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Fully Automated High Throughput Ion Channel Screening July 2003 Adrian Kinkaid, PhD Head of Biology 1 BioFocus plc.
Drug Discovery with Vision Essential expertise for gene to pre-clinical drug discovery Assay Development and Screening Medicinal and Synthetic Chemistry Chemoinformatics and Bioinformatics
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
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
BioFocus Biology Expertise ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Assay Target HTS Hit Evaluation   Lead Optimisation  45 lab-based staff
Ion Channels ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Requirements for an Ion Channel assay ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Ion Channel screening technologies  (used for hERG) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
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
Rubidium efflux assays ,[object Object],[object Object],[object Object],[object Object],[object Object]
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
Typical (hERG) assay protocol ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Sample Processing Hollow cathode lamp source Spray chamber and nebulizer Flame Monochromator Processing  electronics Data processing and instrument control Photomultiplier detector
Sample Processing ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Theory of Atomic Spectroscopy Energy n=1 n=2 n=3 n=4 Ground state Light Beer’s Law: Absorbance    Atom Concentration Excitation
Theory of Atomic Emission Spectroscopy Energy n=1 n=2 n=3 n=4 Ground state Light Beer’s Law: Emission    Atom Concentration Emission
Pros and cons of Rubidium efflux ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Advantages of AAS over Radiometric Flux ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Ion Channel Screening ,[object Object],[object Object],[object Object],[object Object]
AAS-AES Movie clip
IC 50  =90 nM IC 50  =102 nM Radiometric and non-radiometric flux assays are equivalent Comparison of radiometric and non-radiometric flux % Inhibition
hERG blocker dose-response curves E4031, Cisapride, Terfenadine, Risperidone, Astemizole, Haloperidol E4031 Risperidone Terfenadine Astemizole Haloperidol Cisapride
Ion Channel Screening: Screen Statistics ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Ion Channel Screening ,[object Object],[object Object],[object Object],[object Object]
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”
High Throughput Ion Channel Screening Platform: Reader platform
High Throughput Ion Channel Screening Platform: Reader platform
High Throughput Ion Channel Screening Platform: Reader platform
Ion-Channel Screening Capabilities at BioFocus ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
AAS Results Correlate With Electrophysiology K +  Channel
Na +  Channel: Comparison of flux and patch clamp IC 50    M ,[object Object],WCPC Li flux
Ion-Channel Screening Capabilities at BioFocus ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Drug Discovery with Vision

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Fully Automated High Throughput Ion Channel Screening

  • 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.
  • 6.
  • 7.
  • 8.
  • 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.
  • 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.
  • 13. Sample Processing Hollow cathode lamp source Spray chamber and nebulizer Flame Monochromator Processing electronics Data processing and instrument control Photomultiplier detector
  • 14.
  • 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.
  • 18.
  • 19.
  • 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.
  • 24.
  • 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.
  • 30. AAS Results Correlate With Electrophysiology K + Channel
  • 31.
  • 32.