Ziaco, Barbara (2009) DESIGN AND CHARACTERIZATION OF MOLECULAR SCAFFOLDS. [Tesi di dottorato] (Unpublished)
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Item Type: | Tesi di dottorato |
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Resource language: | English |
Title: | DESIGN AND CHARACTERIZATION OF MOLECULAR SCAFFOLDS |
Creators: | Creators Email Ziaco, Barbara barbaraziaco@libero.it |
Date: | 2009 |
Number of Pages: | 111 |
Institution: | Università degli Studi di Napoli Federico II |
Department: | Scienze biologiche |
Scuola di dottorato: | Scienze chimiche |
Dottorato: | Scienze chimiche |
Ciclo di dottorato: | 22 |
Coordinatore del Corso di dottorato: | nome email Vitagliano, Aldo UNSPECIFIED |
Tutor: | nome email Pedone, Carlo UNSPECIFIED |
Date: | 2009 |
Number of Pages: | 111 |
Keywords: | peptide, protein interaction, scaffold, helix, EPL |
Settori scientifico-disciplinari del MIUR: | Area 03 - Scienze chimiche > CHIM/03 - Chimica generale e inorganica |
Additional information: | Indirizzo del dottorato: Struttura e proprietà chimico-fisiche di molecole e sistemi di interesse biologico |
Date Deposited: | 03 Aug 2010 14:44 |
Last Modified: | 31 Oct 2014 10:44 |
URI: | http://www.fedoa.unina.it/id/eprint/4294 |
Collection description
In the post-genomic era the study of the interactions between biomolecules and in particular protein-protein interactions is of growing interest, since they are the basis of all the physiological processes mediated by the formation of complexes between biomolecules. Therefore, detailed knowledge of the molecular mechanisms responsible for these interactions is essential to develop molecules capable of modulating the biological activity of the protein target and then its cellular processes. Currently, the identification of molecules that inhibit or promote protein-protein interactions or protein-nucleic acids is one of the greatest challenges of drug discovery. Unlike the traditional approach based on the design of molecules modeled on specific substrates for enzymatic active sites, the development of compounds able to modulate protein-protein interactions is a more complex process. The interface molecules involved is usually an extended area that includes more non-contiguous regions lacking suitable pockets of binding to small molecules. Moreover, these regions often have elements of secondary structure that once isolated from their context, not having the protein native conformation. So far, several classes of compounds have been used to modulate protein-protein interactions: antibodies, peptides and small organic molecules and rarely miniproteine [1]. The latter, in fact, while constituting the majority of the active ingredients currently on the market are unsuitable to work with very large surface protein. The antibodies show high specificity and are widely used but have high production costs. The peptides are considered, however, good candidates for developing new compounds that interfere with protein-protein recognition [2]. The main approaches currently used to develop compounds of peptidic nature consists in the screening of phage libraries, parallel synthesis of peptides on membrane and rational design. Latter requires that the structural and biochemical information available on at least one of the two interacting partners and have been identified residues involved in the bond [3]. If the design of peptides that mimic the structural organization of the segments involved in the interaction is expected to introduce first stage of waste in order to stabilize the secondary structure and a second in which you must enter the residues responsible for interaction in the right spatial orientation. An alternative approach is to use scaffold [4] molecules structurally stable that already have the desired secondary structure, which can be directly introduced into the residues in the correct spatial orientation. The aim of this PhD project was to design and characterize molecular scaffold, such as peptides and mini-proteins that can modulate the interactions between biomolecules. This aim was addressed by three different approaches: • The development of a new synthetic strategy for obtaining polypeptides, functionally active protein expressed by ligation; • The bio-physical characterization of a helical peptide scaffold; • The transfer of functional epitopes on a scaffold protein known. The first strategy consists in developing a procedure for binding by stable covalent bonds the C-terminus of two polypeptide fragments obtained by recombinant expression in bacteria. This strategy allows to obtain peptide model systems [5] and mini-proteins that retain the same functionality of the target protein but whose dimensions are considerably reduced. The synthetic strategy provides for the chemical ligation reaction between two polypeptides activated as the C-terminal thioester and a bifunctional linker is characterized by the presence of two cysteines in position pseudo N-terminal. The linker was synthesized from dall'etilendiammina which they were linked to two cysteine residues via a peptide bond. The mini-protein to the C-terminal thioester was obtained using expression vectors containing inteine [6]. The synthetic procedure was developed using as a model system for the mini-protein, the sequence coding for the cloning site of the vector