Novel 2,5-Diketopiperazines with In Vitro Activities against Protozoan Parasites of Tropical Diseases.

Malaria, Chagas disease, and leishmaniasis are tropical diseases caused by protozoan parasites of the genera Plasmodium, Trypanosoma and Leishmania, respectively. These diseases constitute a major burden on public health in several regions worldwide, mainly affecting low-income populations in economically poor countries. Severe side effects of currently available drug treatments and the emergence of resistant parasites need to be addressed by the development of novel drug candidates. Natural 2,5-Diketopiperazines (2,5-DKPs) constitute N-heterocyclic secondary metabolites with a wide range of biological activities of medicinal interest. Its structural and physicochemical properties make the 2,5-DKP ring a versatile, peptide-like, and stable pharmacophore attractive for synthetic drug design. In the present work, twenty-three novel synthetic 2,5-DKPs, previously synthesized through the versatile Ugi multicomponent reaction, were assayed for their anti-protozoal activities against P. falciparum, T. cruzi, and L. infantum. Some of the 2,5-DKPs have shown promising activities against the target protozoans, with inhibitory concentrations (IC50) ranging from 5.4 to 9.5 µg/mL. The most active compounds also show low cytotoxicity (CC50), affording selectivity indices ≥ 15. Results allowed for observing a clear relationship between the substitution pattern at the aromatic rings of the 2,5-DKPs and their corresponding anti-Plasmodium activity. Finally, calculated drug-like properties of the compounds revealed points for further structure optimization of promising drug candidates.

A functional assay using human whole blood and flow cytometry analysis to evaluate cytotoxicity and immunomodulatory effect of anti-Trypanosoma cruzi drugs.

The discovery and development of new drugs for the treatment of Chagas disease is urgent due to the high toxicity and low cure efficacy, mainly during the chronic phase of this disease. Other chemotherapeutic approaches for Chagas disease treatment are being researched and require screening assays suitable for evaluating the effectivity of new biologically active compounds. This study aims to evaluate a functional assay using the internalization of epimastigotes forms of Trypanosoma cruzi by human peripheral blood leukocytes from healthy volunteers and analyses by flow cytometry of cytotoxicity, anti-T. cruzi activity, and immunomodulatory effect of benznidazole, ravuconazole, and posaconazole. The culture supernatant was used to measure cytokines (IL-1-ß, IL-6, INF-γ, TNF and IL-10) and chemokines (MCP-1/CCL2, CCL5/RANTES and CXCL8/IL-8). The data showed a reduction in the internalization of T. cruzi epimastigote forms treated with ravuconazole, demonstrating its potential anti-T. cruzi activity. In addition, an increased amount of IL-10 and TNF cytokines was observed in the supernatant of cultures upon the addition of the drug, mainly IL-10 in the presence of benznidazole, ravuconazole and posaconazole, and TNF in the presence of ravuconazole and posaconazole. Moreover, the results revealed a decrease in the MCP-1/CCL2 index in cultures in the presence of benznidazole, ravuconazole, and posaconazole. A decrease in the CCL5/RANTES and CXCL8/IL-8 index in cultures with BZ, when compared to the culture without drugs, was also observed. In conclusion, the innovative functional test proposed in this study may be a valuable tool as a confirmatory test for selecting promising compounds identified in prospecting programs for new drugs for Chagas disease treatment.

Pamidronate, a promising repositioning drug to treat leishmaniasis, displays antileishmanial and immunomodulatory potential.

