Functionalized 1,2,3-triazolium salts as potential agents against visceral leishmaniasis.

Visceral leishmaniasis (VL) is the most severe clinical form of leishmaniasis, being fatal if untreated. In search of a more effective treatment for VL, one of the main strategies is the development and screening of new antileishmanial compounds. Here, we reported the synthesis of seven new acetyl functionalized 1,2,3-triazolium salts, together with four 1,2,3-triazole precursors, and investigated their effect against different strains of L. infantum from dogs and humans. The 1,2,3-triazolium salts exhibited better activity than the 1,2,3-triazole derivatives with IC50 range from 0.12 to 8.66 µM and, among them, compound 5 showed significant activity against promastigotes (IC50 from 4.55 to 5.28 µM) and intracellular amastigotes (IC50 from 5.36 to 7.92 µM), with the best selective index (SI ~ 6-9) and reduced toxicity. Our findings, using biochemical and ultrastructural approaches, demonstrated that compound 5 targets the mitochondrion of L. infantum promastigotes, leading to the formation of reactive oxygen species (ROS), increase of the mitochondrial membrane potential, and mitochondrial alteration. Moreover, quantitative transmission electron microscopy (TEM) revealed that compound 5 induces the reduction of promastigote size and cytoplasmic vacuolization. Interestingly, the effect of compound 5 was not associated with apoptosis or necrosis of the parasites but, instead, seems to be mediated through a pathway involving autophagy, with a clear detection of autophagic vacuoles in the cytoplasm by using both a fluorescent marker and TEM. As for the in vivo studies, compound 5 showed activity in a mouse model of VL at 20 mg/kg, reducing the parasite load in both spleen and liver (59.80% and 26.88%, respectively). Finally, this compound did not induce hepatoxicity or nephrotoxicity and was able to normalize the altered biochemical parameters in the infected mice. Thus, our findings support the use of 1,2,3-triazolium salts as potential agents against visceral leishmaniasis.

Vitamin D increases killing of intracellular Leishmania amazonensis in vitro independently of macrophage oxidative mechanisms.

Vitamin D has been reported to activate macrophage microbicidal mechanisms by inducing the production of antimicrobial peptides and nitric oxide (NO), but conversely has been shown to contribute to a greater susceptibility to Leishmania amazonensis infection in mice. Thus, this study aimed to evaluate the role of vitamin D during intracellular infection with L. amazonensis by examining its effect on macrophage oxidative mechanisms and parasite survival in vitro. Vitamins D2 and D3 significantly inhibited promastigote and amastigote growth in vitro. Vitamin D3 was not able to induce NO and reactive oxygen species (ROS) production in uninfected macrophages or macrophages infected with L. amazonensis. In addition, vitamin D3 in combination with interferon (IFN)-γ did not enhance amastigote killing and in fact, significantly reduced NO and ROS production when compared with the effect of IFN-γ alone. In this study, we demonstrated that vitamin D directly reduces parasite growth in infected macrophages (approximately 50-60% at 50 µm) but this effect is independent of the activation of macrophage oxidative mechanisms. These findings will contribute to a better understanding of the role of vitamin D in cutaneous leishmaniasis.

Leishmania infantum amastigote nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1): Its inhibition as a new insight into mode of action of pentamidine.

Nucleoside triphosphate diphosphohydrolase (NTPDase) 1 from intracellular amastigotes of Leishmania infantum-infected macrophage was identified by immunocytochemistry and confocal laser scanning microscopy using antibodies that specifically recognize its B-domain. This enzyme was previously characterized in Leishmania promastigote form, and here it is shown to be susceptible to pentamidine isethionate (PEN). In initial assays, this antileishmanial compound (100 µM) reduced 60% phosphohydrolytic activity of promastigotes preparation. An active NTPDase 1 was then isolated by non-denaturing gel electrophoresis, and PEN (10 µM) inhibited 74% and 35% of the ATPase and ADPase activities, respectively, of this pure protein. In addition, PEN 0.1-1 µM inhibited 56% potato apyrase activity, a plant protein that shares high identity with Leishmania NTPDase 1. In contrast, amphotericin B, fluconazole, ketoconazole or allopurinol did not significantly affect phosphohydrolytic activity of either promastigotes preparation or potato apyrase. This work suggests amastigote NTPDase 1 as a new molecular target, and inhibition of its catalytic activity by pentamidine can be part of the mode of action of this drug contributing with the knowledge of its antileishmanial effect.

