Trypanothione Reductase Activity Research Articles

Identification of host antioxidant effectors as thioridazine targets: Impact on cardiomyocytes infection and Trypanosoma cruzi-induced acute myocarditis

Phenothiazines inhibit antioxidant enzymes in trypanosomatids. However, potential interferences with host cell antioxidant defenses are central concerns in using these drugs to treat Trypanosoma cruzi-induced infectious myocarditis. Thus, the interaction of thioridazine (TDZ) with T. cruzi and cardiomyocytes antioxidant enzymes, and its impact on cardiomyocytes and cardiac infection was investigated in vitro and in vivo. Cardiomyocytes and trypomastigotes in culture, and mice treated with TDZ and benznidazole (Bz, reference antiparasitic drug) were submitted to microstructural, biochemical and molecular analyses. TDZ was more cytotoxic and less selective against T. cruzi than Bz in vitro. TDZ-pretreated cardiomyocytes developed increased infection rate, reactive oxygen species (ROS) production, lipid and protein oxidation; similar catalase (CAT) and superoxide dismutase (SOD) activity, and reduced glutathione's (peroxidase - GPx, S-transferase – GST, and reductase – GR) activity than infected untreated cells. TDZ attenuated trypanothione reductase activity in T. cruzi, and protein antioxidant capacity in cardiomyocytes, making these cells more susceptible to H2O2-based oxidative challenge. In vivo, TDZ potentiated heart parasitism, total ROS production, myocarditis, lipid and protein oxidation; as well as reduced GPx, GR, and GST activities compared to untreated mice. Benznidazole decreased heart parasitism, total ROS production, heart inflammation, lipid and protein oxidation in T. cruzi-infected mice. Our findings indicate that TDZ simultaneously interact with enzymatic antioxidant targets in cardiomyocytes and T. cruzi, potentiating the infection by inducing antioxidant fragility and increasing cardiomyocytes and heart susceptibility to parasitism, inflammation and oxidative damage.

Assessing proteases and enzymes of the trypanothione system in subpopulations of Leishmania (Viannia) braziliensis Thor strain during macrophage infection.

Leishmania (Viannia) braziliensis Thor strain exhibits a heterogeneous composition comprised of subpopulations with varying levels of infectivity. Clonal subpopulations were previously obtained from the strain Thor by sorting single-parasites and proceeding cultivation. The subpopulations used in this study are named Thor03, Thor 10 and Thor22. Phenotypic characteristics of the parasite, specially focusing on virulence factors and resistance to the antimicrobial mechanisms of macrophages, were investigate in these subpopulations. Cellular and molecular biology, as well as biochemistry approaches were applied to obtain the data analysed in this study. Relative quantification of gene expression was measured for calpain, cysteine protease B (CPB), and subtilisin proteases but no significant differences in these genes' expression among subpopulations was observed. However, subtilisin and CPB proteins were assessed as more abundant in Thor03 by fluorescence-labelled flow cytometry technique. Western Blotting assays, as semi-quantitative analysis in gel, showed higher concentrations of subtilisin (110 to 50 kDa) and CPB (40 to 18 kDa) in extract of intracellular amastigotes from subpopulations Thor03 and Thor10 and calpain (60 to 25 kDa) showed no significant differences among subpopulations. Complementary, higher trypanothione reductase activity was observed in Thor10 intracellular amastigotes and assays of susceptibility to hydrogen peroxide-inducing agents and nitric oxide donors conducted with promastigotes revealed greater resistance to in vitro oxidative stress induction for Thor10, followed by Thor03. The data obtained for the virulence factors explored here suggest how multiple coexisting phenotypic-distinct subpopulations may contribute in adaptability of a single L. (V.) braziliensis strain during infection in the host cells.

Pharmacological potential of new metronidazole/eugenol/dihydroeugenol hybrids against Trypanosoma cruzi in vitro and in vivo.

