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Refining efficacy: allosterism and signal integration in GPCR signaling
Marie-Laure Rives
G protein-coupled receptors are involved in many physiological processes
GPCRs are modulated by diverse signaling molecules and activate a variety of signaling pathways, including kinases, ion channels and gene expression. GPCRs are the targets of ~ 30% of the medication currently on the market.
Fine-tuning the activity of GPCRs: functional selectivity and allosteric modulators
GPCRs as allosteric proteins. Intermolecular interactions between GPCRs and other proteins or small molecules in their environment can alter the conformational equilibrium of the receptor in ways that
change its reactivity toward guest probes, e.g., ligands or cytosolic effectors. Kenakin, T.
Can stabilize differential conformations, e.g. activate
different signaling pathways
Functional selectivity
Can modify the affinity of ligands on the associated protomer and/or modify
signaling
Can stabilize active or inactive conformation
Understanding how the activity of GPCRs of interest is regulated in a relevant physiological system and/or disease state will facilitate the development of better and safer therapeutics
Interacting proteins can modulate the activity of the
receptor
Lateral allostery: Detection of antigen interactions ex vivo by proximity ligation assay
Extensive evidence for D2R-A2AR heteromerization in heterologous expressionIn the striatum, an antagonistic functional interaction between these receptors has been shown both at the
electrophysiological and behavioral levels using in vivo pharmacological approaches (Fuxe et al., 2005; Agnati et al., 2010; Agnati et al., 2004; Ferre et al., 2004)
Heteromerization, thought to be responsible for the antagonistic functional interaction observed in vivoCan we detect dimers ex vivo?
Use of the PLA technology to identify and quantify D2R-A2AR dimers ex vivo
Advantages of this technology:* High sensitivity due to the amplification step
• Allows quantifications of interaction and localization (each dot corresponds to a dimer)
• Reduced background (fluorescence only occurs after complementation of the probes)
Applications:* Protein-protein interactions and localization ex vivo
• Biomarker analysis
Principles:Two primary antibodies raised in different species + Species-specific secondary antibodies, called PLA
probes, each with a unique short DNA strand attached to it.When the PLA probes are in close proximity (<40 nm), the DNA strands interact through a subsequent
addition of two other circle-forming DNA oligonucleotides. After ligation and amplification, labeled complementary oligonucleotide probes highlight the product.
Lateral allostery: Detection of endogenous dopamine D2-adenosine A2A receptor complexes in the striatum (Trifilieff, Rives et al., 2011)
Extensive evidence for D2R-A2AR heteromerization in heterologous expressionIn the striatum, an antagonistic functional interaction between these receptors has been shown both at the
electrophysiological and behavioral levels using in vivo pharmacological approaches (Fuxe et al., 2005; Agnati et al., 2010; Agnati et al., 2004; Ferre et al., 2004)
Heteromerization, thought to be responsible for the antagonistic functional interaction observed in vivoCan we detect dimers ex vivo?
Fine-tuning the activity of GPCRs: functional selectivity and allosteric modulators
GPCRs as allosteric proteins. Intermolecular interactions between GPCRs and other proteins or small molecules in their environment can alter the conformational equilibrium of the receptor in ways that
change its reactivity toward guest probes, e.g., ligands or cytosolic effectors. Kenakin, T.
Can stabilize differential conformations, e.g. activate
different signaling pathways
Functional selectivity
Can modify the affinity of ligands on the associated protomer and/or modify
signaling
Can stabilize active or inactive conformation
Understanding how the activity of GPCRs of interest is regulated in a relevant physiological system and/or disease state will facilitate the development of better and safer therapeutics
Interacting proteins can modulate the activity of the
receptor
The therapeutic potential of kappa opioid receptor ligandsThe kappa opioid receptor is widely expressed in the CNS:- Including the nucleus accumbens, VTA and Raphe nucleus where it regulates dopamine
and serotonine reuptake- DRG, Spinal cord, where it regulates the synaptic transmission of noxious stimulus
Niikura et al., 2010
Kappa antagonists for addiction and affective disorders?
Nalmefene (Selincro), approved for the treatment of alcohol dependence in EU
Kappa activation decreases dopamine release
The therapeutic potential of kappa opioid receptor ligandsThe kappa opioid receptor is widely expressed in the CNS:- Including the nucleus accumbens, VTA and Raphe nucleus where it regulates dopamine
and serotonine reuptake- DRG, Spinal cord, where it regulates the synaptic transmission of noxious stimulus
Can we develop KOR agonists devoid of aversive effects?
