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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?.

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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 GENERAZIONE DI ANIMALI IN CUI SI POSSANO MISURARE EVENTI MOLECOLARI SPECIFICI IN TEMPO REALE?IMAGING


Functional Magnetic Nuclear Resonance GENERAZIONE DI ANIMALI IN CUI SI POSSANO MISURARE EVENTI MOLECOLARI SPECIFICI IN TEMPO REALE?

Nuclear imaging

MicroPET

Positron emission tomography

Micro SPECT

single-photon emission

computerized tomography

Bioluminescence

Optical imaging

Fluorescence

Ultrasound

2/06

MULTI-MODALITY MOLECULAR IMAGING IN LIVING SMALL ANIMALS


LA GENERAZIONE DI UN TOPO REPORTER GENERAZIONE DI ANIMALI IN CUI SI POSSANO MISURARE EVENTI MOLECOLARI SPECIFICI IN TEMPO REALE?

La scelta del:

sistemadiingegneriaanimale

reporter bioluminescenza, PET, NMR

costrutto

reporter


Reporter bioluminescenti e fluorescenti GENERAZIONE DI ANIMALI IN CUI SI POSSANO MISURARE EVENTI MOLECOLARI SPECIFICI IN TEMPO REALE?


GENE GENERAZIONE DI ANIMALI IN CUI SI POSSANO MISURARE EVENTI MOLECOLARI SPECIFICI IN TEMPO REALE?

B-Galattosidasi (Batterica)

Ben caratterizzata, stabile, rilevazione automatizzabile

Utile per studi di tipo anatomico-funzionale

Luciferasi (Lucciola)

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

Ligandi per sostanze radiomarcate

Difficoltà nel generare i ligandiradiomarcati

Utilizzabili per PET

fluorescent proteins (GFP, varianti RFP, BFP)

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

I geni reporter

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 GENERAZIONE DI ANIMALI IN CUI SI POSSANO MISURARE EVENTI MOLECOLARI SPECIFICI IN TEMPO REALE?

La scelta del:

sistemadiingegneriaanimale

reporter bioluminescenza, PET, NMR

costrutto

reporter


Studio GENERAZIONE DI ANIMALI IN CUI SI POSSANO MISURARE EVENTI MOLECOLARI SPECIFICI IN TEMPO REALE?dellafunzionedipromotore

reporter gene

reporter gene


I sistemi reporter nello studio di rilascio/sintesi di trasduttori del segnale (complementazione)



+/- trasduttori del

+/-

ERE 2x

INSULATOR

( MAR )

firefly luciferase

INSULATOR

( MAR )

TK

light

luciferin + ATP = oxyluciferin + AMP +

ERE-Luc reporter mouse

ERE

The ERE-Luc reporter mouse: a model

tostudyof ER transcriptionalactivity

kinase-dependent

activation

Ciana et al., 2001


Evaluation of ER transcriptional activity trasduttori del

20 min.

i.p. of D-luciferin

5 min. after the acquisition


13.5 trasduttori del

14.5

15.5

16.5

18.5

ERE-Luc mouse

dpc (day post conception)

ER istranscriptionallyactive at day 14.5 pc


ectoderm trasduttori del

endoderm

mesoderm

13.5

14.5

16.5

18.5

12.5

P1

dpc (day post conception)

post-natal day 1

ERE-Luc mouse

imaging

IHC

F

C

D

A

B

C – intestine 20x

D – bone 40x

E – heart 40x

F – forebrain 10x

G – moustache 40x

H – skin 20x

G

E

H

16.5 dpc

16.5 dpc

GP Rando, 2007


6 trasduttori del

6

4

4

bone

bone

ovaries

ovaries

4

4

2

2

2

2

0

0

0

0

9

9

20

20

brain

brain

uterus

uterus

7

7

15

15

5

5

10

10

3

3

5

5

1

1

0

0

LUCIFERASE ACTIVITY (RLU)

4

4

12

12

thymus

thymus

hypothalamus

3

3

2

2

6

6

E

M

D

P

1

1

0

0

0

0

20

20

16

16

intestine

intestine

liver

liver

12

12

10

10

8

8

4

4

0

0

P

E

M

D2

0

0

P

P

E

E

M

D2

D

P

P

E

E

D

D2

ERE-Luc mouse

Luciferase activity in adult, cycling females

/ml)

ESTRUS

S

50

5

pg

0

40

(

4

E2 (pg/ml)

