Download Application of label-free detection to HTS using live cells

Application of label-free detection to
HTS using live cells expressing
endogenous GPCRs
Magalie Rocheville
Biological Reagents & Assay Development
GSK - UK
Presentation Outline
Label-free detection and GSK drug discovery
Introduction to the Epic System®
Hit ID and characterisation in non-recombinant cellular
systems :
– A431 cells
– U2OS cells
Conclusions and future perspective
Evaluation performed in collaboration with
Corning, Fontainebleau
GPCR targets in drug discovery
GPCRs are intensively pursued as
tractable drug targets across the
pharmaceutical industry
– Encompasses >30% of marketed drugs
GPCRs are associated with numerous
assay readouts
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Ligand binding
Intracellular calcium (FLIPR, aequorin)
cAMP accumulation (TR-FRET)
G protein activation (S35-GTPgS binding)
Non-classical pathways i.e. Gα12/13, βarrestin, GIRK channels etc…
Conversely, readouts are tailored to
specific pathways
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‘Reductionist’ approach?
Potential gaps in screening format portfolio?
Activation of multiple pathways addressed?
Recombinant vs. native settings?
Hopkins & Groom, Nat Rev Drug Discovery 2002
Scopes underlying LF technologies
• Phenotypic end-point or
summation of signalling events
• Unique kinetic response
• Readout independent of
signalling route(s)
What can LF detection offer?
Physiological relevance and mechanistic ‘texture’ in screening data
LF could impact on most GSK/pharma drug discovery programs:
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Novel hit ID strategy (HTS)
Enable novel assay readouts
Reagent validation
Native/primary cell confirmations of recombinant HTS/SAR data
Additional information on existing chemotypes
Impactful MoA
Receptor panning (& endogenous system characterisation)
Gaps in screening approaches (i.e. non-rad Gαi GPCR)
Differentiation of late-stage compounds vs. multiple signalling pathways
Target ID
Hit ID
Lead
optimisation
Candidate
Selection
drug discovery and development pipeline
Pre-clinical
Dev
Clinical phases
Utility of label-free
Further increases in throughput
enabling hit ID and characterisation
Primarily offering improved/increased
physiological relevance earlier in cascade
Throughput
uHTS
HTS
Currently spans typically discrete activities
1o
SAR
Immediately applicable for 2o functional screening/triage
Label
Free MoA
Commonality of platform (Biochemical & Cellular apps)
1o
culture
Whole
Organ
Physiological Relevance
in
vivo
Introduction to the Epic® System
Cell- and biochemical-based assay platform
Optical
Real time kinetic readout
Contains plate carousel
Temperature control (26oC)
– Plates are lidded
– Thermal equilibration on carousel
– Granite insulation
– Temp sensors
– Cooling system
Scheduling software for integration
– Reader User Interface
– Data Viewer for offline view
User-friendly data extraction software
package
3x scans per read per well
5x lines per scan
Principle of dynamic mass redistribution (DMR)
Phenotypic cumulative endpoint
384-well microplate biosensor
– Resonant Waveguide Grating
– Sensitivity ~ 150mm from plate surface
Indirect measurement of cytoskeletal rearangement, protein recruitment,
endocytosis and recycling, apoptosis, exocytosis …
Cell-based assay methodology
Assay start (26oC throughout)
Compounds
3 mins
Seed cells
(18-24hrs)
Serum-starve
(18-24hrs)
Buffer exchange *
& incubation (2hrs)
EC80 agonist
3-50 mins
3-50 mins
Epic reading: Kinetic or end-point mode
(pre & post-cpd reads *)
Plate cells overnight (cell density optimisation required)
Serum-starve cells (Biomek Fx)
Replace medium with assay buffer (Biomek Fx)
Allow cells to equilibrate for 120 mins
Measure baseline for 3 min
Add compound to wells (Cybiwell)
Measure response following compound addition
Add compound to wells (Cybiwell)
Bench-top system
Measure response following compound addition
integration
Data extracted and exported as required
* DMSO content needs optimisation & is then kept constant throughout assay
Example 1: A431 cells
