The biological in vitro effect and selectivity of aromatic dicationic compounds on Trypanosoma cruzi

Trypanosoma cruzi is a parasite that causes Chagas disease, which affects millions of individuals in endemic areas of Latin America. One hundred years after the discovery of Chagas disease, it is still considered a neglected illness because the available drugs are unsatisfactory. Aromatic compounds represent an important class of DNA minor groove-binding ligands that exhibit potent antimicrobial activity. This study focused on the in vitro activity of 10 aromatic dicationic compounds against bloodstream trypomastigotes and intracellular forms of T. cruzi. Our data demonstrated that these compounds display trypanocidal effects against both forms of the parasite and that seven out of the 10 compounds presented higher anti-parasitic activity against intracellular parasites compared with the bloodstream forms. Additional assays to determine the potential toxicity to mammalian cells showed that the majority of the dicationic compounds did not considerably decrease cellular viability. Fluorescent microscopy analysis demonstrated that although all compounds were localised to a greater extent within the kinetoplast than the nucleus, no correlation could be found between compound activity and kDNA accumulation. The present results stimulate further investigations of this class of compounds for the rational design of new chemotherapeutic agents for Chagas disease

Experimental Chemotherapy for Chagas Disease: A Morphological, Biochemical, and Proteomic Overview of Potential Trypanosoma cruzi Targets of Amidines Derivatives and Naphthoquinones

Chagas disease (CD), caused by Trypanosoma cruzi, affects approximately eight million individuals in Latin America and is emerging in nonendemic areas due to the globalisation of immigration and nonvectorial transmission routes. Although CD represents an important public health problem, resulting in high morbidity and considerable mortality rates, few investments have been allocated towards developing novel anti-T. cruzi agents. The available therapy for CD is based on two nitro derivatives (benznidazole (Bz) and nifurtimox (Nf)) developed more than four decades ago. Both are far from ideal due to substantial secondary side effects, limited efficacy against different parasite isolates, long-term therapy, and their well-known poor activity in the late chronic phase. These drawbacks justify the urgent need to identify better drugs to treat chagasic patients. Although several classes of natural and synthetic compounds have been reported to act in vitro and in vivo on T. cruzi, since the introduction of Bz and Nf, only a few drugs, such as allopurinol and a few sterol inhibitors, have moved to clinical trials. This reflects, at least in part, the absence of well-established universal protocols to screen and compare drug activity. In addition, a large number of in vitro studies have been conducted using only epimastigotes and trypomastigotes instead of evaluating compounds' activities against intracellular amastigotes, which are the reproductive forms in the vertebrate host and are thus an important determinant in the selection and identification of effective compounds for further in vivo analysis. In addition, due to pharmacokinetics and absorption, distribution, metabolism, and excretion characteristics, several compounds that were promising in vitro have not been as effective as Nf or Bz in animal models of T. cruzi infection. In the last two decades, our team has collaborated with different medicinal chemistry groups to develop preclinical studies for CD and investigate the in vitro and in vivo efficacy, toxicity, selectivity, and parasite targets of different classes of natural and synthetic compounds. Some of these results will be briefly presented, focusing primarily on diamidines and related compounds and naphthoquinone derivatives that showed the most promising efficacy against T. cruzi

Influence of Physical Exercise on Advanced Glycation End Products Levels in Patients Living With the Human Immunodeficiency Virus

Introduction: Combined antiretroviral therapy (cART) used to treat acquired immunodeficiency virus (HIV) induces a number of adverse effects, such as insulin resistance and dyslipidemia, which ultimately increases the cardiovascular risk. Advanced glycation end products (AGEs) have been implicated in the etiology of cardiovascular diseases, diabetes and other chronic diseases. It is known that physical exercise improves the lipid profile, insulin resistance and reduces the risk of cardiovascular diseases. However, the impact of physical exercise on AGE levels in HIV-infected patients has not been so far investigated. Therefore, this study compared AGEs levels in people with and without HIV and verified the effect of physical training on serum AGE levels.Methods: Participants were initially assigned into three groups: healthy control (CTL, n = 35), physically inactive HIV-infected (In-HIV, n = 33) and physically active HIV-infected (Ac-HIV, n = 19). The In-HIV group underwent physical training for 3 months, consisting of 60-min sessions of multimodal supervised exercise (aerobic, resistance and flexibility) with moderate intensity (50–80% heart rate reserve), performed 3 times/week. AGEs were measured in serum by fluorescence spectrometry.Results: At baseline, serum AGEs fluorescence level was significantly higher in inactive HIV-patients when compared to controls or active HIV-patients (In-HIV: 0.93 ± 0.08 vs. controls: 0.68 ± 0.13 and Ac-HIV: 0.59 ± 0.04 A.U.; P < 0.001). Triglycerides were also higher in In-HIV than CTL (182.8 ± 102 vs. 132.8 ± 52.3 mg/dL; P < 0.05). Waist circumference was lower in Ac-HIV, compared to In-HIV and controls (83.9 ± 10.4 vs. 92.9 ± 13.5 and 98.3 ± 12.4, respectively; P < 0.05). Body mass, fasting blood glucose, LDL, HDL, and total cholesterol were similar between groups. After training, AGE levels decreased (Baseline: 0.93 ± 0.08 vs. 3 months follow-up: 0.59 ± 0.04 AU; P < 0.001), no further difference being detected vs. CTL or Ac-HIV. Conclusion: HIV-infected patients under cART exhibited elevated AGEs levels compared to healthy individuals and physically active patients. Short-term aerobic training of moderate intensity counteracted this condition

