Label-free optical biosensor for probing GPCR ligand ...

Label-free optical biosensor for
probing GPCR ligand-directed
functional selectivity

Ann M. Ferrie and Ye Fang

Abstract

Realization of the complexity of G protein coupled receptor (GPCR) signaling and
ligand-directed functional selectivity demands high resolution tools for studying
GPCR behavior. Given its well-characterized agonist binding site and the availability
of a wealth of structurally related ligands with functionally diverse properties, β2-
adrenergic receptor (β2AR) has been used as an excellent model system for
studying the mechanism of GPCR activation and signaling. Here we use non-
invasive resonant waveguide grating (RWG) biosensor to systematically examine
the kinetic profiles of human epidermoid carcinoma A431 cells in response to
stimulation with diverse arrays of β2AR ligands. The RWG biosensor offers an
integrated signal, termed as dynamic mass redistribution (DMR) signal, which is in
close proximity to a global representation of GPCR signaling in native cells. Multi-
parameter analysis of agonist-induced DMR signals indicates the presence of
multiple ligand-specific states of the β2AR. The implications of DMR signals
screening of biased ligands are discussed.

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Corning® Epic® System

The Corning Epic System is a high-throughput, label-free detection platform that
consists of SBS-standard 384-well microplates with optical sensors inside each well,
an HTS-compatible microplate reader and a set of label-independent assay
protocols. The Epic System is applicable to both biochemical and cell-based assays,
and enables high-throughput screening of “intractable” targets.

+= Response (pm) 8

Microplate Plate Reader 6

• 384-well format • Compatible w/ HTS automation 4
• Optical biosensor in each well • ≥ 40,000 wells/8hrs
• Surface chemistry • Sensitivity of 5pg/mm2 KD = 53 nM
(300Da drug to 75kDa target)
2
3
R2 = 0.9707

0
0 500 1000 1500 2000 2500 3000
[Acetazolamide] (nM)

Binding Data

• Manipulated and
analyzed by
customer

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Operating Principle: Cell-based Assays

ƒ Measures changes in local index of refraction resulting from the ligand-induced
dynamic mass redistribution (DMR) within the bottom region (~150nm) of the cell
monolayer.

ƒ Change in index is manifested by a shift in resonant wavelength

Baseline Measurement Compound Measurement

Cell Cell Mass
redistribution
surface Stimulation
waveguide Detectable
substrate DMR

Broadband Reflected Broadband Reflected
Source wavelength Source wavelength

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Classical Model of GPCR Drug Action

• In classical models of drug action, the
primary event is binding of a ligand to its
receptor, which, in turn, leads to a
cellular effect whose magnitude
depends on the intrinsic efficacy of the
ligand.

• Classical receptor-occupancy theory
defines the efficacy of ligands as their
ability to alter the equilibrium between
inactive and active states of the
receptor, assuming that all GPCR
activities are correlated.

Kenakin, T. (2007) Trends Pharmacol. Sci., 28, 407-415

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New Paradigm in GPCR Ligand Pharmacology – Ligand
Directed Functional Selectivity

• Parathyroid hormone (PTH) occupies the receptor, produces an active receptor state that goes on to induce
a cellular response, and causes receptor desensitization and phosphorylation upon prolonged activation and,
finally, internalization of the receptor into the cytosol.

• Amino-truncated peptide analogues (antagonists PTH(7–34) and PTH(7–84)) occupy the receptor (produce
antagonism of responses to PTH), do not produce activation but do induce receptor internalization. These
analogues therefore possess some, but not all, of the efficacy properties of PTH.

Kenakin, T. (2005) Nat. Rev. Drug Discov., 4, 919-927.
Violin, J.D., & Lefkowitz, R. J. (2007) Trends Pharmacol. Sci. 28, 416-422.

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Ligand Directed Functional Selectivity –
Pleiotropic Nature of GPCR Signaling

Galandrin, S., Oligny-Longpre, G., and Bouvier, M. (2007) Trends in Pharmacol. Sci. 28, 423-430.

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Multitudes of Ligand Efficacy Demands
High Resolution Pharmacological Tools

Gilchrist, A. (2007) Trends in Pharmacol. Sci. 28, 431-437

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Corning® Epic® System Measures Stimulus-Induced Dynamic
Mass Redistribution (DMR) in Living Cells

• DMR signal is
– A novel physiologically relevant cellular response
– An integrated cellular response consisting of many cellular events downstream the
receptor activation
– A real time kinetic cellular response with rich information
– A native cellular response
– A cell signaling/network interaction-sensitive response

Transition time τ Elevated level
epinephrine

Kinetics κ and t1/2

baseline P-DMR

N-DMR

Fang, Y., et al. (2006) Biophys. J. 91, 1925-1940
Fang, Y. (2006) Assays Drug Dev. Tech. 4, 583-595
Fang, Y. and Ferrie, A.M. (2008) FEBS Lett., 582, 558-564

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Endogenous β2-Adrenergic Receptor in A431 as a Model
System

Kobilka, B. (2004) Mol. Pharmacol. 65, 1060-1062
Swaminath, G. et al. (2005) J. Biol. Chem. 280, 22165-22171

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Epic® Cell Assays Differentiate Ligand-Directed Functional
Selectivity Acting on β2-Adrenergic Receptor in A431

