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Development of Screening Tools to
Identify Nicotinic Subtype-Selective
Compounds
Glenn Kirsch, PhD
Discovery Services │Charles River
Cleveland, Ohio USA
Aurora Ion Channel Retreat
July 7 - 9, 2015
Tobacco Control Act – US FDA
• Signed into law 2009
• Authorized the FDA to regulate tobacco products to
protect public health
• The Center for Tobacco Products (CTP) established
to oversee implementation - including research
• CTP contracted with ChanTest to run the NicScreen
project in August 2013, as part of an initiative to
provide scientific support for developing tobacco
product regulations
2
NicScreen Project Objectives
• Primary NicSreen Objective: Develop and validate
bioassay screening tools for rapid identification of tobacco
product constituents with nAChR subtype selective activity.
• Phase I (9/2013 – 8/2014) - Develop cell lines and high-
throughput assays
• Phase II (9/2015 – 8/2016) – Pilot testing to determine the
pharmacological profiles of different tobacco products
• Phase III (9/2016 – 8/2017) – Proof of concept study for
high-throughput screening to evaluate tobacco product
constituents for subtype selectivity. Develop methods for
evaluating similarities and differences in the pharmacologic
activity of marketed tobacco products and constituents
3
Cognition impairment in schizophrenia and
Alzheimer’s - α7 Agonists and PAMs in
clinical trials (e.g., DMXB-A, galantamine,
EVP-6124).
Parkinson’s disease – transdermal nicotine
and varenicline in clinical trials.
Depression – α4β2 antagonist to augment
SSRIs in clinical trials (e.g., mecamylamine).
Tobacco addiction - α4β2 partial agonist
(varenicline), marketed. Varenicline for
treatment of alcohol, cocaine and
methamphetamine addiction in clinical trials.
Inflammatory diseases (e.g. inflammatory
bowel disease) - α7 receptor activation is
anti-inflammatory in animal models.
Nicotinic Acetylcholine Receptors Are Therapeutic Targets
4
• Neuronal nicotinic receptors - pentameric ligand-gated cation-selective channels
• Subtypes defined by α and β subunit composition, and functional properties (agonist-sensitivity, pharmacologic profile, Ca2+ permeability, and desensitization kinetics)
5
Ion Channel HTS Screening & Profiling
Platforms for Nicotinic Receptor Assays • Screening/Profiling Assays
– Ligand Gated Channels
• Ion Works Barracuda (IWB): automated
patch clamp
• FLIPR: ion- or voltage-sensitive dyes
– Voltage Gated Channels
• IWB: automated patch clamp
• FLIPR: ion-sensitive dyes (Ca2+, Tl+)
• Assay Modes
– agonist
– antagonist
– positive or negative allosteric modulator
IonWorks Barracuda
FLIPRTETRA
Assay
Validation
~2 weeks
Cell Line and Assay Validation Workflow
6
Assay
Development
& Optimization
~3 weeks
Validated
Assay ~2
weeks
Optimize recording
conditions:
• Adjust stimulus parameters
• Adjust intra- and
extracellular solutions,
holding potential
• Conduct stimulus/response
testing
• Optimize cell density
• Increase signal window and
minimize variability (Z’
factor optimization)
• Evaluate DMSO tolerance
Functional Validation:
• Signal stability/uniformity
• 2-Day plate uniformity
tests (intra- and inter-day
variability)
• 2-Day reference
compound testing: 3 – 10
cpds, 8 and 16 pt CRC
Reporting:
• Data
analysis
• Data QC
• Report
writing
• Format
• Report QC
Cell line
production:
• cDNA cloning &
transfection
• Clone selection
• Expression
confirmation
• Functional
characterization
• Stability and
Scale-up
Cell Line
Development
& Validation
~3 months
Assay Acceptance Criteria Plate Level Parameters (FLIPR & IWB) Value
Z’ factor ≥ 0.5
CV for MAX Control ≤ 20%
CV for MIN Control ≤ 20%
Signal Window (SW) ≥ 3
EC50 for reference agonists ≤ 0.5 log*
IC50 for reference antagonists ≤ 0.5 log*
*from historical mean
Validation data are obtained in independent experiments conducted on separate days
Well Level Parameter (IWB only) Value
Rseal > 100 MΩ
Current Amplitude > 0.2 nA
Rseal Stability < 50% decrease
Current Stability < 50% decrease
Success rate (% valid wells) ≥90%
7
8
Ligand-Gated Assays in IonWorks Barracuda (IWB)
Throughput (manual plate handling)
• 10 plates/day/instrument
• 3200 compounds/day
• 16,000 compounds/week
Timeline for screening 100,000 cpd
library ~7 weeks
• Population Patch Clamp (PPC) and single hole
recording modes (384-well patch plate)
• Controls single cell membrane potential and measures
ionic currents in single-cell or cell population (≤ 64
cells/well).