The biological in vitro effect and selectivity of aromatic dicationic compounds on Trypanosoma cruzi

Trypanosoma cruzi is a parasite that causes Chagas disease, which affects millions of individuals in endemic areas of Latin America. One hundred years after the discovery of Chagas disease, it is still considered a neglected illness because the available drugs are unsatisfactory. Aromatic compounds represent an important class of DNA minor groove-binding ligands that exhibit potent antimicrobial activity. This study focused on the in vitro activity of 10 aromatic dicationic compounds against bloodstream trypomastigotes and intracellular forms of T. cruzi. Our data demonstrated that these compounds display trypanocidal effects against both forms of the parasite and that seven out of the 10 compounds presented higher anti-parasitic activity against intracellular parasites compared with the bloodstream forms. Additional assays to determine the potential toxicity to mammalian cells showed that the majority of the dicationic compounds did not considerably decrease cellular viability. Fluorescent microscopy analysis demonstrated that although all compounds were localised to a greater extent within the kinetoplast than the nucleus, no correlation could be found between compound activity and kDNA accumulation. The present results stimulate further investigations of this class of compounds for the rational design of new chemotherapeutic agents for Chagas disease

In Vitro and In Vivo Investigation of the Efficacy of Arylimidamide DB1831 and Its Mesylated Salt Form - DB1965 - against Trypanosoma cruzi Infection

Chagas disease is caused by infection with the intracellular protozoan parasite Trypanosoma cruzi. At present, nifurtimox and benznidazole, both compounds developed empirically over four decades ago, represent the chemotherapeutic arsenal for treating this highly neglected disease. However, both drugs present variable efficacy depending on the geographical area and the occurrence of natural resistance, and are poorly effective against the later chronic stage. As a part of a search for new therapeutic opportunities to treat chagasic patients, pre-clinical studies were performed to characterize the activity of a novel arylimidamide (AIA - DB1831 (hydrochloride salt) and DB1965 (mesylate salt)) against T.cruzi. These AIAs displayed a high trypanocidal effect in vitro against both relevant forms in mammalian hosts, exhibiting a high selectivity index and a very high efficacy (IC50 value/48 h of 5–40 nM) against intracellular parasites. DB1965 shows high activity in vivo in acute experimental models (mouse) of T.cruzi, showing a similar effect to benznidazole (Bz) when compared under a scheme of 10 daily consecutive doses with 12.5 mg/kg. Although no parasitological cure was observed after treating with 20 daily consecutive doses, a combined dosage of DB1965 (5 mg/kg) with Bz (50 mg/kg) resulted in parasitaemia clearance and 100% animal survival. In summary, our present data confirmed that aryimidamides represent promising new chemical entities against T.cruzi in therapeutic schemes using the AIA alone or in combination with other drugs, like benznidazole

Atividade, seletividade e mecanismos de ação de diamidinas aromáticas e análogos sobre Trypanosoma cruzi: um enfoque sobre o kDNA

