Nuclear receptors: in vitro and in vivo approaches Parma, June 13-15 2017

Adriana Maggi University of Milan www.cend.unimi.it

Intracellular Receptors, Androgen (AR; NR3C4) Aryl Hydrocarbon Receptor (AhR) a family of 48 members Constitutive Androstane Receptor (CAR; NR1I3) in humans Alpha (ERα; NR3A1) (ERβ; NR3A2) Estrogen Related Receptor Alpha (ERRα; (PXR; NR1I2) NR3B1) (PGR; NR3C3) Estrogen Related Receptor Gamma (ERRɣ; Receptor Alpha (RARα; NR1B1) NR3B3) Beta (RARβ; NR1B2) (FXR; NR1H4) Retinoic Acid Receptor Gamma (RARɣ; NR1B3) (GR; NR3C1) RAR-related Alpha (RORα; Liver Receptor Homolog-1 (LRH-1; NR5A2) NR1F1) Alpha (LXRα; NR1H3) RAR-related Orphan Receptor Gamma (RORɣ; (LXRβ; NR1H2) NR1F3) Mineralocorticoid Receptor (MR; NR3C2) Alpha (RXRα; NR2B1) Peroxisome Proliferator-Activated Receptor (RXRb; NR2B2) Alpha (PPARα; NR1C1) Retinoid X Receptor Gamma (RXRɣ; NR2B3) Peroxisome Proliferator-Activated Receptor Thyroid Alpha (TRα; Delta (PPARδ; NR1C2) NR1A1) Peroxisome Proliferator-Activated Receptor Beta (TRβ; NR1A2) Gamma (PPARɣ; NR1C3) (VDR; NR1I1)

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 IP3k cAMP

SP1 NFKB AP1 THE TOOLS TO STUDY ACTIVITIES

INTRACELLULAR RECEPTORS : THE TOOLS

INTRACELLULAR RECEPTORS : THE TOOLS FUNCTIONAL CHARACTERIZATION OF ER LIGANDS

BINDING ASSAY FUNCTIONAL CHARACTERIZATION OF ER LIGANDS

ER cDNA

ERE LUC

TRANSFECTION ASSAY

ER ER Units LUC LUC LUC

CTR E2 CTR FUNCTIONAL CHARACTERIZATION OF ER LIGANDS TRANSFECTION ASSAY

LigandsLigand

NR NR

NR

NR Reporter green fluorescent dye

4 ACTIVATION OF ERE-LUC REPORTER IN MOUSE HEPATOCYTES

15 minutes 2 hours 18 hours 1 week

2500 2400 + *** 2400 EC =4.89nM Vehicle EC =4.89nM 50 2000 50 ** 1600 1500 1600

E2 1 nM g Protein g

g Protein g 1000 ***

g Protein g

 

**  E 5 nM 800

2 500 800

RLU/

RLU/ Photon Emission Photon E 10 nM 0 RLU/ 2 vehicle 0.01 0.05 0.1 0.5 1 5 10 0.01 0.1 1 10 0.01 0.1 1 10 - E [ ]nM E2 [ ]nM 2 E2 [ ]nM

800 800 600 600

400

400

g Protein g

g Protein g

 

200 200

RLU/ RLU/ 0 0

M M M M M M M M M 2 2 WME Vehicle EAA+E Valine EAA 1 E+NE 1 NEAA+E LeucineLysine EAA 100 EAA500 NEAA 1 Arginine Histidine NEAA 100NEAA500 E+NE 100E+NE 500 ThreonineTryptophanMethionine Isoleucine Phenylalanine FUNCTIONAL CHARACTERIZATION OF ER LIGANDS

