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Evaluating the IonWorks Barracuda for ligand- gated ion channel compound profiling Andrew Powell Ion Channel Retreat 2014
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Page 1: IonWorks Barracuda

Evaluating the IonWorks Barracuda for ligand-gated ion channel compound profiling Andrew Powell Ion Channel Retreat 2014

Page 2: IonWorks Barracuda

Introduction

• IonWorks Barracuda (Molecular Devices) capabilities

− Utilisation within GSK for voltage-gated channels

• Ligand-gated channel assays on IW-Barracuda:

− GABA-A

− nAChRa1

− TRPV1, TRPV4

− pH-gated channel

• Summary

25 June 2014 IC Retreat

Page 3: IonWorks Barracuda

IonWorks Barracuda Technology Summary

• High throughput planar array electrophysiology platform

− MegaΩ seals, Perforated Patch Clamp (HT or PPC)

− Merging of technologies (IonWorks Quattro/PatchXpress/FLIPR)

− 384 Electrodes/ Fluidic Tips & 4 x plenum electrodes (3.8cm2)

• Continuous Voltage Clamp

− Simultaneous addition & recordings

− Ligand-Gated Ion Channels (LGIC) & Voltage-Gated Ion

Channels (VGIC)

• Multiple Addition Capabilities

− Up to 10 compound additions

− Cross-plate & through-plate modes

− Sonicating Wash Station

• More complex protocols than IW-Quattros

− Multi-protocol functionality (AD)

− Run more pulse trains (≤200) for Use-Dependent compounds

25 June 2014 IC Retreat

• Experiment run time = 15 - 30mins (3x throughput of IonWorks-Quattro)

• Patch-plate consumable costs comparable to IonWorks-Quattro

− Cost per datapoint 5pt cross-plate CRC = £3.50

− Cost per datapoint 6pt CRC in through-plate mode = £0.70

Page 4: IonWorks Barracuda

IonWorks Quattro versus Barracuda: Well view

IonWorks Quattro

PatchPlate (HT)

IonWorks Quattro

PatchPlate (PPC) IonWorks Barracuda

PatchPlate (PPC)

1-2μm pore Low-resistance

pathway

Antibiotic Antibiotic Antibiotic Intracellular

Common Ground Electrode Common Ground Electrode Common Ground Electrode (s)

MΩ resistance seal

Extracellular

Adapted from Nature Reviews Drug Discover: Dunlop et al (2008), Volume 8, pp358-368

Patch Plate

(Extracellular)

Plenum

(Intracellular)

25 June 2014 IC Retreat

Page 5: IonWorks Barracuda

IonWorks Barracuda: Output and export

• 384-well image of response

• Filters: Resistance (<20MΩ in PPC is excluded). Exclude poorly clamped cells, Hole Test failed wells

(blocked, air bubbles)

Page 6: IonWorks Barracuda

IonWorks Barracuda and Early Safety Profiling at GSK

25 June 2014 IC Retreat

0.0 0.5 1.0 1.5 2.0 2.5 3.00

20

40

60

80

100

hERG Peak Tail Current (nA)

Co

un

t

• Voltage-gated ion channel assays established on IW-Barracuda for early detection of

potential cardiac liabilities

• CaV1.2 & KV1.5 assays also established

• NaV1.5 & KCNQ1/KCNE1 assay development in progress

• Ligand-gated neuronal targets: GABA-A & nAChRa1

hERG IW-Barracuda assay Through-plate mode, 6pt CRCs per well, upto 117 triplicate IC50s per run

Mean peak current

= 1.60 +/- 0.18nA

(n=384)

First Pass Screening

High-through-put imaging/binding

assays has be used to flag

ion channel liability

Automated Patch Clamp

Qpatch/PatchXpress

Candidate Selection

eXP: Ion ChannelsFirst Pass Screening

Electrophysiology Platforms that

generate more physiologically

relevant/cost effective data

Page 7: IonWorks Barracuda

0

50

100

150

200

250

300

350

clone #4

10 tested

100% +ve

Cur

rent

Den

sity

(pA

/pF)

