CORSO DI LAUREA SPECIALISTICA IN BIOTECNOLOGIE DEL
FARMACO AA 2010-11
Adriana Maggi
LEZIONE 13
INGEGNERIA ANIMALE
APPLICAZIONI
I SISTEMI REPORTER POSSONO ESSERE
UTILIZZATI PER LA GENERAZIONE DI
ANIMALI IN CUI SI POSSANO MISURARE
EVENTI MOLECOLARI SPECIFICI IN TEMPO
REALE?
IN VIVO IMAGING
MULTI-MODALITY MOLECULAR IMAGING IN LIVING
SMALL ANIMALS
Functional Magnetic Nuclear Resonance
Nuclear imaging
MicroPET
Positron emission tomography
Micro SPECT
single-photon emission
computerized tomography
Optical imaging
Bioluminescence
Fluorescence
Ultrasound
2/06
LA GENERAZIONE DI UN TOPO REPORTER
La scelta del:
sistema di ingegneria animale
reporter bioluminescenza, PET, NMR
costrutto
Reporter bioluminescenti e fluorescenti
I geni reporter
GENE
B-Galattosidasi
(Batterica)
Luciferasi
(Lucciola)
Ligandi per
sostanze
radiomarcate
fluorescent
proteins (GFP,
varianti RFP, BFP)
Ben caratterizzata, stabile,
rilevazione automatizzabile
Presenza di attività endogena in
cellule di mammifero, enzima
tetramerico (risposta non
lineare)
Alta attività specifica, mancanza di
attività endogena (basso bkg)
Richiede l’aggiunta di cofattore,
O2, ATP.
Difficoltà nel generare i ligandi
radiomarcati
Monomerico, non richiede
substrato, assente nei mammiferi,
varianti con diversa l di
fluorescenza. Bassa sensibilità per
mancanza di amplificazione
Utile per studi di tipo anatomicofunzionale
Applicabile a studi di
bioluminescenza in vivo
Utilizzabili per PET
Applicabile a studi di
fluorescenza in vivo
LA GENERAZIONE DI UN TOPO REPORTER
La scelta del:
sistema di ingegneria animale
reporter bioluminescenza, PET, NMR
costrutto
Studio della funzione di promotore
reporter gene
reporter gene
I sistemi reporter nello studio di rilascio/sintesi di
trasduttori del segnale (complementazione)
I sistemi reporter nello studio di interazioni tra proteine
The ERE-Luc reporter mouse: a model
to study of ER transcriptional activity
+/-
+/INSULATOR
( MAR )
kinase-dependent
activation
ERE 2x
TK
INSULATOR
firefly luciferase
luciferin + ATP = oxyluciferin + AMP +
( MAR )
light
ERE-Luc reporter mouse
Ciana et al., 2001
Evaluation of ER transcriptional activity
20 min.
i.p. of D-luciferin
5 min. after the acquisition
ERE-Luc mouse
13.5
14.5
15.5
16.5
18.5
dpc (day post conception)
ER is transcriptionally active at day 14.5
pc
ERE-Luc mouse
13.5
14.5
12.5
dpc (day post conception)
imaging
A
IHC
B
16.5
endoderm
C
18.5
P1
post-natal
day 1
ectoderm
mesoderm
D
F
G
E
H
16.5 dpc
GP Rando, 2007
16.5 dpc
C – intestine 20x
D – bone
40x
E – heart
40x
F – forebrain 10x
G – moustache 40x
H – skin
20x
4
ERE-Luc mouse
ovaries
6
bone
4
2
2
(pg/ml)
Estradiol
E2 (pg/ml)
M
50
5
040
4
30
30
020
2
0
10
10
D
day
M 1
P
day
D 2
day3
P
E
day
E 4
LUCIFERASE ACTIVITY (RLU)
Luciferase activity in adult, cycling females
0
9
7
5
3
1
12
uterus
0
20
brain
15
10
5
hypothalamus
0
4
6
3
2
0
20
1
0
16
liver
10
thymus
intestine
12
8
4
0
Ciana et al, Nature Ned., 2003
0
P
E
D2
M D
P
E
M D
D2
ERE-Luc mice to understand ER involvement in mammals
physiopathology
Pregnancy
&
Embryo
Development
Suckling
Mice
19.5
DAY 1
18.5
DAY 10
DAY 18.5
17.5
DAY 16.5
16.5
CYCLE
DAY 15.5
15.5
14.5
LIFE
DAY 14.5
13.5
Immature
Mice
DAY 13.5
12.5
Adult Mice
THE COMPLEXITY OF
ESTROGEN ACTION
THE COMPLEXITY OF
ESTROGEN TARGETS
• REPRODUCTIVE SYSTEM
- male and female gonads
- hypothalamus and pituitary
G-protein,
IP3K...
