Forskolin-Free Camp Assay for Gi-Coupled Receptors

Biochemical Pharmacology 98 (2015) 381–391

Contents lists available at ScienceDirect

Biochemical Pharmacology

journal homepage: www.elsevier.com/locate/biochempharm

Forskolin-free cAMP assay for Gi-coupled receptors

a,b a a a b

Julie Gilissen , Pierre Geubelle , Nadine Dupuis , Céline Laschet , Bernard Pirotte , a,b,

Julien Hanson *

Laboratory of Molecular Pharmacology, GIGA-Signal Transduction Unit, University of Liège, 11, Avenue de l'hôpital, 4000 Liège, Belgium

Laboratory of Medicinal Chemistry, Centre for Interdisciplinary Research on Medicines (CIRM), University of Liège, 15, Avenue Hippocrate, 4000 Liège,

Belgium

A R T I C L E I N F O A B S T R A C T

Article history: G protein-coupled receptors (GPCRs) represent the most successful receptor family for treating human

Received 21 July 2015

diseases. Many are poorly characterized with few ligands reported or remain completely orphans.

Accepted 11 September 2015

Therefore, there is a growing need for screening-compatible and sensitive assays. Measurement of

Available online 16 September 2015

intracellular cyclic AMP (cAMP) levels is a validated strategy for measuring GPCRs activation. However,

agonist ligands for Gi-coupled receptors are difﬁcult to track because inducers such as forskolin (FSK)

Keywords:

must be used and are sources of variations and errors.

GPCR

We developed a method based on the GloSensor system, a kinetic assay that consists in a luciferase

SUCNR1

fused with cAMP binding domain. As a proof of concept, we selected the succinate receptor 1 (SUCNR1 or

cAMP

Forskolin GPR91) which could be an attractive drug target. It has never been validated as such because very few

GPR91 ligands have been described.

Following analyses of SUCNR1 signaling pathways, we show that the GloSensor system allows real

time, FSK-free detection of an agonist effect. This FSK-free agonist signal was conﬁrmed on other

Gi-coupled receptors such as CXCR4. In a test screening on SUCNR1, we compared the results obtained

with a FSK vs FSK-free protocol and were able to identify agonists with both methods but with fewer false

positives when measuring the basal levels.

In this report, we validate a cAMP-inducer free method for the detection of Gi-coupled receptors

agonists compatible with high-throughput screening.

This method will facilitate the study and screening of Gi-coupled receptors for active ligands.

1. Introduction also many intracellular partners such as arrestins [3]. There are

four main families of G proteins: Gi/o, Gs, Gq/11 and G12/13, which

G protein-coupled receptors (GPCRs) are characterized by seven differ in the signaling pathways they couple to [4].

transmembrane domains and represent the largest family of The efﬁcient identiﬁcation of original ligands for unknown and

proteins in the human genome [1]. They are currently the target for poorly characterized receptors remains a major challenge. To reach

30% of marketed drugs and thus the most successful receptor this goal, a plethora of assays have been developed, in response to

family for treating human diseases. However, among the 350 non- the high demand for ligands both for therapeutic and research

olfactory members, many are poorly characterized with few perspective. More recently, it has been reported that some ligands

ligands reported or remain completely orphans (around 100 in the could selectively activate discrete signaling pathways when

most recent IUPHAR list) [2]. GPCRs signal through G proteins but binding to a receptor [5]. This pharmacological property called

functional selectivity is now being considered when selecting an

assay for screening campaigns. Therefore, there is a renewed

Abbreviations: AC, adenylate cyclase; AUC, area under the curve; cAMP, cyclic interest in screening-compatible and sensitive assays directed

adenosine monophosphate; ERK, extracellular signal regulated kinases; FSK, selectively toward a pathway of interest.

forskolin; GPCRs, G-protein coupled receptors; PTX, pertussis toxin; SA, succinic

Measurement of intracellular cyclic adenosine monophosphate

acid.

(cAMP) levels is a validated strategy for such pathway speciﬁc

* Corresponding author at: Laboratory of Molecular Pharmacology, GIGA-Signal

Transduction Unit, University of Liège, CHU, B34, Tour GIGA(+4), Avenue de l’hôpital, approach [6]. This prominent second messenger is the product of

11, 4000 Liège, Belgium. Tel.: +32 43664748. adenylate cyclase (AC) activity that is directly regulated by Gs- and

E-mail address: [email protected] (J. Hanson).

http://dx.doi.org/10.1016/j.bcp.2015.09.010

382 J. Gilissen et al. / Biochemical Pharmacology 98 (2015) 381–391

Gi-proteins. The vast majority of GPCR activation can be monitored SUCNR1 coding sequence was ampliﬁed from human genomic

with the changes in cAMP levels. The Gs-coupled receptors DNA and cloned into the pIRESpuro expression vector (Clontech

activation is relatively easy and straightforward to detect because Laboratories, Mountain View, California, USA).

they activate AC and consequently increase cAMP levels. Accord- The pGloSensor -22F cAMP (cAMP GloSensor) plasmid was

ingly, many examples of successful screening campaigns on obtained from Promega Corporation (Madison, Wisconsin, USA).

