High Speed Time-lapse Video Platform
     for Analyzing Effects of GPCR and Ion-channel
       Modulators on Cardiomyocyte...
Synta overview
  • 190 employees in Lexington, MA

  • Origins: Lan Bo Chen (DFCI), Susumu Tonegawa (MIT),
    joint ventu...
Pipeline
 • Five small-molecule                              Lead   Pre-   Ph 1   Ph 2   Ph 3
                            ...
Synta’s Live Cell
                Drug Discovery Strategies

      – Label free phase-contrast cellular imaging
      – Dy...
Primary, Endogenous, Live!




                                   (Images from
                                   various ...
Cardiomyocyte Contraction Platform
             Study Goals
  1. Establish high speed video-microscopy-based assay
     sy...
Presentation Outline
     1. Cardiomyocyte contraction regulators
     2. Cardiomyocyte isolation
     3. High speed phase...
Cardiomyocyte Contraction Regulators
                         (Epi;          K+ Channel   (TEA)
                          ...
Cardiomyocyte Isolation
  • Animal and reagents:
       – Newborn day 1-3 SD rats (Charles River
         Laboratory)
    ...
Cardiomyocyte Treatment
  Assay validation:
  • Calcium: 1.8mM
  • Calcium depletion: 20mM EGTA
  • Calcium recovery: 40mM...
…Treatment Notes
    1. Cardiomyocytes were cultured in 10% FBS containing medium for
       7 days before the expts.

   ...
Video Acquisition
       • MetaMorph
          –Stream Acquisition Module
       • Hamamatsu EM CCD Camera
          –Up t...
Video Analysis
                                      MATLAB

       • MATLAB-based software


       • In-house developed ...
Cardiomyocyte Imaging Platform




 CONFIDENTIAL
DDT, Boston
Cardiomyocyte Culture Chamber




 CONFIDENTIAL
DDT, Boston
Typical Movie
                          • Temperature: 36.5C
                          • CO2: 5-6%
                       ...
Quantitative Analysis of Movie
                         by MATLAB
                       MATLAB




        Avi movie



 ...
Data Format:
                Video + MATLAB Quantitation




                              Normalized image intensity

   ...
Activators of Contraction
                     Epinephrine or      Norepinephrine or
                    Adrenaline or Epi...
Spontaneous and Epi-Activated Contractions

                Normal medium   Epi 10uM


                   1               ...
Spontaneous and Activated-Contraction
                Normal medium   Epi 10uM


                     1              2



...
Selective Cellular Calcium
                      Depletion Agent

                EDTA            EGTA




 CONFIDENTIAL
D...
Calcium Depletion Blocks Activated
                      Contraction

                      1           2
                ...
Voltage Gated Calcium Channel Blocker

                Verapamil




 CONFIDENTIAL
DDT, Boston
Verapamil Blocks Spontaneous Contraction
                Normal medium   Verapamil 20uM


                   1            ...
Verapamil Blocks Epinephrine Activated
                   Contraction
                Normal medium   Verapamil 20uM + Epi...
Verapamil Blocks Norepinephrine Activated
                 Contraction
                Normal medium   Verapamil 20uM + NE...
Effect of EGTA and Verapamil on Contraction


                1         2
                                 EGTA 10 mM
Norm...
Effect of EGTA and Verapamil on Contraction
             - Video Quantitation
                   1        2
    Normal    ...
Voltage Gated Potassium Channel
                   Blocker
                Tetraethylammonium (TEA)




 CONFIDENTIAL
DDT,...
TEA Stimulates Contraction

                     1          2
       Normal                          TEA 20mM
       mediu...
CRAC channel
      (calcium-release-activated channel)




 CONFIDENTIAL
DDT, Boston
Known CRAC Blocker 1
                                                                              1
                     ...
SKF-96365’s effects reported
      1. Inhibitor of receptor-mediated calcium entry.
      2. Inhibitor of voltage-gated Ca...
SKF-96365 Blocks Spontaneous Cardiomyocyte
         Contraction at Low Concentration
                Normal medium   SKF 5...
SKF-96365 Blocks Spontaneous Cardiomyocyte
                     Contraction

                Normal medium     SKF 5uM


 ...
Dose Response of Contraction to Low and High
            Concentrations of SKF-96365


                         1      2
 ...
Dose Response of Contraction to Low and High
            Concentrations of SKF-96365

                    1            2
 ...
Known CRAC Blocker 2
                                                     2-APB effect
    2-Aminoethoxydiphenyl          ...
2-APB’s effects reported
        1. Modulator of IP3-induced Ca2+ release
        2. IP3 receptor-independent inhibition o...
Dose Response of Contraction
           to Low and High Concentrations of 2-APB

                        1           2
   ...
Effect of 2-APB on Spontaneous Contraction

  Normal
                     1                   2
  Medium                  ...
Synta’s calcium-release activated
       calcium channel inhibitors (CRACi)

                - Selectivity analysis




 C...
Low Concentration of Compound A on Epi
                        Activation

                       1          2
 Normal med...
High Concentration of Compound A on Epi
                         Activation

