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DNA binding, DNA Methylation and Cell Toxicity of Estradiol Conjugated DNA Methylating Compounds

Estrogen Receptor Binding :FRET AssayCell Toxicity DNA MethylationDesign of Molecules Acknowledgments

Matthew Powella, Sean Millera, Rigel Kishtona, Charles Kellya, Kelly Mastroa, Astrid Linaresb, Manoj Chelvanambib, Vinayak K. Gorec, Giridhar R. Akkarajub Sridhar Varadarajana,*aDepartment of Chemistry and Biochemistry, University of North Carolina Wilmington, Wilmington, NC, bDepartment of Biology, Texas Christian University, Fort Worth, TX, cQuality Chemical Laboratories, Wilmington, NC.

Abstract Estradiol conjugated compounds that can target estrogen receptor positive cells and produce lethal N3-methyladenine adducts in these cells have been synthesized and investigated1. Prior studies with these DNA methylating compounds had shown that the ability of these molecules to produce N3-methyladenine adducts was crucially dependent on the composition of the linker unit connecting the DNA methylating moiety to the estradiol unit. The DNA binding properties of this class of molecules was investigated using stable non-methylating analogs, and the variation in the DNA binding ability of the compounds was compared to the corresponding DNA methylating ability. Cell toxicity studies were conducted using MCF-7 breast cancer cells in order to determine any correlation between toxicity and DNA methylation and estrogen receptor binding. The results of these studies are presented.

ReferencesCompoundIC50 (nM)Relative Binding AffinityEstradiol0.58100%1b0.9958%2b1.4839%3b1.1152%

Synthesis of Molecules3

Toxicity of compounds was determined in MCF-7 breast cancer cells that overexpress estrogen receptor-.

All methyl-sulfonates were toxic to MCF-7 cells, with compound 2a being the most toxic (EC50 40 M ), followed by 3a, and then 1a.

All sulfone analogues were not toxic to MCF-7 cells.

3Me-A is the predominant adduct formed by all three compoundsCompound 2a produces significantly higher levels of 3-MeA relative to the other compounds.

Possible explanation for the variation in DNA binding of compounds.Franza, Gilberto, and Barry Gold. "The Biological Effects of N3-methyladenine."Journal of Cellular Biochemistry19.2 (2004): 250-257. Web. Aug. & Sept. 2013.Charles KellyKishton, Rigel, Sean E. Miller, Heather Perry, Tera Lynch, Mayur Patel, Vinayak K. Gore, and Giridhar R. Akkaraju. "DNA Site-specific N3-adenine Methylation Targeted to Estrogen Receptor-positive Cells."Biorganic and Medicinal Chemistry195093-5102.

This project was funded in part by: the NC Biotechnology Center Research Grant (#CC6425), the Research Corporation (#2008BRG1214), the GlaxoSmithKline Summer Undergraduate Research Scholarship, and the UNCW CSURF Research Supplies award, UNCW CSURF Undergraduate Fellowships, and the UNCW Charles L. Cahill Award. We also thank Dr. Suzanne Wardell and Dr. Donald McDeonnell for help with gene transcription studies.

Compound 2Compound 1Table 1DNA adduct levels obtained upon reaction of compounds with calf thymus DNAaComp-oundconcMnetropMadduct levelmol adduct/mol DNA3-MeA7-MeG1251615 45194 35501764 17280 361001922 35404 109100100180 10482 572256523 36393 315011131 30253 1010014449 161495 35100100769 42409 3325699 19147 2050922 31204 251001206 23318 15100100173 16420 73Me-lex254599 385185 59508234 542399 10810014280 1250774 73100100657 12714 67MMS50001799 3918067 7650001001239 8818935 502a DNA (1 mM) was reacted with methylating compounds, in the presence or absence of netropsin, for 24 h at RT in 10 mM sodium cacodylate buffer (pH 7.0) containing 10% DMSO.

DNA Binding:Fluorescent Intercalator Displacement Assay

Estrogen Receptor Gene Transcription

Duplex DNA Ethidium bromide intercalated DNA exhibits fluorescence enhancement. EtBr is displaced by ligand and fluorescence diminishes.

Compounds act as SERMS.

Stereoview of compound 3a bound in the minor groove of DNA2

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