Gs-coupled receptors have been published [7,8]. In contrast, Human arrestin 2 was ampliﬁed from cDNA prepared from

agonist ligands for Gi-coupled receptors are much more difﬁcult HEK293 cells and cloned into the pIREShygro expression vector

to track with cAMP measurement. This is due to the fact that basal (Clontech Laboratories, Mountain View, California, USA). Arrestin 3

AC activity and cAMP levels in the cell are relatively low [9]. was ampliﬁed from b-arrestin 2 GFP WT (#35411, Addgene,

Inducers such as forskolin (FSK) [10] or similar stratagem [11] must Cambridge, Massachusetts, USA) and cloned into the pIREShygro

be used when assessing a putative agonist. The artiﬁcial expression vector. The pIRES.hygro.FnLARR2, pIRES.hygro.

manipulation of the signal complicates the assay by increasing FnLARR3, pIREShygroFnLARR2.pIRESpuroSUCNR1 and pIREShy-

the sources of variation and errors [12]. groFnLARR3.pIRESpuroSUCNR1 were developed based on Taka-

In this report, we describe and validate a cAMP-inducer free kura et al. [24]. Briefly, the 1-415 first amino acids of firefly

method for the detection of Gi-coupled receptors agonists luciferase were fused with N-Arrestin2 or 3 (FN-Arr2 or 3) and the

compatible with high-throughput screening. The method is based 413-549 amino acids were fused with C-SUCNR1 (SUCNR1-FC).

on the GloSensor system, a live cell, homogenous and kinetic assay

that consists in a luciferase fused with cAMP binding domain [13]. 2.3. Flow cytometry analysis

As a proof of concept, we selected the succinate receptor 1

(SUCNR1, previously termed GPR91) that is coupled to Gi [14,15]. Cells (2 10 cells per tube) were incubated with monoclonal

The receptor-ligand pair has been described as a metabolism ANTI-FLAG M2 (1:1000) for 45 min at 4 C. After wash, cells were

sensor because succinic acid (SA) is a citric acid cycle intermediate incubated with anti-Mouse IgG (H + L), F(ab )2 Fragment (Alexa

that is released outside the cell in case of oxygen deprivation [16]. A Fluor 488 Conjugate; 1:1000) for 45 min at 4 C in the dark. Data

lot of studies have addressed the roles of SUCNR1 and demon- were acquired on BD FACSCalibur 2 lasers (Becton Dickinson, New

strated its implication in the enhancement of immunity [17], Jersey, USA) and analyzed with Cellquest pro. The gate on living

retinal angiogenesis [18], hypertension [15,16,19], liver damage cells was made using the SSC/FSC dot plot.

[20] and platelet aggregation [21,22]. Collectively, these data

suggest that SUCNR1 could be an attractive drug target in several 2.4. Immunoﬂuorescence staining and confocal microscopy

pathologies. However, no synthetic agonists and very few ligands

have been described [23]. HEK293 cells lines were grown on poly-(D-lysine)-treated glass

Herein, we analyze SUCNR1 signaling pathways and show that coverslips (VWR, 20 20 mm) at 37 C in 5% CO2 for 24 h. The cells

SUCNR1 couples preferentially to Gi. We further demonstrate that were incubated on ice 1 h in HBSS (120 mM NaCl, 5.4 mM KCl,

the GloSensor system allows FSK-free detection of an agonist 0.8 mM MgSO4, 10 mM HEPES; pH 7.4; 10 mM glucose) containing

effect. Moreover, we show that the performance of the assay was ANTI-FLAG M2 (1:1000). After several washing steps, cells were

not modiﬁed by the addition of FSK. When we compared the incubated 10 min in HBSS at 37 C, ﬁxed for 5 min on ice and 15 min

results obtained in a test screening with a FSK vs. FSK-free protocol, at room temperature (RT) in PBS containing 4% paraformaldehyde.

we identiﬁed SUCNR1 agonists with both methods but with fewer Cells were blocked and permeabilized at RT for 30 min with PBS

false positive when measuring the basal levels. containing 2% BSA and 0.12% Triton X-100. After wash, cells were

incubated with PBS containing 2% BSA, 0.12% Triton X-100 and anti-

0 1

2. Material and methods Mouse IgG (H + L), F(ab )2 Fragment (Alexa Fluor 488 Conjugate;

1:1000) for 1 h and 45 min at RT in the dark. Cells were washed and

2.1. Material glass coverslips mounted on slides (Marienﬁeld, Germany) with

Prolong Gold Antifade reagent containing dapi (Thermo Fischer

All chemicals used were from Sigma–Aldrich (St. Louis, Misouri, Scientiﬁc/Life Technologies, Waltham, Massachusetts, USA).

USA) unless otherwise stated. CXL12 (300-28A) was from Images were acquired using confocal microscope (Nikon A1R).

PeproTech (Rocky Hill, New Jersey, USA). The following commer-

cially available antibodies were used for several applications: 2.5. cAMP assay

monoclonal anti-FLAG clone M2 (F3165) from Sigma–Aldrich (St.

Louis, Misouri, USA); anti-Mouse IgG (H + L), F(ab )2 Fragment HEK293 cells stably transfected with plasmid containing cAMP

(#4408, Alexa Fluor 488 Conjugate) from Cell Signaling GloSensor or pGlo and pIRESpuroSUCNR1 were selected with

Technology (Danvers, Massachusetts, USA); rabbit monoclonal hygromycin 200 mg mL (A.G. scientiﬁc, San Diego, California,

anti-phospho-p42/44 MAPK antibody (Thyr202/Thyr204, USA) and puromycin 2 mg mL . Prior to the experiment, cells were

D13.14.4E) from Cell Signaling Technology (Danvers, Massachu- starved for 5 h with 1% FBS. Cells from a conﬂuent T175 ﬂask were

setts, USA); rabbit polyclonal IgG anti-Hsp90 a/b antibody (H-114) detached and incubated 1 h in the dark at RT in assay buffer HBSS

from Santa Cruz Biotechnology (Dallas, Texas, USA) and anti-rabbit (120 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 10 mM HEPES; pH 7.4,

IgG, HRP-linked antibody (#7074) from Cell Signaling Technology 10 mM glucose) containing IBMX (300 mM) and luciferin in HEPES

(Danvers, Massachusetts, USA). buffer (GloSensor reagent, Promega) according to manufacturer

instructions. Cells were distributed into 96-well plates

2.2. Cell culture (150,000 cells per well or 37,000 cells per well in 384-well plates,

white Lumitrac , Greiner) containing the ligands at different

Human embryonic kidney 293 (HEK293) cells were from concentrations. After 1 min agitation at 1200 rpm and 9 min

American Type Culture Collection (ATCC, USA) and grown in DMEM incubation with compounds, basal luminescence level was

adjusted to contain 10% fetal bovine serum (FBS, Biochrom AG, recorded. Similarly, luminescence was recorded following injec-

Berlin, Germany), 1% penicillin and streptomycin (Lonza, Verviers, tion of FSK (40 measures; 500 ms integration time). The

Belgium), 1% L-glutamine (Lonza, Verviers, Belgium) at 5% CO2 and luminometer was a Fluoroskan Ascent FL plate reader (Thermo

37 C. Electron Corp., ascent software version 2.6) equipped with

J. Gilissen et al. / Biochemical Pharmacology 98 (2015) 381–391 383

2 dispensers. In the experiment with PTX, cells were incubated incubated in assay buffer (HBSS: 120 mM NaCl, 5.4 mM KCl, 0.8 mM

overnight with 100 ng mL of PTX (Calbiochem/Merck Millipore, MgSO4, 10 mM HEPES; pH 7.4, 10 mM glucose) containing 5 mM

USA) prior to assay. coelenterazine h (regis technologies, USA) for 1 h in the dark at

37 C. Before stimulation with ligands, the coelenterazine-con-

2.6. Intracellular calcium mobilization assay taining buffer was replaced by assay buffer supplemented with

1.8 mM CaCl2. Luminescence was followed for 8 s (40 measures;

The assay has been conducted according to previous description 200 ms integration) immediately upon ligand addition. Measure-

[25]. Briefly, cells from a confluent T175 flask were detached and ments were acquired with a Fluoroskan Ascent FL (Thermo

Fig. 1. Cell line characterization, arrestin binding and [Ca ]i mobilization.

(A) Cell-surface receptor expression analyzed by ﬂow cytometry on HEK293.pGlo.SUCNR1 cells (not labeled in grey compared to Flag labeled in white). (B) SUCNR1 is

internalized in a constitutive manner in HEK293 cells expressing Flag-tagged SUCNR1 at 37 C (right) compare to 0 C (left). (C) SUCNR1 is able to recruit arrestin 3

(EC50 > 1 mM) and arrestin 2 (EC50 > 2 mM) when stimulated with SA in ARR3.SUCNR1 and ARR2.SUCNR1 cells, respectively; SA has no effect on Mock transfected cells. (D)

Calcium mobilization induced by SA (EC50 = 292.9 0.9 mM) in HEK293.G5A.SUCNR1 cells using an aequorin assay. Pretreatment with PTX completely abolished the calcium

mobilization induced by SA. Data are expressed as mean SEM of at least 3 independent experiments. (E) Activation of ERK1/2 by stimulation of untreated and PTX-treated

HEK293.pGlo.SUCNR1 cells with SA at 500 mM during 3 min. Shown is representative of at least 4 experiments.

384 J. Gilissen et al. / Biochemical Pharmacology 98 (2015) 381–391

Electron Corp., ascent software version 2.6, equipped with 2 at 37 C. Cells were immediately put on ice, lysed with ice-cold

dispensers). RIPA Buffer (25 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 1% sodium

deoxycholate, 0.1% SDS; pH 7.6) supplemented with protease

2.7. Arrestin complementation assay inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland).

Cell lysate were analyzed by SDS page electrophoresis followed by

HEK293 cells stably transfected with pIREShygroFnLARR2 or immunoblotting. ERK1/2 phosphorylation was detected with a

pIREShygroFnLARR3 and pIRESpuroSUCNR1 were selected with rabbit monoclonal anti-phospho-p42/44 MAPK antibody

1 1

hygromycin 200 mg mL (or 400 mg mL ) and puromycin 1 mg (Thyr202/Thyr204, D13.14.4E, Cell Signaling, 1:2000), and Hsp90

mL . Cells suspension (HBSS with 20 mM HEPES, pH 7.4, 10 mM was detected with a rabbit polyclonal IgG anti-Hsp90 a/b antibody

glucose) were incubated into 96-well plates (100,000 cells per (Santa Cruz Biotechnology, 1:5000). The membranes were then

well) containing the ligands at different concentrations for 10 min probed with corresponding HRP-conjugated secondary antibody

at RT. Following injection of 50 mM luciferin (Synchem, Germany), (Cell Signaling Technologies, 1:2000).

luminescence was recorded for 30 min using a high sensitivity

luminometer (Berthold technologies, Centro XS LB 960, MicroWin 2.9. Data analysis and statistical procedure

2000 software, equipped with 2 dispensers).

All data analyses were performed using computer software

2.8. Determination of ERK phosphorylation (GraphPad Prism version 5.0 for Windows).

Statistical analyses of differences between 2 groups were

HEK293 cells stably transfected with pIRESpuroSUCNR1 performed by non-parametric, unpaired, 2-tailed Mann–Whitney

(selected with puromycin 1 mg mL ) were plated in 6-well plates, test. P values less than 0.05 were considered as statistically

starved with 1% FBS and pretreated with 100 ng mL PTX or signiﬁcant.

vehicle overnight. Cells were incubated with succinic acid for 3 min

A B

300 300 pGlo pGlo 250 250 MOCK MOCK

200 200

150 150 R.L.U. R.L.U. (% of ctrl)

(% of ctrl) 100 100

50 50

0 0

-8 -7 -6 -5 -4 -10 -9 -8 -7 -6 -5

Log [FSK] Log [Isoproterenol]

C D

100 SUCNR1 - PTX

SUCNR1 + PTX 80 * MOCK 20

(% of ctrl) 10

R.L.U. 40 (% of ctrl) cAMP inhibition cAMP 20 0

-7 -6 -5 -4 -3

Vehicle + - + - + -

SA 500µM - + - + - + Log [Succinic Acid]

PTX - - - - + +

MOCK SUCNR1

Fig. 2. cAMP inhibition mediated by SUCNR1 activation.

