Scognamiglio, Pasqualina Liana (2011) Identification of peptide inhibitors of protein-protein interactions as new potential drugs. [Tesi di dottorato] (Unpublished)
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|Item Type:||Tesi di dottorato|
|Uncontrolled Keywords:||protein-protein interactions|
|Date Deposited:||06 Dec 2011 11:35|
|Last Modified:||17 Jun 2014 06:04|
The innovative concept that proteins exert their function by establishing intricate contact networks rather than acting as independent entities is of outstanding importance both in the comprehension of molecular mechanisms underlying biological processes and in modern drug discovery. Protein-protein interactions are the ensemble of fine tuned recognition events that take place at protein surfaces. These contacts frequently involve large protein surface areas, which comprise several contact sites but, alternatively, they can be mediated by “hot spots” represented by few crucial amino acids. Many studies have highlighted the role of peptide molecules as powerful tools for the characterization and the regulation of these interactions. Peptides are particularly suitable as models for proteins that are not fully folded in their isolated states but that achieve conformational stability upon the formation of complexes with other partners. The characterization of the structural determinants of protein-protein recognition represents an important step for the development of molecular entities able to modulate these interactions. The identification of compounds able to modulate protein-protein interactions represents a major goal for modern drug discovery. Both structure-based design and screening of combinatorial collections have been successfully applied, in this instance often small peptides and peptidomimetics have been selected as inhibitors of protein complexes. In many cases, the lack of sufficient structural information on protein interacting regions renders the combinatorial approach the most suitable method, but the use of completely random peptide libraries often produces redundant chemical species that complicate the deconvolution phases of screening and puzzles the way to the identification of active compounds. Thus, in order to avoid the synthesis of too large combinatorial libraries in two case-studies during this doctoral thesis a simplified approach was followed in it libraries of short peptides, made of a small subset of amino acids were prepared and screened. This approach has been applied to the identification of inhibitors of the protein complex between PED/PEA15 and PLD1 that is involved in the molecular mechanisms of insulin resistance occurring in type 2 diabetes. This study led to the identification of several short peptide inhibitors rich in aromatic and H-donor donor features through the employment of ELISA and SPR binding assay. These short sequences will be converted in small organic molecules following medicinal chemistry rules and actually can be considered as interesting starting scaffold for the design of new therapeutic agents in type 2 diabetes. On the other hand, the screening of small but focused libraries is increasingly used to target specific systems. This method has been applied to the identification of mimicking peptides of KIR region of SOCS1 that is involved in JAK2 recognition. This interaction plays a key role in molecular mechanisms of psoriasis. After the identification of a shorter SOCS1-KIR domain, through an Ala-scanning investigation, focused peptide library in which non essential residues were randomized in a simplified approach were built and assayed. This screening led to the selection of new peptides more effective than natural sequences; indeed one decapeptide resulted binds to JAK2 with a dissociation constant in the nanomolar range and was able to block STAT1 phosphorylation. Actually cellular and in vivo experiments are ongoing in order to investigate its therapeutic applicability. Rational approaches for the identification of protein interacting regions provide deeper insights into the recognition molecular mechanism This rational approach has been applied to other two projects in this thesis. The complex between proteins CypA and AIF is involved in apoptotic cell death, but at structural level only a docked theoretical model is available. In this model few molecular contacts between proteins are detectable, thus a set of sequences were designed in order to map crucial residues of AIF domains involved in the interaction with CypA. The selective disruption of the CypA-AIF complex with these peptides mimicking the protein surfaces was analysed by competition and direct binding SPR experiments. These experiments allowed to identify an active sequence that, once properly modified, could be as a potential suppress the pro-apoptotic action of AIF. This minimalistic approach was successfully applied to another protein complex IB-α/Tat, two proteins involved in the replication of HIV-1. Here IB-α-based peptides were designed and tested in SPR assays. Also in this project the interacting region of IB-α was restricted to a shorter region (26 residues) representing a promising starting point for the development of molecules able to block HIV-1 infection. The characterization of IB-α peptides has provided novel insights into the intrinsic properties of IB-α fragments and structural determinants of IB-α/Tat recognition. In the investigation of protein folding determinants and then in the design of specific modulators of proteic folding and biological function, the protein reassembly through host-chemistry strategy could represent a valid approach. During a period of six months spent at Cambridge University, a protein splitting approach was applied to the small a regulator protein Ubiqutin. Two dissected protein regions were obtained by chemical synthesis: at the C-terminal fragment (residues 47-76) was inserted a glycine as a spacer and then a tryptophan indole group, for the binding to the macrocycle CB; instead at the C-terminus of N-terminal fragment (residues 1-46), a cysteine was inserted to allow the connection with "methylviologen", which has an aromatic ring strongly depleted of electrons for the formation of the ternary complex with CB. The preliminary synthetic results obtained resulted promising for the future development of this project that will be structural studies of individual fragments and the structural and functional characterization of the ternary complex.
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