Estudo da resistência de Plasmodium falciparum à artemisinina e derivados: desenvolvimento e implementação de testes in vitro para avaliação da viabilidade

Malária ou paludismo é uma doença parasitária provocada por protozoários do género Plasmodium e transmitida por fêmeas de mosquitos do género Anopheles. Há cinco espécies de Plasmodium capazes provocar malária em humanos (P. falciparum , P. vivax , P. ovale, P. malariae e P. knowlesi), destas espécies as mais prevalentes são P. falciparum e P. vivax, e a mais letal é P. falciparum. Apesar de a malária ser uma doença que se pode prevenir e tratar, esta continua a ter um impacto devastador na saúde mundial. De acordo com o World Malaria Report de 2015, cerca de 3.2 biliões de pessoas encontravam-se em risco de contrair malária. Em 2015, foram estimados 214 milhões de novos casos de malária e cerca de 438 000 mortes, a maioria em crianças com menos de 5 anos na África Subsariana. Tendo em conta a situação atual da malária, já foram delineadas campanhas que têm por objetivo a erradicação desta doença. Os estudos demonstram o aumento da resistência aos antimaláricos mais utilizados, incluindo a artemisinina e seus derivados. A rápida disseminação da resistência aos antipalúdicos tornou prioritária a vigilância da susceptibilidade dos parasitas aos fármacos. É crucial o desenvolvimento de metodologias inovadoras que facilitem uma avaliação rápida, eficaz e rigorosa do efeito dos fármacos e compostos sintéticos ou naturais, no ciclo e metabolismo de Plasmodium, para que possam ser delineadas estratégias que limitem a propagação da resistência e o desenvolvimento de novos compostos e vacinas. Neste trabalho, padronizamos o uso do fluorímetro de microplacas e resazurina, para avaliar in vitro a viabilidade de P. falciparum quando sujeito a um pulso de seis horas de dihidroartemisinina. Este teste de susceptibilidade, de fácil execução, produz resultados quantitativos, rápidos e fiáveis, o que o torna uma alternativa eficaz ao ensaio RSA0-3h, uma vez que consegue ultrapassar a subjetividade e trabalho árduo, inerentes à observação de esfregaços corados com Giemsa, por dois ou mais microscopistas experientes que contabilizam parasitas viáveis.Malaria is a parasitic disease caused by protozoa of the genus Plasmodium and transmitted by mosquito females of genus Anopheles. There are five species of Plasmodium that are capable to causing malaria in humans (P. falciparum , P. vivax , P. ovale, P. malariae e P. knowlesi) , P. falciparum and P. vivax are the most prevalent species and P. falciparum is the most lethal . Although malaria is a disease that can be prevented and treated, at the same time malaria continues to have a devastating impact on world health. According to the World Malaria Report 2015, about 3.2 billion people remain at risk of malaria. In 2015, there were an estimated 214 million new cases of malaria and 438 000 deaths. mostly of children under five years old in sub-Saharan Africa. Given the current situation of malaria, they have been outlined campaigns which are focused on eradication of this disease. Studies have shown increased resistance to commonly used antimalarial drugs, including artemisinin and its derivatives. The fast spread of resistance to antimalarial became the surveillance of susceptibility of parasites to drugs a priority. It is crucial to the develop innovative methodologies which can favour fast, efficient and accurate evaluation of the effect of drugs and synthetic or natural compounds in the cycle and metabolism of Plasmodium, so they can be drawn strategies that limit the spread of resistance and the development of new compounds and vaccines. In this study, we standardized microplate spectrophotometer and resazurin use to assess in vitro viability of P. falciparum when subjected to a pulse of six hours of dihydroartemisinin. This susceptibility test, easy to perform, produce quantitative, fast and reliable results, which makes it an effective alternative to RSA0-3h since it can overcome the subjectivity and hard work inherent in the observation of smears stained with Giemsa by two or more experienced microscopists that account viable parasites

On the ordeal of quinolone preparation via cyclisation of aryl-enamines; synthesis and structure of ethyl 6-methyl-7-iodo-4-(3-iodo-4-methylphenoxy)-quinoline-3-carboxylate

