®
Application of the Corning Epic
System to label-free functional
GPCR assays
Kathy Dodgson
Lead Generation Biology
AstraZeneca R&D Charnwood
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Overview
•
•
•
•
Outline of the technology
Application to all classes of G-protein
Endogenous receptors
Establishing a screening assay for a
muscarinic receptor
• Summary
®
®
Corning Epic System
• A label-free drug screening system
¾ An SBS-standard 384-well microplate with optical biosensors
¾ An HTS-compatible microplate reader
LOPAC Screening Plate number 1
1uM LOPAC (1st addition)
400.0
300.0
+
=
Response (pm)
200.0
100.0
0.0
0
10
20
30
40
50
60
-100.0
-200.0
-300.0
Compounds by column
Epic® Microplate
• 384-well format
• Optical biosensor
in each well
Epic® Microplate Reader
• Compatible w/ HTS automation
• ≥ 40,000 wells/8hrs
Assay Results
70
80
90
100
The Biosensor
• The
substrate grating is covered with a thin dielectric layer; this forms the
resonant waveguide grating.
• The sensor is illuminated with many wavelengths.
• Only a “single” wavelength that is resonant with the waveguide grating
structure is strongly reflected.
• A change in the index of refraction at the sensor surface causes the
resonance wavelength to shift
• The bottom of each well in the
Transmitted
wavelengths
Waveguide
Substrate with
grating
Broadband
(many wavelengths)
illumination
Resonantly reflected
wavelength
Epic® 384-well microplate contains a
biosensor
The Biosensor
Cell-Based Assays
Cell
Broadband
Source
Mass
Redistribution
Reflected
wavelength
• The Epic® System measures refractive
index changes at the sensor surface
¾ Light of the resonant wavelength couples into, and
propagates down the waveguide.
¾ A portion of the resonant wavelength extends into
the first few hundred nanometers (~150 nm)
above the surface of the waveguide,which defines
the sensing region.
¾ Cells can be plated onto the EPIC plate and their
response to specific stimuli measured.
¾ Dynamic mass redistribution within a cell causes
index of refraction changes, resulting in a
wavelength shift
• The Epic® System reports shifts, relative
to an initial (baseline) measurement.
Integrated Epic® System
Cell-based assay examples
Assay Procedure
•
Cells are plated onto the EPIC assay plate and allowed to adhere O/N
¾ Uncoated standard plates for endogenous cells
¾ Fibronectin coated plates for HEK and CHO clonal lines
•
Prior to assay the cell media is aspirated (Tecan Power Washer) and
replaced with assay buffer (HBSS, 20mM HEPES +/- 0.1%BSA)
•
Compounds and agonists are prepared in the same buffer system.
•
The plates are then equilibrated in the system incubator for 1hr.
•
The EPIC reader measures an initial baseline
•
The compounds are added to the assay plate using an integrated
CyBiWell pipettor
•
The agonist is added and further scans are made
Example of a Gq Receptor
EC80
400
buffer
350
mean
stdev
Z'
Agonist
308
20
0.72
buffer
-8
10
wavelegth shift (pm)
300
250
200
150
100
EC50 = 19nM
50
0
-50
0.01
0.1
1
10
100
[agonist] (nM)
1000
10000
100000
CHO WT comparison
CHO Gq
300
300
250
response (pm)
200
100
200
150
CHO clone EC50 = 24nM
CHO WT EC50 = IA
100
50
0
0
1E-4
1E-3
0.01
0.1
1
10
[agonist] (uM)
CHO WT
100
0
• CHO WT cells did not generate a
response to the agonist.
