Count the Ways:

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Count the Ways Automated Kinase Counter
Screening Gordon Alton, Ph.D. Automation
Project Leader Department of Biochemistry Pfize
r Global Research and Development La Jolla
Laboratories
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Outline

• Introduction
• Kinases
• Automated Ki determination
• Automated library screening

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Protein Kinases Ser, Thr, or Tyr Phosphorylation
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Protein Kinases in Human Biology
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Reminder of why we do Drug Discovery Letter
received at Pfizer HQ Sept 19, 2002
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Protein Kinases in Human Biology Cancer
Disease Progression
Cancer Deaths by Site and Sex Male Female Lung 8
9,300 67,600 Prostate 31,900 Breast 40,800 Colo
rectal 27,800 28,500 Pancreas
13,700 14,500 NHL 13,700 12,400 Ovary 14,000
Glivec
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Protein Kinases Necessity for Counter-Screening
500-600 kinases in Human genome
Upstate
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Commercial Kinase Assays Suitable for Automation
Fluorescence Polarization Panvera-
CoreHTS Molecular Devices- IMAP Chromagen-
Polaris Evotec- competitive FP Fluorescence Pie
rce- IQ Luminex- Beadlyte Caliper-
LabChip Perkin Elmer- LANCE
Chemiluminescence DiscoverX- Hithunter Biowhitak
er- Lumitech ELISA MBL- Mesacup Perkin Elmer-
DELFIA Radioactive Amersham- SPA Discovery
Partners- uARCS
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Custom Assays Potentially Suitable for Automation
Spectrophotometric continuous-coupled
Ligand-displacement Fluorescent-inhibitor
conjugate Radioactive 33P filtration (Biomek
Fx)
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Biochemistry Counter-Screen Assay Support
Automated Ki determination to guide MedChem SAR
Highly precise dose response curves
(Ki) Common detection for multiple kinases 5 -
40 compounds per week Continuous
read Open-access
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Spectrophotometric continuous-coupled (rate _at_ 340
nm)
kinase
Peptide-OH
Peptide-OP
ATP
ADP
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Robotics Core Facility Liquid Handling
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Positive and Negative Controls 20x
concentration (2 mL volume each)
DMSO or Compound diluent (50 mL volume)
A1 A2 A3
A4 A5 A6
Plate reader
Dead plates
B1 B2 B3
B4 B5 B6
Test compounds 200x concentration (10 uL volume)
Coupling Enzymes 1.1x concentration (22 mL volume)
Test Enzyme 20x conc. (2 mL volume)
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PKCa Automated Ki Determination Raw Rate Data
Inhibitors
OD _at_ 340 nm
Maximum Rate (DMSO PKCa)
Time (sec)
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PKCa Automated Ki Determination Data
100 50 0
control
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PKCa Automated Ki Determination Results
Ki 6.3 0.3 nM (staurosporine) Ki 0.8 0.1
nM (bis indolylmaleimide) Ki 327 29 nM
(fluorescein - bis indolylmaleimide) CVs
typically lt 10 Ki reproducibility within 15
(day-to-day) One research associate can generate
64 Kis / day Caveat expensive, uses lots of
enzyme, temp. control required
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Biochemistry Counter-Screen Assay Support SBDD
Inhibition of Focused Libraries (combi-chem)
Common detection for multiple kinases Ki and Km
correlate with spec. assay 1-50 plates (96 well),
periodic use Robust and easy Cost-effective End
point
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Robotics Core Facility Liquid Handling
96-channel 384-well indexing 75 plate capacity
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Core Facility Liquid Handling
Cost-effective Easy to use
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Robotics Core Facility Analytical
Monochromator, 384 well
Fluorescence
UV-Vis
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Commercial Assay Kits Evaluated for Library
Screening
Evaluation Criteria (standard inhibitor
Ki) Robustness, Ease of automation, FTE
allocation, Cost and Data quality Pierce IQ
(fluorescence quench) MBL- Mesacup (colorimetric
ELISA) Panvera CoreHTS (FP with
antibody) Chromagen Polaris (FP with
biotin-avidin) DiscoverX HitHunter
(chemiluminescent reporter)
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PKC Assay Kit Evaluation Results
approximations, depends on of points,
substrates, enzymes and corporate discounts
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Principal of the IQ Assay
Rhodamine peptide lex 560 nm, lem 590 nm
Quenched phosphopeptide
P
RFARKGSLRQKNV
RFARKGSLRQKNV
RFARKGSLRQKNV
RFARKGSLRQKNV
P
RFARKGSLRQKNV
RFARKGSLRQKNV
Rhodamine peptide and phosphopeptide
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IQ Assay Vendor Protocol Conditions
Ki 4.3 0.8 nM (bis indolylmaleimide), spec
0.8 nM
ATP 1 mM, PKCa 1 nM, 90 min
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IQ Assay Enzyme Kinetics
Km 40.7 6.7 uM (spec assay- 45 uM)
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IQ Assay Enzyme Concentration vs. Time
ATP 50 uM
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IQ Calibrator Peptides Dilution of Working
Solution
1000 mX 1X working solution (A B)
1
dilution

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IQ PKCa Assay Ki Determination
Ki 1.0 0.3 nM (bis indolylmaleimide) spec
0.8 nM Ki 3.7 0.5 nM (staurosporine) spec
6.3 nM
ATP 50 uM, PKCa 2 nM, 60 min, 1/32x working
solution
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IQ Library Screening Validation with new
conditions
1 uM staurosporine
Z 0.55
PKCa DMSO
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Conclusions of Case Study

• Continuous-coupled assay is Gold Standard,
  excellent data quality
• but expensive and not suited for libraries
• All commercial kits tested were easy to use and
  vendors
• extremely anxious to please
• In my lab IQ assay performed better for my
  applications
• Out-of-the-box protocols provide reasonably
  robust data,
• however optimization of conditions will always
  be important
• Inexpensive robotics works well

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Acknowledgements

• Jordan Jensen
• Steve Grant
• Brion Murray

La Jolla
Pierce Chromagen DiscoverX Panvera MBL Ramon
Gallo Beckman
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