|Tipo di tesi||Tesi di laurea magistrale|
|Titolo||Studi di virtual screening per l'identificazione di nuovi inibitori allosterici della proteina chinasi EGFR|
|Titolo in inglese|
|Struttura||Dipartimento di Scienze della Vita|
|Corso di studi||CHIMICA E TECNOLOGIA FARMACEUTICHE (D.M. 270/04)|
|Data inizio appello||2017-11-06|
|Disponibilità||Accessibile via web (tutti i file della tesi sono accessibili)|
Le proteine chinasi fanno parte della famiglia delle chinasi e sono responsabili della fosforilazione delle proteine, uno dei principali meccanismi di regolazione cellulare. Una sua alterazione può portare allo sviluppo di numerose patologie tra cui tumori, ipertensione, malattie infiammatorie ed autoimmuni.
Protein kinases are part of the large kinase family and are responsible for protein phosphorylation. Protein phosphorylation is one of the major mechanisms of cellular regulation, and its alteration can lead to the development of many pathologies including tumors, hypertension, inflammatory and autoimmune diseases. The focus of my thesis work was the study of the kinase domain of the Epidermal Growth Factor Receptor (EGFR). This protein contributes, along with other factors, to the progression of non-small cell lung cancer (NSCLC). This type of cancer is very invasive and aggressive, leading to a quickly fatal outcome shortly after diagnosis (8-12 months). EGFR inhibitors are currently available in the clinic.. However, the rapid onset of resistance by tumor cells, particularly when these drugs are administered as monotherapy, represents a major limitation. It has been observed that this phenomenon is due to the development of point mutations in the EGFR receptor, where the most frequent are L858R (leucine to arginine) and T790M (threonine to methionine). These mutations lead to drug resistance by significantly increasing the binding affinity for ATP or by activating the receptor even in the absence of growth factors. Research and development of allosteric inhibitors (molecules that bind to an allosteric pocket close to the ATP binding site) of EGFR can potentially provide biologically active compounds with higher selectivity compared to ATP-competitive inhibitors. These compounds are also less susceptible to drug resistance. My thesis work focused on identifying new allosteric inhibitors selective for mutated forms T790M and/or T790M/L858R of EGFR. This research was carried out through virtual screening studies, using several crystallographic structures of the kinase domain of EGFR and large databases (about 2.5 million molecules) of commercially available compounds. Virtual screening was performed using a molecular docking protocol trough Glide software. I used as a reference the recent crystallographic structure of EGFR in complex with the allosteric ligand EAI001 (PDB code: 5d41). After an initial docking protocol setup procedure, other EGFR crystal structures carrying mutations of our interest (PDB code: 3w2r, 2rgp and 3w32) were included in the study. The compounds were filtered based on the docking score parameter and binding interactions. The most promising compounds were subjected to a more thorough analysis of the ligand-protein complexes using the BEAR program developed within the laboratory where I did my thesis project. This procedure allows to calculate more accurately the binding energies of these complexes. All the studied compounds were evaluated on wild-type structures, either with or without ATP, and on crystallographic structure with mutations T790M and T790M/L858R to predict their potential selectivity towards the mutant forms. The compounds were filtered by cross-referencing the data obtained from the various procedures and then visually analyzed. To facilitate visual inspection of the ligand-protein complexes, ligands were grouped according to the 2D chemical scaffold structure using the Canvas software. Each group was then thoroughly analyzed to select the most promising and representative compounds. At the end of my thesis work, 14 compounds were selected to be purchased and submitted to in-vitro biological evaluation.