Antileishmanial activity of a 4-hydrazinoquinoline derivative: Induction of autophagy and apoptosis-related processes and effectiveness in experimental cutaneous leishmaniasis.

Currently, available treatment options for leishmaniasis are limited and unsatisfactory. In a previous study, a quinoline derivative (AMQ-j), exhibited a strong effect against Leishmania amazonensis and its antileishmanial activity was preliminarily associated with mitochondrial dysfunction. The present study further explores the antileishmanial effect of this compound against L. amazonensis, as well as determines the main cellular processes involved in the death of the parasite. Moreover, this study evaluated the in vivo effect of the AMQ-j in BALB/c mice experimentally infected by L. amazonensis. The results showed that the compound AMQ-j induces a set of morphological and biochemical features that could correlate with both autophagy-related and apoptosis-like processes, indicating intense mitochondrial swelling, a collapse of the mitochondrial membrane potential, an abnormal chromatin condensation, an externalization of phosphatidylserine, an accumulation of lipid bodies, a disorganization of cell cycle, a formation of autophagic vacuoles, and an increase of acidic compartments. Treatment with AMQ-j through an intralesional route was effective in reducing the parasite burden and size of the lesion. No significant increase in the serum levels of hepatic or renal damage toxicity markers was observed. These findings contribute to the understanding of the mode of action of quinoline derivatives involved in the death of Leishmania parasites and encourage new studies in other experimental models of Leishmania infection.

Antileishmanial activity of a naphthoquinone derivate against promastigote and amastigote stages of Leishmania infantum and Leishmania amazonensis and its mechanism of action against L. amazonensis species.

Leishmaniasis has become a significant public health issue in several countries in the world. New products have been identified to treat against the disease; however, toxicity and/or high cost is a limitation. The present work evaluated the antileishmanial activity of a new naphthoquinone derivate, Flau-A [2-(2,3,4-tri-O-acetyl-6-deoxy-ß-L-galactopyranosyloxy)-1,4-naphthoquinone], against promastigote and amastigote-like stages of Leishmania amazonensis and L. infantum. In addition, the cytotoxicity in murine macrophages and human red cells was also investigated. The mechanism of action of Flau-A was assessed in L. amazonensis as well as its efficacy in treating infected macrophages and inhibiting infection of pretreated parasites. Results showed that Flau-A was effective against promastigotes and amastigote-like forms of both parasite species, as well as showed low toxicity in mammalian cells. Results also highlighted the morphological and biochemical alterations induced by Flau-A in L. amazonensis, including loss of mitochondrial membrane potential, as well as increased reactive oxygen species production, cell shrinkage, and alteration of the plasma membrane integrity. The present study demonstrates for the first time the antileishmanial activity of Flau-A against two Leishmania species and suggests that the mitochondria of the parasites may be the main target organelle. Data shown here encourages the use of this molecule in new studies concerning treatment against Leishmania infection in mammalian hosts.

Aminoquinoline compounds: Effect of 7-chloro-4-quinolinylhydrazone derivatives against Leishmania amazonensis.

In this study, we have investigated the antileishmanial activity of ten 7-chloro-4-quinolinylhydrazone derivatives. Among the compounds tested, compounds 2a and 2j presented activity against promastigotes (IC50 values of 52.5 and 21.1 µM, respectively) and compounds 2a and 2c were active against intracellular amastigotes (IC50 of 8.1 and 15.6 µM, respectively) of Leishmania amazonensis. The majority of compounds did not show toxicity against murine macrophages. Compound 2a exhibited low cytotoxicity to human erythrocytes and induced an oxidative imbalance in promastigote forms, reflected by an increase in the formation of reactive oxygen species (ROS) and a reduction of mitochondrial membrane potential. No alteration in the plasma membrane integrity of parasites was observed. Taken together, these results suggest that compound 2a is a selective antileishmanial agent, and preliminary observations suggest that its effects appear to be mediated by mitochondrial dysfunction.

The effect of the phytol-rich fraction from Lacistema pubescens against Leishmania amazonensis is mediated by mitochondrial dysfunction.

