Riassunto analitico
The Hippo pathway is an intracellular signaling pathway that represents a possible target for anticancer therapies. Specifically, this pathway is represented by a phosphorylation cascade that determines the sequential activation of kinases: the final event of this pathway is the gene expression of proteins responsible of tissue regeneration. The Hippo pathway activity is regulated by phosphorylation/dephosphorylation processes, but in particular the best-known process concerns the YAP molecule (Yes-Associated Protein). In fact, YAP in its non-phosphorylated form enters in the nucleus to activate the transcription factor TEAD (Transcription Enhancer Associate Domain) forming the YAP-TEAD complex. In this way, the YAP-TEAD complex can induce the expression of various target genes related to cell growth, such as cell adhesion promoters, cell cycle progression promoters and apoptosis inhibitors. When there is a genetic alteration, the cell becomes a cancer cell or alters its metabolism with a loss of function. Therefore, the interest on this altered signaling pathway is focused on the identification of new therapeutic targets and on the development of new compounds acting effectively on this system. One of the most relevant targets is the TEAD protein which has three binding surfaces: a lipidic site binding the myristic/palmitic acid, the interface 2 under-researched nowadays and the interface 3 which represents the binding site for the Ω-loop of YAP. The high hydrophobic character of interface 3 and the lack of binding sites with pocket structure make the drug discovery process difficult. However, after studies carried out at the Drug Discovery and Biotechnology laboratory (Unimore), some compounds derived from the molecule D361-0054 ((4-(3-metossi-4-((2-metilbenzill)ossi)fenill)-3-metil-2,4,5,7-tetraidro-6H-pirazolo[3,4-b]piridin-6-one) have been identified. Subsequently, the biological activity of this library of compounds was evaluated through a cytotoxicity assay conducted on two cell lines: A2780/CP (cisplatin-resistant ovarian cancer cell line) and HT29 (5FU-resistant colorectal adenocarcinoma cell line). At the end of these experiment three interesting compounds emerged: YAPT-12, YAPT-21 and YAP-26 in part. The aim of my thesis is to confirm the cell growth reduction activity of these compounds by the selectively inhibit capacity the transcriptional action of TEAD. For this purpose, I have carried out the following activities:
1.Purification of compounds by chromatography; 2. Study of the cell growth inhibition effect of purified inhibitors and other compounds with known activity on the Hippo pathway (chemical probe) through cytotoxicity assay (Crystal Violet assay); 3. Study of the inhibitor’s effects on the function of TEAD by RT-PCR and Western Blot assay.
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