Nuclear receptors: in vitro and in vivo · Peroxisome Proliferator-Activated Receptor Gamma...
Transcript of Nuclear receptors: in vitro and in vivo · Peroxisome Proliferator-Activated Receptor Gamma...
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