From well-delimited immunomodulatory, redox and antimicrobial properties; metronidazole and eugenol were used as structural platforms to assembly two new molecular hybrids (AD06 and AD07), whose therapeutic relevance was analyzed on T. cruzi infection in vitro and in vivo. Non-infected, T. cruzi-infected H9c2 cardiomyocytes, and mice non-treated and treated with vehicle, benznidazole (Bz - reference drug), AD06 and AD07 were investigated. Parasitological, prooxidant, antioxidant, microstructural, immunological, and hepatic function markers were analyzed. Our findings indicated that in addition to having a direct antiparasitic effect on T. cruzi, metronidazole/eugenol hybrids (especially AD07) attenuated cellular parasitism, reactive species biosynthesis and oxidative stress in infected cardiomyocytes in vitro. Although AD06 and AD07 exerted no relevant impact on antioxidant enzymes activity (CAT, SOD, GR and GPx) in host cells, these drugs (especially AD07) attenuated trypanothione reductase activity in T. cruzi, which increased parasite's susceptibility to in vitro pro-oxidant challenge. AD06 and AD07 were well tolerated and do not determine humoral response suppression, mortality (100% survival) or hepatotoxicity in mice, as indicated by transaminases plasma levels. AD07 also induced relevant in vivo antiparasitic and cardioprotective effects, attenuating parasitemia, cardiac parasite load and myocarditis in T. cruzi-infected mice. Although this cardioprotective response is potentially related to AD07 antiparasitic effect, a direct anti-inflammatory potential of this molecular hybrid cannot be ruled out. Taken together, our findings indicated that the new molecular hybrid AD07 stood out as a potentially relevant candidate for the development of new, safe and more effective drug regimens for T. cruzi infection treatment.

Assessing the effect of antimony pressure on trypanothione reductase activity in Leishmania (Viannia) braziliensis

Leishmania parasites have an oxidative and chemical defense mechanism called trypanothione system (T[SH]2), the most abundant thiol system in trypanosomatids. This system has a central role in processing pentavalent antimony and resistance has been related to a better capacity to metabolize it through the activation of T[SH]2 enzymatic cascade. A biochemical approach was applied to assess the effect of trivalent (SbIII) and pentavalent antimony (SbV) on Trypanothione Reductase (TR) activity of two Leishmania (Viannia) braziliensis clinical isolates, which were labeled as responder (R) and non-responder (NR) after patient treatment with Glucantime®. Both isolates were characterized based on in vitro susceptibility to SbIII and SbV and trypanothione reductase (TR) activity. SbIII and SbV discriminated susceptibility profiles in all parasite forms, since isolate NR had significantly higher EC50 values than isolate R. Differences were observed in TR activity between promastigotes, axenic amastigotes and intracellular amastigotes: R (0.439 ± 0.009, 0.103 ± 0.01 and 0.185 ± 0.01AU.min−1.μg of protein−1) and NR (1.083 ± 0.04, 0.914 ± 0.04 and 0.343 ± 0.04 AU. min−1.μg of protein−1), respectively. Incubation with SbIII and SbV using each form EC50 value caused a time-dependent differential effect on TR activity suggesting that oxidative defense is related to the antimony susceptibility phenotype. Data gathered here shows a biochemical approach able to discriminate two L. (V.) braziliensis clinical isolates measurements TR activity of promastigotes, axenic amastigotes and intracellular amastigotes.

Innovative Approach for a Classic Target: Fragment Screening on Trypanothione Reductase Reveals New Opportunities for Drug Design.

Trypanothione reductase (TR) is a key factor in the redox homeostasis of trypanosomatid parasites, critical for survival in the hostile oxidative environment generated by the host to fight infection. TR is considered an attractive target for the development of new trypanocidal agents as it is essential for parasite survival but has no close homolog in humans. However, the high efficiency and turnover of TR challenging targets since only potent inhibitors, with nanomolar IC50, can significantly affect parasite redox state and viability. To aid the design of effective compounds targeting TR, we performed a fragment-based crystal screening at the Diamond Light Source XChem facility using a library optimized for follow-up synthesis steps. The experiment, allowing for testing over 300 compounds, resulted in the identification of 12 new ligands binding five different sites. Interestingly, the screening revealed the existence of an allosteric pocket close to the NADPH binding site, named the “doorstop pocket” since ligands binding at this site interfere with TR activity by hampering the “opening movement” needed to allow cofactor binding. The second remarkable site, known as the Z-site, identified by the screening, is located within the large trypanothione cavity but corresponds to a region not yet exploited for inhibition. The fragments binding to this site are close to each other and have some remarkable features making them ideal for follow-up optimization as a piperazine moiety in three out of five fragments.