Evaluation of endogenous cellular responses in
single shot mode
Pharmacology check
on A431 cells
A431 cells are human carcinoma cells reported to display
β2AR-mediated functional activity
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Prototypical Gαs-coupled receptor (↑ icAMP)
A fully-developed assay in dual-mode was available at
Corning using the Epic System® :
GSK robustness set
~1400 cpds @ 5uM FAC
(10 plates)
day 1 vs. day 2
GSK validation set
~10,000 cpds @ 5uM FAC
(54 plates)
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Agonist format configured by 1st addition of compounds
Antagonist format configured by subsequent 2nd addition of
epinephrine (EC80), a non-selective βAR full agonist
Fresh cells
Endpoint read mode
Validation testing is used to assess assay quality and
suitability for screening:
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day 1 vs. day 2
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Cpd set representative of the GSK screening collection in
terms of diversity, structural chemotypes and organic
chemistry
Results help to predict hit rate, assay sensitivity/cut-off,
screen performance and triage strategy during HTS
Robustness testing (1.4K)
Orthogonal straight
line fit
x=y
100% Activation
(20nM epinephrine)
0% Activation
Replicate
Robust
Mean
Robust
SD
Robust
Cut-off
N hits
HR(%)
Set 1
1.41
6.71
21.53
36
2.56
Set 2
0.07
11.16
33.56
24
1.70
Robustness testing (1.4K)
Orthogonal straight
line fit
x=y
+ Epinephrine 2nM
100% Inhibition
(10nM propranolol)
0% Inhibition
Replicate
Robust
Mean
Robust
SD
Robust
Cut-off
N hits
HR(%)
Set 1
4.27
9.04
31.40
13
0.92
Set 2
10.51
13.76
51.78
33
2.34
Antagonism vs. receptor desensitisation
Bearing in mind that actives may/ or may not act via β2AR…
Single shot validation stage (10K)
Agonist mode
Change in
Refractive Index
(pm)
200
150
0.6
0.5
0.4
0.3
100
50
0
-50
0.2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 0.1
0
Change in
Refractive Index
(pm)
0.9
0.8
0.7
Robust Z Prime
250
200
0.9
0.8
150
0.7
0.6
0.5
100
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
-50
AVERAGE_LOW
0.1
0
Plate number
Plate number
AVERAGE_HIGH
0.4
0.3
0.2
50
Robust Z Prime
b2AR Agonist Set 2
b2AR Agonist Set 1
AVERAGE_HIGH
Z_PRIME_1
AVERAGE_LOW
Z_PRIME_1
Antagonist mode
b2AR Antagonist Set 1
200
150
100
50
0
300
Change in
Refractive Index
(pm)
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
250
Robust Z Prime
Change in
Refractive Index
(pm)
300
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
250
200
150
100
50
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Plate number
Plate number
AVERAGE_HIGH
AVERAGE_LOW
Z_PRIME_1
AVERAGE_HIGH
AVERAGE_LOW
Z_PRIME_1
Robust Z Prime
b2AR Antagonist Set 2
Single shot validation stage (10K)
Sample wells
DMSO wells
Single shot validation stage (10K)
Sample wells
DMSO wells
edge-effect?
Summary of validation stats
Replicate
Rob
Mean
Rob
SD
Rob
Cut-off
N hits
HR(%)
b2AR assay agonist Set 1
-0.78
5.72
16.40
143
1.50
b2AR agonist assay Set 2
-2.22
5.90
15.80
133
1.40
b2AR antagonist assay Set 1
2.87
9.36
30.94
345
3.60
b2AR antagonist assay Set 2
5.40
9.34
33.49
443
4.70
Example 2: U2OS cells
Evaluation of endogenous cellular responses in
single shot mode
New assay development
and optimisation
Frozen reagent, DMSO tolerance,
pharmacology etc…
U2OS cells are human osteosarcoma cells reported to
display histamine H1-mediated functional activity
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Prototypical Gαq-coupled receptor (↑ [Ca2+]i )
The assay first needed to be developed in single addition
format on the Epic System®:
GSK robustness set
~1400 cpds @ 5uM FAC
(10 plates)
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Short optimisation phase overall
Histamine used as positive control
Frozen cell approach (V2P or vial-to-plate)
day 1 vs. day 2
Assay performance and reproducibility tested with
robustness compound set:
Active follow-up
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Full temporal profile determination
Results compared with alternative technologies i.e. FLIPR
and TR-FRET cAMP
Kinetic response resolution … cost in throughput!
