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  • The Estrogen ReceptorJulie Davey Dalsgaard Lund

    Bachelors ThesisFebruary 2005

    Department of ChemistryUniversity of Aarhus

    Denmark

  • Contents

    1 Introduction 1

    2 Estrogen Receptor 2

    3 Ligands 43.1 Estradiol and Estriol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.2 Raloxifen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.3 Tamoxifen E and Z . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.4 Genistein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.5 Fulvestrant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.6 Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

    4 Methods 94.1 Overview of Glide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.2 Scoring Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104.3 Ligand Conformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

    5 Results 135.1 Selection of ER Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135.2 Protonation States of Histidin 524 . . . . . . . . . . . . . . . . . . . . . . . . . . 135.3 Intermolecular Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155.4 Results of the Dockings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

    5.4.1 Known ER Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155.4.1.1 Estradiol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175.4.1.2 Estriol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185.4.1.3 Raloxifen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195.4.1.4 4-hydroxytamoxifen E and Z . . . . . . . . . . . . . . . . . . . 215.4.1.5 Genistein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225.4.1.6 Fulvestrant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

    5.4.2 Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255.4.2.1 Primary Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . 255.4.2.2 Secondary Pesticides . . . . . . . . . . . . . . . . . . . . . . . 27

    6 Conclusions 29

    ii

  • 7 Experiments 307.1 Selection of ER Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307.2 Preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317.3 Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

    A Pesticides 35

    B Hydrogen Bonds 37B.1 Estradiol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38B.2 Estriol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38B.3 Raloxifen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39B.4 4-hydroxytamoxifen E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39B.5 4-hydroxytamoxifen Z . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39B.6 Genistein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40B.7 Fulvestrant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

  • Chapter 1

    Introduction

    This project is about the estrogen receptor and the different effects estrogen-like moleculescan have on the receptor. The estrogen receptor is located in the nucleus or the cytoplasmaof the cell. This means that the receptor can have a direct effect on the DNA transcription,when an estrogen enters the cell and binds to the estrogen receptor. In this case, the receptoris released from an inhibitory protein and undergo a conformational change and can nowhave an effect on specific DNA sequences; see [3]. The binding of other compounds than thenatural estrogens can have similar effects. This means that other compounds can have aneffect on the estrogen receptor and cause disruptions in the human body. For that reason, itis very important to be able to determine whether a given molecule has any estrogenic activ-ity or not. Unfortunately, there are many different estrogens and since their structure do notneed to resemble the structure of estradiol the natural agonist for the estrogen receptor forthe molecule to have the same effects, the experimental process is very time-consuming. Theprimary goal of this project is to determine if it is possible to use computer-aided dockingsimulations to speed up the process. However, since I have examined pesticides in additionto known agonists and antagonists for both alpha and beta estrogen receptors, the secondarygoal is determine if any of the examined pesticides have effects on either of the estrogen re-ceptors. I have performed the docking simulations using the computer program Maestro1.The program docks the different compounds in the ligand binding domains of different es-trogen receptors, which I have chosen using the Protein Data Bank2 (PDB).

    1http://www.schrodinger.com/Products/maestro.html2http://www.pdb.org/

    1

  • Chapter 2

    Estrogen Receptor

    The estrogen receptor (ER) is a member of a large family of nuclear receptor transcriptionfactors with specific domains associated with transactivation, DNA, and ligand binding; see[11]. The ER is a twelve helix protein, that is located in the nucleus of the cell, where it func-tions as a ligand-activated transcriptional regulator. Since the receptor is in the nucleus ofthe cell, it can only be affected by molecules that are small enough to pass through the cellmembrane. Such molecules affect ERs to form dimers and affect the DNA through transcrip-tion of DNA within the nucleus; see [6].

    The ER is located in several places in the female body, where it is the target of drugsagainst menopause and some cancer types as described in [11]. The receptor is located inthe hypothalamo pituitary axis, breast tissue, liver, uterus, vagina, and in bone tissue. Thelocation of the receptor affects the response of the receptor: ERs in different locations yieldsdifferent physiological effects. Another thing that influences the effects of the ER is the kindof estrogens which bind to the receptor.

    The binding pocket of the ER is a hydrophobic pocket. When a ligand binds to the ligandbinding domain (LBD), there is a conformational change in the receptor which eventuallyleads to activation or deactivation of responsive genes. Every ligand interacts with a uniqueset of amino-acid residues in the hormone binding cavity and induces a specific orientationof helix 12. When an antagonist binds in the cavity, the bulky side chain of the ligand willprevent helix 12 from covering the bound ligand. On the other hand, when an agonist bindsin the binding cavity, helix 12 covers the agonist and the binding cavity. Figure 2.1 on thefollowing page shows the conformational state of the ER when binding an agonist and anantagonist.

    There are two different ERs: ER and ER; see [11]. ER and ER have different re-sponses and they are located in different tissue. It was thought for many years that therewas only one ER, but it was noticed that estradiol had an effect on tissue where no ERswere located. Today, it is known that there are more than one ER and this explains whyestrogens can have an effect on tissue without ERs. Some ligands bind to both receptorsbut have different effects on them. A ligand can be an agonist for ER and an antagonist forER. The structures of ER and ER are only 47 percent identical. There is a difference inligand binding ability and transactivational ability in the two ERs. The N-terminal transacti-

    2

  • CHAPTER 2. ESTROGEN RECEPTOR 3

    Figure 2.1 The conformations of ER and ER LBD in the presence of an agonist (E2), apartial agonist (GEN), and an antagonist (RAL). Helix 12 is shown as a green rod. The boundligands are shown in space-filling form. Reprinted from [12].

    vation (AF-1) domains have no similarities, but the ERs have quite similar DNA and ligandbinding domains. The C-terminal LBD is multi-functional: It contains the ligand recognitionsite and regions for receptor dimerization and ligand-dependent (AF-2) transactivation; see[11].

    The most examined receptor is ER; see [8]. ERs are found in the liver, uterus, vagina,bone tissue, hypothalamo pituitary axis, and in breast cancer tumors. The structure of ER isdifferent depending on whether an agonist or an antagonist is bound to it. When an agonistis bound to the receptor, helix 12 covers the binding site and the bound agonist. When anantagonist is bound to the receptor, helix 12 is flipped away from the binding site, becausethe antagonist makes it impossible for helix 12 to fold into place. For the receptor to have aneffect, helix 12 has to be in the right position; see figure 2.1.

    ER is found in different parts of the body than ER. ERs are located in the prostate,testis, ovary, and in some areas of the brain. Male and female bodies react differently whenexposed to natural estrogens: Women are at risk of developing breast and endometrial can-cer, whereas men can get gynecomastia. Furthermore, estrogens can disturbe the normalfunction of the male hypothalamus-hypophyseal-gonadal axis, which may lead to decreasedlibido, impotence, drop in androgen levels, and drop in sperm count; see [13]. If human em-bryos are disposed to estrogens, they may develop cryptorchidism, persistance of Mellerianin men, enlarged prostate, vaginal adrenosis, malformations of the female genital tract, andclear cell adenocarcinoma of the vagina.

    ER also has helix 12, but when an agonist binds to the receptor, helix 12 does not overlaythe LBD as it is the case for ER; see figure 2.1 and [11]. The position of helix 12 in ER witha bound agonist is closer to t

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