Slas 2013 poster_wood_m
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This document summarizes research using a live cell kinetic cAMP assay (GloSensor) to screen for positive allosteric modulators (PAMs) of G-protein coupled receptors (GPCRs). Over 80,000 compounds were tested and 300 were confirmed to selectively potentiate GPCR activity. Three confirmed hits were identified as PAMs that increased the potency of an endogenous agonist without direct agonist effects. Further testing identified stereoisomers that selectively modulated the target receptor allosterically in an enantioselective manner. The assay proved robust for high-throughput screening and enabled differentiation of PAMs from agonists through a dual-phase kinetic response measurement.
![The benefits of live cell kinetic cAMP assay (GloSensor) in screening for
positive allosteric modulators
Martyn Wood, Maggi Burton, Evelyne Malray, Michel Famelart, David Urbain and Michel Gillard
UCB Pharma S.A., Discovery Research, Braine-lAlleud, 1420 Belgium
1. Introduction 4. Hits identified and confirmed on PAM activity
G-protein coupled receptors (GPCRs) are the largest class of cell surface 80,000 compounds were tested at 10袖M
receptors and are the targets of many therapeutic agents. There has been much 300 batches were selected as potential selective PAMs for confirmation at
10袖M on both target GPCR expressing cells and parental cells
interest in the identification and development of allosteric modulators of GPCRs
100 hits confirmed and tested in potentiation of orthosteric agonist receptor
as potential novel drugs (Conn et al, 2009; Kenakin & Miller, 2010). This interest stimulated [35S]-GTPS binding
is due to a number of theoretical advantages which allosteric modulators have 3 hits confirmed:
compared to orthosteric drugs (Christopoulos, 2002):
Selectivity - orthosteric site may be conserved across receptor subtypes Compound lacks Compound increases
direct/agonist effects potency of
Ability to modulate rather than drive effects saturability of allosteric effects
endogenous agonist
Ability to tune response only active when and where agonist is release
We have used a novel, live-cell kinetic assay (GloSensor, Promega) for
monitoring changes in intracellular cAMP in real time to identify PAMs of Gi
coupled GPCRs. Such GPCRs act to lower cAMP levels and we have used
forskolin to raise cAMP levels. The GloSensor assay uses a luciferase-based
biosensor fused with a cAMP binding domain.
2. Kinetic assay allows differentiation of PAMs and agonists
Assay preformed in HEK-293 cells transiently transfected with GPCR & Glosensor
Freshly defrosted cells were seeded in 384-well plates (5K per well). Luciferin
substrate was then added according to manufacturers instructions.
Cells were incubated for 10 min with test compound (10 mM) for any direct effect
Cells were then stimulated with forskolin (0.1 mM) for 15 min to elavate cAMP from this we have gone on to identify stereoisomers which show
(Phase 1). Any agonists at Gi-coupled CPCRs would inhibit this stimulation enantioselective allosteric effects at 10 mM on target receptor:
30 min later, an EC20 concentration of dopamine is added (Phase 2):
Dual phase Phase 1 Phase 2 20000
[35S] GTPS bound (cpm)
assay Detects agonism Detects PAM control
+ enantiomer
increases potency
15000 - enantiomer
decrease response
10000
-10 -9 -8 -7 -6 -5
PAM potentiates DA
inhibition of forskloin basal log[agonist].M
Agonist inhibits forskolin
response in absence of DA Conclusions
We have successfully used Glosensor technology as a live-cell real-time assay
in HTS format to identify novel PAMs at GPCRs
The assay has proven to be robust with little variation in agonist EC20
This assay has therefore been used to run HTS on a number of other GPCR
3. GloSensor in HTS format generates robust data targets
Phase 1 Zaverage (FSK/FSK+agonist at [EC100]): 0.72 A major advantage of the Glosensor technonology is the ability to follow
Phase 2 Zaverage (FSK+agonist at [EC20]/FSK+agonist at [EC100]): 0.70 kinetics of the response and double addition, enabling differentiation between
agonists (Phase 1) and PAMs (Phase 2) in a single screen. An end-point assay
would need an additional step to separate these activities in any hits.