(A) HEK293 cells stably transfected with the GloSensor cAMP biosensor (HEK293.pGlo) show concentration-dependent increase in Relative Luminescent Units (R.L.U.) when

treated with increasing concentrations of the adenylate cyclase activator forskolin (EC50 = 870.5 89.0 nM). (B) Effect of isoproterenol, adrenoceptors agonist, on cAMP

production using HEK293.pGlo cells (EC50 = 153.6 4.1 nM). (C) End point measure of the effect of SA at 500 mM on intracellular cAMP stimulated with 1 mM forskolin using

HEK293.pGlo.SUCNR1 cells (n = 3; p < 0.05). (D) SA decreases cAMP levels stimulated with forskolin 1 mM in HEK293.pGlo.SUCNR1 cells in a PTX-sensitive and concentration

dependent manner (EC50 = 79 0.1 mM). Data are expressed as mean SEM of at least 3 experiments.

J. Gilissen et al. / Biochemical Pharmacology 98 (2015) 381–391 385

2.10. Calculation of Z factor (Fig. 2D) [15]. Cells preincubated with PTX or devoid of receptor did

not respond to SA (Fig. 2C and D).

Z values were determined to monitor assay quality and

were calculated according to the formula: 3.3. Real time analysis of cAMP levels modulation mediated by

Z = 1((3sc+ + 3sc)/|mc+ mc|) [26]. SUCNR1 activation

2.11. Hit selection and activity cut-off criteria The GloSensor system is compatible with kinetic measurement

[13] and we were able to follow the evolution of signal upon

We set up two criteria for hit selection: (1) a positive activity on addition of FSK (Fig. 3A). The effect of SA was stable over time

SUCNR1 expressing cells > negative control (vehicle) mean + 6s during the experiment (40 min) and already visible at 10 mM

and (2) activity on Mock cells (HEK293.pGlo) comprised within (Fig. 3A), as expected from the concentration-response curve

negative control (vehicle) mean 3s. Compounds fulﬁlling these 2 (Fig. 2D). We observed that the levels of cAMP were already

criteria were selected for secondary screening. The number of decreased in the presence of SA at the ﬁrst measure. This effect was

compounds selected represented approximately 0.15% of the concentration-dependent and we reasoned that it could be the

collection (Hit rate). Following cherry picking, compounds were direct effect of SA on the system. In order to validate this

assayed in triplicate at one concentration on SUCNR1 and mock hypothesis, we followed the basal levels of cAMP for 30 min and

cells. Compounds showing statistically signiﬁcant activity on injected SA at the concentration of 500 mM (Fig. 3B). Although the

SUCNR1 were selected for complete concentration–response signal was stable before the addition of SA, it immediately dropped

curves. further below the baseline (Fig. 3B). We reversed the experiment

and analyzed the effect of the addition of the agonist after the

injection of FSK (Fig. 3C). The signal induced by FSK was inhibited

3. Results

by SA although the level did not go back to basal but reached a

2+ plateau (Fig. 3C). When we measured the integration over time

3.1. Cell line characterization, arrestin binding and [Ca ]i mobilization

(Area under the curve or AUC) for 5 min on basal levels (Fig. 3D,

grey bars) or for 40 min post-addition of FSK (Fig. 3D, black bars),

We generated HEK293 cell line stably expressing N-terminus

the effect of SA reached signiﬁcance in both conditions (p < 0.01).

ﬂag tagged SUCNR1 and veriﬁed its expression at the cell

We further conﬁrmed the activity of SA on basal cAMP levels

membrane by FACS analysis (Fig. 1A) and its ability to internalize

through SUCNR1 activation with the determination of a complete

in a constitutive manner at 37 C (Fig. 1B). Since arrestins are

concentration–response curve (Fig. 3E). We calculated an EC50 =

reported as being responsible for GPCRs internalization [27], we

22.83 0.03 mM for SA decrease of basal cAMP levels and an

analyzed the capacity of activated SUCNR1 to recruit arrestin 2 and

EC50 = 45.79 0.08 mM for the inhibitory effect of SA on FSK

3. Using a protein complementation strategy validated with the

induced cAMP (Fig. 3E) that were signiﬁcantly different (p < 0.05).

b2-adrenoceptor (data not shown), we showed that arrestin 3

Interestingly, the Emax (Emax = 52.3 2.7% of control) obtained on

(EC50 > 1 mM) and arrestin 2 (EC50 > 2 mM) could be recruited upon

cAMP basal level was signiﬁcantly (p < 0.05) greater than maximal

SA binding (Fig. 1C). These results were consistent with the

inhibition in the presence of FSK (Emax = 38.0 1.5% of control). We

literature [15,28]. We further conﬁrmed the ability of SA to induce

2+ investigated a range of FSK concentrations (0.1–1 mM) and

[Ca ]i mobilization (Fig.1D, EC50 = 292.9 0.9 mM) in an aequorin-

observed that SA EC50 and Emax were dependent on FSK

based assay. The signal was abolished when the cells were

1 concentration (Fig. 3F).

preincubated overnight with pertussis toxin (PTX, 100 ng ml ). In

In order to exclude that the effect on cAMP basal levels was

addition, we detected an increase in phosphorylated extracellular

limited to SUCNR1 or heterologously expressed receptors, we

signal-regulated kinases (ERK) in HEK293.SUCNR1 cells upon SA

determined agonist potency of CXCL12 on endogenous CXCR4

addition (Fig. 1E). This response was not detectable following PTX

[29,30] with the same methodology (Fig. 3G). We calculated an

overnight incubation.

