Applying An Understanding of Applying An Understanding of the Mechanisms of Action of the Mechanisms of Action of

Estrogen and SERMs to Patient Estrogen and SERMs to Patient and Treatment Selection in and Treatment Selection in

Clinical PracticeClinical Practice

Donald P. McDonnell, PhDDonald P. McDonnell, PhD

Professor of Pharmacology and Cancer BiologyProfessor of Pharmacology and Cancer BiologyDirector of Graduate Studies, PharmacologyDirector of Graduate Studies, Pharmacology

Duke University Medical CenterDuke University Medical CenterDurham, North CarolinaDurham, North Carolina

The Estrogen Receptor Is aThe Estrogen Receptor Is aLigand-Dependent Transcription FactorLigand-Dependent Transcription Factor

Altered Cell

Function

Cell MembraneCell Membrane Nuclear MembraneNuclear Membrane

New Protein

3’ mRNA5’ERE

ERE= estrogen response element; mRNA= messenger RNA. Graphic courtesy of Donald P. McDonnell, PhD.

The Potential Fates of Agonist-activated ERThe Potential Fates of Agonist-activated ER

NF-B

ERCoA

ERCoA

ERCoA

ER CoA

ER CoA

ER CoA

X

ERE

AP1

p50 p65

Genomic ActionsNon-genomic Actions

ER ER

ER

ER

c-SRC

ERMNAR

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 ER

A Simple Model to ExplainA Simple Model to ExplainEstrogen Receptor PharmacologyEstrogen Receptor Pharmacology

Agonist

Antagonist

ER ER

ER = estrogen receptor. Graphic courtesy of Donald P. McDonnell, PhD.

Clinical Validation of the Clinical Validation of the SERM ContextSERM Context

Love RR, et al. N Engl J Med. 1992;362:852. Copyright © 1992. Massachusetts Medical Society. All rights reserved.

MonthsMonths

% C

han

ge

in S

pin

al B

MD

% C

han

ge

in S

pin

al B

MD

fro

m B

asel

ine

fro

m B

asel

ine

BaselineBaseline 33 66 1212 1818 2424

-3-3

-2-2

-1-1

00

11

22

33

TamoxifenTamoxifenPlaceboPlacebo

BMD = bone mineral density.

Clomiphene1962

Tamoxifenb

1972

Toremifene1985

Lasofoxifene 1998 Arzoxifene 1998

Empiricaldiscovery

Mechanism-based discovery

1965 1970 1980 1990 2000

Fulvestrantb

1992

aBased on date of first publication; bFDA-approved.

SERMs—A Historical PerspectiveSERMs—A Historical Perspectiveaa

Bazedoxifene 2001

DroloxifeneRaloxifeneb

1983GW5638

1994

Evolution of SERMs/ER ModulatorsEvolution of SERMs/ER Modulators

EstradiolEstradiol

TamoxifenTamoxifen

RaloxifeneRaloxifene

BazedoxifeneBazedoxifene

TSECTSEC

BoneBone Uterus Uterus BrainBrain BreastBreast (VMS)(VMS)

+ +

+

+

+

+

+ +

+

–

–

–

+ +

–

–

–

–

+ +

–

–

–

++

TSEC = tissue selective estrogen complex; VMS = vasomotor symptoms.Graphic courtesy of Donald P. McDonnell, PhD. + = effect; – = no effect.

27-Hydroxycholesterol (27HC)27-Hydroxycholesterol (27HC)

Cholic and Chenodeoxycholic acidsCholic and Chenodeoxycholic acids+ Propionic Acid+ Propionic Acid

Cla

ssi

c (N

eutr

al)

Cla

ssi

c (N

eutr

al)

Pat

hw

ayP

ath

way

HHOOOOHH

HHOOOO

HHOO

Lithocholic acidLithocholic acid Chenodeoxycholic Chenodeoxycholic acidacid

Yam

asak

i P

ath

way

Yam

asak

i P

ath

way

Aci

dic

Pat

hw

ayA

cid

ic P

ath

way

CholesterolCholesterol 27HC27HC Cholestenoic acidCholestenoic acid

CYP27A1CYP27A1 CYP7B1CYP7B1

HOHO

CYP7B1CYP7B1CYP7B1CYP7B1

Produced primarily outside the entero-hepatic axisProduced primarily outside the entero-hepatic axis11