Leishmaniasis is a complex disease caused by protozoan parasite Leishmania and the treatment remains a serious problem since the available drugs exhibited high toxicity and side effects. Plant-derived natural products are promising leads for the development of novel chemotherapeutics. In this work the phytol-rich hexane fraction (PRF) from the leaves of Lacistema pubescens was obtained and identified by GC-MS analysis. When assayed for antileishmanial effects, PRF was active against promastigote and amastigote forms of Leishmania amazonensis (IC50 values of 44.0 and 25.8 µg/mL respectively). Furthermore, PRF did not show significant cytotoxicity on peritoneal macrophages being more destructive to the intracellular parasite than to mammalian cells. In addition, possible targets of PRF were investigated against L. amazonensis promastigotes. The results showed that PRF exerted its antipromastigote activity by marked depolarization of the mitochondrial membrane potential followed by the increase of ROS levels in L. amazonensis promastigotes. During these events, no rupture of the cell membrane integrity was observed. Our results indicated that PRF was effective and selective against L. amazonensis, and that this effect was mainly mediated by mitochondrial dysfunction associated to ROS production.

Gold complexes with benzimidazole derivatives: synthesis, characterization and biological studies.

Synthesis, characterization, DFT studies and biological assays of new gold(I) and gold(III) complexes of benzimidazole are reported. Molecular and structural characterizations of the compounds were based on elemental (C, H and N) and thermal (TG-DTA) analyses, and FT-IR and UV-Visible spectroscopic measurements. The structures of complexes were proposed based DFT calculations. The benzimidazole compounds (Lig1 and Lig2) and the gold complexes were tested against three Leishmania species related to cutaneous manifestations of leishmaniasis. The free benzimidazole compounds showed no leishmanicidal activity. On the other hand, the gold(I and III) complexes have shown to possess significant activity against Leishmania in both stages of parasite, and the gold(III) complex with Lig2 exhibited expressive leishmanicidal activity with IC50 values below 5.7 µM. Also, the gold complexes showed high leishmania selectivity. The gold(I) complex with Lig1, for example, is almost 50 times more toxic for the parasite than for macrophages. Besides the leishmanicidal activity, all complexes exhibited toxic effect against SK-Mel 103 and Balb/c 3T3, cancer cells.

Synthesis, biological evaluation and mechanism of action of benzothiazole derivatives with aromatic hydrazone moiety, a new class of antileishmanial compounds.

Leishmaniasis is a disease caused by protozoa Leishmania spp., considered as a significant and urgent public health problem mainly in developing countries. In the absence of an effective vaccine, the treatment of infected people is one of the most commonly prophylactic measures used to control this disease. However, the therapeutic arsenal is reduced to a few drugs, with serious side effects and variability in efficacy. Attempting to this problem, in this work, a series of benzothiazole derivatives was synthetized and assayed against promastigotes and intracellular amastigotes of L. amazonensis, as well as the toxicity on macrophages. In addition, studies about the mechanism of action were also performed. Among the synthesized molecules, the substitution at position 4 of the aromatic ring appears to be critical for activity. The best compound exhibited IC50 values of 28.86 and 7.70 µM, against promastigotes and amastigotes of L. amazonensis, respectively, being more active than miltefosine, used as reference drug. The in silico analysis of physicochemical and pharmacokinetic (ADMET) properties of this compound suggested a good profile of oral bioavailability and safety. In conclusion, the strategy of using benzothiazole nucleous in the search for new antileishmanial agents was advantageous and preliminar data provide information about the mechanism of action as well as in silico parameters suggest a good profile for preclinical studies.

4-Quinolinylhydrazone analogues kill Leishmania (Leishmania) amazonensis by inducing apoptosis and mitochondria-dependent pathway cell death.

