1. Applying An Understanding of the Mechanisms of Action of Estrogen and SERMs to Patient and Treatment Selection in Clinical Practice Donald P. McDonnell, PhD Professor of Pharmacology and Cancer Biology Director of Graduate Studies, Pharmacology Duke University Medical Center Durham, North Carolina

2. The Estrogen Receptor Is a Ligand-Dependent Transcription Factor Altered Cell Function Cell Membrane Nuclear Membrane New Protein 3’ mRNA 5’ ERE ERE= estrogen response element; mRNA= messenger RNA. Graphic courtesy of Donald P. McDonnell, PhD.

3. The Potential Fates of Agonist-activated ER NF-  B ER CoA ER CoA ER CoA ER CoA ER CoA ER CoA X ERE AP1 p50 p65 Genomic Actions Non-genomic Actions ER c-SRC ER MNAR ER = estrogen receptor; CoA = coactivator; AP = activator protein; MNAR = modulator of nongenomic action of estrogen receptor; NF-kB = nuclear factor kB. Reprinted from McDonnell DP. EJC Supplements. 2004;2:35, with permission from Elsevier. ER

4. A Simple Model to Explain Estrogen Receptor Pharmacology Agonist Antagonist ER ER = estrogen receptor. Graphic courtesy of Donald P. McDonnell, PhD.

5. Clinical Validation of the SERM Context Love RR, et al. N Engl J Med. 1992;362:852. Copyright © 1992. Massachusetts Medical Society. All rights reserved. Months % Change in Spinal BMD from Baseline Baseline36121824 -3 -2 0 1 2 3 Tamoxifen Placebo BMD = bone mineral density.

6. Clomiphene 1962 Tamoxifen b 1972 Toremifene 1985 Lasofoxifene 1998 Arzoxifene 1998 Empirical discovery Mechanism-based discovery 1965 1970 1980 19902000 Fulvestrant b 1992 a Based on date of first publication; b FDA-approved. SERMs—A Historical Perspective a Bazedoxifene 2001 Droloxifene Raloxifene b 1983 GW5638 1994

7. Evolution of SERMs/ER Modulators EstradiolTamoxifenRaloxifeneBazedoxifeneTSEC Bone Uterus Brain Breast (VMS) (VMS) + – + – + – ++ TSEC = tissue selective estrogen complex; VMS = vasomotor symptoms. Graphic courtesy of Donald P. McDonnell, PhD. + = effect; – = no effect.

8. 27-Hydroxycholesterol (27HC) Cholic and Chenodeoxycholic acids + Propionic Acid Classic (Neutral) Pathway HO OH HO O HO Lithocholic acid Chenodeoxycholic acid Yamasaki Pathway Acidic Pathway Cholesterol 27HC Cholestenoic acid CYP27A1 CYP7B1 HO CYP7B1 CYP7B1 Produced primarily outside the entero-hepatic axis 1 Produced primarily outside the entero-hepatic axis 1 Most prevalent circulating oxysterol 1 Most prevalent circulating oxysterol 1 Inhibits estrogen action in the cardiovascular system 1 Inhibits estrogen action in the cardiovascular system 1 1. Umetani M, et al. Nat Med. 2007;13:1185. Graphic courtesy of Carolyn D. DuSell, and Donald P. McDonnell, PhD.

9. 0 10 20 30 40 50 60 70 80 90 456 789101112 - Log [M] Normalized Response E2 27HC 0.5nM E2 + 27HC 27HC Functions as an ER Partial Agonist 27HC = 27-hydroxycholesterol; ER = estrogen receptor; E2 = 17ß-estradiol Reprinted from DuSell CD, et al. Mol Endocrinol. 2008;22:65, with permission from The Endocrine Society.

10. pS2 0 2 4 6 8 10 12 567891011 Fold Induction PR 0 2 4 6 8 10 12 14 567891011 -Log [M] Fold Induction E2 WISP2 0 2 4 6 8 10 12 567891011 Fold Induction ERBB4 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 567891011 27HC Activates Transcription of ER  Target Genes 27HC E2 27HC E2 27HC E2 27HC Adapted from DuSell CD, et al. Mol Endrinol. 2008;22:65, with permission from The Endocrine Society. -Log [M]

11. CYP7B1 Expression is Correlated with Increased Survival In Breast Cancer Disease-Free Years Percent Survival DuSell CD, et al. Unpublished data. Graphic courtesy of Donald P. McDonnell, PhD.

12. Building the Case for 27HC as a “Naturally” Occurring SERM Circulates at levels high enough to activate ER 1 Circulates at levels high enough to activate ER 1 – Range: 0.075 - 0.73 µM – Males > Females Elevated concentration (millimolar range) in atherosclerotic lesions 1,2 Elevated concentration (millimolar range) in atherosclerotic lesions 1,2 Attenuates the positive effect of estrogen on the cardiovascular system 1 Attenuates the positive effect of estrogen on the cardiovascular system 1 K m of 27HC for its catabolic enzyme CYP7B1 (24  M ) 3 is greater than the K d of 27HC for ER (0.5  m) 3 K m of 27HC for its catabolic enzyme CYP7B1 (24  M ) 3 is greater than the K d of 27HC for ER (0.5  m) 3 1. Umetani M, et al. Nat Med. 2007;13:1185. 2. Brown AJ, et al. Atherosclerosis. 1999;142:1. 3. DuSell CD, et al. Mol Endocrinol. 2008;22:65. K m = Michaelis constant; K d = dissociation constant.