0

30

3

Estradiol

0

20

2

0

10

10

day 1

M

day 2

D

day3

P

day 4

E

Ciana et al, Nature Ned., 2003


Pregnancy trasduttori del

&

Embryo

Development

19.5

DAY 1

18.5

DAY 10

DAY 18.5

LIFE

17.5

DAY 16.5

CYCLE

16.5

DAY 15.5

15.5

DAY 14.5

14.5

13.5

Immature

DAY 13.5

Mice

12.5

AdultMice

ERE-Lucmicetounderstand ER involvement in mammalsphysiopathology

SucklingMice


THE COMPLEXITY OF trasduttori del

ESTROGEN ACTION

THE COMPLEXITY OF ESTROGEN TARGETS

  • REPRODUCTIVE SYSTEM

    • - male and femalegonads

    • - hypothalamus and pituitary

  • SKELETAL SYSTEM

  • VASCULAR SYSTEM

    • - endothelium

    • smoothmusclecells

  • RESPIRATORY SYSTEM

  • IMMUNE SYSTEM

  • NERVOUS SYSTEM

    • - central

    • - peripheral

G-protein,

IP3K...

SP1

NFKB

AP1

ERE


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

CNS

cardiovascular

reproductive

growthfactors

P

bone

SP1

NFKB

AP1

tissue-specific

coregulators

ERE


ESTROGEN REPLACEMENT THERAPY trasduttori del

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 trasduttori del bioluminscence

in the ERE-Luc reporter mouse

Thymic area

Hepatic area

Bioluminescenceafter

6h treatment with

15b-estradiol (50ug/kg)

Reproductiveorgans

(mammari glands and

vagina)

Intestine

Muscle-Skeletal System


days of treatment trasduttori del

pellet

CONTROLS

REFERENCE DRUG

DRUG OF INTEREST

In vivo analysis of photon emission

Manual

Automatic

Rando et al. 2009


REPORTER MICE TO STUDY DRUG ACTION “IN VIVO” trasduttori del

VAGINA

CHEST

Photonemission

Luciferaseenzymaticactivity

Vehicle

PCUD 3mg/kg

BZA 10mg/kg

BZA 10mg/kg + PCUD 3mg/kg

Raloxifene 10mg/kg

Biserni et al, in preparation


Effects trasduttori del ofSERMs on ER activity – in vivo imaging

CHRONIC treatment (21 days)

**

°

Vehicle

PCUD 3mg/kg

BZA 10mg/kg

BZA 10mg/kg + PCUD 3mg/kg

Raloxifene 10mg/kg

Biserni et al, in preparation


Effects trasduttori del ofSERMs on ER activity – in vivo imaging

CHRONIC treatment (21 days)

Vehicle

PCUD 3mg/kg

BZA 10mg/kg

BZA 10mg/kg + PCUD 3mg/kg

Raloxifene 10mg/kg

Biserni et al, in preparation


TAIL trasduttori del

LIMB

ACUTE

CHRONIC

Rando et al, Mol. Endocrinol. 2010


HEPATIC AREA trasduttori del

ABDOMEN

ACUTE

CHRONIC

Rando et al, Mol. Endocrinol. 2010


SERCHING FOR NOVEL MODALITIES TO MEASURE trasduttori del

THE EFFICACY OF SERMs

N° peaks, amplitude, frequency

AUC


GENITAL AREA trasduttori del

SKELETAL AREA

*

*

*

*

*

A

Peaks/21d

*

*

*

*

Amplitude

B

*

Period (d)

C

*

*

*

*

*

AUC

*

D

Rando et al, Mol. Endocrinol. 2010

*

*

*

*

E

Potency

*

*


PHENETICS OF DRUG ACTION trasduttori del

THE APPLICATION OF AGGLOMERATIVE HIERARCHICAL CLUSTERING

(AGGLOMERATIVE NESTING version 1.02 )


DEGREE OF FUNCTIONAL CORRELATION AMONG THE PARAMETERS SELECTED

a

b

Rando et al, Mol. Endocrinol. 2010


Space-temporal analysis of drug action in living animals SELECTED

clustering data to generate novel families of compounds

Genital area

Skeletal area

Rando et al, Mol. Endocrinol. 2010


C SELECTED

Reverse Medicinal Chemistry

A

Genital area

B

Skeletal area

Cl

Rando et al, Mol. Endocrinol. 2010


CONCLUSION 1 SELECTED

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


INTACT SELECTED

OVX

Biserni et al. in preparation


TISSUE SPECIFIC EFFECT OF OVX ON SELECTED

THE AMPLITUDE AND FREQUENCY OF ER ACTIVITY


CONCLUSION 2 SELECTED

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


University of Milan SELECTED

Center of Excellence on

Neurodegenerative Diseaseas

Paolo Ciana

ElisabettaVegeto

GianpaoloRando

Valeria Benedusi

Sara Della Torre

Cristina Vantaggiato

Cristian Ibarra

BalajiRamachandran

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 SELECTED

“The whole is more than the sum of its parts “

Aristotle (384 BC – 322 BC)

Methapysics

MODERN PHARMACOLOGY

NEEDS TO REVISIT ANIMAL MODELS


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