Assay Development of Multi-Subunit Ion Channels for ...

Assay Development of Multi-Subunit Ion Channels for Drug Discovery Screening Using Electroporation

GABA-A Receptors Tyrrell Norris Innovative Medicines & Early Development Discovery Sciences Reagents and Assay Development

Cell-Based Assay Development Rapid generation of fit-for-purpose cellular reagents and assays for drug discovery • Target class

• Cell type • Assay technology

• Cell supply scale • Stable or transient expression

• Continuous cell culture or cryopreserved cells 2

AstraZeneca - Discovery Sciences | 2013

Reagents and Assay Development – T.Norris

Continuous Culture or Cryopreserved Cryopreservation uncouples cell culturing from drug-screening activities and allows use of cells as reagents, just like enzymes in biochemical assays. (Guido Zaman, et al, Drug Discovery Today, v12, July 2007) • Decreases day-to-day variation • Eliminates passage effects • Improves consistency 2001 - AstraZeneca aspired to have > 75% of cell-based assays use cryopreserved cells 3



Rapid Generation of Cellular Reagents Transient transfection + cryopreservation • Rapid and flexible work-flow for generating cells for assays for drug discovery • Eliminates continuous cell culture • Facilitates expression of ‘toxic’ targets • Reduces gene-to-assay time • Provides flexibility in running assays Challenge • Cost-effective, large scale, transfection 4



g-Aminobutyric Acid (GABAA) Receptors Major inhibitory neurotransmission in CNS Occurs at all levels of CNS

Are targets for neurological and psychiatric disorders • Sedation • Anesthesia • Epilepsy • Anxiety • Muscle Relaxation 5


Basic and clinical pharmacology, Katzung BG,2001:364


g-Aminobutyric Acid (GABAA) Receptors Pentameric, ligand-gated Cl- ion channel family 18 different subunits

GABA

GABA

BZD

Composed of two a, two b and one g subunits GABA binding site at a / b interface Benzodiazepine (BZD) site at a / g interface 6


ClPharmacological Reviews, v62, 2010


GABA-A Assays for Drug Discovery Identification of GABA-A subtype selective modulators has been slowed by the lack of • Cells expressing receptors with the desired subtype composition • Assays amenable to HTS

Develop flexible, scalable platform for screening • Cryopreserved transiently transfected cells • Functional receptors with disease relevant subunit composition • a1b2g2 and a2b3g2 7



Assay Formats – Options Membrane Potential - Voltage Sensitive Dye (VSD) • False positives • Cl- efflux (opposite direction) • VSD potentiation of GABA-A channels(*)

Electrophysiology-based Assay • Planar patch (Q Patch)

Halide Sensor • Yellow Fluorescent Protein • Consistent co-transfection of expression of 4 constructs • I- influx (non-cognate anion) (*) Mennerick, et al, J Neurosci. 2010;30(8):2871-9 8



Membrane Potential VSD Assay Indicator dye detects changes in membrane potential Membrane depolarization increases fluorescence • Dye enters cell following cations as Cl- flow outwards • Cl- free extracellular buffer (gluconate) to drive efflux Resting State

Hyperpolarized

Signal Decrease

9


Depolarized

Signal Increase

Molecular Devices


VSD - Initial Studies Subunit composition

Buffer

GABA

VSD Signal (RFU)

Full agonist effect requires expression of at least a and b subunits

GABA-A a2 Receptor

Partial, non-agonistmediated, activity from b3 only expression HEK293s cells: lipid-based transfected, cryopreserved 10



VSD - Initial Studies GABA-A a2 Receptor

Benzodiazepine-site modulation requires a, b and g subunits HEK293s cells: lipid-based transfected, cryopreserved 11


a2b3

a2b3g2

VSD Signal (RFU)

Chloride channel activity requires a and b subunits

+

Buffer + GABA + + Diazepam + + DMCM + + Picrotoxin +

+

-

+ +


+ +

Optimization of Expression & Function Design of Experiment (DoE) • Subunit composition • Stiochiometry • Pharmacological response

