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Introduction
G-protein-coupled receptors (GPCR) constitute one of
the largest and most versatile families of cell surface
receptors[1]. GPCR recognize and respond to a variety of
extracellular stimulants and endogenous ligands, including light,
odors, taste substances, hormones, chemotactic factors, and
neurotransmitters[2]. Due to the variety of physiological and
pathological functions regulated by GPCR, they were
considered the most promising drug targets in the
pharmaceutical industry. It is estimated that over 50% of the marketed
drugs are modulators of GPCR
functions[3,4]. With the completion of the Human Genome Project, approximately 1000 genes
encoding GPCR were identified, but only about 200 have
known ligands and functions[5]. Searching for ligands of the
orphan GPCR and better modulators of known receptors will
provide new opportunities in future drug discovery.
GPCR are 7 transmembrane proteins with the amino
terminal and carboxy terminal located in the extracellular and
intracellular spaces, respectively[6]. After ligand stimulation,
GPCR undergo conformational change and activate the
intracellular G-proteins, which are composed of α,
β, and γ-subunits, and then initiate signaling to the cell
interior[7]. Based upon the structure and downstream signaling cascade,
the Gα subunit is mainly divided into 4 families:
Gαs, Gαi/o, Gαq, and Gα12[8]. GPCR coupling to
Gαs (including Gαs[s], Gαs[l], Gαs[xl], Gαs[xxl], and
Gαolf) activate adenylate cyclase, which catalyses cAMP production. Contrarily,
Gαi/o (including Gαt[r], Gαt[c], Gαgust, Gαi1, Gαi2,
Gαi3, Gαo1, Gαo2, Gαo3, and Gαz) inhibit cAMP production. GPCR
coupling to Gαq (including Gαq, Gα11, Gα14, and Gα15/16)
activate phospholipase Cβ, which catalyzes the generation of
IP3 and calcium release from intracellular
store[9,10]. Gα12 (including Gα12 and
Gα13) is believed to be related to the activation of Ras, Raf, and ERK
pathway[11].
Based on the signal transduction cascade of GPCR,
several assay techniques for GPCR ligand screening, such
as radioligand binding, [35S]-GTPγS binding, reporter gene,
cAMP detection, and calcium mobilization are commonly
used. Radiometric techniques not only require an advanced
laboratory, but also generate environment pollution and
impair people's health. So non-radiometric assays, especially
cell-based functional assays, played more important roles in
primary screening[12]. However, these assays can only be
applied for selected Gα subtypes. For example, cAMP assay
can only be used for Gαs and Gαi/o-coupled GPCR, and
calcium mobilization only for Gαq-coupled receptors. These
assays require well-characterized signaling pathway of the
receptors, so they would be difficult to apply to orphan GPCR.
It is therefore apparent that a universal high-throughput
screening (HTS) approach for GPCR ligand screening would
be valuable. Previous studies have demonstrated that most
receptors promiscuously couple to several Gα subtypes, but
because one of the G-proteins occupied the dominant status,
it is hard to detect signals induced by other Gα
subtypes[13]. Overexpression of certain
Gα subunits can shift the original coupling pathway of GPCR to the new
one[13,14]. In the present paper, we tested the coupling of the promiscuous G-protein
Gα15/16 (mouse/human orthologs,
respectively[15]) with various receptors that originally coupled to the
Gαs, Gαi, or Gαq pathways. We found out for all the receptors tested,
Gα15/16 shifted the receptors coupling to the calcium
mobilization pathway, and intracellular calcium change
could be easily detected with a Fluo-4 fluorescent indicator.
Ligand efficacy measured by this method was comparable
with the value obtained using traditional methods. This
assay was validated with the δ-opioid receptor (DOR), which
originally coupled to Gαi and may play important roles in
pain, neurodegenerative, and autoimmune
diseases[16-18]. A large-scale screening of 48 000 compounds was performed
based on this system. Several new modulators (including
both agonists and antagonists) were identified and
confirmed with the traditional
[35S]-GTPγS binding assay. This cell-based calcium assay was proved to be robust and easy
to automate, and could be used as a universal method for
the search of GPCR modulators.