Despite efforts, available alternatives for the treatment of leishmaniasis are still scarce. In this work we tested a class of 15 quinolinylhydrazone analogues and presented data that support the use of the most active compound in cutaneous leishmaniasis caused by Leishmania amazonensis. In general, the compounds showed activity at low concentrations for both parasitic forms (5.33-37.04 µM to promastigotes, and 14.31-61.98 µM to amastigotes). In addition, the best compound (MHZ15) is highly selective for the parasite. Biochemical studies indicate that the treatment of promastigotes with MHZ15 leads the loss of mitochondrial potential and increase in ROS levels as the primary effects, which triggers accumulation of lipid droplets, loss of plasma membrane integrity and apoptosis hallmarks, including DNA fragmentation and phosphatidylserine exposure. These effects were similar in the intracellular form of the parasite. However, in this parasitic form there is no change in plasma membrane integrity in the observed treatment time, which can be attributed to metabolic differences and the resilience of the amastigote. Also, ultrastructural changes such as vacuolization suggesting autophagy were observed. The in vivo effectiveness of MHZ15 in the experimental model of cutaneous leishmaniasis was carried out in mice of the BALB/c strain infected with L. amazonensis. The treatment by intralesional route showed that MHZ15 acted with great efficiency with significantly reduction in the parasite load in the injured paws and draining lymph nodes, without clinical signs of distress or compromise of animal welfare. In vivo toxicity was also evaluated and null alterations in the levels of hepatic enzymes aspartate aminotransferase, and alanine aminotransferase was observed. The data presented herein demonstrates that MHZ15 exhibits a range of favorable characteristics conducive to the development of an antileishmanial agent.

Lantana camara L. induces a multi-targeted cell death process in Leishmania amazonensis.

ETNOPHARMACOLOGICAL RELEVANCE: Lantana camara L. is a species known for its broad spectrum of bioactivities and is commonly used in folk therapy to address inflammatory, dermatological, gastrointestinal, intestinal worms and protozoan diseases. It boasts a diverse array of secondary metabolites such as terpenes, flavonoids, and saponins. However, despite its rich chemical profile, there remains a scarcity of studies investigating its antileishmanial properties. AIM OF THE STUDY: This research aims to explore the antileishmanial potential of L. camara, focusing also on its mechanism of action against Leishmania amazonensis. MATERIAL AND METHODS: The ethanolic extract of L. camara leaves (LCE) was obtained through static maceration, and its phytoconstituents were identified using UFLC-QTOF-MS. The colorimetric MTT method was conducted to determine the effect of LCE on promastigotes of L. amazonensis and murine macrophages. The anti-amastigote activity was evaluated by counting intracellular parasites in macrophages after Giemsa staining. Additionally, investigations into the mechanisms underlying its action were conducted using cellular and biochemical approaches. RESULTS: LCE exhibited significant activity against both promastigotes and intracellular amastigotes of L. amazonensis, with IC50 values of 12.20 µg/mL ± 0.12 and 7.09 µg/mL ± 1.24, respectively. These IC50 values indicate very promising antileishmanial activity, comparable to those found for the positive control miltefosine (5.10 µg/mL ± 1.79 and 8.96 µg/mL ± 0.50, respectively). Notably, LCE exhibited negligible cytotoxicity on macrophages (IC50 = 223.40 µg/mL ± 47.02), demonstrating selectivity towards host cells (SI = 31.50). The antileishmanial activity of LCE involved a multi-targeted cell death process, characterized by morphological and ultrastructural alterations observed through SEM and TEM analyses, as well as oxidative effects evidenced by the inhibition of trypanothione reductase, elevation of ROS and lipid levels, and mitochondrial dysfunction evaluated using DTNB, H2DCFDA, Nile red, and JC-1 assays. Additionally, extraction of ergosterol and double labeling with annexin V and PI revealed modifications to the organization and permeability of the treated parasite's plasma membrane. LCE was found to consist predominantly of terpenes, with lantadenes A, B, and C being among the eleven compounds identified through UFLC-QTOF-MS analysis. CONCLUSIONS: The extract of L. camara presents a diverse array of chemical constituents, prominently featuring high terpene content, which may underlie its antileishmanial properties through a combination of apoptotic and non-apoptotic mechanisms of cell death induced by LCE. This study underscores the therapeutic potential of L. camara as a candidate for antileishmanial treatment, pending further validation.

Insights into the bioactive potential of the Amazonian species Acmella oleracea leaves extract: A focus on wound healing applications.