Submitted by Anderson Silva ([email protected]) on 2012-10-18T20:17:52Z No. of bitstreams: 1 anissa_daliry_ioc_bcm_0031_2011.pdf: 14613668 bytes, checksum: 93e5077e14be3ffeb54129fc214799c9 (MD5)Made available in DSpace on 2012-10-18T20:17:52Z (GMT). No. of bitstreams: 1 anissa_daliry_ioc_bcm_0031_2011.pdf: 14613668 bytes, checksum: 93e5077e14be3ffeb54129fc214799c9 (MD5) Previous issue date: 2011Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, BrasilA doença de Chagas, causada pelo Trypanosoma cruzi, é endêmica na América Latina, afetando mais de 15 milhões de pessoas. Como seu tratamento apresenta eficácia limitada e considerável toxicidade novas drogas são necessárias. Neste contexto, com a colaboração de grupos de química medicinal, nosso objetivo é a investigação da atividade tripanocida, seletividade, alvos celulares e mecanismos de ação de diamidinas aromáticas (DAs) e análogos. Inicialmente avaliamos o efeito tripanocida de onze DAs sobre as formas tripomastigotas e amastigotas intracelulares, assim como sua toxicidade para células de mamíferos e localização intracelular no parasito. Entre estes onze compostos, 2, 5 e 7 foram os mais ativos, com valores de IC50 na faixa micromolar e um alto índice de seletividade sobre as duas formas de T. cruzi. Através de microscopia de fluorescência (MF) foi possível localizar todos os compostos em organelas ricas em DNA, núcleo e mitocôndria (kDNA) e a análise ultraestrutural utilizando os compostos 5 e 7 revelou que estes compostos levam a danos mitocondriais, incluindo desorganização do kDNA em formas tripomastigotas. O acúmulo das diamidinas foi maior no kDNA do que no núcleo, porém tal acúmulo não está correlacionado a uma maior atividade tripanocida. A seguir, visando um melhor entendimento do mecanismo de ação de diamidinas e análogos, investigamos uma possível correlação entre as propriedades de ligação ao kDNA de treze compostos com a atividade tripanocida, através de estudos de desnaturação térmica (Tm) e dicroísmo circular (DiC). Nossos resultados mostram tanto com kDNA purificado de epimastigotas como com uma sequência conservada de 22-mer presente em minicírculos de T. cruzi, que a forte interação de amidinas ao kDNA não é o fator determinante para desencadear sua atividade tripanocida. Nosso próximo passo foi a avaliação de alterações topológicas induzidas por dez compostos aromáticos sobre o kDNA de T. cruzi através de alteração da mobilidade em gel e MF. Os estudos eletroforéticos foram conduzidos pela incubação de fragmentos de kDNA, obtidos pela digestão com as endonucleases EcoRI e CvQI, com os compostos por análise em gel de poliacrilamida. As diamidinas DB889 e DB185 induziram consideráveis alterações na mobilidade dos fragmentos. Além disso, incubando a rede intacta e purificada de kDNA com a DB75 e monitorando o efeito por MF, obervarmos a capacidade desta DA de induzir um grande aumento da área da rede. Em resumo, nossos resultados revelam que diamidinas e congêneres são capazes de induzir profundas alterações na topologia do kDNA do T. cruzi sugerindo que esta estrutura pode ser um dos possíveis alvos destes compostos. Entretanto, a localização preferencial dos compostos no kDNA, assim como sua afinidade à esta estrutura e capacidade de alterar a mobilidade dos fragmentos em géis de poliacrilamida não está correlacionada com sua atividade tripanocida. Estes dados sugerem fortemente que outros fatores podem estar envolvidos no mecanismo de ação destes compostos, operando de modo primário ou secundário a interação composto:kDNA. Outros estudos serão necessários para melhor identificar os mecanismos envolvidos na ação destes compostos, visando contribuir para o desenho racional de compostos líderes para o tratamento da doença de Chagas.Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America, affecting more de 15 million people. Due to the limited efficacy and considerable toxicity, the therapy for Chagas disease is far from being considerable ideal and thus alternative drugs are urgently needed. In this context, with the collaborations of medicinal chemistry groups, our aim was to investigate the trypanocidal activity, selectivity, cellular targets and mechanisms of action of aromatic diamidines (ADs) and analogues. First, we tested the trypanocidal effect of eleven ADs upon intracellular amastigotes and trypomastigotes, their toxicity towards mammalian cells and their sub-cellular localization. Compounds 2, 5 and 7 were the most active, presenting IC50 values on the micromolar range and displaying high selectivity indexes for both T. cruzi forms. Using fluorescence microscopy (FM), all the compounds were localized in organelles rich in DNA, nucleus and mitochondrion (kDNA) and in bloodstream forms treated with the compounds 5 and 7 we observed ultrastructural damages on mitochondrial organelle, including kDNA disorganization. The accumulation of the diamidines was higher in the kDNA than in the nucleus, but such accumulation could not be correlated with a higher trypanocidal activity. Next, aiming to better understand the mechanism of action of diamidines and analogues, we investigated the possible correlation between kDNA binding properties of thirteen compounds with their anti-T. cruzi effect, through thermal denaturation (T m) and circular dichroism (CiD) studies. Our data demonstrated using the purified kDNA of epimastigotes or a conserved synthetic parasite sequence of 22-mer present in T. cruzi minicircles, that the strong interaction of the amidines with the kDNA is not a determinant factor to the triggering of the trypanocidal activity. Our next step was the evaluation of topological changes induced by ten aromatic compounds on T. cruzi kDNA, through gel mobility shifts and FM. The eletrophoretic studies were conducted by the incubation of kDNA fragments obtained by digestion with the endonucleases EcoRI e CvQI with the compounds and analysis by poliacrylamide gel. The diamidines DB889 and DB185 induced substantial mobility shifts in the fragmented bands. Additionally, by FM of whole kDNA network incubated with DB75 we observed the ability of this diamidine to cause a striking expansion of the kDNA network area. Taken together our results suggest that diamidines and related compounds provoke profound alterations in the normal topology of T. cruzi kDNA suggesting that this structure may represent one of the potential targets of these compounds. However, the compound preferential accumulation in kDNA, as well as their affinity and capability of inducing topological changes in that structure is not correlated with their anti-T. cruzi activity. These findings strongly suggest that other molecular mechanisms may be also operating primarily or secondarily to the drug:KDNA interaction. Further studies are needed to better identify the mechanisms involved in the activity of this class of chemical, aiming to contribute to the rational design of lead compounds for the treatment of Chagas disease

