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Projeto de investigação
Molecular, Structural and Cellular Microbiology – Instituto de Tecnologia Química e Biológica António Xavier
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Publicações
N-heterocyclic carbene iron complexes as anticancer agents
Publication . Lenis-Rojas, Oscar A.; Cordeiro, Sandra; Horta-Meireles, Marta; Fernández, Jhonathan Angel Araujo; Vila, Sabela Fernández; Rubiolo, Juan Andrés; Cabezas-Sainz, Pablo; Sanchez, Laura; Fernandes, Alexandra R.; Royo, Beatriz; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; MDPI - Multidisciplinary Digital Publishing Institute
Cisplatin and its derivatives are commonly used in chemotherapeutic treatments of cancer, even though they suffer from many toxic side effects. The problems that emerge from the use of these metal compounds led to the search for new complexes capable to overcome the toxic side effects. Here, we report the evaluation of the antiproliferative activity of Fe(II) cyclopentadienyl complexes bearing n-heterocyclic carbene ligands in tumour cells and their in vivo toxicological profile. The in vitro antiproliferative assays demonstrated that complex Fe1 displays the highest cytotoxic activity both in human colorectal carcinoma cells (HCT116) and ovarian carcinoma cells (A2780) with IC50 values in the low micromolar range. The antiproliferative effect of Fe1 was even higher than cisplatin. Interestingly, Fe1 showed low in vivo toxicity, and in vivo analyses of Fe1 and Fe2 compounds using colorectal HCT116 zebrafish xenograft showed that both reduce the proliferation of human HCT116 colorectal cancer cells in vivo.
In Vitro and In Vivo Biological Activity of Ruthenium 1,10-Phenanthroline-5,6-dione Arene Complexes
Publication . Lenis-Rojas, Oscar A.; Roma-Rodrigues, Catarina; Carvalho, Beatriz; Cabezas-Sainz, Pablo; Fernández Vila, Sabela; Sánchez, Laura; Baptista, Pedro V.; Fernandes, Alexandra R.; Royo, Beatriz; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; MDPI - Multidisciplinary Digital Publishing Institute
Ruthenium(II) arene complexes exhibit promising chemotherapeutic properties. In this study, the effect of the counter anion in Ru(II) complexes was evaluated by analyzing the biological effect of two Ru(II) p-cymene derivatives with the 1,10-phenanthroline-5,6-dione ligand of general-formula [(η6-arene)Ru(L)Cl][X] X = CF3SO3 (JHOR10) and PF6 (JHOR11). The biological activity of JHOR10 and JHOR11 was examined in the ovarian carcinoma cell line A2780, colorectal carcinoma cell line HCT116, doxorubicin-resistant HCT116 (HCT116-Dox) and in normal human dermal fibroblasts. Both complexes JHOR10 and JHOR11 displayed an antiproliferative effect on A2780 and HCT116 cell lines, and low cytotoxicity in fibroblasts. Interestingly, JHOR11 also showed antiproliferative activity in the HCT116-Dox cancer cell line, while JHOR10 was inactive. Studies in A2780 cells showed that JHOR11 induced the production of reactive oxygen species (ROS) that trigger autophagy and cellular senescence, but no apoptosis induction. Further analysis showed that JHOR11 presented no tumorigenicity, with no effect in the cellular mobility, as evaluated by thye wound scratch assay, and no anti- or pro-angiogenic effect, as evaluated by the ex-ovo chorioallantoic membrane (CAM) assay. Importantly, JHOR11 presented no toxicity in chicken and zebrafish embryos and reduced in vivo the proliferation of HCT116 injected into zebrafish embryos. These results show that these are suitable complexes for clinical applications with improved tumor cell cytotoxicity and low toxicity, and that counter-anion alteration might be a viable clinical strategy for improving chemotherapy outcomes in multidrug-resistant (MDR) tumors.
