ABSTRACT
American trypanosomiasis is a worldwide health problem that requires attention due to ineffective treatment options. We evaluated n-butyl and isobutyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives against trypomastigotes of the Trypanosoma cruzi strains NINOA and INC-5. An in silico analysis of the interactions of 1,4-di-N-oxide on the active site of trypanothione reductase (TR) and an enzyme inhibition study was carried out. The n-butyl series compound identified as T-150 had the best trypanocidal activity against T. cruzi trypomastigotes, with a 13% TR inhibition at 44 µM. The derivative T-147 behaved as a mixed inhibitor with Ki and Ki' inhibition constants of 11.4 and 60.8 µM, respectively. This finding is comparable to the TR inhibitor mepacrine (Ki = 19 µM).
Subject(s)
Chagas Disease , Trypanocidal Agents , Trypanosoma cruzi , Humans , Trypanocidal Agents/pharmacology , Trypanocidal Agents/chemistry , Quinoxalines/chemistry , Oxides/pharmacology , NADH, NADPH Oxidoreductases , Chagas Disease/drug therapy , Enzyme Inhibitors/chemistryABSTRACT
Herein, relationships between the structures of 1-aminoethyl-substituted chromenes and their antimalarial activities were thoroughly investigated. At first, the methyl moiety in the side chain was removed to eliminate chirality. The hydrogenation state of the benzopyran system, the position of the phenolic OH moiety, and the distance of the basic amino moiety toward both aromatic rings were varied systematically. 1-Benzopyran-5-ol 8b (IC50 = 10 nM), 1-benzopyran-7-ol 9c (IC50 = 38 nM), and the aminoalcohol 19c (IC50 = 17 nM) displayed antiplasmodial activity with IC50 values below 50 nM. To identify the mechanism of action, inhibition of three key enzymes by 9c was investigated. 9c was not able to reduce the number of Plasmodia in erythrocytes of mice. This low in vivo activity was explained by fast clearance from blood plasma combined with rapid biotransformation of 9c. Three main metabolites of 9c were identified by liquid chromatography-mass spectrometry (LC-MS) methods.
Subject(s)
Antimalarials/chemistry , Antimalarials/pharmacology , Benzopyrans/chemistry , Benzopyrans/pharmacology , Biological Products/chemistry , Plasmodium/drug effects , Alkylation , Animals , Antimalarials/chemical synthesis , Benzopyrans/chemical synthesis , Biomimetic Materials/chemical synthesis , Biomimetic Materials/chemistry , Biomimetic Materials/pharmacology , Chemistry Techniques, Synthetic , Erythrocytes/drug effects , Erythrocytes/parasitology , Kinetics , Mice , Structure-Activity RelationshipABSTRACT
Hydroperoxide reduction in African trypanosomes relies on 2-Cys-peroxiredoxins (Prxs) and glutathione peroxidase-type enzymes (Pxs) which both obtain their reducing equivalents from the trypanothione/tryparedoxin couple and thus act as tryparedoxin peroxidases. While the cytosolic forms of the peroxidases are essential, the mitochondrial mPrx and Px III appear dispensable in bloodstream Trypanosoma brucei. This led to the suggestion that in this developmental stage which is characterized by a mitochondrion that lacks an active respiratory chain, only one of the two peroxidases might be required. Here we show that bloodstream cells in which the Px III gene is deleted and mPrx is down-regulated by RNA interference, proliferate as the parental cells indicating that both mitochondrial peroxidases are dispensable. However, when we raised the culture temperature to 39 °C, mPrx-depleted cells died indicating that under conditions mimicking a fever situation in the mammalian host, the protein becomes essential. In contrast, depletion of mPrx in insect stage procyclic T. brucei causes a proliferation defect under standard conditions at 27 °C, in the absence of any stress. In the absence of mPrx, a tryparedoxin-coupled roGFP2 biosensor expressed in the mitochondrial matrix is unable to respond to antimycin A treatment. Thus mPrx reduces mitochondrial H2O2 with the generation of trypanothione disulfide and acts as peroxidase. However, mPrx-depleted procyclic cells neither display any alteration in the cytosolic or mitochondrial trypanothione redox state nor increased sensitivity towards exogenous oxidative stressors suggesting that the peroxidase activity is not the crucial physiological function. After prolonged mPrx-depletion, the cells almost stop proliferation and display a highly elongated shape and diminished MitoTracker Red staining. In contrast to the situation in the mammalian bloodstream T. brucei and Leishmania, mPrx appears to play a constitutive role for the morphology, mitochondrial function and proliferation of the insect stage of African trypanosomes.
