In the quest of chemical probes for MBD2: nucleoside and dimer analogues of 5-methylcytosine interact with hMBD2

DNA methylation is a well-known epigenetic modification that modulates gene expression without altering the DNA sequence of the genome. Aberrant DNA methylation patterns characterize a wide range of diseases, from cancer to neurological disorders, and serve as both validated therapeutic targets and diagnostic tools. Among the proteins involved in the regulation of DNA methylation are the DNA methylation reader proteins (MBPs), which bind methylated DNA and initiate a signalling cascade involving different protein complexes, predominantly resulting in gene repression, thereby playing a significant role in gene regulation. Notably, the Methyl-CpG Binding Domain 2 (MBD2) protein exhibits a strong preference for methylated DNA and is implicated in tumour formation and maintenance. Here, we developed chemical compounds designed to interfere with MBD2 and its interaction with DNA. Building on our previous findings, we chemically modified 5-methylcytosine at position N4 and synthesized the corresponding deoxynucleosides and CpG dimers through convergent chemical pathways. We tested 60 compounds in two optimized screening assays to assess their interaction with MBD2 and their ability to disrupt the DNA-MBD2 complex. The incorporation of the modified nucleobase into the CpG dimers significantly enhanced the biological activity of these compounds. Furthermore, we investigated the impact of the configuration (D/L) of the modified nucleoside on their activity. NMR experiments confirmed that the compounds interact with the amino acids involved in DNA binding. This approach has led to the identification of the first compounds capable of interfering with the MBD domain of MBD2 and disrupting its interaction with DNA.