Materials and methods
Reagents Mammalian expression vectors encoding
cannabinoid receptors 1 and 2 (CB1 and CB2), α1a adrenergic
receptor (α1aAR), α2b adrenergic receptor (α2bAR),
dopamine receptor 5 (DRD5), and Gα15/16 were purchased from
UMR cDNA Resource Center (Rolla, MO, USA). Plasmids
encoding chemokine receptors CCR5, CXCR4, δ-opioid
receptor, and β2 adrenergic receptor (β2AR) were kindly
provided by Dr Gang Pei from Shanghai Institutes for Biological
Sciences (Shanghai, China). Fluo-4 AM was purchased from
Invitrogen (Carlsbad, CA, USA). FlashBlue GPCR
scintillation beads and
[35S]-GTPγS were products of PerkinElmer
(Boston, MA, USA). SDF-1 was purchased from GL Biochem
(Shanghai, China). Sulfinpyrazone, RANTES, DPDPE,
isoproterenol, phenylephrine, dopamine, noradrenalin,
TIPP-ψ, naltrindole, [D-Ala2]-deltorphin II and DADLE were
purchased from Sigma_Aldrich (St Louis, MO, USA). Other
reagents and solvents used in the experiments were of
analytical grade.
Cell transfection CHO-K1 or HEK293 cells were obtained
from ATCC (Manassas, VA, USA) and maintained in F12
nutritional medium or Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 100 mg/L
penicillin, and 100 mg/L streptomycin at 37 °C in a humidified
atmosphere of 5% CO2. For transient transfection,
approximately 1×106 cells were mixed with 2 µg plasmids in 200 µL
transfection buffer, and electroporation was carried out with
a Scientz-2C electroporation apparatus (Scientz Biotech,
Ningbo, China). The experiments were carried out 24 h after
transfection. For stable cell line generation, the transfected
cells were seeded into 10 cm dishes, and proper antibiotics
(500 µg/mL G418 and/or 20 µg/mL blasticidin) were added to
the culture medium the next day. The selection medium was
changed every 3 d until colonies were formed. The single
colony was picked up, expanded, and tested for the
expression of transfected genes.
Calcium mobilization assay CHO cells co-transfected
with receptors and Gα15/16 were plated onto 96-well plates
at a density of 30 000 cells/100 µL per well and incubated
overnight. The cells were loaded with 2 µmol/L Fluo-4 AM
in Hanks' balanced salt solution (HBSS; containing 5.4
mmol/L KCl, 0.3 mmol/L
Na2HPO4, 0.4 mmol/L
KH2PO4, 4.2 mmol/L
NaHCO3, 1.3 mmol/L CaCl2, 0.5 mmol/L
MgCl2, 0.6 mmol/L MgSO4, 137 mmol/L NaCl, 5.6 mmol/L
D-glucose, and 250 µmol/L sulfinpyrazone, pH 7.4) at
37 °C for 50 min. After removal of the excess dye, the
cells were rinsed with HBSS once. In the antagonist mode,
50 µL HBSS containing known antagonists (positive
control), compounds of interest, or DMSO (negative
control, final concentration 1%) were added. After
incubation at room temperature for 10 min, 25 µL agonists were
dispensed into the well with a FlexStation II micro-plate
reader (Molecular Devices, Sunnyvale, CA, USA), and
intracellular calcium change was recorded with an excitation
wavelength of 485 nm and emission wavelength of 525 nm.
In the agonist mode, 50 µL HBSS was added to the
dye-loaded cells, and 25 µL of known agonists (positive control),
compounds of interest, or DMSO (negative control, final
concentration 1%) were added with FlexStation II, and
calcium change was measured.