Subject(s)
Hydrogen Peroxide , Trypanosoma brucei brucei , Animals , Hydrogen Peroxide/metabolism , Mitochondria/metabolism , Oxidation-Reduction , Peroxiredoxins/genetics , Peroxiredoxins/metabolismABSTRACT
Trypanosomes have a trypanothione redox metabolism that provides the reducing equivalents for numerous essential processes, most being mediated by tryparedoxin (Tpx). While the biosynthesis and reduction of trypanothione are cytosolic, the molecular basis of the thiol redox homeostasis in the single mitochondrion of these parasites has remained largely unknown. Here we expressed Tpx-roGFP2, roGFP2-hGrx1 or roGFP2 in either the cytosol or mitochondrion of Trypanosoma brucei. We show that the novel Tpx-roGFP2 is a superior probe for the trypanothione redox couple and that the mitochondrial matrix harbors a trypanothione system. Inhibition of trypanothione biosynthesis by the anti-trypanosomal drug Eflornithine impairs the ability of the cytosol and mitochondrion to cope with exogenous oxidative stresses, indicating a direct link between both thiol systems. Tpx depletion abolishes the cytosolic, but only partially affects the mitochondrial sensor response to H2O2. This strongly suggests that the mitochondrion harbors some Tpx and, another, as yet unidentified, oxidoreductase.
Trypanosoma brucei are single-celled parasites that cause human sleeping sickness and animal diseases. Like in other organisms, the parasite contains different compartments, each having several specific roles. The mitochondrion is the compartment that provides most of the energy needed to keep the cell alive. Many cellular processes, such as those that happen in the mitochondrion, produce compounds including hydrogen peroxide that can cause 'oxidative damage'. To counteract this, cells make small molecules called thiols. These thiols provide 'reducing' power to chemically balance out the oxidative damage. Trypanosomes have an unusual thiol system that relies on a molecule called trypanothione. Trypanosoma brucei cells make trypanothione in the cytosol, the fluid which surrounds all cellular compartments; here it is also used up with the help of a protein called tryparedoxin. However, it was not known which thiols are present in the mitochondrion. Ebersoll et al. have now made a molecular sensor that can detect trypanothione. The sensor includes a fluorescent protein, which changes its brightness based on its oxidation state, fused to the tryparedoxin protein. This probe could either be put in the cytosol or mitochondrion of Trypanosoma brucei cells. Treating the cells with hydrogen peroxide changed the fluorescence of the biosensor. Trypanosoma brucei cells without tryparedoxin protein in their cytosol still responded to an oxidative challenge in the mitochondrion. The experiments reveal that trypanosomes do have a mitochondrial trypanothione system. This new fluorescent biosensor will be used to study how other cellular compartments deal with oxidative conditions. The tests will reveal how different compartments communicate with each other to counteract the stress. The sensor could also be used to determine how anti-parasite drugs affect the cells' trypanothione system.