Role of Maternal Microbiota and Nutrition in Early-Life Neurodevelopmental Disorders

The rise in the prevalence of obesity and other related metabolic diseases has been paralleled by an increase in the frequency of neurodevelopmental problems, which has raised the likelihood of a link between these two phenomena. In this scenario, maternal microbiota is a possible linking mechanistic pathway. According to the “Developmental Origins of Health and Disease” paradigm, environmental exposures (in utero and early life) can permanently alter the body’s structure, physiology, and metabolism, increasing illness risk and/or speeding up disease progression in offspring, adults, and even generations. Nutritional exposure during early developmental stages may induce susceptibility to the later development of human diseases via interactions in the microbiome, including alterations in brain function and behavior of offspring, as explained by the gut–brain axis theory. This review provides an overview of the implications of maternal nutrition on neurodevelopmental disorders and the establishment and maturation of gut microbiota in the offspring

Cerebral microvascular dysfunction in metabolic syndrome is exacerbated by ischemia–reperfusion injury

Abstract Background Metabolic syndrome (MetS) is associated with an increased risk of cerebrovascular diseases, including cerebral ischemia. Microvascular dysfunction is an important feature underlying the pathophysiology of cerebrovascular diseases. In this study, we aimed to investigate the impacts of ischemia and reperfusion (IR) injury on the cerebral microvascular function of rats with high-fat diet-induced MetS. Results We examined Wistar rats fed a high-fat diet (HFD) or normal diet (CTL) for 20 weeks underwent 30 min of bilateral carotid artery occlusion followed by 1 h of reperfusion (IR) or sham surgery. Microvascular blood flow was evaluated on the parietal cortex surface through a cranial window by laser speckle contrast imaging, functional capillary density, endothelial function and endothelial–leukocyte interactions by intravital videomicroscopy. Lipid peroxidation was assessed by TBARs analysis, the expression of oxidative enzymes and inflammatory markers in the brain tissue was analyzed by real-time PCR. The cerebral IR in MetS animals induced a functional capillary rarefaction (HFD IR 117 ± 17 vs. CTL IR 224 ± 35 capillary/mm2; p < 0.05), blunted the endothelial response to acetylcholine (HFD IR −16.93% vs. CTL IR 16.19% from baseline inner diameter p < 0.05) and increased the endothelial–leukocyte interactions in the venules in the brain. The impact of ischemia on the cerebral microvascular blood flow was worsened in MetS animals, with a marked reduction of cerebral blood flow, exposing brain tissue to a higher state of hypoxia. Conclusions Our results demonstrate that during ischemia and reperfusion, animals with MetS are more susceptible to alterations in the cerebral microcirculation involving endothelial dysfunction and oxidative stress events

Active Transport of Glutamate in Leishmania (Leishmania) amazonensis

Leishmania spp. are the causative agents of leishmaniasis, a complex of diseases with a broad spectrum of clinical manifestations. Leishmania (Leishmania) amazonensis is a main etiological agent of diffuse cutaneous leishmaniasis. Leishmania spp., as other trypanosomatids, possess a metabolism based significantly on the consumption of amino acids. However, the transport of amino acids in these organisms remains poorly understood with few exceptions. Glutamate transport is an important biological process in many organisms. In the present work, the transport of glutamate is characterized. This process is performed by a single kinetic system (K-m=0.59 +/- 0.04 mM, V-max=0.123 +/- 0.003 nmol/min per 20 x 10(6) cells) showing an energy of activation of 52.38 +/- 4.7 kJ/mol and was shown to be partially inhibited by analogues, such as glutamine, aspartate, alpha-ketoglutarate and oxaloacetate, methionine, and alanine. The transport activity was sensitive to the extracellular concentration of H+ but not to Na+ or K+. However, unlike other amino acid transporters presently characterized, the treatment with specific ionophores confirmed the participation of a K+, and not H+ membrane gradient in the transport process.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[03/13257-8]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Pesquisa (CNPq)FAPESPFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP