Transcript Label free – why is it important to PKI and you? Label

Synergistic validation of
biochemical and cellular targets
using luminescent/fluorescent
and label free technologies
Roger Bossé, Ph.D.
June 15th, 2011
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© 2011
2009 PerkinElmer
Detection of Biological Events
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Label-Free Modalities: Isothermal Titration Calorimetry
ΔG = -RTlnK = ΔH-TΔS
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Label-Free Modalities: Cellular Dielectric Spectroscopy
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Label-Free Modalities: Surface Plasmon Resonance
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Label-Free Modalities: EPIC (waveguide grating structure)
When the sensor is illuminated with broadband light, only a single wavelength that is
resonant with the waveguide grating structure is strongly reflected.
Epic System measures the wavelength reflected by the sensor. This wavelength is
determined by the optical properties of the sensing zone ~150nm above the sensor.
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Cellular & Biochemical Assays Validated on Epic® Technology
Biochemical Applications
Assay Development
Aggregation
Direct Binding Assay With Nuclear Receptors
Fragment Screening
Functional Enzyme Assays
HTS
Kinases & Proteases
Microarray
Protein-Oligo (DNA / RNA) Interactions
Protein:Protein
Receptor – Antibody Binding
Antibody – Antibody Binding
Small Molecule Binding
Antibodies / Biologics
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Cell-Based Applications
Antibodies / Biologics
Cells:
Endogenous & Recombinant
Primary & Stem
Adherent & Suspension
Freshly Cultured & Frozen
Patient Samples
Cell Adhesion
Chemotaxis
Cytotoxicity
GPCRs
GPCR Heterodimerization
Ion channels
Nuclear Hormone Receptors
Phagocytosis
Receptor Tyrosine Kinases
Toll-like Receptors (TLRs)
Transient Transfections
Viral infection
Label-Free: Small Compound Binding Detection
Molecular
Weight
(Da)
EnSpire
KD [nM]
Biacore**
KD [nM]
Epic
KD [nM]
BSF
157
983
848
nt
CBS
201
790
893
700
Acetazolamide
222
55
19
27
Dansylamide
250
701
760
513
Furosemide
330
639
513
550
Sulpiride
341
96 M
186 M
29 M
Carbonic Anhydrase II Binding To 6 Small Molecule Inhibitors Using
High-Sensitivity Biochemical Plate
Compound
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Benzenesulfonamide (157)
20
Carbachol (183)
CBS (201)
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Acetazolamide (222)
10
Dansyl Amide (250)
Furosemide (330)
5
Sulpiride (341)
0
Response (pm)
Benzenesulfonamide (157 Da), KD = 983 nM
5
0
0
2500
5000
7500
5
0
0
Furosemide (330 Da), KD = 639 nM
7500
15
10
Furosemide
5
0
0
2500
5000
Conc (nM)
7500
10000
10
5
Acetazolamide
0
0
2500
5000
7500
10000
Carbachol (182 Da), No Binding
30
10
Sulpiride
5
20
10
Carbachol
0
0
0
-5
200
Conc (M)
400
600
15
10
Dansyl Amide
5
-5
Conc (nM)
-5
15
20
0
10000
20
20
15
Sulpiride (341 Da), KD = 96 nM
Response (pm)
Response (pm)
5000
25
25
8
2500
Conc (nM)
-5
30
-5
CBS
10000
Conc (nM)
-5
10
Response (pm)
Response (pm)
Response (pm)
25
Benzenesulfonamide
10
30
20
15
15
Dansyl Amide (250 Da), KD = 701 nM
Acetazolamide (222 Da), KD = 55 nM
CBS (201 Da), KD = 790 nM
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Response (pm)
-5
Response (pm)
Response (pm)
30
0
5000
Conc (nM)
10000
0
2500
5000
Conc (nM)
7500
10000
Label Free: Biologics Binding Detection
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Label-Free Detection of Cellular Activity
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How Epic® Label-free Technology Works
t
surface
waveguide
substrate
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A
0
B
N-DMR
d
E
D
2
P -DMR
Response (x102 pm)
3
F
-1
C
-2
0
2000
