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Projeto de investigação
Strategic Project - UI 4013 - 2011-2012
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Publicações
Squaric acid/4-aminoquinoline conjugates: novel potent antiplasmodial agents
Publication . Ribeiro, Carlos J. A.; Kumar, S. P.; Gut, Jiri; Gonçalves, Lidia M.; Rosenthal, Philip J.; Moreira, Rui; Santos, Maria M. M.
We report the synthesis and structure–activity relationship (SAR) analysis of a series of hybrid compounds containing a squaric moiety conjugated with heterocyclic moieties from well-known antimalarials. This novel series of compounds presents improved antiplasmodial activity compared with the squaric derivatives described in our previous work. Three compounds, 8b (IC50 = 99 nM), 8c (IC50 = 95 nM), and 8d (IC50 = 105 nM) had greater in vitro potency than chloroquine 1 (IC50 = 140 nM) against chloroquine resistant Plasmodium falciparum. In addition, they were noncytotoxic against NIH 3T3 and Hek 293T cells.
Pyruvate dehydrogenase complex deficiency : mutational spectrum in Portugal, molecular mechanisms underlying pathogenic variants and potential therapeutic effect of Arginine
Publication . Pavlu-Pereira; Rivera, Isabel Antolin; Vicente, João Filipe Bogalho
Pyruvate dehydrogenase complex (PDC) occupies an essential position in cellular energy production. The oxidative decarboxylation of pyruvate to acetyl-coenzyme A is conducted by a highly organized multienzyme system. Impaired PDC activity leads to a metabolic disorder affecting mainly the tissues with a high demand for ATP.
This work is dedicated to a comprehensive study of PDC and PDC deficiency at diverse levels. We described and discussed the clinical, biochemical and genotypic findings from thirteen Portuguese PDC deficient patients, we submitted the clinically relevant variants to the detailed structural and functional analyses, and we evaluated the ability of arginine and/or thiamine to restore PDC function in patient-derived cell lines.
All patients from our cohort had the clinical onset between the neonatal period and infancy, manifested different degrees of neurological involvement and, interestingly, most of them reached adulthood. The mutational spectrum revealed ten different mutations in the following genes coding for the respective subunits PDHA1 (PDC-E1α), PDHX (PDC-E3BP), and DLD (PDC-E3). The most striking evidence was a relatively high incidence of E3BP deficiency, nevertheless, PDHA1 mutations were predominant.
The selected pathogenic PDC-E1α variants with amino acid substitutions in different structural regions were analyzed by biochemical and biophysical methodologies, combined with molecular dynamics simulations, revealing a limited impact of the mutations on the conformational stability and presenting a significant functional impairment in terms of reduced residual PDC-E1 enzymatic activity and lower affinity for the thiamine pyrophosphate cofactor.
Applying identical methodologies, we evaluated the effect of arginine supplementation on recombinant PDC-E1 variants. Furthermore, we conducted a study assessing the impact of arginine and/or thiamine treatment in patient-derived cell lines. The rescue efficacy of these stabilizing molecules is clearly determined by the mutation per se. Remarkably, the major effect of arginine was observed in p.R253G variant, at all levels of this study. The beneficial effect of arginine upon the recombinant protein and the considerable response to arginine and thiamine supplementation in the patient´s fibroblast, confirmed previous observations demonstrating the efficacy of arginine treatment in improving the clinical phenotype of the patient carrying the p.R253G mutation.
Enhancing Macrocyclic Diterpenes as Multidrug-Resistance Reversers: Structure–Activity Studies on Jolkinol D Derivatives
Publication . Reis, Mariana; Ferreira, Ricardo J.; Santos, Maria M. M.; Molnár, J.; Santos, Daniel J. V. A.; Ferreira, M. J. U.
The phytochemical study of Euphorbia piscatoria yielded jolkinol D (1) in a large amount, whose derivatization gave rise to 12 ester derivatives (2–13) and hydrolysis to compound 14. The in vitro modulation of P-gp of compounds 1–14 was evaluated through a combination of transport and chemosensitivity assays, using the L5178 mouse T lymphoma cell line transfected with the human MDR1 gene. Apart from jolkinol D, all derivatives (2–14) showed potential as MDR reversal agents. In this small library of novel bioactive macrocyclic lathyrane diterpene derivatives, designed to evaluate structure–activity relationships essential in overcoming multidrug resistance (MDR), some correlations between MDR reversal and molecular weight, accessible solvent areas, and octanol/water partition coefficient were identified that can contribute to the development of new selective P-gp reversal agents.
Intracellularly-Targeted Nanoplatform to Target Dendritic Cells and for Immunomodulation
Publication . Peres, Carina Sofia Gonçalves; Florindo, Helena F.; Graça, Luís; Préat, Véronique
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Immunotherapy, notably cancer vaccines and immune checkpoint modulators, has emerged as a promising alternative therapy. However, limited efficacy has been obtained for cancer vaccines and severe immune-mediated side effects have been related to immune checkpoint inhibitors under clinical development. Thus, to overcome the main disadvantages presented by these two therapeutic options when used individually, and considering the heterogeneity of TNBC, combination therapies are under clinical development to treat this specific breast cancer subtype.
We hypothesized that the development of a multifunctional nanovaccine could re-shape the tumour microenvironment (TME), sensitizing TNBC to the agonist immune checkpoint OX40, and thus, improving the overall anti-tumour immune response.
For this purpose, a poly(lactic acid) (PLA) nanoparticle (NP)-based nanovaccine was designed and synthesized to target dendritic cells (DC) and the TME by incorporating TNBC-associated antigens, toll-like receptor ligands CpG and Poly(I:C), and siRNA to downregulate the potent immunosuppressive cytokine transforming growth factor-β (TGF-β). NP surface was modified by hyaluronic acid to promote DC activation, but also to potentiate their delivery to the TME by targeting CD44 receptor, overexpressed in TNBC cells.
