Post on 13-Feb-2017
Evaluating the IonWorks Barracuda for ligand-gated ion channel compound profiling Andrew Powell Ion Channel Retreat 2014
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
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
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)
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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)
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
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)
• 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)
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
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
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
• 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
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
• 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
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)
• 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
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
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
Back-up Slides
19 25 June 2014 IC Retreat
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
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