Nuclear receptors: in vitro and in vivo approaches

Parma, June 13-15 2017

Adriana Maggi

University of Milan www.cend.unimi.it

Androgen Receptor (AR; NR3C4) Aryl Hydrocarbon Receptor (AhR) Constitutive Androstane Receptor (CAR; NR1I3) Estrogen Receptor Alpha (ERα; NR3A1) Estrogen Receptor Beta (ERβ; NR3A2) Estrogen Related Receptor Alpha (ERRα; NR3B1) Estrogen Related Receptor Gamma (ERRɣ; NR3B3) Farnesoid X Receptor (FXR; NR1H4) Glucocorticoid Receptor (GR; NR3C1) Liver Receptor Homolog-1 (LRH-1; NR5A2) Liver X Receptor Alpha (LXRα; NR1H3) Liver X Receptor Beta (LXRβ; NR1H2) Mineralocorticoid Receptor (MR; NR3C2) Peroxisome Proliferator-Activated Receptor Alpha (PPARα; NR1C1) Peroxisome Proliferator-Activated Receptor Delta (PPARδ; NR1C2) Peroxisome Proliferator-Activated Receptor Gamma (PPARɣ; NR1C3)

Pregnane X Receptor (PXR; NR1I2) Progesterone Receptor (PGR; NR3C3) Retinoic Acid Receptor Alpha (RARα; NR1B1) Retinoic Acid Receptor Beta (RARβ; NR1B2) Retinoic Acid Receptor Gamma (RARɣ; NR1B3) RAR-related Orphan Receptor Alpha (RORα; NR1F1) RAR-related Orphan Receptor Gamma (RORɣ; NR1F3) Retinoid X Receptor Alpha (RXRα; NR2B1) Retinoid X Receptor Beta (RXRb; NR2B2) Retinoid X Receptor Gamma (RXRɣ; NR2B3) Thyroid Hormone Receptor Alpha (TRα; NR1A1) Thyroid Hormone Receptor Beta (TRβ; NR1A2) Vitamin D Receptor (VDR; NR1I1)

Intracellular Receptors, a family of 48 members in humans

NR Functional Classification

The endogenous ligands for Intracellular Receptors

MECHANISM OF ACTION 1.

MECHANISM OF ACTION 2.

THE PHARMACOLOGY OF INTRACELLULAR RECEPTORS

THE PHARMACOLOGY OF INTRACELLULAR RECEPTORS 2.

THE COMPLEXITY OF ESTROGEN ACTION

ERalpha

ERbeta

SP1

NFKB

AP1

IP3k

cAMP

THE TOOLS TO STUDY INTRACELLULAR RECEPTOR

ACTIVITIES

INTRACELLULAR RECEPTORS : THE TOOLS

INTRACELLULAR RECEPTORS : THE TOOLS

FUNCTIONAL CHARACTERIZATION OF ER LIGANDS

BINDING ASSAY

ER cDNA

ERE LUC

CTR CTR E2

LUC

Un

its

LUC

ER ER

FUNCTIONAL CHARACTERIZATION OF ER LIGANDS

TRANSFECTION ASSAY

Reporter

green fluorescent dye

NR NR NR

Ligands Ligands Ligand

NR

NR NR

4

FUNCTIONAL CHARACTERIZATION OF ER LIGANDS

TRANSFECTION ASSAY

15 minutes 2 hours 18 hours 1 week

ACTIVATION OF ERE-LUC REPORTER IN MOUSE HEPATOCYTES

0.01 0.1 1 10

800

1600

2400

E2 [ ]nM

RL

U/

g P

rote

in EC50=4.89nMVehicle

E2 1 nM

E2 5 nM

E210 nM

Ph

oto

n E

mis

sio

n

vehicle 0.01 0.05 0.1 0.5 1 5 100

500

1000

1500

2000

2500

E2 [ ]nM

RL

U/

g P

ro

tein

0.01 0.1 1 10

800

1600

2400

E2 [ ]nM

RL

U/

g P

ro

tein

Vehicle

WM

E M

EAA 1M

EAA 100

M

EAA500 M

NEAA 1M

NEAA 100

M

NEAA500 M

E+NE 1M

E+NE 100

M

E+NE 500

2

EAA+E 2

NEAA+E

0

200

400

600

800

RL

U/

g P

ro

tein

Argin

ine

Phenylala

nine

Threonin

e

Trypto

phan

Met

hionin

e

Histid

ine

Valine

Leucin

e

Lysin

e

Isole

ucine

0

200

400

600

800

RL

U/

g P

ro

tein

EC50=4.89nM***

*****

**

-

+

Reporter

green fluorescent dye

NR NR NR

Ligands Ligands Ligand

NR

NR NR

4

FUNCTIONAL CHARACTERIZATION OF ER LIGANDS

REPORTER MICE, THE ULTIMATE FUNCTIONAL ASSAY

INSULATOR ( MAR )

