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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).

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.

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., Graphical abstract Image 1, Highlights • In bloodstream T. brucei, both mitochondrial tryparedoxin peroxidases are dispensable. • Heat-stressed bloodstream cells require the mitochondrial peroxiredoxin (mPrx). • In procyclic (PC) T. brucei, mPrx plays a constitutive role for proliferation. • Lack of mPrx affects the structure and mitochondrial membrane potential of PC cells.

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 (K; i; ) against Trypanosoma (T.) brucei TR and T. cruzi TR and in vitro activities (half-maximal inhibitory concentration, IC; 50; ) 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.

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.

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.

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&mdash, most prominently free cysteine thiol groups in proteins&mdash, 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&ndash, 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.

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 73 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 50 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.

Trypanosomatids are protozoan parasites with a unique trypanothionebased thiol redox metabolism. Mediated by the small oxidoreductase tryparedoxin, trypanothione is the universal electron donor for the biosynthesis of DNA precursors, reduction of methionine sulfoxides as well as detoxification of hydroperoxides by distinct thiol peroxidases. The fact that nearly all enzymes involved are essential renders the pathway attractive for drug development approaches against these parasites.

In order to identify new compounds to treat Chagas disease during the acute phase with higher activity and lower toxicity than the reference drug benznidazole (Bz), a series of tetraamine-based compounds was prepared and their trypanocidal effects against Trypanosoma cruzi were evaluated by light microscopy through the determination of IC50 values. Cytotoxicity was determined by flow cytometry assays against Vero cells. In vivo assays were performed in BALB/c mice, in which the parasitemia levels were quantified by fresh blood examination; the assignment of a cure was determined by PCR and reactivation of blood parasitemia levels after immunosuppression. The mechanism of action was elucidated at metabolic and ultra-structural levels by (1)H NMR and TEM studies. Finally, as tetraamines are potentially capable of casuing oxidative damage in the parasites, the study was completed by assessing their activity as potential iron superoxide dismutase (Fe-SOD) and trypanothione reductase (TR) inhibitors. High-selectivity indexes observed in vitro were the basis of promoting three of the tested compounds to in vivo assays. The tests on the murine model for the acute phase of Chagas disease showed better parasitemia inhibition values than those found for Bz. Tetraamines 2 and 3 induced a remarkable decrease in the reactivation of parasitemia after immunosuppression and curative rates of 33 and 50%, respectively. Tetraamine 3 turned out to be a great inhibitor of Fe-SOD and TR. The high anti-parasitic activity and low toxicity render these tetraamines appropriate molecules for the development of an affordable anti-Chagas agent.

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., eLife digest Plants, animals and fungi all belong to a group of organisms known as eukaryotes. Their cells host a variety of compartments, with each having a specific role. For example, mitochondria are tasked with providing the energy that powers most of the processes that keep the cell alive. Membranes delimit these compartments, as well as the cells themselves. Iron is an element needed for chemical reactions that are essential for the cell to survive. Yet, the byproducts of these reactions can damage – ‘oxidize’ – the lipid molecules that form the cell’s membranes, including the one around mitochondria. Unless enzymes known as peroxidases come to repair the oxidized lipids, the cell dies in a process called ferroptosis. Scientists know that this death mechanism is programmed into the cells of humans and other complex eukaryotes. However, Bogacz and Krauth-Siegel wanted to know if ferroptosis also exists in creatures that appeared early in the evolution of eukaryotes, such as the trypanosome Trypanosoma brucei. This single-cell parasite causes sleeping sickness in humans and a disease called nagana in horses and cattle. Before it infects a mammal, T. brucei goes through an ‘insect stage’ where it lives in the tsetse fly; there, it relies on its mitochondrion to produce energy. Bogacz and Krauth-Siegel now show that if the parasites in the insect stage do not have a specific type of peroxidases, they die within a few hours. In particular, problems in the membranes of the mitochondrion stop the compartment from working properly. These peroxidases-free trypanosomes fare better if they are exposed to molecules that prevent iron from taking part in the reactions that can harm lipids. They also survive more if they are forced to create large amounts of an enzyme that relies on iron to protect the mitochondrion against oxidation. Finally, using drugs that prevent ferroptosis in human cells completely rescues these trypanosomes. Taken together, the results suggest that ferroptosis is an ancient cell death program which exists in T. brucei; and that, in the insect stage of the parasite's life cycle, this process first damages the mitochondrion. This last finding could be particularly relevant because the role of mitochondria in ferroptosis in mammals is highly debated. Yet, most of the research is done in cells that do not rely on this cellular compartment to get their energy. During their life cycle, trypanosomes are either dependent on their mitochondria, or they can find their energy through other sources: this could make them a good organism in which to dissect the precise mechanisms of ferroptosis.

