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

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