Abstract
Abstract Aberrant DNA methylation of gene promoters can be involved in silencing tumor suppressor genes in cancer. The protein ubiquitin-like containing PHD and ring finger domains 1 (UHRF1) is an essential component in the cellular machinery for DNA methylation maintenance and can couple DNA methylation and other epigenetic histone modifications. UHRF1 is thought to bind newly synthesized hemimethylated DNA and recruit DNA methyltransferases to copy the methylation pattern to the daughter DNA strand. Interfering with the ability of UHRF1 to bind methylated DNA can lead to the re-expression of epigenetically silenced genes including tumor suppressors, which positions UHRF1 as a target for anticancer drug development. We have created a high-throughput, time-resolved fluorescence resonance energy transfer (TR-FRET)-based assay to screen for inhibitors capable of disrupting the interaction between the UHRF1 SRA domain and hemi-methylated DNA. The assay measures the degree of TR-FRET between a 5′-fluorescein (FAM) labeled hairpin oligonucleotide and a terbium-conjugated antibody bound to the 6xHis-tag of the UHRF1_SRA domain polypeptide. In 384-well plate format, the assay achieved a Z' factor of 0.74. We successfully screened the Library of Pharmacologically Active Compounds (LOPAC) for inhibitors of UHRF1_SRA binding to hemimethlylated DNA, and validated 6 hit compounds in a dose-response series. These include: aurintricarboxylic acid, NF449, 6-hydroxy-DL-DOPA, NF023, PPNDS and mitoxantrone. Additionally, we observed a decrease in methylation one week after treatment of DU145 cells with mitoxantrone at a concentration near its IC50 from the TR-FRET binding assay. This suggests that mitoxantrone, a known DNA topoisomerase II poison and general DNA intercalator, may interfere with methylation maintenance. In summary, we have established a novel method to identify small molecule inhibitors of the UHRF1-methylated DNA interface, and have shown that one hit compound, mitoxantrone, can perturb methylation maintenance near its IC50 for inhibition of SRA domain binding to methylated DNA. Citation Format: David A. Walker, Nicolas Wyhs, Hugh Giovinazzo, Srinivasan Yegnasubramanian, William G. Nelson. Development of a high-throughput screening assay to identify UHRF1 inhibitors via time-resolved fluorescence resonance energy transfer (TR-FRET). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5390. doi:10.1158/1538-7445.AM2014-5390
Published Version
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