African trypanosomes express three virtually identical glutathione peroxidase (Px)-type enzymes that occur in the cytosol (Px I and II) and mitochondrion (Px III) and detoxify fatty acid-derived hydroperoxides. Selective deletion of the genes revealed that procyclic Trypanosoma brucei lacking either the cytosolic or mitochondrial enzyme proliferate nearly as wild-type parasites, whereas the knockout of the complete genomic locus is lethal. Flow cytometry and immunofluorescence analyses revealed that the Px I-III-deficient parasites lose their mitochondrial membrane potential, which is followed by a loss of the lysosomal signal but not the glycosomal one. Mitochondrial damage and cell lysis are prevented by Trolox, ubiquinone derivatives and the iron chelator deferoxamine, whereas starch-deferoxamine is inefficient. In glucose-rich medium, cell death is attenuated suggesting that oxidants generated by the respiratory chain contribute to the lethal phenotype. Thus, the Px-type peroxidases protect procyclic cells from an iron-mediated oxidative membrane damage that originates at the mitochondrion. This contrasts with the situation in bloodstream cells, where the lysosome is the primarily affected organelle. Strikingly, either the cytosolic or the mitochondrial form of the peroxidases is required and sufficient to protect the mitochondrion and prevent cell lysis.

Malaria and African trypanosomiasis are tropical diseases caused by the protozoa Plasmodium and Trypanosoma, respectively. The parasites undergo complex life cycles in the mammalian host and insect vector, during which they are exposed to oxidative and nitrosative challenges induced by the host immune system and endogenous processes. Attacking the parasite’s redox metabolism is a target mechanism of several known antiparasitic drugs and a promising approach to novel drug development. Apart from this aspect, oxidation of cysteine residues plays a key role in protein-protein interaction, metabolic responses to redox events, and signaling. Understanding the role and dynamics of reactive oxygen species and thiol switches in regulating cellular redox homeostasis is crucial for both basic and applied biomedical approaches. Numerous techniques have therefore been established to detect redox changes in parasites including biochemical methods, fluorescent dyes, and genetically encoded probes. In this review, we aim to give an insight into the characteristics of redox networks in the pathogens Plasmodium and Trypanosoma, including a comprehensive overview of the consequences of specific deletions of redox-associated genes. Furthermore, we summarize mechanisms and detection methods of thiol switches in both parasites and discuss their specificity and sensitivity.

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., Graphical abstract fx1, Highlights • Bloodstream T. brucei lacking glutaredoxin 2 are fully viable in vitro and in vivo. • A temperature rise shifts the cellular redox state to more oxidizing conditions. • Glutaredoxin 2-deficiency confers bloodstream cells with thermo-tolerance. • The insect stage requires redox-active glutaredoxin 2 for viability and morphology.

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.

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.

none 20 si 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. Uliassi, Elisa; Fiorani, Giulia; Krauth-Siegel, R. Luise; Bergamini, Christian; Fato, Romana; Bianchini, Giulia; Carlos Menéndez, J.; Molina, Maria Teresa; López-Montero, Eulogio; Falchi, Federico; Cavalli, Andrea; Gul, Sheraz; Kuzikov, Maria; Ellinger, Bernhard; Witt, Gesa; Moraes, Carolina B.; Freitas-Junior, Lucio H.; Borsari, Chiara; Costi, Maria Paola; Bolognesi, Maria Laura* Uliassi, Elisa; Fiorani, Giulia; Krauth-Siegel, R. Luise; Bergamini, Christian; Fato, Romana; Bianchini, Giulia; Carlos Menéndez, J.; Molina, Maria Teresa; López-Montero, Eulogio; Falchi, Federico; Cavalli, Andrea; Gul, Sheraz; Kuzikov, Maria; Ellinger, Bernhard; Witt, Gesa; Moraes, Carolina B.; Freitas-Junior, Lucio H.; Borsari, Chiara; Costi, Maria Paola; Bolognesi, Maria Laura*

