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BioSens-All™: A New Technology Dedicated to
GPCR Biased Ligand Drug Discovery
Billy Breton,1 Nathalie Rouleau,1 Mélanie Frauli,2 and Stephan Schann2
1. Domain Therapeutics NA Inc, NeoMed Institute, 7171 Frederick Banting, Montréal Québec H4S 1Z9, Canada.
2. Domain Therapeutics, Blvd Sébastien Brant, 67400 Strasbourg-Illkirch, FRANCE. [email protected]
INTRODUCTION
GPCRs have proven to be a valuable target family for drug discovery and development with more than 30% of marketed drugs acting through this receptor superfamily.
However, numerous GPCR members remain challenging with no selective and druggable ligands being successfully developed. For these difficult targets, novel strategies
consisting in developing allosteric modulators and biased ligands (BLs) are now emerging. Indeed, all GPCR agonists do not always uniformly activate all signalling
pathways mediated by a given receptor. BLs therefore constitute a novel approach to more selectively activate a receptor for the development of safer drugs. This concept
of ligand bias, also referred to as functional selectivity, has now been validated in vitro and in vivo, and several GPCR BLs are currently under clinical investigations.
Despite these promising features, uncovering and profiling BLs still remains challenging as no specific technology has been proposed for this task. Cell-system dependency
of the multiple functional analyses for instance makes it difficult to guide medicinal chemists in their optimization programs toward active BLs. To meet this expectation, the
dedicated BioSens-All™ technology was generated. BioSens-All™ consists in a set of multiple biosensors each engineered to follow one specific functional pathway. The
parallel characterization of GPCR orthosteric or allosteric ligands with theses biosensors, in a homogenous format, enables the generation of signalling signatures and
consistent comparative data for chemists.
The present poster will present and exemplified how BioSens-All™ can be used to support medicinal chemists in their quest for GPCR biased ligands.
1. Biased Ligands: The Theory
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2. A fully Integrated Platform to Identify Biased Ligand
GPCRs constitute complex receptors that can engage multiple signaling pathways upon
Specific binder selection using a FRET-based binding assay: DTect-All™
•
activation (a).
Use as a filter to focus only on binders of the GPCR
•
Therapeutic efficacy is often driven by a single pathway, adverse events by others.
•
Biased ligands selectively activate one pathway given rise to safer drug candidates (b). This is
Wide functional profiling with multiple BRET-based biosensors: BioSens-All™
•
Enable clustering of binders based on their signalling signatures
done by stabilization of specific receptor conformation.
Biased clusters assessed using native assays for efficacy and side effect profile
•
Deliver fully validated biased hits / leads
Small molecules, peptides
or antibodies
Validated hits for
ready for
optimization
Galandrin S et al, TiPS 2007
3. Domain’s New Technology: BioSens-All™
•
Native systems
characterization
BRET-based biosensors each designed to follow activation of one functional pathway
Cluster
#1
• Profiling of multiple signaling pathways done in parallel in a same cellular background
Cluster
#2
Cluster
#3
…
• Simple transfection of non-modified GPCR with intracellular BRET signal
• Profiling in homogenous assay format enabling direct comparison for biased ligand characterization
• Determination of signaling signature of each GPCR binder
Wide functional
profiling
FRET
•
•
•
•
•
•
•
•
•
•
Gs
Gi1/2/3
GoA/B
Gq
Gz
G11
G12
G13
G14
G15/16
GFP
GFP
•
•
•
•
•
b-arrestin-1
b-arrestin-2
•
•
•
•
•
•
•
Ca2+
cAMP
DAG
Dye
TPR1
GRK2/3
PKC
p63
Merlin
p115
Internalization
C-ter
domain
C-ter
domain
C-ter
domain
Step 1
PROXIMAL
Step 2
•
Was shown to activate alternative pathways such as Gq/Ca2+/PKC, beta-arrestin
2
100
90
80
E C 5 0 = 5 6 .9 p M
PTH
% o f n o n s tim u la t e d
Class B GPCR activating the canonical Gs/cAMP/PKA pathway
100
T y r1 -P T H
80
60
-1 6
-1 4
-1 2
-1 0
-8
-6
-4
-1 6
lo g [ P e p t id e ] ( M )
[Tyr1]PTH(1-34)
0,6nM (100%)
0,7nM (100%)
0,01nM (full)
0,06nM (full)
0,06nM (full)
3nM (full)
95
P T H E C 5 0 = 3 5 .1 n M
T y r 1 -P T H
-1 4
-1 2
Beta-arrestin
1,3nM (100%)
27nM (56%)
Internalization
5nM (100%)
104nM (75%)
-6
-4
E C 50= 16 2 nM
B-arrestin recruitment biosensor
PTH + PTX
T y r 1 -P T H
150
100
-1 0
-8
-6
-1 6
-4
-1 4
-1 2
-1 0
-8
-6
-4
lo g [ P e p t id e ] ( M )
b  a r r 2 b io s e n s o r
> 10µM
13,4nM
> 10µM
1,1nM (full)
34,2nM (partial)
2
90
80
P T H E C 5 0 = 1 3 .4 n M
-1 6
-1 4
-1 2
PTH
E C 5 0 = 1 .1 n M
T y r 1 -P T H
E C 5 0 = 3 4 .2 n M
300
200
100
T y r 1 -P T H
70
% o f n o n s t i m u la t e d
162nM (full)
100
BRET
> 10µM
2
400
% o f n o n s t i m u la t e d
Go
PTH
lo g [ P e p t id e ] ( M )
> 10µM
> 10µM
% o f n o n s t i m u la t e d
2
100
G o b io s e n s o r
35,1nM
16nM (100%)
-8
90
BRET
30nM (100%)
-1 0
E R K -N L S 1 5 m in
BRET
2
PTH(1-34)
Gq
ERK 1/2
BRET
[Tyr1]PTH(1-34)
Gs
IP1
BioSens-All™
PTH(1-34)
-1 2
200
% o f n o n s t i m u la t e d
Cupp ME et al, JPET 2013
-1 4
lo g [ P e p tid e ] (M )
G q b io s e n s o r
profiled with BioSens-All™ compared with the endogenous PTH(1-34)
E C 5 0 = 5 5 .4 p M
T y r 1 -P T H E C 5 0 = 3 0 0 0 p M
70
Numerous BLs were already described for PTH1R: [Tyr1]PTH(1-34) was chosen and
G-protein activation biosensors
P T H E C 5 0 = 9 .7 p M
recruitment or ERK(1/2) phosphorylation
cAMP
Step 3
c A M P b io s e n s o r
BRET
PTH1 receptor (PTH1R) plays key role in bone remodeling
% o f n o n s t i m u la t e d
BRET
2
G s b io s e n s o r
•
•
C-ter
domain
DISTAL
4. Illustrative example
•
GFP
Dye
-1 0
-8
lo g [ P e p t id e ] ( M )
-6
-4
-1 6
-1 4
-1 2
-1 0
-8
-6
-4
Same level of potency and similar bias
as that described in the literature was
found using BioSens-All™ technology
lo g [ P e p t id e ] ( M )
Conclusion
Biased ligands represent a novel research strategy for the development of safer drug candidates targeting GPCRs. This class of agonists / PAMs / antagonists or NAMs are
specifically activating / potentiating / blocking only one or a fewer signaling pathway(s) of the receptor. A new technology called BioSens-All™ was specifically designed for
their identification and characterization. BRET-based BioSens-All™ can be used as a stand alone tool or in a fully integrated manner in combination with the FRET-based
DTect-All™ binding assay.