SP1
NFKB
AP1
• SKELETAL SYSTEM
• VASCULAR SYSTEM
- endothelium
- smooth muscle cells
• RESPIRATORY SYSTEM
• IMMUNE SYSTEM
• NERVOUS SYSTEM
- central
- peripheral
ER COMPLEXITY OF ACTION
and A NEW CLASS OF
DRUGS: Selective Estrogen
Recepor Modulators
CNS
cardiovascular
growth
factors
reproductive
P
bone
SP1
NFKB
AP1
tissue-specific
coregulators
ESTROGEN REPLACEMENT THERAPY
The efficacy of SERMs on estrogen receptor
transcriptional activity was measured in a model
of surgical menopause (ovx mice)
SERMs ability to replace the natural hormone was
evaluated by comparison with ER activity in
healthy, cycling mice
Measuring bioluminscence
in the ERE-Luc reporter mouse
Hepatic area
Reproductive organs
(mammari glands and
vagina)
Intestine
Muscle-Skeletal System
Thymic area
Bioluminescence after
6h treatment with
15b-estradiol (50ug/kg)
pellet
days of treatment
CONTROLS
In vivo analysis of photon emission
Manual
Automatic
REFERENCE DRUG
DRUG OF INTEREST
Rando et al. 2009
REPORTER MICE TO STUDY DRUG ACTION “IN VIVO”
Photon emission
1000000
500000
0
8000000
AUC
20
VAGINA
RLU
1500000
Luciferase enzymatic activity
UTERUS
15
10
5
0
CHEST
500
6000000
4000000
2000000
LIVER
300
200
100
0
0
CHEST
2
Photon emission
Photon
p/s/cm /sr
emission
p/s/cm2/sr
100000
70000
40000
0
VAGINA
400000
300000
200000
100000
10000
Biserni et al, in preparation
Vehicle
PCUD 3mg/kg
BZA 10mg/kg
BZA 10mg/kg + PCUD 3mg/kg
Raloxifene 10mg/kg
400
RLU
AUC
2000000
1
0
2
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Days
Days
Effects of SERMs on ER activity – in vivo imaging
CHRONIC treatment (21 days)
2000000
100000
**
°
1500000
70000
AUC
Photon emission
p/s/cm2/sr
REPRODUCTIVE AREA
40000
1000000
500000
10000
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21
0
Days
Biserni et al, in preparation
Vehicle
PCUD 3mg/kg
BZA 10mg/kg
BZA 10mg/kg + PCUD 3mg/kg
Raloxifene 10mg/kg
Effects of SERMs on ER activity – in vivo imaging
CHRONIC treatment (21 days)
100000
2000000
80000
1500000
AUC
Photon emission
p/s/cm2/sr
LIMBS
60000
1000000
40000
500000
20000
0
1
2
3
4
5
6
7
8
0
9 10 11 12 13 14 15 16 17 18 19 20 21
Days
Vehicle
PCUD 3mg/kg
BZA 10mg/kg
BZA 10mg/kg + PCUD 3mg/kg
Raloxifene 10mg/kg
100000
2000000
80000
1500000
AUC
Photon emission
p/s/cm2/sr
TAIL
60000
40000
1000000
500000
20000
0
1
2
3
4
5
6
Biserni et al, in preparation
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21
Days
0
ovx
cyc
E2
CE
Rando et al, Mol. Endocrinol. 2010
CE+BZA BZA
150
0
0
RAL2
RAL10
LAS
OSP
LA
S
TA
M
A
L2
R
ZA
0
LA
S
A
L1
SP
O
A
L2
R
50
ZA
100
B
100
R
300
SP
250
O
200
0
B
TAIL
E2
limb
PC
U
D
P+
B
ZA
50
E2
100
cy
c
150
ov
x
200
cts/cm2 s (vs cyc=100%)
limb
PC
U
P+ D
B
Z
R A
A
L1
0
200
AUC
250
ov
x
cy
c
TA
M
R
A
L2
LA
S
SP
O
400
E2
PC
U
D
B
ZA
LA
S
O
S
P+ P
B
ZA
R
A
L2
R
A
L1
0
cy
c
0
E2
ZA
A
L1
0
PC
U
D
B
ZA
CHRONIC
R
500
ov
x
cts/cm2 s (vs cyc=100%)
ACUTE
B
P+
ov
x
cy
c
AUC
LIMB
tail
600
500
400
300
200
150
100
50
tail
TAM
HEPATIC
AREA
hepatic
ovx
50
CE
CE+BZA BZA
Rando et al, Mol. Endocrinol. 2010
RAL2
ZA
PC
U
D
P+
B
ZA
R
A
L1
0
R
A
L2
B
E2
LA
S
SP
RAL10
LAS
B
ZA
R
A
L2
O
SP
LA
S
R
O
