Isoprenoid alcohols utilization by malaria parasites.

Plasmodium falciparum is the etiological agent of human malaria, one of the most widespread diseases in tropical and subtropical regions. Drug resistance is one of the biggest problems in controlling the disease, which leads to the need to discover new antimalarial compounds. One of the most promissory drugs purposed is fosmidomycin, an inhibitor of the biosynthesis of isoprene units by the methylerythritol 4-phosphate (MEP) pathway, which in some cases failed in clinical studies. Once formed, isoprene units are condensed to form longer structures such as farnesyl and geranylgeranyl pyrophosphate, which are necessary for Heme O and A formation, ubiquinone, and dolichyl phosphate biosynthesis as well as for protein isoprenylation. Even though the natural substrates of polyprenyl transferases and synthases are polyprenyl pyrophosphates, it was already demonstrated that isoprenoid alcohols (polyprenols) such as farnesol (FOH) and geranylgeraniol (GGOH) can rescue parasites from fosmidomycin. This study better investigated how this rescue phenomenon occurs by performing drug-rescue assays. Similarly, to FOH and GGOH, it was observed that phytol (POH), a 20-carbon plant isoprenoid, as well as unsaponifiable lipid extracts from foods rescue parasites from the antimalarial effect of fosmidomycin. Contrarily, neither dolichols nor nonaprenol rescue parasites from fosmidomycin. Considering this, here we characterized the transport of FOH, GGOH, and POH. Once incorporated, it was observed that these substances are phosphorylated, condensed into longer isoprenoid alcohols, and incorporated into proteins and dolichyl phosphates. Through proteomic and radiolabelling approaches, it was found that prenylated proteins are naturally attached to several isoprenoids, derived from GGOH, dolichol, and POH if exogenously added. Furthermore, the results suggest the presence of at least two promiscuous protein prenyltransferases in the parasite: one enzyme which can use FPP among other unidentified substrates and another enzyme that can use GGPP, phytyl pyrophosphate (PPP), and dolichols, among other substrates not identified here. Thus, further evidence was obtained for dolichols and other isoprenoid products attached to proteins. This study helps to better understand the apicoplast-targeting antimalarial mechanism of action and a novel post-translational modification of proteins in P. falciparum.

Repurposing topical triclosan for cutaneous leishmaniasis: Preclinical efficacy in a murine Leishmania (L.) amazonensis model.

Leishmaniasis remains an important neglected tropical infection caused by the protozoan Leishmania and affects 12 million people in 98 countries. The treatment is limited with severe adverse effects. In the search for new therapies, the drug repositioning and combination therapy have been successfully applied to neglected diseases. The aim of the present study was to evaluate the in vitro and in vivo anti-Leishmania (Leishmania) amazonensis potential of triclosan, an approved topical antimicrobial agent used for surgical procedures. in vitro phenotypic studies of drug-treated parasites were performed to evaluate the lethal action of triclosan, accompanied by an isobolographic ex-vivo analysis with the association of triclosan and miltefosine. The results showed that triclosan has activity against L. (L.) amazonensis intracellular amastigotes, with a 50% inhibitory concentration of 16 µM. By using fluorescent probes and transmission electron microscopy, a pore-forming activity of triclosan toward the parasite plasma membrane was demonstrated, leading to depolarization of the mitochondrial membrane potential and reduction of the reactive oxygen species levels in the extracellular promastigotes. The in vitro interaction between triclosan and miltefosine in the combination therapy assay was classified as additive against intracellular amastigotes. Leishmania-infected mice were treated with topical triclosan (1% base cream for 14 consecutive days), and showed 89% reduction in the parasite burden. The obtained results contribute to the investigation of new alternatives for the treatment of cutaneous leishmaniasis and suggest that the coadministration of triclosan and miltefosine should be investigated in animal models.

Efficacy of sertraline against Trypanosoma cruzi: an in vitro and in silico study.

