marie-laure rives allostery functional selectivity kor columbia university

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?








signaling




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)

http://www.ncbi.nlm.nih.gov/pubmed/23223282

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


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


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 (%

)



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



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








signaling




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


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


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


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


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

marie-laure rives allostery functional selectivity kor columbia university

Documents