ETHNOPHARMACOLOGICAL RELEVANCE: Acmella oleracea is traditionally used by Amazonian folks to treat skin and mucous wounds, influenza, cough, toothache, bacterial and fungal infections. Its phytoconstituents, such as alkylamides, phenolic compounds, and terpenes, are reported to produce therapeutic effects, which justify the medicinal use of A. oleracea extracts. However, the scientific evidence supporting the application A. oleracea bioactive products for wound treatment of remains unexplored so far. OBJECTIVE: This work aimed to characterize the phytochemical composition of methanolic extract of A. oleracea leaves (AOM) and to investigate their antioxidant, anti-inflammatory, antimicrobial and healing potential focusing on its application for wound healing. MATERIAL AND METHODS: The dried leaves from A. oleracea submitted to static maceration in methanol for 40 days. The phytochemical constitution of AOM was analyzed based on the total phenolic dosage method and by UFLC-QTOF-MS analysis. Antioxidant activity was assessed by DPPH and NO scavenging activities, as well as MDA formation, evaluation of ROS levels, and phosphomolybdenum assays. In vitro anti-inflammatory activities were assessed by reduction of NO, IL-6, and TNF-α production and accumulation of LDs in peritoneal macrophages cells. Antimicrobial activity was evaluated by determining MIC and MBC/MFC values against P. aeruginosa, E. coli, S. epidermidis, S. aureus and C. albicans, bacterial killing assay, and biofilm adhesion assessment. In vitro wound healing activity was determined by means of the scratch assay with L929 fibroblasts. RESULTS: Vanillic acid, quercetin, and seven other alkamides, including spilanthol, were detected in the UFLC-QTOF-MS spectrum of AOM. Regarding the biocompatibility, AOM did not induce cytotoxicity in L929 fibroblasts and murine macrophages. The strong anti-inflammatory activity was evidenced by the fact that AOM reduced the cellular production of inflammatory mediators IL-6, TNF-α, NO, and LDs in macrophages by 100%, 96.66 ± 1.95%, 99.21 ± 3.82%, and 67.51 ± 0.72%, respectively. The antioxidant effects were confirmed, since AOM showed IC50 values of 44.50 ± 4.46 and 127.60 ± 14.42 µg/mL in the DPPH and NO radical inhibition assays, respectively. Additionally, AOM phosphomolybdenium reducing power was 63.56 ± 13.01 (RAA% of quercetin) and 104.01 ± 21.29 (RAA% of rutin). Finally, in the MDA quantification assay, AOM showed 63,69 ± 3.47% of lipid peroxidation inhibition. It was also observed that the production of ROS decreased by 69.03 ± 3.85%. The MIC values of AOM ranged from 1000 to 125 µg/mL. Adhesion of S. aureus, P. Aeruginosa, and mixed biofilms was significantly reduced by 44.71 ± 4.44%, 95.50 ± 6.37 %, and 51.83 ± 1.50%, respectively. AOM also significantly inhibited the growth of S. aureus (77.17 ± 1.50 %) and P. aeruginosa (62.36 ± 1.01%). Furthermore, AOM significantly enhanced the in vitro migration of L929 fibroblasts by 97.86 ± 0.82% compared to the control (P < 0.05). CONCLUSIONS: This study is the first to report total antioxidant capacity and intracellular LD reduction by AOM. The results clearly demonstrated that AOM exerts potent anti-inflammatory, antioxidant, antimicrobial, and wound healing effects, encouraging its further investigation and promising application in wound treatment.

Exploring the repositioning of the amodiaquine as potential drug against visceral leishmaniasis: The in vitro effect against Leishmania infantum is associated with multiple mechanisms, involving mitochondria dysfunction, oxidative stress and loss of cell cycle control.