Al-Hasani and Bruchas, 2011
Kappa opioid receptor activation induces aversive events involved in the re-instatement of addictive behaviors
Kappa opioid receptor activation reduces synaptic transmission of noxious stimulus
Functional selectivity at the kappa opioid receptor (KOR), a promising concept for the development of safer therapeutics
Kenakin, 2007
Therapeutic effects? side effects?
GPCRs can activate several signaling pathways in a ligand-dependent manner. Some of these pathways are responsible for the therapeutic effects of one given drug, whereas the other pathways
might trigger the adverse effects.
p38 activation is responsible for the aversive effects of kappa agonists
Inhibition of the dysphoric effects of a kappa agonist in
p38SERTKO mice
(Lemos et al., 2013)
S369AGRK3
KOR-mediated activation of p38 is GRK3- and arrestin-
dependent
(Bruchas et al., 2006)
J Neurosci. Stress produces aversion and potentiates cocaine reward by releasing endogenous dynorphins in the ventral
striatum to locally stimulate serotonin reuptake.Schindler et al., 2012
Stress-induced activation of the dynorphin / kappa opioid system in the brain increases dysphoria through a p38αMAPK-dependent
translocation of SERT in Nac (Lemos et al., 2013)
Development of selective biased ligands at the kappa opioid receptor (KOR)
* 6’GNTI is a G-protein biased KOR agonist that does not recruit arrestin
* Discovery of a novel selective kappa-opioid receptor agonist using crystal structure-based
virtual screening
Trevena, Inc
p38SERT
6’GNTI is a G-protein biased hKOR agonist that does not recruit arrestin
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
0
25
50
75
100
EKC6'GNTIU50488
basallog [drug]
G p
rote
in a
ctiv
atio
n (%
)
i/o Arrestin3
6’GNTI is a partial agonist at hKOR for G protein activation but acts as an antagonist for arrestin recruitment
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
0
25
50
75
100
basal
U50488EKC6'GNTI
log [drug]
Arr
estin
recr
uitm
ent (
%)
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
0
25
50
75
100
basal
+ 6'GNTIU504886'GNTI
100 nM1 M
EKC
log [drug]
Arr
estin
recr
uitm
ent (
%)
6’GNTI is a G-protein biased hKOR agonist that does not recruit arrestin
Arrestin3GRK3
GRK3 induces a >2-fold increase in EKC- and U50488-induced arrestin
recruitment to hKOR
But still no significant recruitment to hKOR after 6’GNTI activation
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
0
50
100
150
200
250
300
basal
+ GRK3
EKC6'GNTIU50488
log [drug]
Arr
estin
recr
uitm
ent (
%)
6’GNTI does not induce KOR phosphorylation and blocks hKOR internalization
6’GNTI does not induce the internalization of hKOR and can block the internalization
induced by EKC
0
25
50
75
100
Veh
EKC1 M
+ 6'GNTI500 nM
6'GNTI1 M
U5048810 M
*** ***
ns ns
Cel
l sur
face
exp
ress
ion
(%)
Flag
ratKOR
S369
6’GNTI does not induce the phosphorylation of ratKOR at S369
(-)-cyclaz. But Lev 6’GNTI U50 EKC Veh
6’GNTI acts as an antagonist at the GRK/arrestin pathway
G protein-biased KOR agonists for safe pain relief?
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
0
200000
400000
600000
800000U504886'GNTILevorphanolEKCNorBNIJDTic
CHO-FhKOR plated in 96-well plateStarved ON in DMEM/F12 + 0.025% acid ascorbic and 25 mMHepes + PTX 100 ng/mL2 minutes stimulation with drugs
5 min HRP detection
log [drug]
Phos
pho
p38
Cell-based phospho-p38 ELISAIn inducible stable CHO cell lines (hKOR, rKOR, rKOR-S369A)
+ PTX
6’GNTI activates p38 in a PTX-dependent manner
p38 activation in vitro seems G protein-dependent, not arrestin dependent
Would a G protein-biased KOR agonist be devoid of aversive effects?
Stress-induced activation of the dynorphin / kappa opioid system in the brain increases dysphoria through a p38αMAPK-dependent
translocation of SERT in Nac
(Lemos et al., 2013)
Is 6’GNTI a safer analgesic?
