Electrochemical, Spectrophotometric and Liquid-Chromatographic Approaches for Analysis of Tropical Disease Drugs

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Mapping the risk of soil-transmitted helminthic infections in the Philippines

Background In order to increase the efficient allocation of soil-transmitted helminth (STH) disease control resources in the Philippines, we aimed to describe for the first time the spatial variation in the prevalence of A. lumbricoides, T. trichiura and hookworm across the country, quantify the association between the physical environment and spatial variation of STH infection and develop predictive risk maps for each infection. Methodology/Principal Findings Data on STH infection from 35,573 individuals across the country were geolocated at the barangay level and included in the analysis. The analysis was stratified geographically in two major regions: 1) Luzon and the Visayas and 2) Mindanao. Bayesian geostatistical models of STH prevalence were developed, including age and sex of individuals and environmental variables (rainfall, land surface temperature and distance to inland water bodies) as predictors, and diagnostic uncertainty was incorporated. The role of environmental variables was different between regions of the Philippines. This analysis revealed that while A. lumbricoides and T. trichiura infections were widespread and highly endemic, hookworm infections were more circumscribed to smaller foci in the Visayas and Mindanao. Conclusions/Significance This analysis revealed significant spatial variation in STH infection prevalence within provinces of the Philippines. This suggests that a spatially targeted approach to STH interventions, including mass drug administration, is warranted. When financially possible, additional STH surveys should be prioritized to high-risk areas identified by our study in Luzon.

Avaliação citotóxica, mutagênica e genotóxica dos análogos do Megazol

A doença de Chagas é uma doença tropical infecciosa e negligenciada responsável por um grande número de pessoas infectadas e em risco de infecção, principalmente nas regiões pobres da América Latina. No momento, apenas duas drogas, Benzonidazol e Nifurtimox, estão disponíveis para o tratamento da doença de Chagas, mas são ineficazes por apresentarem baixa taxa de cura. O Megazol é um importante representante da classe dos nitroimidazóis e é uma alternativa promissora devido ao seu potencial tripanocida com um perfil superior de ação quando comparado ao tratamento disponível. No entanto, o Megazol não é utilizado clinicamente uma vez que possui atividade mutagênica e carcinogênica relatada. O Instituto de Tecnologia em Fármacos (Farmanguinhos) desenvolveu três análogos do Megazol: PTAL 05-02 (3-amino-5-(1-metil-5-nitro-1H-imidazol-2-il)-1H-1,2,4-triazol), PAMT 09 (2-amino-N-(1-metil-4-nitro-1H-imidazol-5-il)-5-(trifluorometil)-1H-1,2,4-triazol) e PTAL 04-09 (1-(1-metil-4-nitro-1H-imidazol-5-il)-1H-pirazol). O objetivo deste trabalho é apresentar novas moléculas análogas do Megazol com atividade tripanocida, desenvolvidas a partir de estratégias racionais de desenvolvimento de substâncias bioativas ao manter o perfil farmacodinâmico do Megazol enquanto tenta diminuir ou remover o efeito genotóxico. Testes genotóxicos na avaliação segura de novas substâncias bioativas foram utilizados, de acordo com as diretrizes da OECD. O teste da Salmonella/microssoma foi utilizado na avaliação mutagênica e citotóxica, utilizando linhagens de Salmonella enterica sorovar Typhimurium, deficientes e supercompetentes na síntese de enzimas nitroredutase e acetiltransferase. O análogo PAMT 09 não foi mutagênico em nenhuma concentração e linhagem utilizada. Os análogos PTAL 05-02 e PTAL 04-09 foram mutagênicos, na ausência de S9 mix, para a linhagem TA98/1,8-DNP6. Na avaliação de citotoxicidade, os três análogos foram citotóxicos, independente de metabolização exógena S9 mix. O teste do micronúcleo, utilizando células de macrófago de rato, foi realizado para a avaliação genotóxica dos análogos do Megazol. Os três análogos foram capazes de induzir a formação de micronúcleos e apresentaram efeito citotóxico.

The genome of the blood fluke Schistosoma mansoni

Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. Here we present analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and new families of micro-exon genes that undergo frequent alternative splicing. As the first sequenced flatworm, and a representative of the Lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, and the identification of membrane receptors, ion channels and more than 300 proteases provide new insights into the biology of the life cycle and new targets. Bioinformatics approaches have identified metabolic chokepoints, and a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.

