In vitro anti-Leishmania activity and molecular docking of spiro-acridine compounds as potential multitarget agents against Leishmania infantum.

Leishmaniasis is an infectious disease with several limitations regarding treatment schemes. This work reports the anti-Leishmania activity of spiroacridine compounds against the promastigote (IC50 = 1.1 to 6.0 µg / mL) and amastigote forms of the best compounds (EC50 = 4.9 and 0.9 µg / mL) inLeishmania (L.) infantumand proposes an in-silico study with possible selective therapeutic targets for L. infantum. The substituted dimethyl-amine compound (AMTAC 11) showed the best leishmanicidal activity in vitro, and was found to interact with TryRandLdTopoI. comparisons with standard inhibitors were performed, and its main interactions were elucidated. Based on the biological assessment and the structure-activity relationship study, the spiroacridine compounds appear to be promisinganti-leishmaniachemotherapeutic agents to be explored.

Methyl gallate: Selective antileishmanial activity correlates with host-cell directed effects.

Leishmaniasis is a widespread tropical infection caused by different species of Leishmania protozoa. Many of the available drugs against the disease are toxic and in certain cases parasite drug resistance is developed. The discovery of drugs for the treatment of leishmaniasis is a pressing concern. In the present work, we describe in vitro studies of the phenolic compound methyl gallate (MG) against Leishmania (Leishmania) amazonensis and its possible mechanisms of action. The in vitro activity of MG was assayed against L. amazonensis (promastigotes, axenic amastigotes, and intramacrophagic amastigotes). Cytotoxicity tests were performed with J774A.1 macrophages and THP-1 cell derived macrophages. To evaluate mechanisms of action, we analyzed cellular TNF-α, IL-12, IFN-γ, IL-10, IL-6, NO, ROS levels, arginase activity, and structural mechanisms (phagocytic and lysosomal activities) involving macrophage activation. Meglumine antimoniate and amphotericin B were used as reference drugs. It was observed that MG effectively inhibited the growth of both promastigote (IC50 5.71 µM) and amastigote-like forms (EC50 5.39 µM), with much higher selectivity indexes than the reference drugs, being more benign towards J774A.1 macrophages than meglumine antimoniate and amphotericin B, at 1631- and 70.92-fold respectively, with respect to the promastigote form. Additionally, MG proved to be even more active against intracellular amastigotes of the parasite (EC50 4.24 µM). Our results showed that antileishmania activity was associated with increased TNF-α, IL-12, NO and ROS levels, as well as decreased IL-6 and decreased arginase activity. In addition, MG induced increased phagocytic capability, and lysosomal volume in macrophages; structural parameters of microbicidal activity. Taken together, our results suggest that MG may be a promising candidate for new drug development against leishmaniasis.

Biological activity of Morita-Baylis-Hillman adduct homodimers in L. infantum and L. amazonensis: anti-Leishmania activity and cytotoxicity.

This study is a report on the anti-Leishmania activity of Morita-Baylis-Hillman (MBH) homodimers adducts against the promastigote and axenic amastigote forms of Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis and on the cytotoxicity of these adducts to human blood cells. Both studied homodimers, MBH 1 and MBH 2, showed activity against the promastigote forms of L. infantum and L. amazonensis, which are responsible for visceral and cutaneous leishmaniasis, respectively. Additionally, the homodimers presented biological activity against the axenic amastigote forms of these two Leishmania species. The adducts exhibited no hemolytic activity to human peripheral blood mononuclear cells or erythrocytes at the tested concentrations and achieved higher selectivity indices than amphotericin B. Evaluation of cell death by apoptosis revealed that the homodimers had better apoptosis/necrosis profiles than amphotericin B in the promastigote forms of both L. infantum and L. amazonensis. In conclusion, these Morita-Baylis-Hillman adducts had anti-Leishmania activity in an in vitro model and may thus be promising molecules in the search for new drugs to treat leishmaniasis.

SB-83, a 2-Amino-thiophene derivative orally bioavailable candidate for the leishmaniasis treatment.