250 phenylethylamine xamoterol alprenolol
NH2 ON
200 HO
OH H
150 ON NH O

100 catechol OH H N
50 HO
0 O
-50 HO

250 H CGP12177
N ON
200 O
N OH H
H

150

100 labetalol
HO
50 dopamine Tyramine

0 HO HO H2N N
O OH H
-50 HO NH2 NH2

250 Halostachine HO salbutamol H S(-)Pindolol
N HO N N
200
HO H OH H ON
150 OH H
(-)isoproterenol
100 norepinephrine HO H Pindolol
50 HO HO N N

0 HO NH2 HO H ON
-50 HO OH H

250

200

150 (-)-epinephrine HO salmeterol
100 HO OH
OH
NH

50 HO N O
0 HO H

-50

60min

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The DMR Signals Are Specific to β2AR: Propranolol Inhibition

• Only three ligands show off-target effects: dopamine (>100μM),
phenylethylamine and tyramine

• The β-blocker propranolol dose-dependently attenuated the DMR induced by all
other agonists examined at their corresponding EC100, yielding similar IC50s.

Ligands (conc.) IC50

Isoproterenol (0.1nM) 1.2±0.3nM
Epinephrine (2nM) 2.8±1.2nM
Norepinephrine (100nM) 3.5±1.3nM

Dopamine (10μM) 3.6±0.9nM

Catachol (500μM) 1.4±0.4nM (~50%)

Halostachine (100μM) 1.9±0.3nM
Salmeterol (100nM) 58.7±5.7nM
Salbutamol (10nM) 2.5±1.1nM
Labetalol (100nM) 9.1±2.4nM
CGP12177 (1nM) 2.1±0.3nM
Alprenolol (10nM) 1.2±0.5nM
S(-)pindolol (1nM) 3.2±1.9nM
Pindolol (1nM) 8.3±2.3nM
Xamoterol (250nM) 3.6±1.7nM

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DMR Signal Offers New Dimensions for Analyzing Receptor
Signaling and Ligand Biased Potencies and Efficacies

ligand Ligand -logKi (ref.) -logEC50±S.E. P-DMR(pm) N-DMR(pm) τ (s) t1/2 (s)
11.07±0.07 534±32
Isoproterenol 6.97 (72) 10.13±0.06 232±15 37±5 130±20 521±25
7.99±0.07 232±12 37±7 132±20 559±15
Epinephrine 7.16 (29) 5.96±0.06 209±16 29±5 180±17 480±23
3.30±0.07 214±32 31±4 252±15 289±42
Norepinephrine 5.40 (29) 4.63±0.05 152±13 0±3 130±15 349±41
9.68±0.07 208±21 20±7 200±32 370±45
Dopamine 4.35 (29) 6.90±0.10 160±14 0±4 290±17 n.a.
9.07±0.04 244±23 40±11 152±20 539±32
Catachol 3.80 (17) 8.05±0.05 209±13 32±3 132±20 544±46
9.95±0.07 149±17 8±4 250±15 582±32
Halostachine 5.08 (29) 10.23±0.09 123±14 5±5 238±26 540±26
10.85±0.06 124±16 0±4 233±15 580±56
Salmeterol 8.89 (72) 9.97±0.04 137±12 0±3 249±26 516±47
7.32±0.08 142±19 0±4 250±15 974±150
Salbutamol 5.35 (73) 75±18 0±5 260±26
Labetalol 7.97 (74)
CGP12177 9.20 (71)
Alprenolol 9.49 (74)
S(-)pindolol 10.16 (75)
Pindolol 9.15 (74)
Xamoterol 6.06 (76)

Fang, Y. and Ferrie, A.M. (2008) FEBS Lett., 582, 558-564

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Multi-Parameter Analysis Differentiates Ligand-Directed
Functional Selectivity: Efficacy

isoproterenol salbutamol dopamine

300 epinephrine nonepinephrine Halostachine

Response (pm) 200

100

0

-13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3

[agonist], log M

TOP epinephrine salbutamol isoproterenol nonepinephrine dopamine Halostachine
LOGEC50 231.9 209.6 231.6 208.6 212.4 207.9
EC50 -10.13 -9.068 -11.07 -7.992 -5.962 -4.626
7.3651e-011 8.5581e-010 8.4299e-012 1.0188e-008 1.0919e-006 2.3634e-005

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Multi-Parameter Analysis Differentiates Ligand-Directed
Functional Selectivity: Potency Shift

• The shifts in apparent potency relative to affinity have been attributed to the
differential coupling efficiencies of ligands

• The weaker the agonist is, the closer to kd the EC50 is; and the greater shift in
EC50 indicates that the ligand is more effective in causing cAMP accumulation

Xamoterol log Ki
Pindolol log EC50

S(-)pindolol

Alprenolol

CGP12177
Labetalol

Salbutamol
Salmeterol

Halostachine -10 -8 -6 -4 -2 0
Catachol log EC50 versus log Ki
Dopamine

Norepinephrine
Epinephrine

Isoproterenol

-12

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Multi-Parameter Analysis Differentiates Ligand-Directed
Functional Selectivity: Dose-Dependent Responses

• Salmeterol is a long acting β-agonist with low intrinsic activity, and exhibits dual
efficacies – a weak partial agonist for producing an effective interaction between
the receptor and β-arrestin 2, and a full agonist for causing cAMP accumulation

300Response (pm)300120024003600
250 Response (pm)200
200 Response (pm)
150 100
0

-100
0

Time (sec)

100 300

200

50 100

0

0 -100

0 1200 2400 3600

-50 Time (sec)

-13 -12 -11 -10 -9 -8 -7 -6 -5 -4

[salmeterol], log M

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