INGEGNERIA ANIMALEAPPLICAZIONI

CORSO DI LAUREA SPECIALISTICA IN BIOTECNOLOGIE DEL FARMACO AA 2010-11

Adriana MaggiLEZIONE 13

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

Optical imagingBioluminescence

Ultrasound

MicroPETPositron emission tomography

Nuclear imaging

Micro SPECTsingle-photon emission

computerized tomography

2/06

Fluorescence

LA GENERAZIONE DI UN TOPO REPORTER

La scelta del:

sistema di ingegneria animale

reporter bioluminescenza, PET, NMR

costrutto

reporter

Reporter bioluminescenti e fluorescenti

I geni reporter

Monomerico, non richiede substrato, assente nei mammiferi, varianti con diversa l di fluorescenza. Bassa sensibilità per mancanza di amplificazione

fluorescent proteins (GFP, varianti RFP, BFP)

Utilizzabili per PETDifficoltà nel generare i ligandi radiomarcati

Ligandi per sostanze radiomarcate

Alta attività specifica, mancanza di attività endogena (basso bkg) Richiede l’aggiunta di cofattore, O2, ATP.

Luciferasi (Lucciola)

Utile per studi di tipo anatomico-funzionale

Ben caratterizzata, stabile, rilevazione automatizzabile

B-Galattosidasi (Batterica)

GENE

Presenza di attività endogena in cellule di mammifero, enzima tetramerico (risposta non lineare)

Applicabile a studi di bioluminescenza in vivo

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

reporter

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

INSULATOR( MAR )

INSULATOR

( MAR )

lightluciferin + ATP = oxyluciferin + AMP +

TKERE 2x

firefly luciferase

+/- +/-

ERE-Luc reporter mouse

The ERE-Luc reporter mouse: a model to study of ER transcriptional activity

Ciana et al., 2001

ERE

kinase-dependent activation

i.p. of D-luciferin

20 min.

Evaluation of ER transcriptional activity

5 min. after the acquisition

18.516.514.513.5 15.5

ERE-Luc mouse

ER is transcriptionally active at day 14.5 pc

dpc (day post conception)

ERE-Luc mouse

P118.516.514.513.512.5dpc (day post conception) post-natal

day 1

endoderm mesoderm ectodermimaging IHC

A B C D

E

F

G

H

C – intestine 20xD – bone 40xE – heart 40xF – forebrain 10xG – moustache 40x H – skin 20x

16.5 dpc 16.5 dpc

GP Rando, 2007

ERE-Luc mouse bone

0

2

4

6

thymus

01

23

4

brain

0

5

10

15

20

P E M D2

intestine

0

48

1216

bone

0

2

4

6

thymus

01

23

4

brain

0

5

10

15

20

LUCI

FERA

SE A

CTIV

ITY

(RLU

)

P E D

intestine

0

48

1216

liver

0

10

20

P E M D2

ovaries

0

2

4

uterus

13579

0

6

12

liver

0

10

20

P E D2P E D

ovaries

0

2

4

uterus

13579

0

6

12 hypothalamus

M D P E

Estr

adio

l(pg/m

l)

1020

30

40

50

day 1 day 2 day3 day 4

SESTRUS

E2

(p

g/m

l)

1020304050

M D P E

Luciferase activity in adult, cycling females

Ciana et al, Nature Ned., 2003

Suckling Mice

Pregnancy &

EmbryoDevelopment

13.5

14.5

15.5

16.5

17.5

18.5

19.5

12.5

DAY 18.5

DAY 16.5

DAY 13.5

DAY 14.5

DAY 15.5

DAY 1

DAY 10

Immature Mice

Adult Mice

LIFE

CYCLE

ERE-Luc mice to understand ER involvement in mammals physiopathology

THE COMPLEXITY OFESTROGEN ACTION

ERE

G-protein,

IP3K...

SP1NFKBAP1

THE COMPLEXITY OF ESTROGEN TARGETS

• REPRODUCTIVE SYSTEM- male and female gonads- hypothalamus and pituitary

• SKELETAL SYSTEM• VASCULAR SYSTEM

- endothelium- smooth muscle cells

• RESPIRATORY SYSTEM• IMMUNE SYSTEM• NERVOUS SYSTEM

- central- peripheral

CNS

cardiovascular

reproductive

bone

ERE

SP1NFKBAP1

tissue-specificcoregulators

growthfactors P

ER COMPLEXITY OF ACTION and A NEW CLASS OF DRUGS: Selective Estrogen Recepor Modulators

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

Reproductive organs(mammari glands and vagina)

Muscle-Skeletal System

Hepatic area

Measuring bioluminscence in the ERE-Luc reporter mouse

Thymic area

Intestine

Bioluminescence after6h treatment with

15b-estradiol (50ug/kg)

pellet days of treatment

CONTROLS

REFERENCE DRUG

DRUG OF INTEREST

Manual

In vivo analysis of photon emission

Automatic

Rando et al. 2009

REPORTER MICE TO STUDY DRUG ACTION “IN VIVO”

