Leishmania amazonensis and Leishmania chagasi: In vitro leishmanicide activity of Virola surinamensis (rol.) warb.

Virola species have been used in traditional medicine as healing in skin infections. From V. surinanensis oil were isolated several sesquiterpene as the nerolidol which showed activity against species of Leishmania. The current study aimed to evaluate the leishmanicide activity and toxicity of extracts, fractions and surinamesin obtained from leaves of Virola surinamensis. Hexane, Ethyl Acetate, and Methanol extracts were obtained from powder of dry leaves of V. surinamensis. The hexane and ethyl acetate extracts were fractionated by silica gel column chromatography and increasingly polar gradient. The viability of L. chagasi and L. amazonensis promastigotes was assessed by tetrazolium salt assay (MTT). Peritoneal macrophages were exposed to L. amazonensis promastigotes. The treatment was performed with the extracts for 24 h. Then, the coverslips were stained and the infection index was determined. Cytotoxicity was determined in macrophage cells by peritoneum viability assay (MTT). The selectivity index was calculated as the product of cytotoxic concentration 50% and inhibitory concentration 50%. The hexane extract showed leishmanicide activity in promastigotes. The ethyl acetate, methanol extracts and fractions (C1-C6), were inactive against promastigote form of L. chagasi and L. amzazonensis. None extract showed effect on L. amazonensis amastigotes. All samples tested showed low cytotoxicity (CC50 > 500 µg/mL). The selectivity index of the hexane extract was greater than 5. The hexane extract of V. surinamensis was active against L. chagasi and L. amazonensis promastigotes. The extract fractionation did not increase significantly its antipromastigote activity. The surinamensin is probably not responsible for the activity. The extracts were inactive against amastigotes of L. amazonensis.

Antiprotozoan lead discovery by aligning dry and wet screening: prediction, synthesis, and biological assay of novel quinoxalinones.

Protozoan parasites have been one of the most significant public health problems for centuries and several human infections caused by them have massive global impact. Most of the current drugs used to treat these illnesses have been used for decades and have many limitations such as the emergence of drug resistance, severe side-effects, low-to-medium drug efficacy, administration routes, cost, etc. These drugs have been largely neglected as models for drug development because they are majorly used in countries with limited resources and as a consequence with scarce marketing possibilities. Nowadays, there is a pressing need to identify and develop new drug-based antiprotozoan therapies. In an effort to overcome this problem, the main purpose of this study is to develop a QSARs-based ensemble classifier for antiprotozoan drug-like entities from a heterogeneous compounds collection. Here, we use some of the TOMOCOMD-CARDD molecular descriptors and linear discriminant analysis (LDA) to derive individual linear classification functions in order to discriminate between antiprotozoan and non-antiprotozoan compounds as a way to enable the computational screening of virtual combinatorial datasets and/or drugs already approved. Firstly, we construct a wide-spectrum benchmark database comprising of 680 organic chemicals with great structural variability (254 of them antiprotozoan agents and 426 to drugs having other clinical uses). This series of compounds was processed by a k-means cluster analysis in order to design training and predicting sets. In total, seven discriminant functions were obtained, by using the whole set of atom-based linear indices. All the LDA-based QSAR models show accuracies above 85% in the training set and values of Matthews correlation coefficients (C) vary from 0.70 to 0.86. The external validation set shows rather-good global classifications of around 80% (92.05% for best equation). Later, we developed a multi-agent QSAR classification system, in which the individual QSAR outputs are the inputs of the aforementioned fusion approach. Finally, the fusion model was used for the identification of a novel generation of lead-like antiprotozoan compounds by using ligand-based virtual screening of 'available' small molecules (with synthetic feasibility) in our 'in-house' library. A new molecular subsystem (quinoxalinones) was then theoretically selected as a promising lead series, and its derivatives subsequently synthesized, structurally characterized, and experimentally assayed by using in vitro screening that took into consideration a battery of five parasite-based assays. The chemicals 11(12) and 16 are the most active (hits) against apicomplexa (sporozoa) and mastigophora (flagellata) subphylum parasites, respectively. Both compounds depicted good activity in every protozoan in vitro panel and they did not show unspecific cytotoxicity on the host cells. The described technical framework seems to be a promising QSAR-classifier tool for the molecular discovery and development of novel classes of broad-antiprotozoan-spectrum drugs, which may meet the dual challenges posed by drug-resistant parasites and the rapid progression of protozoan illnesses.

Structural characterization of a galectin isolated from the marine sponge Chondrilla caribensis with leishmanicidal potential.

