title: Tandem virtual screening targeting the SRA domain of UHRF1 identifies a novel chemical tool modulating DNA methylation.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Panepistimiopolis Zografou, Greece; PharmaGnose S.A., Papathanassiou 24, Chalcis 34100, Greece.
authors.forename: Vassilios
authors.initials: V
authors.lastname: Myrianthopoulos
authors.affiliation: Centre de Recherche en Cancérologie Nantes-Angers, INSERM, U892 and LaBCT, Institut de Cancérologie de l'Ouest, Nantes, France.
authors.forename: Pierre Francois
authors.initials: PF
authors.lastname: Cartron
authors.affiliation: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Zita
authors.initials: Z
authors.lastname: Liutkevičiūtė
authors.affiliation: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Saulius
authors.initials: S
authors.lastname: Klimašauskas
authors.affiliation: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Daumantas
authors.initials: D
authors.lastname: Matulis
authors.affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS, INSERM, Université de Strasbourg, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Bronner
authors.affiliation: CNRS UMR 7272, Institut de Chimie, Université de Nice-Sophia Antipolis, Nice, France.
authors.forename: Nadine
authors.initials: N
authors.lastname: Martinet
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Panepistimiopolis Zografou, Greece. Electronic address: mikros@pharm.uoa.gr.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Mikros
mesh_terms: D022762:CCAAT-Enhancer-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D006801:Humans; D061986:MCF-7 Cells; D008958:Models, Molecular; D015394:Molecular Structure; D000072417:Protein Domains / Q000187:drug effects; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D044767:Ubiquitin-Protein Ligases
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D054852:Small Molecule Libraries; C406364:UHRF1 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: Compound NSC232003; DNA demethylation; Epigenetics; Ligand-based screening; QM-docking; Structure-based screening