Fully Automated High Throughput Ion Channel Screening
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Fully Automated High Throughput Ion Channel Screening

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Process Analysis & Automation were commissioned by Biofocus Ltd. To design and build an automated multi-atomic absorption spectrometer system. Using four Thermo Electron SOLAAR AA spectrometers......

Process Analysis & Automation were commissioned by Biofocus Ltd. To design and build an automated multi-atomic absorption spectrometer system. Using four Thermo Electron SOLAAR AA spectrometers (with associated Gilson autosamplers), a Hamilton Microlab SWAP robot and a Kendro stacker, a fully automated and reliable workcell was produced. OVERLORD dynamic scheduler (Overlord2) was used control the workcell, with NetLORD used to control the four AA control PCs. We believe that this is the only fully commercially available automated AA system.

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  • 1. Fully Automated High Throughput Ion Channel Screening July 2003 Adrian Kinkaid, PhD Head of Biology 1 BioFocus plc.
  • 2. Drug Discovery with Vision Essential expertise for gene to pre-clinical drug discovery Assay Development and Screening Medicinal and Synthetic Chemistry Chemoinformatics and Bioinformatics
  • 3. Drug Discovery with Vision collaborative, target-based drug discovery programmes Target Selection Assay Development Discovery Library Access HTS Hit-to-Lead Expansion Lead Optimisation Diverse Library Synthesis Targeted Library Synthesis Library design Molecular Modelling Data Analysis Bioinformatics flexible options for customised projects
  • 4. BioFocus’ 3 UK Science Centres Cambridge Science Park Assay Development HTS Bioinformatics Chesterford Park Synthetic Chemistry Computational Chemistry Business Development UK Offices Sittingbourne Research Centre Synthetic Chemistry Computational Chemistry Headquarters and Registered Offices Total staff = 180
  • 5. BioFocus Biology Expertise
    • Bioinformatics
    • Molecular
    • biology
    • Expression studies
    • Stable cell generation
    • Potency
    • Selectivity
    • Multiple
    • platforms
    • Multiple
    • readouts
    • 'Big pharma'
    • systems
    • Library choice
    • Functional effects
    • Target-related
    • HERG
    • Cytotoxicity
    • Apoptosis
    • Cell proliferation
    • CYP450
    • Drug discovery process
    Assay Target HTS Hit Evaluation Lead Optimisation 45 lab-based staff
  • 6. Ion Channels
    • Represent 5% of Molecular Targets
    • Proven Drugs already available on the market
    • Relevant targets for many therapeutic areas:
      • Cancer - Stroke
      • Arthritis - Alzheimer’s Disease
      • Cardiovascular Disease - Cystic Fibrosis?
    • Functional
    • Integral Membrane protein complexes
    • Movement of ions difficult to follow…?
  • 7. Requirements for an Ion Channel assay
    • High-throughput
    • Low false-positive rate
    • Low false-negative rate
    • Direct measure of function
    • Good correlation with electrophysiology
    • Reliability
    • Reproducibility
    • Amenable to miniaturization
    • Low cost
    • hERG used as a model channel
  • 8. Ion Channel screening technologies (used for hERG)
    • Fluorescence-based assays
      • Membrane potential-sensitive dyes
    • Radioligand binding assays
      • [ 3 H]Dofetilide
    • Automated electrophysiology
      • Automated two-electrode voltage clamp systems
      • Automated whole-cell patch clamp systems
      • Planar patch clamp techniques
    • Rubidium efflux assays
      • Cerenkov counting of 86 Rb +
      • Atomic absorption spectrometry of 85 Rb +
  • 9. Summary of Ion Channel Platforms Redistribution of High Medium Low Compound voltage-dependent dyes Interference FRET-based technology High Medium/High High Radioligand binding High Low Low Non-functional/ Radioactive Automated two-electrode Low/Medium High High Low efficacy voltage clamp Automated whole-cell Low/Medium High High Cell dialysis patch clamp Planar patch clamp Medium/High High High Cell dialysis Radiometric ion flux High Medium Low Radioactive Non-radiometric ion flux High Medium Low Throughput Information quality Cost Comments
  • 10. Rubidium efflux assays
    • Atomic absorption spectrometry of 85 Rb +
      • Hollow cathode Rubidium lamp
      • Air/acetylene flame
    • Cerenkov counting of 86 Rb +
      • Liquid scintillation counter (Perkin Elmer ‘Topcount’)
  • 11. K + ATPase HERG Rb + Loading Inhibitor K + ATPase HERG K + ATPase HERG Pre-Incubation Inhibitor K + ATPase HERG K + ATPase HERG Stimulus DEPOLARISATION Rb + Flux Assay Theory Radiometric: Cerenkov counting of 86 Rb + flux Non-radiometric: atomic absorption spec. of 85 Rb + flux
  • 12. Typical (hERG) assay protocol
    • Cells in 96 well plates
    • Add dilute compound and incubate