Visceral leishmaniasis (VL) is a progressive, debilitating, and potentially fatal disease if left untreated. As a neglected tropical disease (NTD), the available treatment is restricted to a few drugs, which typically must be administered over a long period but are associated with serious adverse effects and have variability in efficacy. In this sense, drug repositioning has been considered an excellent strategy in the search for alternative treatments, especially in reducing the time and cost of the research. In this work, the repositioning potential of amodiaquine (AQ), a well-known antimalarial drug, was investigated for the treatment of VL. AQ showed significant and selective activity against promastigotes (IC50 = 11.6 µg/mL) and intracellular amastigotes (IC50 = 2.4 µg/mL) of L. infantum, being 10 times more destructive to the intracellular parasites than the host cell. In addition, pre-treatment of macrophages with AQ caused a significant reduction in the infection index, indicating a prophylactic effect of this drug. SEM images showed that AQ induces strong shape alterations of the promastigotes with an increase in cell volume with rounding and ribbing (vertical ridges), as well as a shortened flagellum. In addition, AQ induced depolarization of the ΔΨm, an increase in ROS and neutral lipids levels, and changes in the cell cycle in promastigotes, without alterations to the permeability of the parasite plasma membrane. L. infantum-infected macrophages treated with AQ induced the activation of oxidative mechanisms by infected host cells, with an increase in ROS and NO levels. Finally, in vitro interactions between AQ and miltefosine were found to have an additive effect in both biological stages of the parasite, with the ∑FIC50 values ranging from 0.74 to 1.16 µg/mL and 0.54-1.11 µg/mL for promastigotes and intracellular amastigotes, respectively. Overall, these data highlight the utility of drug repurposing and indicate future preclinical testing for AQ itself or in combination as a potential VL treatment.

Synthesis of 1,2,3-Triazole-Containing Methoxylated Cinnamides and Their Antileishmanial Activity against the Leishmania braziliensis Species.

Leishmaniasis is a group of infectious diseases caused by protozoan parasites that belong to the genus Leishmania. Currently, there is no human vaccine, and the available treatments are associated with toxicity, high cost, and the emergence of resistant strains. These factors highlight the need to identify new antileishmanial candidates. In this study, we synthesized twenty-four methoxylated cinnamides containing 1,2,3-triazole fragments and evaluated their antileishmanial activity against the Leishmania braziliensis species, which is the main etiological agent responsible for American Tegumentary Leishmaniasis (ATL). The cinnamides were synthetically prepared using nucleophilic acyl substitution and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions. The compounds were characterized using infrared, nuclear magnetic resonance, and high-resolution mass spectrometry techniques. We performed preliminary studies to evaluate the biological activity of these compounds against L. braziliensis promastigotes and axenic amastigotes. Compound 28, N-((1-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1H-1,2,3-triazole-4-yl) methyl)-3,4-dimethoxy cinnamide, demonstrated relevant antileishmanial activity with low toxicity in murine cells. The selectivity index values for this compound were superior compared with data obtained using amphotericin B. Furthermore, this cinnamide derivative reduced the infection percentage and number of recovered amastigotes in L. braziliensis-infected macrophages. It also induced an increase in reactive oxygen species production, depolarization of the mitochondrial potential, and disruption of the parasite membrane. Taken together, these findings suggest that this synthetic compound holds potential as an antileishmanial candidate and should be considered for future studies in the treatment of ATL.

Leishmania (Viannia) braziliensis nucleoside triphosphate diphosphohydrolase (NTPDase 1): localization and in vitro inhibition of promastigotes growth by polyclonal antibodies.

Nucleoside triphosphate diphosphohydrolase (NTPDase) activity was recently characterized in Leishmania (Viannia) braziliensis promastigotes (Lb), and an antigenic conserved domain (r82-121) from the specific NTPDase 1 isoform was identified. In this work, mouse polyclonal antibodies produced against two synthetic peptides derived from this domain (LbB1LJ, r82-103; LbB2LJ, r102-121) were used. The anti-LbB1LJ or anti-LbB2LJ antibodies were immobilized on protein A-sepharose and immunoprecipitated the NTPDase 1 of 48 kDa and depleted approximately 40% of the phosphohydrolytic activity from detergent-homogenized Lb preparation. Ultrastructural immunocytochemical microscopy identified the NTPDase 1 on the parasite surface and in its subcellular cytoplasmic vesicles, mitochondria, kinetoplast and nucleus. The ATPase and ADPase activities of detergent-homogenized Lb preparation were partially inhibited by anti-LbB1LJ antibody (43-79%), which was more effective than that inhibition (18-47%) by anti-LbB2LJ antibody. In addition, the immune serum anti-LbB1LJ (67%) or anti-LbB2LJ (33%) was cytotoxic, significantly reducing the promastigotes growth in vitro. The results appoint the conserved domain from the L. braziliensis NTPDase as an important target for inhibitor design and the potential application of these biomolecules in experimental protocols of disease control.