NPs presented a spherical-shape with an average diameter close to 200 nm, displaying narrow polydispersity index, near-neutral surface charge, and entrapment efficiencies (EE) superior to 85%. NPs were extensively taken up by both DC and TNBC cells, without affecting their viability. In addition, NPs induced DC activation and maturation, increasing significantly the expression of the surface markers. 4T1 tumour-bearing animals treated with the multifunctional nanovaccine combined with αOX40 showed a noteworthy tumour remission, with a higher overall survival and a tumour volume 4-fold lower than those obtained for αOX40-treated mice. The nanovaccine re-shaped the immune profiling within the TME, which correlated with the overall anti-tumour effect obtained in this combinatorial scheme. This study revealed the synergy between a multi-targeting nanovaccine and αOX40 in TNBC, providing important insights for the establishment of novel combination regimens against this tumour.
Actividade antibacteriana dos constituintes químicos da planta medicinal africana Cleistochlamys kirkii
Publication . Monteiro, Ângelo Filipe Almeida; Ferreira, Maria José Umbelino; Fernandes, Ana Margarida Monteiro Madureira
O trabalho apresentado nesta dissertação teve como objectivo principal a obtenção de compostos com actividade antibacteriana (Staphylococcus aureus) a partir da planta medicinal africana C. kirkii (Annonaceae). Nesse sentido utilizaram-se duas abordagens: i) isolamento dos constituintes maioritários de uma fracção do extracto de metanol; e ii) derivatização molecular de um composto isolado em grande quantidade.
A partir de uma fracção, resultante do fracionamento bioguiado do extracto de metanol de C. kirkii, foram isolados, por técnicas cromatográficas, um novo composto com o esqueleto do ciclo-hexeno, denominado cleistinol (2.2), um novo alcalóide aporfínico, a cleistonina (2.7), e quatro derivados do heptano, três dos quais são novos compostos, o (R)-(E)-melodorinol (2.4), o (R)-(E)-acetilmelodorinol (2.5) e o kirkiinolato (2.6), e umheptano nunca isolado anteriormente de fontes naturais, o (R)-(Z)-melodorinol (2.3). Foi ainda isolada a dichamanetina (2.1), uma flavanona já descrita na literatura.
Por derivatização molecular da dichamanetina (2.1), isolada em grande quantidade e utilizando vários derivados de aminas, foram obtidas nove iminas, nomeadamente: uma oxima (2.8), quatro hidrazonas (2.9 a 2.12), três carbo-hidrazidas (2.13 a 2.15) e uma semicarbazona (2.16). Preparou-se também um derivado tri-acetilado da dichamanetina (2.17).
As estruturas químicas dos compostos foram estabelecidas com base nas suas características físicas ([α]22D) e espectroscópicas (ressonância magnética unidimensional – 1H-RMN e 13C-RMN – e bidimensional - 1H-1H-COSY, HMQC, HMBC e NOESY) e por comparação com dados descritos na literatura.
A actividade antibacteriana dos compostos foi avaliada em estirpes de Staphyloccocus aureus, nomeadamente numa estirpe sensível (ATCC 6538), numa estirpe resistente à meticilina (ATCC 43300) e ainda na estirpe CIP 106414 meticilina resistente com resistência intermédia à vancomicina. Entre os compostos isolados, a dichamanetina (2.1) demonstrou ser um potente agente antibacteriano em todas as estirpes (CMI = 0,49 μg mL-1). O composto 2.5 apresentou valores de CMI = 15,63 e 62,5 μg mL-1, na estirpe sensível e na ertirpe resistente CIP 106414, respectivamente. Os compostos 2.3 e 2.4 apresentaram um valor de CMI = 62,5 μg mL- na estirpe sensível. O composto 2.3 exibiu também CMI = 62,5 μg mL-1 na estirpe resistente CIP 106414.
Relativamente aos derivados obtidos a partir da dichamanetina, os valores mais baixos de CMI verificaram-se para os compostos 2.8, 2.9 e 2.11 em todas as estirpes do Staphylococcus aureus (CMI = 0,98 a 1,95 μg mL-1). As hidrazonas 2.10 e 2.13 exibiram valores de CMI entre 7,81 e 31,25 μg mL-1 para todas as estirpes. De salientar ainda a actividade das carbo-hidrazidas 2.14 e 2.16 nas estirpes ATCC6538 e CIP106414 (CMI = 3,9 e 15,63 μg mL-1, respectivamente). No entanto, nenhum dos derivados de imina revelou ser mais potente que a própria dichamanetina, evidenciando a importância do grupo carbonilo em C4 para actividade antibacteriana, assim como a presença dos grupos hidroxilo livres, uma vez que a sua acetilação levou a uma perda da actividade.
Posteriormente foi avaliada a interacção entre os compostos 2.1 a 2.17 e alguns antibióticos de referência. Um total de oito compostos, três isolados (2.3, 2.4 e 2.5) e cinco sintetizados por derivatização (2.9, 2.10, 2.12, 2.14 e 2.15) foram capazes de exercer um efeito modulador na actividade dos três antibióticos ensaiados (amoxicilina, ofloxacina e vancomicina) nas mesmas estirpes utilizadas para avaliar a actividade antibacteriana. De salientar o composto 2.12, um composto inactivo, que se revelou um forte efeito modulador na actividade dos três antibióticos aqui testados, em todas as estirpes usadas.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
6817 - DCRRNI ID
Número da atribuição
PEst-OE/SAU/UI4013/2011