forms of the T. brucei protein were reduced by tryparedoxin and trypanothione, whereas glutathione cleaved only the protein disulfide. In the glutathione peroxidase-type tryparedoxin peroxidase III of T. brucei, either Cys(47) or Cys(95) became glutathionylated but not both residues in the same protein molecule. T. brucei thioredoxin contains a third cysteine (Cys(68)) in addition to the redox active dithiol/disulfide. Treatment of the reduced protein with GSSG caused glutathionylation of Cys(68), which did not affect its capacity to catalyze reduction of insulin disulfide. Reduced T. brucei tryparedoxin possesses only the redox active Cys(32)-Cys(35) couple, which upon reaction with GSSG formed a disulfide. Also glyoxalase II and Trypanosoma cruzi trypanothione reductase were not sensitive to thiolation at physiological GSSG concentrations.

Arylfurans as potential Trypanosoma cruzi trypanothione reductase inhibitors

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 microM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60% at 20 microg/ml (59 and 90 microM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 microg/ml (0.6-1.5 mM).

Vanadate as a Futile, Superoxide Ion-producing Substrate of Trypanothione Reductase from Trypanosoma cruzi

Abstract Recombinant Trypanosoma cruzi trypanothione reductase can oxidize NADPH in a futile cycle in the presence of vanadate ion as a result of the generation of reactive oxygen and other radical species from the active site dithiol and the NADPH. This process is inhibited by competitive inhibitors of trypanothione reductase catalyzed reduction of trypanothione disulfide.

Trypanosoma cruzi trypanothione reductase inhibitors: phenothiazines and related compounds modify experimental Chagas' disease evolution.

Chagas' disease affects about 18 million people and 25% of the population of Latin America is at risk of acquiring Chagas' disease. The chemotherapy of Chagas' disease is still an open field and remains as an unsolved problem. Nifurtimox and benznidazole are currently used to treat this disease, however, both drugs have high toxicity and are mutagenic with the result that the patients frequently fail to follow treatment. T. cruzi enzimes such as trypanothione reductase, represent potential drug targets because they play an essential role in the life of this organism. This enzyme has been isolated, purified and studied by X ray crystallography. Phenothiazines and related compounds inhibit trypanothione reductase and a specially favoured fit is a phenothiazine with a 2- substitued with 2- chloro and 2- trifluoromethyl with a remote hydrophobic patch. The essential phenothiazine nucleus can adopt more than one inhibitory orientation in its binding site. Phenothiazines and related compounds are drugs used in psychiatric treatments. These anti-depressants inhibit trypanothione reductase through the peroxidase/ H2O2/ system, and also exert other trypanocidal effects upon epimastigotes and tripomastigotes forms: clomipramine through an anticalmodulin action; trifluopherazine and thioridazine induced disruption of mitochondria and prometazine provoked serious cell membrane disorganization. Clomipramine and thioridazine were also effective in treatment of mice with experimental Chagas' disease, significantly modifying the natural evolution of the infection; cardiac function and survival of infected and treated animals were not different from non infected animals. Phenothiazines and related compounds are promising trypanocidal agents for treatment of Chagas' disease. Other trypanocidal agents as nifurtimox, benznidazol,Allopurinol, cystein protease inhibitors and others, are also discussed.

Trypanosoma Cruzi trypanothione reductase is inactivated by peroxidase-generated phenothiazine cationic radicals

Trypanosoma cruzi trypanothione reductase (TR) was irreversibly inhibited by peroxidase/H2O2/phenothiazine (PTZ) systems. TR inactivation depended on (a) time of incubation with the phenothiazine system; (b) the peroxidase nature and (c) the PTZ structure and concentration. With the most effective systems, TR inactivation kinetics were biphasic, with a relatively fast initial phase during which about 75% of the enzyme activity was lost, followed by a slower phase leading to total enzyme inactivation. GSH prevented TR inactivation by the peroxidase/H2O2/PTZ+· systems. Production of PTZ+· cation radicals by PTZ peroxidation was essential for TR inactivation. Horseradish peroxidase, leukocyte myeloperoxidase (MPO) and the pseudo-peroxidase myoglobin (Mb) were effective catalysts of PTZ+· production. Promazine, thioridazine, chlorpromazine, propionylpromazine prochlorperazine, perphenazine and trimeprazine were effective constituents of the HRP/H2O2/PTZ system. The presence of substituents at the PTZ nucleus position 2 exerted significant influence on PTZ activity, as shown by the different effects of 2-trifluoromethyl and 2-H or 2-chlorophenothiazines. The PTZ+· cation radicals disproportionation regenerated the non-radical PTZ molecule and produced the PTZ sulfoxide that was inactive on TR. Thiol compounds including GSH interacted with PTZ+· cation radicals transferring an electron from the sulfide anion to the PTZ+·, thus nullifying the PTZ+· biological and chemical activities.

Ajoene is an inhibitor and subversive substrate of human glutathione reductase and Trypanosoma cruzi trypanothione reductase: crystallographic, kinetic, and spectroscopic studies.

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.

New spermine and spermidine derivatives as potent inhibitors of Trypanosoma cruzi Trypanothione Reductase

Several spermine and spermidine derivatives containing 2-amino diphenylsulfide substituents were prepared and tested for their inhibiting effects on Trypanosoma cruzi trypanothione reductase. IC 50 values were assessed between 0.3 and 3 μM. Compound 32 ( K i = 0.4 μM) is the most potent TR inhibitor described so far.

Crystal structure of theTrypanosoma cruzi trypanothione reductase·mepacrine complex

The three-dimensional structure of the complex between Trypanosoma cruzi trypanothione reductase (TR) (EC 1.6.4.8) and the antiparasitic drug mepacrine (quinacrine) has been solved at 2.9 angstoms resolution. Mepacrine is a competitive inhibitor of TR but does not affect human glutathione reductase (GR), a closely related host enzyme. Of particular importance for inhibitor binding are four amino acid residues in the disulfide substrate-binding site of TR that are not conserved in human GR, namely, Glu-18 (Ala-34 in GR), Trp-21 (Arg-37), Ser-109 (Ile-113), and Met-113 (Asn-117). The acridine ring of mepacrine is fixed at the active site close to the hydrophobic wall formed by Trp-21 and Met-113. Specific pairwise interactions between functional groups of the drug and amino acid side chains include the ring nitrogen and Met-113, the chlorine atom and Trp-21, and the oxymethyl group and Ser-109. The alkylamino chain of mepacrine points into the inner region of the active site and is held in position by a solvent-mediated hydrogen bond to Glu-18. The structure of the complex shows for the first time the atomic interactions between TR and an inhibitory ligand. This is a crucial step towards the rational design of inhibitors that might be suited as drugs against Chagas' disease.

Inhibiting effects of spermidine derivatives on Trypanosoma cruzi trypanothione reductase.

Trypanothione reductase is a vital component of the antioxidant defenses of trypanosomes. This enzyme reduces trypanothione, a spermidine-glutathione conjugate. The inhibitory effects of several spermidine derivatives on the reduction of trypanothione by Trypanosoma cruzi trypanothione reductase were assessed. Spermidine derivatives containing hydrophobic aromatic substituents were found to be competitive inhibitors of trypanothione reductase. N4-acylated spermidine derivatives were less effective inhibitors than the corresponding N4-alkylated derivatives. The most effective compounds studied were N1, N8-bis(2-naphthylmethyl)spermidine and N4-(2-naphthylmethyl)spermidine, with Ki values of 9.5 and 108 microM, respectively.