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This work aims to develop poly(d,l-lactide-co-glycolide) (PLGA)-nanospheres containing amphotericin B (AmB) with suitable physicochemical properties and anti-parasitic activity for visceral leishmaniasis (VL) therapy. When compared with unloaded-PLGA-nanospheres, the AmB-loaded PLGA-nanospheres displayed an increased particle size without affecting the polydispersity and its negative surface charge. AmB stability in the PLGA-nanospheres was >90% over 60-days at 30°C. The AmB-PLGA-nanospheres demonstrated significant in vitro and in vivo efficacy and preferential accumulation in the visceral organs. In addition, an immune-modulatory effect was observed in mice treated with AmB-PLGA-nanospheres, correlating with improved treatment efficacy. The in vitro cytotoxic response of the T-lymphocytes revealed that AmB-PLGA-nanospheres efficacy against VL infection was strictly due to the action of CD8+- but not CD4+-T lymphocytes. Overall, we demonstrate a crucial role for CD8+ cytotoxic T lymphocytes in the efficacy of AmB-PLGA nanospheres, which could represent a potent and affordable alternative for VL therapy. [ABSTRACT FROM AUTHOR]

Abstract: Perkinsus atlanticus is a pathogenic protist that infects the clam Ruditapes decussatus. Although it was recently proposed that the genus Perkinsus belongs to a new phylum, Perkinsozoa, in the infra-kingdom Alveolata, there remain different opinions about whether this genus should form a phylum on its own and consequently divergent views about its taxonomic characterization. In this work, we have identified nine chromosomes by pulsed field gel electrophoresis (PFGE) combined with densitometry analysis. The obtained karyotype of Perkinsus atlanticus, like that of other early branches of the dinoflagellate lineage, displays a more conventional chromosome organization, different from that of most dinoflagellates. [Copyright &y& Elsevier]

Three polyamine derivatives assigned as bis-naphthalimidopropyl putrescine (BNIPPut), spermidine (BNIPSpd) and spermine (BNIPSpm) were studied to determine their effects on the proliferation of murine splenocytes and human peripheral blood mononuclear cells (PBMC) induced by the mitogens, Con A, LPS and PHA. All compounds showed a dose dependent inhibitory effect on mouse and human T cell proliferation induced by the mitogens, with BNIPPut exhibiting the most potent antiproliferative activity, followed by BNIPSpd and by BNIPSpm, respectively (Put>Spd>Spm), when considering human T cells. This suppressive activity also affects the capacity of mouse spleen cells to produce Th1 cytokines, namely IL-2 and INF-γ after in vitro stimulation with Con A. The polyamine-induced inhibition also occurred in the case of LPS-stimulated B cells with a marked decrease of CD69 expression by these cells. Furthermore, the ability for these polyamine derivatives to induce apoptosis on Con A-stimulated splenocytes could be related to their antiproliferative activity. [Copyright &y& Elsevier]

The intracellular protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas'' disease. We have previously characterized a T. cruzi virulence factor named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family. Single mutant parasite clones (Tc52+/−) exhibiting low virulence in vitro and in vivo were obtained by targeted Tc52 gene replacement. In this report, we have extended our study to analyze the immune response and the disease phenotype in Tc52+/−-infected BALB/c mice, during the acute and chronic phases of the disease. Significantly lower parasitemia were found in Tc52+/−-infected mice, as compared to wild-type parasite (WT)-infected ones. However, the expansion of all classes of lymphocytes and macrophages was similar for both clones. Furthermore, except for IgG2b levels which were higher in the case of WT-infected mice, all classes of Ig presented no significant difference for WT and Tc52+/−-infected animals. Interestingly, a lack of suppression of IL-2 production and of T-cell proliferation inhibition was observed in the case of spleen cells from Tc52+/−-infected mice. Finally, the pattern of inflammation process was different and characterized as diffused in the case of Tc52+/−-infected mice, or presenting numerous foci in the case of WT-infected mice. Localization of the Tc52 protein in tissue sections and infected heart cell primary cultures by immunofluorescence and immunogold labeling, respectively, revealed the presence of Tc52 at the amastigote surface and associated to aggregates within host cell vesicles. Taken together, these results reinforce the notion of Tc52 being a virulence factor playing a role in the phenotype of the immune response associated to the infection and on the course of the disease. [Copyright &y& Elsevier]