Visceral leishmaniasis (VL) is an infectious disease caused by Leishmania infantum (L. infantum). Currently, there are no vaccines and/or prophylactic therapies against VL, and the recentpharmacological approaches come from the drug repositioning strategy. Here, we evaluated the anticancer drug pamidronate (PAM) to identify a new therapeutic option for the treatment of human VL. We assessed its in vitro antileishmanial activity against the promastigote and amastigote forms of L. infantum by evaluating cell cytotoxicity. The antileishmanial and immunomodulatory activities were assessed using human peripheral blood leukocytes ex vivo. PAM induced the formation of vacuoles in the cytoplasm of the promastigotes and alterations in the morphology of the kinetoplast and mitochondria in vitro, which indicates anti-promastigote activity. PAM also reduced the number of infected macrophages and intracellular amastigotes in a concentration-dependent manner, with cell viability above 70%. In ex vivo, PAM reduced the internalized forms of L. infantum in the classical monocyte subpopulation. Furthermore, it enhanced IL-12 and decreased IL-10 and TGF-ß by monocytes and neutrophils. Increased IFN-γ and TNF levels for CD8- and CD8+ T lymphocytes and B lymphocytes, respectively, were observed after the treatment with PAM, as well as a reduction in IL-10 by the lymphocyte subpopulations evaluated. Taken together, our results suggest that PAM may be eligible as a potential therapeutic alternative for drug repurposing to treat human visceral leishmaniasis.

Novel 2-Nitroimidazole and Imidazooxazole Derivatives and their Activity against Trypanosoma cruzi and Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB) is one of the top ten causes of death worldwide, while Chagas disease (CD) is the parasitic disease that kills the largest number of people in the Americas. TB is the leading cause of death for patients with AIDS; it kills 1.5 million people and causes 10 million new cases every year. The lack of newly developed chemotherapeutic agents and insufficient access to health care services for a diagnosis increase the incidence of multidrug-resistant TB (MDRTB) cases. Although CD was identified in 1909, the chronic stages of the disease still lack adequate treatment. OBJECTIVE: The purpose of this work was to design and synthesize two new series of 2-nitroimidazole 5a-e and imidazooxazoles 6a-e with 1H-1,2,3-triazolil nucleus and evaluate their activities against Tc and Mycobacterium tuberculosis (Mtb). METHODS: Two series of five compounds were synthesized in a 3 or 4-step route in moderated yields, and their structures were confirmed by NMR spectral data analyses. The in vitro antitrypanosomal evaluation of products was carried out in an intracellular model using L929 cell line infected with trypomastigotes and amastigote forms of Tc of ß-galactosidase-transfected Tulahuen strain. Their antimycobacterial activity was evaluated against Mtb strain H37Rv. RESULTS: In general, 2-nitroimidazolic derivatives proved to be more potent in regard to antitrypanocidal and antimycobacterial activity. The non-cytotoxic 2-nitroimidazole derivative 5b was the most promising with a half maximum inhibitory concentration of 3.2 µM against Tc and a minimum inhibitory concentration of 65.3 µM against Mtb. CONCLUSION: Our study reinforced the importance of 2-nitroimidazole and 1H-1,2,3-triazole nuclei in antimicrobial activity. In addition, derivative 5b proved to be the most promising, presenting important activity against Tc and Mtb and could be used as a starting point for the development of new agents against these diseases.

Synthesis, Antitrypanosomal and Antimycobacterial Activities of Coumarin N-acylhydrazonic Derivatives.

BACKGROUND: Near to 5-7 million people are infected with T. cruzi in the world, and about 10,000 people per year die of problems associated with this disease. METHODS: Herein, the synthesis, antitrypanosomal and antimycobacterial activities of seventeen coumarinic N-acylhydrazonic derivatives have been reported. RESULTS: These compounds were synthesized using methodology with reactions global yields ranging from 46%-70%. T. cruzi in vitro effects were evaluated against trypomastigote and amastigote, forming M. tuberculosis activity towards H37Rv sensitive strain and resistant strains. DISCUSSION: Against T. cruzi, the more active compounds revealed only moderate activity IC50/96h~20 µM for both trypomastigotes and amastigotes intracellular forms. (E)-2-oxo-N'- (3,4,5-trimethoxybenzylidene)-2H-chromene-3-carbohydrazide showed meaningful activity in INH resistant/RIP resistant strain. CONCLUSION: These compound acting as multitarget could be good leads for the development of new trypanocidal and bactericidal agents.

Diversity, ecology, and bioprospecting of culturable fungi in lakes impacted by anthropogenic activities in Maritime Antarctica.