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., Author summary African trypanosomes are the causative agents of human sleeping sickness and Nagana cattle disease. These strictly extracellular pathogens multiply in the blood and body fluids of their mammalian hosts and the tsetse fly vector, where efficient redox regulation is essential for parasite survival. While most organisms use the glutathione/glutathione reductase and thioredoxin/thioredoxin reductase couples to maintain cellular redox balance, trypanosomes rely on a unique trypanothione-based thiol metabolism to survive exogenous and endogenous oxidative stresses. Despite the lack of thioredoxin reductases, the Trypanosoma brucei genome encodes thioredoxins, raising questions for their biological function. Our work is the first report on T. brucei thioredoxin-2 (Trx2). We show that Trx2 is located in the mitochondrion and its absence affects parasite proliferation and infectivity. Recombinant Trx2 lacks protein disulfide reductase activity but protects proteins against aggregation and maintains them folding-competent. Remarkably, a mutant that is devoid of any cysteine residues is able to fully substitute for the authentic protein under in vitro and in vivo conditions. Our data reveal that Trx2 does not function as a classical thioredoxin but acts as a chaperone that plays a crucial role in the mitochondrion of T. brucei.

Background: Trypanothione synthetase catalyzes the conjugation of spermidine with two GSH molecules to form trypanothione. Results: The kinetic parameters were measured under in vivo-like conditions. A mathematical model was developed describing the entire kinetic profile. Conclusion: Trypanothione synthetase is affected by substrate and product inhibition. Significance: The combined kinetic and modeling approaches provided a so far unprecedented insight in the mechanism of this parasite-specific enzyme., In pathogenic trypanosomes, trypanothione synthetase (TryS) catalyzes the synthesis of both glutathionylspermidine (Gsp) and trypanothione (bis(glutathionyl)spermidine (T(SH)2)). Here we present a thorough kinetic analysis of Trypanosoma brucei TryS in a newly developed phosphate buffer system at pH 7.0 and 37 °C, mimicking the physiological environment of the enzyme in the cytosol of bloodstream parasites. Under these conditions, TryS displays Km values for GSH, ATP, spermidine, and Gsp of 34, 18, 687, and 32 μm, respectively, as well as Ki values for GSH and T(SH)2 of 1 mm and 360 μm, respectively. As Gsp hydrolysis has a Km value of 5.6 mm, the in vivo amidase activity is probably negligible. To obtain deeper insight in the molecular mechanism of TryS, we have formulated alternative kinetic models, with elementary reaction steps represented by linear kinetic equations. The model parameters were fitted to the extensive matrix of steady-state data obtained for different substrate/product combinations under the in vivo-like conditions. The best model describes the full kinetic profile and is able to predict time course data that were not used for fitting. This system's biology approach to enzyme kinetics led us to conclude that (i) TryS follows a ter-reactant mechanism, (ii) the intermediate Gsp dissociates from the enzyme between the two catalytic steps, and (iii) T(SH)2 inhibits the enzyme by remaining bound at its product site and, as does the inhibitory GSH, by binding to the activated enzyme complex. The newly detected concerted substrate and product inhibition suggests that TryS activity is tightly regulated.

Trypanosomes and Leishmania, the causative agents of African sleeping sickness, Chagas disease, and Leishmaniasis, are responsible for over half a million human deaths per year in subtropical and tropical regions around the world. Only a handful of chemotherapeutics are available, and their efficacy suffers from drug resistance and serious side-effects. Therefore, new inhibiting compounds of essential targets are urgently needed. Trypanosomes lack both glutathione reductase and thioredoxin reductase, but possess the phylogenetically related trypanothione reductase (TR), a validated drug target and key enzyme of the unique trypanothione-based thiol metabolism. By inhibiting this essential flavoenzyme the parasite becomes vulnerable against oxidative stress induced by the host defense system and drug treatment. Here, we provide an overview of published TR screenings, and present an alternative approach combining in silico and in vitro experiments. Starting from an in vitro screen of 2816 highly diverse compounds, 21 novel TR actives could be identified. These inhibitors were used as the starting point to identify similar substances within a compound library of 200 000 available chemicals by a ligand-based in silico screening. In vitro screening of the resulted in silico-enriched dataset led to the identification of 61 additional TR actives. All 82 TR inhibitors showed IC50 values down to the nanomolar range. Subsequently, the dataset of known actives was used to implement a novel structure-based in silico screening approach that might identify further potential novel TR inhibitors.