Leishmania infected macrophages have conditions to produce adenosine. Despite its known immunosuppressive effects, no studies have yet established whether adenosine alter Leishmania parasitic burden upon macrophage infection. This work aimed at investigating whether endogenous adenosine exerts an autocrine modulation of macrophage response towards Leishmania infection, identifying its origin and potential pharmacological targets for visceral leishmaniasis (VL), using THP-1 differentiated macrophages. Adenosine deaminase treatment of infected THP-1 cells reduced the parasitic burden (29.1 ± 2.2%, P < 0.05). Adenosine A 2A and A 2B receptor subtypes expression was confirmed by RT-qPCR and by immunocytochemistry and their blockade with selective adenosine A 2A and A 2B antagonists reduced the parasitic burden [14.5 ± 3.1% (P < 0.05) and 12.3 ± 3.1% (P < 0.05), respectively; and 24.9 ± 2.8% (P < 0.05), by the combination of the two antagonists)], suggesting that adenosine A 2 receptors are tonically activated in infected THP-1 differentiated macrophages. The tonic activation of adenosine A 2 receptors was dependent on the release of intracellular adenosine through equilibrative nucleoside transporters (ENT1/ENT2): NBTI or dipyridamole reduced (~25%) whereas, when ENTs were blocked, adenosine A 2 receptor antagonists failed to reduce and A 2 agonists increase parasitic burden. Effects of adenosine A 2 receptors antagonists and ENT1/2 inhibitor were prevented by L-NAME, indicating that nitric oxide production inhibition prevents adenosine from increasing parasitic burden. Results suggest that intracellular adenosine, released through ENTs, elicits an autocrine increase in parasitic burden in THP-1 macrophages, through adenosine A 2 receptors activation. These observations open the possibility to use well-established ENT inhibitors or adenosine A 2 receptor antagonists as new therapeutic approaches in VL. • THP-1 macrophages express adenosine receptor (AR) subtypes A 1 , A 2A and A 2B. • Both A 2A and A 2B AR are tonically activated in infected THP-1 macrophages. • A 2 AR activation is dependent on the released of adenosine through the ENTs. • A 2 AR activation inhibits nitric oxide production in infected macrophages. [ABSTRACT FROM AUTHOR]

Pteridine reductase 1 (PTR1) is a catalytic protein belonging to the folate metabolic pathway in Trypanosmatidic parasites. PTR1 is a known target for the medicinal chemistry development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. In previous studies, new nitro derivatives were elaborated as PTR1 inhibitors. The compounds showing a diamino-pyrimidine core structure were previously developed but they showed limited efficacy. Therefore, a new class of phenyl-, heteroaryl- and benzyloxy-nitro derivatives based on the 2-nitroethyl-2,4,6-triaminopyrimidine scaffold were designed and tested. The compounds were assayed for their ability to inhibit T. brucei and L. major PTR1 enzymes and for their antiparasitic activity towards T. brucei and L. infantum parasites. To understand the structure-activity relationships of the compounds against Tb PTR1, the X-ray crystallographic structure of the 2,4,6-triaminopyrimidine (TAP) was obtained and molecular modelling studies were performed. As a next step, only the most effective compounds against T. brucei were then tested against the amastigote cellular stage of T. cruzi , searching for a broad-spectrum antiprotozoal agent. An early ADME-Tox profile evaluation was performed. The early toxicity profile of this class of compounds was investigated by measuring their inhibition of h ERG and five cytochrome P450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), cytotoxicity towards A549 cells and mitochondrial toxicity. Pharmacokinetic studies (SNAP-PK) were performed on selected compounds using hydroxypropyl-β-cyclodextrins (50 % w/v) to preliminarily study their plasma concentration when administered per os at a dose of 20 mg/kg. Compound 1p , showed the best pharmacodynamic and pharmacokinetic properties, can be considered a good candidate for further bioavailability and efficacy studies. [Display omitted] • Trypanosomiasis still represents a Neglected Infectious Disease and a medical need. • Pteridine reductase (PTR1) is a target specific for trypansomatdic infections. • 2-nitroethyl-2,4,6-triaminopyrimidine derivatives show low nanoMolar potency against PTR1. • 5-(1-(benzo[ b ]thiophen-3-yl)-2-nitroethyl) derivative show a suitable pharmacokinetic in mice. [ABSTRACT FROM AUTHOR]