* 6’GNTI is a G-protein biased KOR agonist that does not recruit arrestin
* Analgesic efficacy of 6’GNTI in a model of peripheral pain
However, 6’GNTI does not cross the blood brain barrier and cannot be used as a tool compound to assess whether or not a G protein-biased agonist at KOR would be devoid of
aversive effects
Need to develop better compounds
Berg et al., 2011
p38?
Development of novel KOR chemotypes
Wu et al., 2012Structure of the human k-opioid receptor in complex with JDTic.
Virtual screen of 4.5 million commercially available, “lead-
like” small molecules
22 selected tested molecules
Development of novel KOR chemotypes
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 220
50
100
100 M 10 M
MCKK-
**
***
***
***
% o
f spe
cifi
c3H
-dip
reno
rphi
ne b
indi
ng
Binding to the human kappa opioid receptor
Development of novel KOR chemotypes
G protein activation assay at hKOR
MCKK-17 is a selective kappa agonist
-13 -11 -10 -9 -8 -7 -5-6-12 -4 -13 -11 -10 -9 -8 -7 -5-6-12 -4
MCKK-17
0
50
100
EKC
MCKK-17-R/S
DOP MOP
G p
rote
in a
ctiv
atio
n (%
)
Development of novel KOR chemotypes
G protein activation assay at hKOR
0
50
100
EKC
MCKK-17-R/SMCKK-17-RMCKK-17-S
DOP MOP
G p
rote
in a
ctiv
atio
n (%
)
G protein activation assay at MOR and DOR
Interestingly, MCKK-17-R is the active enantiomer at DOR
-13 -11 -10 -9 -8 -7 -5-6-12 -4
MCKK-17-S is the active enantiomer
-13 -11 -10 -9 -8 -7 -5-6-12 -4
Development of novel KOR chemotypes
G protein activation assay at hKOR
MCKK-17-S is the active enantiomer
Arrestin recruitment at hKOR
EKC
0
50
100
MCKK-17-R/SMCKK-17-R
MCKK-17-S
Arr
estin
recr
uitm
ent (
%)
But it is not G-protein biased
-13 -11 -10 -9 -8 -7 -5-6-12 -4 -13 -11 -10 -9 -8 -7 -5-6-12 -4
Deciphering molecular determinants of signaling bias Biased ligands interact strongly with I2946.55, but less so with Y1393.33 and Y3207.43
Marta Filizola Ana Negri and Davide Provasi
Fine-tuning the activity of GPCRs: functional selectivity and allosteric modulators
GPCRs as allosteric proteins. Intermolecular interactions between GPCRs and other proteins or small molecules in their environment can alter the conformational equilibrium of the receptor in ways that
change its reactivity toward guest probes, e.g., ligands or cytosolic effectors. Kenakin, T.
Can stabilize differential conformations, e.g. activate
different signaling pathways
Functional selectivity
Can modify the affinity of ligands on the associated protomer and/or modify
signaling
Can stabilize active or inactive conformation
Understanding how the activity of GPCRs of interest is regulated in a relevant physiological system and/or disease state will facilitate the development of better and safer therapeutics
Interacting proteins can modulate the activity of the
receptor
Fine-tuning the activity of GPCRs with allosteric modulators
Normal state Disease state: Neurotransmitter
release
Disease state + PAM: Restored neuronal
activity
PAM affinity PAM affinity and/or efficacy
Ago-PAM Ago-NAM
• Spatio-temporal control of activation: Allosteric modulators only exert their actions in presence of the endogenous agonist, allowing to fine-tune the activity of a given receptor potentially impaired in the disease state
• PAM ligands may also exhibit allosteric agonist activity, termed ago-potentiators, a feature that may prove advantageous in certain CNS diseases
• Functional selectivity: it may be possible to tailor allosteric development to target specific downstream receptor pathways.
Challenges for drug discovery: Demonstrate in vivo target engagement Wootten et al., 2013
Advantages of allosteric modulators of GPCRs:• Subtype selectivity: The orthosteric site is often highly conversed across subtypes. Allosteric modulators often yield
more selectivity
Agonist
Allosteric Modulator
Fine-tuning the activity of specific synapses with mGluRs PAM
Dopamine depletion associated with PD in the nigrostriatal pathway leads to hyperactivity of inhibitory projections from the striatum to the globus pallidus, the first synapse in the basal ganglia “indirect pathway”, contributing to motor dysfunction and DA neuronal in PD patients. mGlu4 is expressed presynaptically at the striato-pallidal synapse and mGlu4 PAMs are hypothesized to act by reducing activity within the indirect pathway. Additionally, L-AP4 and other mGlu4 receptor agonists reverse motor symptoms in preclinical rodent models of PD (Jeff Conn).