BACKGROUND: Solving primary structure of lectins leads to an understanding of the physiological roles within an organism and its biotechnological potential. Only eight sponge lectins have had their primary structure fully determined. METHODS: The primary structure of CCL, Chondrilla caribensis lectin, was determined by tandem mass spectrometry. The three-dimensional structure was predicted and the protein-carbohydrate interaction analysed by molecular docking. Furthermore, the anti-leishmanial activity was observed by assays with Leishmania infantum. RESULTS: The amino acid sequence consists of 142 amino acids with a calculated molecular mass of 15,443 Da. The lectin has a galectin-like domain architecture. As observed in other sponge galectins, the signature sequence of a highly conserved domain was also identified in CCL with some modifications. CCL exhibits a typical galectin structure consisting of a ß-sandwich. Molecular docking showed that the amino acids interacting with CCL ligands at the monosaccharide binding site are mostly the same as those conserved in this family of lectins. Through its interaction with L. infantum glycans, CCL was able to inhibit the development of this parasite. CCL also induced apoptosis after eliciting ROS production and altering the membrane integrity of Leishmania infantum promastigote. CONCLUSIONS: CCL joins the restricted group of sponge lectins with determined primary structure and very high biotechnological potential owing to its promising results against pathogens that cause Leishmaniasis. GENERAL SIGNIFICANCE: As the determination of primary structure is important for biological studies, now CCL can become a sponge galectin with an exciting future in the field of human health.

A Molecular Modeling Approach to Identify Potential Antileishmanial Compounds Against the Cell Division Cycle (cdc)-2-Related Kinase 12 (CRK12) Receptor of Leishmania donovani.

The huge burden of leishmaniasis caused by the trypanosomatid protozoan parasite Leishmania is well known. This illness was included in the list of neglected tropical diseases targeted for elimination by the World Health Organization. However, the increasing evidence of resistance to existing antimonial drugs has made the eradication of the disease difficult to achieve, thus warranting the search for new drug targets. We report here studies that used computational methods to identify inhibitors of receptors from natural products. The cell division cycle-2-related kinase 12 (CRK12) receptor is a plausible drug target against Leishmania donovani. This study modelled the 3D molecular structure of the L. donovani CRK12 (LdCRK12) and screened for small molecules with potential inhibitory activity from African flora. An integrated library of 7722 African natural product-derived compounds and known inhibitors were screened against the LdCRK12 using AutoDock Vina after performing energy minimization with GROMACS 2018. Four natural products, namely sesamin (NANPDB1649), methyl ellagic acid (NANPDB1406), stylopine (NANPDB2581), and sennecicannabine (NANPDB6446) were found to be potential LdCRK12 inhibitory molecules. The molecular docking studies revealed two compounds NANPDB1406 and NANPDB2581 with binding affinities of -9.5 and -9.2 kcal/mol, respectively, against LdCRK12 which were higher than those of the known inhibitors and drugs, including GSK3186899, amphotericin B, miltefosine, and paromomycin. All the four compounds were predicted to have inhibitory constant (Ki) values ranging from 0.108 to 0.587 µM. NANPDB2581, NANPDB1649 and NANPDB1406 were also predicted as antileishmanial with Pa and Pi values of 0.415 and 0.043, 0.391 and 0.052, and 0.351 and 0.071, respectively. Molecular dynamics simulations coupled with molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) computations reinforced their good binding mechanisms. Most compounds were observed to bind in the ATP binding pocket of the kinase domain. Lys488 was predicted as a key residue critical for ligand binding in the ATP binding pocket of the LdCRK12. The molecules were pharmacologically profiled as druglike with inconsequential toxicity. The identified molecules have scaffolds that could form the backbone for fragment-based drug design of novel leishmanicides but warrant further studies to evaluate their therapeutic potential.

Antiparasitic activity of new gibbilimbol analogues and SAR analysis through efficiency and statistical methods.

Chagas' disease and leishmaniasis are parasitic infections enrolled among the neglected tropical diseases, which urge for new treatments. In the search for new chemical entities as prototypes, gibbilimbols A/B have shown antiparasitic activity against Trypanosoma cruzi and Leishmania infantum, and then a set of analogues (LINS03 series) of this natural product were synthesized and evaluated in vitro against the parasites. In the present paper we reported five new compounds with activity against these protozoan parasites, and quite low cytotoxicity. Moreover, the interference of plasma membrane permeability of these analogues were also evaluated. We found that [(4-methoxyphenyl)methyl]octylamine (4) was noteworthy due to its high activity against the amastigote form of both parasites (IC50 1.3-5.8 µM) and good selectivity index. In order to unveil the SAR for this chemotype, we also presented a group efficiency analysis and PCA and HCA study, which indicated that the methoxyl provides good activity with lower cytotoxicity to mammalian cells. The results from SAR analyses suggest different mechanisms of action between the neutral and basic compounds. In summary, the analogues represent important activity against these parasites and must be prototypes for further exploitation.