• 384-channel pipettor, integrated 384-channel electronic
head
• Validated assays for multiple subtypes in ligand-
gated 5-HT3R, GABAAR, nAChR, NMDAR and TRP
channels
IonWorks Barracuda
Continuous voltage-clamp current
measurement with rapid solution addition for
fast-desensitizing, ligand-gated channels
electrode pipette
cells
Nicotinic Cell Lines and Assays
nAChR Subtype Parental Cell Assays
α3β4 CHO IonWorks Barracuda, FLIPR
α4β2 CHO IonWorks Barracuda, FLIPR
α3β4α5 CHO IonWorks Barracuda, FLIPR
α7 CHO IonWorks Barracuda, QPatch, FLIPR
α6/3β2β3V273S HEK293 IonWorks Barracuda, FLIPR
9
α4β2: Ionic Current Distribution in IonWorks
Barracuda (Single Cell Mode)
333 mM ACh 333 mM ACh
Untransfected CHO Cells a4b2-CHO Cells Clone #26
Stimulation – 0.3 mM ACh
Mean peak ±SD = 1.21 ± 0.82 nA
α4β2: Signal Uniformity
Io n W o rk s B a r ra c u d a U n ifo rm ity - D a y s 1 a n d 2
0 2 /1 7 /2 0 1 4
P la te 1 P la te 4 P la te 2 P la te 5 P la te 3 P la te 6
0 .0 0
0 .2 5
0 .5 0
0 .7 5
1 .0 0
1 .2 5
Mean
Std. Deviation
Std. Error of Mean
Plate 1
-0.001421
0.002791
0.0006977
Plate 4
-0.002157
0.0009560
0.0002390
Plate 2
0.6130
0.03558
0.008896
Plate 5
0.5803
0.04661
0.01165
Plate 3
1.061
0.03884
0.009709
Plate 6
0.9834
0.04179
0.01045
5 m M (-) -N ic o t in e
1 0 0 m M (-) -N ic o t in e
V e h ic le C o n tro l
M IN
M ID
M A X
S ig n a l L e v e l
Cu
rre
nt
(nA
)
D a y 1 D a y 2
Z ` = 0 .5 2 0 .5 7
S W = 3 .4 4 4 .0 8
IonWorks Barracuda (PPC)
FLIPR
α4β2: Agonist Assay 16 point CRC
1E-5 1E-4 1E-3 0.01 0.1 1 10 100 1000
-20
0
20
40
60
80
100
120
140
160
180 Acetylcholine
Nicotine
Epibatidine
IonWorks Barracuda
[Agonist], mM
Ac
tiva
tio
n, %
Reference
Agonist
EC50, μM
IWB FLIPR Xenopus*
Acetylcholine 9.4 0.15 4
Nicotine 5.7 0.12 1
Epibatidine 0.17 0.0024 ND
12
nAChR a4/b2 Response to AgonistsCurve Fit Min and Max Constrained
[Agonist] (mM)
Are
a U
nd
er
Cu
rve
Flu
ore
sce
nce
(R
LU)
-200,000
0
200,000
400,000
600,000
800,000
1,000,000
1.210 0 6
0.0001 0.001 0.01 0.1 1 10 100 1,000
Acetylcholine chlorideEC50 = 150 nM
(-)-Nicotine ditartrateEC50 = 122 nM
()-EpibatidineEC50 = 2.35 nM
10E-510E-6
FLIPR MP-Assay
*Moroni et al., 2006
Max Response, PNU120596 (6 μM)
IWB: α7-CHO Agonist Assay
0.01 0.1 1 10 100 1000
0
20
40
60
80
100
120
140 Nicotine, EC
50 = 86.7 mM
Acetylcholine, EC50
= 95.3 mM
Epibatidine, EC50
= 9.1 mM
[Concentration], μM
No
rma
lize
d P
ea
k C
urr
en
t, %
1E-4 1E-3 0.01 0.1 1 10 100
0
20
40
60
80
100
120 Nicotine
Cytisine
Epibatidine
Acetylcholine
PNU 282987
[Concentration], μM
No
rma
lize
d P
ea
k C
urr
en
t, %
EC50, μM
Nicotine 1.3
Cytisine 1.4
Epibatidine 0.03
Acetylcholine 2.7
PNU282987 0.07
Max Response, no PAM
13
α7 Positive Allosteric Modulators
Reference Compound
EC50, µM
IWB FLIPR Published
NS 1738 21.7 ND 12.5*
PNU 120596 0.53 3.6 0.216†