Leishmaniasis, affecting more than 12 million people worldwide has become a severe public-health problem. The therapeutic arsenal against leishmaniasis is mainly administered by parenteral route; it is toxic, expensive, and associated with recurrence risk. The need for further therapeutic compounds research is pressing. In previous studies, we demonstrated the antileishmanial activities of ten 2-amino-thiophene derivatives, which evidenced the action of a compound, called SB-83, having expressive antileishmania activity in an in vitro infection model. In the present work, we describe preclinical studies of the thiophenic derivative SB-83, such as acute toxicity, genotoxicity, in vivo oral efficacy in a murine model, and in vitro antileishmanial activity against an L. amazonensis SbIII-resistant strain. Determining acute preclinical toxicity, the LD50 of SB-83 was estimated at 2500 mg/kg orally, with few behavioral changes in Swiss mice. Further, treatment with 2000 mg/kg of SB-83 did not induce in vivo genotoxic activity in the peripheral blood micronucleus assay. In 7 weeks of oral treatment, SB-83 reduced paw lesion size in L. amazonensis infected mice by 52.47 ± 5.32%, and decreased the parasite load of the popliteal lymph node and spleen at the highest dose tested (200 mg/kg) respectively by 42.57 ± 3.14%, and 100%, without presenting weight change or other changes of clinical importance in the biochemical and hematological profiles. The treatment of promastigotes and intracellular amastigotes of SbIII sensitive and resistant strains with SB-83 did not produce differences in antileishmania activity, which suggests no cross-resistance. Thus, this work demonstrated that SB-83 has potential as a new active drug candidate even when orally administered, which may become a new therapeutic alternative for the treatment of leishmaniasis.

New thiophene-acridine compounds: Synthesis, antileishmanial activity, DNA binding, chemometric, and molecular docking studies.

In this study, we synthesized eight new compounds containing the 2-amino-cycloalkyl[b]thiophene and acridine moieties (ACT01 and ACS01 -ACS07 ). None tested compounds presented human erythrocyte cytotoxicity. The new compounds presented antipromastigote activity, where ACS01 and ACS02 derivatives presented significant antileishmanial activity, with better performance than the reference drugs (tri and pentavalent antimonials), with respective IC50 values of 9.60 ± 3.19 and 10.95 ± 3.96 µm. Additionally, these two derivatives were effective against antimony-resistant Leishmania (Leishmania) amazonensis strains. In addition, binding and fragmentation DNA assays were performed. It was observed that the antileishmanial activity of ACS01 is not associated with DNA fragmentation of the promastigote forms. However, it interacted with DNA with a binding constant of 104  m-1 . In partial least-squares studies, it was observed that the most active compounds (ACS01 and ACS02 ) showed lower values of amphiphilic moment descriptor, but there was a correlation between the lipophilicity of the molecules and antileishmanial activity. Furthermore, the docking molecular studies showed interactions between thiophene-acridine derivatives and the active site of pyruvate kinase enzyme with the major contribution of asparagine 152 residue for the interaction with thiophene moiety. Thus, the results suggested that the new thiophene-acridine derivatives are promising molecules as potential drug candidates.

Synthesis and In Vitro Anti Leishmania amazonensis Biological Screening of Morita-Baylis-Hillman Adducts Prepared from Eugenol, Thymol and Carvacrol.

Leishmaniasis represents a series of severe neglected tropical diseases caused by protozoa of the genus Leishmania and is widely distributed around the world. Here, we present the syntheses of Morita-Baylis-Hillman adducts (MBHAs) prepared from eugenol, thymol and carvacrol, and their bioevaluation against promastigotes of Leishmania amazonensis. The new MBHAs are prepared in two steps from essential oils in moderate to good yields and present IC50 values in the range of 22.30-4.71 µM. Moreover, the selectivity index to the most potent compound is very high (SIrb > 84.92), far better than that of Glucantime® (SIrb 1.39) and amphotericin B (SIrb = 22.34). Conformational analysis were carried out at the M062X//6-31+G(d,p) level of theory to corroborate a hypothesis about the nitroaromatic bioreduction mechanism.