In this study, we accessed culturable fungal assemblages present in the sediments of three lakes potentially impacted anthropogenically in the Fildes Peninsula, King George Island, Antarctica and identified 63 taxa. Cladosporium sp. 2, Pseudeurotium hygrophilum, and Pseudogymnoascus verrucosus were recovered from the sampled sediments of all lakes. High concentrations of metals and the lowest fungal diversity indices were detected in the sediments of the Central Lake, which can be influenced by human activities due to their proximity to research stations to those of the other two lakes, which were far from the Antarctic stations. At least one type of biological activity was demonstrated by 40 fungal extracts. Among these, P. hygrophilum, P. verrucosus, Penicillium glabrum, and Penicillium solitum demonstrated strong trypanocidal, herbicidal, and antifungal activities. Our results suggest that an increase of the anthropogenic activities in the region might have affected the microbial diversity and composition. In addition, the fungal diversity in these lakes may be a useful model to study the effect of anthropogenic activities in Antarctica. We isolated a diverse group of fungal taxa from Antarctic lake sediments, which have the potential to produce novel compounds for the both the medical and agriculture sectors.

Diversity and bioprospecting of cultivable fungal assemblages in sediments of lakes in the Antarctic Peninsula.

We recovered 195 fungal isolates from the sediments of different lakes in the Antarctic Peninsula, which were screened to detect bioactive compounds. Forty-two taxa belonging to the phyla Ascomycota, Basidiomycota, and Mortierellomycota were identified. Thelebolus globosus, Antarctomyces psychrotrophicus, Pseudogymnoascus verrucosus, Vishniacozyma victoriae, and Phenoliferia sp. were found to be the most prevalent. The fungal assemblages showed high diversity and richness, but low dominance values. However, the diversity indices and fungal distribution ranged according to the different lake sediments. Sixty fungal extracts displayed at least one biological activity against the evaluated targets. Among them, Pseudogymnoascus destructans showed selective trypanocidal activity, Cladosporium sp. 1 and Trichoderma polysporum showed antifungal activity, and Pseudogymnoascus appendiculatus and Helotiales sp. showed high herbicidal activity. We detected a rich and diverse fungal community composed of cold cosmopolitan and psychrophilic endemic taxa recognized as decomposers, symbiotics, pathogens, and potential new species, in the sediments of Antarctic lakes. The dynamics and balance of this fungal community represents an interesting aquatic web model for further ecological and evolutionary studies under extreme conditions and potential climate changes in the regions. In addition, we detected fungal taxa and isolates able to produce bioactive compounds that may represent the source of prototype molecules for applications in medicine and agriculture.

Cultivable fungi present in deep-sea sediments of Antarctica: taxonomy, diversity, and bioprospecting of bioactive compounds.

We accessed the culturable mycobiota present in marine sediments at different depths in Antarctica Ocean. Acremonium fusidioides, Penicillium allii-sativi, Penicillium chrysogenum, Penicillium palitans, Penicillium solitum, and Pseudogymnoascus verrucosus were identified. Penicillium allii-sativi was the dominant species. At least one isolate of each species was capable to present antifungal, trypanocidal, leishmanicidal, antimalarial, nematocidal, or herbicidal activities. Penicillium produced extracts with strong trypanocidal and antimalarial activities, and the extracts of P. solitum and P. chrysogenum demonstrated strong antimalarial activities. Acremonium fusidioides and P. verrucosus displayed strong selective herbicidal properties. The 1H NMR signals for extracts of A. fusidioides, P. chrysogenum, and P. solitum indicated the presence of highly functionalized secondary metabolites, which may be responsible for the biological activities detected. In the deep marine Antarctic sediments, we detected fungal assemblages in which the Penicillium species were found to be dominant and demonstrated capabilities to survive and/or colonise that poly-extreme habitat. Penicillium being a polyextremophile Antarctic species, exhibited strong biological activities and the presence of aromatic compounds in its extracts may indicate that they are wild ancient strains with high genetic and biochemical potentials that enable them to produce bioactive compounds which can be researched in further studies and used in the chemotherapy of neglected tropical diseases as well as in agriculture.