RNA interference targeting cathepsin B of the carcinogenic liver fluke, Opisthorchis viverrini

Functional genomics have not been reported for Opisthorchis viverrini or the related fish-borne fluke, Clonorchis sinensis. Here we describe the introduction by square wave electroporation of Cy3-labeled small RNA into adult O. viverrini worms. Adult flukes were subjected to square wave electroporation employing a single pulse for 20 ms of 125V in the presence of 50 µg/ml of Cy3-siRNA. The parasites tolerated this manipulation and, at 24 and 48 h after electroporation, fluorescence from the Cy3-siRNA was evident throughout the parenchyma of the worms, with strong fluorescence evident in the guts and reproductive organs of the adult worms. Second, other worms were treated using the same electroporation settings with double stranded RNA targeting an endogenous papain-like cysteine protease, cathepsin B. This manipulation resulted in a significant reduction in specific mRNA levels encoding cathepsin B, and a significant reduction in cathepsin B activity against the diagnostic peptide, Z-Arg-Arg-AMC. This appears to be the first report of introduction of reporter genes into O. viverrini and the first report of experimental RNA interference (RNAi) in this fluke. The findings indicated the presence of an intact RNAi pathway in these parasites which, in turn, provides an opportunity to probe gene functions in this neglected tropical disease pathogen.

Optimal control and numerical optimization applied to epidemiological models

A relação entre a epidemiologia, a modelação matemática e as ferramentas computacionais permite construir e testar teorias sobre o desenvolvimento e combate de uma doença. Esta tese tem como motivação o estudo de modelos epidemiológicos aplicados a doenças infeciosas numa perspetiva de Controlo Ótimo, dando particular relevância ao Dengue. Sendo uma doença tropical e subtropical transmitida por mosquitos, afecta cerca de 100 milhões de pessoas por ano, e é considerada pela Organização Mundial de Saúde como uma grande preocupação para a saúde pública. Os modelos matemáticos desenvolvidos e testados neste trabalho, baseiam-se em equações diferenciais ordinárias que descrevem a dinâmica subjacente à doença nomeadamente a interação entre humanos e mosquitos. É feito um estudo analítico dos mesmos relativamente aos pontos de equilíbrio, sua estabilidade e número básico de reprodução. A propagação do Dengue pode ser atenuada através de medidas de controlo do vetor transmissor, tais como o uso de inseticidas específicos e campanhas educacionais. Como o desenvolvimento de uma potencial vacina tem sido uma aposta mundial recente, são propostos modelos baseados na simulação de um hipotético processo de vacinação numa população. Tendo por base a teoria de Controlo Ótimo, são analisadas as estratégias ótimas para o uso destes controlos e respetivas repercussões na redução/erradicação da doença aquando de um surto na população, considerando uma abordagem bioeconómica. Os problemas formulados são resolvidos numericamente usando métodos diretos e indiretos. Os primeiros discretizam o problema reformulando-o num problema de optimização não linear. Os métodos indiretos usam o Princípio do Máximo de Pontryagin como condição necessária para encontrar a curva ótima para o respetivo controlo. Nestas duas estratégias utilizam-se vários pacotes de software numérico. Ao longo deste trabalho, houve sempre um compromisso entre o realismo dos modelos epidemiológicos e a sua tratabilidade em termos matemáticos.

Sequence and phylogenetic analysis of viper venom serine proteases

Snakebites are a major neglected tropical disease responsible for as many as 95000 deaths every year worldwide. Viper venom serine proteases disrupt haemostasis of prey and victims by affecting various stages of the blood coagulation system. A better understanding of their sequence, structure, function and phylogenetic relationships will improve the knowledge on the pathological conditions and aid in the development of novel therapeutics for treating snakebites. A large dataset for all available viper venom serine proteases was developed and analysed to study various features of these enzymes. Despite the large number of venom serine protease sequences available, only a small proportion of these have been functionally characterised. Although, they share some of the common features such as a C-terminal extension, GWG motif and disulphide linkages, they vary widely between each other in features such as isoelectric points, potential N-glycosylation sites and functional characteristics. Some of the serine proteases contain substitutions for one or more of the critical residues in catalytic triad or primary specificity pockets. Phylogenetic analysis clustered all the sequences in three major groups. The sequences with substitutions in catalytic triad or specificity pocket clustered together in separate groups. Our study provides the most complete information on viper venom serine proteases to date and improves the current knowledge on the sequence, structure, function and phylogenetic relationships of these enzymes. This collective analysis of venom serine proteases will help in understanding the complexity of envenomation and potential therapeutic avenues.