Tracking W-Formate Dehydrogenase Structural Changes During Catalysis and Enzyme Reoxidation
Publication . Vilela-Alves, Guilherme; Manuel, Rita Rebelo; Oliveira, Ana Rita; Pereira, Inês Cardoso; Romão, Maria João; Mota, Cristiano; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); MDPI - Multidisciplinary Digital Publishing Institute
Metal-dependent formate dehydrogenases (Fdh) catalyze the reversible conversion of CO2 to formate, with unrivalled efficiency and selectivity. However, the key catalytic aspects of these enzymes remain unknown, preventing us from fully benefiting from their capabilities in terms of biotechnological applications. Here, we report a time-resolved characterization by X-ray crystallography of the Desulfovibrio vulgaris Hildenborough SeCys/W-Fdh during formate oxidation. The results allowed us to model five different intermediate structures and to chronologically map the changes occurring during enzyme reduction. Formate molecules were assigned for the first time to populate the catalytic pocket of a Fdh. Finally, the redox reversibility of DvFdhAB in crystals was confirmed by reduction and reoxidation structural studies.
Spectroscopic and Structural Characterization of Reduced Desulfovibrio vulgaris Hildenborough W-FdhAB Reveals Stable Metal Coordination during Catalysis
Publication . Oliveira, Ana Rita; Mota, Cristiano; Klymanska, Kateryna; Biaso, Frederic; Romão, Maria Joao; Guigliarelli, Bruno; Pereira, Inês Cardoso; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; ACS - American Chemical Society
Metal-dependent formate dehydrogenases are important enzymes due to their activity of CO2reduction to formate. The tungsten-containing FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough is a good example displaying high activity, simple composition, and a notable structural and catalytic robustness. Here, we report the first spectroscopic redox characterization of FdhAB metal centers by EPR. Titration with dithionite or formate leads to reduction of three [4Fe-4S]1+clusters, and full reduction requires Ti(III)-citrate. The redox potentials of the four [4Fe-4S]1+centers range between -250 and -530 mV. Two distinct WVsignals were detected, WDVand WFV, which differ in only the g2-value. This difference can be explained by small variations in the twist angle of the two pyranopterins, as determined through DFT calculations of model compounds. The redox potential of WVI/Vwas determined to be -370 mV when reduced by dithionite and -340 mV when reduced by formate. The crystal structure of dithionite-reduced FdhAB was determined at high resolution (1.5 Å), revealing the same structural alterations as reported for the formate-reduced structure. These results corroborate a stable six-ligand W coordination in the catalytic intermediate WVstate of FdhAB.
Studies on the Interaction between Model Proteins and Fluorinated Ionic Liquids
Publication . Alves, Márcia M. S.; Melo, Manuel N.; Mertens, Haydyn D. T.; Pereiro, Ana B.; Archer, Margarida; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); DQ - Departamento de Química; MDPI AG
Proteins are inherently unstable, which limits their use as therapeutic agents. However, the use of biocompatible cosolvents or surfactants can help to circumvent this problem through the stabilization of intramolecular and solvent-mediated interactions. Ionic liquids (ILs) have been known to act as cosolvents or surface-active compounds. In the presence of proteins, ILs can have a beneficial effect on their refolding, shelf life, stability, and enzymatic activities. In the work described herein, we used small-angle X-ray scattering (SAXS) to monitor the aggregation of different concentrations of ILs with protein models, lysozyme (Lys) and bovine serum albumin (BSA), and fluorescence microscopy to assess micelle formation of fluorinated ILs (FILs) with Lys. Furthermore, coarse-grained molecular dynamics (CG-MD) simulations provided a better understanding of Lys–FIL interactions. The results showed that the proteins maintain their globular structures in the presence of FILs, with signs of partial unfolding for Lys and compaction for BSA with increased flexibility at higher FIL concentrations. Lys was encapsulated by FIL, thus reinforcing the potential of ILs to be used in the formulation of protein-based pharmaceuticals.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
6817 - DCRRNI ID
Número da atribuição
UIDB/04612/2020