Assay optimisation parameters
Cell density and adherence
Plating time and need for serum starvation
DMSO tolerance
Reading time period (50’ sequence)
Method of data extraction i.e. Max response over time vs. AUC over time
vs. single end-point
Max response
pEC50 = 6.84
Hill = 1.49
0
-20
1500
1000
pEC50 = 6.58
300
Wave shift (pM)
20
360
2000
Wave shift (pM)
Wave shift (pM)
40
AUC start to peak
AUC across all sequence
60
Hill = 2.36
500
-9
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-7
-6
-5
log [histamine (M)]
-4
-3
180
Hill = 1.09
120
60
0
0
-10
pEC50 = 6.12
240
-10
-9
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-7
-6
-5
-4
-3
log [histamine (M)]
- histamine pEC50 in FLIPR = 5.94
- histamine inactive in cAMP
-10
-9
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-7
-6
log [histamine (M)]
-5
-4
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Histamine temporal responses
Time to Peak (mSec)
4000
Compound-dependent
Concentration-dependent
3000
High control
40uM
2000
1000
0
-11
-10
-9
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-5
-4
-3
log [histamine (M)]
Histamine CRCs
15 mins
DMSO plate + low/high controls
50 mins
Robustness plate controls (15’ end-point)
100
Day 1
Day 2
Wave Shift (pM)
80
60
Lows
40
Highs
20
0
1
2
3
4
5
6
7
8
9
10
-20
Plate #
Signal-to-noise for histamine was consistent within- and across days
Good correlation between days
End-point reads
Replicate
Rob Mean
Rob SD
Rob Cut-off
N hits
HR(%)
15’ end-point day 1
0.27
3.60
11.08
11
0.78
15’ end-point; day 2
-0.16
3.24
9.56
10
0.71
50’ end-point day 1
-2.4
11.87
33.19
17
1.21
50’ end-point day 2
-1.93
10.11
28.40
13
0.92
Controls & samples more widespread at 50’min time point
Temporal profiles from diverse chemotypes
Plate 4_1
‘potential ‘active X’
cherry-picked for follow-up
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Actives with mix of temporal & magnitude profiles (+ve, -ve)
Potentially different signalling events
Analysis method - max response? AUC?
Profile correlation between actives
Plate 3 on day 1
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Plate 3 on day 2
Low hit rate; CR ~ 0.6
Visual matching of hits between duplicates i.e. reproducible
Different hits at early time-point vs. late time-point
Activity confirmation assays
Epic
cAMP
Other(s)
FLIPR
Drug?
Profile original cell host (i.e. A431 or U2OS cells) in alternative assay formats
Activity cross-check with recombinant line expressing target of interest
Specificity assays (lacking primary target)
Example of ‘histamine-like’ hit follow-up
Epic ‘hit’
in U2OS
Temporal profile matching
that of histamine (H1)
cAMP – Gs
cAMP - Gi
FLIPR
Inactive in cAMP format + /- fsk (U2OS)
Inactive in FLIPR (U2OS)
Inactive when tested in FLIPR against H1-CHO stable cell line
Alternative signalling route? Slow-onset binder at Gq? False +ve or false –ve?