LARVICIDAL ACTIVITY OF GRANDISIN AGAINST AEDES AEGYPTI

Dengue is a tropical disease caused by an arbovirus transmitted by the mosquito Aedes aegypti. Because no effective vaccine is available for the disease, the strategy for its prevention has focused on vector control by the use of natural insecticides. The aim of this study was to evaluate the larvicidal activity of the lignan grandisin, a leaf extract from Piper solmsianum, against Ae. aegypti.

Community-based study on knowledge, attitude and practice on the mode of transmission, prevention and treatment of the Buruli ulcer in Ga West District, Ghana

Buruli ulcer disease (BUD), a devastating tropical disease caused by Mycobacterium ulcerans, occurs in more than 80% of the administrative districts of Ghana. To elucidate community perceptions and
understanding of the aetiology of BUD, attitudes towards Buruli patients and treatment-seeking behaviours, we conducted a survey with 504 heads of households and seven focus group discussions in Ga West District, Ghana. Although 67% of participants regarded BUD as a health problem, 53% did not know its cause. Sixteen per cent attributed the cause to drinking non-potable water, 8.1% mentioned poor personal hygiene or dirty surroundings, and 5.5% identified swimming or wading in ponds as a risk factor. About 5.2% thought that witchcraft and curses cause BUD, and 71.8% indicated that BU sufferers first seek treatment from herbalists and only refer to the hospital as a last resort. The main
reasons were prospects of prolonged hospital stay, cost of transport, loss of earnings and opportunity associated with parents attending their children’s hospitalization over extended period, delays in being
attended by medical staff, and not knowing the cause of the disease or required treatment. The level of acceptance of BUD sufferers was high in adults but less so in children. The challenge facing health workers is to break the vicious cycle of poor medical outcomes leading to poor attitudes to hospital treatment in the community. Because herbalists are often the first people consulted by those who contract the disease, they need to be trained in early recognition of the pre-ulcerative stage of Buruli lesions.

Structural and Functional Studies of a Bothropic Myotoxin Complexed to Rosmarinic Acid: New Insights into Lys49-PLA(2) Inhibition

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Clonagem e expressão da glicoproteína transmembrana do retrovírus HTLV-1 em células de mamíferos

The retrovirus HTLV-1 is the etiological agent of the adult T-cell leukemia and HTLV-1 associated myelopathy/tropical spastic paraparesis. The proviral genome has 9,032 base pairs, showing regulatory and structural genes. The env gene encodes for the transmembrane glycoprotein gp 21. The development of methodologies for heterologous protein expression, as well as the acquisition of a cellular line that constituently expresses the recombinant, were the main goals of this work. The DNA fragment that encodes for gp 21 was amplified by nested-PCR and cloned into a pCR2.1-TOPO vector. After which, a sub-cloning was realized using the expressing vector pcDNA3.1+. The transfection of mammalian cells HEK 293 was performed transitorily and permanently. Production of the recombinant gp 21 was confirmed by flux cytometry experiments and the cell line producing protein will be used in immunogenicity assays.

Synthesis, cytotoxicity and in vitro antileishmanial activity of naphthothiazoles

The leishmaniasis is a spectral disease caused by the protozoan Leishmania spp., which threatens millions of people worldwide. Current treatments exhibit high toxicity, and there is no vaccine available. The need for new lead compounds with leishmanicidal activity is urgent. Considering that many lead leishmanicidal compounds contain a quinoidal scaffold and the thiazole heterocyclic ring is found in a number of antimicrobial drugs, we proposed a hybridization approach to generate a diverse set of semi-synthetic heterocycles with antileishmanial activity. We found that almost all synthesized compounds demonstrated potent activity against promastigotes of Leishmania (Viannia) braziliensis and reduced the survival index of Leishmania amastigotes in mammalian macrophages. Furthermore, the compounds were not cytotoxic to macrophages at fivefold higher concentrations than the EC50 for promastigotes. All molecules fulfilled Lipinski's Rule of Five, which predicts efficient orally absorption and permeation through biological membranes, the in silico pharmacokinetic profile confirmed these characteristics. The potent and selective activity of semi-synthetic naphthothiazoles against promastigotes and amastigotes reveals that the 2-amino-naphthothiazole ring may represent a scaffold for the design of compounds with leishmanicidal properties and encourage the development of drug formulation and new compounds for further studies in vivo. © 2013 John Wiley & Sons A/S.

Dinâmica populacional de minicírculos de cinetoplastos em Leishmania infantum chagasi

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)