Planejamento e desenvolvimento de novos compostos com atividade frente a Leishmania spp: Síntese, avaliação da atividade e toxicidade de derivados 5-nitro-2-furfurilidênicos e estudos de relações estrutura-atividade / Design of new compounds against Leishmania spp: synthesis, biological evaluation and toxicity of 5-nitro-2-furfurylidene derivates and structure-activity relationship studies

A leishmaniose é uma zoonose de ampla distribuição mundial, causada pelos parasitas tripanossomatídeos do gênero Leishmania. Infelizmente, o arsenal terapêutico disponível é precário, mas vê-se crescente o interesse científico pela busca do potencial de derivados nitroheterocíclicos como alternativas terapêuticas. Nesse contexto, este trabalho analisou o potencial de derivados 5-nitro-2-furfurilidênicos contra diferentes cepas de Leishmania, assim como investigou um possível modo de ação para esta classe de nitrocompostos. Para tal, a quimioteca foi sintetizada de acordo com publicações prévias do grupo. O potencial de inibição de crescimento das culturas de promastigotas de L. (L.) infantum (Linf) e L. (L.) major (Lmaj) foi determinado, utilizando miltefosina (MILT) (Linf - IC50: 8,28±0,33 µM), anfotericina B (AMB) (Linf - IC50: 0,02±0,002 µM) e nifurtimox (NFX) (Lmaj - IC50: 3,5±0,09 µM) como referência. A maioria dos compostos apresentaram maior potencial que as referênias, destacando o composto 40 (Linf - IC50: 0,2±0,019 µM/ Lmaj - IC50: 0,087 ± 0,001 µM) como mais eficaz. Contra as formas amastigotas intracelulares, para Linf os compostos 40, 13 e 15 foram mais eficazes em reduzir a carga parasitária dos macrófagos infectados que fármacos de referência. Para Lmajor, o composto 40 (IC50: 0,006 ± 0,0003 µM) foi mais ativo que o NFX (IC50: 2,15 ± 0,01 µM). Também foi determinada a atividade da quimioteca frente a enzima nitrorredutase (NTR1), utilizando cepas de T. brucei superexpressantes de NTR1, e os compostos analisados foram até 18 vezes mais eficazes que à cepa wild-type. Ademais, a partir da análise exploratória de dados por análise de componentes principais (PCA) e de grupamentos hierárquicos (HCA), foi reconhecida a influência das propriedades relacionadas com o equilíbrio hidrófilo-lipófilo e da natureza estérica/geométrica das moléculas para atividade anti-Leishmania

Leishmaniasis is a worldwide zoonosis caused by trypanosomatid parasites of the genus Leishmania. Unfortunately, the available therapeutic arsenal is precarious, but there is growing scientific interest in searching the potential of nitroheterocyclic derivatives as therapeutic alternatives. In this context, this work analyzed the potential of 5-nitro-2-furfurylidene derivatives against different Leishmania strains, as well as investigated the potential mode of action for this nitro compounds class. To this end, the chemolibrary was synthesized according to our group's previous publications. The growth inhibitory potential potential for promastigote cultures of L. (L.) infantum (Linf) and L. (L.) major (Lmaj) was determined using miltefosine (MILT) (Linf - IC50: 8.28±0.33 µM), amphotericin B (AMB) (Linf - IC50: 0.02±0.002 µM) and nifurtimox (NFX) (Lmaj - IC50: 3.5±0.09 µM) as reference. Most of the compounds were more potent than the references, highlighting compound 40 (Linf - IC50: 0.2±0.019 µM/ Lmaj - IC50: 0.087 ± 0.001 µM) as the most effective. Against intracellular amastigote, for Linf, compounds 40, 13 and 15 were more effective in reducing the parasite load of infected macrophages than reference drugs. For Lmajor, compound 40 (IC50: 0.006 ± 0.0003 µM) was more active than NFX (IC50: 2.15 ± 0.01 µM). The activity against nitroreductase (NTR1) enzyme was determined using overexpressing NTR1 mutant T. brucei strains, and the analyzed compounds were up to 18 times more effective than wild-type. Furthermore, exploratory data analysis using principal component analysis (PCA) and hierarchical clustering (HCA) methods were used. The influence of properties related to the hydrophiliclipophilic balance and the steric/geometric nature of the molecules was associated with the anti-Leishmanial activity

