USING SYNTHETIC APPROCHES TO GLYCOCONJUGATES TO UNDERSTAND CARBOHYDRATE-MEDIATED BIOCHEMICAL INTERACTIONS
Creators: name: VETRO, MARIA
Publisher: name: Universit� degli Studi di Milano
Year: 2014

description: This PhD Thesis deals with the synthesis of carbohydrates derivatives as analogues of natural compounds and their biological evaluation as tools for the study of biochemical regulatory pathways. Carbohydrates, together with proteins, nucleic acids and lipids, are the building blocks on which complex living systems are based. This spectacular class of compounds acts as energetic sources, signaling regulators and components of cell surface. So, they are involved in many biological processes essential for the living cell, as immune response, inflammation, cell growth, cell-cell adhesion and so forth. The possibility to develop biochemical tools based on carbohydrates structures could be used to explain their functional role in many of these mechanisms and how to intervene to conceive new drugs systems for the treatment of several pathologies like cancers and bacterial infections, generally due to altered carbohydrate-mediated interactions. The topic of this PhD thesis can be divided into two parts. The first one, composed by Chapter 1, 2 and 3, deals with the synthesis of glycosylglycerols ligands of Akt, a protein kinase overexpressed in many cancer cells, while the second part, resumed in Chapter 4, is oriented to the study of the opportunity to employ gold nanoparticles as alternative carriers for saccharide antigens in the vaccinology field. Protein kinase are enzymes involved in the regulation of many crucial cellular processes like proliferation, differentiation and apoptosis [1]. Among them, the serine/threonine protein kinase B, also known as Akt, plays a key role as a component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis, which is implicated in aberrant tumor cell signaling. Inappropriate activation of the Akt kinase is a common event in human tumors making Akt a critical player in cell survival and, consequently, inhibitors that target Akt are potentially relevant for cancer therapy. Akt is a soluble cytosolic protein composed of three parts, a N-terminal pleckstrin homology (PH) domain, a central catalytic kinase domain and a C-terminal regulatory domain [2]. The key second messenger phosphatidylinositol 3,4,5 triphosphate [PtdIn(3,4,5)P3 or PIP3], generated by the phosphatidylinositol-3 kinase (PI3K), binds to the PH domain inducing a conformational change of Akt which exposes two site of phosphorylation, specifically threonine 308 and serine 473. Their phosphorylation results in a fully activation of Akt that dissociates from the plasma membrane and can phosphorylate many substrates in the cytoplasm and nucleus [3]. The structure of the phosphatidylinositol PIP3, the natural ligand of Akt PH domain, is composed by an inositol, with a phosphate group in position 3 and a glycerol moiety in position 1 carrying long acyl chains. Sulfoquinovosylacylglycerol (SQDG) analogues, synthesized in our laboratory, show structural features similar to PIP3, as an acylglycerol portion and a negative head. Furthermore the inositol ring can be easily reconducted to a glucose scaffold in which the pyranosidic oxygen replaces the hydroxyl group in position 2 of the inositol. These likeness prompted us to evaluate them as potential Akt inhibitors both in vitro and in vivo. An ELISA test was performed and the obtained results demonstrated the ability of our compounds to inhibit the Akt. In particular, the presence of the anionic group and a long chain lipophilic portion were necessary for their activity. The most active in vitro compounds were also tested for their antiproliferative activity on cell systems confirming these preliminary results. Following our interest in the research of potential Akt inhibitor able to bind selectively the PH domain, we decided to further explore the potentiality of different anionic glycolipids. Thus we prepared a new class of compounds with the carboxylate instead of the sulfonate group on the sugar, the glucuronosyldiacylglycerol (GlcADG) analogues. The Akt inhibitory activity of the GlcADG analogues was evaluated both in vitro and in vivo, indicating again the importance of a negative charge and long acyl chains on the glycerol moiety. To better understand the action mechanism of Akt inhibition by studying their cell localization, part of this PhD thesis was also directed to the preparation of two fluorescent glucuronides structurally related to the most active GlcADGs. In particular the 7-nitro-1,2,3-benzoxadiazole (NBD) was chosen as the fluorophore and was linked to the glycerol moiety through a N-spacer. The biological evaluation of both compound is now underway. The second part of this PhD thesis reports the potential application of gold nanoparticles as carrier for saccharide antigens related to Streptococcus pneumoniae. The carbohydrate's world and that of nanotechology combine together seeking to improve the development of fully synthetic carbohydrate vaccines against Streptococcus pneumoniae. Infection of Streptococcus. pneumoniae is one of major cause of morbidity and mortality in the world, causing pneumonia, bacteremia and meningitis [4]. The S. pneumoniae bacterial cell is surrounded by an outer capsule that consists of structurally diverse polysaccharides (CPSs) which elicit specific antibodies able to confer protection against the bacterium. So, they have been used in the preparation of vaccines, but when polysaccharide are used