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  • Influenza DNA vaccine purification using pHEMA cryogel support
    Publication . Santos, Tiago; Brito, Andreia; Boto, Renato; Sousa, Pedro; Almeida, Paulo; Cruz, Carla; Tomaz, C. T.
    Influenza virus is a huge financial and social burden for health care systems over the world. Currently, traditionalapproaches are not effective in the fight of the epidemy and new alternatives like DNA vaccines have been developed. However, the downstream process of DNA vaccines is a constant challenge in the biotechnology industry. Cryogels has several advantages over traditional supports and have been tested as stationary phase in chromatographic separations. In this work, a method based on poly(2-hydroxyethyl methacrylate) cryogel was used to purify the plasmid NTC7482-41H-VA2 HA, which express the Influenza hemagglutinin gene. For this purpose, the cryogel was synthesized by cryo-polymerization of 2-hydroxyethyl methacrylate and characterized by scanning electron microscopy. The purification of supercoiled isoform of the plasmid NTC7482-41H-VA2 HA from a clarified lysate sample was achieved in a two-step experiment using NaCl and the dynamic binding capacity of pHEMA cryogel was determined. The assessment of DNA vaccine allowed to conclude that the level of contaminants such as proteins, genomic DNA, RNA and endotoxins are in accordance with FDA agency.
  • Polyazamacrocycles as potential antitumor agents for human prostate cancer cells
    Publication . Cruz, Carla; Cairrão, Elisa; Lourenço, Olga; Almeida, Paulo; Verde, Ignacio; Queiroz, João
    Polyazamacrocycles are currently being studied and used in a variety of applications beyond their traditional place in supramolecular and co-ordination chemistry. This study suggests additional applications of these compounds with particular emphasis on their use as antiproliferative agents that could be potentially used to treat cancer. Four polyazamacrocycles were tested in human prostate cancer LNCaP and prostate epithelial PNTA1 cells to analyze changes in cell proliferation and cell death capabilities. Their intracellular localization was also evaluated by confocal microscopy. The results show a decrease in proliferation rate and cell viability of LNCaP and PNTA1, after treatment with these compounds. The decrease in the number of viable cells is similar for the majority of the compounds studied, and at higher concentration, the proliferation efficiency decreased significantly in the cell lines studied. Also, our results suggest that L and L2 induce early apoptosis in PNTA1 cells and late apoptosis/necrosis in LNCaP cells. The compounds did not induce a significant increase in necrosis of both cell types. Although the compounds did not localize in a unique organelle, all of them have as main target the Golgi apparatus and other localization profiles differed depending on the cell line.
  • Trisubstituted barbiturates and thiobarbiturates: Synthesis and biological evaluation as xanthine oxidase inhibitors, antioxidants, antibacterial and anti-proliferative agents
    Publication . Figueiredo, Joana; Serrano, João L.; Cavalheiro, Eunice Cerdeira Soares; Keurulainen, Leena Maria; Yli-Kauhaluoma, Jari Tapani; Moreira, Vânia M; Ferreira, Susana; Domingues, F.C.; Silvestre, Samuel; Almeida, Paulo
    Barbituric and thiobarbituric acid derivatives have become progressively attractive to medicinal chemists due to their wide range of biological activities. Herein, different series of 1,3,5-trisubstituted barbiturates and thiobarbiturates were prepared in moderate to excellent yields and their activity as xanthine oxidase inhibitors, antioxidants, antibacterial agents and as anti-proliferative compounds was evaluated in vitro. Interesting bioactive barbiturates were found namely, 1,3-dimethyl-5-[1-(2-phenylhydrazinyl)ethylidene]pyrimidine-2,4,6(1H,3H,5H)-trione (6c) and 1,3-dimethyl-5-[1-[2-(4-nitrophenyl)hydrazinyl]ethylidene]pyrimidine-2,4,6(1H,3H,5H)-trione (6e), which showed concomitant xanthine oxidase inhibitory effect (IC50 values of 24.3 and 27.9 μM, respectively), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (IC50 values of 18.8 and 23.8 μM, respectively). In addition, 5-[1-(2-phenylhydrazinyl)ethylidene]pyrimidine-2,4,6(1H,3H,5H)-trione (6d) also revealed DPPH radical scavenger effect, with an IC50 value of 20.4 μM. Moreover, relevant cytotoxicity against MCF-7 cells (IC50 = 13.3 μM) was observed with 5-[[(2-chloro-4-nitrophenyl)amino]methylene]-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (7d). Finally, different 5-hydrazinylethylidenepyrimidines revealed antibacterial activity against Acinetobacter baumannii (MIC values between 12.5 and 25.0 μM) which paves the way for developing new treatments for infections caused by this Gram-negative coccobacillus bacterium, known to be an opportunistic pathogen in humans with high relevance in multidrug-resistant nosocomial infections. The most promising bioactive barbiturates were studied in silico with emphasis on compliance with the Lipinski's rule of five as well as several pharmacokinetics and toxicity parameters.
  • 5-Hydrazinylethylidenepyrimidines effective against multidrug-resistant Acinetobacter baumannii: Synthesis and in vitro biological evaluation of antibacterial, radical scavenging and cytotoxic activities
    Publication . Figueiredo, Joana; Serrano, João L.; Soares, MN; Ferreira, Susana; Domingues, F.C.; Almeida, Paulo; Silvestre, Samuel
    Acinetobacter baumannii has emerged as an important nosocomial pathogen in recent years, with infectious outbreaks caused by multidrug-resistant strains increasing worldwide. Thus, new antibacterial treatments for multidrug-resistant A. baumannii strains are needed. In this work, a series of 5-hydrazinylethylidenepyrimidines were synthesized and in vitro evaluated against two multidrug-resistant A. baumannii strains (AcB 13/10 and AcB 73/10). Minimum inhibitory concentration results demonstrated that generally the compounds in study presented values in a low micromolar range. In the determination of in vitro bacterial growth at 24 h, it was observed that the pyrimidines 3a and 3c, with an unsubstituted hydrazinylphenyl, have bacteriostatic activity in both multidrug-resistant A. baumannii strains, with a concentration-dependent action. In general, an additive effect occurred in the combination of these compounds with gentamicin, rifampicin and polymyxin B, for both strains. Furthermore, all 5-hydrazinylethylidenepyrimidines under study presented a good 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, generally low xanthine oxidase inhibition and low cytotoxicity in normal human dermal fibroblasts as well as potential favorable drug-likeness properties. Thus, these molecules can be considered attractive for the future development of antibacterial agents against multidrug-resistant A. baumannii.