Unraveling of interacting protein network of chaperonin TCP1 gamma subunit of Leishmania donovani.

T-complex polypeptide-1 (TCP1) is a group II chaperonin that folds various cellular proteins. About 10% of cytosolic proteins in yeast have been shown to flux through the TCP1 protein complex indicating that it interacts and folds a plethora of substrate proteins that perform essential functions. In Leishmania donovani, the gamma subunit of TCP1 (LdTCP1γ) has been shown to form a homo-oligomeric complex and exhibited ATP-dependent protein folding activity. LdTCP1γ is essential for the growth and infectivity of the parasite. The interacting partners of L. donovani TCP1γ, involved in many cellular processes, are far from being understood. In this study, we utilized co-immunoprecipitation assay coupled with liquid chromatography-mass spectrometry (LC-MS) to unravel protein-protein interaction (PPI) networks of LdTCP1γ in the L. donovani parasite. Label-free quantification (LFQ) proteomic analysis revealed 719 interacting partners of LdTCP1γ. String analysis showed that LdTCP1γ interacts with all subunits of TCP1 complex as well as other proteins belonging to pathways like metabolic process, ribosome, protein folding, sorting, and degradation. Trypanothione reductase, identified as one of the interacting partners, is refolded by LdTCP1γ. In addition, the differential expression of LdTCP1γ modulates the trypanothione reductase activity in L. donovani parasite. The study provides novel insight into the role of LdTCP1γ that will pave the way to better understand parasite biology by identifying the interacting partners of this chaperonin.

Trypanosuppressive effects of Kolaviron may be associated with down regulation of Trypanothione reductase in Trypanosoma congolense infection.

Trypanothione reductase is a key enzyme that upholds the redox balance in hemoflagellate protozoan parasites such as T. congolense. This study aims at unraveling the potency of Kolaviron against trypanothione reductase in T. congolense infection using Chrysin as standard. The experiment was performed using three different approaches; in silico, in vitro and in vivo. Kolaviron and Chrysin were docked against trypanothione reductase, revealing binding energies (-9.3 and -9.0 kcal/mol) and Ki of 0.211μM and 0.151μM at the active site of trypanothione reductase as evident from the observed strong hydrophobic/hydrogen bond interactions. Parasitized blood was used for parasite isolation and trypanothione reductase activity assay using standard protocol. Real-time PCR (qPCR) assay was implored to monitor expression of trypanothione reductase using primers targeting the 177-bp repeat satellite DNA in T. congolense with SYBR Green to monitor product accumulation. Kolaviron showed IC50 values of 2.64μg/ml with % inhibition of 66.78 compared with Chrysin with IC50 values of 1.86μg/ml and % inhibition of 53.80. In vivo studies following the administration of these compounds orally after 7 days post inoculation resulted in % inhibition of Chrysin (57.67) and Kolaviron (46.90). Equally, Kolaviron relative to Chrysin down regulated the expression trypanothione reductase gene by 1.352 as compared to 3.530 of the infected group, in clear agreement with the earlier inhibition observed at the fine type level. Overall, the findings may have unraveled the Kolaviron potency against Trypanosoma congolense infection in rats.

Novel N,N-di-alkylnaphthoimidazolium derivative of β-lapachone impaired Trypanosoma cruzi mitochondrial electron transport system

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, a neglected tropical disease that is endemic in Latin America and spreading worldwide due to globalization. The current treatments are based on benznidazole and nifurtimox; however, these drugs have important limitations and limited efficacy during the chronic phase, reinforcing the necessity of an alternative chemotherapy. For the last 30 years, our group has been evaluating the biological activity of naphthoquinones and derivatives on T. cruzi, and of the compounds tested, N1, N2 and N3 were found to be the most active in vitro. Here, we show the synthesis of a novel β-lapachone-derived naphthoimidazolium named N4 and assess its activity on T. cruzi stages and the mechanism of action. The new compound was very active on all parasite stages (IC50/24 h in the range of 0.8–7.9 μM) and had a selectivity index of 5.4. Mechanistic analyses reveal that mitochondrial ROS production begins after short treatment starts and primarily affects the activity of complexes II-III. After 24 h treatment, a partial restoration of mitochondrial physiology (normal complexes II-III and IV activities and controlled H2O2 release) was observed; however, an extensive injury in its morphology was still detected. During treatment with N4, we also observed that trypanothione reductase activity increased in a time-dependent manner and concomitant with increased oxidative stress. Molecular docking calculations indicated the ubiquinone binding site of succinate dehydrogenase as an important interaction point with N4, as with the FMN binding site of dihydroorotate dehydrogenase. The results presented here may be a good starting point for the development of alternative treatments for Chagas disease and for understanding the mechanism of naphthoimidazoles in T. cruzi.