SB-206553 >5 µM ND 45‡ *Friis et al. 2008. QPatch ionic current †Hurst et al. 2005. FLIPR Ca2+ flux ‡Roncarati et al., 2008. FLIPR Ca2+ flux PNU 120596 was the most effective PAM
IonWorks Barracuda
14
FLIPR
1E-3 0.01 0.1 1 10 100
0
20
40
60
80
100
120 NS 1738
PNU 120596
SB-206553
[Concentration], mM
Ac
tiva
tio
n, %
Agonist Profiles
0.1
1
10
100
Acetylcholine Nicotine
EC
50,
μM
IWB Agonist
α3β4
α3β4α5
α4β2
α7
α6*
0.001
0.01
0.1
1
10
100
Acetylcholine NicotineE
C50,
μM
FLIPR Agonist
α3β4
α3β4α5
α4β2
α7
α6*
15
IWB: Subtype Selectivity Pilot Study: ENZO Library Agonist
Mode SP Screen, 786 Drugs @ 60 µM
16
FLIPR: Subtype Selectivity Pilot Study: ENZO Library Agonist Mode
SP Screen, 786 Drugs @ 12 µM
-10
10
30
50
70
90
110α4β2 %Activation
-10
10
30
50
70
90
110α3β4 %Activation
-10
10
30
50
70
90
110α3β4α5 %Activation
-10
10
30
50
70
90
110α7 %Activation
17
IWB: Enzo Lib. Screen (60 µM) – PAM Mode
α7-Selective PAMs
PM1 antineoplastic, EGFR inhibitor
PM2 antineoplastic, kinase inhibitor
PM3 phosphodiesterase inhibitor
PM4 microtubule disruptor
PM5 corticosteroid
α3β4-Selective PAM
PM6 antibacterial, KCNQ1 channel activator
18
α7 PAM Confirmation CRC: EGFR Inhibitor
PM1
Concentration, µM
0.01 1
% A
cti
va
tio
n
-50
50
150
250
350
450
550
AUC (5 s)
Peak
19
0.01 μM
0.3 μM
1 μM
30 μM
FLIPR vs. IWB Antagonist Screen
0
50
100
% In
hib
itio
n @
10
μM
α7: FLIPR vs. IWB
IWB
FLIPR
20
IWB: Antagonist Evaluation
0.1
1.0
10.0
100.0
IC5
0, μ
M
IWB: nAChR Subtype Selectivity
α3β4
α4β2
α7
IWB: Evidence for Pore Blocking by Nondepolarizing Neuromuscular Blocking Agents in α7 Receptors
Pancuronium
Conc (uM)0.01 1
%In
hib
itio
n
-50
-20
10
40
70
100
Vercuronium
Conc (uM)0.01 1
%In
hib
itio
n
-50
-20
10
40
70
100
Rocuronium
Conc (uM)0.01 1
%In
hib
itio
n
-50
-20
10
40
70
100
Concentration-Response Curves: Peak current (blue) vs. AUC (green)
Drug IC50, μM (AUC) IC50, μM (Peak Current)
Pancuronium 0.17 0.57
Rocuronium 0.21 0.59
Vecuronium 0.19 0.50
22
nAChR Summary • IWB and FLIPR platforms are suitable for high-throughput
nAChR assays, but have different agonist/antagonist
sensitivities
• IWB Agonist & antagonist sensitivity/selectivity of the α3β4,
α3β4α5 and α4β2 IWB assays were similar to that of
conventional e-phys.
• IWB Sensitivity of α4β2 and α7 subtypes positive allosteric
modulators was similar to conventional e-phys.
• α6/3 β2 β3V273S Cell line showed the expected high sensitivity
to agonists. EC50 values were similar to conventional e-phys.
• α7 Subtype-selective, novel agonists and PAMs were
identified in the Enzo Library.
23
Acknowledgements • Cell and Molecular Biology
Luke Armstrong, Head
• Zhiqi Liu
• Abby Sewell
• Amy Wright
• Discovery Services
Jessica Brimecombe, Head
• Yuri Kuryshev
• Nikolai Fedorov
• Caiyun Wu
• Xiaoyi Du
• Peter Hawryluk
• Support
• US FDA, Center for Tobacco Products
24
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