EC50 = 16.01 1.07 nM and Emax = 62.9 0.2 (% of control) for

CXCL12-induced decrease of basal cAMP levels and an EC50 = 13.93

3.2. cAMP inhibition mediated by SUCNR1 activation 1.12 nM and Emax = 16.9 2.4 (% of control) for the inhibitory

effect of CXCL12 on FSK-induced cAMP production. The differences

We set up a GloSensor cAMP bioassay by stably transfecting the between Emax were statistically signiﬁcant (p < 0.0007).

plasmid pGloSensor-22F into HEK293 cells. We determined the The observed differences in EC50 and Emax may be partially

EC50 of different cAMP inducers in order to determine the explained by variations in luciferin concentration in assay buffer,

concentrations that should be used in our system. We could before and after FSK injection. We tested several assay buffers with

detect a robust signal with increasing levels of FSK (EC50 = 870.5 different luciferin content but did not see any effect on SA response

89.0 nM) and isoproterenol (EC50 = 153.6 4.1 nM), a potent (data not shown).

agonist for endogenous adrenoceptors coupled to Gs in HEK293

cells (Fig. 2A and B). We used this HEK293.pGlo cell line to stably 3.4. Optimization of a screening protocol and assay performance

transfect SUCNR1 subcloned in a bicistronic IRES vector allowing

the simultaneous expression of two proteins from the same We designed a protocol compatible with the screening of

transcript. The generated cell line HEK293.pGlo.SUCNR1 displayed chemical libraries (Fig. 4A). In addition, we wanted to compare the

a stable expression of the receptor over time, when the cells were effects of compounds on basal and FSK-induced cAMP levels. 1 ml

grown in a selection medium. We validated the cell line by of drug solutions from the library were distributed in 96-well

performing end-point assays in the presence of FSK used at the plates. 100 ml of a cell suspension was added and mixed

concentration of 1 mM, the approximate EC50 in our system as thoroughly. The mixture between drugs and cells was incubated

determined by titration experiment (Fig. 2A). Incubation of SA at RT for 10 min (Fig. 4A). The basal level of each well was measured

(500 mM) and FSK (1 mM) for 40 min resulted in a 28.3 1.4% for 5 min and the AUC was determined. These results constituted

decrease compared to control upon activation of SUCNR1 (Fig. 2C). the “basal level screen”, pictured in gray in Fig. 4A. Forskolin at

–

Complete concentration response curves permitted the calcula- 1 mM ﬁnal concentration was added to each well and a kinetic

tion of an EC50 of 79 0.1 mM, consistent with published literature measurement was taken for 40 min. The signal was integrated and

386 J. Gilissen et al. / Biochemical Pharmacology 98 (2015) 381–391

A B

3.5 0.5 Drug Addition Vehicle Vehicle 3.0 SA 10µM 0.4 SA 500µM 2.5 0.3 2.0

1.5 0.2 SA 500µM (R.L.U.) (R.L.U.) cAMP levels cAMP cAMP levels cAMP 1.0 0.1 0.5

0.0 0.0

0 400 800 1200 1600 0 1000 2000 3000 3850 Time (s) Time (s)

C D

2.5 FSK Drug 2500 Addition Addition Vehicle

2.0 2000 **

SA 500µM ** 1.5 1500

(R.L.U.) 1.0 1000 cAMP levels cAMP cAMP levels cAMP ** (AUC, R.L.U.) 0.5 500

0.0 0

0 1000 2000 3000 3850

Vehicle + - + -

Time (s) FSK 1µM - - + +

SA 500µM - + - + E F

80 50 - FSK Vehicle + FSK FSK 0.1µM 40 60 FSK 0.5µM FSK 1µM 30 40 20 (% of ctrl) (% of ctrl) 20

cAMP inhibition cAMP 10 cAMP inhibition cAMP

0 0

-8-7 -6 -5 -4 -3 -7 -6 -5 -4 -3 Log [Succinic Acid] Log [Succinic Acid]

- FSK 60 + FSK + PTX 40

20 (% of ctrl) cAMP inhibition cAMP

-9 -8 -7

Log [CXCL12]

Fig. 3. Real time analysis of cAMP levels modulation mediated by SUCNR1 activation.

(A) SA effect is stable over the time of experiment (40 min) and already visible at 10 mM. At the ﬁrst measure, cAMP levels were already below control baseline. (B) Basal cAMP

levels dropped below the baseline after injection of SA at 500 mM. (C) cAMP levels induced by 1 mM forskolin are inhibited by SA at 500 mM although the level did not go back

to basal but reached a plateau. (D) Comparison of the integration over time (expressed as area under the curve or AUC) on basal levels (white bars) or for 40 min post-addition

of FSK (black bars) in presence of SA at 500 mM (p < 0.01). (E) Concentration–response curve for SA on HEK293.pGlo.SUCNR1 cells stimulated with forskolin 1 mM

(EC50 = 45.79 0.08 mM; Emax = 38.0 1.5% compared to control) or not stimulated (EC50 = 22.83 0.03 mM; Emax = 52.3 2.7% of control). (F) Concentration–response curve

J. Gilissen et al. / Biochemical Pharmacology 98 (2015) 381–391 387

A B Drug FSK 8 Addition Addition 240 Vehicle 200 Vehicle 6 160 SA 500µM 4 120 (R.L.U.)

cAMP level cAMP SA 500µM cAMP level cAMP 80

2 (AUC, R.L.U.) 40 Z’=0.81

0 0

0 5 10 15 20 25 30 35 40 45 50 0 6 12 18 24 30 36 42 48 Time (min) Well #

C D

12000 125

Vehicle 10000 100

8000 Vehicle 75 6000 SA 500µM 50 cAMP level cAMP 4000 level cAMP (AUC, R.L.U.) (AUC, R.L.U.) SA 500µM 2000 Z’=0.74 25 Z’=0.75

0 0

0 6 12 18 24 30 36 42 48 0 24 48 72 96 120 144 168 192 Well # Well #

3500

3000 Vehicle 2500

2000

1500 cAMP level cAMP

(AUC, R.L.U.) 1000 SA 500µM

500 Z’=0.61

0 24 48 72 96 120 144 168 192

Well #

Fig. 4. Optimization of a screening protocol and assay performance.