Polymerase chain reaction for the evaluation of Schistosoma mansoni infection in two low endemicity areas of Minas Gerais, Brazil.

This study aimed to evaluate the occurrence of schistosomiasis in areas with low endemicity using polymerase chain reaction (PCR) as a diagnostic method. We analysed faecal samples from 219 individuals residing in Piau and Coronel Pacheco, state of Minas Gerais, Brazil, using a single faecal sample from each individual and two slides of the Kato-Katz technique as a gold standard. Fifteen out of the 219 samples were positive with both methods of diagnosis. One sample was diagnosed as positive by the Kato-Katz technique only and 61 were diagnosed only by PCR. The positivity rates were 7.3% with the Kato-Katz method and 34.7% with PCR. When both techniques were assumed to have 100% specificity and positive individuals were identified by both methods, the sensitivity of the Kato-Katz method was 20.8% and the PCR sensitivity was 98.7%. The Kappa index between the two techniques was 0.234, suggesting weak agreement. The assessment of a single faecal sample by PCR detected more cases of infection than the analysis of one sample with two slides using the Kato-Katz technique, suggesting that PCR can be a useful diagnostic tool, particularly in areas with low endemicity.

Rational-Based Discovery of Novel ß-Carboline Derivatives as Potential Antimalarials: From In Silico Identification of Novel Targets to Inhibition of Experimental Cerebral Malaria.

Malaria is an infectious disease widespread in underdeveloped tropical regions. The most severe form of infection is caused by Plasmodium falciparum, which can lead to development of cerebral malaria (CM) and is responsible for deaths and significant neurocognitive sequelae throughout life. In this context and considering the emergence and spread of drug-resistant P. falciparum isolates, the search for new antimalarial candidates becomes urgent. ß-carbolines alkaloids are good candidates since a wide range of biological activity for these compounds has been reported. Herein, we designed 20 chemical entities and performed an in silico virtual screening against a pool of P. falciparum molecular targets, the Brazilian Malaria Molecular Targets (BRAMMT). Seven structures showed potential to interact with PfFNR, PfPK7, PfGrx1, and PfATP6, being synthesized and evaluated for in vitro antiplasmodial activity. Among them, compounds 3−6 and 10 inhibited the growth of the W2 strain at µM concentrations, with low cytotoxicity against the human cell line. In silico physicochemical and pharmacokinetic properties were found to be favorable for oral administration. The compound 10 provided the best results against CM, with important values of parasite growth inhibition on the 5th day post-infection for both curative (67.9%) and suppressive (82%) assays. Furthermore, this compound was able to elongate mice survival and protect them against the development of the experimental model of CM (>65%). Compound 10 also induced reduction of the NO level, possibly by interaction with iNOS. Therefore, this alkaloid showed promising activity for the treatment of malaria and was able to prevent the development of experimental cerebral malaria (ECM), probably by reducing NO synthesis.

A simple quinoline salt derivative is active in vitro against Plasmodiumfalciparum asexual blood stages and inhibits the development of cerebral malaria in murine model.

Chloroquine (CQ) was the most effective and widely used drug for the prophylaxis and treatment of severe and non-severe malaria. Although its prophylactic use has led to resistance to P. falciparum in all endemic countries, CQ still remains the drug of choice for the treatment of vivax malaria. Otherwise, the speed in which parasite resistance to available antimalarials rises and spreads in endemic regions points to the urgent need for the development of new antimalarials. Quinoline derivatives have been used as a tool in the search for new drugs and were investigated in the present study in an attempt to produce a HIT compound to avoid the cerebral malarial (CM). Seven compounds were synthesized, including three quinoline derivate salts. The cytotoxicity and antiplasmodial activity were assayed in vitro, highlighting compound 3 as a HIT, which also showed interaction with ferriprotoporphyrin IX similarly to CQ. Physicochemical and pharmacokinetic properties of absorption were found to be favorable when analyzed in silico. The in vivo assays, using the experimental cerebral malaria (ECM) model, showed important values of parasite growth inhibition on the 7th day-post infection (Q15 15 mg/kg: 76.9%, Q30 30 mg/kg: 90,1% and Q50 50 mg/kg: 92,9%). Compound 3 also showed significant protection against the development of CM, besides hepatic and renal parameters better than CQ. In conclusion, this quinoline derivative demonstrated promising activity for the treatment of malaria and was able to avoid the development of severe malaria in mice.