• GABA-A a1b2g2 • Vector ratio = 3 : 1 : 3

• GABA-A a2b3g2 • Vector ratio = 10 : 1 : 10 • Low b3 ratio required to control leaky channel 12



Optimization of Expression & Function • Hill slope ~2 • 2 GABA binding sites

Benzodiazepine-site • Positive and negative modulation • Receptors contain g subunit Consistent with a stoichiometry of two a, two b and one g subunits 13


GABA-A a2b3g2 Receptor

VSD Signal (RFU)

GABA response

GABA

Diazepam DMCM

-8

-7

-6

-5

GABA log [M]


-4

Overcoming Limits of Lipid Transfection MaxCyte STX Static / Flow Electroporation • Flexible, scalable, cost-effective

Cells + DNA

Processing Assembly (PA)

Electroporate

Recovery

Cryopreserve MaxCyte, Inc

• Single Batch - up to 2x1010 cells • Consistency - between batches, pooled cell batches • Variability - high quality DNA is important 14



Improved VSD Assay - Electroporation

• CHO-K1 cells – improved signal, reproducibility

Agonist and modulators detectable Scalable platform for assay development & screening 15


GABA-A a2b3g2 / CHO-K1 500

VSD Signal (RFU)

HEK293s and CHO-K1 cell lines

400

Diazepam

300

DMCM

200

GABA

100

-8

-7

-6

-5

GABA log [M]


-4

Patch Clamp Electrophysiology Assay Measure currents across the cell membrane Considered the ‘Gold Standard’ for ion channel studies

Excellent signal resolution Sophion Q-Patch automated planar patch clamp instrument 16



Q-Patch Electrophysiology Assay Expected pharmacology

GABA-A a1b2g2

GABA-A a2b3g2

Cells functional in multiple assay platforms • VSD • Plate-based automated electrophysiology

EC50 = 4.1uM

17


EC50 = 4.4uM


Benchmarking Transient vs Stable Cells Comparison of Transient MaxCyte vs Stable cell line Q-Patch

Q-Patch: 100uM GABA @ -80mV Cells

% Record

% Respond

a1b2g2 MaxCyte

94

56

-3.0 nA

a1b2g2 Stable

56

38

-0.2 nA

a2b3g2 MaxCyte

96

50

-1.0 nA

a2b2g2 Stable

92

71

-0.8 nA

VSD: GABA a1b2g2 MaxCyte Stable Cell Line

VSD Signal (RFU)

VSD assay

Transient and Stable cells yield similar results

-8

Stable cell line: Chantest EZ-cells 18


Avg Response

-7

-6

-5

GABA log [M] Reagents and Assay Development – T.Norris

-4

YFP Halide Sensor Assay • Intracellular sensor of anionic flux • Mutant YFP-H148Q / I152L • Increased sensitivity to halide concentration • F- > I- > NO3- > Cl- > Br- > formate- > acetate• Fluorescence quench is proportional to ion flux Resting

Iodide

Outside

Stimulated

Iodide Quench

YFP

Inside

Invitrogen

19



YFP Assay Agonist-Independent Signal Quench

Is this GABA-A Receptor-mediated?

GABA-A a2b3g2

60mM NaI

Agonist-independent YFP signal quench

GABA-A a1b2g2

120mM NaI

High [NaI] used based on literature

A = 0 uM GABA + NaI B = 100 uM GABA + NaI 20



YFP Assay Agonist-Independent Signal Quench GABA-A a2b3g2

Fluorescence quench

40

• Proportional to [NaI]

• Picrotoxin blocks YFP quench

30 % Quench

• GABA-A receptor dependent

20

10

0 NaI (mM) 0 GABA -

21


Buffer Picrotoxin

5 -

10 -

20 -

40 -


10 EC80

YFP Assay – [NaI] Optimization % Baseline

Minimal Basal YFP Quench

Time

Maximal Signal Window

Effect on Potency

GABA EC50 0-40mM NaI

Quench % YFP % Quench

34

0mM 5mM 10mM 20mM 40mM

40mM 20mM

30 30

10mM

26 22

5mM

20 18 14

10 10

0 mM

6

0

2 1x10

-8

-8

-7

1x10 1x10 GABA Conc. (M)

