RESUMO
BACKGROUND: Chagas disease and leishmaniasis affect a significant portion of the Latin American population and still lack efficient treatments. In this context, natural products emerge as promising compounds for developing more effective therapies, aiming to mitigate side effects and drug resistance. Notably, species from the Amaryllidaceae family emerge as potential reservoirs of antiparasitic agents due to the presence of diverse biologically active alkaloids. PURPOSE: To assess the anti-Trypanosoma cruzi and anti-Leishmania infantum activity of five isolated alkaloids from Hippeastrum aulicum Herb. (Amaryllidaceae) against different life stages of the parasites using in silico and in vitro assays. Furthermore, molecular docking was employed to evaluate the interaction of the most active alkaloids. METHODS: Five natural isoquinoline alkaloids isolated in suitable quantities for in vitro testing underwent preliminary in silico analysis to predict their potential efficacy against Trypanosoma cruzi (amastigote and trypomastigote forms) and Leishmania infantum (amastigote and promastigote forms). The in vitro antiparasitic activity and mammalian cytotoxicity were investigated with a subsequent comparison of both analysis (in silico and in vitro) findings. Additionally, this study employed the molecular docking technique, utilizing cruzain (T. cruzi) and sterol 14α-demethylase (CYP51, L. infantum) as crucial biological targets for parasite survival, specifically focusing on compounds that exhibited promising activities against both parasites. RESULTS: Through computational techniques, it was identified that the alkaloids haemanthamine (1) and lycorine (8) were the most active against T. cruzi (amastigote and trypomastigote) and L. infantum (amastigote and promastigote), while also revealing unprecedented activity of alkaloid 7methoxy-O-methyllycorenine (6). The in vitro analysis confirmed the in silico tests, in which compound 1 presented the best activities against the promastigote and amastigote forms of L. infantum with half-maximal inhibitory concentration (IC50) 0.6 µM and 1.78 µM, respectively. Compound 8 exhibited significant activity against the amastigote form of T. cruzi (IC50 7.70 µM), and compound 6 demonstrated activity against the trypomastigote forms of T. cruzi and amastigote of L. infantum, with IC50 values of 89.55 and 86.12 µM, respectively. Molecular docking analyses indicated that alkaloids 1 and 8 exhibited superior interaction energies compared to the inhibitors. CONCLUSION: The hitherto unreported potential of compound 6 against T. cruzi trypomastigotes and L. infantum amastigotes is now brought to the forefront. Furthermore, the acquired dataset signifies that the isolated alkaloids 1 and 8 from H. aulicum might serve as prototypes for subsequent structural refinements aimed at the exploration of novel leads against both T. cruzi and L. infantum parasites.
Assuntos
Alcaloides , Amaryllidaceae , Isoquinolinas , Leishmania infantum , Simulação de Acoplamento Molecular , Trypanosoma cruzi , Trypanosoma cruzi/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Amaryllidaceae/química , Alcaloides/farmacologia , Alcaloides/química , Alcaloides/isolamento & purificação , Isoquinolinas/farmacologia , Isoquinolinas/química , Isoquinolinas/isolamento & purificação , Animais , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Humanos , Antiparasitários/farmacologia , Antiparasitários/química , Antiparasitários/isolamento & purificação , Antiprotozoários/farmacologia , Antiprotozoários/química , Antiprotozoários/isolamento & purificaçãoRESUMO
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)
Assuntos
Trypanosoma cruzi , Basidiomycota , Bioensaio , Membrana Celular , Doença de Chagas , Pleurotus , Ergosterol , MitocôndriasRESUMO
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.
RESUMO
The neglected tropical diseases Chagas disease and leishmaniasis affect together more than 20 million people living mainly in developing countries. The mainstay of treatment is chemotherapy, however the drugs of choice, which include benznidazole and miltefosine, are toxic and have numerous side effects. Safe and effective therapies are urgently needed. Marine alpha-pyrones have been previously identified as scaffolds with potential antiprotozoan activities. In this work, using a phenotypic screen, twenty-seven examples of 3-substituted 4-hydroxy-6-methyl alpha-pyrones were synthesized and their antiparasitic efficacy evaluated against Leishmania (L.) infantum and Trypanosoma cruzi in order to evaluate structure-activity relationships within the series. The mechanism of action and the in vivo efficacy of the most selective compound against T. cruzi were evaluated using different techniques. In vitro data indicated that compounds 8, 15, 25, 26 and 28 presented IC50 values in the range between 13 and 54 µM against L. infantum intracellular amastigotes. Among them, hexanoyl substituted pyrone 8 was the most selective and potent, with a Selectivity Index (SI) > 14. Fifteen of the alpha-pyrones were effective against T. cruzi trypomastigotes, with 3-undecanoyl (11) and 3-tetradecanoyl (12) substituted pyrones being the most potent against trypomastigotes, with IC50 values of 1 and 2 µM, respectively, and SI higher than 70. Using flow cytometry and fluorescent-based assays, pyrone 12 was found to
As doenças tropicais negligenciadas A doença de Chagas e a leishmaniose afetam juntas mais de 20 milhões de pessoas que vivem principalmente nos países em desenvolvimento. O suporte principal do tratamento é a quimioterapia, no entanto, os medicamentos de escolha, que incluem benznidazol e miltefosina, são tóxicos e têm inúmeros efeitos colaterais. Terapias seguras e eficazes são urgentemente necessárias. As alfa-pironas marinhas foram previamente identificadas como andaimes com potenciais atividades antiprotozoárias. Neste trabalho, usando uma tela fenotípica, sintetizaram-se 27 exemplos de 4-hidroxi-6-metil alfa-pironas 3-substituídas e avaliou-se sua eficácia antiparasitária contra Leishmania (L.) infantum e Trypanosoma cruzi para avaliar a estrutura relações de atividade dentro da série. O mecanismo de ação e a eficácia in vivo do composto mais seletivo contra T. cruzi foram avaliados por diferentes técnicas. Dados in vitro indicaram que os compostos 8, 15, 25, 26 e 28 apresentaram valores de IC50 na faixa entre 13 e 54 µM contra amastigotas intracelulares de L. infantum. Entre elas, a pirona 8 substituída com hexanoílo foi a mais seletiva e potente, com um índice de seletividade (SI)> 14. Quinze das alfa-pironas foram eficazes contra as tripomastigotas de T. cruzi, com 3-undecanoil (11) e 3-tetradecanoil ( 12) pironas substituídas sendo as mais potentes contra tripomastigotas, com valores de IC50 de 1 e 2 µM, respectivamente, e SI superiores a 70. Usando citometria de fluxo e ensaios à base de fluorescência, a pirona 12 foi encontrada para