10 tested

100% +ve

clone #3

1 mM GABA 1 mM GABA

Clone #3 Clone #4

0

50

100

150

200

250

300

350

clone #4

10 tested

100% +ve

Cur

rent

Den

sity

(pA

/pF)

10 tested

100% +ve

clone #3

1 mM GABA1 mM GABA 1 mM GABA1 mM GABA

Clone #3 Clone #4

Ligand-gated channels on IW-Quattro

• IW-Quattro could be used to record currents from slow ligand-gated channels (Hollands et

al 2009. J. Biomol. Screening 14: 769)

• Currents measured at 0mV - absence of leak current

Before Barracuda

25 June 2014 IC Retreat

-60mV

0mV

Voltage

Current

0.5nA

100ms

0nA

control (pre)

GABA, 30M (post)

post-pre

1nA

200ms

control (pre)

Agonist (post)

post-pre

Agonist

1nA

Record time

0.1 1 10 100

0.0

0.5

1.0

1.5

1

2

3

4

post

-pre

cu

rren

t (n

A)

[GABA] (M)

Individual wells in addition order

Agonist EC50 =

1.6 ± 0.3μM (n=8)

But literature agonist

EC50 = 10 – 30 μM

(measured at peak)

Agonist (μM)

Page 8: IonWorks Barracuda

• Antagonists, agonists and

positive modulators carry major

neuronal liability

• Early safety profiling assay

• Penta-heteromeric receptors

(2:2:1 stoichiometry)

• BacMam virus transduced CHO

cells (α1, β3, γ2 subunits)

Ligand-gated channels on IW-Barracuda: GABA-A

Medium tip height gives consistent resolution of current GABAA: Variance of Desensitisation

-5 -4 -3 -2 -1-4

-3

-2

-1

0

2s

1s

r2 = 0.91

r2 = 0.70

Peak Current (nA)

Desen

sit

isin

g C

urr

en

t (n

A)

Tip height impacts

current kinetics Medium tip height

t activation

27.6 ± 6.8 ms (n=16)

t desensitisation

926.6 ± 120.6 ms (n=16)

25 June 2014 IC Retreat

From: Tija C et al (2008). Nature Reviews

Neuroscience 9: 331-343

100M GABA

0.2nA

2000ms

GABAA Response: Trace

0 1000 2000 3000 4000-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

Time (ms)

Cu

rren

t (n

A)

Curr

en

t (n

A)

-6 -5 -4 -3 -2 -1 00

10

20

30

40 Peak

1s

2s

Best-fit values

AMPLITUDE

MEAN

SD

Std. Error

AMPLITUDE

MEAN

SD

2s

29.01

-1.453

0.3163

0.2676

0.004349

0.003092

1s

18.50

-2.630

-0.5159

0.5722

0.01842

0.01847

Peak

15.36

-3.111

0.6027

0.8327

0.03773

0.03774

GABAA Peak Current (nA)

Co

un

t

Bin size = 0.5nA (n=48)

Mean peak current =

3.1 +/- 0.6nA (n=48)

Page 9: IonWorks Barracuda

GABA-A Barracuda Assay: Tool Pharmacology

GABA current variability at

high concentrations

Consistent EC50 (23.9μM, n=16)

Reproducible positive

modulator response

Diazepam & pentobarbital potentiated

GABA EC20 response

Diazepam EC50 = 0.43 ± 0.25 μM

Pentobarbital EC50 = 50 ± 40 μM

25 June 2014 IC Retreat

GABA-A Tools:

Positive Modulators

-10 -8 -6 -4 -2-20

0

20

40

60

80

100 Diazepam (Buffer)

Diazepam (EC20 GABA)

Pentobarbital (Buffer)

Pentobarbital (EC20 GABA)

log [Compound] M

Resp

on

se (

% M

axim

um

Acti

vati

on

)

Z’ = 0.48EC50 =23.9 4.5 μM

Agonist correlation with

GABA-A Q-patch data

Page 10: IonWorks Barracuda

GABA-A Barracuda Assay: Tool Pharmacology

SEMs shown 25 June 2014 IC Retreat

pA2 = equilibrium dissociation constant

Competitive and non-competitive antagonist pharmacology GABA-A Schild Plot:Bicuculline vs. GABA