Synthesis of New Thiosemicarbazones and Semicarbazones Containing the 1,2,3-1H-triazole-isatin Scaffold: Trypanocidal, Cytotoxicity, Electrochemical Assays, and Molecular Docking.

BACKGROUND: Chagas disease, also known as American trypanosomiasis, is classified as one of the 17 most important neglected diseases by the World Health Organization. The only drugs with proven efficacy against Chagas disease are benznidazole and nifurtimox, however both show adverse effects, poor clinical efficacy, and development of resistance. For these reasons, the search for new effective chemical entities is a challenge to research groups and the pharmaceutical industry. OBJECTIVE: Synthesis and evaluation of antitrypanosomal activities of a series of thiosemicarbazones and semicarbazones containing 1,2,3-1H triazole isatin scaffold. METHOD: 5'-(4-alkyl/aryl)-1H-1,2,3-triazole-isatins were prepared by Huisgen 1,3-dipolar cycloaddition and the thiosemicarbazones and semicarbazones were obtained by the 1:1 reactions of the carbonylated derivatives with thiosemicarbazide and semicarbazide hydrochloride, respectively, in methanol, using conventional reflux or microwave heating. The compounds were assayed for in vitro trypanocidal activity against Trypanosoma cruzi, the aetiological agent of Chagas disease. Beyond the thio/semicarbazone derivatives, isatin and triazole synthetic intermediates were also evaluated for comparison. RESULTS: A series of compounds were prepared in good yields. Among the 37 compounds evaluated, 18 were found to be active, in particular thiosemicarbazones containing a non-polar saturated alkyl chain (IC50 = 24.1, 38.6, and 83.2 µM; SI = 11.6, 11.8, and 14.0, respectively). To further elucidate the mechanism of action of these new compounds, the redox behaviour of some active and inactive derivatives was studied by cyclic voltammetry. Molecular docking studies were also performed in two validated protein targets of Trypanosoma cruzi, i.e., cruzipain (CRZ) and phosphodiesterase C (TcrPDEC). CONCLUSION: A class of thio/semicarbazones structurally simple and easily accessible was synthesized. Compounds containing thiosemicarbazone moieties showed the best results in the series, being more active than the corresponding semicarbazones. Our results indicated that the activity of these compounds does not originate from an oxidation-reduction pathway but probably from the interactions with trypanosomal enzymes.

Cultivable fungi present in Antarctic soils: taxonomy, phylogeny, diversity, and bioprospecting of antiparasitic and herbicidal metabolites.

Molecular biology techniques were used to identify 218 fungi from soil samples collected from four islands of Antarctica. These consisted of 22 taxa of 15 different genera belonging to the Zygomycota, Ascomycota, and Basidiomycota. Mortierella, Antarctomyces, Pseudogymnoascus, and Penicillium were the most frequently isolated genera and Penicillium tardochrysogenum, Penicillium verrucosus, Goffeauzyma gilvescens, and Mortierella sp. 2 the most abundant taxa. All fungal isolates were cultivated using solid-state fermentation to obtain their crude extracts. Pseudogymnoascus destructans, Mortierella parvispora, and Penicillium chrysogenum displayed antiparasitic activities, whilst extracts of P. destructans, Mortierella amoeboidea, Mortierella sp. 3, and P. tardochrysogenum showed herbicidal activities. Reported as pathogenic for bats, different isolates of P. destructans exhibited trypanocidal activities and herbicidal activity, and may be a source of bioactive molecules to be considered for chemotherapy against neglected tropical diseases. The abundant presence of P. destructans in soils of the four islands gives evidence supporting that soils in the Antarctic Peninsula constitute a natural source of strains of this genus, including some P. destructans strains that are phylogenetically close to those that infect bats in North America and Europe/Palearctic Asia.

Ornithine decarboxylase or gamma-glutamylcysteine synthetase overexpression protects Leishmania (Vianna) guyanensis against antimony.

