N6-methyladenosine (m6A) is one of the most common reversible RNA modifications, conserved in all eukaryotes from yeast to humans. It is mainly observed in the 3’UTR, the 5’UTR and near the Transcription Start Sites (TSS), inside a consensus sequence identified as [G/A/U][A/G][m6A-C][U/A/C. The m6A modification is involved in the regulation of many different biological phenomena, starting from development, stemness, adipogenesis, spermatogenesis and many others. Its misregulation has been found to be the driving force of different pathologies, most importantly has been widely described as having a major role in carcinogenesis. The methylation machinery is composed of methyltransferases, erasers and readers, the latter recognizing the modification upon target RNAs and guiding them to different fates. The proteins that act as readers of the methylation belong to the YTH-domain family, with four cytoplasmic isoforms (YTHDF1-3;YTHDC2), involved in the regulation of translation and decay of m6A-containing mRNAs, and one nuclear isoform (YTHDC1), involved in alternative splicing. The YTH domain proteins have been identified to have major roles in cancer development, mainly causing misregulation of cancer-related RNA targets. However, its role in the development of the pediatric tumor Neuroblastoma was still largely unknown. In my PhD project I focused on characterizing the role of the YTHDF1 protein in Neuroblastoma development, reporting that YTHDF1 supports Neuroblastoma aggressiveness by enhancing its proliferation, tumour-sphere forming ability and its invasiveness. To address whether modulating the YTHDF1-mediated regulation of m6A transcripts signaling through YTHDF1 could represent a new pharmacological strategy target in NB, I performed for the first time an High Throughput Screening aiming to interfere with the RNA binding ability of the YTH domain, testing a 2000 compound library of small molecules. Among others, only one molecule was identified for as a strong binder, and consequently demonstrated its direct interaction with the binding pocket of the YTHD by different biochemical approaches. For the first time has been proposed an encouraging proof-of-concept of m6A readers as potential pharmacological targets for the treatment of NB, that led to the repurposing of an already FDA approved drug as an inhibitor for this novel target.
Identification and validation of small molecules targeting the YTH domain / Micaelli, Mariachiara. - (2020 Oct 19), pp. 1-108.
|Titolo:||Identification and validation of small molecules targeting the YTH domain|
|Anno di pubblicazione:||2020-10-19|
|Struttura:||Dipartimento di Biologia Cellulare, Computazionale e Integrata - CIBIO|
|Corso di dottorato:||Biomolecular Sciences|
|Tesi in cotutela:||no|
|Settore Scientifico Disciplinare:||Settore BIO/11 - Biologia Molecolare|
|Digital Object Identifier (DOI):||10.15168/11572_276926|
|Appare nelle tipologie:||08.1 Tesi di dottorato (Doctoral Thesis)|
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