Synthesis and in-vitro evaluation of YTH reader protein inhibitors for neuroblastoma treatment

Neuroblastoma, a common pediatric cancer, often presents with high heterogeneity and poor prognosis in high-risk cases. This thesis explores the potential of targeting the YTHDF1 protein, an m6A RNA reader, for neuroblastoma treatment. The aim of my thesis work was a multidisciplinary approach in the study of molecules related to m6A71, a compound identified through in-silico screening for its high affinity to YTHDF1. Seven analogs were designed to optimize interactions with the YTHDF1 domain, incorporating variations in the chain length and terminal aromatic moieties. The compounds were synthesized, purified and structurally characterized by extensive NMR and high-resolution ESI-Ms analysed under the supervision of Prof. Ines Mancini in the Laboratory of Bioorganic Chemistry, Department of Physics at Trento university. The binding affinities and inhibitory potentials of these analogs were evaluated through a series of biochemical assays, including Homogeneous Time-Resolved Fluorescence (HTRF) and RNA Electrophoresis Mobility Shift Assay (REMSA). The compounds demonstrated varying degrees of inhibition, with two of them (3 and 4, with S-alkyl functionalized chains) showing enhanced activity over m6A71. ADME prediction indicated favorable pharmacokinetic profiles for all analogs, mainly for compounds 5 and 7 having an N-alkyl functionalized chain and a methoxyl group, respectively. Cell viability assays on CHP134 neuroblastoma cell lines further validated the biological activity of the top-performing compounds, highlighting their impact on neuroblastoma cell proliferation. These biological experiments were carried out at the at the Cellular, computational and Integrative Biology (CIBIO) Department of Trento University, under the supervision of prof. Alessandro Quattrone. Starting from the qualitative structure activity relationship study performed in this thesis work, future investigations should focus on comprehensive cell-based studies and optimization of the lead compounds to enhance their therapeutic efficacy and bioavailability. The insights gained from this research contribute to the broader understanding of m6A modifications in cancer and specifically underscores the significance of m6A RNA modifications in neuroblastoma pathogenesis, presenting a promising approach for targeted therapy through YTHDF1 inhibition.