Basing on a library of thiadiazole derivatives showing anti-trypanosomatidic activity, we have considered the thiadiazoles opened forms and reaction intermediates, thiosemicarbazones, as compounds of interest for phenotypic screening against Trypanosoma brucei (Tb), intracellular amastigote form of Leishmania infantum (Li) and Trypanosoma cruzi (Tc) . Similar compounds have already shown interesting activity against the same organisms. The compounds were particularly effective against T. brucei and T. cruzi . Among the 28 synthesized compounds, the best one was (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene) hydrazinecarbothioamide ( A14 ) yielding a comparable anti-parasitic activity against the three parasitic species ( Tb EC 50  = 2.31 μM, Li EC 50  = 6.14 μM, Tc EC 50  = 1.31 μM) and a Selectivity Index higher than 10 with respect to human macrophages, therefore showing a pan-anti-trypanosomatidic activity. (E)-2-((3'.4′-dimethoxy-[1.1′-biphenyl]-3-yl)methyle ne) hydrazinecarbothioamide ( A12) and (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene)hydrazine carbothioamide ( A14 ) were able to potentiate the anti-parasitic activity of methotrexate (MTX) when evaluated in combination against T. brucei , yielding a 6-fold and 4-fold respectively Dose Reduction Index for MTX. The toxicity profile against four human cell lines and a panel of in vitro early-toxicity assays (comprising h ERG, Aurora B, five cytochrome P450 isoforms and mitochondrial toxicity) demonstrated the low toxicity for the thosemicarbazones class in comparison with known drugs. The results confirmed thiosemicarbazones as a suitable chemical scaffold with potential for the development of properly decorated new anti-parasitic drugs. [ABSTRACT FROM AUTHOR]

Abstract: Bisnaphthalimidopropyl (BNIP) derivatives were recently identified as inhibitors of the Leishmania Silent Information Regulator 2 (SIR2) NAD+-dependent deacetylase. In this report we have for the first time, determined the potential of these compounds to treat visceral leishmaniasis using BALB/c mice chronically infected with Leishmania infantum as a model. These experiments led to the identification of BNIPdiaminooctane (BNIPDaoct) as an effective compound able to induce significant reduction of the parasite load in the spleen and in the liver. Indeed, at a dose of 1mg/kg, BNIPDaoct was more effective to treat leishmaniasis in a short course treatment (3 or 6 drug administrations) than the standard amphotericin B. Moreover, no indications of hematological toxicity were detected as evaluated by the hemoglobin, hematocrit, white and red blood cell counts, hence making BNIPDaoct a potential therapeutic agent against leishmaniasis. [Copyright &y& Elsevier]