                  1            2
   Normal   ...
Sequential Treatment of Compound A and
                         Verapamil

                       1         2
          No...
Sequential Treatment of Compound A and Verapamil

                    1           2
                                      ...
Sequential Treatment of Compound B and
                         Verapamil

                         1         2
          ...
Sequential Treatment of Compound B and Verapamil


                   1           2
                                      ...
Concluding Remark on
        Synta CRAC Inhibitor’s Selectivity

              Unlike SKF-96365 and 2-APB,
           Synt...
Summary of Effects of Modulators Tested
  Assay validation:
  • Calcium depletion: 20mM EGTA (Stopped contraction)
  • Con...
Cardiomyocyte Contraction Regulators
                         (Epi;          K+ Channel   (TEA)
                          ...
Conclusions on the Platform
  1. The in vivo effects of cardiac contraction modulators are
     reproducible in this in vi...
Future Transformation
  Incorporating the following fine technologies:
  1. Sensitive and automated imaging system capable...
Useful Structures for Imaging Contraction Strength




                Dr Birgitte Lygren   Abcam
 CONFIDENTIAL
DDT, Boston
Acknowledgement
         Synta Biology Department         MVI (Micro Video
           Mats Holmqvist (now Novartis    Inst...
Upcoming SlideShare
Loading in...5
×

Cardiomyocyte Video Talk At 2008 Ddt In Boston

1,199

Published on

Published in: Business
0 Comments
1 Like
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total Views
1,199
On Slideshare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
0
Comments
0
Likes
1
Embeds 0
No embeds

No notes for slide

Cardiomyocyte Video Talk At 2008 Ddt In Boston

  1. 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. 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. 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. 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. 5. Primary, Endogenous, Live! (Images from various Web Sources) CONFIDENTIAL DDT, Boston
  6. 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. 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. 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. 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. 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. 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. 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. 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. 14. Cardiomyocyte Imaging Platform CONFIDENTIAL DDT, Boston
  15. 15. Cardiomyocyte Culture Chamber CONFIDENTIAL DDT, Boston
  16. 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. 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. 18. Data Format: Video + MATLAB Quantitation Normalized image intensity 15 sec 52 BPM CONFIDENTIAL DDT, Boston
  19. 19. Activators of Contraction Epinephrine or Norepinephrine or Adrenaline or Epi Noradrenaline or NE CONFIDENTIAL DDT, Boston
  20. 20. Spontaneous and Epi-Activated Contractions Normal medium Epi 10uM 1 2 CONFIDENTIAL DDT, Boston
  21. 21. Spontaneous and Activated-Contraction Normal medium Epi 10uM 1 2 52 BPM 162 BPM CONFIDENTIAL DDT, Boston
  22. 22. Selective Cellular Calcium Depletion Agent EDTA EGTA CONFIDENTIAL DDT, Boston
  23. 23. Calcium Depletion Blocks Activated Contraction 1 2 NE 20uM Normal medium 3 BPM EGTA 20mM 8 160 0 CONFIDENTIAL DDT, Boston
  24. 24. Voltage Gated Calcium Channel Blocker Verapamil CONFIDENTIAL DDT, Boston
  25. 25. Verapamil Blocks Spontaneous Contraction Normal medium Verapamil 20uM 1 2 32 BPM 0 BPM CONFIDENTIAL DDT, Boston
  26. 26. Verapamil Blocks Epinephrine Activated Contraction Normal medium Verapamil 20uM + Epi 20uM 1 2 CONFIDENTIAL 20 BPM 0 BPM DDT, Boston
  27. 27. Verapamil Blocks Norepinephrine Activated Contraction Normal medium Verapamil 20uM + NE 20uM 1 2 CONFIDENTIAL 20 BPM 0 BPM DDT, Boston
  28. 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. 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. 30. Voltage Gated Potassium Channel Blocker Tetraethylammonium (TEA) CONFIDENTIAL DDT, Boston
  31. 31. TEA Stimulates Contraction 1 2 Normal TEA 20mM medium 3 BPM Epi 20uM 27 68 190 CONFIDENTIAL DDT, Boston
  32. 32. CRAC channel (calcium-release-activated channel) CONFIDENTIAL DDT, Boston
  33. 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. 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. 35. SKF-96365 Blocks Spontaneous Cardiomyocyte Contraction at Low Concentration Normal medium SKF 5uM 1 2 CONFIDENTIAL DDT, Boston
  36. 36. SKF-96365 Blocks Spontaneous Cardiomyocyte Contraction Normal medium SKF 5uM 1 2 56 BPM 0 BPM CONFIDENTIAL DDT, Boston
  37. 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. 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. 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. 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. 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. 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. 43. Synta’s calcium-release activated calcium channel inhibitors (CRACi) - Selectivity analysis CONFIDENTIAL DDT, Boston
  44. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 55. Useful Structures for Imaging Contraction Strength Dr Birgitte Lygren Abcam CONFIDENTIAL DDT, Boston
  56. 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

×