Subject(s)
Glutathione/analogs & derivatives , Mitochondria/metabolism , Spermidine/analogs & derivatives , Thioredoxins/metabolism , Trypanosoma brucei brucei/metabolism , Biosensing Techniques , Eflornithine/pharmacology , Glutathione/biosynthesis , Glutathione/metabolism , Green Fluorescent Proteins/metabolism , Homeostasis , Hydrogen Peroxide/pharmacology , Oxidation-Reduction , Oxidative Stress/drug effects , Spermidine/biosynthesis , Spermidine/metabolism , Trypanocidal Agents/pharmacology , Trypanosoma brucei brucei/drug effectsABSTRACT
In the course of our investigations on the antitrypanosomal potential of sesquiterpene lactones (STL), we have recently reported on the exceptionally strong activity of 4,15-iso-Atriplicolide tiglate, which demonstrated an IC50 value of 15 nM against Trypanosoma brucei rhodesiense, the etiologic agent responsible for East African human trypanosomiasis (HAT). Since STLs are known to often interact with their biological targets (e.g., in anti-inflammatory and anti-tumor activity) by means of the covalent modification of biological nucleophiles-most prominently free cysteine thiol groups in proteins-it was a straightforward assumption that such compounds might interfere with the trypanothione-associated detoxification system of trypanosomes. This system heavily relies on thiol groups in the form of the dithiol trypanothione (T(SH)2) and in the active centers of enzymes involved in trypanothione metabolism and homeostasis. Indeed, we found in the present study that 4,15-iso-atriplicolide tiglate, as well as its structural homologues, the corresponding methacrylate and isobutyrate, are inhibitors of trypanothione reductase (TR), the enzyme serving the parasites to keep T(SH)2 in the dithiol state. The TR inhibitory activity was demonstrated to be time-dependent and irreversible. Quite interestingly, of the several further STLs with different core structures that were also tested, none inhibited TR at a significant level. Thus, the TR inhibitory effect by the 4,15-iso-atriplicolide esters appears to be specific for this particular type of furanoheliangolide-type STL. Some structure-activity relationships can already be deduced on the basis of the data reported here, which may serve as the starting point for searching further, possibly more potent, TR inhibitors.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Lactones/chemistry , Lactones/pharmacology , NADH, NADPH Oxidoreductases/antagonists & inhibitors , Sesquiterpenes/chemistry , Animals , Dose-Response Relationship, Drug , Humans , Molecular Structure , Structure-Activity RelationshipABSTRACT
Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes.
Subject(s)
Protozoan Proteins/metabolism , Thioredoxins/metabolism , Trypanosoma brucei brucei/metabolism , Animals , Gene Knockdown Techniques , Genes, Protozoan , Humans , Mitochondrial Proteins/antagonists & inhibitors , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Molecular Chaperones/antagonists & inhibitors , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Mutation , Oxidation-Reduction , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Thioredoxins/antagonists & inhibitors , Thioredoxins/genetics , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/pathogenicityABSTRACT
Trypanothione reductase (TR) plays a key role in the unique redox metabolism of trypanosomatids, the causative agents of human African trypanosomiasis (HAT), Chagas' disease, and leishmaniases. Introduction of a new, lean propargylic vector to a known class of TR inhibitors resulted in the strongest reported competitive inhibitor of Trypanosoma (T.) brucei TR, with an inhibition constant Ki of 73â nm, which is fully selective against human glutathione reductase (hGR). The best ligands exhibited in vitro IC50 values (half-maximal inhibitory concentration) against the HAT pathogen, T. brucei rhodesiense, in the mid-nanomolar range, reaching down to 50â nm. X-Ray co-crystal structures confirmed the binding mode of the ligands and revealed the presence of a HEPES buffer molecule in the large active site. Extension of the propargylic vector, guided by structure-based design, to replace the HEPES buffer molecule should give inhibitors with low nanomolar Ki and IC50 values for in vivo studies.