Conclusions & remaining challenges
The Epic System® is a novel cell-based LF technology
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Good throughput in 384-well plate format, integrated platform
Robust at scale, very sensitive
Costly (specialised plates (Opex), overall assay time)
Compatibility with endogenously-expressed receptors is a big advantage
All-encompassing phenotypic readout offers unique opportunities
Successfully platform evaluation for single shot mode screening
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Promising performance depending on how you measure
Hit rate (unexpectedly) low?
Cost/benefit of end-point vs. kinetic reads for primary screening
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Big impact on throughput – is kinetic a viable option?
Are we at risk of ‘missing’ interesting hits without high content read?
Potentially identify ‘extra hits’ missed in non-label free screens?
Data is quantitative & qualitative but more work required to define
how (if) it correlates with existing assay formats
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Follow-up ongoing
Conclusions & remaining challenges
Data analysis is difficult enough, and data interpretation even
more so
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Only through detailed analysis and interpretation can you exploit the potential
of the technology
Data interpretation: opening Pandora’s box?
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Complex biology
Novel readout (cell shape/holistic)
A target may have single vs. multiple signalling routes
Functional selectivity
Temporal profile complicates analysis
Can we exploit these data to further drug discovery?
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Use of phenotypic assays may have improved physiological relevance
Value of LF in compound progression remains to be addressed
Enabling? How predictive is this technology of in vivo readouts?
Acknowledgements
Corning
Volker Eckelt
Alice Gao
Silvie Bailly
Lucinda Gedge
Ute Vespermann
GSK
Isabel Coma
Angela Dunne
Phil Green
Dion Daniels
Alan Wise
Julio Martin
Jeff Jerman
Backup Slides
A431 assay flow chart (endpoint)
Day 1
Plate A431 cells onto EPIC plate (15k, 30ul/well), Incubate @ 37oC, 5% CO2
for 24 hours
Day 2
Day 3
Wash cells in serum-free media and incubate in 20ul/well
serum free media @ 37oC, 5% CO2 for 24 hours
Wash cells in PBS. Incubate in 20ul/well Assay Buffer (HBSS, 2% HEPES,
1% DMSO) for 90 mins
BASELINE READ
Dilute 0.5ul 1mM cpd plate in 50ul/well Assay Buffer
Add 20ul/well to cell plate (FAC 5uM, 1% DMSO). Incubate for 50 mins
AGONIST END POINT READ
Add 20ul/well 2nM (EC80) Epinepherine, Incubate for 50 mins
ANTAGONIST END POINT READ
Baseline Read
Agonist Read
Antagonist Read
U2OS assay flow chart (kinetic)
Seeding medium: DMEM/F12+10%FBS (‘M1-GSK media)
Assay buffer: HBSS+20mM HEPES + DMSO
50 mins read
time per plate
Recover frozen cell vial stored at -140oC
Thaw, add media , spin, discard media
Day 1
Prepare cell suspension in seeding medium
Plate with FX (20000 cells/well, 30uL)
Incubate overnight
Replace medium with asssay buffer
(include 1x wash), leave final vol of 30 uL
Day 2
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Load assay plate (26oC)
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Allow to incubate for 2hrs
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Select the program and start
baseline measurements for 4.5mins
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Unload assay plate, add cpds 10ul
with CybiWell
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Reload assay plate (26oC)
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Start post-scan in kinetic mode
through appropriate duration ~ 50
mins
30ul assay buffer
with 2% DMSO
2hrs
10ul compound
@ 2% DMSO
Thaw 1ul compound/well stampouts
Dilute by adding 50ul assay buffer
to match DMSO (2%)
Revisiting ‘hit rate’ across reading time
FLIPR vs Epic
FLIPR vs FLIPR
140
120
Max response
100
120
80
100
60
40
80
20
60
0
40
0
20
40
60
80
100
120
% response_2
20
0
-200
-150
-100
-50
0
% response_2
1 - FLIPR
50
(Epic)
2 - Epic
100
150
Non-overlap in actives
between both technologies
140
Bench-top system integration
Biomek Fx used for buffer exchange
CybiWell used for compound addition
Cytomat incubator for plate incubation