Fully Automated High Throughput Ion Channel Screening
July 2003
Adrian Kinkaid, PhDHead of Biology 1BioFocus plc.
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…?
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
Ion Channel screening technologies (used for hERG)
•Fluorescence-based assaysMembrane potential-sensitive dyes
•Radioligand binding assays[3H]Dofetilide
•Automated electrophysiologyAutomated two-electrode voltage clamp systemsAutomated whole-cell patch clamp systemsPlanar patch clamp techniques
•Rubidium efflux assaysCerenkov counting of 86Rb+
Atomic absorption spectrometry of 85Rb+
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
Summary of Ion Channel Platforms
Rubidium efflux assays
Atomic absorption spectrometry of 85Rb+
Hollow cathode Rubidium lampAir/acetylene flame
Cerenkov counting of 86Rb+
Liquid scintillation counter (Perkin Elmer ‘Topcount’)
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 86Rb+ fluxNon-radiometric: atomic absorption spec. of 85Rb+ flux
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
Sample ProcessingSample Processing
Hollow cathodelamp source
Spray chamberand nebulizer
Flame
Monochromator
Processing electronics
Data processingand instrumentcontrol
Photomultiplierdetector
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
Theory of Atomic Spectroscopy
Energy
n=1
n=2n=3n=4
Ground state
Light
Beer’s Law: Absorbance Atom Concentration
Excitation
Theory of Atomic Emission Spectroscopy
Energy
n=1
n=2n=3n=4
Ground state
Light
Beer’s Law: Emission Atom Concentration
Emission
Pros and cons of Rubidium efflux
AdvantagesHigh throughput – relative to E-Phys etc.Low costDirect measurement of channel activityCan be performed as a non-radiometric assay
DisadvantagesHigh [K+]o relieves HERG inactivation
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
Ion Channel Screening
• Cells processed using appropriate automation
• Supernatants analysed for Ion Content– Single burner system (low throughput)– Multi burner system
AAS-AES Movie clip
AAS Vs 86Rb
-3 -2 -1 0 1-20
0
20
40
60
80
100
log [M]
cpm
86RbAAS
IC50 =90 nM
IC50 =102 nM
Radiometric and non-radiometric flux assays are equivalent
Comparison of radiometric and non-radiometric flux
% I
nh
ibit
ion
hERG blocker dose-response curves
E4031, Cisapride, Terfenadine, Risperidone, Astemizole, Haloperidol
-1 0 1 2 3
0
50
100
log [astemizole] M
% inhib
itio
nIC50 = 1.5 M
A
-2 -1 0 1
0
50
100
log [cisapride] M
% inhib
itio
n
IC50 = 565 nMC
-2 -1 0 1 2
0
50
100
log [haloperidol] M
% inhib
itio
n
IC50 = 655 nMB
D
-3 -2 -1 0 1
0
50
100
log [E4031] M
% inhib
itio
n
IC50 = 192 nM
-1 0 1 2
0
50
100
log [terfenadine] M
% inhib
itio
n
IC50 = 8.4 MF
-2 -1 0 1 2
0
50
100
log [risperidone] (M)
% inhib
itio
nIC50 = 5.9 M
EE4031 Risperidone Terfenadine
Astemizole Haloperidol Cisapride
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
Ion Channel Screening
• Cells processed using appropriate automation
• Supernatants analysed for Ion Content– Single burner system (low throughput)– Multi burner system
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
• 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 105 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.Proven capability of finding blockers and openers.• Hits validated by Electrophysiology…Hits validated by Electrophysiology…
AAS Results Correlate With Electrophysiology
-2 -1 0 1 2 3-2
-1
0
1
2
3
Electrophysiology IC50 (M)
Rb
+ e
fflu
x IC
50 ( M
)
K+ Channel
Na+ Channel: Comparison of flux and patch clamp
1
10
100
1000
Prenylamine TTX Quinidine Lidocaine
Series1
Series2WCPC
Li flux
IC5
0
M
• Good agreement between flux assay and electrophysiology
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 105 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.Proven capability of finding blockers and openers.• Hits validated by ElectrophysiologyHits validated by Electrophysiology
Drug Discovery with Vision