BACKGROUND: Drug repurposing has been an interesting and cost-effective approach, especially for neglected diseases, such as Chagas disease. METHODS: In this work, we studied the activity of the antidepressant drug sertraline against Trypanosoma cruzi trypomastigotes and intracellular amastigotes of the Y and Tulahuen strains, and investigated its action mode using cell biology and in silico approaches. RESULTS: Sertraline demonstrated in vitro efficacy against intracellular amastigotes of both T. cruzi strains inside different host cells, including cardiomyocytes, with IC50 values between 1 to 10 µM, and activity against bloodstream trypomastigotes, with IC50 of 14 µM. Considering the mammalian cytotoxicity, the drug resulted in a selectivity index of 17.8. Sertraline induced a change in the mitochondrial integrity of T. cruzi, resulting in a decrease in ATP levels, but not affecting reactive oxygen levels or plasma membrane permeability. In silico approaches using chemogenomic target fishing, homology modeling and molecular docking suggested the enzyme isocitrate dehydrogenase 2 of T. cruzi (TcIDH2) as a potential target for sertraline. CONCLUSIONS: The present study demonstrated that sertraline had a lethal effect on different forms and strains of T. cruzi, by affecting the bioenergetic metabolism of the parasite. These findings provide a starting point for future experimental assays and may contribute to the development of new compounds.

Terapias alternativas e combinadas para o tratamento da Leishmaniose Cutânea Americana: uma abordagem experimental in vitro e in vivo

A leishmaniose é uma doença tropical causada por protozoários do gêneroLeishmania que afeta 12 milhões de pessoas em 98 países. Seu tratamentoconta com um restrito arsenal terapêutico e exige a administração defármacos tóxicos por longos períodos. Na busca por novas terapias, oreposicionamento de fármacos e a associação terapêutica têm sidoaplicados com sucesso para doenças negligenciadas. O presente estudoteve como objetivo a avaliação in vitro, ex vivo e in vivo do potencial antiLeishmania(Leishmania) amazonensis dos fármacos amitriptilina, econazol,sertralina e triclosan, bem como o estudo de associações terapêuticas invitro e/ou ex vivo e mecanismo de ação in vitro dos fármacos amitriptilina etriclosan. Os resultados demonstraram que todos os fármacos estudados apresentaram atividade contra formas promastigotas e amastigotasintracelulares de L. (L.) amazonensis, com valores de Concentração Efetiva50% que variam de 1,50 a 51,48 µM. Os resultados obtidos a partir das associações entre os fármacos estudados e fármacos padrões foram classificados como aditivos ou indiferentes. Por meio da investigação domecanismo de ação leishmanicida, foi possível concluir que a mitocôndria é uma organela alvo do fármaco amitriptilina, enquanto que o fármaco triclosaninduz danos à membrana plasmática parasitária. Quando tratados comeconazol por via oral (10 mg/kg/dia por 28 dias consecutivos) ou triclosanpor via tópica (creme 1% por 14 dias consecutivos), houve uma redução de75 a 89% da carga parasitária dos camundongos infectados com L. (L.) amazonensis. Os resultados obtidos contribuem para a investigação de novas alternativas para o tratamento da leishmaniose cutânea e sugerem que novos estudos utilizando associação ou coadministração dessesfármacos com fármacos padrões podem ser promissores em modelosanimais.

Leishmaniasis is a tropical disease caused by protozoa of the genusLeishmania that affects 12 million people in 98 countries. There is a limitedtherapeutic arsenal and the treatment requires the administration of toxicdrugs for long periods. In the search for new therapies, the drug repositioningand therapeutic association have been successfully applied to neglecteddiseases. The aim of the present study was to evaluate in vitro, ex vivo andin vivo anti-Leishmania (Leishmania) amazonensis potential of the drugsamitriptyline, econazole, sertraline and triclosan, as well as the study of invitro and / or ex vivo therapeutic associations and mechanism of action of thedrugs amitriptyline and triclosan. The results showed that all studied drugshave activity against L. (L.) amazonensis promastigotes and intracellularamastigotes, with 50% Effective Concentration values ranging from 1.50 to51.48 μM. The results obtained from the combination between the studieddrugs and standard drugs were classified as additives or indifferent. Throughthe investigation of the leishmanicial mechanism of action, it was possible toconclude that the mitochondria is a target organelle of the drug amitriptyline,whereas the drug triclosan induces damage to the parasitic plasmamembrane. When treated with oral econazole (10 mg/kg/day for 28consecutive days) or triclosan topically (1% cream for 14 consecutive days),there was a 75 - 89% reduction in the parasite load of the mice infected withL. (L.) amazonensis. The results obtained contribute to the investigation ofnew alternatives for the treatment of cutaneous leishmaniasis and suggestthat new studies using association or coadministration of these drugs withstandard drugs may be promising in animal models.