Most prevalent circulating oxysterolMost prevalent circulating oxysterol11

Inhibits estrogen action in the cardiovascular systemInhibits estrogen action in the cardiovascular system11

1. Umetani M, et al. Nat Med. 2007;13:1185. Graphic courtesy of Carolyn D. DuSell, and Donald P. McDonnell, PhD.

0102030405060

708090

456789101112

- Log [M ]

No

rmal

ized

Res

po

nse

E2

27HC

0.5nM E2 + 27HC

27HC Functions as an 27HC Functions as an ER Partial AgonistER 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.

pS2

0

2

4

6

8

10

12

567891011

Fo

ld In

du

ctio

n

PR

02468

101214

567891011 -Log [M]

Fo

ld In

du

ctio

n E2

WISP2

0

2

4

6

8

10

12

567891011

Fo

ld In

du

ctio

n

ERBB4

00.10.20.30.40.50.60.70.8

567891011

Fo

ld In

du

ctio

n

27HC Activates Transcription of ER27HC Activates Transcription of ER Target Genes Target Genes

27HC

E227HC

E227HC

E227HC

Adapted from DuSell CD, et al. Mol Endrinol. 2008;22:65, with permission from The Endocrine Society.

-Log [M]

-Log [M] -Log [M]

CYP7B1 Expression is Correlated with CYP7B1 Expression is Correlated with Increased Survival In Breast CancerIncreased Survival In Breast Cancer

Disease-Free Years

Per

cent

Sur

viva

l

DuSell CD, et al. Unpublished data. Graphic courtesy of Donald P. McDonnell, PhD.

Building the Case for 27HC as a Building the Case for 27HC as a “Naturally” Occurring SERM“Naturally” Occurring SERM

Circulates at levels high enough to activate ERCirculates at levels high enough to activate ER11

– Range: 0.075 - 0.73 µM Range: 0.075 - 0.73 µM

– Males > Females Males > Females

Elevated concentration (millimolar range) in Elevated concentration (millimolar range) in atherosclerotic lesionsatherosclerotic lesions1,21,2

Attenuates the positive effect of estrogen on the Attenuates the positive effect of estrogen on the cardiovascular systemcardiovascular system11

KKmm of 27HC for its catabolic enzyme CYP7B1 of 27HC for its catabolic enzyme CYP7B1

(24 (24 MM))33 is greater than the K is greater than the Kdd of 27HC for ER of 27HC for ER

(0.5 (0.5 m)m)33

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.

Km = Michaelis constant; Kd = dissociation constant.

Factors That Influence the Molecular Factors That Influence the Molecular Pharmacology of ER LigandsPharmacology of ER Ligands

Presence or absence of a coexpressed Presence or absence of a coexpressed

progesterone receptorprogesterone receptor

Relative expression level of 2 estrogen Relative expression level of 2 estrogen

receptor (ER) subtypesreceptor (ER) subtypes

Impact of “estrogen” on structure of ERImpact of “estrogen” on structure of ER

Ability of differently conformed receptors Ability of differently conformed receptors

to interact with factors needed for activityto interact with factors needed for activity

0

20

40

60

80

100

120

140

160

Blank 10-11 10-10 10-9 10-8 10-7 10-6

ER and PR Regulate Each OtherER and PR Regulate Each Other

Progesterone (M)

ER

Ac

tivi

ty

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.

Factors That Influence the Molecular Factors That Influence the Molecular Pharmacology of ER LigandsPharmacology of ER Ligands

Presence or absence of a coexpressed Presence or absence of a coexpressed progesterone receptorprogesterone receptor

Relative expression level of 2 estrogen Relative expression level of 2 estrogen receptor (ER) subtypesreceptor (ER) subtypes

Impact of “estrogen” on structure of ERImpact of “estrogen” on structure of ER

Ability of differently conformed receptors Ability of differently conformed receptors to interact with factors needed for activityto interact with factors needed for activity

Primary Structure of ERPrimary Structure of ERαα and ER and ERββaa

DNA-DNA- bindingbindingdomaindomain

179 89 38 251 43NHNH22 COOHCOOH

Ligand-bindingLigand-bindingdomaindomain

ERERαα

AF-1AF-1 AF-2AF-2

142 84 28 248 28NHNH22 COOHCOOH ERERββ

18% 24% 58% Amino acid Amino acid homologyhomology

12%97%

aNumbers 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.