In vitro activity of the hydroethanolic extract and biflavonoids isolated from Selaginella sellowii on Leishmania (Leishmania) amazonensis

This study is the first phytochemical investigation of Selaginella sellowii and demonstrates the antileishmanial activity of the hydroethanolic extract from this plant (SSHE), as well as of the biflavonoids amentoflavone and robustaflavone, isolated from this species. The effects of these substances were evaluated on intracellular amastigotes of Leishmania (Leishmania) amazonensis, an aetiological agent of American cutaneous leishmaniasis. SSHE was highly active against intracellular amastigotes [the half maximum inhibitory concentration (IC50) = 20.2 µg/mL]. Fractionation of the extract led to the isolation of the two bioflavonoids with the highest activity: amentoflavone, which was about 200 times more active (IC50 = 0.1 μg/mL) and less cytotoxic than SSHE (IC50 = 2.2 and 3 μg/mL, respectively on NIH/3T3 and J774.A1 cells), with a high selectivity index (SI) (22 and 30), robustaflavone, which was also active against L. amazonensis (IC50 = 2.8 µg/mL), but more cytotoxic, with IC50 = 25.5 µg/mL (SI = 9.1) on NIH/3T3 cells and IC50 = 3.1 µg/mL (SI = 1.1) on J774.A1 cells. The production of nitric oxide (NO) was lower in cells treated with amentoflavone (suggesting that NO does not contribute to the leishmanicidal mechanism in this case), while NO release was higher after treatment with robustaflavone. S. sellowii may be a potential source of biflavonoids that could provide promising compounds for the treatment of cutaneous leishmaniasis.

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.

Visceral leishmaniasis (kala-azar): challenges ahead.

Indian visceral leishmaniasis (VL) is a parasitic disease caused by a haemoflagellete Leishmania donovani and transmitted by the bite of sand fly Phlebotomus argentipes. It affects various age groups. In India about 1,00,000 cases of VL are estimated to occur annually; of these, the State of Bihar accounts for over than 90 per cent of the cases. Diagnosis of VL typically relies on microscopic examination of tissue smears but serology and molecular methods are better alternatives currently. Notwithstanding the growing incidence of resistance, pentavalent antimony complex has been the mainstay for the treatment of VL during the last several decades. The second line drugs such as amphotericin B, lipid formulations of amphotericin B, paromomycin and recently developed miltefosine are the other alternatives. In spite of significant development in various areas of Leishmania research, there is a pressing need for the technological advancement in the understanding of immune response, drug resistance and the pathogenesis of leishmaniasis that could be translated into field applicable and affordable methods for diagnosis, treatment, and control of the disease.

Visceral leishmaniasis - current therapeutic modalities.

Major therapeutic obstacles in the treatment of visceral leishmaniasis (VL) include the alarming increase in antimonial unresponsiveness especially in Bihar, India and relapses in HIV-Leishmania co-infected patients. The therapeutic armamentarium for VL is currently plagued with several limitations as the available drugs are toxic, majority are effective only parenterally and need to be administered for extended periods. The first orally effective drug, miltefosine has been approved for treating VL. In antimony refractory zones, pentavalent antimony has been largely replaced by amphotericin B deoxycholate, but prolonged hospitalization, toxic effects, and requirement for monitoring greatly hamper its widespread application in endemic regions. Lipid formulations of amphotericin B, a remarkable advance in amphotericin B therapy, have greatly reduced toxicity enabling large doses to be delivered over a short period. Even a single dose treatment with liposomal amphotericin B cures > 90 per cent patients; however, the stumbling block is its prohibitive cost that precludes its widespread accessibility in endemic countries. Studies using paromomycin in VL are encouraging, and judging by the preliminary results of a recently concluded phase III trial, it could be an extremely useful and affordable antileishmanial drug. Other orally effective drugs include the azoles and allopurinol but these have met with limited success owing to either poor efficacy or unacceptable toxicity. Sitamaquine has undergone limited evaluation, and the data suggest effective antileishmanial activity; its role has to be delineated for which additional developmental studies are proposed. This review highlights the progress made in the treatment of VL, including the multiple mechanisms of action of antileishmanial drugs with a view to enable the researcher to undertake the challenge of providing affordable and effective chemotherapy.

Drug unresponsiveness & combination therapy for kala-azar.