Subject(s)
Drug Design , Enzyme Inhibitors/chemistry , NADH, NADPH Oxidoreductases/antagonists & inhibitors , Protozoan Proteins/antagonists & inhibitors , Trypanosoma brucei brucei/enzymology , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Inhibitory Concentration 50 , Ligands , Molecular Dynamics Simulation , NADH, NADPH Oxidoreductases/metabolism , Protozoan Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Trypanosomal and leishmanial infections claim tens of thousands of lives each year. The metabolism of these unicellular eukaryotic parasites differs from the human host and their enzymes thus constitute promising drug targets. Tryparedoxin (Tpx) from Trypanosoma brucei is the essential oxidoreductase in the parasite's hydroperoxide-clearance cascade. Inâ vitro and inâ vivo functional assays show that a small, selective inhibitor efficiently inhibits Tpx. With X-ray crystallography, SAXS, analytical SEC, SEC-MALS, MD simulations, ITC, and NMR spectroscopy, we show how covalent binding of this monofunctional inhibitor leads to Tpx dimerization. Intra- and intermolecular inhibitor-inhibitor, protein-protein, and inhibitor-protein interactions stabilize the dimer. The behavior of this efficient antitrypanosomal molecule thus constitutes an exquisite example of chemically induced dimerization with a small, monovalent ligand that can be exploited for future drug design.
Subject(s)
Antiprotozoal Agents/chemistry , Bacterial Proteins/chemistry , Enzyme Inhibitors/chemistry , Oxidoreductases/chemistry , Thioredoxins/chemistry , Trypanosoma brucei brucei/enzymology , Animals , Antiprotozoal Agents/metabolism , Drug Design , Enzyme Inhibitors/metabolism , Glutathione/analogs & derivatives , Glutathione/chemistry , Humans , Hydrogen Peroxide/metabolism , Molecular Dynamics Simulation , Oxidation-Reduction , Protein Binding , Protein Conformation , Protein Multimerization , Spermidine/analogs & derivatives , Spermidine/chemistry , Trypanosoma/metabolism , Trypanosoma/parasitologyABSTRACT
Tryparedoxin peroxidases, distant relatives of glutathione peroxidase 4 in higher eukaryotes, are responsible for the detoxification of lipid-derived hydroperoxides in African trypanosomes. The lethal phenotype of procyclic Trypanosoma brucei that lack the enzymes fulfils all criteria defining a form of regulated cell death termed ferroptosis. Viability of the parasites is preserved by α-tocopherol, ferrostatin-1, liproxstatin-1 and deferoxamine. Without protecting agent, the cells display, primarily mitochondrial, lipid peroxidation, loss of the mitochondrial membrane potential and ATP depletion. Sensors for mitochondrial oxidants and chelatable iron as well as overexpression of a mitochondrial iron-superoxide dismutase attenuate the cell death. Electron microscopy revealed mitochondrial matrix condensation and enlarged cristae. The peroxidase-deficient parasites are subject to lethal iron-induced lipid peroxidation that probably originates at the inner mitochondrial membrane. Taken together, ferroptosis is an ancient cell death program that can occur at individual subcellular membranes and is counterbalanced by evolutionary distant thiol peroxidases.
Subject(s)
Apoptosis , Iron/metabolism , Peroxidases/metabolism , Protozoan Proteins/metabolism , Trypanosoma brucei brucei/enzymology , Adenosine Triphosphate/metabolism , Animals , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cytoprotection , Lipid Peroxidation , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Oxidants/metabolism , Parasites/metabolism , Phenotype , Superoxide Dismutase/metabolism , Trypanosoma brucei brucei/ultrastructureABSTRACT
The tropical diseases human African trypanosomiasis, Chagas disease, and the various forms of leishmaniasis are caused by parasites of the family of trypanosomatids. These protozoa possess a unique redox metabolism based on trypanothione and trypanothione reductase (TR), making TR a promising drug target. We report the optimization of properties and potency of cyclohexylpyrrolidine inhibitors of TR by structure-based design. The best inhibitors were freely soluble and showed competitive inhibition constants (Ki ) against Trypanosoma (T.) brucei TR and T.â cruzi TR and inâ vitro activities (half-maximal inhibitory concentration, IC50 ) against these parasites in the low micromolar range, with high selectivity against human glutathione reductase. X-ray co-crystal structures confirmed the binding of the ligands to the hydrophobic wall of the "mepacrine binding site" with the new, solubility-providing vectors oriented toward the surface of the large active site.
