Selectivity of Binding to Estrogen Receptors Α and Β as Determined by Fluorescence Polarization

Selectivity of binding to estrogen receptors α and β as determined by fluorescence polarization results and conclusions: ER α compounds tested for estrogenicity 120 50 0 0 1 2 3 50 0 0 • 50 = 54.2 nM • Ki = 47.8 nM IC Cl O Br O O O Permethrin O O • All three compounds used in agriculture and ranching applications • Permethrin also found in household products like tick repellent and pet shampoos flourescence polarization assay -4 -2 0 2 Fenvalerate 180 160 140 140 140 120 120 120 100 100 80 60 -6 -4 -2 0 2 4 100 80 60 -6 -4 -2 0 2 4 80 60 -6 -4 -2 • No binding 0 2 4 log[Fenvalerate] (uM) log [Deltamethrin] (uM) Log[Permethrin] (uM) • No binding • No binding esults and conclusions: ER β PPT DPN PPt Bisphenol A BPA dPn 300 300 300 250 250 250 200 200 200 150 150 150 100 100 100 50 0 0 1 2 3 Polarization (mP) Polarization (mP) Fluorescence polarization (FP) measures the amount of polarized light emitted by a fluorescent molecule when excited by polarized light. The larger the mol• 50 = 54.2 nM • No binding • 50 = 54.2 nM ecule, the slower is rotates in solution • Ki = 47.8 nM • Ki = 47.8 nM and the more polarized light (the higher Permethrin Deltamethrin Fenvalerate polarization) it will have. A small molecule rotates more quickly and will have a lower polarization value. The ability of the compounds shown above to bind to the ER and displace a fluormone (a fluorescent ER ligand) from ER α or • •N • ER β was measured using FP. If the compound did not bind to For all assays, the polarization of the ER fluormone complex is the high polarization standard the ER, the fluormone stayed bound to the ER and the polarizashown in red, while the polarization of the displaced fluormone is the low standard shown in tion of the large complex was high. If the competitor did bind to the ER, it displaced the fluormoe and the polarization of the fluormone alone was low. These assays were conducted in 96 and 384 well plates. A variety of concentrations of the competitors were run with the ER fluormone complex. The polarization was graphed versus the concentration to get a binding curve. Polarization (mP) 50 0 0 1 2 3 50 0 -4 -2 0 2 4 Log[BPA] (uM) log[DPN] (nM) IC IC Log[PPT] (nM) 300 300 250 250 200 200 150 150 240 220 200 180 160 140 100 80 60 -4 -3 -2 -1 0 1 2 3 4 Polarization (mP) 120 Polarization (mP) Polarization (mP) 100 50 -4 -2 0 2 Log[Deltamethrin] (uM) N Log[Permethrin] (uM) N Various synthetic compounds ubiquitous in the environment have been implicated as xenoestrogens, or chemicals that can bind to the Er thus mimicking estrogen. they have been linked to many health problems including various forms of cancer, sexual and developmental defects, and reproductive abnormalities. Because the Er has a large binding pocket, a diverse range of chemicals can act as xenoestrogens. though these chemicals are not as potent in binding as natural estrogen, the total xenoestrogen burden due to their ubiquity is cause for concern. this study also examines the binding of bisphenol A and three pyrethroid pesticides, Permethrin, fenvalerate and deltamethrin, with both Er α and ER β. -6 E R a lp h a F e n va le ra te 6 h o u rs 160 260 Binding modes of SERMs within the ligand binding pocket of ER α (blue) and ER β (green): The effect of the different shape pocket on ER selectivity is shown. 40 • 50 = 31.7 uM • Ki = 14.07 uM Deltamethrin 160 CN 60 Log [BPA] (uM) Polarization (mP) O O 3 E R a lp h a a n d D e lta m e th rin 6 h o u rs Er alpha Permethrin 6 hours Polarization (mP) Br 2 • No binding 180 OH 1 80 log[DPN] (nM) Log[PPT] (nM) • Found in plastics and epoxy coatings used in food containers Polarization (mP) • ER β selective SERM 100 Polarization (mP) CN HO OH 100 100 Polarization (mP) Small molecules have been identified which bind each receptor with differing binding affinities. These selective estrogen receptor modulators (SERMs) hold the potential to be pharmacologically effective in treating diseases specific to one type of estrogen receptor while not affecting the other. For example, ER α and ER β are both present in breast tissue, and the ratio of ER β to ER α is being examined as one indicator in determining the likelihood of successful treatment of breast cancer by certain drugs. Here we use changes in the fluorescence polarization to calculate binding affinities for DPN and PPT, two SERMs, to ER α and ER β. 140 200 N Cl Cl 160 250 200 Pyrethroid Pesticides: Permethrin, Deltamethrin, and Fenvalerate CN 180 300 150 HO O 200 150 HO Ligand Binding Domain of ER α and ER β: The residues that differ in the binding pocket between the two receptors are shown in black. These differences are largely responsible for ER ligand selectivity. 350 250 OH Bisphenol Bisphenol A A dPn IC N DPN OH DPN PPt 300 Polarization (mP) Estrogen’s role in cell growth and proliferation has long been appreciated both in the normal development of secondary sexual characteristics and in diseased states in cancers of the breast, ovaries and uterus. We are beginning to appreci- PPT ate estrogen’s expanded role in maintaining such diverse functions as the skin’s elasticity, the health of the central nervous system, bone density and cardiac health. Estrogen plays out its roles in varied tissues by binding to two major li• ER α selective SERM gand activated nuclear receptors, estrogen receptor alpha (ER α) and estrogen Bisphenol A receptor beta (ER β). Though the ligand binding cavities of the two receptors differ by only two amino acids, the overall degree of homology between ER α and ER β is low. The body uses the receptor selectivity to its advantage by dispersing the receptors in varying ratios to different tissues. PPT R introduction 4 6 100 50 -4 -3 -2 -1 0 1 Log[Fenvalerate] (uM) 2 3 4