Prevalence and risk factors for giardiasis and soil-transmitted helminthiasis in three municipalities of Southeastern Minas Gerais State, Brazil: risk factors for giardiasis and soil-transmitted helminthiasis.

Giardiasis and soil-transmitted helminthiasis (STH) are parasitic diseases that are among the major health concerns observed in economically disadvantaged populations of developing countries, and have clear social and environmental bases. In Brazil, there is a lack of epidemiologic data concerning these infections in the study area, whose inhabitants have plenty of access to health care services, including good dwelling and adequate sanitary conditions. In this survey we investigated the risk factors for giardiasis and STH in three municipalities with good sanitation, situated in Minas Gerais state, Brazil. A cross-sectional survey was conducted in the municipalities of Piau, Coronel Pacheco and Goianá, in both urban and rural areas. The fieldwork consisted of a questionnaire and the examination of 2,367 stool samples using the Hoffmann, Pons and Janer method. Of all individuals from the population sample, 6.1% were found infected with the parasitic diseases included in this work. Hookworm infection was the most prevalent disease, followed by giardiasis, trichuriasis and ascariasis. Infection was more prevalent in males (8.1%, p < 0.001; odds ratio [OR] = 1.975) and in individuals living in rural areas (8.6%, p = 0.003; OR = 1.693). Multivariate analysis showed that variables such as inadequate sewage discharge (p < 0.001), drinking of unsafe water (p < 0.001), lack of sanitary infrastructure (p = 0.015), and host sex (p < 0.001) were the risk factors more strongly associated with infection status (95% confidence interval [CI]). In this study we demonstrate that giardiasis and STH still persist, infecting people who have good housing conditions and free access to public health care and education.

PF-429242, a Subtilisin Inhibitor, Is Effective in vitro Against Leishmania infantum.

PF-429242 is an inhibitor of subtilisin, an important protease found in Leishmania. However, studies regarding the effect of PF-429242 on Leishmania are scarce. In this work we evaluated the antileishmanial effect of PF-429242 against Leishmania infantum and the mechanism involved in the death of the parasite. PF-429242 had low toxicity against mammalian cells (peritoneal macrophages) (CC50 = 189.07 µM) and presented activity against L. infantum promastigotes (IC50 = 2.78 µM) and intracellular amastigotes (IC50 = 14.07 µM), indicating selectivity toward the parasite. Transmission electron microscopy (TEM), as well as staining of L. infantum promastigotes with MitoTracker® Red, rhodamine 123 and MitoSOX, revealed that the mitochondria was a potential target of PF-429242. In addition, PF-429242 caused an accumulation of neutral lipids in promastigotes, which was demonstrated by Nile Red staining and TEM, and induced oxidative stress (H2DCFDA staining). Furthermore the formation of autophagic vacuoles in L. infantum promastigotes was observed by MDC staining and TEM. However, the killing induced by PF-429242 in L. infantum promastigotes appeared to be unrelated to apoptosis and/or necrosis as there was no phosphatidylserine externalization, DNA fragmentation or alterations in the permeability of the parasite plasma membrane, as assessed by annexin V-FITC, TUNEL and propidium iodide staining, respectively. The morphological and ultrastructural evaluation of the promastigotes by optical microscopy, scanning electron microscopy (SEM) and TEM, revealed the presence of parasites with flagellar defects. TEM analysis of the intracellular amastigotes indicated that mitochondrial damage and autophagy could also be involved in the death of these forms after treatment with PF-429242. In addition, PF-429242 treatment stimulated NO production from infected macrophage, but only at a high concentration (100 µM), as well as an increase of TNF levels after treatment with 10 µM of PF-429242. The compound did not stimulate ROS or IL-10 production. Together, these data highlight the antileishmanial potential of PF-429242, inducing several cellular alterations in the parasite, such as mitochondrial damage, neutral lipids accumulation, oxidative stress and autophagy which culminate in the death of L. infantum, as well as modulating host cellular responses that favor the development of an immune response against the parasite.