ERERββ Is a Key Component of the Cellular Is a Key Component of the Cellular Processes That Regulate Cellular Processes That Regulate Cellular

Sensitivity to AgonistsSensitivity to Agonists

ERα

ERβ

ERα/ERβ

0

50

100

150

200

250

nh 12 11 10 9 8 7 6-Log [M] E2

No

rmal

ized

Re

spo

nse

Reprinted from Hall JM, et al. Endocrinology. 1999;140:5566, with permission from The Endocrine Society.

ERERββ Agonists May Have Utility as Agonists May Have Utility asTreatment for Inflammatory Bowel DiseaseTreatment for Inflammatory Bowel Disease

Reprinted from Harris HA, et al. Endocrinology. 2003;144:4241, with permission from The Endocrine Society.

DiarrheaDiarrhea

NormaNormall

0.5

1

1.5

2

2.5

3

3.5

0 2 4 6 8Day of Treatment

Sto

ol S

core

Sto

ol S

core

Vehicle

20 mg/kg

10 mg/kg

5 mg/kg

1 mg/kg

0.3 mg/kg

0.1 mg/kg

Factors That Influence the Molecular Factors That Influence the Molecular Pharmacology of ER LigandsPharmacology of ER Ligands

Presence or absence of a coexpressed Presence or absence of a coexpressed progesterone receptorprogesterone receptor

Relative expression level of 2 ER subtypesRelative expression level of 2 ER subtypes

Impact of “estrogen” on structure of Impact of “estrogen” on structure of estrogen receptor (ER)estrogen receptor (ER)

Ability of differently conformed receptors Ability of differently conformed receptors to interact with factors needed for activityto interact with factors needed for activity

Crystallography Has Confirmed the Relationship Crystallography Has Confirmed the Relationship Between the Conformation of ER-ligand Complexes Between the Conformation of ER-ligand Complexes

and Their Biologic Activity and Their Biologic Activity

Color Ligand

Green IOS974/GW5638

Yellow 4-hydroxytamoxifen

Blue RaloxifeneGraphic courtesy of Virginia Carnahan and Donald P. McDonnell, PhD.

Different Compounds InduceDifferent Compounds InduceDifferent Structural Alterations WithinDifferent Structural Alterations Within

the Estrogen Receptorsthe Estrogen Receptors

InactiveInactive Fully ActivatedFully Activated

PurePureAntagonistsAntagonists

SERMsSERMs PurePureAgonistsAgonists

Adapted from McDonnell DP, et al. Mol Endocrinol. 1995;9:659, with permission from The Endocrine Society.

Factors That Influence the Molecular Factors That Influence the Molecular Pharmacology of ER LigandsPharmacology of ER Ligands

Presence or absence of a coexpressed Presence or absence of a coexpressed progesterone receptorprogesterone receptor

Relative expression level of 2 estrogen Relative expression level of 2 estrogen receptor (ER) subtypesreceptor (ER) subtypes

Impact of “estrogen” on structure of ERImpact of “estrogen” on structure of ER

Ability of differently conformed receptors Ability of differently conformed receptors to interact with factors needed for activityto interact with factors needed for activity

An Updated Model of Nuclear Receptor Pharmacology

CoA CoA

NRE

X

NRE

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.

ConclusionsConclusions

Receptor conformation is determined byReceptor conformation is determined bythe nature of the bound ligandthe nature of the bound ligand

Differences in receptor conformation dictate Differences in receptor conformation dictate coactivator binding preferencescoactivator binding preferences

The biologic activity of different NR-cofactor The biologic activity of different NR-cofactor complexes is not equivalentcomplexes is not equivalent

Screening for ligands which facilitate specific Screening for ligands which facilitate specific NR-cofactor interactions allows process/cell-NR-cofactor interactions allows process/cell-specific modulators to be developedspecific modulators to be developed