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