Identification and evaluation of small molecule inhibitors of the YTHDF protein readers in m6A epitranscriptomic signalling

N6-methyladenosine (m6A) is the most abundant post-transcriptional internal modification of eukaryotic mRNA and a key regulator of RNA metabolism, playing a major role in transcript stability, splicing, nuclear export, translation, and decay. The YTH proteins are the key readers of this modification, with the three cytoplasmic YTHDF paralogues (YTHDF1/2/3) influencing mRNA stability and translation. In recent years, the role of the YTHDF proteins in various types of cancer has been highlighted by multiple publications, displaying that these proteins can be critical drivers of cancer development and aggression. More specifically, work performed by our group shows that depletion of all three YTHDF proteins simultaneously significantly impairs neuroblastoma cell survival and clonogenic potential. Despite their therapeutic potential, no potent small molecule inhibitors capable of targeting all the YTHDF readers without discrimination has been developed. The main objective of this work was to identify and characterise small molecule inhibitors of the YTHDF proteins and evaluate their potential as therapeutic candidates in neuroblastoma. Firstly, the recombinant YTH domains of all YTH proteins were produced and purified, followed by the optimisation of a high-throughput screening platform, based on homogeneous time-resolved fluorescence (HTRF) and the RNA electrophoretic mobility shift assay (REMSA). Using this platform, m6A71, a novel YTHDF inhibitor, was identified and validated. This compound showed little selectivity among the YTH domains of the three YTHDF paralogues, but, unfortunately, also inhibited the YTH domains of the two YTHDC paralogues. Cellular thermal shift assay confirmed target engagement in neuroblastoma cells, where m6A71 treatment led to a reduction in viability, proliferation and clonogenic potential, and an increase in apoptosis. Proteomic profiling revealed that m6A71 disrupts regulatory pathways associated with oxidative phosphorylation, cell growth and survival. In parallel, we launched a medicinal chemistry campaign with the aim of generating analogues of m6A71 with improved characteristics: potency, selectivity profile, and pharmacokinetic parameters. This led to the identification of the essential pharmacophore groups and the identification of some structural changes that could be beneficial to the compound’s potency, but, mainly, to its selectivity in the attempt to abolish the ability to inhibit the YTH domains of the YTHDC proteins. Lastly, following up on a previous screening campaign, a series of novel ebselen analogues was developed and evaluated, but no analogues showed difference in the inhibitory potency compared to ebselen, showing that the covalent binding of this compound is the most integral part of the compound-protein interaction. Additionally, a DNA-encoded library screening was attempted, yielding additional candidate scaffolds, whose activity was, unfortunately, not validated in further biochemical assays. Overall, this work provides a comprehensive discovery pipeline for YTHDF inhibitors, establishing m6A71 as a chemical probe that can be used as an inhibitor of the YTH proteins. The reported findings show that m6A71 has the potential to be developed into a small molecule inhibitor, which could be crucial for therapeutic interventions in cancers involving the m6A signalling pathway.