Research ArticleDRUG DESIGN

Design of pathway preferential estrogens that provide beneficial metabolic and vascular effects without stimulating reproductive tissues

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Sci. Signal.  24 May 2016:
Vol. 9, Issue 429, pp. ra53
DOI: 10.1126/scisignal.aad8170
  • Fig. 1 Structures and molecular and binding properties of E2 and four PaPEs, and a model of E2 and PaPE-1 binding to ERα-LBD.

    (A) MW is molecular weight; cLogP is log10 of the calculated octanol-water partition coefficient; volume is molecular volume; and polar surface area is a measure of compound polarity. All values were obtained using ChemBioDraw Ultra (version 13.0.0.3015). RBA values were determined by competitive radiometric binding assays (18). E2 is set at 100 on both ERs. Inhibition constant (Ki) values were calculated as Ki = Kd (for E2) × (100/RBA), where Kd of E2 is 0.2 nM (ERα) and 0.5 nM (ERβ). Values are average of three to four determinations with coefficient of variation <0.3. (B) Computational model comparing PaPE-1 and E2 in the ligand-binding pocket of ERα. The model of ERα + E2, based on a crystal structure [Protein Data Bank (PDB) ID: 1GWR], has E2 and helical elements shown in silver gray and the pocket volume contour in slate blue. The model for PaPE-1 was generated from the ERα + E2 structure by progressive transformation of the ligand structure from E2 to PaPE-1, partnered with progressive minimization of the ligand and the LBD. The resulting positions of the PaPE-1 ligand and hydrogen-bonding residues are shown in yellow.

  • Fig. 2 Comparison of regulation of gene expression, cell proliferation, and pathway signaling by PaPE-1 and E2.

    (A) PaPE-1 preferentially activates extranuclear-initiated genes (LRRC54) over nuclear genes (PgR) compared to E2 in MCF-7 cells. Cells were treated with control vehicle (Veh), E2, or PaPE-1, and gene expression was monitored by quantitative polymerase chain reaction (qPCR) (n = 3 biological replicates). (B) MCF-7 cells were pretreated with ICI 182,780 (ICI) and then treated with vehicle, E2, or PaPE-1 in the presence or absence of ICI 182,780. RNA was isolated and subjected to qPCR analysis for the indicated genes (n = 3 biological replicates). (C) MCF-7 cells transfected with siControl (siCtrl), siERα, or siGPR30 were treated with vehicle, E2, or PaPE-1. RNA was isolated and subjected to qPCR analysis (n = 3 biological replicates). (D) MCF-7 cells were treated with vehicle, E2, or the indicated concentrations of PaPE-1 for 6 days, and proliferation was monitored by WST assay (n = 4 biological replicates). OD, optical density. (E) PaPE-1 selectively activated mTOR and MAPK signaling in MCF-7 cells. Left: Cells were treated with control vehicle (V) or the indicated concentrations of E2 or PaPE-1 for 15 min (upper panel) and 45 min (lower panel), and Western blots were done to assess the activation of signaling proteins and phosphorylation (p) of Ser118 in ERα. Total ERα was monitored, and total ERK2 was used as a loading control (n = 3 biological replicates). Right: Total amount of these factors in cells receiving the indicated treatments (n = 3 biological replicates). (F) PaPE-1 induces interaction between ERα and RAPTOR. MCF-7 cells were treated with E2 or PaPE-1. Cells were crosslinked and incubated with ERα and RAPTOR antibodies overnight, and PLA was performed. Quantitation of signal intensities is shown in the graph (n = 3 biological replicates). Two-way analysis of variance (ANOVA), Bonferroni posttest, *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 50 μm.

  • Fig. 3 PaPE-1 and E2 regulate common as well as distinct groups of genes in MCF-7 cells.

    (A) Cells were treated with E2 or PaPE-1 for 4 and 24 hours. RNA was isolated, and RNA-seq was performed. Regulated genes are considered to be those with P < 0.05 and expression fold change >2 (n = 2 biological replicates). (B) PaPE-1–mediated gene expression changes are sensitive to mTOR and MAPK pathway inhibitors. Effect of mTOR and MAPK inhibitors on gene regulation by E2 and PaPE-1 in MCF-7 cells is shown. Cells were pretreated with PP242 or AZD6244 before treatment with E2 or PaPE-1 in the presence or absence of inhibitors. RNA was isolated, and RNA-seq was performed. (P < 0.05, fold change >2; n = 2 biological replicates). (C) PaPE-1 does not induce recruitment of ERα or ERK2 to chromatin but stimulates recruitment of RNA pol II. MCF-7 cells were treated with E2 or PaPE-1. ChIP-seq was performed for the indicated proteins. UCSC (University of California, Santa Cruz) genome tracks of cistromes in the presence of E2 or PaPE-1 are shown (right panel) (n = 3 biological replicates that were pooled). bp, base pairs.