the distribution of controls from
day to day screen is an indicator
of screen robustness/variance
-Blue dots: EC100 agonist controls
- Grey dots: From 4048 EC20 agonist
References
controls, 81% ranges from 10 to 30%, Christopoulos A (2002) Nature Rev Drug Discovery 1: 198-210
16%>30%, only 3%>40%
Conn PJ, Christopoulos A, Lindsley CW (2009) Nature Rev Drug Discovery 8: 41-54
-Pink dots: O% effect: antagonist 1袖M Kenakin T, Miller LJ (2010) Pharmacol Rev 62: 265-304
-the variance in control values
between plates and across days
was low
Screening Days 1 2 3 4 5 6 7 8 9 10 11 12 13 14
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Slas 2013 poster_wood_m
- 1. The benefits of live cell kinetic cAMP assay (GloSensor) in screening for positive allosteric modulators Martyn Wood, Maggi Burton, Evelyne Malray, Michel Famelart, David Urbain and Michel Gillard UCB Pharma S.A., Discovery Research, Braine-lAlleud, 1420 Belgium 1. Introduction 4. Hits identified and confirmed on PAM activity G-protein coupled receptors (GPCRs) are the largest class of cell surface 80,000 compounds were tested at 10袖M receptors and are the targets of many therapeutic agents. There has been much 300 batches were selected as potential selective PAMs for confirmation at 10袖M on both target GPCR expressing cells and parental cells interest in the identification and development of allosteric modulators of GPCRs 100 hits confirmed and tested in potentiation of orthosteric agonist receptor as potential novel drugs (Conn et al, 2009; Kenakin & Miller, 2010). This interest stimulated [35S]-GTPS binding is due to a number of theoretical advantages which allosteric modulators have 3 hits confirmed: compared to orthosteric drugs (Christopoulos, 2002): Selectivity - orthosteric site may be conserved across receptor subtypes Compound lacks Compound increases direct/agonist effects potency of Ability to modulate rather than drive effects saturability of allosteric effects endogenous agonist Ability to tune response only active when and where agonist is release We have used a novel, live-cell kinetic assay (GloSensor, Promega) for monitoring changes in intracellular cAMP in real time to identify PAMs of Gi coupled GPCRs. Such GPCRs act to lower cAMP levels and we have used forskolin to raise cAMP levels. The GloSensor assay uses a luciferase-based biosensor fused with a cAMP binding domain. 2. Kinetic assay allows differentiation of PAMs and agonists Assay preformed in HEK-293 cells transiently transfected with GPCR & Glosensor Freshly defrosted cells were seeded in 384-well plates (5K per well). Luciferin substrate was then added according to manufacturers instructions. Cells were incubated for 10 min with test compound (10 mM) for any direct effect Cells were then stimulated with forskolin (0.1 mM) for 15 min to elavate cAMP from this we have gone on to identify stereoisomers which show (Phase 1). Any agonists at Gi-coupled CPCRs would inhibit this stimulation enantioselective allosteric effects at 10 mM on target receptor: 30 min later, an EC20 concentration of dopamine is added (Phase 2): Dual phase Phase 1 Phase 2 20000 [35S] GTPS bound (cpm) assay Detects agonism Detects PAM control + enantiomer increases potency 15000 - enantiomer decrease response 10000 -10 -9 -8 -7 -6 -5 PAM potentiates DA inhibition of forskloin basal log[agonist].M Agonist inhibits forskolin response in absence of DA Conclusions We have successfully used Glosensor technology as a live-cell real-time assay in HTS format to identify novel PAMs at GPCRs The assay has proven to be robust with little variation in agonist EC20 This assay has therefore been used to run HTS on a number of other GPCR 3. GloSensor in HTS format generates robust data targets Phase 1 Zaverage (FSK/FSK+agonist at [EC100]): 0.72 A major advantage of the Glosensor technonology is the ability to follow Phase 2 Zaverage (FSK+agonist at [EC20]/FSK+agonist at [EC100]): 0.70 kinetics of the response and double addition, enabling differentiation between agonists (Phase 1) and PAMs (Phase 2) in a single screen. An end-point assay would need an additional step to separate these activities in any hits. the distribution of controls from day to day screen is an indicator of screen robustness/variance -Blue dots: EC100 agonist controls - Grey dots: From 4048 EC20 agonist References controls, 81% ranges from 10 to 30%, Christopoulos A (2002) Nature Rev Drug Discovery 1: 198-210 16%>30%, only 3%>40% Conn PJ, Christopoulos A, Lindsley CW (2009) Nature Rev Drug Discovery 8: 41-54 -Pink dots: O% effect: antagonist 1袖M Kenakin T, Miller LJ (2010) Pharmacol Rev 62: 265-304 -the variance in control values between plates and across days was low Screening Days 1 2 3 4 5 6 7 8 9 10 11 12 13 14 www.ucb.com