In mice cynaropicrin (CYN) potently inhibits the proliferation of Trypanosoma brucei-the causative agent of Human African Trypanosomiasis-by a so far unknown mechanism. We hypothesized that CYNs α,β-unsaturated methylene moieties act as Michael acceptors for glutathione (GSH) and trypanothione (T(SH)2), the main low molecular mass thiols essential for unique redox metabolism of these parasites. The analysis of this putative mechanism and the effects of CYN on enzymes of the T(SH)2 redox metabolism including trypanothione reductase, trypanothione synthetase, glutathione-S-transferase, and ornithine decarboxylase are shown. A two step extraction protocol with subsequent UPLC-MS/MS analysis was established to quantify intra-cellular CYN, T(SH)2, GSH, as well as GS-CYN and T(S-CYN)2 adducts in intact T. b. rhodesiense cells. Within minutes of exposure to CYN, the cellular GSH and T(SH)2 pools were entirely depleted, and the parasites entered an apoptotic stage and died. CYN also showed inhibition of the ornithine decarboxylase similar to the positive control eflornithine. Significant interactions with the other enzymes involved in the T(SH)2 redox metabolism were not observed. Alongside many other biological activities sesquiterpene lactones including CYN have shown antitrypanosomal effects, which have been postulated to be linked to formation of Michael adducts with cellular nucleophiles. Here the interaction of CYN with biological thiols in a cellular system in general, and with trypanosomal T(SH)2 redox metabolism in particular, thus offering a molecular explanation for the antitrypanosomal activity is demonstrated. At the same time, the study provides a novel extraction and analysis protocol for components of the trypanosomal thiol metabolism.

Malaria causes millions of death cases per year. Since Plasmodium falciparum rapidly develops drug resistance, it is of high importance to investigate potential drug targets which may lead to novel rational therapy approaches. Here we report on the interaction of translationally controlled tumor protein of P. falciparum (PfTCTP) with the anti-malarial drug artemisinin. Furthermore, we investigated the crystal structure of PfTCTP. Using mass spectrometry, bioinformatic approaches and surface plasmon resonance spectroscopy, we identified novel binding sites of artemisinin which are in direct neighborhood to amino acids 19-46, 108-134 and 140-163. The regions covered by these residues are known to be functionally important for TCTP function. We conclude that interaction of artemisinin with TCTP may be at least in part explain the antimalarial activity of artemisinin.

Px IV is a distant relative of the known glutathione peroxidase-type enzymes of African trypanosomes. Immunofluorescence microscopy of bloodstream cells expressing C-terminally Myc6-tagged Px IV revealed a mitochondrial localization. Recombinant Px IV possesses very low activity as glutathione peroxidase but catalyzes the trypanothione/tryparedoxin-dependent reduction of hydrogen peroxide and, even more efficiently, of arachidonic acid hydroperoxide. Neither overexpression in bloodstream cells nor the deletion of both alleles in bloodstream or procyclic parasites affected the in vitro proliferation. Trypanosoma brucei Px IV shares 58% of all residues with TcGPXII. The orthologous enzymes have in common their substrate preference for fatty acid hydroperoxides. However, the T. cruzi protein has been reported to be localized in the endoplasmic reticulum and to be specific for glutathione as reducing agent. Taken together, our data show that Px IV is a low abundant tryparedoxin peroxidase of T. brucei that is not essential, at least under culture conditions.

Parasitic infections continue to be a major problem for global human health. Vaccines are practically not available and chemotherapy is highly unsatisfactory. One approach toward a novel antiparasitic drug development is to unravel pathways that may be suited as future targets. Parasitic organisms show a remarkable diversity with respect to the nature and functions of their main low-molecular-mass antioxidants and many of them developed pathways that do not have a counterpart in their mammalian hosts.Work of the last years disclosed the individual antioxidants employed by parasites and their distinct pathways. Entamoeba, Trichomonas, and Giardia directly use cysteine as main low-molecular-mass thiol but have divergent cysteine metabolisms. Malarial parasites rely exclusively on cysteine uptake and generate glutathione (GSH) as main free thiol as do metazoan parasites. Trypanosomes and Leishmania have a unique trypanothione-based thiol metabolism but employ individual mechanisms for their cysteine supply. In addition, some trypanosomatids synthesize ovothiol A and/or ascorbate. Various essential parasite enzymes such as trypanothione synthetase and trypanothione reductase in Trypanosomatids and the Schistosoma thioredoxin GSH reductase are currently intensively explored as drug target molecules.Essentiality is a prerequisite but not a sufficient property of an enzyme to become a suited drug target. The availability of an appropriate in vivo screening system and many other factors are equally important.The current organism-wide RNA-interference and proteome analyses are supposed to reveal many more interesting candidates for future drug development approaches directed against the parasite antioxidant defense systems.