as antigen, they give a T-independent response and consequently result unable to generate a long immunological memory [5]. Step forward have been made when carbohydrate antigens were conjugate to a protein carrier. Indeed, this conjugation induce T-dependent switch with the production of IgG antibodies and the generation of a long-lasting memory response [6]. However, other problems of saccharide-based vaccines need a solution, e.g. no-specific methods of conjugation of the antigens to the protein carrier, risks of carrier-induced epitope suppression and the process of CPSs isolation and purification from natural sources, which require many checks at different stages raising the cost of the final vaccine. The possibility to use the minimum immunological portion obtained by synthetic methodology can solve this latter, while the search for new carriers might overcome the hurdles encountered with the use of proteins as carrier. In the last years, gold nanoparticles functionalized with oligosaccharides, named gold glyconanoparticles [7] (GNPs), have been developed in order to mimic the natural presentation of the carbohydrate coating which is present at the cell or pathogen surface. GNPs are characterized to be non-cytotoxic, soluble in biological media and easy to prepare and purify. Recently, GNPs coated with the synthetic tetrasaccharide repeating unit of Streptococcus pneumoniae serotype 14, identified as the smallest structure necessary to evoke an immunological response [8], OVA323-339 peptide as T helper cell, and ?-D-glucose to assist water solubility, were prepared. They resulted able to induce IgG antibodies against native polysaccharide of S. Pneumoniae serotype 14, and represent the first example of a fully synthetic carbohydrate vaccine against the bacterium [9]. According to this result, we decided to prepare different GNPs bearing the trisaccharide repeating unit of the 19F serotype of S. pneumoniae, alone or together with the tetrasaccharide related to serotype 14, with the aim to develop new multiantigenic systems displaying different carbohydrate antigens arranged on a single nanoparticle. As a first step, the trisaccharide repeating unit related to Pn19F and ?-D-glucose suitably functionalized with a thiol-ending linker were synthesized during the six months spent in the Laboratory of GlycoNanotechology of CIC biomaGUNE center (San Sebastian-Spain). Then, all the GNPs were prepared, characterized for their physical properties and tested in vivo in mice to evaluate their ability to induce specific IgG antibodies against native capsular polysaccharides of S. pneumoniae type 14 and 19F, by Dr. Dodi Safari at Eijkman Institute for Molecular Biology in Jakarta. Mice were immunized intracutaneously with GNPs in mixture with Quillaja purified saponin as adjuvant. ELISA test with polysaccharide Pn14 and Pn19F as coating material were performed on sera of immunized mice to measure the antibodies against native polysaccharide of Streptococcus pneumoniae. However, IgG able to recognize the native polysaccharide were found only in sera of mice immunized with GNPs carrying the tetrasaccharide Pn14, suggesting that the length of trisaccharide Pn19F is not enough to induce an immune response in vivo. Moreover additional ELISA test using GNPs to cover the plate were also performed [10]. They demonstrated that all the GNPs have been able to induce a specific immune response in vivo, thus demonstrating the antigenicity of the system and confirming the ability of GNPs to act as carrier of saccharide antigens. 1. Cohen, P., Protein kinases ? the major drug targets of the twenty-first century? Nat. Rev.Drug Discov. 2002, 1, 309?315. 2. Brazil, D. P., Hemmings, B.A., Ten years of protein kinase B signalling: a hard Akt to follow. Trends Biochem. Sci. 2001, 26, 657-664. 3. Sale, E. M., Sale, G.J., Protein kinase B: signalling roles and therapeutic targeting. Cell. Mol. Life Sci. 2008, 65, 113-127.4. Bryce, J., Boschi-Pinto, C., Shibuya K., Black, R.E., WHO estimates of the causes of death in children Lancet 2005, 365, 1147-1152. 5. Mond, J. J., Lees, A., Snapper, C.M., T Cell-Independent Antigens Type 2. Annu. Rev. Immunol. 1995, 13, 655-692. 6. Avery, O. T., Goebel, W.F., Chemo-immunological studies on conjugated carbohydrate-protein: The immunological specifity of an antigen prepared by combining the capsular polysaccharide of type III pneumococcus with foreign protein. J. Exp. Med. 1931, 54, 437-447. 7. de la Fuente, J. M., Barrientos, A. G., Rojas, T. C., Rojo, J., Canada, J., Fern�ndez, A., Penad�s, S., Gold glyconanoparticles as water-soluble polyvalent models to study carbohydrate interaction. Angew. Chem. Int. Ed. Engl 2001, 40, 2258-2261. 8. Safari, D., Dekker, H. A. T., Joosten, J. A. F., Michalik, D., carvalho de Souza, A., Adamo, R., Lahmann, M., Sundgren, A., Oscarson, S., Kamerling, J. P., Snippe, H, Identification of the smallest structure capable of evoking opsonophagocytic antibodies against Streptococcus pneuminiae type 14. Infect. Immun. 2008, 76, 4615-4623. 9. Safari, D., Marradi, M., Chiodo, F., Dekker, H. A. T., Shan, Y., Adamo, R., Oscarson, S., Rijkers, G. T., Lahmann, M., Kamerling, J. P., Penad�s, S., Snippe, H., Gold nanoparticles as carriers for a synthetic Streptococcus pneuminiae type 14 conjugate vaccine. Nanomedicine 2012, 651-662. 10. Chiodo, F., Marradi, M., Tefsen, B., Snippe, H., van Die, I., Penad�s, S., High Sensitive Detection of Carbohydrate Binding Proteins in an ELISA-Solid Phase Assay Based on Multivalent Glyconanoparticles. PLoS ONE 2013, 8, 1-11.