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