Preclinical Gold Complexes as Oral Drug Candidates to Treat Leishmaniasis Are Potent Trypanothione Reductase Inhibitors

The drugs currently used to treat leishmaniases have limitations concerning cost, efficacy, and safety, making the search for new therapeutic approaches urgent. We found that the gold(I)-derived complexes were active against L.infantum and L.braziliensis intracellular amastigotes with IC50 values ranging from 0.5 to 5.5 μM. All gold(I) complexes were potent inhibitors of trypanothione reductase (TR), with enzyme IC50 values ranging from 1 to 7.8 μM. Triethylphosphine-derived complexes enhanced reactive oxygen species (ROS) production and decreased mitochondrial respiration after 2 h of exposure, indicating that gold(I) complexes cause oxidative stress by direct ROS production, by causing mitochondrial damage or by impairing TR activity and thus accumulating ROS. There was no cross-resistance to antimony; in fact, SbR (antimony-resistant mutants) strains were hypersensitive to some of the complexes. BALB/c mice infected with luciferase-expressing L.braziliensis or L.amazonensis and treated orally with 12.5 mg/kg/day of AdT Et (3) or AdO Et (4) presented reduced lesion size and parasite burden, as revealed by bioimaging. The combination of (3) and miltefosine allowed for a 50% reduction in miltefosine treatment time. Complexes 3 and 4 presented favorable pharmacokinetic and toxicity profiles that encourage further drug development studies. Gold(I) complexes are promising antileishmanial agents, with a potential for therapeutic use, including in leishmaniasis caused by antimony-resistant parasites.

Mitochondrial disfunction and ROS production are essential for anti-Trypanosoma cruzi activity of β-lapachone-derived naphthoimidazoles

Chagas disease is caused by the hemoflagellate protozoa Trypanosoma cruzi and is one of the most important neglected tropical diseases, especially in Latin American countries, where there is an association between low-income populations and mortality. The nitroderivatives used in current chemotherapy are far from ideal and present severe limitations, justifying the continuous search for alternative drugs. Since the1990s, our group has been investigating the trypanocidal activity of natural naphthoquinones and their derivatives, and three naphthoimidazoles (N1, N2 and N3) derived from β-lapachone were found to be most effective in vitro. Analysis of their mechanism of action via cellular, molecular and proteomic approaches indicates that the parasite mitochondrion contains one of the primary targets of these compounds, trypanothione synthetase (involved in trypanothione production), which is overexpressed after treatment with these compounds. Here, we further evaluated the participation of the mitochondria and reactive oxygen species (ROS) in the anti-T. cruzi action of naphthoimidazoles. Preincubation of epimastigotes and trypomastigotes with antioxidants (α-tocopherol and urate) strongly protected the parasites from the trypanocidal effect of naphthoimidazoles, decreasing the ROS levels produced and reverting the mitochondrial swelling phenotype. The addition of pro-oxidants (menadione and H2O2) before the treatment induced an increase in parasite lysis. Despite the O2 uptake and mitochondrial complex activity being strongly reduced by N1, N2 and N3, urate partially restored the mitochondrial metabolism only in N1-treated parasites. In parallel, MitoTEMPO, a mitochondrial-targeted antioxidant, protected the functionality of the mitochondria in N2- and N3-treated parasites. In addition, the trypanothione reductase activity was remarkably increased after treatment with N1 and N3, and molecular docking demonstrated that these two compounds were positioned in pockets of this enzyme. Based on our findings, the direct impairment of the mitochondrial electron transport chain by N2 and N3 led to an oxidative misbalance, which exacerbated ROS generation and led to parasite death. Although other mechanisms cannot be discounted, mainly in N1-treated parasites, further investigations are required.

