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Projeto de investigação
Strategic Project - LA 6 - 2011-2012
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Antibody-Conjugated Nanoparticles for Therapeutic Applications
Publication . Cardoso, Maria Margarida Canas Mendes de Almeida; Peça, Inês N.; Roque, Ana Cecília Afonso; DQ - Departamento de Química; CQFB-REQUIMTE - Centro de Química Fina e Biotecnologia (Lab. Associado REQUIMTE); Bentham Science Publishers
A great challenge to clinical development is the delivery of chemotherapeutic agents, known to cause severe toxic effects, directly to diseased sites which increase the therapeutic index whilst minimizing off-target side effects. Antibody-conjugated nanoparticles offer great opportunities to overcome these limitations in therapeutics. They combine the advantages given by the nanoparticles with the ability to bind to their target with high affinity and improve cell penetration given by the antibodies. This specialized vehicle, that can encapsulate several chemotherapeutic agents, can be engineered to possess the desirable properties, allowing overcoming the successive physiological conditions and to cross biological barriers and reach a specific tissue or cell. Moreover, antibody-conjugated nanoparticles have shown the ability to be internalized through receptor-mediated endocytosis and accumulate in cells without being recognized by the P-glycoprotein, one of the main mediators of multi-drug resistance, resulting in an increase in the intracellular concentration of drugs. Also, progress in antibody engineering has allowed the manipulation of the basic antibody structure for raising and tailoring specificity and functionality. This review explores recent developments on active drug targeting by nanoparticles functionalized with monoclonal antibodies (polymeric micelles, liposomes and polymeric nanoparticles) and summarizes the opportunities of these targeting strategies in the therapy of serious diseases (cancer, inflammatory diseases, infectious diseases, and thrombosis).
Dextran-Coated Magnetic Supports Modified with a Biomimetic Ligand for IgG Purification
Publication . Santana, Sara D. F.; Dhadge, Vijaykumar L; Roque, Ana Cecília Afonso; CQFB-REQUIMTE - Centro de Química Fina e Biotecnologia (Lab. Associado REQUIMTE); DQ - Departamento de Química; ACS - American Chemical Society
Dextran-coated iron oxide magnetic particles modified with ligand 22/8, a protein A mimetic ligand, were prepared and assessed for IgG purification. Dextran was chosen as the agent to modify the surface of magnetic particles by presenting a negligible level of nonspecific adsorption. For the functionalization of the particles with the affinity ligand toward antibodies, three methods have been explored. The optimum coupling method yielded a theoretical maximum capacity for human IgG calculated as 568 ± 33 mg/g and a binding affinity constant of 7.7 × 10⁴ M⁻¹. Regeneration, recycle and reuse of particles was also highly successful for five cycles with minor loss of capacity. Moreover, this support presented specificity and effectiveness for IgG adsorption and elution at pH 11 directly from crude extracts with a final purity of 95% in the eluted fraction.
Kinetic and Structural Studies of Aldehyde Oxidoreductase from Desulfovibrio gigas Reveal a Dithiolene-Based Chemistry for Enzyme Activation and Inhibition by H2O2.
Publication . Marangon, Jacopo; Correia, Hugo D.; Brondino, Carlos D.; Moura, José João Galhardas de; Romão, Maria João; Gonzalez, Pablo Javier; Santos-silva, Teresa Sacadura; CQFB-REQUIMTE - Centro de Química Fina e Biotecnologia (Lab. Associado REQUIMTE); DQ - Departamento de Química; PLOS - Public Library of Science
Mononuclear Mo-containing enzymes of the xanthine oxidase (XO) family catalyze the oxidative hydroxylation of aldehydes and heterocyclic compounds. The molybdenum active site shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. The XO family member aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is an exception as presents in its catalytically competent form an equatorial oxo ligand instead of the sulfido ligand. Despite this structural difference, inactive samples of DgAOR can be activated upon incubation with dithionite plus sulfide, a procedure similar to that used for activation of desulfo-XO. The fact that DgAOR does not need a sulfido ligand for catalysis indicates that the process leading to the activation of inactive DgAOR samples is different to that of desulfo-XO. We now report a combined kinetic and X-ray crystallographic study to unveil the enzyme modification responsible for the inactivation and the chemistry that occurs at the Mo site when DgAOR is activated. In contrast to XO, which is activated by resulfuration of the Mo site, DgAOR activation/inactivation is governed by the oxidation state of the dithiolene moiety of the pyranopterin cofactor, which demonstrates the non-innocent behavior of the pyranopterin in enzyme activity. We also showed that DgAOR incubation with dithionite plus sulfide in the presence of dioxygen produces hydrogen peroxide not associated with the enzyme activation. The peroxide molecule coordinates to molybdenum in a η(2) fashion inhibiting the enzyme activity.
Exploring new protein-based scaffolds for bioengineering applications
Publication . Dias, Ana Margarida Gonçalves Carvalho; Roque, Ana; Casanova, Olga
The potential of WW domains as proteins scaffolds for the development of purification affinity reagents has been explored.
Minimal versions of native human Pin1 (hPin1_WW) and human YAP65 (hYAP65_WW) WW domains were produced through chemical synthesis using solid-phase peptide synthesis. After purification, characterization by mass spectrometry and circular dichroism demonstrated, respectively, the correct molecular mass, and the correct folding of WW domains with thermal stability comparable to the extended version expressed biologically. These peptides were further immobilized in chromatographic supports. Adsorbents bearing hPin1_WW captured phosphorylated peptides and proteins, whereas those modified with hYAP65_WW captured Proline-rich peptides. In both cases the binding and elution of the target peptides was achieved under mild conditions.
Encouraged by these results, a näive library based on the sequence of WW prototype was designed and generated. The library was further evolved in vitro through phage and ribosome display methodologies against Human Serum Albumin (HSA) and Immunoglobulin G (IgG). The phage display library leads to the identification of CW3S (a WW domain derived from Clone 3) as a potential HSA binder. This ligand was produced biologically in co-expression with GFP, and was also chemically synthesized. The affinity between CW3S and HSA was determined as Ka of 8.37x106 M-1 (KD=119nM) by ELISA. The chemically synthesized peptide was characterized by circular dichroism showing the folding and thermal stability similar to other native WW domains. This peptide was also immobilized in agarose and captured HSA (0.291μg
protein/mg support at 4°C).
This work strongly demonstrated the robustness of the WW domains to withstand
modifications and mutations, therefore possessing a clear potential for application as a protein scaffold.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
6820 - DCRRNI ID
Número da atribuição
PEst-C/EQB/LA0006/2011