Antiparasitic effect of Dinoponera quadriceps giant ant venom.

Neglected tropical diseases (NTD) are treated with toxic therapy of limited efficacy. Previously, we studied the antimicrobial effect of Dinoponera quadriceps venom (DqV) against bacteria. To continue the study, we report in this short communication the antimicrobial effect of DqV against Leishmania amazonensis and Trypanosoma cruzi. DqV inhibits the promastigote forms of L. amazonensis and all T. cruzi developmental forms, with low toxicity in host cells. DqV causes cell death in T. cruzi through necrotic and apoptotic mechanisms observed by staining the cells with annexin V-FITC (AX) and propidium iodide (PI), loss of mitochondrial membrane potential by flow cytometry analyses and confocal microscopy and morphological alterations, such as loss of membrane integrity and cell shrinkage by scanning electron microscopy (SEM). In conclusion, we suggest there is an antimicrobial effect also on parasites.

Synthesis and Antileishmanial Activity of Natural Dehydrodieugenol and Its Mono- and Dimethyl Ethers.

The study of chemistry of naturally occurring compounds and the synthesis of their derivatives is fundamentally important for the development of new drugs. In this work, dehydrodieugenol (DHDE) was obtained through oxidative coupling of eugenol, promoted by an aqueous mixture of potassium ferricyanide (K3 [Fe(CN)6 ]) and NH3  · H2 O. The partial methoxylation of DHDE with MeI and K2 CO3 mainly resulted in the molecular-shaped monomethyl ether (DHDE-1MeO) and its dimethyl ether derivative (DHDE-2MeO). The products from the reactions were characterized by (1) H- and (13) C-NMR spectroscopy. Additionally, these studies have reported the antileishmanial activity of DHDE against Leishmania amazonensis (IC50 value of 42.20 µg ml(-1) ) and shown that partial methoxylation of DHDE results in a significant increase in its antiparasitic activity (IC50 value of 13.68 µg ml(-1) ). Based on in vitro bioassays, DHDE-1MeO has shown the highest leishmanicidal activity in promastigota form. Production by direct one-step synthesis of this monomethoxylated compound can be considered to be a cost-effective and environmentally friendly method with a short reaction time.

2-Amino-thiophene derivatives present antileishmanial activity mediated by apoptosis and immunomodulation in vitro.

This study evaluated the effects of 2-amino-thiophene derivatives on the promastigote and amastigote forms of Leishmania (Leishmania) amazonensis and their possible mechanisms of action. Initially, we evaluated the antileishmanial activity of ten 2-amino-thiophene derivatives on promastigote and axenic amastigote forms of Leishmania amazonensis and their cytotoxicity against murine macrophages and human red blood cells. Three promising compounds were selected for studies of the cell death process using flow cytometry analysis and a DNA fragmentation assay. The effects of the compounds were assessed on intramacrophagic amastigotes, and the modulation of cytokine and NO production was investigated. All thiophene derivatives showed antileishmanial activity against promastigotes and axenic amastigotes with less toxicity for murine macrophages and human red blood cells. The best values were obtained for compounds containing a lateral indole ring. Docking studies suggested that these compounds played an important role in inhibiting trypanothione reductase (TryR) activity. The selected compounds SB-200, SB-44, and SB-83 induced apoptosis in promastigotes involving phosphatidylserine externalization and DNA fragmentation in a pattern similar to that observed for the positive control. Additionally, SB-200, SB-44, and SB-83 significantly reduced the infection index of macrophages by the parasites; for compounds SB-200 and SB-83 this reduction was associated with increased TNF-α, IL-12, and NO levels. This study demonstrated the effective and selective action of 2-amino-thiophene derivatives against L. amazonensis, resulting in apoptosis-like cell death and immunomodulation in vitro. The results suggest that they are promising compounds for the development of new leishmanicidal drugs.