Efficacy of sertraline against Trypanosoma cruzi: an in vitro and in silico study

Drug repurposing has been an interesting and cost-effective approach, especially for neglected diseases, such as Chagas disease. Methods: In this work, we studied the activity of the antidepressant drug sertraline against Trypanosoma cruzi trypomastigotes and intracellular amastigotes of the Y and Tulahuen strains, and investigated its action mode using cell biology and in silico approaches. Results: Sertraline demonstrated in vitro efficacy against intracellular amastigotes of both T. cruzi strains inside different host cells, including cardiomyocytes, with IC50 values between 1 to 10 µM, and activity against bloodstream trypomastigotes, with IC50 of 14 µM. Considering the mammalian cytotoxicity, the drug resulted in a selectivity index of 17.8. Sertraline induced a change in the mitochondrial integrity of T. cruzi, resulting in a decrease in ATP levels, but not affecting reactive oxygen levels or plasma membrane permeability. In silico approaches using chemogenomic target fishing, homology modeling and molecular docking suggested the enzyme isocitrate dehydrogenase 2 of T. cruzi (TcIDH2) as a potential target for sertraline. Conclusions: The present study demonstrated that sertraline had a lethal effect on different forms and strains of T. cruzi, by affecting the bioenergetic metabolism of the parasite. These findings provide a starting point for future experimental assays and may contribute to the development of new compounds.(AU)

Ergosterol isolated from the basidiomycete Pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria.

BACKGROUND: Major drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds. METHODS: Fruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS). RESULTS: The most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC50 value of 51.3 µg/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 µg/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite. CONCLUSIONS: The selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.

Ergosterol isolated from the basidiomycete Pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria

Background Major drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds. Methods Fruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS). Results The most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC50 value of 51.3 μg/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 μg/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite. Conclusions The selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.(AU)

Ergosterol isolated from the basidiomycete pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria

Background: Major drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds. Methods: Fruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS). Results: The most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC50 value of 51.3 mu g/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 mu g/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite. Conclusions: The selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.

Ergosterol isolated from the basidiomycete Pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria

Abstract Background Major drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds. Methods Fruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS). Results The most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC50 value of 51.3 g/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 g/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite. Conclusions The selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.

Investigation of Calcium Channel Blockers as Antiprotozoal Agents and Their Interference in the Metabolism of Leishmania (L.) infantum.

Leishmaniasis and Chagas disease are neglected parasitic diseases endemic in developing countries; efforts to find new therapies remain a priority. Calcium channel blockers (CCBs) are drugs in clinical use for hypertension and other heart pathologies. Based on previous reports about the antileishmanial activity of dihydropyridine-CCBs, this work aimed to investigate whether the in vitro anti-Leishmania infantum and anti-Trypanosoma cruzi activities of this therapeutic class would be shared by other non-dihydropyridine-CCBs. Except for amrinone, our results demonstrated antiprotozoal activity for fendiline, mibefradil, and lidoflazine, with IC50 values in a range between 2 and 16 µM and Selectivity Index between 4 and 10. Fendiline demonstrated depolarization of mitochondrial membrane potential, with increased reactive oxygen species production in amlodipine and fendiline treated Leishmania, but without plasma membrane disruption. Finally, in vitro combinations of amphotericin B, miltefosine, and pentamidine against L. infantum showed in isobolograms an additive interaction when these drugs were combined with fendiline, resulting in overall mean sum of fractional inhibitory concentrations between 0.99 and 1.10. These data demonstrated that non-dihydropyridine-CCBs present antiprotozoal activity and could be useful candidates for future in vivo efficacy studies against Leishmaniasis and Chagas' disease.