Molecular docking study on the interaction between 2-substituted-4,5-difuryl Imidazoles with different Protein Target for antileishmanial activity

Molecular docking based inhibition of Trypanothione reductase activity by Taxifolin novel target for antileishmanial activityRavi Kumar Gundampati and Medicherla V. Jagannadham

Synthesis and 2D-QSAR studies of neolignan-based diaryl-tetrahydrofuran and -furan analogues with remarkable activity against Trypanosoma cruzi and assessment of the trypanothione reductase activity

Two series of diaryl-tetrahydrofuran and -furan were synthesised and screened for anti-trypanosomal activity against trypomastigote and amastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. Based on evidence that modification of a natural product may result in a more effective drug than the natural product itself, and using known neolignan inhibitors veraguensin 1 and grandisin 2 as templates to synthesise simpler analogues, remarkable anti-trypanosomal activity and selectivity were found for 3,5-dimethoxylated diaryl-furan 5c and 2,4-dimethoxylated diaryl-tetrahydrofuran 4e analogues with EC50 0.01 μM and EC50 0.75 μM, respectively, the former being 260-fold more potent than veraguensin 1 and 150-fold better than benznidazole, the current available drugs for Chagas disease treatment. The ability of the most potent anti-trypanosomal compounds to penetrate LLC-MK2 cells infected with T. cruzi amastigotes parasite was tested, which revealed 4e and 5e analogues as the most effective, causing no damage to mammalian cells. In particular, the majority of the derivatives were non-toxic against mice spleen cells. 2D-QSAR studies show the rigid central core and the position of dimethoxy-aryl substituents dramatically affect the anti-trypanosomal activity. The mode of action of the most active anti-trypanosomal derivatives was investigated by exploring the anti-oxidant functions of Trypanothione reductase (TR). As a result, diarylfuran series displayed the strongest inhibition, highlighting compounds 5d-e (IC50 19.2 and 17.7 μM) and 5f-g (IC50 8.9 and 7.4 μM), respectively, with similar or 2-fold higher than the reference inhibitor clomipramine (IC50 15.2 μM).

Cyclobenzaprine Raises ROS Levels in Leishmania infantum and Reduces Parasite Burden in Infected Mice.

BackgroundThe leishmanicidal action of tricyclic antidepressants has been studied and evidences have pointed that their action is linked to inhibition of trypanothione reductase, a key enzyme in the redox metabolism of pathogenic trypanosomes. Cyclobenzaprine (CBP) is a tricyclic structurally related to the antidepressant amitriptyline, differing only by the presence of a double bond in the central ring. This paper describes the effect of CBP in experimental visceral leishmaniasis, its inhibitory effect in trypanothione reductase and the potential immunomodulatory activity.Methodology/Principal FindingsIn vitro antileishmanial activity was determined in promastigotes and in L. infantum-infected macrophages. For in vivo studies, L. infantum-infected BALB/c mice were treated with CBP by oral gavage for five days and the parasite load was estimated. Trypanothione reductase activity was assessed in the soluble fraction of promastigotes of L. infantum. For evaluation of cytokines, L. infantum-infected macrophages were co-cultured with BALB/c splenocytes and treated with CBP for 48 h. The supernatant was analyzed for IL-6, IL-10, MCP-1, IFN-γ and TNF-α. CBP demonstrated an IC50 of 14.5±1.1μM and an IC90 of 74.5±1.2 μM in promastigotes and an IC50 of 12.6±1.05 μM and an IC90 of 28.7±1.3 μM in intracellular amastigotes. CBP also reduced the parasite load in L. infantum-infected mice by 40.4±10.3% and 66.7±10.5% in spleen at 24.64 and 49.28 mg/kg, respectively and by 85.6±5.0 and 89.3±4.8% in liver at 24.64 and 49.28mg/kg, after a short-term treatment. CBP inhibited the trypanothione reductase activity with a Ki of 86 ± 7.7 μM and increased the ROS production in promastigotes. CBP inhibited in 53% the production of IL-6 in infected macrophages co-culture.Conclusion/SignificanceTo the best of our knowledge, this study is the first report of the in vivo antileishmanial activity of the FDA-approved drug CBP. Modulation of immune response and induction of oxidative stress in parasite seem to contribute to this efficacy.