Traditional uses, chemical constituents, and biological activities of Bixa orellana L.: a review.

Bixa orellana L., popularly known as "urucum," has been used by indigenous communities in Brazil and other tropical countries for several biological applications, which indicates its potential use as an active ingredient in pharmaceutical products. The aim of this work was to report the main evidence found in the literature, concerning the ethnopharmacology, the biological activity, and the phytochemistry studies related to Bixa orellana L. Therefore, this work comprises a systematic review about the use of Bixa orellana in the American continent and analysis of the data collected. This study shows the well-characterized pharmacological actions that may be considered relevant for the future development of an innovative therapeutic agent.

Genetic diversity of Leishmania infantum field populations from Brazil.

Leishmania infantum (syn. Leishmania chagasi) is the etiological agent of visceral leishmaniasis (VL) in Brazil. The epidemiology of VL is poorly understood. Therefore, a more detailed molecular characterization at an intraspecific level is certainly needed. Herein, three independent molecular methods, multilocus microsatellite typing (MLMT), random amplification of polymorphic DNA (RAPD) and simple sequence repeats-polymerase chain reaction (SSR-PCR), were used to evaluate the genetic diversity of 53 L. infantum isolates from five different endemic areas in Brazil. Population structures were inferred by distance-based and Bayesian-based approaches. Eighteen very similar genotypes were detected by MLMT, most of them differed in only one locus and no correlation was found between MLMT profiles, geographical origin or the estimated population structure. However, complex profiles composed of 182 bands obtained by both RAPD and SSR-PCR assays gave different results. Unweighted pair group method with arithmetic mean trees built from these data revealed a high degree of homogeneity within isolates of L. infantum. Interestingly, despite this genetic homogeneity, most of the isolates clustered according to their geographical origin.

Genetic diversity of Leishmania infantum field populations from Brazil

Leishmania infantum (syn. Leishmania chagasi) is the etiological agent of visceral leishmaniasis (VL) in Brazil. The epidemiology of VL is poorly understood. Therefore, a more detailed molecular characterization at an intraspecific level is certainly needed. Herein, three independent molecular methods, multilocus microsatellite typing (MLMT), random amplification of polymorphic DNA (RAPD) and simple sequence repeats-polymerase chain reaction (SSR-PCR), were used to evaluate the genetic diversity of 53 L. infantum isolates from five different endemic areas in Brazil. Population structures were inferred by distance-based and Bayesian-based approaches. Eighteen very similar genotypes were detected by MLMT, most of them differed in only one locus and no correlation was found between MLMT profiles, geographical origin or the estimated population structure. However, complex profiles composed of 182 bands obtained by both RAPD and SSR-PCR assays gave different results. Unweighted pair group method with arithmetic mean trees built from these data revealed a high degree of homogeneity within isolates of L. infantum. Interestingly, despite this genetic homogeneity, most of the isolates clustered according to their geographical origin.

Isolamento e caracterização de parasitas responsáveis pela leishmaniose visceral em João Pessoa - Paraíba - Brasil / Isolation and characterization of visceral leishmaniosis in João Pessoa - Paraíba - Brazil

Objetivo: Isolar parasitas de Leishmania a partir de humanos e cães com quadro clínico de leishmaniose visceral da cidade de João Pessoa e estudar estes isolados através da técnica de RAPD-PCR. Metodologia: Os isolados de Leishmania de casos humanos foram obtidos através de punção de medula óssea de pacientes com quadro clínico de leishmaniose visceral e os de origem canina foram obtidos de amostras do baço de cães sintomáticos. Todas as amostras foram inoculadas em meio NNN/BHI e incubadas a 25°C. Foi realizada extração de DNA de todos os isolados de Leishmania e monitoradas as reações de RAPD-PCR. Os produtos destas reações foram aplicados em gel de agarose e os padrões de bandas analisados comparativamente. Resultados: Foram obtidos três isolados de Leishmania de origem humana e quatro isolados de origem canina. A análise dos padrões de bandas da molécula de DNA dos isolados obtidos com a técnica de RAPD demonstrou que os isolados de origem canina compartilharam o mesmo número de bandas com a cepa de referência Leishmania chagasi e os de origem humana apresentaram diferenças quanto ao número de bandas compartilhadas com esta cepa. Conclusão: Existe diversidade genética entre os parasitas de Leishmania que causam a leishmaniose visceral humana e canina na cidade de João Pessoa, sugerindo a presença de mais de uma cepa deste parasita nesta localidade