PC
U
E2
E2
0
ov
x
cy
c
0
SP
A
L1
P+ 0
B
ZA
R
A
L2
TA
M
B
ZA
LA
S
50
E2
100
D
100
PC
U
P+ D
B
Z
R A
A
L1
0
TA
M
AUC
150
200
cyc
abdomen
0
250
E2
PC
U
D
O
SP
R
A
L1
0
B
ZA
LA
S
P+
B
ZA
R
A
L2
cy
c
100
200
300
ov
x
cy
c
AUC
hepatic
0
150
O
100
200
cy
c
200
250
ov
x
300
3500
2500
1500
500
CHRONIC
cts/cm2 s (vs cyc=100%)
2000
1500
1000
500
400
ov
x
cts/cm2 s (vs cyc=100%)
ACUTE
ABDOMEN
abdomen
OSP
TAM
SERCHING FOR NOVEL MODALITIES TO MEASURE
THE EFFICACY OF SERMs
N° peaks, amplitude, frequency
Photon emission
p/s/cm2/sr
400000
CHEST
300000
200000
AUC
100000
0
0
1
2
3
4
5
6
7
8
9
10
11
Days
12
13
14
15
16
17
18
19
20
21
GENITAL AREA
C
8
6
6
4
4
2
2
0
0
12
12
Peaks/21d
Amplitude
B
8
9
6
Period (d)
A
*
**
*
6
3
0
0
6
6
5
5
4
4
3
3
2
2
AUC
200
*
*
200
*
150
50
0
200
600
450
300
200
150
100
50
0
100
* **
100
0
Potency
E
*
250
100
150
* * * **
9
3
800
600
400
300
D
SKELETAL AREA
*
**
50
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
(AGGLOMERATIVE NESTING version 1.02 )
period
DEGREE OF FUNCTIONAL CORRELATION AMONG THE PARAMETERS SELECTED
a
8
b
period
0.04
amplitude
0.05
<0.01
AUC
0.03
0.05
0.96
potency
0.10
0.05
0.76
0.46
period amplitude
AUC
6
4
amplitude
2
1000
peaks
number
100
10
1
0.1
10000
AUC
**
p<0.01
1000
100
10
100
**
potency
**
p<0.01
p<0.01
10
1
0
2
4
6
peak number
Rando et al, Mol. Endocrinol. 2010
8 2
4
6
period
8 0.1
1
10
amplitude
100 10
100 1000 10000
AUC
Space-temporal analysis of drug action in
living animals
clustering data to generate novel families of
compounds
Genital area
Skeletal area
Rando et al, Mol. Endocrinol. 2010
ovx
cyc
E2
CE
CE+BZA BZA
RAL2
RAL10
LAS
OSP
TAM
A
Genital area
B
Skeletal area
C Reverse Medicinal Chemistry
Cl
ovx
cyc
E2
CE
CE+BZA BZA
Rando et al, Mol. Endocrinol. 2010
RAL2
RAL10
LAS
OSP
TAM
CONCLUSION 1
Adding the time dimension to the
study of drug activity leads to a
novel ability to define drug efficacy
INTACT
8000
6000
LIMB
4000
GENITALAREA
HEPATIC AREA
2000
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22
80000
OVX
60000
LIMB
40000
GENITAL AREA
HEPATIC AREA
20000
0
1
2
3
4
5
Biserni et al. in preparation
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22
TISSUE SPECIFIC EFFECT OF OVX ON
THE AMPLITUDE AND FREQUENCY OF ER ACTIVITY
CONCLUSION 2
The possibility to measure in vivo the
activity of estrogenic compounds on
their target, may lead to the
identification of novel and more
efficacious therapies for the postmenopause
University of Milan
Center of Excellence on
Neurodegenerative Diseaseas
Paolo Ciana
Elisabetta Vegeto
Gianpaolo Rando
Valeria Benedusi
Sara Della Torre
Cristina Vantaggiato
Cristian Ibarra
Balaji Ramachandran
Andrea Biserni
Monica Rebecchi
Clara Meda
Collaborators at Milan University:
Paola Campadelli
David Horner
Funding: EU Strep EWA EWA LSHM-CT-2005-518245
EU IP CRESCENDO LSHM-CT-2005-018652
EU NoE DIMI LSHB-CT-2005-512146
NIH RO1(AG027713)
The real impact of molecular
engineering on drug discovery
“The whole is more
than the sum of its
parts “
Aristotle (384 BC – 322 BC)
Methapysics
MODERN PHARMACOLOGY
NEEDS TO REVISIT ANIMAL
MODELS
Scarica

Diapositiva 1