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 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

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

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…?

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

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 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 +

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 +

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 Atomic absorption spectrometry of 85 Rb + Hollow cathode Rubidium lamp Air/acetylene flame Cerenkov counting of 86 Rb + Liquid scintillation counter (Perkin Elmer ‘Topcount’)

Atomic absorption spectrometry of 85 Rb +

Hollow cathode Rubidium lamp

Air/acetylene flame

Cerenkov counting of 86 Rb +

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 86 Rb + flux Non-radiometric: atomic absorption spec. of 85 Rb + 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

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 Processing Hollow cathode lamp source Spray chamber and nebulizer Flame Monochromator Processing electronics Data processing and instrument control Photomultiplier detector

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

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=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 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

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

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

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

Cells processed using appropriate automation

Supernatants analysed for Ion Content

Single burner system (low throughput)

Multi burner system

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 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

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

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 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…

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…

AAS Results Correlate With Electrophysiology K + Channel

Na + Channel: Comparison of flux and patch clamp IC 50  M Good agreement between flux assay and electrophysiology WCPC Li flux

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 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

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

Drug Discovery with Vision