Trypanosomatids present a unique mechanism for detoxification of peroxides that is dependent on trypanothione (bisglutathionylspermidine). Ornithine decarboxylase (ODC) and γ-glutamylcysteine synthetase (GSH1) produce molecules that are direct precursors of trypanothione. In this study, Leishmania guyanensis odc and gsh1 overexpressor cell lines were generated to investigate the contribution of these genes to the trivalent antimony (SbIII)-resistance phenotype. The ODC- or GSH1-overexpressors parasites presented an increase of two and four-fold in SbIII-resistance index, respectively, when compared with the wild-type line. Pharmacological inhibition of ODC and GSH1 with the specific inhibitors α-difluoromethylornithine (DFMO) and buthionine sulfoximine (BSO), respectively, increased the antileishmanial effect of SbIII in all cell lines. However, the ODC- and GSH1-overexpressor were still more resistant to SbIII than the parental cell line. Together, our data shows that modulation of ODC and GSH1 levels and activity is sufficient to affect L. guyanensis susceptibility to SbIII, and confirms a role of these genes in the SbIII-resistance phenotype.

Novel gold(I) complexes with 5-phenyl-1,3,4-oxadiazole-2-thione and phosphine as potential anticancer and antileishmanial agents.

The current anticancer and antileishmanial drug arsenal presents several limitations concerning their specificity, efficacy, costs and the emergence of drug-resistant cells lines, which encourages the urgent need to search for new alternatives. Inspired by the fact that gold(I)-based compounds are promising antitumoral and antileishmanial drug candidates, we synthesized novel gold(I) complexes containing phosphine and 5-phenyl-1,3,4-oxadiazole-2-thione and evaluated their anticancer and antileishmanial activities. Synthesis was performed by reacting 5-phenyl-1,3,4-oxadiazole-2-thione derivatives with chloro(triphenylphosphine)gold(I) and chloro(triethylphosphine)gold(I). The novel compounds were characterized by infrared, Raman, 1H, 13C nuclear magnetic resonance, high-resolution mass spectra, and x-ray crystallography. The coordination of the ligands to gold(I) occurred through the exocyclic sulfur atom. All gold(I) complexes were active at low micromolar or nanomolar range with IC50 values ranging from <0.10 to 1.66 µM against cancer cell lines and from 0.9 to 4.2 µM for Leishmania infantum intracellular amastigotes. Compound (6-A) was very selective against murine melanoma B16F10, colon cancer CT26.WT cell lines and L. infantum intracellular amastigotes. Compound (7-B) presented the highest anticancer activity against both cancer cell lines while the promising antileishmanial lead was compound (6-A). Tiethylphosphine gold(I) complexes were more active than the conterparts triphenylphosphine derivatives for both anticancer and antileishmanial activities. Triethylphosphine gold(I) derivatives presented antimony cross-resistance in L. guyanensis demonstrating their potential to be used as chemical tools to better understand mechanisms of drug resistance and action. These findings revealed the anticancer and antileishmanial potential of gold(I) oxadiazole phosphine derivatives.

Involvement of nucleoside diphosphate kinase b and elongation factor 2 in Leishmania braziliensis antimony resistance phenotype.