  • Fig. 4 Unlike E2, PaPE-1 does not change uterus or thymus weight and does not induce mammary gland ductal branching, but like E2, PaPE-1 reduces mammary gland adipocyte area.

    (A) PaPE-1 does not affect uterus or thymus weight. C57BL/6 mice were ovariectomized and then were given daily subcutaneous injections of E2 or were implanted with PaPE-1 pellets for 4 days. Weights of uterus and thymus were monitored (n = 8 mice per treatment). Scale bars, 5 mm. (B) PaPE-1 stimulates minimal mammary ductal elongation but it greatly reduces adipocyte size in mammary gland. Ovariectomized C57BL/6 mice were implanted with E2 or PaPE-1 pellets. Whole-mount stain (scale bars, 1 mm) and H&E stain (scale bars, 200 μm) of mammary gland are shown. (C) Mammary gland adipocyte area was calculated from the H&E images (n = 4 mice per treatment). A one-way ANOVA model was fitted to assess the contribution of ligand treatment on uterine weight, thymus weight, or mammary gland adipocyte area. When the main effects were statistically significant at α = 0.05, pairwise t tests with a Bonferroni correction were used to identify the treatments that were significantly different from each other. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 5 Like E2, PaPE-1 reduces the increase in body weight after ovariectomy and reduces adipose stores and blood triglyceride concentrations.

    (A) PaPE-1 is effective in normalizing body weight after ovariectomy. Ovariectomized C57BL/6 mice were implanted with pellets containing E2 or PaPE-1 or vehicle control for 3 weeks (n = 8 mice per group). Animals were on a normal chow diet. Two-way ANOVA, Bonferroni posttest, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, comparing treatments to vehicle. (B) Food consumption for each mouse from (A) was monitored weekly. (C) Body composition for each mouse from (A) was monitored using EchoMRI at the end of 3 weeks. One-way ANOVA, Newman-Keuls posttest, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (D) H&E staining of perigonadal adipose tissue (AT). Images are representative of eight mice per group. Scale bar, 500 μm. (E) Weights of various adipose tissue depots after 3 weeks of control vehicle or ligand exposure. (F) Triglycerides were measured in the blood of animals (n = 6 mice per group) at the end of 3 weeks of vehicle, E2, or PaPE-1 treatment. (G) H&E staining (upper two rows; scale bars, 100 μm) and Oil Red O staining (lower panel; scale bar, 20 μm) of the liver after 3 weeks of treatment. Images are representative of eight mice per group. (H) Gene expression analysis of SREBP1c and FASN in the liver at 3 weeks (upper panel) (n = 12 mice per group), and time course of FASN and ACACA expression in the livers of E2- and PaPE-1–treated mice (n = 3 mice per group). A two-way ANOVA model was fitted to assess the contribution of ligand treatment and time of ligand treatment on body weight, food consumption, or gene expression. A one-way ANOVA model was fitted to assess the contribution of ligand treatment on fat mass, lean mass, water mass, triglyceride concentrations, weight of different fat depots, or gene expression. When the main effects were statistically significant at α = 0.05, pairwise t tests with a Bonferroni correction were used to identify the treatments that were significantly different from each other. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 6 Gene regulation and signaling pathway activations by PaPE-1 and E2 in tissues in vivo.

    (A) Ovariectomized C57BL/6 mice were implanted with pellets containing E2 and PaPE-1. Liver, skeletal muscle, perigonadal fat, pancreas, and uterus were harvested. RNA was isolated, and qPCR was performed for the indicated genes (n = 8 mice per treatment). (B) PaPE-1 activates mTOR signaling (as monitored by increased phosphorylation of S6) in liver and skeletal muscle. Ovariectomized C57BL/6 mice were injected with E2 (E) or PaPE-1 (P) for 2 hours. The indicated tissues were collected and subjected to Western blot analysis for phosphorylated S6 and MAPK1/2. β-Actin and total ERK2 were used as loading controls. Total MAPK1/2 and total P70S6K are also shown (n = 3 mice per treatment).

  • Fig. 7 Like E2, PaPE-1 elicits repair of the vascular endothelium after injury, an effect that is prevented by the antiestrogen ICI 182,780.

    (A) Carotid artery reendothelialization after an injury that denudes the endothelial layer in ovariectomized mice treated with PaPE-1 or E2 in the absence or presence of the antiestrogen ICI 182,780 (n = 6 to 9 mice per group). *P < 0.05 compared to basal control. Scale bar, 400 μm. (B) E2, but not PaPE-1, increases uterine weight, an effect that is blocked by ICI 182,780 (n = 7 mice per treatment). (C) eNOS stimulation by E2 and PaPE-1 in the presence and absence of the antiestrogen ICI 182,780 in bovine aortic endothelial cells (BAECs) (n = 4 biological replicates; *P < 0.05 compared to control).