Pentavalent antimonials (SbV) have been successfully used for treatment of kala-azar since last six decades. Since 1970s its conventional dosages have failed to achieve with 60 per cent unresponsiveness reported with WHO regimen in Bihar (India). Pentamidine initially used as a second line of drug, acquired resistance (25%) even with prolonged dosage. Newer oral drug miltefosine is a potent antileishmanial drug with longer half-life, a property likely to acquire resistance. Paromomycin has undergone extensive clinical trials in Indian kala-azar patients. Being an aminoglycoside, acquired resistance is likely to occur when used as a monotherapy. To encounter the problem of treatment failure in kala-azar and to reduce length of therapy, combination of at least two effective antileishmanial agents is a desirable option. In India sodium stibogluconate (SSG) in standard dose has been combined with other antileishmanial agents including paromomycin without encouraging result. Infection with Leishmania donovani depresses cell-mediated immunity. Immunological balance is tilted in favour of Th2 suppressive cytokines over Th1 producing protective cytokines. Interferon gamma (IFN-gamma) has been used in combination with SbV in Indian kala-azar patients with unexpectedly discouraging results. Combination of two most potent leishmanicidal drugs amphotericin B and miltefosine which are not dependent on host immune system, may shorten the course of therapy besides encountering unresponsiveness. A combination therapy should be preferred when treating kala-azar associated with HIV/AIDS. Immunotherapy with exogenous Th1 stimulating cytokines or use of antileishmanial vaccine in combination with a potent chemotherapeutic agent is a future option.

Animal models for vaccine studies for visceral leishmaniasis.

Visceral leishmaniases (VL) or kala-azar is the most dreaded and devastating amongst the various forms of leishmaniases. The disease, though localized in certain areas only, has gained immense importance because of high mortality rate, mainly in children. The parasite is responsible for a spectrum of clinical syndromes, which can, in most extreme cases, go from an asymptomatic infection to a fatal form of VL. Chemotherapeutic measures, alone are not sufficient to control and contain the disease. As an alternate strategy, vaccination is also under experimental and clinical trails. The situation unquestionably demands the use of proper screening system, rationale chemical synthesis, vaccine development and targeted vaccine delivery. Thus, development of an acceptable vaccine is not an easy task. While the factors, which determine clinical outcomes, are in part, a feature of the parasite, it is the nature of the host and its genetic make up and immune status that play crucial role. The prerequisite of reliable animal model is that it should have a considerably good correlation with the clinical situation and is expected to mimic the pathological features and immunological responses observed in humans when exposed to a variety of Leishmania spp. with different pathogenic characteristics. Many experimental animal models like rodents, dogs and monkeys have been developed, each with specific features, but none accurately reproduces what happens in humans. In addition to the nature of the host, the major difference between natural and experimental infections is the parasite inoculum; in natural conditions, the infected sand fly vector deposits a few hundred metacyclic promastigotes into the dermis of the host, whereas experimental infections are induced by the injection (subcutaneous or intravenous) of millions of promastigotes grown in axenic cultures in vitro or amastigotes recovered from infected spleens. In public health terms, VL is the disease of humans and dogs (which may be considered secondary or 'accidental' hosts in the leishmanial life cycle) who often exhibit severe clinical signs and symptoms when infected, whereas reservoir hosts generally show a few, minor or no signs. This situation makes the definition of a suitable laboratory model a difficult one since the various experimental hosts may behave either like a reservoir or an accidental host. This review discusses the concept of animal models for VL and provides a critical evaluation of the most common experimental models and their respective advantages and disadvantages. Particular emphasis is given to the value of using mouse, hamster, dog and primate models, especially in the context of testing potential antileishmanial vaccines.

Apoptosis in Leishmania species & its relevance to disease pathogenesis.