Abstract: Northeastern Portugal is a region where canine leishmaniasis (CanL) is endemic. In this study, a sero-epidemiological survey was conducted in 654 dogs from that geographical area. Serum samples were evaluated by the direct agglutination test (DAT) and also by enzyme-linked immunosorbent assay (ELISA) using five different defined antigens. Seroprevalence of infection was 21.3% based on the assumption that seropositive animals were positive for at least three tests. A high degree of agreement was found between DAT and LAM-ELISA (89%; kappa value [κ]=0.67). A statistically significant difference (p <0.05) of seropositivity was found between adult (23.4%) and juvenile dogs (12.2%), apparently healthy (14.8%) and sick dogs (40.2%), vaccinated (19.7%) and non-vaccinated (41.2%) animals, seropositive (26.9%) and seronegative (18.0%) for Toxoplasma gondii, living in rural (18.5%) or urban (32.6%) areas, and between animals living exclusively outdoors (18.2%) and those living in a mixed habitat (27.5%). Risk factors for canine Leishmania infection, as defined by multiple logistic regression analysis, were of clinical status (odds ratio [OR]=3.1) and Toxoplasma infection (OR=1.5). [Copyright &y& Elsevier]

Abstract: Dendritic cells (DC) are professional antigen-presenting cells with a unique capacity to initiate and modulate immune responses by their ability to prime naïve T-cells. Upon stimuli, DC experience several morphologic, phenotypic and functional changes in a process referred to as maturation. This process is crucial to the biological functions of DC since their maturation status confer them the ability to polarize distinct T-cell subsets. In this work we explored the relevance of PI3-Kinase, Mitogen-Activated Protein Kinases (MAPKs) and NF-kappaB on cytokines/chemokines and co-stimulatory molecules expression. As experimental model, we used a fetal skin-derived dendritic cell line (FSDC) induced to mature by treatment with lipopolysaccharide (LPS). Morphology and ultrastructure were analyzed by confocal and electron microscopies, respectively. Levels of phosphorylated proteins were evaluated by Western blot, production of cytokines/chemokines was analyzed by protein arrays and the expression of surface molecules was evaluated by flow cytometry. The effect of specific inhibitors of the studied signaling pathways on the transcription of cytokines/chemokines and co-stimulatory molecules was accessed by Quantitative Real-Time RT-PCR. The results showed that LPS induces significant morphological and ultrastructural changes in FSDC. Western blot analysis revealed that LPS challenge promotes an early and transient activation of NF-κB, ERK1/2, p38 MAPK, along with a more sustained PI3 kinase/AKT activation. The co-stimulatory CD40, CD80, CD86 and antigen-presenting MHC class I and II molecules were increased and among secreted molecules, interleukin IL-6, CCL5, G-CSF, CCL2, CXCL2 were strongly up-regulated. Using a pharmacological approach we observed that LPS-induced increase of these molecules was differentially regulated by the distinct signaling pathways. Moreover, the polarizing Th2 cytokines/chemokines induced by LPS in FSDC were found to be positively regulated by NF-κB and ERK and negatively modulated by p38 MAPK. Altogether these results suggest that the use of pharmacological inhibitors to manipulate DC maturation, namely the polarizing Th1/Th2 cytokine/chemokine profile, may be useful in the development of more specific immunotherapeutic protocols. [Copyright &y& Elsevier]

Abstract: We recently described a delivery system that is composed of a chitosan core to which the hepatitis B surface antigen (HBsAg) was adsorbed and subsequently coated with sodium alginate. In this present work, alginate coated chitosan nanoparticles were evaluated as a subcutaneous adjuvant for HBsAg. HBsAg loaded, alginate coated or uncoated chitosan nanoparticles, associated or not with CpGODN were subcutaneously administered to mice and several immunological parameters were evaluated. A high anti-HBsAg IgG titer (2271±120 mIU/ml), with the majority of antibodies being of Th2 type, was observed within group I, vaccinated with HBsAg loaded onto coated nanoparticles. However, regarding cellular immune response, no significant differences were observed for antigen-specific splenocyte proliferation or for the secretion of IFN-γ and IL-4, when compared to the control group. The co-delivery of antigen-loaded nanoparticles in the presence of the immunopotentiator, CpG ODN 1826, resulted in an increase of anti-HBsAg IgG titers that was not statistically different from the first group; however, an increase of the IgG2a/IgG1 ratio from 0.1 to 1.0 and an increase (p <0.01) of the IFN-γ production by the splenocytes stimulated with the HBV antigen was observed. The enhancement of the immune response observed with the antigen-loaded nanoparticles demonstrated that chitosan is a promising platform for parenteral HBsAg delivery and, when co-administered with the CpG ODN, resulted in a mixed Th1/Th2 type immune response. [Copyright &y& Elsevier]

