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Cardiomyocyte Video Talk At 2008 Ddt In Boston

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  • 1. High Speed Time-lapse Video Platform for Analyzing Effects of GPCR and Ion-channel Modulators on Cardiomyocyte Contraction Mei Zhang, M.D., Ph.D. Senior Scientist Synta Pharmaceuticals CONFIDENTIAL DDT, 2008, Boston
  • 2. Synta overview • 190 employees in Lexington, MA • Origins: Lan Bo Chen (DFCI), Susumu Tonegawa (MIT), joint venture 1992-2002 with Japanese pharma and photography companies to discover new drugs in cancer and inflammatory diseases • Today: Pipeline of five novel mechanism, small-molecule drugs in cancer, chronic inflammation – all discovered and developed internally • Long-term view of drug discovery and development CONFIDENTIAL DDT, Boston
  • 3. Pipeline • Five small-molecule Lead Pre- Ph 1 Ph 2 Ph 3 Synta opt Clin drugs in cancer, chronic Ownership inflammation ONCOLOGY 1. Elesclomol Melanoma Shared • All NCEs with distinct, Other TBA with GSK first/ best-in-class 2. STA-9090 100% mechanisms 3. STA-9584 100% • All discovered and INFLAMM developed internally, from Synta compound 4. STA-5326 100% library and Synta 5. CRAC 100% chemistry platform CONFIDENTIAL DDT, Boston
  • 4. Synta’s Live Cell Drug Discovery Strategies – Label free phase-contrast cellular imaging – Dynamic time-lapse video analysis – Whole cell-based screen and primary cell-based confirmation – High content imaging-based mechanism of action study – Interactive analysis between in vitro and in vivo assays CONFIDENTIAL DDT, Boston
  • 5. Primary, Endogenous, Live! (Images from various Web Sources) CONFIDENTIAL DDT, Boston
  • 6. Cardiomyocyte Contraction Platform Study Goals 1. Establish high speed video-microscopy-based assay system for analyzing cardiomyocyte contraction. 2. Explore the predictability of the system for the effects of known GPCR and ion channel modulators on cardiomyocyte function. 3. Examine selectivity of Synta’s calcium-release activated calcium channel inhibitors (CRACi) on cardiomyocytes. CONFIDENTIAL DDT, Boston
  • 7. Presentation Outline 1. Cardiomyocyte contraction regulators 2. Cardiomyocyte isolation 3. High speed phase-contrast imaging system 4. MetaMorph module and MATLAB program 5. High content analysis of cardiomyocyte contraction videos 6. Future development of the assay system CONFIDENTIAL DDT, Boston
  • 8. Cardiomyocyte Contraction Regulators (Epi; K+ Channel (TEA) NE) (2-APB) b-AR CX40, 43, 45 (EGTA) (2-APB) (Verapamil, SKF96365) Modified from Dr Birgitte Lygren, University of Oslo, Norway CONFIDENTIAL DDT, Boston
  • 9. Cardiomyocyte Isolation • Animal and reagents: – Newborn day 1-3 SD rats (Charles River Laboratory) – Cardiomyocyte isolation Kit – SureCoat solution (Cellutron) – Collagen I coated cell culture plate (BD Labware) • Neonatal rat ventricular cardiomyocyte isolation (CelluTron instructions): – Coat plates – Anesthetize neonatal rats – Remove and trim the heart – Stir the hearts in digestion buffer, transfer all the cell suspension, until all the tissue is digested – Filter cells (0.22um) – Seed the cells at 20k/well/100ul (96-well) – At day 7 of culture, cells are ready for the sequential treatment experiments (Longer time may be required for gap-junction formation and synchronized contraction.) CONFIDENTIAL DDT, Boston