• Both cell types responded to ATP
(not shown)
Example of a Gi Receptor
EC80
300
response (pm)
250
buffer
200
150
EC50 = 2.2nM
100
50
0
10
20
30
Dose response
0
0.01
0.1
1
10
[agonist] (nM)
• HEK cell line
• Chemokine receptor
• EC50 similar to other assay formats
0
10
20
30
Effect of Pertussis on Gi response
• Response decreased
by pre-treatment with
pertussis toxin
100
% Inhibition
80
untreated
100ng/ml pertussis
200ng/ml pertussis
60
• 200 ng/ml PTX
decreased the response
by 70%
40
20
0
0.01
0.1
1
[agonist] (nM)
10
Example of a Gs Receptor
Effect of formoterol on H292 cells (15K/well)
70
50
60
response (pm)
50
0
40
30
20
EC50 = 130pM
10
0
-10
-20
1E-4
0
10
20
30
40 mins
1E-3
0.01
0.1
1
10
[agonist] (nM)
• H292 is an epithelial cell line
• Cells adhere to uncoated plate
• Endogenous Gs response for
beta2 receptor
Summary of GPCR Responses
Gq response
Gi response
Gs response
400
50
300
200
200
100
0
100
0
0
0
-50
5
10
15
®
0
10
20
30
0
10
20
• The EPIC is able to measure distinct responses for
the 3 types of GPCR
30
40
Endogenous Receptors
Human Neutrophils
• Human neutrophils were prepared by density
gradient centrifugation
• They were resuspended in HBSS/HEPES
plus 0.1% BSA and plated onto uncoated
EPIC assay plates at 80 to 50K/well
• The plates were centrifuged briefly
• Agonists were prepared in the same buffer
system
• The effect of IL8 and GROα was measured
using the Epic® System
Neutrophil Response to IL8
25nM
12.5nM
1200
3.2nM
1000
1.6nM
0.8nM
Response (pm)
800
600
EC50 = 1nM
400
200
0
-200
0.01
0.1
1
[IL8] (nM)
60K neutrophils/well
FLIPR EC50 = 1nM
10
Neutrophil Response to GROα
1600
1400
25nM
1200
12.5nM
3.2nM
1.6nM
Response (pm)
1000
800
600
EC50 1.4nM
400
200
0.8nM
0
-200
0.01
0.1
1
[GROalpha] (nM)
60K neutrophils/well
FLIPR EC50 = 0.5nM
10
Screening Assay
Muscarinic Antagonist Screening
Antagonist
• Cells plated out O/N
• Cells washed and
assay buffer added
• Initial baseline read
• Compounds added
to adherent cells
• 0.25% DMSO final
• Agonist then added
(0.25% DMSO)
Buffer controls
EC80 control
Antagonist
(O – P)
(L – N)
( B – K)
CHO antagonist rank order
10
9.5
9
EPIC
8.5
Run 2
9
8.5
8
8
7.5
7.5
Run 1
7.5
8
8.5
9
• Five standard compounds were
tested on two days
• Equivalent pIC50 values were
obtained
7
7
7.5
8
8.5
9
9.5
10
FLIPR
• The pIC50 values correlated well
with FLIPR data
CHO Gq Screening Test
120
• 20 wells were spiked
with a standard
compound at 2
concentrations
100
% Inhibition
80
60
• All the spiked wells were
identified
40
20
• No false positives >20%
0
0
50
100
150
-20
Well
200
250
• The Z’ value was >0.6
Correlation of Hits
150
• A compound plate
containing 320
compounds was tested
in duplicate on the
same day
125
Correlated hits
100
75
• Line of correlation
R2 = 0.81
50
25
0
-25
-25
0
25
50
32726
75
100
125
150
Summary
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• We have shown that the Epic System can measure
specific functional responses to cloned Gi, Gs and
Gq GPCRs, with distinct temporal relationships.
• The technology is also sensitive enough to measure
endogenous responses offering the possibility of
performing assays in a biologically relevant system
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• We are using the Epic System for orthogonal
screening post HTS. We intend to screen the first
HTS on the system next year.
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• We have also employed the EPIC for
compound/protein binding assays (See Philip
Rawlins’ poster).
Acknowledgements
AstraZeneca
• Philip Rawlins
• Martin Coldwell
• Malbinder Fagura
• Dave Laughton
CORNING
• Denis To Van
• Alice Gao
• Ute Vespermann