Abstract: Canine visceral leishmaniasis (CVL) is characterized by a high incidence of asymptomatic infections. Because of the high prevalence of asymptomatic dogs in the endemic areas of visceral leishmaniasis (VL), a sensitive test is required for an accurate diagnosis. In this study, we evaluated the detection of symptomatic and asymptomatic Leishmania infantum infection in dogs using the secreted LicTXNPx antigen (Leishmania infantum cytosolic tryparedoxin peroxidase) in an ELISA format and compared it to soluble Leishmania antigens from promastigote or amastigote forms (SPLA and SALA) and two other unrelated secreted Leishmania proteins (LiTXN1 and TDR1). Moreover, we evaluated the diagnostic potential using the promastigote or amastigote-flow cytometric methodologies. The assays utilized sera collected from a cohort of L. infantum experimentally infected dogs, in which the intravenous or intradermal parasite injection mimics a symptomatic or asymptomatic pattern of infection, respectively. Our study indicated that anti-LicTXNPx antibodies were present in both symptomatic and asymptomatic experimental infections. Among the different Leishmania recombinant proteins tested, LicTXNPx showed a good predictive correlation with total soluble promastigote or amastigote Leishmania antigens, suggesting this antigen as a good candidate for a marker in either symptomatic or asymptomatic infection. The use of flow cytometry using both forms of live parasites was also tested with the same group of dogs. Amastigotes were shown to have more advantages than promastigotes for the serological diagnostic in both symptomatic and asymptomatic dogs, since higher continuous levels of anti-amastigote antibodies were detected during the course of experimental infection. Moreover, additional studies were done using sera from non-infected dogs and clinically asymptomatic and symptomatic dogs with confirmed naturally occurring L. infantum infections. The sensitivities of amastigote and promastigote flow cytometry were 96% vs. 89%, respectively, while the specificity for both was 93.2%. Therefore, our findings showed for the first time the potential of amastigote-flow cytometry regarding their applicability to detect both symptomatic and asymptomatic VL canine infections. [Copyright &y& Elsevier]

In this work, fluorescence measurements were performed using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to evaluate the effects of the interaction of nonsteroidal anti-inflammatory drugs—NSAIDs (meloxicam, lornoxicam, and nimesulide) with several membrane systems (liposomes with and without cholesterol, mouse splenocytes, mouse macrophages cell line—J774, human leukemia monocyte cell line—THP-1, and human granulocytes and mononuclear cells). DPH fluorescence quenching studies revealed that the NSAIDs studied were able to efficiently quench the probe located in membrane hydrocarbon region. Fluorescence anisotropy measurements were also made to investigate the effects on membrane fluidity resulting from the interaction between the drugs and membrane systems. All the anti-inflammatory drugs studied show an increase in the membrane fluidity in a concentration dependent manner. Results obtained provide an insight into NSAIDs capacity to be inserted in lipid bilayers and alter the lipid dynamics. The induced changes in lipid dynamics may modulate the activity of inflammatory enzymes or may be related with deleterious topical action of NSAIDs on gastric phospholipid fluidity. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97: 3195–3206, 2008 [ABSTRACT FROM AUTHOR]