Subject(s)
Enzyme Inhibitors/pharmacology , NADH, NADPH Oxidoreductases/antagonists & inhibitors , Pyrrolidines/pharmacology , Trypanocidal Agents/pharmacology , Trypanosoma/drug effects , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Ligands , Models, Molecular , Molecular Structure , NADH, NADPH Oxidoreductases/metabolism , Oxidation-Reduction , Parasitic Sensitivity Tests , Pyrrolidines/chemical synthesis , Pyrrolidines/chemistry , Solubility , Trypanocidal Agents/chemical synthesis , Trypanocidal Agents/chemistry , Trypanosoma/metabolismABSTRACT
Trypanosoma brucei glutaredoxin 2 (Grx2) is a dithiol glutaredoxin that is specifically located in the mitochondrial intermembrane space. Bloodstream form parasites lacking Grx2 or both, Grx2 and the cytosolic Grx1, are viable in vitro and infectious to mice suggesting that neither oxidoreductase is needed for survival or infectivity to mammals. A 37⯰C to 39⯰C shift changes the cellular redox milieu of bloodstream cells to more oxidizing conditions and induces a significantly stronger growth arrest in wildtype parasites compared to the mutant cells. Grx2-deficient cells ectopically expressing the wildtype form of Grx2 with its C31QFC34 active site, but not the C34S mutant, regain the sensitivity of the parental strain, indicating that the physiological role of Grx2 requires both active site cysteines. In the procyclic insect stage of the parasite, Grx2 is essential. Both alleles can be replaced if procyclic cells ectopically express authentic or C34S, but not C31S/C34S Grx2, pointing to a redox role that relies on a monothiol mechanism. RNA-interference against Grx2 causes a virtually irreversible proliferation defect. The cells adopt an elongated morphology but do not show any significant alteration in the cell cycle. The growth retardation is attenuated by high glucose concentrations. Under these conditions, procyclic cells obtain ATP by substrate level phosphorylation suggesting that Grx2 might regulate a respiratory chain component.
Subject(s)
Adaptation, Physiological/genetics , Glutaredoxins/genetics , Trypanosoma brucei brucei/metabolism , Trypanosomiasis, African/metabolism , Adenosine Triphosphate/metabolism , Alleles , Animals , Catalytic Domain , Cell Proliferation/genetics , Cytosol/metabolism , Glutaredoxins/chemistry , Glutaredoxins/metabolism , Hot Temperature , Humans , Mice , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/parasitology , Mitochondrial Membranes/metabolism , Mutation , Oxidation-Reduction , Trypanosoma brucei brucei/pathogenicity , Trypanosomiasis, African/parasitology , Trypanosomiasis, African/pathologyABSTRACT
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.