Uso da miltefosina no tratamento clínico de cães com leishmaniose visceral: revisão de literatura / Use of miltefosin in the clinical treatment of dogs with visceral leishmaniosis / Uso de miltefosina en el tratamiento clínico de perros con leishmaniosis visceral: revisión de la literatura

A leishmaniose visceral canina é uma doença de caráter zoonótico, acometendo os seres humanos e diversas espécies de animais silvestres e domésticos. Objetivou-se com o presente estudo realizar uma revisão de literatura sobre o uso da miltefosina no tratamento clínico de cães com leishmaniose visceral. Trata- se de uma revisão de literatura, a qual foi realizada por meio de consultas à periódicos e livros presentes na biblioteca do Cesmac. Foram utilizadas bases de dados como: portal Capes, SCIELO, Google Acadêmico; pesquisa em monografias, teses e dissertações. Causada pelo protozoário Leishmania chagasi, sendo o cão doméstico o principal reservatório desse protozoário. Por representar um problema grave de saúde pública e ser considerada uma doença potencialmente fatal (quando não tratada precocemente e adequadamente), faz- se importante que o clínico esteja familiarizado com os sinais clínicos, exames complementares e principais protocolos terapêuticos, em especial a utilização da miltefosina no tratamento da leishmaniose visceral em cães. Por ser uma zoonose que causa graves problemas de saúde pública e que vem crescendo cada vez mais no Brasil, cabe aos médicos veterinários assumirem o compromisso na conscientização sobre a importância do diagnóstico precoce além de promoverem o bem-estar animal e a saúde pública.(AU)

Canine visceral leishmaniasis is a zoonotic disease, affecting humans and several species of wild and domestic animals. The objective of the present study was to carry out a literature review on the use of miltefosine in the clinical treatment of dogs with visceral leishmaniasis. This is a literature review, which was carried out through consultations with periodicals and books present in the Cesmac library. Databases such as: Capes portal, SCIELO, Google Scholar; research in monographs, theses and dissertations. Caused by the protozoan Leishmania chagasi, with the domestic dog being the main reservoir of this protozoan. As it represents a serious public health problem and is considered a potentially fatal disease (when not treated early and properly), it is important that the clinician is familiar with the clinical signs, complementary exams and main therapeutic protocols, especially the use of miltefosine in the treatment of visceral leishmaniasis in dogs. As it is a zoonosis that causes serious public health problems and that has been growing more and more in Brazil, it is up to veterinarians to make a commitment to raise awareness of the importance of early diagnosis in addition to promoting animal welfare and public health.(AU)

La leishmaniosis visceral canina es una enfermedad zoonótica que afecta a los seres humanos y a varias especies de animales salvajes y domésticos. El objetivo de este estudio fue realizar una revisión bibliográfica sobre el uso de la miltefosina en el tratamiento clínico de perros con leishmaniosis visceral. Se trata de una revisión bibliográfica, que se realizó mediante consultas a publicaciones periódicas y libros presentes en la biblioteca del Cesmac. Se utilizaron bases de datos como: portal Capes, SCIELO, Google Académico; investigación en monografías, tesis y disertaciones. Causada por el protozoo Leishmania chagasi, siendo el perro doméstico el principal reservorio de este protozoo. Dado que representa un grave problema de salud pública y se considera una enfermedad potencialmente mortal (cuando no se trata de forma temprana y adecuada), es importante que el clínico esté familiarizado con los signos clínicos, las pruebas adicionales y los principales protocolos terapéuticos, especialmente el uso de miltefosina en el tratamiento de la leishmaniosis visceral en perros. Siendo una zoonosis que causa graves problemas de salud pública y que viene creciendo cada vez más en Brasil, corresponde a los veterinarios asumir el compromiso de concienciar sobre la importancia del diagnóstico precoz y promover el bienestar animal y la salud pública.(AU)

Evaluación de la actividad leishmanicida in vitro de extractos de Annona cherimolioides / Assessment of the in vitro leishmanicide activity of Annona cherimolioides extracts

INTRODUCCIÓN: la utilización de nuestra flora permite un acercamiento en la búsqueda de nuevas sustancias con alto nivel de bioactividad característico de la familia Annonaceae, en donde se reportan una serie de metabolitos secundarios muy promisorios para combatir estos patógenos. OBJETIVO: evaluar la actividad leishmanicida sobre amastigotes intracelulares de Leishmania (V.) panamensis y la actividad tóxica en macrófagos humanos y Artemia salina. MÉTODOS: se aplicó cromatografía de columna y cromatografía en capa preparativa para la extracción y el aislamiento de los alcaloides presentes en la corteza de tallo de Annona cherimolioides Triana & Planch. Extractos y fracciones alcaloidales se evaluaron para determinar la actividad leishmanicida por medio de citometría de flujo, además de la actividad citotóxica sobre la línea celular U937 y sobre macrófagos peritoneales de hámster. La actividad citotóxica in vivo fue evaluada sobre Artemia salina. RESULTADOS: se obtuvo un compuesto depurado, en el cual se determinó la presencia de un núcleo aporfínico según resonancia magnética nuclear 1-H y espectrofotometría. No se encontró actividad contra los parásitos de Leishmania. El extracto crudo mostró mayor toxicidad sobre Artemia salina, mientras que el compuesto depurado mostró una citotoxicidad moderada sobre la línea celular U937. CONCLUSIONES: se encontró una actividad citotóxica selectiva contra las células tumorales (línea celular U937), en comparación con la toxicidad observada en células en cultivo primario (no tumorales). Debido a la citotoxicidad mostrada sobre la línea celular U937, no se pudo determinar la concentración efectiva de los extractos contra amastigotes intracelulares, por lo que se hace necesario continuar las evaluaciones en los sistemas in vitro utilizando células no tumorales.

INTRODUCTION: the use of our flora allows a approach to search of new substances with a high level of bioactivity characteristic of the Annonaceae family where it is reported a series of secondary metabolites very promissory to attack these pathogens. OBJECTIVE: to assess the leishmanicidal activity on intracellular amastigotes of Leishmania (V) panamensis and the toxic activity on human macrophages and Artemia salina. METHODS: it was applied the column chromatography and the preparative layer chromatography for extraction and isolation of alkaloids present in the stem bark of Annona cherimolioides Triana & Planch. Alkaloid extracts and fractions were assessed to determine the leishmanicidal activity by flow cytometry as well as cytotoxic activity on the U937 cellular line and on the peritoneal macrophages of hamster. The in vivo cytotoxic activity was assessed on Artemia salina. RESULTS: a purifying compound was obtained in which the presence of an aporphine nucleus was determined by 1-H nuclear magnetic resonance and spectrophotometry. There wasn't activity against the Leishmania parasites. The crude extract showed the highest toxicity on Artemia salina whereas the purifying compound showed a moderate toxicity on the U937 cell line. DISCUSSION: there was a selective cytotoxic activity against tumoral cells (U397 cell line) compared to toxicity seen in the primary culture (non-tumoral). Due to the cytotoxicity showed on the above cell line, it was impossible to determine the effective concentration of extracts against intracellular amastigotes, thus it is necessary to continue the assessments of in vitro systems using non-tumoral cells.

Evaluation of the leishmanicide action of ethanol extracts of Crotalaria retusa L (Fabaceae) / Avaliação da ação leishmanicida do extrato etanólico da Crotalaria retusa L (Fabaceae)

The purpose of the present work is to conduct an evaluation of the cytotoxicity of ethanol extracts and the total alkaloid fraction (TAF) from Crotalaria retusa for procyclic promastigotes cells of Leishmania chagasi. The kinetic study of extraction assisted by ultrasound of the total alkaloids present in Crotalaria retusa made it possible the optimization of the extraction parameters. It was evaluated the leishmanicide action of the TAF which did not show toxic activity for cells of the parasite in high concentrations. It was observed a powerful leishmanicide action of the ethanol extracts (10 and 30 percent) after the concentration of 5.6 mg/mL of Crotalaria retusa, and the ethanol present in the extractive solution (10 and 30 percent) in the concentration from 70 and 210 x 10-4 percent, respectively. These results suggest that the cytotoxicity of the ethanol extract of Crotalaria retusa at 10 and 30 percent for cells of Leishmania chagasi, can be associated only to the concentration of the alcohol present in the extract.

O presente trabalho se propõe avaliar a atividade citotóxica do extrato etanólico bruto e da fração dos alcalóides totais (FAT) da planta Crotalaria retusa para células promastigotas metacíclicas de Leishmania chagasi. O estudo da cinética de extração por ultra-som para os alcalóides totais da Crotalaria retusa, tornou possível a otimização dos parâmetros de extração. Foi avaliada a ação leishmanicida da FAT da planta em estudo, a qual não mostrou atividade citotóxica em altas concentrações. Foi observado uma potente ação leishmanicida para os extratos etanólicos (10 e 30 por cento) após a concentração de 5,6 mg/mL de Crotalaria retusa e do etanol presente na solução extrativa (10 e 30 por cento) nas concentrações de 70 e 210 x 10-4 por cento, respectivamente. Estes resultados sugerem a citotoxicidade do extrato etanólico da Crotalaria retusa de 10 a 30 por cento para células de Leishmania chagasi, associada possivelmente à concentração do etanol presente no extrato.