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![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 +](https://image.slidesharecdn.com/automatedionchannels-090930235835-phpapp02/75/Fully-Automated-High-Throughput-Ion-Channel-Screening-8-2048.jpg)
![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](https://image.slidesharecdn.com/automatedionchannels-090930235835-phpapp02/75/Fully-Automated-High-Throughput-Ion-Channel-Screening-12-2048.jpg)
![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](https://image.slidesharecdn.com/automatedionchannels-090930235835-phpapp02/75/Fully-Automated-High-Throughput-Ion-Channel-Screening-17-2048.jpg)
Fully Automated High Throughput Ion Channel Screening
- 1. Fully Automated HighThroughput Ion Channel Screening July 2003 Adrian Kinkaid, PhD Head of Biology 1 BioFocus plc.
- 2. Drug Discovery withVision Essential expertise for gene to pre-clinical drug discovery Assay Development and Screening Medicinal and Synthetic Chemistry Chemoinformatics and Bioinformatics
- 3. Drug Discovery withVision 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 UKScience 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 ExpertiseBioinformatics 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 Represent5% 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 anIon 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 screeningtechnologies (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 IonChannel 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 assaysAtomic 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) assayprotocol 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 Hollowcathode lamp source Spray chamber and nebulizer Flame Monochromator Processing electronics Data processing and instrument control Photomultiplier detector
- 14. Sample Processing Dissolvedsalt 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 AtomicSpectroscopy Energy n=1 n=2 n=3 n=4 Ground state Light Beer’s Law: Absorbance Atom Concentration Excitation
- 16. Theory of AtomicEmission Spectroscopy Energy n=1 n=2 n=3 n=4 Ground state Light Beer’s Law: Emission Atom Concentration Emission
- 17. Pros and consof 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 AASover 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 ScreeningCells processed using appropriate automation Supernatants analysed for Ion Content Single burner system (low throughput) Multi burner system
- 20.
- 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-responsecurves 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 ScreeningCells processed using appropriate automation Supernatants analysed for Ion Content Single burner system (low throughput) Multi burner system
- 25. High Throughput IonChannel 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 IonChannel Screening Platform: Reader platform
- 27. High Throughput IonChannel Screening Platform: Reader platform
- 28. High Throughput IonChannel Screening Platform: Reader platform
- 29. Ion-Channel Screening Capabilitiesat 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 CorrelateWith 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 Capabilitiesat 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 withVision