Abstract
DNA methylation can mediate epigenetic silencing of tumor suppressor and cancer protective genes. The protein ubiquitin-like containing PHD and ring finger domains 1 (UHRF1) is an essential component in cells for DNA methylation maintenance. The SET- and RING-associated (SRA) domain of UHRF1 can bind hemimethylated DNA, and mediate recruitment of DNA methyltransferases to copy the methylation pattern to the newly synthesized daughter strand. Loss of UHRF1 function can lead to demethylation and re-expression of epigenetically silenced tumor suppressor genes and can reduce cancer cell growth and survival. We created a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay to screen for inhibitors capable of disrupting the interaction between the UHRF1-SRA domain and hemimethylated DNA. Using this assay (Z’ factor of 0.74 in 384-well format) we screened the Library of Pharmacologically Active Compounds (LOPAC) for UHRF1-SRA inhibitors, and validated 7 hit compounds. These compounds included the anthracycline derivatives idarubicin and mitoxantrone, which are commonly used chemotherapeutic drugs known to mediate cytotoxicity by acting as class II topoisomerase (TOP2) poisons. In a panel of additional known topoisomerase poisons, only the anthracycline derivatives showed dose responsive inhibition of UHRF1-SRA. Additionally, mitoxantrone and doxorubicin showed dose-responsive global DNA demethylation and demonstrated a synergistic growth inhibition of multiple cancer cell lines when combined with the DNA methyltransferase (DNMT) inhibitor decitabine. These data validate a novel TR-FRET assay for identification of UHRF1 inhibitors, and revealed unexpected epigenetic properties of commonly used chemotherapeutic drugs that showed synergistic cytotoxicity of cancer cells when combined with a demethylating agent.
Highlights
Cytosine methylation at cytosine-phospho-guanine (CpG) dinucleotides is a common epigenetic alteration in virtually all human somatic tissues
We have developed a robust time-resolved fluorescence resonance energy transfer (TR-FRET) assay to identify compounds that inhibit the binding of the SET- and RING-associated (SRA) domain of UHRF1 to hemimethylated DNA
We developed a TR-FRET assay to measure binding of the SRA domain of UHRF1 to various DNA substrates (Figure 1A)
Summary
Cytosine methylation at cytosine-phospho-guanine (CpG) dinucleotides is a common epigenetic alteration in virtually all human somatic tissues. Aberrations in DNA methylation patterns are found in most human cancers; hypermethylation of gene promoters has been causally implicated in epigenetic repression of key tumor suppressor genes [1]. These hypermethylation events can be stably maintained, and be subject to clonal selection during cancer progression [2, 3]. The only FDA-approved drugs targeting the DNA methylation machinery are the nucleoside analogs azacitidine and decitabine Both drugs inhibit DNA methyltransferases through incorporation into genomic DNA in lieu of a native cytosine, leading to covalent trapping and degradation of DNMTs [4]. A nonnucleoside inhibitor of the methylation machinery would achieve the same therapeutic effect but with fewer side effects, and for this reason, multiple efforts for developing non-nucleoside DNMT inhibitors are currently underway [6,7,8,9,10]
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