-9 -8 -7 -6 -5 -4 -3

0

25

50

75

100

log[Bicuculline] M

Resp

on

se (

% M

axim

um

Acti

vati

on

)

IC50 = 9.37μM

GABA-A Antagonism:Determining pA2 from Schild Plot

3.5 4.0 4.5 5.0 5.5 6.0 6.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0pA2 = 5.84

Slope = -0.84 +/- 0.06

r2 = 0.98

pA2 = equilibrium dissociation constant

-Log[Bicuculline] M

Lo

g(E

C50 G

AB

A'/E

C50 G

AB

A)-

1)

• 3min incubation followed by EC80 GABA

• Bicuculline IC50 = 9.37μM

• Schild plot: competitive

− pA2 = 5.84

− Slope = -0.84

• Schild analysis carried out on multiple

concentration responses across one

Barracuda run

Page 11: IonWorks Barracuda

GABA-A Production Assay Formats on Barracuda

• Development of two separate assays:

– Agonist:Antagonist dual (Test compound addition, then EC80 GABA)

• Incorporates 3m incubation of antagonists.

– PAM (EC20)

• 5 point CRC, 64 cmpds per plate – sufficient definition to flag GABA-A activity early in hit

compound triage

25 June 2014 IC Retreat

Page 12: IonWorks Barracuda

• The α1β1δε nicotinic acetylcholine receptor has a critical role in triggering muscle contraction

– Agonists and antagonists cause muscular side effects such as paralysis or spasms

• HEK293 α1β1δε stable cell line

25 June 2014 IC Retreat

Ligand-gated channels on IW-Barracuda: nAChRa1

nAChRa11 Pharmacology

BIOCAT #138074

-8 -7 -6 -5 -4 -3 -2 -1-25

0

25

50

75

100

125

150

175

log[Acetylcholine] M

Resp

on

se (

% M

axim

um

Acti

vati

on

)

-2.5 -2.0 -1.5 -1.0 -0.50

5

10

15

20

Bin size = 0.2nA

Best-fit values

AMPLITUDE

MEAN

SD

Std. Error

AMPLITUDE

MEAN

SD

14.67

-1.335

0.1782

2.280

0.03166

0.03230

nAChR Peak Current (nA)

Co

un

t

Mean peak current = 1.3 ± 0.2nA

Bin size = 0.2nA (n=43)

EC50 = 49.23M

• Plate-based FLIPR-Ca2+ assay has

high false-positive rate

• Preliminary data demonstrates IW-

Barracuda assay will mitigate this

− Hit triage before patch-clamp /

Q-Patch

• Speed of response may influence

spread in current heterogeneity

• Reproducible antagonist

pharmacology

nAChRa11 Pharmacology

BIOCAT #138074

-10 -9 -8 -7 -6 -5 -4 -3-25

0

25

50

75

100

125

Best-fit values

BOTTOM

TOP

LOGEC50

HILLSLOPE

EC50

Span

Std. Error

BOTTOM

LOGEC50

-5.569

= 100.0

-7.309

-0.7329

4.907e-008

= 105.6

2.258

0.06259

log[Pancuronium] M

Resp

on

se (

% M

axim

um

Acti

vati

on

)

IC50 = 49.1 ± 6.6nM

10mM3.33mM1.11mM0.37mM0.12mM0.04mM0.014mM

500ms

0.5nA

ACh

0.5nA

500ms

Page 13: IonWorks Barracuda

Reagent optimisation for IW-Barracuda

GABA-A a13g2 nAChR a11

0

0.5

1

1.5

2

2.5

control 18 37 6 19 1 23

Sig

nal S

ize n

A

Clone

Selecting optimal cell line for platform Optimising cell plating schedule for

BacMam transduction

25 June 2014 IC Retreat

Page 14: IonWorks Barracuda

• Transient Receptor Potential family of ion channels – TRPV1

• Expressed in sensory neurons – involved in nociception - activated by heat and chemical ligands

