Intracellular Receptor A site of Drug Action

Heny Ekowati

Department of Pharmacy,

Faculty of Medicine and Health Sciences

UNSOED, 2011

Nuclear Receptors1. Proteins interact with steroids and other

hormones that diffuse through the cell membrane.

2. Form hormone-receptor complexes that function as activators by binding to enhancers hormone response elements.

3. Sex hormones: estrogens and androgens; glucocorticoids, cortisol, vitamin D Ca2+ metabolism; thyroid hormone, retinoic acid developmental factors.

Steroid Hormone Receptor Signaling

Steroid hormones are produced by endocrine glands

Essential regulators of: reproduction, secondary sex characteristics

Development, differentiation

Glucose metabolism

Response to stress and salt balance

Nuclear Receptor Superfamily

large family of structurally related ligand-inducible transcription factors, including: steroid receptors (SRs), thyroid/retinoids receptors (TR, RARs and RXRs),

vitamin D receptors (VDR), estrogen receptors (ERa and ERb), and orphan receptors for which no ligand has

been yet identified. While having in common a modular structure, they

are activated by distinct lipophilic small molecules such as glucocorticoids, progesterone, estrogens, retinoids, and fatty acid derivatives

All steroids are synthesized from cholesterol.

Characterized by the presence of 3 functional domains: DNA-binding domain (DBD): 2 Zn finger motifscentrally located

Ligand-binding domain (LBD): C-terminal, 12 a helicesLBD: 1. Ligand-dependent nuclear translocation signal

2. Chaperone protein binding 3. Dimerization interface

4. Ligand-depndent activation function domain (AF-2) Transactivation domain: ligand–independent AF-1

AF domains are a-helical structures that can recruit co-activators.

Steroid Hormone Receptors

Receptor activation induces the modulation of transcription of specific genes that has hormone receptor elements (HRE).

Responses to hydrophobic hormones are mediated by intracellular

receptors

Transcription

Translation

Cytoplasm

Nucleus

Nuclear envelope

Plasma membrane Lipophilic hormone carried

in blood

Hormone binds intracellular receptor inducing receptor dimerization and activation

Complex is imported into nucleus

Binds to “hormone response element” to regulate gene expression

Intracellular receptor

Promoter Target gene“Hormone response element”

Target cell

LipophillicHormone

Fig.15-12

Thyroid hormone receptors exist as dimers

Estrogen Reseptor

Estrone  [53-16-7]

Estradiol-17b  [50-28-2]

Estradiol-17a  [57-91-0]

Primary estrogen produced by the ovaries.

Estriol  [50-27-1]

Formed from estradiol, weak estrogen, predominant estrogen after menopause.

Weak estrogen, used to treat menopause symptoms.

Estrogens(oestrogen)

Estrogen Action Stimulates tissue growth by:

promoting cell proliferation (DNA synthesis and cell division) in female sex organs (breasts, uterus),

promoting hypertrophy, or increasing a cell's size, such as occurs in female breast and male muscle during puberty and

initiating synthesis of specific proteins. Gene transcription of: myc, cyclin D, TGFa, Bcl2,

Cathepsin D, NOS

Estrogen Receptors ER-a

Uterus, testis, pituitary, ovary, epididymis, and adrenal gland.

ER-b (Kuiper et al. 1996) brain, kidney, prostrate, ovary, lung, bladder, intestine,

and epididymis. 88% identity with rat ER-b;

47% identity with human ER-a

Both ERs are localized to membrane, cytosol, and nucleus.

ERa and b differ in C-terminal ligand binding domains and N-terminal transactivation domains. Highest homology in DNA binding domain.

Estrogen-related orphan receptors (ERR) , , a b g

Estrogen Receptors

http://www.bio.cmu.edu/Courses/BiochemMols/ER/#ERchime

Estrogen Receptor

DNA pol a

Cyclins E,A

B-Myb

I-Classical Mechanism: ERE dependent Gene Transactivation

Mechanism

Osborne, C. K.; Zhao, H.; Fuqua, S. A. Selective estrogen receptor modulators: structure, function, and clinical use. J Clin Oncol 2000, 18, 3172-3186.

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Molecular Action of Estrogen

Adopted from Stanley J Birge et al

AP I – activator protein

CRP – co regulator protein

ER – estrogen receptor

ERE – estrogen response element

Poly II – polymerase II

TATA- adenine-thymine-rich sequence important for gene transcription

21SERMs - Prof.S.N.Panda - 45th. AICOG

Molecular Action of EstrogenDifferent response in different

tissues

Adopted from Lewis J. Kleinsmith Ph.D, Donna Kerrigan M.S., Jeanne Kelly

ER effects on different cell types

Copyright ©2005 The Endocrine Society

Bjornstrom, L. et al. Mol Endocrinol 2005;19:833-842

Fig. 2. Schematic Illustration of How Genomic and Nongenomic Actions of ERs on a Target Gene Promoter May Converge

24SERMs - Prof.S.N.Panda - 45th. AICOG

Effects of Estrogen at Various Sites in the Body

Tissue Effect of Estrogen Stimulation

Clinical Effect of Stimulation

Clinical Effect of Absence of Stimulation

Bone Increased deposits of calcium into bone

Increased bone density Osteoporosis

Brain Blocks the release of ovarian estrogen

NoneHot flashes, sleep disorders, mood changes, problems with memory? Alzheimer’s disease??