BACKGROUND: Nucleoside diphosphate kinase b (NDKb) is responsible for nucleoside triphosphates synthesis and it has key role in the purine metabolism in trypanosomatid protozoans. Elongation factor 2 (EF2) is an important factor for protein synthesis. Recently, our phosphoproteomic analysis demonstrated that NDKb and EF2 proteins were phosphorylated and dephosphorylated in antimony (SbIII)-resistant L. braziliensis line compared to its SbIII-susceptible pair, respectively. METHODS: In this study, the overexpression of NDKb and EF2 genes in L. braziliensis and L. infantum was performed to investigate the contribution of these proteins in the SbIII-resistance phenotype. Furthermore, we examined the role of lamivudine on SbIII susceptibility in clones that overexpress the NDKb gene, and the effect of EF2 kinase (EF2K) inhibitor on the growth of EF2-overexpressing parasites. RESULTS: Western blot analysis demonstrated that NDKb and EF2 proteins are more and less expressed, respectively, in SbIII-resistant line of L. braziliensis than its wild-type (WTS) counterpart, corroborating our previous phosphoproteomic data. NDKb or EF2-overexpressing L. braziliensis lines were 1.6 to 2.1-fold more resistant to SbIII than the untransfected WTS line. In contrast, no difference in SbIII susceptibility was observed in L. infantum parasites overexpressing NDKb or EF2. Susceptibility assays showed that NDKb-overexpressing L. braziliensis lines presented elevated resistance to lamivudine, an antiviral agent, but it did not alter the leishmanicidal activity in association with SbIII. EF2-overexpressing L. braziliensis clone was slightly more resistant to EF2K inhibitor than the WTS line. Surprisingly, this inhibitor increased the antileishmanial effect of SbIII, suggesting that this association might be a valuable strategy for leishmaniasis chemotherapy. CONCLUSION: Our findings represent the first study of NDKb and EF2 genes overexpression that demonstrates an increase of SbIII resistance in L. braziliensis which can contribute to develop new strategies for leishmaniasis treatment.

Molecular characterization of lipoamide dehydrogenase gene in Trypanosoma cruzi populations susceptible and resistant to benznidazole.

Lipoamide dehydrogenase (LipDH) is a flavin-containing disulfide oxidoreductase from the same group of thioredoxin reductase, glutathione reductase and trypanothione reductase. This enzyme is found in the mitochondria of all aerobic organisms where it takes part in at least three important multienzyme complexes from the citric acid cycle. In this study, we performed a phylogenetic analysis comparing the amino acid sequence of the LipDH from Trypanosoma cruzi (TcLipDH) with the LipDH from other organisms. Subsequently, the copy number of the TcLipDH gene, the mRNA and protein levels, and the enzymatic activity of the LipDH were determined in populations and strains of T. cruzi that were either resistant or susceptible to benznidazole (BZ). In silico analysis showed the presence of two TcLipDH alleles in the T. cruzi genome. It also showed that TcLipDH protein has less than 55% of identity in comparison to the human LipDH, but the active site is conserved in both of them. Southern blot results suggest that the TcLipDH is a single copy gene in the genome of the T. cruzi samples analyzed. Northern blot assays showed one transcript of 2.4 kb in all T. cruzi populations. Northern blot and Real Time RT-PCR data revealed that the TcLipDH mRNA levels were 2-fold more expressed in the BZ-resistant T. cruzi population (17LER) than in its susceptible pair (17WTS). Western blot results revealed that the TcLipDH protein level is 2-fold higher in 17LER sample in comparison to 17WTS sample. In addition, LipDH activity was higher in the 17LER population than in the 17WTS. Sequencing analysis revealed that the amino acid sequences of the TcLipDH from 17WTS and 17LER populations are identical. Our findings show that one of the mechanisms associated with in vitro-induced BZ resistance to T. cruzi correlates with upregulation of LipDH enzyme.

Silver and Nitrate Oppositely Modulate Antimony Susceptibility through Aquaglyceroporin 1 in Leishmania (Viannia) Species.

Antimony (Sb) resistance in leishmaniasis chemotherapy has become one of the major challenges to the control of this spreading worldwide public health problem. Since the plasma membrane pore-forming protein aquaglyceroporin 1 (AQP1) is the major route of Sb uptake in Leishmania, functional studies are relevant to characterize drug transport pathways in the parasite. We generated AQP1-overexpressing Leishmania guyanensis and L. braziliensis mutants and investigated their susceptibility to the trivalent form of Sb (Sb(III)) in the presence of silver and nitrate salts. Both AQP1-overexpressing lines presented 3- to 4-fold increased AQP1 expression levels compared with those of their untransfected counterparts, leading to an increased Sb(III) susceptibility of about 2-fold. Competition assays using silver nitrate, silver sulfadiazine, or silver acetate prior to Sb(III) exposure increased parasite growth, especially in AQP1-overexpressing mutants. Surprisingly, Sb(III)-sodium nitrate or Sb(III)-potassium nitrate combinations showed significantly enhanced antileishmanial activities compared to those of Sb(III) alone, especially against AQP1-overexpressing mutants, suggesting a putative nitrate-dependent modulation of AQP1 activity. The intracellular level of antimony quantified by graphite furnace atomic absorption spectrometry showed that the concomitant exposure to Sb(III) and nitrate favors antimony accumulation in the parasite, increasing the toxicity of the drug and culminating with parasite death. This is the first report showing evidence of AQP1-mediated Sb(III) susceptibility modulation by silver in Leishmania and suggests the potential antileishmanial activity of the combination of nitrate salts and Sb(III).