13. Factors That Influence the Molecular Pharmacology of ER Ligands Presence or absence of a coexpressed progesterone receptor Presence or absence of a coexpressed progesterone receptor Relative expression level of 2 estrogen receptor (ER) subtypes Relative expression level of 2 estrogen receptor (ER) subtypes Impact of “estrogen” on structure of ER Impact of “estrogen” on structure of ER Ability of differently conformed receptors to interact with factors needed for activity Ability of differently conformed receptors to interact with factors needed for activity

14. 0 20 40 60 80 100 120 140 160 Blank10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 ER and PR Regulate Each Other Progesterone (M) ER Activity ER ER + PR-A All samples treated with estradiol. ER = estrogen receptor; PR = progesterone receptor. Adapted from Wen DX, et al. Mol Cell Biol. 1994;14:8356, with permission from the American Society for Microbiology.

15. Factors That Influence the Molecular Pharmacology of ER Ligands Presence or absence of a coexpressed progesterone receptor Presence or absence of a coexpressed progesterone receptor Relative expression level of 2 estrogen receptor (ER) subtypes Relative expression level of 2 estrogen receptor (ER) subtypes Impact of “estrogen” on structure of ER Impact of “estrogen” on structure of ER Ability of differently conformed receptors to interact with factors needed for activity Ability of differently conformed receptors to interact with factors needed for activity

16. Primary Structure of ERα and ERβ a DNA- binding bindingdomain 179893825143 NH 2 COOH Ligand-bindingdomain ERα AF-1 AF-2 142842824828 NH 2 COOH ERβ 18%24%58% Amino acid homology 12%97% a Numbers in boxes indicate number of amino acids. Reprinted from Hall JM, et al. Mol Interv. 2005;5:343, with permission from The American Society for Pharmacology and Experimental Therapeutics.

17. ERβ Is a Key Component of the Cellular Processes That Regulate Cellular Sensitivity to Agonists ERα ERβ ERα/ERβ 0 50 100 150 200 250 nh 12 11 10 9 8 7 6 -Log [M] E2 Normalized Response Reprinted from Hall JM, et al. Endocrinology. 1999;140:5566, with permission from The Endocrine Society.

18. ERβ Agonists May Have Utility as Treatment for Inflammatory Bowel Disease Reprinted from Harris HA, et al. Endocrinology. 2003;144:4241, with permission from The Endocrine Society. Diarrhea Normal 0.5 1 1.5 2 2.5 3 3.5 02468 Day of Treatment Stool Score Vehicle 20 mg/kg 10 mg/kg 5 mg/kg 1 mg/kg 0.3 mg/kg 0.1 mg/kg

19. Factors That Influence the Molecular Pharmacology of ER Ligands Presence or absence of a coexpressed progesterone receptor Presence or absence of a coexpressed progesterone receptor Relative expression level of 2 ER subtypes Relative expression level of 2 ER subtypes Impact of “estrogen” on structure of estrogen receptor (ER) Impact of “estrogen” on structure of estrogen receptor (ER) Ability of differently conformed receptors to interact with factors needed for activity Ability of differently conformed receptors to interact with factors needed for activity

20. Crystallography Has Confirmed the Relationship Between the Conformation of ER-ligand Complexes and Their Biologic Activity ColorLigand GreenIOS974/GW5638 Yellow4-hydroxytamoxifen BlueRaloxifene Graphic courtesy of Virginia Carnahan and Donald P. McDonnell, PhD.

21. Different Compounds Induce Different Structural Alterations Within the Estrogen Receptors Inactive Fully Activated PureAntagonists SERMs PureAgonists Adapted from McDonnell DP, et al. Mol Endocrinol. 1995;9:659, with permission from The Endocrine Society.

22. Factors That Influence the Molecular Pharmacology of ER Ligands Presence or absence of a coexpressed progesterone receptor Presence or absence of a coexpressed progesterone receptor Relative expression level of 2 estrogen receptor (ER) subtypes Relative expression level of 2 estrogen receptor (ER) subtypes Impact of “estrogen” on structure of ER Impact of “estrogen” on structure of ER Ability of differently conformed receptors to interact with factors needed for activity Ability of differently conformed receptors to interact with factors needed for activity

23. An Updated Model of Nuclear Receptor Pharmacology CoA NRE X CoR NR NRE SNRMs CoR Antagonist Agonist CoR = corepressor; NRE = nuclear response element; NR = nuclear receptor; SNRM = selective nuclear receptor modulator; CoA = coactivator. Graphic courtesy of Donald P. McDonnell, PhD.

24. Conclusions Receptor conformation is determined by the nature of the bound ligand Receptor conformation is determined by the nature of the bound ligand Differences in receptor conformation dictate coactivator binding preferences Differences in receptor conformation dictate coactivator binding preferences The biologic activity of different NR-cofactor complexes is not equivalent The biologic activity of different NR-cofactor complexes is not equivalent Screening for ligands which facilitate specific NR-cofactor interactions allows process/cell-specific modulators to be developed Screening for ligands which facilitate specific NR-cofactor interactions allows process/cell-specific modulators to be developed