In African trypanosomes, the detoxification of broad spectrum hydroperoxides relies on a unique cascade composed of trypanothione (T(SH)(2)), trypanothione reductase, tryparedoxin (Tpx), and nonselenium glutathione peroxidase-type enzymes. All three proteins are essential for Trypanosoma brucei. Here, we subjected the complete system to a high throughput screening approach with nearly 80,000 chemicals. Twelve compounds inhibited the peroxidase system. All but one carried chloroalkyl substituents. The detailed kinetic analysis showed that two compounds weakly inhibited trypanothione reductase, but none of them specifically interacted with the peroxidase. They proved to be time-dependent inhibitors of Tpx-modifying Cys-40, the first cysteine of its active site WCPPC motif. Importantly, gel shift assays verified Tpx as a target in the intact parasites. T(SH)(2), present in the in vitro assays and in the cells in high molar excess, did not interfere with Tpx inactivation. The compounds inhibited the proliferation of bloodstream T. brucei with EC(50) values down to

The objective of the present work was to evaluate the biological activities of the major bioactive compound, xanthatin, and other compounds from Xanthium strumarium (Asteraceae) leaves. Inhibition of bloodstream forms of Trypanosoma brucei brucei and leukaemia HL-60 cell proliferation was assessed using resazurin as a vital stain. Xanthatin was found to be the major and most active compound against T. b. brucei with an IC50 value of 2.63 µg/mL and a selectivity index of 20. The possible mode of action of xanthatin was further evaluated. Xanthatin showed antiinflammatory activity by inhibiting both PGE2 synthesis (24% inhibition) and 5-lipoxygenase activity (92% inhibition) at concentrations of 100 µg/mL and 97 µg/mL, respectively. Xanthatin exhibited weak irreversible inhibition of parasite specific trypanothione reductase. Unlike xanthatin, diminazene aceturate and ethidium bromide showed strong DNA intercalation with IC50 values of 26.04 µg/mL and 44.70 µg/mL, respectively. Substantial induction of caspase 3/7 activity in MIA PaCa-2 cells was observed after 6 h of treatment with 100 µg/mL of xanthatin. All these data taken together suggest that xanthatin exerts its biological activity by inducing apoptosis and inhibiting both PGE2 synthesis and 5-lipoxygenase activity thereby avoiding unwanted inflammation commonly observed in diseases such as trypanosomiasis. Copyright © 2011 John Wiley & Sons, Ltd.

Hydroperoxide detoxification in African trypanosomes is achieved by 2-Cys-peroxiredoxin (TXNPx)- and non-selenium glutathione peroxidase (Px)-type enzymes which both obtain their reducing equivalents from the unique trypanothione/tryparedoxin system. Previous RNA interference approaches revealed that the cytosolic TXNPx and the Px-type enzymes are essential for Trypanosoma brucei. Because of partially overlapping in vitro substrate specificities and subcellular localisation the physiological function of the individual enzymes was not yet clear. As shown here, TXNPx and Px are expressed at comparable levels and in their active reduced state. Px-overexpressing parasites were less sensitive toward linoleic acid hydroperoxide but not hydrogen peroxide. Kinetic studies confirmed that Px—but not TXNPx—reduces lipophilic hydroperoxides including phospholipids with high efficiency. Most interestingly, the severe proliferation defect of Px-depleted bloodstream cells could be rescued by Trolox, but not by hydrophilic antioxidants, in the medium. This allowed us to knock-out the three Px genes individually and thus to distinguish their in vivo role. Deletion of the cytosolic Px I and II resulted in extremely fast membrane peroxidation followed by cell lysis. Cells lacking specifically the mitochondrial Px III showed a transient growth retardation and cardiolipin peroxidation but adapted within 24 h to normal proliferation.

Summary Lipoamide dehydrogenase (LipDH) is a component of four mitochondrial multienzyme complexes. RNA interference or the deletion of both alleles in bloodstream Trypanosoma brucei resulted in an absolute requirement for exogenous thymidine. In the absence of thymidine, lipdh−/− parasites showed a severely altered morphology and cell cycle distribution. Most probably, in bloodstream cells with their only rudimentary mitochondrion, LipDH is required as component of the glycine cleavage complex which generates methylene-tetrahydrofolate for dTMP and thus DNA synthesis. The essential role of LipDH in bloodstream parasites was confirmed by an in vivo model. Lipdh−/− cells were unable to infect mice. Our data further revealed that degradation of branched-chain amino acids takes place but is dispensable. In cultured bloodstream – but not procyclic – African trypanosomes, the total cellular concentration of LipDH increases with increasing cell densities. In procyclic parasites, LipDH mRNA depletion caused an even stronger proliferation defect that was not reversed by thymidine suggesting that in the fully elaborated mitochondrion of these cells the primary effect is not on the glycine cleavage complex. Since the medium used for the cultivation of procyclic cells was not supplemented with glucose, impairment of the 2-ketoglutarate dehydrogenase complex is probably the main effect of LipDH depletion.