Abstract: The purpose of this work was to assess the ability of recombinant hepatitis B vaccine, encapsulated in alginate-coated chitosan nanoparticles, to induce local and systemic immune responses following oral vaccination. The antigen was administered either alone or in combination with the immunopotentiator, synthetic oligodeoxynucleotide containing immunostimulatory CpG motif (CpG ODN) as adjuvant, and associated or not with the alginate-coated chitosan nanoparticles. After two immunizations the group I (HBsAg associated with nanoparticles) and the group VI (HBsAg and CpG, both associated with nanoparticles) showed enhanced immune responses. Both groups showed significant higher values of the CD69 expression in CD4+ and CD8+ T-lymphocytes and lower values of this marker in B lymphocytes. Moreover, a strongest proliferative response of the splenocytes, ex vivo stimulated with concanavalin A, was observed in the same groups. Although with a presence of non-responder mice within the groups, only mice of the groups I and VI elicited the generation of anti-HBsAg antibodies detected in serum (IgG) and in the intestinal washings (sIgA). The results demonstrated that coated chitosan nanoparticles might have potential for being used as a deliver system for oral vaccination with the recombinant hepatitis B surface antigen. [Copyright &y& Elsevier]

Abstract: Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity on histone and various other cellular substrates. Functional analyses of such proteins have been carried out in a number of prokaryotes and eukaryotes organisms but until now, none have described an essential function for any SIR2 genes. Here using genetic approach, we report that a cytosolic SIR2 homolog in Leishmania is determinant to parasite survival. L. infantum promastigote tolerates deletion of one wild-type LiSIR2 allele (LiSIR2+/−) but achievement of null chromosomal mutants (LiSIR2−/−) requires episomal rescue. Accordingly, plasmid cure shows that these parasites maintain episome even in absence of drug pressure. Though single LiSIR2 gene disruption (LiSIR2+/−) does not affect the growth of parasite in the promastigote form, axenic amastigotes display a marked reduction in their capacity to multiply in vitro inside macrophages and in vivo in Balb/c mice. Taken together these data support a stage specific requirement and/or activity of the Leishmania cytosolic SIR2 protein and reveal an unrelated essential function for the life cycle of this unicellular pathogenic organism. The lack of an effective vaccine against leishmaniasis, and the need for alternative drug treatments, makes LiSIR2 protein a new attractive therapeutic target. [Copyright &y& Elsevier]

Abstract: Diagnosis of leishmaniasis is frequently based on serological methods, such as direct agglutination, immunofluorescence tests and ELISA assays with Leishmania total extracts, as antigen, however due to highly inconclusive results, more reliable tests are needed. In the present study, the prevalence of antibodies to a number of recombinant proteins (LmSIR2, LmS3a, LimTXNPx, LicTXNPx and LiTXN1) highly conserved among Leishmania species, were evaluated by ELISA in Leishmania infantum infected children from an endemic area of Portugal. We found that sera from children patients had antibodies against the different recombinant proteins, LicTXNPx presented the highest immuno-reactivity compared to the other and the most often recognized in the case of acute visceral leishmaniasis (VL). Moreover, in children treated with meglumine antimoniate or amphotericin B, antibodies against some of the recombinant proteins declined, whereas conventional serology using crude extracts showed little or no difference between the pre- and post-treatment values. The highest reduction was observed in the case of antibodies against the LicTXNPx protein. These results suggest that the antibodies against LicTXNPx might be a useful constituent of a defined serological test for the diagnosis and the monitoring of the therapeutic response in VL. The monitoring and follow-up in a large-scale field trials of such marker in areas where leishmaniasis is endemic will lend support to this. [Copyright &y& Elsevier]