  • 10. Cardiomyocyte Treatment Assay validation: • Calcium: 1.8mM • Calcium depletion: 20mM EGTA • Calcium recovery: 40mM CaCl2 • Contractile activation: Epinephrine (Epi) 20uM; Norepinephrine (NE) 20uM • Voltage gated calcium channel blockage: Verapamil 20uM • Voltage gated potassium channel blockage: TEA chloride 20mM New calcium-channel inhibitor analysis: • Known CRAC inhibitors: SKF96365 20uM; 2-ABP 20uM; • Synta CRAC inhibitors: Compound A 20uM; Compound B 20uM CONFIDENTIAL DDT, Boston
  • 11. …Treatment Notes 1. Cardiomyocytes were cultured in 10% FBS containing medium for 7 days before the expts. 2. Cells were imaged in 5% CO2 environment around 37C. 3. Compounds were added as 10x solution in culture media into culturing media directly. 4. Immediate responses were captured. Most videos were captured within 30 sec after adding treatment solution. 5. Same cells were imaged before and after treatment. CONFIDENTIAL DDT, Boston
  • 12. Video Acquisition • MetaMorph –Stream Acquisition Module • Hamamatsu EM CCD Camera –Up to 300 frames/second • Live Cell Culture Environment (Temperature and CO2) • Nikon Inverted Microscope with Motorized XYZ Ludl Stage (LEP MAC5000) CONFIDENTIAL DDT, Boston
  • 13. Video Analysis MATLAB • MATLAB-based software • In-house developed by Mats Holmqvist and Mei Zhang • Whole field and whole movie intensity measurement CONFIDENTIAL DDT, Boston
  • 14. Cardiomyocyte Imaging Platform CONFIDENTIAL DDT, Boston
  • 15. Cardiomyocyte Culture Chamber CONFIDENTIAL DDT, Boston
  • 16. Typical Movie • Temperature: 36.5C • CO2: 5-6% • Objective: 20x Phase Contrast • 300 frames/movie • Duration 15s (15249ms) • Synchronized beating CONFIDENTIAL DDT, Boston
  • 17. Quantitative Analysis of Movie by MATLAB MATLAB Avi movie 158 c ontractions per m inute 10000 8000 6000 4000 2000 0 -2000 -4000 -6000 -8000 0 0.5 1 1.5 2 2.5 3 CONFIDENTIAL DDT, Boston
  • 18. Data Format: Video + MATLAB Quantitation Normalized image intensity 15 sec 52 BPM CONFIDENTIAL DDT, Boston
  • 19. Activators of Contraction Epinephrine or Norepinephrine or Adrenaline or Epi Noradrenaline or NE CONFIDENTIAL DDT, Boston
  • 20. Spontaneous and Epi-Activated Contractions Normal medium Epi 10uM 1 2 CONFIDENTIAL DDT, Boston
  • 21. Spontaneous and Activated-Contraction Normal medium Epi 10uM 1 2 52 BPM 162 BPM CONFIDENTIAL DDT, Boston
  • 22. Selective Cellular Calcium Depletion Agent EDTA EGTA CONFIDENTIAL DDT, Boston
  • 23. Calcium Depletion Blocks Activated Contraction 1 2 NE 20uM Normal medium 3 BPM EGTA 20mM 8 160 0 CONFIDENTIAL DDT, Boston
  • 24. Voltage Gated Calcium Channel Blocker Verapamil CONFIDENTIAL DDT, Boston
  • 25. Verapamil Blocks Spontaneous Contraction Normal medium Verapamil 20uM 1 2 32 BPM 0 BPM CONFIDENTIAL DDT, Boston
  • 26. Verapamil Blocks Epinephrine Activated Contraction Normal medium Verapamil 20uM + Epi 20uM 1 2 CONFIDENTIAL 20 BPM 0 BPM DDT, Boston
  • 27. Verapamil Blocks Norepinephrine Activated Contraction Normal medium Verapamil 20uM + NE 20uM 1 2 CONFIDENTIAL 20 BPM 0 BPM DDT, Boston