0

5

10

15

20 UTERUS

RLU

0

500000

1000000

1500000

2000000

AUC

VAGINA

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2110000

40000

70000

100000

VAGINA

Days

Phot

on e

miss

ion

p/s/

cm2/

sr

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 210

100000

200000

300000

400000

CHEST

Days

Phot

on

emis

sion

p/

s/cm

2/sr

0

2000000

4000000

6000000

8000000

AUC

CHEST

0

100

200

300

400

500 LIVER

RLU

VehiclePCUD 3mg/kgBZA 10mg/kgBZA 10mg/kg + PCUD 3mg/kgRaloxifene 10mg/kg

Luciferase enzymatic activityPhoton emission

Biserni et al, in preparation

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2110000

40000

70000

100000

REPRODUCTIVE AREA

Days

Phot

on e

mis

sion

p/

s/cm

2/sr

0

500000

1000000

1500000

2000000

AUC

°**

VehiclePCUD 3mg/kgBZA 10mg/kgBZA 10mg/kg + PCUD 3mg/kgRaloxifene 10mg/kg

Effects of SERMs on ER activity – in vivo imaging

CHRONIC treatment (21 days)

Biserni et al, in preparation

Effects of SERMs on ER activity – in vivo imaging

CHRONIC treatment (21 days)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2120000

40000

60000

80000

100000

TAIL

Days

Phot

on e

mis

sion

p/

s/cm

2/sr

0

500000

1000000

1500000

2000000

AUC

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2120000

40000

60000

80000

100000

LIMBS

Days

Phot

on e

mis

sion

p/

s/cm

2/sr

0

500000

1000000

1500000

2000000

AUC

VehiclePCUD 3mg/kgBZA 10mg/kgBZA 10mg/kg + PCUD 3mg/kgRaloxifene 10mg/kg

Biserni et al, in preparation

limb

0

100

200

300

400

500

AU

C

tail

0

50

100

150

200

250

AU

C

limb

0

50

100

150

200

250

cts/c

m2 s

(vs c

yc=

100%

)

tail

0

50

100

150

200300400500600

cts

/cm2 s

(vs c

yc=

100%

)

LIMB TAIL

ACUTE

CHRONIC

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

hepatic

0

100

200

300

500150025003500

AU

C

abdomen

0

50

100

150

200

250

AU

C

hepatic

0

100

200

300

400500

100015002000

cts/c

m2 s

(vs

cy

c=

100

%)

abdomen

0

50

100

150

200

250

cts

/cm2 s

(vs c

yc=

100%

)

HEPATIC AREA ABDOMENACUTE

CHRONIC

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

SERCHING FOR NOVEL MODALITIES TO MEASURE THE EFFICACY OF SERMs

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 210

100000

200000

300000

400000

CHEST

Days

Phot

on e

mis

sion

p/

s/cm

2/sr

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/21

dPe

riod

(d)

GENITAL AREA SKELETAL AREA

***

*

*

* ****

2

3

4

5

6

2

3

4

5

6

Ampl

itude

0

50

100

150

200

250

0

100

200

300400600800

**

* ***

050

100150200300450600

0

50

100

150

200

***

*

**

D

A

B

C

E

AUC

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

(AGGLOMERATIVE NESTING version 1.02 )

2

4

6

8

0.1

1

10

100

1000

10

100

1000

10000

**p<0.01

0 2 4 6 81

10

100

2 4 6 8 0.1 1 10 100

**p<0.01

10 100 1000 10000

**p<0.01

peak number period amplitude AUC

per

iod

amp

litu

de

AU

Cp

ote

ncy

a

period 0.04

amplitude 0.05 <0.01

AUC 0.03 0.05 0.96

potency 0.10 0.05 0.76 0.46

peaks number

period amplitude AUC

b

DEGREE OF FUNCTIONAL CORRELATION AMONG THE PARAMETERS SELECTED

Rando et al, Mol. Endocrinol. 2010

Space-temporal analysis of drug action in living animals

clustering data to generate novel families of compounds

Genital area

Skeletal area

ovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAMovx cyc E2 CE CE+BZA BZA RAL10RAL2 LAS OSP TAMRando et al, Mol. Endocrinol. 2010

Genital area

Skeletal area

Reverse Medicinal ChemistryA

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

CONCLUSION 1

Adding the time dimension to the study of drug activity leads to a novel ability to define drug efficacy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 220

2000

4000

6000

8000

LIMBGENITALAREAHEPATIC AREA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 220

20000

40000

60000

80000

LIMBGENITAL AREAHEPATIC AREA

INTACT

OVX

Biserni et al. in preparation

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 post-menopause

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)

University of MilanCenter of Excellence on

Neurodegenerative Diseaseas

Collaborators at Milan University: Paola Campadelli

David Horner

Paolo CianaElisabetta Vegeto

Gianpaolo RandoValeria BenedusiSara Della TorreCristina VantaggiatoCristian IbarraBalaji RamachandranAndrea BiserniMonica RebecchiClara Meda

“The whole is more than the sum of its parts “Aristotle (384 BC – 322 BC)Methapysics

The real impact of molecular engineering on drug discovery

MODERN PHARMACOLOGY NEEDS TO REVISIT ANIMAL

MODELS