How do the alkaloids emetine and homoharringtonine kill trypanosomes? An insight into their molecular modes of action

BackgroundAlthough Trypanosoma brucei causes deadly sleeping sickness, the number of the registered medications is rather limited. Some plant alkaloids are potent trypanocidal agents. PurposeIn this study, we wanted to elucidate the molecular modes of trypanocidal activity of the alkaloids emetine and homoharringtonine against Trypanosoma brucei brucei. MethodsWe investigated the activity of both alkaloids regarding growth recovery from alkaloid-induced stress. We measured the inhibition of protein biosynthesis using the Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay kit. Reduction of mitochondrial membrane potential and cell cycle arrest were measured by means of flow cytometry. Additionally, we determined spectrophotometrically the inhibition of the trypanosome specific enzyme trypanothione reductase activity and DNA intercalation. ResultsBoth alkaloids prevented that parasites could resume normal growth after pretreatment with the alkaloids. They inhibited protein biosynthesis in a time- and concentration-dependent manner. In contrast to homoharringtonine, emetine is also a DNA intercalator. Homoharringtonine decreased the mitochondrial membrane potential. Both alkaloids caused cell cycle arrest. Both alkaloids failed to affect trypanothione reductase, a crucial component of the redox system of trypanosomes. ConclusionWe assume that both alkaloids are primarily inhibitors of protein biosynthesis in trypanosomes, with DNA intercalation as an additional mechanism for emetine. This is the first study that elucidates the molecular mode of trypanocidal action of emetine and homoharringtonine.

Further evidence of the trypanocidal action of eupomatenoid-5: Confirmation of involvement of reactive oxygen species and mitochondria owing to a reduction in trypanothione reductase activity

Our group assays natural products that are less toxic and more effective than available nitroheterocycles as promising therapeutic options for patients with Chagas disease. Our previous study reported the trypanocidal activity of eupomatenoid-5, a neolignan isolated from the leaves of Piper regnellii var. pallescens, against the three main parasitic forms of Trypanosoma cruzi. The present study further characterizes the biochemical and morphological alterations induced by this compound to elucidate the mechanisms of action involved in the cell death of T. cruzi. We show that eupomatenoid-5 induced oxidative imbalance in the three parasitic forms, especially trypomastigotes, reflected by a decrease in the activity of trypanothione reductase and increase in the formation of reactive oxygen species (ROS). A reduction of mitochondrial membrane potential was then triggered, further impairing the cell redox system through the production of more ROS and reactive nitrogen species. Altogether, these effects led to oxidative stress, reflected by lipid peroxidation and DNA fragmentation. These alterations are key events in the induction of parasite death through various pathways, including apoptosis, necrosis, and autophagy.

Molecular docking based inhibition of Trypanothione reductase activity by Taxifolin novel target for antileishmanial activity

Article history: Received on: 04/10/2012 Revised on: 16/10/2012 Accepted on: 21/10/2012 Available online: 29/10/2012 The theoretical docking study, conducted on a sample of previously reported for anti-inflammatory and antioxidant activities of Taxifolin at the binding site of Leishmania infantum trypanothione reductase (Try R) examine interaction energy. Taxifolin is widely used in the traditional medicine have been investigated for their putative chemo preventive and antileishmanial properties for the last few decades. A theoretical docking study, the evaluation of Taxifolin as inhibitor of trypanothione reductase a validated drug target enzyme of the Leishmania parasite. Taxifolin was found to bind at active site of L. infantum TryR with lowest binding energy and RMSD values to be -8.82 Kcal/Mol and 2.0 A respectively. Docking analysis of TryR with ligand enabled us to identify specific residues viz. Ser-14, Ala-47, Ser-162, Thr-336 and Arg-286, within the TryR binding pocket to play an important role in ligand binding affinity. The availability of TryR built model, together with insights gained from docking analysis will promote the rational design of potent and selective TryR inhibitor as antileishmanial therapeutic. The study contributes towards understanding mechanism of antileshmanial effect of the Taxifolin. This compound has shown promising biological activity in preliminary studies by targeting multiple signaling pathways. Thus on the basis of our in silico studies we hypothesize that this compound into Taxifolin can be inhibitory effect on against leishmaniasis.