Trypanothione reductase (TR) is an essential enzyme in the trypanothione-based redox metabolism of trypanosomatid parasites. This system is absent in humans and, therefore, offers a promising target for the development of selective new drugs against African sleeping sickness and Chagas' disease. Over the past two decades, a variety of nonpeptidic small-molecule ligands of the parasitic enzyme were discovered. A current goal is to decipher the binding mode of these known inhibitors in order to optimize their structures. We analyzed the binding mode of recently reported 1-(1-(benzo[b]thiophen-2-yl)cyclohexyl)piperidine (BTCP) analogues using computer modeling methods. This led us to conclude that the analogues occupy a different region of the active site than the diaryl sulfide-based class of inhibitors. A combination of the two motifs significantly increased affinity for the enzyme compared to the respective parent compounds. The newly synthesized conjugates exhibit K(ic) values for TR as low as 0.51+/-0.1 muM and high selectivity for the parasitic enzyme over the related human glutathione reductase (hGR), as was predicted by our molecular modeling studies. In vitro studies showed IC(50) values in the low micromolar to submicromolar range against Trypanosoma brucei rhodesiense, often in combination with low cytotoxicity against mammalian cells. Interestingly, even stronger activities were found against Plasmodium falciparum

Trypanosoma brucei, the causative agent of African sleeping sickness, possesses two dithiol glutaredoxins (Grx1 and Grx2). Grx1 occurs in the cytosol and catalyzes protein deglutathionylations with k(cat)/K(m)-values of up to 2 × 10(5) M(-1) S(-1). It accelerates the reduction of ribonucleotide reductase by trypanothione although less efficiently than the parasite tryparedoxin and has low insulin disulfide reductase activity. Despite its classical CPYC active site, Grx1 forms dimeric iron-sulfur complexes with GSH, glutathionylspermidine, or trypanothione as non-protein ligands. Thus, contrary to the generally accepted assumption, replacement of the Pro is not a prerequisite for cluster formation. T. brucei Grx2 shows an unusual CQFC active site, and orthologues occur exclusively in trypanosomatids. Grx2 is enriched in mitoplasts, and fractionated digitonin lysis resulted in a co-elution with cytochrome c, suggesting localization in the mitochondrial intermembrane space. Grx2 catalyzes the reduction of insulin disulfide but not of ribonucleotide reductase and exerts deglutathionylation activity 10-fold lower than that of Grx1. RNA interference against Grx2 caused a growth retardation of procyclic cells consistent with an essential role. Grx1 and Grx2 are constitutively expressed with cellular concentrations of about 2 μM and 200 nM, respectively, in both the mammalian bloodstream and insect procyclic forms. Trypanothione reduces the disulfide form of both proteins with apparent rate constants that are 3 orders of magnitude higher than those with glutathione. Grx1 and, less efficiently, also Grx2 catalyze the reduction of GSSG by trypanothione. Thus, the Grxs play exclusive roles in the trypanothione-based thiol redox metabolism of African trypanosomes.

Trypanothione reductase (TR) is a flavoenzyme unique to trypanosomatid parasites and a target for lead discovery programs. Various inhibitor scaffolds have emerged in the past, exhibiting moderate affinity for the parasite enzyme. Herein we show that the combination of two structural motifs of known TR inhibitors - diaryl sulfides and mepacrine - enables the simultaneous addressing of two hydrophobic patches in the active site. The binding efficacy of these conjugates is enhanced over that of the respective parent inhibitors. They show K(ic) values for the parasite enzyme down to 0.9+/-0.1 microm and exhibit high selectivity for TR over human glutathione reductase (GR). Despite their considerable molecular mass and in some cases permanent positive charges, in vitro studies revealed IC(50) values in the low micromolar to sub-micromolar range against Trypanosoma brucei rhodesiense and Trypanosoma cruzi, as well as the malaria parasite Plasmodium falciparum, which lack trypanothione metabolism. The inhibitors exhibit strong fluorescence due to their aminoacridine moiety. This feature allows visualization of the drugs in the parasite where high accumulation was observed by fluorescence microscopy even after short exposure times.