Apoptosis is a morphologically distinct form of cell death necessary for embryogenesis, tissue homeostasis and disease control in metazoans. Earlier, it was thought that apoptosis is the prerogative of multicellular organisms. However, it is now evident that unicellular organisms are also capable of undergoing apoptosis. In the context of Leishmania spp., a unicellular eukaryote responsible for causing leishmaniasis, the process of apoptosis is important for successful survival. The flagellated promastigote form of the parasite resides in the midgut of the insect vector, the female sandfly and at this niche; the cell fittest to survive to pass onto the pharynx of the fly is selected by eliminating unfit cells through apoptosis. Within the mammalian host, inside the macrophage, apoptosis is necessary to regulate cell numbers and to minimize immune reactions. With most apoptosis inducing stimuli, L. donovani shows typical features of apoptotic death like cell shrinkage, nuclear condensation and DNA fragmentation. Agents capable of precipitating apoptosis in this parasite include anti-leishmanial drugs like antimony, amphotericin B, pentamidine and miltefosine. Other agents like heat shock, treatment with staurosporine, knocking out centrin gene also precipitate apoptosis of the parasites. A pivotal role in cellular apoptosis is played by the single mitochondrion of Leishmania spp., where a fall or increase in mitochondrial potential leads to cell death by apoptosis. Ca2+ appears to be a vital ion involved in Leishmania apoptosis and partial inhibition of cytosolic Ca2+ increase achieved by chelating extracellular or intracellular Ca2+ during oxidative stress results in significant rescue of the fall of the mitochondrial membrane potential and consequently apoptosis. Elucidation of the molecular events linked to apoptotic death of Leishmania spp. might help define a more comprehensive view of the cell death machinery in terms of evolutionary origin and identify new target molecules for chemotherapeutic drug development and therapeutic intervention.

Leishmania/HIV co-infections in the second decade.

Leishmania-HIV co-infection has been globally controlled in Southern Europe since 1997 because of highly active anti retroviral therapy (HAART), but it appears to be an increasing problem in other countries such as Ethopia, Sudan, Brazil or India where both infections are becoming more and more prevalent. Most of the scientific background on Leishmania/HIV co-infection has been dropped from the Mediterranean experience and although the situations among countries are not fully comparable, it is of high importance to take advantage of this knowledge. In this review several aspects of the Leishmania/HIV co-infection are emphasized viz., epidemiological features, new ways of transmission, pathogenesis, clinical outcome, diagnosis, treatment and secondary prohylaxis. An extensive review of the literature on Leishmania/HIV co-infection has allowed the inclusion of a comprehensive and updated list of bibliographical references.

Caracterização bioquímica e molecular de possíveis alvos terapêuticos do metabolismo de aminoácidos em Tripanossomatídeos / Biochemical and molecular characterization of possible tarepeutic targets against Trypanosomatids in the amino acid metabolism

Trypanosoma cruzi e Leishmania spp. são parasitas de grande relevância médica nas Américas e em países da Europa e Ásia. Esses organismos apresentam um ciclo de vida heteroxeno infectando hospedeiros vertebrados e invertebrados e, portanto, precisam se adaptar às diferentes condições ambientais. Dessa forma, diversas enzimas metabólicas, proteínas e transportadores participam no ajuste do metabolismo às condições ambientais e flutuações na disponibilidade de nutrientes do meio. Nesse aspecto, os aminoácidos são de importância vital para o metabolismo de tripanossomatídeos, porém pouco se sabe da dinâmica do transporte desses nutrientes para dentro da célula e existem algumas lacunas no estudo de algumas enzimas envolvidas no metabolismo de aminoácidos. Portanto, uma dos objetivos desse trabalho foi caracterizar bioquimicamente o transporte de L- glutamato, um aminoácido importante para o ciclo de vida desses organismos e as formas presentes no inseto de diversos tripanossomatídeos utilizam L-glutamato no metabolismo energético. A incorporação de glutamato ao longo do tempo foi estudada em formas promastigotas de Leishmania amazonensis, foi possível observar que a incorporação é saturável e apresenta uma faixa de linearidade durante os primeiros 120 segundos de incubação com aminoácido radioativamente marcado, atingindo um platô após 15 minutos...Os tripanossomatídeos são capazes de metabolizar L-glutamato para produção de energia, o fato de L. amazonensis incorporar L-glutamato com eficiência é um indicativo da relevância desse aminoácido no metabolismo desses parasitas. O outro objetivo do trabalho, se relaciona com a busca de alvos terapeêuticos em potencial no metabolismo de aminoácidos em Trypanosoma cruzi. A asparagina é de importância vital para a síntese correta de protéinas e para processos de modificações pós-traducionais como N-glicosilação...Além disso, também identificamos uma sequência codificante correspondente a enzima L-Asparaginase (L-Asp)...

Leishmania (Viannia) braziliensis growth in vitro culture relies more on folic acid availability than Leihsmania (Leishmania) amazonensis

We compared the in vitro growth of promastigotes from two Leishmania species in TC-100 and Schneider media. Leishmania (Leishmania) amazonensis replication rates were similar in both tissue culture media and reached maximum rates by 48 h. In contrast Leishmania (Viannia) braziliensis growth was significantly greater in TC-100 but maximum rates were achieved by 96 h. Folic acid appears to be the limiting factor and supplementation of Schneider media with this nutrient improved L. (V.) braziliensis replication rates and decreased the time of maximum replication to 48 h.