Subject(s)
Glyceraldehyde-3-Phosphate Dehydrogenases/antagonists & inhibitors , NADH, NADPH Oxidoreductases/antagonists & inhibitors , Quinones/pharmacology , Trypanocidal Agents/pharmacology , Trypanosoma brucei brucei/drug effects , Trypanosoma cruzi/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Humans , Models, Molecular , Molecular Structure , NADH, NADPH Oxidoreductases/metabolism , Parasitic Sensitivity Tests , Quinones/chemical synthesis , Quinones/chemistry , Structure-Activity Relationship , Trypanocidal Agents/chemical synthesis , Trypanocidal Agents/chemistry , Trypanosoma brucei brucei/enzymology , Trypanosoma brucei brucei/growth & development , Trypanosoma cruzi/enzymology , Trypanosoma cruzi/growth & developmentABSTRACT
This paper describes the development of a class of peptide-based inhibitors as novel antitrypanosomal and antimalarial agents. The inhibitors are based on a characteristic peptide sequence for the inhibition of the cysteine proteases rhodesain of Trypanosoma brucei rhodesiense and falcipain-2 of Plasmodium falciparum. We exploited the reactivity of novel unsaturated electrophilic functions such as vinyl-sulfones, -ketones, -esters, and -nitriles. The Michael acceptors inhibited both rhodesain and falcipain-2, at nanomolar and micromolar levels, respectively. In particular, the vinyl ketone 3b has emerged as a potent rhodesain inhibitor (k2nd = 67 × 106 M-1 min-1), endowed with a picomolar binding affinity (Ki = 38 pM), coupled with a single-digit micromolar activity against Trypanosoma brucei brucei (EC50 = 2.97 µM), thus being considered as a novel lead compound for the discovery of novel effective antitrypanosomal agents.
Subject(s)
Antimalarials/pharmacology , Carbamates/pharmacology , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Dipeptides/pharmacology , Phenylalanine/analogs & derivatives , Trypanocidal Agents/pharmacology , Antimalarials/chemical synthesis , Antimalarials/toxicity , Carbamates/chemical synthesis , Carbamates/toxicity , Cathepsin L/metabolism , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/toxicity , Dipeptides/chemical synthesis , Dipeptides/toxicity , HeLa Cells , Humans , Hydrogen Bonding , Malaria/drug therapy , Molecular Docking Simulation , Molecular Dynamics Simulation , Neglected Diseases/drug therapy , Phenylalanine/chemical synthesis , Phenylalanine/pharmacology , Phenylalanine/toxicity , Plasmodium falciparum/drug effects , Stereoisomerism , Structure-Activity Relationship , Trypanocidal Agents/chemical synthesis , Trypanocidal Agents/toxicity , Trypanosoma brucei brucei/drug effects , Trypanosomiasis, African/drug therapyABSTRACT
Human African Trypanosomiasis (HAT) is caused by two subspecies of the genus Trypanosoma, namely Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. The disease is fatal if left untreated and therapy is limited due to only five non-adequate drugs currently available. In preliminary studies, dimeric tacrine derivatives were found to inhibit parasite growth with IC50-values in the nanomolar concentration range. This prompted the synthesis of a small, but smart library of monomeric and dimeric tacrine-type compounds and their evaluation of antiprotozoal activity. Rhodesain, a lysosomal cathepsin-L like cysteine protease of T. brucei rhodesiense is essential for parasite survival and likely target of the tacrine derivatives. In addition, the inhibition of trypanothione reductase by bistacrines was found. This flavoprotein oxidoreductase is the main defense against oxidative stress in the thiol redox system unique for protozoa.
Subject(s)
Tacrine/pharmacology , Trypanocidal Agents/pharmacology , Trypanosoma brucei brucei/drug effects , Trypanosomiasis, African/drug therapy , Animals , Cell Line , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Mice , Molecular Structure , Parasitic Sensitivity Tests , Structure-Activity Relationship , Tacrine/chemistry , Trypanosoma brucei brucei/cytologyABSTRACT
Tryparedoxin (Tpx) is a pivotal protein in the redox-metabolism of trypanosomatid parasites. Tpx has previously been identified as a potential target for drug development in the fight against human African sleeping sickness caused by Trypanosoma brucei. Tpx belongs to the thioredoxin superfamily and acts as an oxidoreductase in the parasite's cytoplasm. It contains a WCPPC active site motif, which enables the protein to undergo thiol-disulfide exchange. To promote future protein-drug interaction analyses, we report the 1H, 13C and 15N backbone chemical shift assignments for both the oxidized and reduced states of Tpx. The redox state of the protein has a significant impact on the chemical shifts of the residues at the active site of the protein, especially on the two redox active site cysteines. The NMR assignments presented here will be a prerequisite for investigating drug binding to Tpx in molecular detail and to drive further drug optimization.