– TRPV4

• Expressed in lung epithelia – role in maintaining cellular volume - activated by mechanical stimuli

and chemical ligands

• BacMam virus transduced HEK293

25 June 2014 IC Retreat

TRPV channel IW-Barracuda assays

TRPV1 Representative

Current

TRPV4

10μM Capsaicin

-2.0 -1.5 -1.0 -0.5 0.0 0.50

5

10

15

20

Best-fit values

AMPLITUDE

MEAN

SD

Std. Error

AMPLITUDE

MEAN

SD

16.78

-0.8252

0.2134

1.802

0.02647

0.02656

TRPV1 Peak Current (nA)

Co

un

t

Mean peak current = 0.83 ± 0.21nA

Bin size = 0.2nA (n=47)

Current

Distribution Representative

Current

-2.0 -1.5 -1.0 -0.5 0.0 0.50

50

100

150

200

250

Best-fit values

AMPLITUDE

MEAN

SD

Std. Error

AMPLITUDE

MEAN

SD

192.4

-0.7293

0.1554

3.128

0.002752

0.003038

TRPV4 Peak Current (nA)

Co

un

t

Mean peak current = 0.73 ± 0.16nA

Bin size = 0.2nA (n=47)

Current

Distribution

TRPV4 agonist

1000ms

1nA

Activation

Buffer

1nA

1000ms 5000ms

0.5nA

Desensitisation

Activation

0.5nA

5000ms

Page 15: IonWorks Barracuda

25 June 2014 IC Retreat

TRPV channel IW-Barracuda assays

TRPV1 TRPV4

TRPV1: Barracuda versus Quattro Data

5 6 7 8 9 10

5

6

7

8

9

10

r2 = 0.80

IonWorks Barracuda (pIC50)

Ion

Wo

rks Q

uatt

ro (

pIC

50)

TRPV1: Barracuda versus FLIPR Data

5 6 7 8 9 10

5

6

7

8

9

10

r2 = 0.90

IonWorks Barracuda (pIC50)

FL

IPR

(p

IC50)

TRPV4: Barracuda versus FLIPR Data

4 5 6 7 8 94

5

6

7

8

9

r2 = 0.78

IonWorks Barracuda (pIC50)

FL

IPR

(p

IC50)

TRPV4 response to agonist TRPV1: Agonist curve

-10 -9 -8 -7 -6 -5 -4-0.5

0.0

0.5

1.0

1.5

Best-fit values

BOTTOM

TOP

LOGEC50

HILLSLOPE

EC50

Span

Std. Error

BOTTOM

TOP

LOGEC50

HILLSLOPE

Span

-0.01166

0.7873

-6.304

1.142

4.960e-007

0.7990

0.01087

0.02899

0.05019

0.1261

0.03315

log[Agonist] M

Resp

on

se (

nA

)

EC50=0.50 ± 0.05µM

(n=32)

TRPV1 response to capsaicin

EC50=0.32

± 0.03 µM

(n=32)

Page 16: IonWorks Barracuda

• BacMam virus transduced CHO cells

• pH-mediated activation

25 June 2014 IC Retreat

pH-gated channel IW-Barracuda assays

pH 6

Mean peak current = 3.20 ± 0.54 nA

Bin size = 0.5nA (n=128)

-10 -8 -6 -4 -2-0.5

0.0

0.5

1.0

1.5

2.0

2.5

Compound X

Compound Y

Best-fit values

BOTTOM

TOP

LOGEC50

HILLSLOPE

EC50

Span

Std. Error

BOTTOM

TOP

LOGEC50

HILLSLOPE

Span

Amiloride

-0.04042

1.539

-4.607

-1.981

2.469e-005

1.580

0.1222

0.07796

0.1042

0.7672

0.1491

Diminazine

-0.03547

1.955

-5.780

-1.443

1.660e-006

1.990

0.09569

0.06843

0.07885

0.3441

0.1251

SDs shown, n=4

log[Compound] M

C

urr

en

t (n

A)