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 2 O 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.

In trypanosomes, the thioredoxin-type protein TXN (tryparedoxin) is a multi-purpose oxidoreductase that is involved in the detoxification of hydroperoxides, the synthesis of DNA precursors and the replication of the kinetoplastid DNA. African trypanosomes possess two isoforms that are localized in the cytosol and in the mitochondrion of the parasites respectively. Here we report on the biological significance of the cTXN (cytosolic TXN) of Trypanosoma brucei for hydroperoxide detoxification. Depending on the growth phase, the concentration of the protein is 3–7-fold higher in the parasite form infecting mammals (50–100 μM) than in the form hosted by the tsetse fly (7–34 μM). Depletion of the mRNA in bloodstream trypanosomes by RNA interference revealed the indispensability of the protein. Proliferation and viability of cultured trypanosomes were impaired when TXN was lowered to 1 μM for more than 48 h. Although the levels of glutathione, glutathionylspermidine and trypanothione were increased 2–3.5-fold, the sensitivity against exogenously generated H2O2 was significantly enhanced. The results prove the essential role of the cTXN and its pivotal function in the parasite defence against oxidative stress.

A significant improvement in the treatment of trypanosomiases has been achieved with the recent development of nifurtimox-eflornithine combination therapy (NECT). As an alternative to drug combinations and as a means to overcome most of the antitrypanosomatid drug discovery challenges, a multitarget drug design strategy has been envisaged. To begin testing this hypothesis, we designed and developed a series of quinone-coumarin hybrids against glyceraldehyde-3-phosphate dehydrogenase/trypanothione reductase (GAPDH/TR). These enzymes belong to metabolic pathways that are vital to Trypanosoma brucei and Trypanosoma cruzi, and have thus been considered promising drug targets. The synthesized molecules were characterized for their dual-target antitrypanosomal profile, both in enzyme assays and in in vitro parasite cultures. The merged derivative 2-{[3-(3-dimethylaminopropoxy)-2-oxo-2H-chromen-7-yl]oxy}anthracene-1,4-dione (10) showed an IC50 value of 5.4 μM against TbGAPDH and a concomitant Ki value of 2.32 μM against TcTR. Notably, 2-{4-[6-(2-dimethylaminoethoxy)-2-oxo-2H-chromen-3-yl]phenoxy}anthracene-1,4-dione (compound 6) displayed a remarkable EC50 value for T.brucei parasites (0.026 μM) combined with a very low cytotoxicity toward mammalian L6 cells (7.95 μM). This promising low toxicity of compound 6 might be at least partially due to the fact that it does not interfere with human glutathione reductase.

As constituents of their unusual trypanothione-based thiol metabolism, African trypanosomes express two dithiol glutaredoxins (Grxs), a cytosolic Grx1 and a mitochondrial Grx2, with so far unknown biological functions. As revealed by gel shift assays, in the mammalian bloodstream form of Trypanosoma brucei, Grx1 is in the fully reduced state. Upon diamide treatment of the cells, Grx1 forms an active site disulfide bridge that is rapidly re-reduced after stress removal; Cys76, a conserved non-active site Cys remains in the thiol state. Deletion of both grx1 alleles does not result in any proliferation defect of neither the procyclic insect form nor the bloodstream form, even not under various stress conditions. In addition, the Grx1-deficient parasites are fully infectious in the mouse model. A functional compensation by Grx2 is unlikely as identical levels of Grx2 were found in wildtype and Grx1-deficient cells. In the classical hydroxyethyl disulfide assay, Grx1-deficient bloodstream cells display 50-60% of the activity of wildtype cells indicating that the cytosolic oxidoreductase accounts for a major part of the total deglutathionylation capacity of the parasite. Intriguingly, at elevated temperature, proliferation of the Grx1-deficient bloodstream parasites is significantly less affected compared to wildtype cells. When cultured for three days at 39°C, only 51% of the cells in the wildtype population retained normal morphology with single mitochondrial and nuclear DNA (1K1N), whereas 27% of the cells displayed ≥2K2N. In comparison, 64% of the Grx1-deficient cells kept the 1K1N phenotype and only 18% had ≥2K2N. The data suggest that Grx1 plays a role in the regulation of the thermotolerance of the parasites by (in)directly interfering with the progression of the cell cycle, a process that may comprise protein (de)glutathionylation step(s).