-6

1x10

-5

-7 -6 -5 GABA log [M]

GABA-A a2b3g2 / CHO-K1 22


10mM NaI Optimal

GABA pEC50

42

40 38

5

10 20 NaI (mM)

40


GABAA Receptor Allosteric Modulators Modulators active in all assay platforms

140

Q-Patch Assay

120 100 80 60 40 20

Identify GABA-A subtype selectivity in all assay platforms

% Diazepam Effect

Similar rank order modulation between assay platforms

0

-20 140

VSD Assay

120 100 80 60 40

20 0

500

YFP Assay

400 300 200 100

GABA-A a1b2g2 / CHO-K1 GABA-A a2b3g2 / CHO-K1 23


0

Diazepam Lorazepam Clobazam Alpidem L- 838,417 TPA- 023 Desmethyl Clobazam


Summary Cryopreserved, transiently transfected cells • Effective approach to evaluate expression and assay feasibility, development and screening • MaxCyte electroporation facilitates cost-effective, largescale transient transfection • Efficient means to generate cells for different assay platforms • Express functional multi-subunit targets • Develop robust assays suitable for drug discovery

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Acknowledgments MaxCyte Cryopreserved Cells & Assays

Helena Peilot Sjögren Teres Johansson Andreas Nolting Mei Ding Lipid-Transfected Cryopreserved Cells & Assays Jay Liu Tongming Chen 25



AstraZeneca Innovative Medicines & Early Development

Discovery Sciences

Reagents and Assay Development

• • • • • 26


Cellular Assay Development Stem / iPS Cells Biochemical Assay Development Protein for Structure Transgenics Reagents and Assay Development – T.Norris

References Yellow fluorescent Protein-Based Assay to Measure GABAA Channel Activation and Allosteric Modulation in CHO-K1 Cells. T.Johansson, T.Norris, H.Peilot-Sjögren. PLOSOne, v8, 2013. Diverse Voltage-Sensitive Dyes Modulate GABAA Receptor Function. S.Mennerick, M.Chisari, S.Hong-Jin, A.Taylor, M.Vasek, L.Eisenman. C.Zorumski. The Journal of Neuroscience, v30, 2010. Regulation of GABAA Receptor Subunit Expression by Pharmacological Agents. M.Uusi-Oukari, E.Korpi, Pharmacological Reviews, v62, 2010. A High-Throughput Functional Assay for Characterization of g-Aminobutyric AcidA Channel Modulators Using Cryopreserved Transiently Transfected Cells. J.Lui, T.Chen, T.Norris, K.Knappenberger, J.Huston, M.Wood, R.Bostwick. Assay and Drug Development Technologies, v6, 2008. Cryopreserved cells facilitate cell-based drug discovery. G.Zaman, J.deRoos, M.Blemenröhr, C.van Koppen, J.Oosteron. Drug Discovery Today, v12, 2007. A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABAA receptor chloride channels. W.Kruger, D.Gilbert, R.Hawthorne, D.Hryciw, S.Frings, P.Poronnik. J.Lynch. Neuroscence Letters, v380, 2005. Green fluorescent protein-based halide indicators with improved chloride and iodide affinities. J.Galietta, P.Haggie, A.Verkman. FEBS Letters, v499, 2001. B.Katzung, S.Masters, A.Trevor, Basic and clinical pharmacology, 2001:364. Mechanism and Cellular Applications of a Green Fluorescent Protein-based Halide Sensor. S.Jayaraman, P.Haggie, R.Wachter, S.Remington, A.Verkman. The Journal of Biological Chemistry, v275, 2000. GABAA receptors are differentially sensitive to zinc: dependence on subunit composition. T.Smart, S.Moss, X.Xie, R.Huganir. British Journal of Pharmacology, v103, 1991.

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