Compound X

IC50 = 24.7 ± 5.3μM (n = 4)

Compound Y

IC50 = 1.6 ± 1.3μM (n = 4)

SDs shown

1000ms

1nA

pH-activation

Buffer

1nA

1000ms -6 -5 -4 -3 -2 -1 0 1

0

20

40

60

80

100 Peak

1.5s

2.5s

Best-fit values

AMPLITUDE

MEAN

SD

Std. Error

AMPLITUDE

MEAN

SD

2.5s

82.81

-1.356

0.3049

0.5360

0.002171

0.002446

1.5s

52.63

-2.579

0.4719

1.216

0.01266

0.01252

Peak

45.08

-3.202

-0.5383

2.379

0.03277

0.03284

pH-activated Current (nA)

Co

un

t

• Greater heterogeneity in peak current for desensitising current

• Antagonist pharmacology reasonably reproducible

Page 17: IonWorks Barracuda

Summary

• Confidence in utility of IW-Barracuda to enable

– early hit triage for safety liability targets

– compound pharmacology for hit-to-lead & lead optimisation

• Requirement for robust cellular reagents

– Moderate heterogeneity can be tolerated in PPC recordings depending

on desired output for assay (hit triage vs SAR)

– Kinetics of ligand addition likely to contribute to heterogeneity • Faster (activating &) desensitising channels seem to show more heterogeneity

• Utilisation of IW-Barracuda for compound MoA on ligand-gated

channels limited to date, but opportunities anticipated

– Opportunities to carry out more detailed ligand-gated channel MoA with more

flexible voltage protocols

• Other ligand-gated channels to be evaluated – including very fast

desensitising channels (e.g. P2X1 or nAChRa7)

25 June 2014 IC Retreat

Page 18: IonWorks Barracuda

Acknowledgements

• Kelly Gatfield

• Metul Patel

• Dave Downie

• David Standing

• Bret Utley

• Michelle Wantoch

• Stephen Brough

• Rob Tanner

• Yu Hua Chen

GSK - Biological Sciences

• Brian Donovan (PX/Q-Patch)

• Chris Nixon

• Tania Bakshi

• Claire Townsend

• Dan Gillie

• Tony Leesnitzer

• Craig McKay (now with MDC)

• Jane Hinton (MDC)

• Mark Jennings (MDC)

25 June 2014 IC Retreat

Page 19: IonWorks Barracuda

Back-up Slides

19 25 June 2014 IC Retreat

Page 20: IonWorks Barracuda

Automated Electrophysiology at GSK

IonWorks Barracuda IonWorks Quattro

Array of 8x8

holes/well

384 well

Population Patch

Clamp (PPC)

plate

Silicon based chip

with microfluidics:

16 or 48 wells in

parallel

10 hole per

measurement

384 well PPC with

discreet amplifiers

and a 384-channel

pipettor

QPatch

Page 21: IonWorks Barracuda

IonWorks Platforms

IonWorks Quattro IonWorks Barracuda

Perforated Patch Clamp Perforated Patch Clamp

MΩ Seals (20-60MΩ in PPC) MΩ Seals (20-60MΩ in PPC)

48 Electrodes (E-head)/ Fluidic Tips 384 Electrodes/ Fluidic Tips

1 x plenum electrode (≤ 0.3cm2) 4 x plenum electrodes (3.8cm2)

Intermittent recordings Continuous recordings

Voltage-Gated Ion Channel (VGIC) but not

fast Ligand-Gated Ion Channel (LGIC)

VGIC and LGIC (fast and slow)

Data written at end of run Data written continuously

Maximum two compound additions Maximum 10 compound additions

(Through-plate)

Experiment run time = 45 - 90mins Experiment run time = 15 - 30mins (3x

throughput)

Consumable costs on Barracuda ~10% more than Quattro

Cost per datapoint = £3.17 (5pt CRC),

£0.63 (SS)

Cost per datapoint = £3.50 (5pt CRC),

£0.70 (SS), £0.70 (6pt CRC, through-

plate mode)

25 June 2014 IC Retreat


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