Subject(s)
Hydrogen Peroxide/metabolism , Nuclear Magnetic Resonance, Biomolecular , Thioredoxins/chemistry , Thioredoxins/metabolism , Trypanosoma , Amino Acid Motifs , Oxidation-ReductionABSTRACT
AIMS: Trypanosomatids have a unique trypanothione-based thiol redox metabolism. The parasite-specific dithiol is synthesized from glutathione and spermidine, with glutathionylspermidine as intermediate catalyzed by trypanothione synthetase. In this study, we address the oxidative stress response of African trypanosomes with special focus on putative protein S-thiolation. RESULTS: Challenging bloodstream Trypanosoma brucei with diamide, H2O2 or hypochlorite results in distinct levels of reversible overall protein S-thiolation. Quantitative proteome analyses reveal 84 proteins oxidized in diamide-stressed parasites. Fourteen of them, including several essential thiol redox proteins and chaperones, are also enriched when glutathione/glutaredoxin serves as a reducing system indicating S-thiolation. In parasites exposed to H2O2, other sets of proteins are modified. Only three proteins are S-thiolated under all stress conditions studied in accordance with a highly specific response. H2O2 causes primarily the formation of free disulfides. In contrast, in diamide-treated cells, glutathione, glutathionylspermidine, and trypanothione are almost completely protein bound. Remarkably, the total level of trypanothione is decreased, whereas those of glutathione and glutathionylspermidine are increased, indicating partial hydrolysis of protein-bound trypanothione. Depletion of trypanothione synthetase exclusively induces protein S-glutathionylation. Total mass analyses of a recombinant peroxidase treated with T(SH)2 and either diamide or hydrogen peroxide verify protein S-trypanothionylation as stable modification. INNOVATION: Our data reveal for the first time that trypanosomes employ protein S-thiolation when exposed to exogenous and endogenous oxidative stresses and trypanothione, despite its dithiol character, forms protein-mixed disulfides. CONCLUSION: The stress-specific responses shown here emphasize protein S-trypanothionylation and S-glutathionylation as reversible protection mechanism in these parasites. Antioxid. Redox Signal. 27, 517-533.
Subject(s)
Glutathione/analogs & derivatives , Glutathione/metabolism , Protein S/metabolism , Spermidine/analogs & derivatives , Trypanosoma brucei brucei/metabolism , Diamide/pharmacology , Humans , Hydrogen Peroxide/pharmacology , Hypochlorous Acid/pharmacology , Oxidative Stress , Proteome/analysis , Protozoan Proteins/analysis , Spermidine/metabolism , Sulfhydryl Compounds/analysisABSTRACT
Chagas disease or American trypanosomiasis is a worldwide public health problem. In this work, we evaluated 26 new propyl and isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives as potential trypanocidal agents. Additionally, molecular docking and enzymatic assays on trypanothione reductase (TR) were performed to provide a basis for their potential mechanism of action. Seven compounds showed better trypanocidal activity on epimastigotes than the reference drugs, and only four displayed activity on trypomastigotes; T-085 was the lead compound with an IC50 = 59.9 and 73.02 µM on NINOA and INC-5 strain, respectively. An in silico analysis proposed compound T-085 as a potential TR inhibitor with better affinity than the natural substrate. Enzymatic analysis revealed that T-085 inhibits parasite TR non-competitively. Compound T-085 carries a carbonyl, a CF3, and an isopropyl carboxylate group at 2-, 3- and 7-position, respectively. These results suggest the chemical structure of this compound as a good starting point for the design and synthesis of novel trypanocidal derivatives with higher TR inhibitory potency and lower toxicity.
