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