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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?
Functional Magnetic Nuclear Resonance 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 La scelta del: sistemadiingegneriaanimale reporter bioluminescenza, PET, NMR costrutto reporter
GENE 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 La scelta del: sistemadiingegneriaanimale reporter bioluminescenza, PET, NMR costrutto reporter
Studio dellafunzionedipromotore reporter gene reporter gene
I sistemi reporter nello studio di rilascio/sintesi di trasduttori del segnale (complementazione)
+/- +/- 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 20 min. i.p. of D-luciferin 5 min. after the acquisition
13.5 14.5 15.5 16.5 18.5 ERE-Luc mouse dpc (day post conception) ER istranscriptionallyactive at day 14.5 pc
ectoderm 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 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 & 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 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: Selective Estrogen Recepor Modulators CNS cardiovascular reproductive growthfactors P bone SP1 NFKB AP1 tissue-specific coregulators ERE
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
Measuringbioluminscence 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 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” 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
EffectsofSERMs 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
EffectsofSERMs 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 LIMB ACUTE CHRONIC Rando et al, Mol. Endocrinol. 2010
HEPATIC AREA ABDOMEN ACUTE CHRONIC Rando et al, Mol. Endocrinol. 2010
SERCHING FOR NOVEL MODALITIES TO MEASURE THE EFFICACY OF SERMs N° peaks, amplitude, frequency AUC
GENITAL AREA SKELETAL AREA * * * * * A Peaks/21d * * * * Amplitude B * Period (d) C * * * * * AUC * D Rando et al, Mol. Endocrinol. 2010 * * * * E Potency * *
PHENETICS OF DRUG ACTION 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 clustering data to generate novel families of compounds Genital area Skeletal area Rando et al, Mol. Endocrinol. 2010
C Reverse Medicinal Chemistry A Genital area B Skeletal area Cl 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
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
University of Milan 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 “The whole is more than the sum of its parts “ Aristotle (384 BC – 322 BC) Methapysics MODERN PHARMACOLOGY NEEDS TO REVISIT ANIMAL MODELS