Design of a screening protocol for direct comparison between the effect of compounds on basal and FSK-induced cAMP levels. (A) The mixture between drugs and cells is

incubated at RT for 10 min. First, the “basal level screen” (in light gray) is performed and expressed as AUC of basal level of each well measured for 5 min. Next, the forskolin at

1 mM ﬁnal concentration is added to each well and a kinetic measurement is taken for 40 min. The signal is integrated and expressed as AUC for each well. These results are

called the “FSK-induced screen” (in black). (B) Assay performance for the two different measurements with 96-wells plates: Z factor calculated for basal level measurement is

0 0 0 0 0

Z = 0.81 (C) and after FSK stimulation is Z = 0.74. (D) Z factor for basal level measurement has been calculated for 384-wells plates to be Z = 0.75 and (E) Z = 0.61 after FSK stimulation.

expressed as AUC for each well. These results were called the “FSK- We determined the Z factor to be 0.81. The values obtained

induced screen”, pictured in black, in Fig. 4A. First, we determined following FSK stimulation are shown in Fig. 4C and the calculated Z

the assay performance for the two different measurements in 96- factor was 0.74. We determined the assay performance with the

wells plates. Basal cAMP level values distribution is given in Fig. 4B. same procedure on 384-wells plates and found a Z factor = 0.75 for

for SA on HEK293.pGlo.SUCNR1 cells stimulated with different concentrations of FSK. Increased concentrations of FSK induced increased EC50 and decreased Emax for SA

(EC50 = 18.86 4.33 mM; Emax = 74.1 1.7% of control without FSK; EC50 = 26.96 5.31 mM; Emax = 72.3 2.3% of control for 0.1 mM FSK; EC50 = 41.73 10.5 mM;

Emax = 58.28 5.9% of control for 0.5 mM FSK; EC50 = 26.96 5.31 mM; Emax = 54.87 9.1% of control for 1 mM FSK). (G) Concentration–response curve for CXCL12 on

HEK293.pGlo cells stimulated with forskolin 1 mM (EC50 = 13.93 1.12 nM; Emax = 16.9 2.4% compared to control) or not stimulated (EC50 = 16.01 1.07 nM; Emax = 62.9

0.2% of control). Pretreatment with PTX completely abolished the effect of CXCL12 on HEK293.pGlo cells. Data are expressed as mean SEM of at least 3 experiments.

388 J. Gilissen et al. / Biochemical Pharmacology 98 (2015) 381–391

SA inhibition of basal cAMP levels (Fig. 4D) and Z factor = 0.61 counter ion (Table 1). We reasoned that we detected an agonist

(Fig. 4E) for SA inhibition of FSK-induced cAMP production. activity because of the presence of the counter ion. We bought

some of the compounds in another chemical form to conﬁrm this

1280TM

3.5. Screening of the Sigma LOPAC library hypothesis. For instance, compound 3E8 in the library, BRL 54443,

a 5-hydroxytryptamine receptor 5-HT1e & 5-HT1F agonist [32],

We applied our protocol on a test screening of the Sigma showed no activity alone in our assay (Fig. 6B) whereas maleic acid

1280TM

LOPAC library, constituted by 1280 compounds of known conﬁrmed its activity and showed an EC50 = 93.8 1.3 mM and

activity [31] distributed in sixteen 96-well plates. The compounds Emax = 32.6 3% when assayed in the presence of FSK. Its EC50 on

were diluted to give a ﬁnal concentration of 100 mM. We basal cAMP levels was 79.4 1.1 mM and Emax = 49.4 3.9%

performed the screening on the HEK293.pGlo.SUCNR1 and a compared to control (Fig. 6C). 7 compounds in the set of 13

counter-screening on the HEK293.pGlo cell line. As expected, many conﬁrmed hits (3E2, 5D3, 11A4, 11C4, 11H3, 11H6 and 15G10) were

compounds had an inﬂuence on both the basal and FSK-induced identiﬁed in the “basal level screen” but remained unnoticed in the

cAMP levels. Therefore, we distributed the results on 2 axis: y axis “FSK-induced screen”.

representing the effect (% of inhibition) of compounds on SUCNR1

and x axis representing the effect (% of inhibition) on HEK293.pGlo 4. Discussion and conclusions

cells (Fig. 5A). Fig. 5A presents the plotted values of the basal

measurements. We set two thresholds for the selection of hits: an In the present study, we demonstrated the feasibility of

inhibition above mean + 6s intra-plate on SUCNR1 cell line and an assaying and detecting agonist ligands of Gi-coupled receptors

activity comprised within mean 3s intra-plates on HEK293.pGlo directly from the inhibition of the basal cellular levels of cAMP.

cells. For the “basal level screen”, 30 compounds met the criteria Bioassays for GPCRs cover a wide range of strategies from

whereas 48 compounds were selected in the “FSK-induced screen”. speciﬁc biosensor for second messenger to binding assays.

11 compounds were common to the two sets (Fig. 5B). Recently, increased attention has been given to pharmacological

assays because of the novel paradigm that some ligand-receptor

3.6. Secondary screening of the selected hits pairs may display functional selectivity that translate in com-

pounds activating only discrete signaling pathways [5]. Therefore,

We performed a cherry pick of the compounds that met our unbiased approaches such as label-free or arresting binding assays

criteria in both conditions (67 compounds) and tested them have been widely publicized as being a superior set up for the

according to the protocol depicted in Fig. 4A, in triplicate. Fig. 6A identiﬁcation of GPCR ligands [33,34]. In theory, they can detect

shows the results for the compounds that displayed a statistically the binding of any activating ligand with a universal read-out. The

signiﬁcant difference between activity on HEK293.pGlo.SUCNR1 advantages of the approach being that the wider the net, the more

and HEK293.pGlo cells. All the 13 conﬁrmed active compounds had substances might be identiﬁed [34]. While there is an evident

succinic acid or maleic acid (a weaker SUCNR1 agonist [15]) as a interest for truly general assays, some receptors have been

A B

100 100 Basal level (-FSK) FSK-Induced 80 Common

60 80

20 60

-20 40 Inhibition of cAMP levels Inhibition of cAMP levels Inhibition of cAMP -40 HEK293.pGlo.SUCNR1 (% of ctrl) HEK293.pGlo.SUCNR1 (% of ctrl)

-60 20

-80

-100 0

-100-80 -60 -40 -20 0 20 40 60 80 100 -25 -20 -15 -10 -5 0 5 10 15 20 25 Inhibition of cAMP levels Inhibition of cAMP levels HEK293.pGlo (% of ctrl) HEK293.pGlo (% of ctrl)

1280TM

Fig. 5. Screening of the Sigma LOPAC library.