    • Add High K + Buffer and incubate
    • Transfer supernatant to deep well block or plate
    • Make up to 1ml or 330ul with 0.1% CsCl Solution
    • [Seal and Store]
    • Read
  • 13. Sample Processing Hollow cathode lamp source Spray chamber and nebulizer Flame Monochromator Processing electronics Data processing and instrument control Photomultiplier detector
  • 14. Sample Processing
    • Dissolved salt RbCl (s) = Rb + (aq) + Cl - (aq)
    • Flame (2000 - 3000 K) solvent evaporates
        • Rb + (aq) + Cl - (aq) = RbCl (s)
    • Solid melt & vaporise RbCl (s) = RbCl (g)
    • Vapour decomposes into individual atoms
    • RbCl (g) = Rb (g) + Cl (g)
    • Individual atoms can absorb energy by collision or ionisation
    • Prevent ionisation by using CsCl ionisation buffer
  • 15. Theory of Atomic Spectroscopy Energy n=1 n=2 n=3 n=4 Ground state Light Beer’s Law: Absorbance  Atom Concentration Excitation
  • 16. Theory of Atomic Emission Spectroscopy Energy n=1 n=2 n=3 n=4 Ground state Light Beer’s Law: Emission  Atom Concentration Emission
  • 17. Pros and cons of Rubidium efflux
      • Advantages
        • High throughput – relative to E-Phys etc.
        • Low cost
        • Direct measurement of channel activity
        • Can be performed as a non-radiometric assay
      • Disadvantages
        • High [K + ] o relieves HERG inactivation
  • 18. Advantages of AAS over Radiometric Flux
    • Health and Safety
    • Ease of handling
    • Cost of components
    • Cost of disposal
    • Environmental Impact
    • Sensitivity
    • No time limits to read samples once prepared
        • Decay or Licence constraints
  • 19. Ion Channel Screening
    • Cells processed using appropriate automation
    • Supernatants analysed for Ion Content
      • Single burner system (low throughput)
      • Multi burner system
  • 20. AAS-AES Movie clip
  • 21. IC 50 =90 nM IC 50 =102 nM Radiometric and non-radiometric flux assays are equivalent Comparison of radiometric and non-radiometric flux % Inhibition
  • 22. hERG blocker dose-response curves E4031, Cisapride, Terfenadine, Risperidone, Astemizole, Haloperidol E4031 Risperidone Terfenadine Astemizole Haloperidol Cisapride
  • 23. Ion Channel Screening: Screen Statistics
    • Signal to Background
      • Dependent on expression levels and cell leakage
      • Aim for 3:1
      • S:B as low as 1.3:1 has been acceptable
    • Precision
      • Analytical chemistry technique: very low CVs
    • Z’-factor
      • Cut-off at 0.3 (typical)
      • Average 0.6
  • 24. Ion Channel Screening
    • Cells processed using appropriate automation
    • Supernatants analysed for Ion Content
      • Single burner system (low throughput)
      • Multi burner system
  • 25. High Throughput Ion Channel Screening Platform: Reader platform initial design SOLAAR S AAS #1 SOLAAR S AAS #2 SOLAAR S AAS #3 SOLAAR S AAS #4 AutoSampler 2 Position #1 AutoSampler 2 Position #2 AutoSampler 2 Position #3 AutoSampler 2 Position #4 Linear Track Robotic arm 80 DWB On-line Storage Operating system e.g. Overlord Data Processing Activity Base All equipment must be “off the shelf”
  • 26. High Throughput Ion Channel Screening Platform: Reader platform
  • 27. High Throughput Ion Channel Screening Platform: Reader platform
  • 28. High Throughput Ion Channel Screening Platform: Reader platform
  • 29. Ion-Channel Screening Capabilities at BioFocus
    • hERG Channel Screening
      • Established and Validated
      • Selectivity screen: low throughput required
      • 100’s to 1000’s of compounds per campaign
    • Potassium Channel Screening
      • n x 10 5 compound screens
      • Uncoupling of slow process (AAS/AES reading) from assay process
      • Full/partial automation of assay process
      • Full automation of AAS/AES readers
    • Sodium Channels
      • As for Potassium Channels
    • Chloride Channels? In theory.
    • Proven capability of finding blockers and openers.
    • Hits validated by Electrophysiology…
  • 30. AAS Results Correlate With Electrophysiology K + Channel
  • 31. Na + Channel: Comparison of flux and patch clamp IC 50  M
    • Good agreement between flux assay and electrophysiology
    WCPC Li flux
  • 32. Ion-Channel Screening Capabilities at BioFocus
    • hERG Channel Screening
      • Established and Validated
      • Selectivity screen: low throughput required
      • 100’s to 1000’s of compounds per campaign
    • Potassium Channel Screening
      • n x 10 5 compound screens
      • Uncoupling of slow process (AAS reading) from assay process
      • Full/partial automation of assay process
      • Full automation of AAS readers
    • Sodium Channels
      • As for Potassium Channels
    • Chloride Channels? In theory.
    • Proven capability of finding blockers and openers.
    • Hits validated by Electrophysiology
  • 33. Drug Discovery with Vision