African trypanosomes express three virtually identical glutathione peroxidase (Px)-type enzymes that occur in the cytosol (Px I and II) and mitochondrion (Px III) and detoxify fatty acid-derived hydroperoxides. Selective deletion of the genes revealed that procyclic Trypanosoma brucei lacking either the cytosolic or mitochondrial enzyme proliferate nearly as wild-type parasites, whereas the knockout of the complete genomic locus is lethal. Flow cytometry and immunofluorescence analyses revealed that the Px I-III-deficient parasites lose their mitochondrial membrane potential, which is followed by a loss of the lysosomal signal but not the glycosomal one. Mitochondrial damage and cell lysis are prevented by Trolox, ubiquinone derivatives and the iron chelator deferoxamine, whereas starch-deferoxamine is inefficient. In glucose-rich medium, cell death is attenuated suggesting that oxidants generated by the respiratory chain contribute to the lethal phenotype. Thus, the Px-type peroxidases protect procyclic cells from an iron-mediated oxidative membrane damage that originates at the mitochondrion. This contrasts with the situation in bloodstream cells, where the lysosome is the primarily affected organelle. Strikingly, either the cytosolic or the mitochondrial form of the peroxidases is required and sufficient to protect the mitochondrion and prevent cell lysis.

Trypanosomatids are the causative agents of African sleeping sickness, Chagas' disease, and the different forms of leishmaniasis. This family of protozoan parasite possesses a trypanothione-based redox metabolism that provides the reducing equivalents for various vital processes such as the biosynthesis of DNA precursors and the detoxification of hydroperoxides. Almost all enzymes of the redox pathway proved to be essential and therefore fulfil one crucial prerequisite for a putative drug target. Trypanothione synthetase and trypanothione reductase are present in all trypanosomatids but absent from the mammalian host which, in addition to the essentiality, renders them highly specific. The chemotherapy research on both enzymes is further supported by the availability of high-throughput screening techniques and crystal structures. In this review we focus on the recent advances and limitations in the development of lead compounds targeting trypanothione synthetase and trypanothione reductase. We present an overview of the available inhibitors and discuss future perspectives including other components of the parasite-specific redox pathway.

A number of phenothiazine-, phenoxazine- and related tricyclics-derived chloroacetamides were synthesized and evaluated in vitro for antiprotozoal activities against Leishmania major (L. major) promastigotes. Several analogs were remarkably potent inhibitors, with antileishmanial activities being comparable or superior to those of the reference antiprotozoal drugs. Furthermore, we explored the structure-activity relationships of N-10 haloacetamides that influence the potency of such analogs toward inhibition of L. major promastigote growth in vitro. With respect to the mechanism of action, selected compounds were evaluated for time-dependent inactivation of Trypanosoma brucei trypanothione reductase. Our results are indicative of a covalent interaction which could account for potent antiprotozoal activities.

With the goal to identify novel trypanothione reductase (TR) inhibitors, we performed a combination of in vitro and in silico screening approaches. Starting from a highly diverse compound set of 2,816 compounds, 21 novel TR inhibiting compounds could be identified in the initial in vitro screening campaign against T. cruzi TR. All 21 in vitro hits were used in a subsequent similarity search-based in silico screening on a database containing 200,000 physically available compounds. The similarity search resulted in a data set containing 1,204 potential TR inhibitors, which was subjected to a second in vitro screening campaign leading to 61 additional active compounds. This corresponds to an approximately 10-fold enrichment compared to the initial pure in vitro screening. In total, 82 novel TR inhibitors with activities down to the nM range could be identified proving the validity of our combined in vitro/in silico approach. Moreover, the four most active compounds, showing IC50 values of, Author Summary Trypanosomatidae are responsible for approximately half a million human fatalities per annum and the situation is compounded by substantial economic losses due to affecting live stock as well. Trypanothione reductase (TR) is an essential key enzyme of the unique trypanothione-based thiol metabolism of the trypanosomatidae and TR is a promising target for the development of selective inhibitors. However, TR is a very hard to attack target in standard drug discovery approaches. Therefore, we developed a combined and iterative in vitro and in silico screening approach, which led to a high number of novel TR inhibitors. 82 of those showed activities down to the nM range against T. cruzi TR. Moreover, the four most active compounds were selected for determining the inhibitor constant. In first on parasite efficacy studies, three of those compounds inhibited the proliferation of bloodstream T. brucei cell line 449 with EC50 values down to 2 μM.