Is PHCCC mGluR4-homodimer specific or is it inducing any functional cross-regulation at the level of the signaling pathways? Path to targeting specifically striato-pallidal synapses for less side effects?
L-AP4 PHCCC R4
-11 -10 -9 -8 -7 -6 -5 -4 -3
020406080
100120140
DMSO10 uMPHCCC
Log agonist, [M]
Per
cent
max
glut
amat
e re
spon
se
PHCCC and VU0155041 PAMs at mGluR4
L-AP4 PHCCC R2_R4
-11 -10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
120
DMSO10 uMPHCCC
Log agonist, [M]
Per
cent
max
glut
amat
e re
spon
se
PHCCC is inactive at mGluR4 in presence of mGluR2
mGlu4
mGlu2/4
PHCCC
PHCCC
VU0155
VU0155
Fine-tuning the activity of specific synapses with mGluRs PAM
Dopamine depletion associated with PD in the nigrostriatal pathway leads to hyperactivity of inhibitory projections from the striatum to the globus pallidus, the first synapse in the basal ganglia “indirect pathway”, contributing to motor dysfunction and DA neuronal in PD patients. mGlu4 is expressed presynaptically at the striato-pallidal synapse and mGlu4 PAMs are hypothesized to act by reducing activity within the indirect pathway. Additionally, L-AP4 and other mGlu4 receptor agonists reverse motor symptoms in preclinical rodent models of PD (Jeff Conn).
Is PHCCC mGluR4-homodimer specific or is it inducing any functional cross-regulation at the level of the signaling pathways? Path to targeting specifically striato-pallidal synapses for less side effects?
L-AP4 PHCCC R4
-11 -10 -9 -8 -7 -6 -5 -4 -3
020406080
100120140
DMSO10 uMPHCCC
Log agonist, [M]
Per
cent
max
glut
amat
e re
spon
se
PHCCC and VU0155041 PAMs at mGluR4
L-AP4 PHCCC R2_R4
-11 -10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
120
DMSO10 uMPHCCC
Log agonist, [M]
Per
cent
max
glut
amat
e re
spon
se
PHCCC is inactive at mGluR4 in presence of mGluR2
mGlu4
mGlu2/4
L-AP4
PHCCC
i/o +
L-AP4
PHCCC
i/o +
Development of BRET sensors to assess at the molecular level the dimer-selectivity of mGluRs PAMs
L-AP4 PHCCC R4
-11 -10 -9 -8 -7 -6 -5 -4 -3
020406080
100120140
DMSO10 uMPHCCC
Log agonist, [M]
Per
cent
max
glut
amat
e re
spon
se
PHCCC and VU0155041 PAMs at mGluR4
L-AP4 PHCCC R2_R4
-11 -10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
120
DMSO10 uMPHCCC
Log agonist, [M]
Per
cent
max
glut
amat
e re
spon
se
PHCCC is inactive at mGluR4 in presence of mGluR2
mGlu4
mGlu2/4
PHCCC
PHCCC
VU0155
VU0155
i/o Venus
RLuc
PHCCC is indeed silent in presence of mGluR2,
whereas VU0155 potentiates mGluR2
coupling to Gi/o
G p
rote
in a
ctiv
atio
n
BRET stands for Bioluminescence resonance energy transfer, a non-radiative transfer of energy that occurs between an excited luminescent enzyme/substrate donor complex and a fluorescent molecular acceptor that are separated by less than 100 Å Allows measuring constitutive and dynamic protein–protein interactions in living cells and has been widely used to study GPCR signaling pathways
Monday, May 1, 2023
Acknowledgments
Jonathan A. Javitch
Prashant DonthamsettiBo Feng
Wesley AsherRachel KolsterYongfang ZhaoCaline Karam
Mahalaxmi AburiEneko Urizar
Philip S. Portoghese
Matthew MetcalfMorgan Le Naour
Marta FilizolaAna Negri
Davide Provasi