REPORTER MICE, THE ULTIMATE FUNCTIONAL ASSAY

LigandsLigand

NR NR

NR

NR Reporter green fluorescent dye

4 ERE-Luc reporter mice to study the dynamics of ER activity in living mice

estrogen +/- +/- INSULATOR INSULATOR ERE 2x TK ( MAR ) firefly luciferase ( MAR )

luciferin + ATP = oxyluciferin + AMP + light

Ciana et al., Mol. Endocrinol. 2001 Ciana et al., Nature Med. 2003 SEMI-QUANTITATIVE ANALYSIS OF ER ACTIVITY I N THE ERE-LUC REPORTER MOUSE

2000 *

TIME 0 emission 3 HOURS 1000

* 6 HOURS

photon

TIME 0 3 HOURS 6 HOURS 24 HOURS 24 HOURS Liver Access to the dynamic of molecular events in complex organisms Access to the dynamic of drug effects

3Rs rule in drug development THE ERE-Luc REPORTER MOUSE A PHARMACOLOGICAL APPLICATION pellet days of treatment

CONTROLS In vivo analysis of photon emission

Manual Automatic

REFERENCE DRUG

DRUG OF INTEREST

Rando et al. 2009 HEPATIC AREA ABDOMEN hepatic abdomen ACUTE 2000 250 1500 1000 200 500 400 150 300

100

s (vs cyc=100%) s (vs cyc=100%)

200 2 2 50

100 cts/cm cts/cm 0 hepatic 0 abdomen

3500 250 E2 E2 ovx cyc ovx cyc OSP BZA LAS OSP LAS BZA 2500 RAL2 PCUD RAL2 PCUD RAL10 P+BZA P+BZA RAL10 CHRONIC 1500 200 500 150

300

AUC AUC 200 100

100 50

0 0

E2 E2 ovx cyc BZA LAS ovx cyc BZA OSP LAS OSP RAL2 TAM TAM RAL2 PCUD RAL10P+BZA PCUDP+BZARAL10

ovx cyc E2 CE CE+BZA BZA RAL2 RAL10 LAS OSP TAM

Della Torre et al. Endocrinol 2011 SERCHING FOR NOVEL MODALITIES TO MEASURE THE EFFICACY OF SERMs

N° peaks, amplitude, frequency CHEST

400000

300000

/sr 2

200000 p/s/cm

100000 Photon emission Photon AUC

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

8 6 6 * * * ** A 4 4 2 2

Peaks/21d 0 0

12 12 9 * * 9 6 * 6 B * 3 3

Amplitude 0 0 6 6 * 5 5 C 4 4

3 3 Period (d) Period 2 2

800 250 600 200 * 400 * 300 * 150 * *

D AUC 200 * 100 100 50 0 0 600

200 450 * 150 * 300 * 200 E 100 * 150 *

Potency 50 100 * 50 0 0 Rando et al, Mol. Endocrinol. 2010 ovx cyc E2 CE CE+BZA BZA RAL2 RAL10 LAS OSP TAM PHENETICS OF DRUG ACTION

THE APPLICATION OF AGGLOMERATIVE HIERARCHICAL CLUSTERING TO THE STUDY OF THE IN VIVO ACTION OF ESTROGENIC COMPOUNDS clustering data Space-temporal analysis of drug action in to generate novelliving families animals of compounds

Genital area

Skeletal area

ovx cyc E2 CE CE+BZA BZA RAL2 RAL10 LAS OSP TAM Rando et al, Mol. Endocrinol. 2010 A Genital area C Reverse Medicinal Chemistry

B Skeletal area

Cl

ovx cyc E2 CE CE+BZA BZA RAL2 RAL10 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

Past Collaborators : Collaborators: Roberta Fontana Elisabetta Vegeto Luisa Ottobrini Paolo Ciana Gianpaolo Rando Gianpaolo Rando

Valeria Benedusi Paolo Ciana Sara Della Torre Funding: Saba Khalilpour Federica Lolli Nicoletta Rizzi Alessandro Villa

Clara Meda Sara Della Torre Monica Rebecchi

Clara Meda “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