  • Fig. 8 Assessment of the activities of PaPE-2, PaPE-3, and PaPE-4 in MCF-7 cells, in BAECs, and in mice.

    (A) Assessment of extranuclear-initiated LRRC54 gene expression compared to direct nuclear PgR gene expression in MCF-7 cells treated for 4 hours with control vehicle, E2, or the indicated PaPE (n = 4 biological replicates). (B) Proliferation of MCF-7 cells after treatment with different concentrations of E2 or the PaPE for 6 days (n = 4 biological replicates). (C) Stimulation of various cell signaling pathways by different concentrations of E2, PaPE-1, or PaPE-4 after 15 min in MCF-7 cells. ERα abundance is also shown (n = 3 biological replicates). (D) Time course of cell signaling pathway activations by PaPE-2 or PaPE-3, monitored at the indicated times. ERα abundance is shown, and total ERK2 is used as a loading control (n = 2 biological replicates). (E) Stimulation of NOS activity during 15-min treatment of BAECs with ligand either alone or with ICI 182,780 (n = 4 biological replicates). (F) The PaPEs and E2 reduce weight gain after ovariectomy in C57BL/6 mice. Ovariectomized animals received pellets of E2, the PaPE, or vehicle, and body weight was monitored over the next 3 weeks. A group of intact non-ovariectomized mice were included for comparison (n = 4 mice per group). A two-way ANOVA model was fitted to assess the contribution of ligand treatment and time of ligand treatment on body weight or food consumption. When the effects were statistically significant at α = 0.05, pairwise t tests with a Bonferroni correction were used to identify the treatments that were significantly different from each other. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (G) Food consumption of the mice in (F) was monitored over time. (H) Assessment of uterine weight gain in the ovariectomized C57BL/6 mice in (F) after 3 weeks of receiving E2 or PaPE-1, PaPE-2, PaPE-3, and PaPE-4. One-way ANOVA, Newman-Keuls posttest, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (I) Fat mass, lean mass, and water mass were measured by EchoMRI at the end of the 3-week treatment period in the mice in (F). A one-way ANOVA model was fitted to assess the contribution of ligand treatment on gene expression, fat mass, lean mass, and water mass. When the main effects were statistically significant at α = 0.05, pairwise t tests with a Newman-Keuls correction were used to identify the treatments that were significantly different from each other. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/429/ra53/DC1

    Fig. S1. ER and coactivator binding and interaction assays with ligands and ligand dissociation rates.

    Fig. S2. Verification of siRNA results in Fig. 2C with another siRNA.

    Fig. S3. PLAs with E2 and PaPE-1.

    Fig. S4. Pharmacokinetic studies for analysis of blood concentrations of PaPE-1 after injection or pellet implantation.

    Fig. S5. Effects of PaPE-1 require ERα.

    Table S1. List of genes differentially expressed by cell treatment with E2 and PaPE-1.

    Table S2. BED files for ERα, ERK2, and pSer5 RNA pol II ChIP-seq data from experiments with MCF-7 cells.

  • Supplementary Materials for:

    Design of pathway preferential estrogens that provide beneficial metabolic and vascular effects without stimulating reproductive tissues

    Zeynep Madak-Erdogan, Sung Hoon Kim, Ping Gong, Yiru C. Zhao, Hui Zhang, Ken L. Chambliss, Kathryn E. Carlson, Christopher G. Mayne, Philip W. Shaul, Kenneth S. Korach, John A. Katzenellenbogen, Benita S. Katzenellenbogen*

    *Corresponding author. Email: katzenel{at}illinois.edu

    This PDF file includes:

    • Fig. S1. ER and coactivator binding and interaction assays with ligands and ligand dissociation rates.
    • Fig. S2. Verification of siRNA results in Fig. 2C with another siRNA.
    • Fig. S3. PLAs with E2 and PaPE-1.
    • Fig. S4. Pharmacokinetic studies for analysis of blood concentrations of PaPE-1 after injection or pellet implantation.
    • Fig. S5. Effects of PaPE-1 require ERα.
    • Legends for tables S1 and S2

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.46 MB

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). List of genes differentially expressed by cell treatment with E2 and PaPE-1.
    • Table S2 (Microsoft Excel format). BED files for ERα, ERK2, and pSer5 RNA pol II ChIP-seq data from experiments with MCF-7 cells.

    Citation: Z. Madak-Erdogan, S. H. Kim, P. Gong, Y. C. Zhao, H. Zhang, K. L. Chambliss, K. E. Carlson, C. G. Mayne, P. W. Shaul, K. S. Korach, J. A. Katzenellenbogen, B. S. Katzenellenbogen, Design of pathway preferential estrogens that provide beneficial metabolic and vascular effects without stimulating reproductive tissues. Sci. Signal. 9, ra53 (2016).

    © 2016 American Association for the Advancement of Science