INSULATOR ( MAR )

light luciferin + ATP = oxyluciferin + AMP +

ERE 2x firefly luciferase

+/- +/-

TK

estrogen

ERE-Luc reporter mice to study

the dynamics of ER activity in living mice

Ciana et al., Mol. Endocrinol. 2001 Ciana et al., Nature Med. 2003

Access to the dynamic of molecular

events in complex organisms

Access to the dynamic of drug effects

3Rs rule in drug development

TIME 0 3 HOURS TIME 0 3 HOURS 24 HOURS TIME 0 3 HOURS 6 HOURS 24 HOURS

TIME 0

3 HOURS

6 HOURS *

* 2000

1000 *

*

Live

r p

ho

ton

em

issi

on

SEMI-QUANTITATIVE ANALYSIS OF ER ACTIVITY I N THE ERE-LUC REPORTER MOUSE

THE ERE-Luc REPORTER MOUSE A PHARMACOLOGICAL APPLICATION

pellet days of treatment

CONTROLS

REFERENCE DRUG

DRUG OF INTEREST

Manual

In vivo analysis of photon emission

Automatic

Rando et al. 2009

hepatic

ovx cyc

PCUD E

2OSP

RAL10

P+B

ZA

RAL2

TAM

BZA

LAS

0

100

200

300

500150025003500

AU

C

abdomen

ovx cyc

PCUD

P+B

ZA

RAL10

TAM E

2BZA

RAL2

OSP

LAS

0

50

100

150

200

250

AU

C

hepatic

ovx cyc E

2

PCUD

OSP

RAL10

BZA

LAS

P+B

ZA

RAL2

0

100

200

300

400500

1000

1500

2000cts

/cm

2 s

(vs c

yc=

100%

)

abdomen

ovx cyc

OSP

LAS E

2BZA

PCUD

P+B

ZA

RAL10

RAL2

0

50

100

150

200

250

cts

/cm

2 s

(vs c

yc=

100%

)

HEPATIC AREA ABDOMEN ACUTE

CHRONIC

ovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAMovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAM

Della Torre et al. Endocrinol 2011

SERCHING FOR NOVEL MODALITIES TO MEASURE THE EFFICACY OF SERMs

0

100000

200000

300000

400000

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Ph

oto

n e

mis

sio

n

p/s

/cm

2/s

r

Days

CHEST

AUC

N° peaks, amplitude, frequency

0

3

6

9

12

0

2

4

6

8

0

3

6

9

12

0

2

4

6

8

Peak

s/2

1d

Pe

rio

d (

d)

GENITAL AREA SKELETAL AREA

* * *

*

*

* * * * *

2

3

4

5

6

2

3

4

5

6

Am

plit

ud

e

0

50

100

150

200

250

0

100

200

300400600800

*

* * * * *

0

50

100

150

200

300

450

600

0

50

100

150

200

* * *

*

*

*

D

A

B

C

E

AU

C

Pote

ncy

ovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAMovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAM

Rando et al, Mol. Endocrinol. 2010

PHENETICS OF DRUG ACTION

THE APPLICATION OF AGGLOMERATIVE

HIERARCHICAL CLUSTERING TO THE STUDY OF THE

IN VIVO ACTION OF ESTROGENIC COMPOUNDS

Space-temporal analysis of drug action in living animals

Genital area

Skeletal area

ovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAMovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAM

Rando et al, Mol. Endocrinol. 2010

clustering data to generate novel families of compounds

Genital area

Skeletal area

Reverse Medicinal Chemistry A

B

C

Cl

ovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAMovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAM

Rando et al, Mol. Endocrinol. 2010

Time and space: the dimensions for

a full understanding of the effect of

drugs and pollutants ability to

interfere with intracellular receptors

activities

CONCLUSION 2

The ERE-Luc reporter mouse

represents a novel tool to identify

synthetic compounds and ED

present in the environment or in the

alimentary chain and to provide a

comprehensive view on their activity

on Ers whole body

University of Milan Center of Excellence on

Neurodegenerative Diseaseas

Elisabetta Vegeto Paolo Ciana Valeria Benedusi Sara Della Torre Saba Khalilpour Federica Lolli Nicoletta Rizzi Alessandro Villa Clara Meda Monica Rebecchi

Funding:

Past Collaborators :

Roberta Fontana Luisa Ottobrini Gianpaolo Rando

Paolo Ciana

Gianpaolo Rando

Sara Della Torre

Clara Meda

Collaborators:

“The whole is more than the sum of its parts “

Aristotle (384 BC – 322 BC)

Methapysics

Intracellular Receptors, are transcription factors able to bind the DNA