Tryparedoxins are components of the hydroperoxide detoxification cascades of Kinetoplastida, where they mediate electron transfer between trypanothione and a peroxiredoxin, which reduces hydroperoxides and possibly peroxynitrite. Tryparedoxins may also be involved in DNA synthesis, by their capacity to reduce ribonucleotide reductase. Here we report on the isolation of two tryparedoxin genes from Leishmania infantum, LiTXN1 and LiTXN2, which share the same genetic locus. These genes are both single copy and code for two active tryparedoxin enzymes, LiTXN1 and LiTXN2, with different biochemical and biological features. LiTXN1 is located to the cytosol and is upregulated in the infectious forms of the parasite, strongly suggesting that it might play an important role during infection. LiTXN2 is the first mitochondrial tryparedoxin described in Kinetoplastida. Biochemical assays performed on the purified recombinant proteins have shown that LiTXN1 preferentially reduces the cytosolic L. infantum peroxiredoxins, LicTXNPx1 and LicTXNPx2, whereas LiTXN2 has a higher specific activity for a mitochondrial peroxiredoxin, LimTXNPx. Kinetically, the two tryparedoxins follow a ping-pong mechanism and show no saturation. We suggest that LiTXN1 and LiTXN2 are part of two distinct antioxidant machineries, one cytosolic, the other mitochondrial, that complement each other to ensure effective defence from several sources of oxidants throughout the development of L. infantum. [Copyright &y& Elsevier]

In Kinetoplastida 2-Cys peroxiredoxins are the ultimate members of unique enzymatic cascades for detoxification of peroxides, which are dependent on trypanothione, a small thiol specific to these organisms. Here we report on two distinct Leishmania infantum peroxiredoxins, LicTXNPx and LimTXNPx, that may be involved in such a pathway. LicTXNPx, found in the cytoplasm, is a typical 2-Cys peroxiredoxin encoded by LicTXNPx, a member of a multicopy gene family. LimTXNPx, encoded by a single copy gene, LimTXNPx, is confined to the mitochondrion and is unusual in possessing an Ile-Pro-Cys motif in the distal redox center, replacing the common peroxiredoxin Val-Cys-Pro sequence, apart from an N-terminal mitochondrial leader sequence. Based on sequence and subcellular localization, the peroxiredoxins of Kinetoplastida can be separated in two distinct subfamilies. As an approach to investigate the function of both peroxiredoxins in the cell, L. infantum promastigotes overexpressing LicTXNPx and LimTXNPx were assayed for their resistance to H2O2 and tert-butyl hydroperoxide. The results show evidence that both enzymes are active as peroxidases in vivo and that they have complementary roles in parasite protection against oxidative stress. [Copyright &y& Elsevier]

The LIBRA compound library is a collection of 522 non-commercial molecules contributed by various Italian academic laboratories. These compounds have been designed and synthesized during different medicinal chemistry programs and are hosted by the Italian Institute of Technology. We report the screening of the LIBRA compound library against Trypanosoma brucei and Leishmania major pteridine reductase 1, Tb PTR1 and Lm PTR1. Nine compounds were active against parasitic PTR1 and were selected for cell-based parasite screening, as single agents and in combination with methotrexate (MTX). The most interesting Tb PTR1 inhibitor identified was 4-(benzyloxy)pyrimidine-2,6-diamine (LIB_66). Subsequently, six new LIB_66 derivatives were synthesized to explore its Structure-Activity-Relationship (SAR) and absorption, distribution, metabolism, excretion and toxicity (ADMET) properties. The results indicate that PTR1 has a preference to bind inhibitors, which resemble its biopterin/folic acid substrates, such as the 2,4-diaminopyrimidine derivatives. Image 1 • LIBRA is a 522 compounds library, highly characterized. • Compound LB_66 has a 2,4-pyrimidine scaffold emerging as preferred selected HIT with selective inhibition against Tb PTR1. • 6 new 2,4-pyrimidine-based compounds showed 1.3–2.3 potentiation index in combination with methotrexate against T. brucei. • PTR1 prefers inhibitors with 2,4-diaminopyrimidine scaffolds that resemble its biopterin/folic acid substrates. [ABSTRACT FROM AUTHOR]