Control de la actividad de la piruvato quinasa en leishmania: una herramienta farmacológica? / Control of activity of the piruvate kinase in leishmania: is an pharmacologic tools?

El estudio del metabolismo energético de Leishmania debe tomar en cuenta su ciclio de vida digenético. Para los promastigotes de algunos Trypanosomatidae, entre ellos Leishmania, se han descritos todas las enzimas de la vía glicolítica y del ciclo de Krebs, un sistema de fosforilación oxidativa y la cadena de transporte de electrones; sin embargo, la glucosa es un substrato de segunda importancia metabólica y la fermentación aeróbica es mas importante en las fases estacionarias que en las fases exponenciales de crecimiento. Estas diferencias con la glicólisis en mamíferos, sugieren que los mecanismos regulatorios complejos clásicos, deben estar disminuidos o ausentes en estos organismos. Sólo la enzima piruvato quinasa parece estar regulada y este hecho, además de su exclusiva localización cristosólica sugieren su posible papel en el control del catabolismo de los carbohidratos y aminoácidos y hacen pensar que pudiera servir como objetivo estratégico para diseñar agentes quimioterapéuticos de forma racional.

The developmental regulation and biosynthesis of GPI-related structures in Leishmania parasites

Most macromolecules on the surface of Leishmania parasites, including the major surface proteins and a complex lipophosphoglycan (LPG) are anchored to the plasma membrane via GPI glycolipids. Free glycoinositol-phospholipids (GIPLs) which are not linked to protein or phosphoglycan are also abundant in the plasma membrane. From structural and metabolic labeling studies it is proposed that most Leishmania species express three distinct pathways of GPI biosynthesis. Some of these pathways (i.e those involved in the protein and LPG anchor biosynthesis) are down-regulated during the differentiation of the insect (promastigote) stage to the mammalian (amastigote) stage. In contrast, the GIPLs are expressed in high copy number in both developmental stages. Based on analysis of the lipid moieties of the different GPI species it is possible that the pathways of GPI anchor and GIPL biosynthesis are located in different subcellular compartments. The relative flux through the GIPL and LPG biosynthetic pathways has been examined in L. Major promastigotes. These studies showed that while the rate of synthesis of the GIPLs and LPG is similar, LPG is shed more rapidly from the plasma membrane and has a higher turnover. The possible metabolic relationship between the GIPL and LPG biosynthetic pathways is discussed

Identificación bioquímica de las leishamanias / Biochemical identification of leishmanias

Las dificultades para clasificarlas leishmanias según su comportamiento en los mamíferos húespedes y en los flebótomos vectores han planteado la necesidad de establecer criterios de clasificación que no dependan de estas complejas relaciones recíprocas. La electroforesis de isoenzimas... es un instrumento taxonómico útil para tal propósito. De hecho, se ha convertido en el método bioquímico más utilizado para distinguir las leishmanias, y ha ayudado mucho a comprender la epidemiología de la leishmaniasis cutánea y la visceral. En esencia, la electroforesis de isoenzimas... consta de los pasos siguientes: se preparan extractos solubres no purificados de los microorganismos en estudio; los extractos se colocan en una placa para electroforesis; los componentes del extracto se dirigen hacia el cátodo o el ánodo según su carga elétrica; la placa se cubre con un sustrato específico susceptible de ser alterado por la enzima escogida; por último, mediante una coloración capaz de revelar la ubicación del sustrato alterado, se determina el desplazamiento de la enzima sobre la placa. Las diferencias en los perfiles isoenzimáticos resultantes indican diferencias en los genes que regulan la producción de las enzimas correspondientes. Si se somete a prueba un número suficiente de enzimas, la ausencia de dichas diferencias indica que las poblaciones de leishmanias estudiadas son muy similares. Una de las complicaciones de esta prueba es que las infecciones mixtas de mamíferos o vectores por más de una especie de leishmania tornan confusa la interpretación de los datos de isoenzimas.... Por conseguiente es aconsejable clonar estirpes estandarizadas y representativas de Leishmania para usarlas al hacer compraciones amplias de isoenzimas...