  • 28. Effect of EGTA and Verapamil on Contraction 1 2 EGTA 10 mM Normal medium 1 2 Normal medium Verapamil 10 μM CONFIDENTIAL DDT, Boston
  • 29. Effect of EGTA and Verapamil on Contraction - Video Quantitation 1 2 Normal EGTA medium 10 mM 56 BPM 0 BPM 1 2 Verapamil Normal 10uM medium CONFIDENTIAL 58 BPM 0 BPM DDT, Boston
  • 30. Voltage Gated Potassium Channel Blocker Tetraethylammonium (TEA) CONFIDENTIAL DDT, Boston
  • 31. TEA Stimulates Contraction 1 2 Normal TEA 20mM medium 3 BPM Epi 20uM 27 68 190 CONFIDENTIAL DDT, Boston
  • 32. CRAC channel (calcium-release-activated channel) CONFIDENTIAL DDT, Boston
  • 33. Known CRAC Blocker 1 1 -80 -60 -40 -20 20 40 60 80 100 SKF-96365 hydrochloride Voltage (mV) Current Density (pA/pF) -1 -2 -3 Current @ -80 mV SKF96365 100 0 Current SKF96365 0 Washout Current (nA) -0.02 0 5 10 15 Start of Trace (CP) (min) CONFIDENTIAL DDT, Boston
  • 34. SKF-96365’s effects reported 1. Inhibitor of receptor-mediated calcium entry. 2. Inhibitor of voltage-gated Ca2+ channels 3. Inhibitor of histamine-induced formation of nitric oxide in human endothelial cells 4. CRAC inhibitor CONFIDENTIAL DDT, Boston
  • 35. SKF-96365 Blocks Spontaneous Cardiomyocyte Contraction at Low Concentration Normal medium SKF 5uM 1 2 CONFIDENTIAL DDT, Boston
  • 36. SKF-96365 Blocks Spontaneous Cardiomyocyte Contraction Normal medium SKF 5uM 1 2 56 BPM 0 BPM CONFIDENTIAL DDT, Boston
  • 37. Dose Response of Contraction to Low and High Concentrations of SKF-96365 1 2 Epi 10 μM Normal medium 3 4 SKF 5 μM SKF 50 μM CONFIDENTIAL DDT, Boston
  • 38. Dose Response of Contraction to Low and High Concentrations of SKF-96365 1 2 Normal medium Epi 10uM 40 BPM 136 BPM 3 4 SKF 5uM SKF 50uM CONFIDENTIAL 0 BPM DDT, Boston 80 BPM
  • 39. Known CRAC Blocker 2 2-APB effect 2-Aminoethoxydiphenyl 50 0 I (mean) borate (2-APB) 0 2_APB Washout I (nA) -0.01 -0.02 0 2 4 6 8 Time (min) CONFIDENTIAL DDT, Boston
  • 40. 2-APB’s effects reported 1. Modulator of IP3-induced Ca2+ release 2. IP3 receptor-independent inhibition of Ca2+ release activated Ca2+ channels 3. Activator of TRPV1, TRPV2 and TRPV3 4. Increasing Orai3 channel pore size 5. Directly inhibits Connexin26 and/or Connexin32 gap-junction channels CONFIDENTIAL DDT, Boston
  • 41. Dose Response of Contraction to Low and High Concentrations of 2-APB 1 2 Normal 2-APB medium 5uM 3 4 2-APB 2-APB 50uM 100uM CONFIDENTIAL DDT, Boston
  • 42. Effect of 2-APB on Spontaneous Contraction Normal 1 2 Medium 2-APB (contaminated 5uM with pacemakers?) 120 BPM 176 BPM 3 4 2-APB 2-APB 50uM 100uM CONFIDENTIAL ~50 BPM ~40 BPM (?) DDT, Boston
  • 43. Synta’s calcium-release activated calcium channel inhibitors (CRACi) - Selectivity analysis CONFIDENTIAL DDT, Boston
  • 44. Low Concentration of Compound A on Epi Activation 1 2 Normal medium Compound A 2uM 3 BPM Epi 40 30 20uM 120 CONFIDENTIAL DDT, Boston
  • 45. High Concentration of Compound A on Epi Activation 1 2 Normal Compound A medium 20uM 3 4 DMSO Epi 20uM BPM 12 16 10 100 CONFIDENTIAL DDT, Boston
  • 46. Sequential Treatment of Compound A and Verapamil 1 2 Normal Compound A medium 10uM 3 4 Epi Verapamil 10uM 10uM CONFIDENTIAL DDT, Boston