(A) The ligands were screened at 100 mM on cells expressing SUCNR1 and counter-screened on mock cell line to estimate their effect on cAMP levels in the absence of SUCNR1.

Compounds with an activity (% of inhibition) superior to mean + 6s compared to vehicle on HEK293.pGlo.SUCNR1 cells and an activity comprised between mean 3s on

HEK293.pGlo cells were selected to be evaluated in a secondary screening. (B) We compared the results obtained for the “basal level screen” (30 compounds identiﬁed as

inverted triangle) and the “FSK-induced screen” (48 compounds identiﬁed by squares), 11 compounds common to the two sets are represented as ﬁlled circles.

J. Gilissen et al. / Biochemical Pharmacology 98 (2015) 381–391 389

- FSK 50 + FSK

(% of ctrl) 20 cAMP inhibition cAMP

0 8 4 SA 3E2 3E 3H3 5C9 5D3 6A9 A4 10H5 11 11B4 11C 11H3 11H6 5G10

1 B C

50 50 - FSK - FSK 40 + FSK 40 + FSK MOCK 30 30

20 20 (% of ctrl) (% of ctrl) cAMP inhibition cAMP cAMP inhibition cAMP 10 10

0 0

-7 -6 -5 -4 -3 -8-7 -6 -5 -4 -3

Log [BRL 54443] Log [Maleic Acid]

Fig. 6. Secondary screening on compounds meeting the criteria.

67 compounds selected with the primary screening were evaluated at 100 mM in triplicates. Compounds with no signiﬁcant activity on HEK293.pGlo.SUCNR1 cells compare to

HEK293.pGlo cells were designated as false positive. (A) Only compounds with succinic acid or maleic acid as counter ions showed signiﬁcant activity either with addition of

forskolin or not on SUCNR1. (B) BRL 54443 without maleate as a counter ion is inactive on SUCNR1. (C) Concentration–response curve of maleic acid on SUCNR1 cells

stimulated with forskolin 1 mM (93.8 1.3 mM; Emax = 32.6 3% compared to control) or not stimulated (EC50 = 79.4 1.1 mM; Emax = 49.4 3.9% compared to control). Data

are expressed as mean SEM of at least 3 independent experiments.

described as being not, or not well, coupled to arrestins or other Sundstrom et al. proposed that the [Ca ]i mobilization was a

canonical pathways. For instance, prominent examples of GPCRs consequence of PLC-b activation by the dimer Gbg [41]. Our

lacking the ability to bind arrestins include the b3-adrenoceptor, observation that SA elicits a concentration-dependent PTX-

the relaxin family peptide receptor 1 (RXFP1) and 2 (RXFP2), or the sensitive [Ca ]i mobilization is consistent with SUCNR1 being

glucose-dependent insulinotropic polypeptide (GIP) receptor not coupled to Gq, at least when heterologously expressed in

[35–38]. More surprisingly, some GPCRs such as the atypical HEK293 cells. Using an in-house luciferase complementation assay

chemokine receptor ACKR3 or receptors involved in stem cells

biology (LGR5) seem to lack G-protein-coupling [39,40]. Therefore,

Table 1

a general paradigm in terms of signaling is difﬁcult to apply to the

Conﬁrmed hits.

entire family or even sub groups of GPCRs. A ﬁt for purpose

3E2 ()-Brompheniramine maleate

approach could increase the odds of identifying good ligands for

3E8 BRL 54443 maleate

poorly characterized receptors. In fact, it could be speculated that

3H3 (+)-Brompheniramine maleate

ﬁ

the growing observations that all GPCRs do not t into general 5C9 Doxylamine succinate

signaling paradigm might explain why some receptors are still 5D3 5-Carboxamidotryptamine maleate

6A9 N,N-Dipropyl-5-carboxamidotryptamine maleate

orphan, even of surrogate ligands.

10H5 Methylergonovine maleate

We selected SUCNR1 to develop a screening assay focused on

11A4 ()-MK-801 hydrogen maleate

cAMP. The signaling pathways for this receptor remained

11B4 2-Methyl-5-hydroxytryptamine maleate

somehow elusive with some discrepancies in the literature. 11C4 Alpha-methylserotonin maleate

11H3 Dizocilipine maleate

SUCNR1 was originally described as coupled to both Gi and Gq

11H6 Nomifensine maleate

and to be internalized upon SA exposure in HEK293 cells [15]. The

15G10 S()-Timolol maleate

view of SUCNR1 being coupled to Gq has been later challenged.

390 J. Gilissen et al. / Biochemical Pharmacology 98 (2015) 381–391

for arrestin recruitment detection, we show that activated SUCNR1 endogenous CXCR4, a Gi-coupled chemokine receptor expressed in

is able to couple to arrestin 3 and arrestin 2. However the receptor HEK293 cells [29]. It can be hypothesized that technologies

seems to be dramatically less efﬁciently coupled to arrestin 2 previously lacked the required sensitivity and/or dynamic re-

(EC50 > 2 mM) and 3 (EC50 > 1 mM) compared to Gi (EC50 = 22.83 sponse to robustly detect variations in Gi induced decrease of cAMP

0.03 mM), although the level of receptor membrane expression levels. During the preparation of this manuscript, we could ﬁnd

was similar (data not shown). Indeed, we were not able to calculate only few reports of ligands identiﬁed with cAMP-based assays on

the EC50 for arrestin recruitment with this assay because higher Gi receptors [47], and never for direct agonists. This is consistent

concentrations induced non-speciﬁc effects due to acidic nature of with technical limitations that preclude the choice of a cAMP-

SA. This weak coupling is consistent with other reports for arrestin based assay for Gi receptors, despite its advantages.