Comparative proteomics profiling of a gentamicin-attenuated Leishmania infantum cell line identifies key changes in parasite thiol-redox metabolism

We have previously described an attenuated line of Leishmania infantum (H-line), selected by culturing promastigotes in vitro in the presence of gentamicin. To elucidate the molecular basis for this attenuation, we undertook a comparative proteomic analysis using multiplex 2-dimensional (2D) difference gel electrophoresis. Eighteen proteins that showed significant and reproducible changes in expression were identified. Many of these were components of the thiol-redox control system in Leishmania and this observation, validated by Western blot, prompted us to investigate the sensitivity of the attenuated line to oxidative stress. The attenuated line was found to be significantly more susceptible to hydrogen peroxide, a change which may explain the loss of virulence. In a direct assay of trypanothione-dependent peroxidase activity, hydrogen peroxide metabolism in the H-line was significantly lower than in wild type. Furthermore, trypanothione reductase activity was significantly lower in the H-line, suggesting that gentamicin selection may result in pleiotropic affects on thiol metabolism in Leishmania. A putative RNA-binding protein was very strongly up-regulated in the attenuated line, suggesting a possible target for gentamicin in Leishmania.

Inhibitory Effect of Silver Nanoparticles on Trypanothione Reductase Activity and Leishmania infantum Proliferation.

In Leishmania the glutathione/glutathione reductase eukaryotic redox sys-tem is replaced by the unique trypanothione/trypanothione reductase (TR) system. In vitro, silver is a more effective TR inhibitor than antimony, the first line drug against leishmaniasis in most endemic countries, and its mechanism of inhibition is similar to that of Sb(III). In particular, silver binds with high affinity to the catalytic triad Cys52, Cys57, and His461', thereby inhibiting TR. Here, Ag(0) activity was tested on the promastigote and amastigote stages of Leishmania infantum using a drug-delivery system consisting in Ag(0) nanoparticles encapsulated by ferritin molecules (PfFt-AgNPs). These were able to induce an antiproliferative effect on the parasites at metal concentrations lower than those used with antimony.

Complementary medicinal chemistry-driven strategies toward new antitrypanosomal and antileishmanial lead drug candidates

Trypanosomiases and Leishmaniases are neglected tropical diseases that affect the less developed countries. For this reason, they did not and still do not have high visibility in Western societies. The name neglected diseases also refers to the fact that they often received little interest at the level of public investment, research and development. The drug discovery scenario, however, is changing dramatically. After a period in which different socioeconomic factors have prevented massive research efforts in this field, such efforts have increased considerably in the very recent years, with significant scientific advancements. In this context, we have embarked on a new drug discovery project devoted to identification of new small molecules for the treatment of trypanosomal and leishmanial diseases. Two complementary approaches have been pursued and are reported here. The first deals with a structure-based drug design, and a privileged structure-guided synthesis of quinazoline compounds able to modulate trypanothione reductase activity was accomplished. In the second, a combinatorial library, built on a natural product-based strategy, was synthesized. Using whole parasite assays, different quinones have been identified as promising lead compounds. A combination of both approaches to hopefully overcome some of the challenges of anti-trypanosomatid drug discovery has eventually been proposed.

Molecular Basis of Antimony Treatment in Leishmaniasis

Leishmaniasis is a disease that affects 2 million people and kills 70000 persons every year. It is caused by Leishmania species, which are human protozoan parasites of the trypanosomatidae family. Trypanosomatidae differ from the other eukaryotes in their specific redox metabolism because the glutathione/glutathione reductase system is replaced by the unique trypanothione/trypanothione reductase system. The current treatment of leishmaniasis relies mainly on antimonial drugs. The crystal structures of oxidized trypanothione reductase (TR) from Leishmania infantum and of the complex of reduced TR with NADPH and Sb(III), reported in this paper, disclose for the first time the molecular mechanism of action of antimonial drugs against the parasite. Sb(III), which is coordinated by the two redox-active catalytic cysteine residues (Cys52 and Cys57), one threonine residue (Thr335), and His461' of the 2-fold symmetry related subunit in the dimer, strongly inhibits TR activity. Because TR is essential for the parasite survival and virulence and it is absent in mammalian cells, these findings provide insights toward the design of new more affordable and less toxic drugs against Leishmaniasis.