|Tipo di tesi||Tesi di laurea magistrale|
|Titolo||Sviluppo di strumenti biofisici per la caratterizzazione della proteina ricombinante hTEAD4 YBD e gli inibitori in grado di dissociare il complesso hTEAD4:YAP|
|Titolo in inglese||Development of biophysical tools to characterize recombinant hTEAD4 YBD and disrupters of hTEAD4:YAP complex|
|Struttura||Dipartimento di Scienze della Vita|
|Corso di studi||Chimica e tecnologia farmaceutiche (D.M. 270/04)|
|Data inizio appello||2022-10-27|
|Disponibilità||Embargo di 3 anni|
|Data di rilascio||2025-10-27|
Lo sviluppo dei farmaci è ispirato dalla necessità di nuove terapie per le neglected disease o dalla necessità di limitare le interazioni tra farmaci e gli effetti collaterali migliorando la qualità di vita del paziente.
The Drug Discovery process is inspired by the necessity of therapeutics for neglected diseases, or the need to limit drug interactions and side effects improving patience’s quality of life. Once a target is validated as involved in the disease progression, the identification of a druggable pocket is required to proceed with hits identification. In cancer research there has been a growing interest towards Hippo Pathway, an intracellular kinase signalling pathway whose deregulation is considered a cancer hallmark. When active, the kinase cascade determines the phosphorylation of YAP (Yes-associated protein), causing its cytoplasmatic retention and proteasome degradation. Conversely, when the pathway is inactive, the non-phosphorylated form of YAP migrates into the nucleus binding, thus activating, the transcriptional enhanced associated domain (TEAD1-4) transcription factors leading to the expression of cell survival and proliferation related genes. For this reason, the disruption of hTEAD4:YAP complex represents a promising target for a novel class of anticancer drugs. In the last decades, the complex formation has been investigated and the crystal structure solved showing that the C-terminal YBD of hTEAD interacts with the N-terminal region of YAP via three interfaces. The interface 3 of hTEAD4 interacting with the Ω-loop motif of YAP has been demonstrated to be crucial for the complex stability. A lack of knowledge about deep understanding of structural features and assays to study drug:target interactions are at the basis of a slow hit and lead identification achievement. Therefore, our laboratory was involved in a research project with the aim of developing novel and simplified assays to detect hit/lead drug target interactions. In this context, the aim of my thesis was to consolidate the structural knowledge about TEAD YBD and to set-up exploratory assays to study its interactions with substrates and PPI ligands. To reach these objectives I developed the following activities: i. optimization of the expression of recombinant hTEAD4 YBD (aa 217- 434) protein in E.Coli and set up of an optimal purification protocol. ii. optimization of the expression of recombinant hTEAD4 YBD double mutant (aa 217- 434 S222C, E434C) protein in E.Coli and set up of an optimal purification protocol. iii. structural characterization of the obtained protein with circular dichroism (CD). iv. double mutant hTEAD4 YBD conjugation with 5’-thiololigonucleotides 20mer coupled with dithiodipyridine (DTDP) for optical tweezer studies. v. design of fluorescence-based assay to examine disruption of hTEAD4 YBD:YAP complex. In conclusion, this work has led to the optimization of a protocol to express and purify the recombinant hTEAD4 YBD wild type and double mutant in E.Coli ArcticExpress cells (yield 20mg/L of cell culture). Its correct sequence was confirmed through LC/MS analysis while the secondary structure was analysed through CD. The double mutant was successfully conjugated with activated oligonucleotide for optical tweezer studies to discover dynamic pockets, however the complex detection is to be optimized. Furthermore, a FRET assay and fluorescence anisotropy assay were designed, and preliminary results have been obtained demonstrating hTEAD4 YBD and a 15aa YAP-like peptide complex formation. However, they need to be further optimized and validated with a positive control.