Molecular characterization and functional analysis of pteridine reductase in wild-type and antimony-resistant Leishmania lines.

Pteridine reductase (PTR1) is an NADPH-dependent reductase that participates in the salvage of pteridines, which are essential to maintain growth of Leishmania. In this study, we performed the molecular characterization of ptr1 gene in wild-type (WTS) and SbIII-resistant (SbR) lines from Leishmania guyanensis (Lg), Leishmania amazonensis (La), Leishmania braziliensis (Lb) and Leishmania infantum (Li), evaluating the chromosomal location, mRNA levels of the ptr1 gene and PTR1 protein expression. PFGE results showed that the ptr1 gene is located in a 797 kb chromosomal band in all Leishmania lines analyzed. Interestingly, an additional chromosomal band of 1070 kb was observed only in LbSbR line. Northern blot results showed that the levels of ptr1 mRNA are increased in the LgSbR, LaSbR and LbSbR lines. Western blot assays using the polyclonal anti-LmPTR1 antibody demonstrated that PTR1 protein is more expressed in the LgSbR, LaSbR and LbSbR lines compared to their respective WTS counterparts. Nevertheless, no difference in the level of mRNA and protein was observed between the LiWTS and LiSbR lines. Functional analysis of PTR1 enzyme was performed to determine whether the overexpression of ptr1 gene in the WTS L. braziliensis and L. infantum lines would change the SbIII-resistance phenotype of transfected parasites. Western blot results showed that the expression level of PTR1 protein was increased in the transfected parasites compared to the non-transfected ones. IC50 analysis revealed that the overexpression of ptr1 gene in the WTS L. braziliensis line increased 2-fold the SbIII-resistance phenotype compared to the non-transfected counterpart. Furthermore, the overexpression of ptr1 gene in the WTS L. infantum line did not change the SbIII-resistance phenotype. These results suggest that the PTR1 enzyme may be implicated in the SbIII-resistance phenotype in L. braziliensis line.

Biological activities of ophiobolin K and 6-epi-ophiobolin K produced by the endophytic fungus Aspergillus calidoustus.

Endophytic fungi represent ubiquitous microbial organisms able to live in the tissues of different plants around the world and represent a prolific source of bioactive metabolites. In the present study, the endophytic fungus Aspergillus calidoustus was isolated from the medicinal plant Acanthospermum australe (Asteraceae), and identified using molecular, physiological and morphological methods. A methylene chloride crude extract of A. calidoustus has been produced and subjected to antifungal bioassay-directed fractionation which resulted in the isolation of the two bioactive compounds: ophiobolin K and 6-epi-ophiobolin K. These pure compounds displayed antifungal activity against fungal plant pathogens, protozoal activity against Trypanosoma cruzi, and cytotoxic activity against human tumoral cell lines. The results show that A. calidoustus was able to produce the antifungal and cytotoxic metabolites ophiobolin K and 6-epi-ophiobolin K, which may help the fungus to colonise and occupy the substratum as well as survive in natural environments.

Phosphoproteomic analysis of wild-type and antimony-resistant Leishmania braziliensis lines by 2D-DIGE technology.

