In Quest of Chemical Probes for DNA Methylation Reader Proteins: Nucleoside and Dimer Analogues of 5‐Methylcytosine Interact with MBD2

: DNA methylation modulates gene expression without altering the DNA sequence. Aberrant DNA methylation patterns characterise a wide range of diseases, from cancer to neurological disorders, and serve as validated therapeutic targets and diagnostic tools. Among the proteins involved in DNA methylation regulation, the human reader Methyl-CpG Binding Domain 2 protein (MBD2) exhibits a strong preference for methylated DNA and initiates signalling cascades predominantly resulting in gene repression. The full description of this mechanism is still to be elucidated. Here, we developed original chemical compounds to interfere with MBD2 and its interaction with DNA. Through convergent chemical pathways, we modified 5-methylcytosine at position N4 and synthesised the corresponding deoxynucleosides and CpG dimers. We tested 70 compounds in two screening assays to assess their interaction with MBD2 and their ability to disrupt the MBD2/DNA complex. Remarkably, substituting modified nucleobases into CpG dimers significantly boosted the biological activity. Furthermore, we investigated the impact of the configuration (D/L) of the modified nucleosides and identified four dimers (5d, 7e, 15e and 16e) able to disrupt the MBD2/DNA complex, highlighting the advantage of the L-configuration. Importantly, NMR experiments confirmed their interaction with amino acids of MBD2 involved in methylated DNA binding.