Tandem virtual screening targeting the SRA domain of UHRF1 identifies a novel chemical tool modulating DNA methylation.
Ubiquitin-like protein UHRF1 that contains PHD and RING finger domain 1 is a key epigenetic protein enabling maintenance of the DNA methylation status through replication. A tandem virtual screening approach was implemented for identifying small molecules able to bind the 5-methylcytosine pocket of UHRF1 and inhibit its functionality. The NCI/DTP small molecules Repository was screened in silico by a combined protocol implementing structure-based and ligand-based methodologies. Consensus ranking was utilized to select a set of 27 top-ranked compounds that were subsequently evaluated experimentally in a stepwise manner for their ability to demethylate DNA in cellulo using PCR-MS and HPLC-MS/MS. The most active molecules were further assessed in a cell-based setting by the Proximity Ligation In Situ Assay and the ApoTome technology. Both evaluations confirmed that the DNMT1/UHRF1 interactions were significantly reduced after 4 h of incubation of U251 glioma cells with the most potent compound NSC232003, showing a 50% interaction inhibition at 15 μM as well as induction of global DNA cytosine demethylation as measured by ELISA. This is the first report of a chemical tool that targets UHRF1 and modulates DNA methylation in a cell context by potentially disrupting DNMT1/UHRF1 interactions. Compound NSC232003, a uracil derivative freely available by the NCI/DTP Repository, provides a versatile lead for developing highly potent and cell-permeable UHRF1 inhibitors that will enable dissection of DNA methylation inheritance.
['Myrianthopoulos|Vassilios|V|', 'Cartron|Pierre Francois|PF|', 'Liutkevičiūtė|Zita|Z|', 'Klimašauskas|Saulius|S|', 'Matulis|Daumantas|D|', 'Bronner|Christian|C|', 'Martinet|Nadine|N|', 'Mikros|Emmanuel|E|']
D022762:CCAAT-Enhancer-Binding Proteins D019175:DNA Methylation D004305:Dose-Response Relationship, Drug D004353:Drug Evaluation, Preclinical D006801:Humans D061986:MCF-7 Cells D008958:Models, Molecular D015394:Molecular Structure D000072417:Protein Domains D054852:Small Molecule Libraries D013329:Structure-Activity Relationship D044767:Ubiquitin-Protein Ligases
Epigenetics Compound NSC232003 DNA demethylation Ligand-based screening QM-docking Structure-based screening