Protein phosphorylation is one of the most studied post-translational modifications that is involved in different cellular events in Leishmania. In this study, we performed a comparative phosphoproteomics analysis of potassium antimonyl tartrate (SbIII)-resistant and -susceptible lines of Leishmania braziliensis using a 2D-DIGE approach followed by MS. In order to investigate the differential phosphoprotein abundance associated with the drug-induced stress response and SbIII-resistance mechanisms, we compared nontreated and SbIII-treated samples of each line. Pair wise comparisons revealed a total of 116 spots that showed a statistically significant difference in phosphoprotein abundance, including 11 and 34 spots specifically correlated with drug treatment and resistance, respectively. We identified 48 different proteins distributed into seven biological process categories. The category "protein folding/chaperones and stress response" is mainly implicated in response to SbIII treatment, while the categories "antioxidant/detoxification," "metabolic process," "RNA/DNA processing," and "protein biosynthesis" are modulated in the case of antimony resistance. Multiple sequence alignments were performed to validate the conservation of phosphorylated residues in nine proteins identified here. Western blot assays were carried out to validate the quantitative phosphoproteome analysis. The results revealed differential expression level of three phosphoproteins in the lines analyzed. This novel study allowed us to profile the L. braziliensis phosphoproteome, identifying several potential candidates for biochemical or signaling networks associated with antimony resistance phenotype in this parasite.

Diversity and bioprospection of fungal community present in oligotrophic soil of continental Antarctica.

We surveyed the diversity and capability of producing bioactive compounds from a cultivable fungal community isolated from oligotrophic soil of continental Antarctica. A total of 115 fungal isolates were obtained and identified in 11 taxa of Aspergillus, Debaryomyces, Cladosporium, Pseudogymnoascus, Penicillium and Hypocreales. The fungal community showed low diversity and richness, and high dominance indices. The extracts of Aspergillus sydowii, Penicillium allii-sativi, Penicillium brevicompactum, Penicillium chrysogenum and Penicillium rubens possess antiviral, antibacterial, antifungal, antitumoral, herbicidal and antiprotozoal activities. Bioactive extracts were examined using (1)H NMR spectroscopy and detected the presence of secondary metabolites with chemical shifts. Our results show that the fungi present in cold-oligotrophic soil from Antarctica included few dominant species, which may have important implications for understanding eukaryotic survival in cold-arid oligotrophic soils. We hypothesize that detailed further investigations may provide a greater understanding of the evolution of Antarctic fungi and their relationships with other organisms described in that region. Additionally, different wild pristine bioactive fungal isolates found in continental Antarctic soil may represent a unique source to discover prototype molecules for use in drug and biopesticide discovery studies.

Identification of two p23 co-chaperone isoforms in Leishmania braziliensis exhibiting similar structures and Hsp90 interaction properties despite divergent stabilities.

The small acidic protein called p23 acts as a co-chaperone for heat-shock protein of 90 kDa (Hsp90) during its ATPase cycle. p23 proteins inhibit Hsp90 ATPase activity and show intrinsic chaperone activity. A search for p23 in protozoa, especially trypanosomatids, led us to identify two putative proteins in the Leishmania braziliensis genome that share approximately 30% identity with each other and with the human p23. To understand the presence of two p23 isoforms in trypanosomatids, we obtained the recombinant p23 proteins of L. braziliensis (named Lbp23A and Lbp23B) and performed structural and functional studies. The recombinant proteins share similar solution structures; however, temperature- and chemical-induced unfolding experiments showed that Lbp23A is more stable than Lbp23B, suggesting that they may have different functions. Lbp23B prevented the temperature-induced aggregation of malic dehydrogenase more efficiently than did Lbp23A, whereas the two proteins had equivalent efficiencies with respect to preventing the temperature-induced aggregation of luciferase. Both proteins interacted with L. braziliensis Hsp90 (LbHsp90) and inhibited its ATPase activity, although their efficiencies differed. In vivo identification studies suggested that both proteins are present in L. braziliensis cells grown under different conditions, although Lbp23B may undergo post-translation modifications. Interaction studies indicated that both Lbp23 proteins interact with LbHsp90. Taken together, our data suggest that the two protozoa p23 isoforms act similarly when regulating Hsp90 function. However, they also have some differences, indicating that the L. braziliensis Hsp90 machine has features providing an opportunity for novel forms of selective inhibition of protozoan Hsp90.