  • 47. Sequential Treatment of Compound A and Verapamil 1 2 Compound A Normal 10uM medium 64 BPM 66 BPM 3 4 Epi Verapamil 10uM 10uM CONFIDENTIAL 156 BPM 0 BPM DDT, Boston
  • 48. Sequential Treatment of Compound B and Verapamil 1 2 Normal Compound B medium 10uM 3 4 Epi Verapamil 10uM 10uM CONFIDENTIAL DDT, Boston
  • 49. Sequential Treatment of Compound B and Verapamil 1 2 Compound B Normal 10uM medium 32 BPM 40 BPM 3 4 Epi Verapamil 10uM 10uM 146 BPM 0 BPM CONFIDENTIAL DDT, Boston
  • 50. Concluding Remark on Synta CRAC Inhibitor’s Selectivity Unlike SKF-96365 and 2-APB, Synta CRAC inhibitors do not block calcium channels and electrical synapses (gap junctions) of cardiomyocytes. CONFIDENTIAL DDT, Boston
  • 51. Summary of Effects of Modulators Tested Assay validation: • Calcium depletion: 20mM EGTA (Stopped contraction) • Contractile activation: Epinephrine (Epi) 20uM; Norepinephrine (NE) 20uM (Both stimulated contraction) • Voltage gated calcium channel blockage: Verapamil 20uM (Totally blocked contraction) • Voltage gated potassium channel blockage: TEA chloride 20mM (Stimulated contraction while preserved excitability by Epi/NE) New calcium-channel inhibitor analysis: • Known CRAC inhibitor: SKF96365 20uM (Blocked contraction) • Known CRAC inhibitor: 2-ABP 20uM (Disrupted synchronization) • Synta CRAC inhibitors: Compound A 20uM; Compound B 20uM (No effect on contraction) CONFIDENTIAL DDT, Boston
  • 52. Cardiomyocyte Contraction Regulators (Epi; K+ Channel (TEA) NE) (2-APB) b-AR CX40, 43, 45 (EGTA) (2-APB) (Verapamil, SKF96365) Modified from Dr Birgitte Lygren, University of Oslo, Norway CONFIDENTIAL DDT, Boston
  • 53. Conclusions on the Platform 1. The in vivo effects of cardiac contraction modulators are reproducible in this in vitro system. 2. We are able to differentiate Synta’s new CRACi from known non-selective inhibitors for the side-effects on cardiac muscles. 3. Advantages of the platform : – Primary cell – Endogenous GPCR, ion channels and gap-junctions – Synchronized contraction – Quantitative analysis – Self-comparative data before and after treatment – Able to record rhythm, regularity and synchronization CONFIDENTIAL DDT, Boston
  • 54. Future Transformation Incorporating the following fine technologies: 1. Sensitive and automated imaging system capable of HTS (MDC IXMicro system) 2. Stem cell-differentiated cardiomyocyte (Cor.At) 3. Non-toxic labeling reagents (Invitrogen Cell- Trackers) 4. Multi-parameter HCA analysis (rhythm, regularity, synchronization and strength) (MetaExpress, AcuityExpress) CONFIDENTIAL DDT, Boston
  • 55. Useful Structures for Imaging Contraction Strength Dr Birgitte Lygren Abcam CONFIDENTIAL DDT, Boston
  • 56. Acknowledgement Synta Biology Department MVI (Micro Video Mats Holmqvist (now Novartis Instruments) Cambridge) David Claypool Shuzhen Qin Patrick Verdier Long Li (now Celgene) Dan Zhou NEUE Biosciences Michael Xie (now GSK China) Mike Decavalcante Jim Barsoum Molecular Devices Synta IT Department Michele Dahl Michael Ahlfont Sylvia de Bruin Atom Grams CONFIDENTIAL DDT, Boston

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