4000
Time (sec)
Broadband
Source
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Reflected
wavelength
KEY:
A: Equilibration with buffer
B: Compound addition
B to C: bulk index change
C to D: increased mass within the sensing volume
D to E: steady transition state
E to F: decreased mass within the sensing volume
DMR = Dynamic Mass Redistribution
Label Free as Universal Detection Technique to Measure Cellular Activity
Effect of anti-EGFR(528) on32nMEGF-inducedresponse of
quiescent A431cells
Ion Channels
Receptor Tyrosine Kinases
EGF dans A431 cells
200
2
Response
Response
(unit) (pm)
100
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Toll-Like Receptors
Activation dose-dépendante de TLR3 dans A549
36.2nM
18.1nM
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Control
9.06nM
4.53nM
-100
-1
2.27nM
-200
-2
32 nM EGF
1.13nM
-300
-3
0.57nM
-400
-4
-500
-5
0
0
Courtesy of Len Kaczmarek, Yale University
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2000 min
60
Time (s)
Viral Infection
4000
90
6000
Cytotoxicity
Emetidine-induced toxicity on Cardiomyocytes
HeLa cells
Courtesy of Moonsoo Jin, Cornell University
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Label-Free Detection of GPCR Cellular Activity
Conventional assay
LF Assay
Gaq
Gai
Gas
Naïve or transfected cells
Naïve or transfected cells
Ca+2
Release
cAMP
Reduction
cAMP
Production
Receptor oligomerisation
Functional selectivity test
Different ligands activate
different pathways
Ca+2 flux
cAMP (-)
cAMP (+)
Global
assaycell response
assay (phenotypical)
assay
GPCR dimers can stimulate
different pathways
Functional selectivity
dimers
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Performing Label-Free Cell Based Assays is Straightforward
Thaw & Seed!
40 L; CHO Cells at 4-8K per well & 1321N1 Cells at 8-12K per well
Seed Cells
Incubate Cells O/N
Serum Starve Cells O/N (Optional)
Buffer Exchange
Replace Medium With Assay Buffer & Allow Cell Plate To Equilibrate
Incubate Cells 90 min rt
Baseline Scan
(30 min)
Agonist Assay
Scan (60 min)
Antagonist Assay
Scan (30 min)
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Add Compounds at 6X
Data Analysis
Agonist Assay
Scan (60 min)
2
Add Compounds at 5X
Data Analysis
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Detection of GPCR Activity: Distinct Pathways Yield Distinct Traces
Gs-coupled Receptors
Gi-coupled Receptors
2-AR
PAR1
1
A431 cell
response
0
0.5
0.0
-0.5
A431 cell
response
-1.0
-1
0
600
120 20
18030
240
0 10
40300
50 360
60 420
70
0
0
0
0
0
0
Time (min)
Time (sec)
Gq-coupled responses typically have a
initial rapid P-DMR response
An N-DMR period often follows,
especially for endogenous receptors
Engineered cells can produce very large
(>600pm) peak response amplitudes
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Response (unit)
R es pons e (unit)
Response (unit)
2
100pm
100pm
1.0
3
0
MC receptors
2.0
0 600
10 1200
20 1800
30 2400
40 3000
50 3600
60 4200
70
Time (s ec )
Time (min)
Gs-coupled responses often have an
initial N-DMR response
A gradual P-DMR period typically follows
100pm
Gq-coupled Receptors
1.5
1.0
0.5
A431 cell
response
0.0
0
10 20
50 3600
60 4200
70
6000 1200
1800 30
2400403000
Time (min)
(sec)
Time
Response profile is dependent on
specific cell biology
Often Gi-coupled responses have two
phases that resemble portions of the Gq
& Gs responses
Gi-coupled response may also resemble
the Gq response profile, especially in
overexpressed cells
Detection of GPCR Activity: cAMP, Ca2+ and …. Label-Free
A1 &  Opioid, Gi-Pathway
M1, Gq-Pathway
Agonist & Antagonist Effects On Endogenous B2AR In A431 Cells
Agonist & Antagonist Effects On Endogenous A1 In HEK293 Cells
Agonist & Antagonist Effects On Endogenous M1 in HEK293 Cells
50
200
40
150
Isoproterenol (Agonist)
