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
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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
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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
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5. Primary, Endogenous, Live!
(Images from
various Web
Sources)
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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
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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
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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
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13. Video Analysis
MATLAB
• MATLAB-based software
• In-house developed by Mats Holmqvist and
Mei Zhang
• Whole field and whole movie intensity
measurement
CONFIDENTIAL
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14. Cardiomyocyte Imaging Platform
CONFIDENTIAL
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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
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18. Data Format:
Video + MATLAB Quantitation
Normalized image intensity
15 sec
52 BPM
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19. Activators of Contraction
Epinephrine or Norepinephrine or
Adrenaline or Epi Noradrenaline or NE
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20. Spontaneous and Epi-Activated Contractions
Normal medium Epi 10uM
1 2
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21. Spontaneous and Activated-Contraction
Normal medium Epi 10uM
1 2
52 BPM 162 BPM
CONFIDENTIAL
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22. Selective Cellular Calcium
Depletion Agent
EDTA EGTA
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23. Calcium Depletion Blocks Activated
Contraction
1 2
NE 20uM
Normal medium
3 BPM
EGTA 20mM 8 160
0
CONFIDENTIAL
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24. Voltage Gated Calcium Channel Blocker
Verapamil
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25. Verapamil Blocks Spontaneous Contraction
Normal medium Verapamil 20uM
1 2
32 BPM 0 BPM
CONFIDENTIAL
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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
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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
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31. TEA Stimulates Contraction
1 2
Normal TEA 20mM
medium
3
BPM
Epi 20uM
27 68
190
CONFIDENTIAL
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32. CRAC channel
(calcium-release-activated channel)
CONFIDENTIAL
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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
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35. SKF-96365 Blocks Spontaneous Cardiomyocyte
Contraction at Low Concentration
Normal medium SKF 5uM
1 2
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36. SKF-96365 Blocks Spontaneous Cardiomyocyte
Contraction
Normal medium SKF 5uM
1 2
56 BPM 0 BPM
CONFIDENTIAL
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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
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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
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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
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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
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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
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55. Useful Structures for Imaging Contraction Strength
Dr Birgitte Lygren Abcam
CONFIDENTIAL
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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