3 [28] and has at least two consequences. First, it could be Although no original agonists were identiﬁed during the test

postulated that arrestin doesn't play a signiﬁcant physiological role screening, this FSK-free method was able to robustly identify true

in this receptor-ligand system since SA would have to reach agonists of the receptor disseminated in the library as counter ions,

important concentration (above 1 mM) to weakly activate this despite an important proportion of compounds interfering with

pathway. Secondly, for this kind of receptor, a screening based on the system. It was rather unexpected to observe that FSK did not

arrestin assay would not be a good option. Ligands slightly less bring any improvement in our assays, in terms of EC50, Emax or

effective than SA would probably never be detected. Therefore, number of conﬁrmed hits. However, the important artifacts that

SUCNR1 can be considered as a good candidate for a Gi-based FSK can bring are well described, such as its impact on the

screening assay. activation of AC via Gs-coupled receptors [48,49]. In addition, it has

Gi-coupled receptor activity can be assessed by many techni- poor aqueous solubility and the concentration to use must be

ques. Historically, GTP-g-S assays were commonly used but determined since its EC50 depends on the cell type and assay

difﬁcultly amenable to large screenings due to the use of employed [11]. Therefore, the choice of the working concentration

radioactive reagents [42]. More recently, promiscuous G proteins will affect the potency of active compounds and may hide weakly

have been utilized to couple Gi-coupled receptors to phospholipase active compounds or partial agonists resulting in false negatives

C-b (PLC-b) and detect activation with [Ca ]i transient mobiliza- [11]. A conﬁrmation of this assumption is the detection of 7 hits in

tion. Actually, SA has been paired with SUCNR1 using this kind of our “basal level screen” that were not identiﬁed when using FSK.

approach [15]. Although this method has proven its effectiveness These hits can be considered as false negative generated by FSK.

in some deorphanization campaigns [1], it suffers from important In summary, we established a screening-compatible FSK-free

drawbacks. Firstly, some special equipment such as FLIPR is cAMP assay that allows the identiﬁcation of agonist ligands for

required for signal acquisition and puts the technique (in a Gi-coupled receptors. We selected SUCNR1 as a proof of concept

screening setting) out of reach for most academic labs or small given its elusive characterization. For the ﬁrst time, we report a

sized companies. Secondly, the promiscuous G protein is another cAMP level determination method, compatible with high-through-

surrogate that does not effectively couple to all Gi receptors or may put screening that does not require the use of a cAMP inducer with

modify the pharmacology of ligands [43]. Another approach is the Gi-coupled receptor. Our protocol is readily available, easy to set

direct measurement of cAMP that has many advantages. The most up, fast and relatively cheap. Therefore, it should facilitate

obvious one being that it is the endogenous second messenger for screening campaigns for Gi-coupled receptors, especially for

Gi and Gs receptors, leaving the pharmacology and coupling system academic labs and small sized biotech companies that study

of the receptor unaffected. In addition, the signal is stable over Gi receptors and do not have access to the [Ca ]i-FLIPR assay.

time, compared to transient [Ca ]i mobilization. Several end point Facilitating screening of Gi pathway brings also renewed oppor-

assays have been adapted for the direct or indirect detection of tunities to screen Gi-exclusive receptors that are unable to

cAMP in cell lysate, with some variations in protocol and sensitivity efﬁciently couple to promiscuous G proteins and arrestins.

[6]. These assays all rely on competition between endogenous

cAMP and some added labeled cAMP, which is a major disadvan-

Conﬂict of interest

tage. In addition, they are not compatible with real time kinetic

measure [12]. Reporter gene assays based on cAMP sensitive

The authors declare no conﬂict of interest.

transcription factor (CRE) activation are also available and

frequently used [12]. Recently, biosensors have been developed,

Author contributions

opening the possibility of analyzing cAMP ﬂuctuations in living

cells in a real time fashion [12,13]. Biosensors can be considered

JH designed and supervised the study. JG, PG, ND, CL and JH

superior compared to previous techniques on several aspects: they

performed the experiments and acquired the data. JG, JH and BP

are more sensitive, no lysis is required and kinetic measurements

analyzed the data, interpreted the results and wrote the paper.

on living cells are feasible.

The GloSensor system is such a biosensor that has been

developed and marketed in 2008 by Promega Corporation [13]. It Acknowledgments

has been extensively reported in the literature for various uses,

from ligand identiﬁcations for Gs-coupled receptors [44] to the This work was supported by the Fonds pour la Recherche

ﬁ ﬁ

dissection of subtle pharmacological aspects of cAMP regulation Scienti que (F.R.S.-FNRS) Incentive Grant for Scienti c Research

such as biased signaling or endosomal cAMP generation [45]. An (F.4510.14), University of Liège (Crédit de Démarrage-Fonds

improved version with increased dynamic range called “22F” (used Spéciaux) and Léon Fredericq Foundation. JH and CL are F.R.S.-

in this study) was released a couple of years after the initial report FNRS Research Associate and Ph.D. fellow, respectively. ND is a

[46]. It was suggested by the authors that the sensitivity of the FRIA PhD fellow. JG received grant from Léon Fredericq Foundation.

enhanced construct could be sufﬁcient for recording cAMP We thank the GIGA Imaging Platform for technical support in

inhibition of basal levels without FSK [46]. However, to the best confocal image acquisition and FACS analysis. The luminometer

of our knowledge, it is the ﬁrst time that the ability of this assay to (Berthold technologies, Centro XS LB 960, MicroWin 2000

measure Gi mediated decrease of basal levels is comprehensively software, equipped with 2 dispensers) was provided by JC Twizere.

investigated. Without the use of FSK, we were able to monitor The authors gratefully acknowledge the technical assistance of C.

agonist activity on heterologously expressed SUCNR1 but also Piron.

J. Gilissen et al. / Biochemical Pharmacology 98 (2015) 381–391 391

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