Propranolol (Antagonist)
100
50
0
30
N6-CPA (Agonist)
20
-50
-12
-10
-8
10
-12
-8
-6
Propanolol (Antagonist)
100
0
-14
-12
-10
-8
300
DAMGO (Agonist)
200
CTOP (Antagonist)
100
-12
Log [Ligand], M
Isoproterenol (Agonist)
10000
Propranolol (Antagonist)
5000
0
-14
-12
-10
-8
Log [Ligand], M
-6
200
150
Carbachol (Agonist)
Atropine (Antagonist)
100
50
-8
-6
-12
-4
-10
-8
-6
-4
Log [Ligand], M
Agonist & Antagonist Effects On Recombinant Opioid Mu (OP3) in CHO-K1 Cells
With AlphaScreen-cAMP
Agonist & Antagonist Effects On Recombinant M1 in CHO-K1 Cells
With Ca2+ Flux Assay
AlphaScreen (Counts)
15000
-4
Log [Ligand], M
25000
20000
-10
12000
40000
10000
30000
Carbachol (Agonist)
DAMGO (Agonist)
8000
CTOP (Antagonist)
Signal
Agonist & Antagonist Effects On Endogenous B2AR In A431 Cells With
AlphaScreen-cAMP
-6
-8
0
0
-200
-10
Log [Ligand], M
250
-6
-100
Atropine (Antagonist)
50
Agonist & Antagonist Effects On Recombinant M1 in CHO-K1 Cells
Response (pm)
200
Response (pm)
Isoproterenol (Agonist)
Carbachol (Agonist)
100
-50
Log [Ligand], M
400
300
150
-12
-4
Agonist & Antagonist Effects On Recombinant Opioid Mu (OP3) In CHO-K1 Cells
400
Response (pm)
-10
-10
Log [Ligand], M
200
0
-6
Agonist & Antagonist Effects On Recombinant B2AR In CHO-K1 Cells
AlphaScre e n (Counts)
DPCPX (Antagonist)
0
-14
16
250
Response (pm)
250
Response (pm)
Response (pm)
2AR, Gs-Pathway
Atropine (Antagonist)
20000
10000
6000
0
-12
-10
-8
Log [Ligand], M
-6
-4
-12
-10
-8
-6
Log [Ligand], M
-4
Label-Free Detection of GPCR Activity: Cell Titration
B2AR (CHO, Recombinant), Label-Free
Cell Titration Data With Agonist
400
Isoproterenol 16K cell/well
Signal, pm
300
Isoproterenol 8K cell/well
200
Isoproterenol 4K cell/well
100
Isoproterenol 2K cell/well
0
-14
-10
-8
-6
-4
[Ligand], M
-100
B2AR (CHO, Recombinant), Label-Free
Cell Titration Data With Antagonist
-12
400
Propanolol 16K cell/well
Signal, pm
300
Propanolol 8K cell/well
200
Propanolol 4K cell/well
100
Propanolol 2K cell/well
0
-14
-100
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-12
-10
-8
[Ligand], M
-6
-4
Label-Free Detection of GPCR Activity: Detecting Inverse Agonists
EnSpire Label-Free Analysis:
2 Agonists & 1 Inverse Agonist in CHO-hCB2 Cells
Response (pm)
100
CP 55,940
50
WIN 55,212-2
Cpd X
0
-14
-12
-10
-8
-6
-4
Conc, [M]
-50
HillSlope
EC50
CP 55,940 WIN 55,212-2
0.8616
1.116
2.328e-009 2.021e-009
Cell Density = 15,000 cells/well
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Cpd X
-3.401
2.823e-008
Label-Free Detection of GPCR Activity in Primary Cells
Neutrophil responses
● Established the feasibility of using primary cells for secondary
screening
IL8
fMLFF
ATP
1200
180
1600
1000
EC50 = 1nM
400
200
1000
800
600
EC50 = 10nM
400
200
0
0
Wavelength shift (pm)
600
Wavelength shift (pm)
1200
800
Response (pm)
160
1400
140
120
100
EC50 = 1.5uM
80
60
-200
-200
0.01
40
0.1
1
[IL8] (nM)
Philip Rawlins & Kathryn Dodgson
SBS Lille, 2009
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10
1
10
fmlff
100
0.1
1
ATP (uM)
10
100
Label-Free Detection of GPCR Activity: Ligand Functional Selectivity
Ligand-Directed Functional Selectivity Acting on β2-Adrenergic Receptor in A431
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Functional Selectivity: Signal Deconvolution with G-protein Toxins
 Chemokine receptor expressed
in HEK-293
 200 ng/ml Ptox = 70% inhibition
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K.Dodgson, Astrazeneca (LRIG, October 2007)
Summary
Various LF modalities
 Biochemical assays with purified components
 Cellular
 Both biochemical & cellular
Pros & Cons
 Pros: native biological components, “unbiased”
 Cons: throughput, price
LF is not everything…
 Complement existing modalities
 Orthogonal validation technique
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