BreastStimulates growth of breast tissue

Bigger breasts,? Increased risk of breast cancer, increased sensitivity of the breast,

Smaller breasts

Blood Clotting

Increased risk of blood clots No change in clotting

Blood Fats Increased HDL, decreased LDL, decreased Cholesterol,

Decreased HDL, increased LDL, increased Cholesterol

Skin Increased fat deposits in skin Softer skin Thinner skin, liver spots, dry skin

Uterus Increased stimulation of uterine lining and muscle

Heavier cycles, increased risk of uterine cancer No periods

VaginaIncreased thickening of skin, better blood supply to tissue

Vaginal discharge, feelings of pelvic congestion

Dryness, vaginal infections, painful sex, incontinence of urine, pelvic weakness

ORMELOXIFENE

The individual elements of the molecular structure give a tissue selectivity- different

DNA transcriptions in different tissues

Estrogen agonist

Estrogen antagonist

Chemical Name- Trans -7-methyl-2-2-dimethyl-3-phenyl-4(4-(2-

pyroldinoethoxy)phenyl(-chroman hydrochloride), related to

centchroman

The perfect SERM

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The perfect SERMORMELOXIFENE

Enhanced tissue selectivity Basic amine side chain – uterine AE

action Pyrolidine base – highest degree of

antagonistic action Benzopyran group – agonistic action &

binding affinity Very strong binding affinity to ER

Quick & potent action Slow nuclear build up & prolonged

retention of ER Long half life & prolonged action

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An optimally designed potent SERM with Varied Tissue ResponseOestrogen Antagonist in UTERUS &

BREASTMild Oestrogenic action on Vagina, Bone mineral density, CNS and Serum LipidsNo action on Hypothalamic Pituitary Ovarian function, Thyroid, Adrenal.

The perfect SERM

ORMELOXIFENE

No Progestational, Androgenic or Antiandrogenic properties

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ORMELOXIFENE

Special benefit in perimenopausal women – Relief of PMS

Currently indicated for the treatment of Dysfunctional Uterine Bleeding at ANY AGE.Not suitable for women desiring pregnancy

Approved for inclusion in National Family Welfare Program, for social marketing.

The perfect SERM

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ORMELOXIFENE

Currently being evaluated for use in the treatment and prevention of: -• Breast Cancer• Osteoporosis Possible future use: -• Menopause management• Fibromyoma• Endometriosis and Adenomyosis• Contraceptive

The perfect SERM

Glucocorticoid

Receptor

Formation of a receptor/ligand complex can occur in the cytosol or the nucleus and typically leads to dimerization of the occupied receptor.Dimerized receptor binds to the hormone regulatory element (HRE, 8-15 base pairs) on the target DNA molecule changes in gene transcription

Positive and Negative Transcriptional Effects of Steroid Receptors

Binding of a receptor dimer imme-diately adjacent to a transcriptionfactor leads to synergistic activa-tion of transcription

Binding of a receptor dimer to anegative hormone response ele-ment may displace a positive transcription factor.

Protein-protein interaction between receptordimer and a positive transcription factor such as AP 1 may block AP 1/DNA bindingand repress the transcriptional response.Figure 22.15 – Devlin, Textbook of

Biochemistry

NF-B Proteins: Latent Gene Regulatory Proteins Pivotal to Most Inflammatory Responses

• Proinflammatory cytokines, suchas TNF-, bind to their specificmembrane receptors and initiatea pathway that activates NF-B,normally sequestered in aninactive state through associationwith IB proteins.

• Once activated, NF-B turns onthe transcription of > 60 genesthat participate in inflammatoryresponses.

• Degradation of IB exposes a nuclear localization signal on NF-B, which move into the nucleus.

• A TNF-/receptor interactioninitiates of pathway that marksIB for degradation.

A glucocorticoid interaction with its receptor results in increasing the transcriptionof the protein IB, which binds and inhibits the activity of NF-B, a a transcriptionalactivator that stimulates transcription of genes for inflammatory cytokines.

Glucocorticoid induction of IB synthesis through GCbinding to its intracellularreceptor and stimulating trans-cription of the gene.

IB binds toand inhibits thenuclear translo-cation of NF-B.

NF-B stimulates theultimate production ofinflammatory cytokinesTumor necrosis factor (TNF)

binding to its receptor leads tothe ultimate degradation of IB

How glucocorticoids suppress immune and inflammatory reactions mediated by cytokines

Thank you for your attention