Recent studies directed to the design of compounds targeting the bc(1) protein complex of Plasmodium falciparum, the parasite responsible for most lethal cases of malaria, identified quinolones (4-oxo-quinolines) with low nanomolar inhibitory activity against both the enzyme and infected erythrocytes. The 4-oxo-quinoline 3-ester chemotype emerged as a possible source of potent bc(1) inhibitors, prompting us to expand the library of available analogs for SAR studies and subsequent lead optimization. We now report the synthesis and structural characterization of unexpected ethyl 6-methyl-7-iodo-4-(3-iodo-4-methylphenoxy)quinoline-3-carboxylate, a 4-aryloxy-quinoline 3-ester formed during attempted preparation of 6-methyl-7-iodo-4-oxo-quinoline-3-carboxylate (4-oxo-quinoline 3-ester). We propose that the 4-aryloxy-quinoline 3-ester derives from 6-methyl-7-iodo-4-hydroxy-quinoline-3-carboxylate (4-hydroxy-quinoline 3-ester), the enol form of 6-methyl-7-iodo-4-oxo-quinoline-3-carboxylate. Formation of the 4-aryloxy-quinoline 3-ester confirms the impact of quinolone/hydroxyquinoline tautomerism, both on the efficiency of synthetic routes to quinolones and on pharmacologic profiles. Tautomers exhibit different cLogP values and interact differently with the enzyme active site. A structural investigation of 6-methyl-7-iodo-4-oxo-quinoline-3-carboxylate and 6-methyl-7-iodo-4-hydroxy-quinoline-3-carboxylate, using matrix isolation coupled to FTIR spectroscopy and theoretical calculations, revealed that the lowest energy conformers of 6-methyl-7-iodo-4-hydroxy-quinoline-3-carboxylate, lower in energy than their most stable 4-oxo-quinoline tautomer by about 27 kJ mol(-1), are solely present in the matrix, while the most stable 4-oxo-quinoline tautomer is solely present in the crystalline phase.Fundacao para a Ciencia e Tecnologia (FCT - Portugal) [UID/Multi/04326/2013]; QREN-COMPETE-UE; CCMAR; FCT [SFRH/BD/81821/2011, RECI/BBB-BQB/0230/2012, UI0313/QUI/2013, UID/FIS/04564/2016]; FEDER/COMPETE-UE; [PTDC/QEQ-QFI/3284/2014 - POCI-01-0145-FEDER-016617]info:eu-repo/semantics/publishedVersio

Synthesis and Base Pairing Properties of Platinum(II) Nucleosides Based on Uracil

The synthesis and base pairing properties of platinum complexes based on uridine and deoxyuridine nucleosides are described. The synthesis was performed by C–I oxidative addition with protected and unprotected nucleosides. The metallated compounds feature an agostic interaction at H6. Uridine complexes undergo self-base pairing and also establish base pairs with adenosine. The formation of an intermolecular N-H-Pt bond is also observe

Sacsin Deletion Induces Aggregation of Glial Intermediate Filaments

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disorder commonly diagnosed in infants and characterized by progressive cerebellar ataxia, spasticity, motor sensory neuropathy and axonal demyelination. ARSACS is caused by mutations in the SACS gene that lead to truncated or defective forms of the 520 kDa multidomain protein, sacsin. Sacsin function is exclusively studied on neuronal cells, where it regulates mitochondrial network organization and facilitates the normal polymerization of neuronal intermediate filaments (i.e., neurofilaments and vimentin). Here, we show that sacsin is also highly expressed in astrocytes, C6 rat glioma cells and N9 mouse microglia. Sacsin knockout in C6 cells (C6Sacs−/−) induced the accumulation of the glial intermediate filaments glial fibrillary acidic protein (GFAP), nestin and vimentin in the juxtanuclear area, and a concomitant depletion of mitochondria. C6Sacs−/− cells showed impaired responses to oxidative challenges (Rotenone) and inflammatory stimuli (Interleukin-6). GFAP aggregation is also associated with other neurodegenerative conditions diagnosed in infants, such as Alexander disease or Giant Axonal Neuropathy. Our results, and the similarities between these disorders, reinforce the possible connection between ARSACS and intermediate filament-associated diseases and point to a potential role of glia in ARSACS pathology

Primaquine-based ionic liquids as a novel class of antimalarial hits

info:eu-repo/semantics/publishedVersio

Synthesis, Base Pairing Properties, and Biological Activity Studies of Platinum(II) Complexes Based on Uracil Nucleosides

The synthesis and base pairing properties of platinum complexes based on uridine and deoxyuridine nucleosides and preliminary studies of their antiproliferative activity are described. Platinum(II) uridine and deoxyuridine complexes were synthesized by C–I oxidative addition to Pt(0)(PPh3)4. First, the synthesis was performed with protected nucleosides to generate complexes 1 and 2, which were deprotected under basic conditions, affording complexes 3 and 4 in good yields. The synthesis with the unprotected nucleosides was also performed and provided complexes 3 and 4 effectively. Base pairing interactions were measured for complex 1, either for self-base pairing or for the Watson–Crick base pair. Complex 1 undergoes self-base pairing in CDCl3, and this aggregation was found not to be dependent on metalation. Contrastingly, for the Watson–Crick base pair with adenine, base pairing was also observed, but metalation was found to affect hydrogen bonding considerably. Complexes 3 and 4 and the corresponding ligand precursors were evaluated for their antiproliferative activity against human glioblastoma cell line U-251. The compounds showed IC50 values of 3.30 (3) and 1.84 (4) μM but are also toxic for nontumorous cell lines