Aspectos Socioeconômicos da Leishmaniose Visceral em João Pessoa - Paraíba - Brasil / Socio-Economic Aspects of Visceral Leishmaniasis in João Pessoa - Paraíba - Brazil

Objetivo: Avaliar o perfil socioecônomico dos casos humanos de leishmaniose visceral ocorridos no biênio 1999/2000 na cidade de João Pessoa/Paraíba/Brasil. Material e Metodos: Através dos arquivos da Fundação Nacional de Saúde (FUNASA) foi realizada a coleta das fichas de notificação de todos os casos registrados da doença e dos seus respectivos endereços. Foi elaborado e aplicado um questionário na residência dos casos identificados abordando aspectos socioecônomicos como o grau de escolaridade, renda familiar e condições de moradia. Resultados: A análise dos dados mostrou que 72,5 por cento dos casos de leishmaniose visceral em João Pessoa ocorreram em crianças menores de cinco anos cuja maioria dos pais não havia concluído o ensino fundamental. A renda familiar em 47,8 por cento dos casos foi no máximo de um salário mínimo e em nenhuma das residências havia presença de rede de esgoto, sendo que em 52 por cento das residencias foi constatado o acúmulo de lixo no peridomicílio. Conclusão: Houve um predomínio da leishmaiose visceral na população com baixos índices de escolaridade, baixa renda familiar e precárias condições sanitárias de moradia, sugerindo uma provável relação entre estes parâmetros e um maior risco de desenvolver a leishmaiose visceral

Influence of microbiota in experimental cutaneous leishmaniasis in Swiss mice

Neste trabalho, estudamos a infeccao experimental de camundongos Suicos/NIH com Leishmania major em comparacao com camundongos isogenicos C57BL/6 e BALB/c que sao resistentes e suceptiveis a esta infeccao, respectivamente. Camundongos Suicos mostraram lesoes que se curaram espontaneamente e se restringiram ao local de inoculacao. Celulas linfoides derivadas destes animais desenvolveram uma resposta do tipo Th1, caracterizada pela producao de niveis altos de IFN-gama e niveis baixos de IL-4. Com o objetivo de estudar a importancia da microbiota no desenvolvimento da leishmaniose cutanea neste modelo, camundongos Suicos sem germes foram infectados na pata com promastigotas de L. major, convencionalizados apos 3 semanas de infeccao e as lesoes comparadas com as observadas em camundongos convencionais...

Isolamento e caracterizaçäo de bacteriófagos de Lactococcus lactis e Lactococcus cremosis / Isolation and characterization of lactococcal bacteriophages from cheese whey

Amostras de soro, coletadas em indústrias de queijo de Minas Gerais, Brasil, foram usada para isolar e caracterizar bacteriófagos de bactérias lácticas. Dez bacteriófagos foram observados em microscópio eletrônico e seus DNAs foram analisados em gel de agarose após a clivagem com várias enzimas de restriçäo. Sete bacteriófagos foram específicos para Lactococcus lactis subsp. cremosis e três para L. lactis subsp. lactis. As micrografias eletrônicas mostraram que todos os bacteriófagos apresentavam morfologia similar, contendo cabeça isométrica e cauda curta näo contrátil. Baseando-se no padräo de restriçäo do DNA e no tamanho do genoma, os bacteriófagos foram classificados em quatro grupos distintos. Todos os três bacteriófagos específicos para L. lactis subsp. lactis se situaram no mesmo grupo.