pTrcHisA. The vector was modified by the insertion dell'inteina MxeGyrA (N198A). The fusion construct was expressed in cells BL21 (DE3) of Escherichia coli and purified by affinity chromatography on chitin resin, the mini-protein thioester was obtained following the dell'inteina splicing in the presence of thiols. Following the mini-protein thioester was used in two separate ligation reactions with the linker, to obtain homodimers and heterodimers. The pure products, characterized by LC-MS, were all obtained with good yields [7]. This synthetic strategy offers the opportunity to unite chemically two protein fragments in a stable manner through the use of a bifunctional linker, which can be suitably modified by varying the length and rigidity of the spacer between the cysteine residues and this has considerable potential for biotechnological applications . This methodology can be used to combine neighboring peptide chains in space but not in sequence, to mimic, for example, discontinuous epitopes, to synthesize scaffolds of small (mini-antibody) as an alternative dimerization domains such as Leucin zipper. For the second approach has made the chemical and physical characterization of a peptide for its possible use as scaffolds for helical structural reasons. It was recently described a peptide, QK, able to mimic in vitro and in vivo biological activity of VEGF [8-10] in aqueous solution which assumes a well-defined helical conformation. Analysis of circular dichroism and NMR data indicate that the peptide QK has a thermal stability is unusual for a peptide composed only of natural amino acids. To assess the structural determinants of this stability, the experimental data have been supplemented with molecular dynamics simulations. Theoretical studies have indicated that the N-terminal region and a hydrophobic contact between the Leu7 and Leu10 are important for the thermal stability of the peptide. To test these predictions have been synthesized 3 peptides similar QK: QK1-12 that lacks the C-terminal; QK4-15 than the N-terminal and QK10A, in which Leu10 was replaced with un'alanina. The analysis of peptides by circular dichroism and NMR showed that QK1-12, unlike QK4-15, maintains a helical structure and thermal stability similar to QK, QK10A has about half the helical content and does not retain 's unusual thermal stability [11]. Finally, using a combination of experimental techniques such as CD, NMR and MD was possible to characterize at atomic one possible pathway for formation of the peptide helix QK10A and provide information sull'inusuale thermal stability of the peptide QK prerequisite for its use as helical scaffold. The third approach has included the development of a mini-biologically active protein from a scaffold known. The biological system chosen was that of VEGF and its receptors. An analysis of three-dimensional structure of the complex between VEGF/Flt-1D2 and mutagenesis have identified residues important for binding to VEGF receptors [12], these data demonstrated that the binding region of VEGF receptor includes the helix 17-25. The scaffold was the chosen Avian Pancreatic Polypeptide (APP), a miniproteina of 36 amino acids, very stable, which owns un'α-helix exposed to solvent and thruster poliprolina type II. Based on the overlap of the propeller of VEGF and the APP scaffold have been designed in which two different molecules have been transferred to the residues responsible for interaction with VEGF receptors and the peptide QK respectively named APP1 and APP_QK. Molecules are designed and the wild type were obtained by recombinant cells BL21 (DE3) of Escherichia coli. The proteins were purified by affinity chromatography and analyzed by LC-MS. Finally, preliminary in vitro biological assays have shown for the molecule APP_QK a similar activity to that of QK peptide. In conclusion, this work by providing different approaches contribute significantly to the development of new scaffolds for targeting protein-protein interactions. BIBLIOGRAFY [1] Cochran A.G., Chem. Bio. 5: 654-659 (2001) [2] Souroujon M.C. and Mochly-Rosen D., Nat. Biotechnol. 16: 919-924 (1998) [3] Cochran A.G., Chem. Bio. 7: R85-94 (2000) [4]Hershberger S.J., Lee S.G. and J. Chmielewski Scaffolds for blocking proteinprotein interactions. Curr. Top Med. Chem. (2007) 7(10):928-42 [5] B.R. Gibney, F. R. and P. L. Dutton, Curr. Opin. Chem. Biol. 1997, 1: 537-542 [6] T. W. Muir, Annu Rev Biochem 2003, 72, 249 [7] B. Ziaco, S. Pensato, L.D.D’Andrea, E.Benedetti and A.Romanelli, Org.Let. 2008, 10(10):1955-58 [8] L.D. D’Andrea, G. Iaccarino, R. Fattorusso, D. Sorriento, C. Carannante, D. Capasso, B. Trimarco and C. Pedone, Proc Natl Acad Sci U S A. 2005, 102, (40):14215-20 [9] Dudar GK, D'Andrea LD, Di Stasi R, Pedone C, Wallace JL. Am J Physiol Gastrointest Liver Physiol. 2008 Aug;295(2):G374-81. Epub 2008 Jun 26. [10] G Santulli, M Ciccarelli, G Palumbo, A Campanile, G Galasso, B Ziaco, GG Altobelli, V Cimini, F Piscione, LD D'Andrea, C Pedone, B Trimarco and G Iaccarino Journal of Translational Medicine 2009, 7:41 [11]D Diana, B Ziaco, G Colombo, G Scarabelli, A Romanelli, C Pedone, R Fattorusso and LD. D’Andrea Chemistry Eur. J. 2008, 14, 4164 – 4166 [12]Wiesmann C., Fuh G., Christinger H.W., Eigenbrot C., Wells J.A., and De Vos A.M., Cell 91: 695-704 (1997)
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