Subject(s)
NADH, NADPH Oxidoreductases/antagonists & inhibitors , Quinoxalines/chemistry , Quinoxalines/pharmacology , Trypanocidal Agents/chemistry , Trypanocidal Agents/pharmacology , Binding Sites , Inhibitory Concentration 50 , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , NADH, NADPH Oxidoreductases/chemistry , Parasitic Sensitivity Tests , Protein Binding , Structure-Activity Relationship , Trypanosoma cruzi/drug effectsABSTRACT
Thirteen new polyamine derivatives coupled to hydroxybenzotriazole have been synthesized and evaluated for their in vitro antikinetoplastid activity. Trypanosoma Trypanothione reductase (TryR) was envisioned as a potential target. Among all tested molecules, only one compound, a N3-spermidine-benzotriazole derivative, displayed relevant inhibitory activity on this enzyme but was not active on parasites. The corresponding Boc-protected spermidine-benzotriazole was however trypanocidal against Trypanosoma brucei gambiense with an IC50 value of 1µM and was completely devoid of cytotoxicity. On the intramacrophage amastigotes of Leishmania donovani, a N2-spermidine conjugate of this series, exhibited an interesting IC50 value of 3µM associated with both low cytotoxicity against axenic Leishmania donovani. These new compounds are promising leads for the development of antikinetoplastid agents and their targets have to be deciphered.
Subject(s)
Antiprotozoal Agents/chemistry , Antiprotozoal Agents/pharmacology , Leishmania donovani/drug effects , Triazoles/chemistry , Triazoles/pharmacology , Trypanosoma brucei brucei/drug effects , Animals , Antiprotozoal Agents/chemical synthesis , Humans , Leishmania donovani/enzymology , Leishmaniasis, Visceral/drug therapy , NADH, NADPH Oxidoreductases/antagonists & inhibitors , NADH, NADPH Oxidoreductases/metabolism , Spermidine/analogs & derivatives , Spermidine/chemical synthesis , Spermidine/pharmacology , Triazoles/chemical synthesis , Trypanocidal Agents/chemical synthesis , Trypanocidal Agents/chemistry , Trypanocidal Agents/pharmacology , Trypanosoma brucei brucei/enzymology , Trypanosomiasis, African/drug therapyABSTRACT
A series of dipeptide nitriles known as inhibitors of mammalian cathepsins were evaluated for inhibition of rhodesain, the cathepsin L-like protease of Trypanosoma brucei. Compound 35 consisting of a Leu residue fitting into the S2 pocket and a triarylic moiety consisting of thiophene, a 1,2,4-oxadiazole and a phenyl ring fitting into the S3 pocket, and compound 33 with a 3-bromo-Phe residue (S2) and a biphenyl fragment (S3) were found to inhibit rhodesain in the single-digit nanomolar range. The observed steep structure-activity relationship could be explained by covalent docking simulations. With their high selectivity indices (ca. 200) and the good antitrypanosomal activity (8µM) the compounds represent promising starting points for new rhodesain inhibitors.
Subject(s)
Antitubercular Agents/pharmacology , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Dipeptides/pharmacology , Nitriles/pharmacology , Trypanosoma brucei brucei/drug effects , Antitubercular Agents/chemical synthesis , Antitubercular Agents/chemistry , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Dipeptides/chemical synthesis , Dipeptides/chemistry , Dose-Response Relationship, Drug , Molecular Structure , Nitriles/chemical synthesis , Nitriles/chemistry , Structure-Activity Relationship , Trypanosoma brucei brucei/enzymologyABSTRACT
Dipeptidyl nitroalkenes are potent reversible inhibitors of cysteine proteases. Inhibitor 11 resulted to be the most potent one with Ki values of 0.49 and 0.44 nM against rhodesain and cruzain, respectively. According to enzymatic dilution and dialysis experiments, as well as computational and NMR studies, dipeptidyl nitroalkenes are tightly binding covalent reversible inhibitors.