An effective in vitro and in vivo antileishmanial activity and mechanism of action of 8-hydroxyquinoline against Leishmania species causing visceral and tegumentary leishmaniasis.

The development of new therapeutic strategies to treat leishmaniasis has become a priority. In the present study, the antileishmanial activity of 8-hydroxyquinoline (8-HQN) was investigated against in vitro promastigotes and in vivo intra-macrophage amastigotes of three Leishmania species: Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis. Studies were performed to establish the 50% Leishmania inhibitory concentration (IC50) of 8-HQN, as well as its 50% cytotoxic concentration (CC50) on murine macrophages and in human red blood cells. The inhibition of macrophages infection was also evaluated using parasites that were pre-treated with 8-HQN. The effects of this compound on nitric oxide (NO) production and in the mitochondrial membrane potential were also evaluated. Finally, the therapeutic efficacy of 8-HQN was assessed in a known murine model, L. amazonensis-chronically infected BALB/c mice. Our results showed that 8-HQN was effective against promastigote and amastigote stages of all tested Leishmania species, presenting a selectivity index of 328.0, 62.0 and 47.0 for L. amazonensis, L. infantum and L. braziliensis, respectively. It was effective in treating infected macrophages, as well as in preventing the infection of these cells using pre-treated parasites. In addition, 8-HQN caused an alteration in the mitochondrial membrane potential of the parasites. When administered at 10mg/kg body weight/day by subcutaneous route, this product was effective in reducing the lesion diameter, as well as the parasite load in evaluated tissues and organs of infected animals. The results showed the in vitro and in vivo efficacy of 8-HQN against three different Leishmania species causing tegumentary and/or visceral leishmaniasis, and it could well be used for future therapeutic optimization studies to treat leishmaniasis.

Bioactivity and chemical composition of the essential oil from the leaves of Guatteria australis A.St.-Hil.

Essential oil from the leaves of Guatteria australis was obtained by hydrodistillation, analyzed by Gas Chromatography coupled to Mass Spectromery (GC-MS) and their antiproliferative, antileishmanial, antibacterial, antifungal and antioxidant activities were also evaluated. Twenty-three compounds were identified among which germacrene B (50.66%), germacrene D (22.22%) and (E)-caryophyllene (8.99%) were the main compounds. The highest antiproliferative activity was observed against NCI-ADR/RES (TGI = 31.08 µg/ml) and HT-29 (TGI = 32.81 µg/ml) cell lines. It also showed good antileishmanial activity against Leishmania infantum (IC50 = 30.71 µg/ml). On the other hand, the oil exhibited a small effect against Staphylococcus aureus ATCC 6538, S. aureus ATCC 14458 and Escherichia coli ATCC 10799 (MIC = 250 µg/ml), as well as small antioxidant activity (457 µmol TE/g) assessed through ORACFL assay. These results represent the first report regarding chemical composition and bioactivity of G. australis essential oil.

Activity of imidazole compounds on Leishmania (L.) infantum chagasi: reactive oxygen species induced by econazole.

Drug repositioning has been considered a promising approach to discover novel treatments against neglected diseases. Among the major protozoan diseases, leishmaniasis remains a public health threat with few therapeutic alternatives, affecting 12 million people in 98 countries. In this study, we report the in vitro antileishmanial activity of the imidazole drugs clotrimazole, and for the first time in literature, econazole and bifonazole and their potential action to affect the regulation of reactive oxygen species (ROS) of the parasites. The lethal action of the imidazoles was investigated using spectrofluorimetric techniques to detect ROS content, plasma membrane permeability, and mitochondrial membrane potential. The imidazoles showed activity against L. (L.) infantum chagasi promastigotes with IC50 values in a range of 2-8 µM; econazole was also effective against Leishmania intracellular amastigotes, with an IC50 value of 11 µM, a similar in vitro effectiveness to miltefosine. Leishmania promastigotes rapidly up-regulated the ROS release after incubation with the imidazoles, but econazole showed a marked increase in ROS content of approximately 1,900 % higher than untreated parasites. When using SYTOX(®) Green as a fluorescent probe, the imidazoles demonstrated considerable interference in plasma membrane permeability at the early time of incubation; econazole resulted in the higher influx of SYTOX(®) Green at 60 min. Despite cellular alterations, no depolarization could be observed to the mitochondrial membrane potential of Leishmania until 60 min. The lethal action of econazole involved strong permeabilization of plasma membrane of promastigotes, with an overloaded ROS content that contributed to the death of parasites. Affecting the ROS regulation of Leishmania via small molecules would be an interesting strategy for new drugs.

Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum.

Studying the cellular death pathways in Leishmania is an important aspect of discovering new antileishmanials. While using a drug repositioning approach, the lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide (NTZ) was investigated against Leishmania (L.) infantum. The in vitro antileishmanial activity and cytotoxicity were assessed using both parasite stages and mammalian NCTC cells, respectively. The lethal action of NTZ was investigated by detecting the phosphatidylserine (PS) exposure, reactive oxygen species (ROS) regulation, plasma membrane permeability, mitochondrial membrane potential and ultrastructural modifications by transmission electron microscopy. NTZ's activity against L. infantum was confirmed, producing IC50 values of 42.71µg/mL against promastigotes and 6.78µg/mL against intracellular amastigotes. NTZ rapidly altered the cellular metabolism of promastigotes by depolarising the mitochondrial membrane and up-regulating the reactive oxygen species (ROS). In addition, the flow cytometry data revealed an intense and time-dependent exposure of PS in promastigotes. When using SYTOX(®) Green as a fluorescent probe, NTZ demonstrated no interference in plasma membrane permeability. The ultrastructural alterations in promastigotes were time-dependent and caused chromatin condensation, plasma membrane blebbing and mitochondrial swelling. These data suggest that NTZ induced oxidative stress in L. (L.) infantum and might be a useful compound for investigating new therapeutic targets.

Mecanismo de ação de fármacos sintéticos e associações terapêuticas em Leishmania (L) infantum / Mechanism of action of synthetic drugs and therapeutic associations in Leishmania (L) infantum

A leishmaniose visceral (LV) é uma doença negligenciada endêmica em 68 países e que se tornou um grande problema na área da saúde pública. Essa doença afeta principalmente populações menos favorecidas, onde o acesso, os cuidados médicos e a terapia medicamentosa são limitados. Visando buscar novas alternativas para o tratamento da LV, o reposicionamento de fármacos oferece uma grande oportunidade para introdução de novas terapias. Por meio desta abordagem, foi realizada em Leishmania (Leishmania) infantum: i) a triagem de oito fármacos nitrogenados, sendo eles: bifonazol, clotrimazol, cloxazolam, econazol, hidroxizina, loratadina, nitazoxanida e quetiapina; ii) o estudo da ação letal dos fármacos loratadina e nitazoxanida, e iii) o estudo in vitro da associação do fármaco nitazoxanida com fármacos padrões. Para isto, os possíveis mecanismos de ação foram estudados por meio de ensaios fluorimétricos, visando avaliar alteração de permeabilidade de membrana plasmática, alteração do potencial de membrana mitocondrial, produção de espécies reativas de oxigênio, exposição de fosfatidilserina, assim como o estudo das alterações ultraestruturais. Os resultados mostram que todos os fármacos apresentaram atividade contra formas promastigotas, com concentração efetiva 50% (CE50) entre 2 e 167 μM; apenas o econazol e a nitazoxanida eliminaram os amastigotas intracelulares, com CE50 de 11 a 22 μM, respectivamente. O fármaco loratadina alterou a permeabilidade da membrana plasmática do parasita, causando poros e levando o parasita a morte. O estudo da ação letal da nitazoxanida em Leishmania sugere que o fármaco tenha como alvo a mitocôndria, considerando a despolarização do potencial de membrana mitocondrial e aumento da produção de espécies reativas de oxigênio, resultando em estresse oxidativo e morte celular. Além disso, a presença de marcadores específicos como a exposição de fosfatidilserina e alterações...