[35S]-GTPγS binding assay Cell membranes were
isolated as previously described[19]. In brief, CHO/DOR cells
were pelleted by centrifugation and resuspended in lysis
buffer (5 mmol/L Tris-HCl, 5 mmol/L EDTA, and 5 mmol/L
EGTA, pH 7.5), and then homogenized with a Dounce
tissue grinder. The lysate was centrifuged at
1000×g for 10 min. After removal of the deposition, crude membranes
were then pelleted by centrifugation at 12
000×g for 15 min at 4 °C. The membranes were resuspended in reaction
buffer (20 mmol/L HEPES, 100 mmol/L NaCl, and 5
mmol/L MgCl2, pH 7.4), and the protein concentration was determined using
the Bradford method[20]. The exchange of
[35S]-GTPγS was measured using a scintillation proximity assay, as previously
described[21]. For each assay point, 5 µg membrane was
incubated in 100 µL reaction buffer for 3 h at
30 °C with 100 µg FlashBlue GPCR beads, 10 µmol/L GDP,
10 µg/mL saponin, 0.2 nmol/L
[35S]-GTPγS, and the indicated concentration of compounds. For non-specific basal
binding measurement, 2 µmol/L GTPγS was added.
Membrane-bound [35S]-GTPγS was measured with a Microbeta
scintillation counter (PerkinElmer, Waltham, MA, USA).
HTS campaign The compound library used for the
screening of DOR modulators was comprised of 48 000
different compounds. A 10 compound pool/well mix was
applied to the primary screening in the antagonistic mode, with
an average final concentration of 4.4 µmol/L for each
compound. This matrix system maximized the advantage of
HTS and allowed duplicate screening of each
compound[22]. In each 96-well plate, 8 wells were used as positive controls
(100 nmol/L TIPP-ψ in 1% DMSO) and another set of 8 wells
as negative controls (1% DMSO). The inhibition rate of 100
nmol/L TIPP-ψ was normalized to 100%, and that of the
negative control was 0. The inhibition rate of each compound
was calculated with the following equation:
Inhibition %=(Calcium peak value
compound_calcium peak value
1%DMSO)/(calcium peak value
TIPP-ψ _calcium peak value
1%DMSO)×100%. The samples showing more than 70%
inhibition were considered "hits" in the primary screening.
Data analysis Data were analyzed with GraphPad Prism
software (GraphPad, San Diego, CA, USA). Non-linear
regression analyses were performed to generate dose-response
curves and calculate EC50 or
IC50 values. Linear regression was used to analyze data reproducibility. Two-tailed
Student's t-test was applied to analyze differences. The Z'
factor was calculated by the following equation:
Z'=1_(3SD++3SD_)/|Ave
+_Ave|, where
SD+ is the standard deviation of the positive control,
SD_ is the standard deviation of the negative control,
Ave+ is the mean value of the positive control, and
Ave_ is the mean value of the negative control.
Results
Gα15/16 can couple to various GPCR and mediate calcium response
In the present study, we first tested whether promiscuous G-protein
Gα15/16 could couple to different types of GPCR and mediate calcium response
upon stimulation. Five Gαi/o-coupled (DOR, CB1, CB2, CCR5,
and CXCR4), 3 Gαs-coupled (α2bAR, β2AR, and DRD5),
and 1 Gαq-coupled (α1aAR) receptors were co-expressed in
CHO-K1 or HEK293 cells with Gα15/16, and calcium assay
was carried out as described earlier. Representative kinetic
and dose-response curves are shown in Figure 1, and the
EC50 of various ligands are summarized in Table 1. For most
of the Gαi/o- and Gαs-coupled receptors, agonist
stimulation caused little or no change in the intracellular calcium
concentration, and the calcium assay could not be used to
measure the EC50 value of ligands (Figure 1; Table 1). When
co-expressed with Gα15/16, all receptors produced a
significant calcium-elevating effect after proper stimulation (Figure
1; Table 1). We also found that overexpression of
Gα15/16 made little difference in the calcium response generated by
Gαq-coupled receptor α1aAR (Figure 1E,1F). One of the
receptor α2bAR mainly coupled to Gαs, was also reported
to induce calcium response by coupling to plasma membrane
calcium channels[23]. Overexpression of
Gα15/16 with α2bAR increased the calcium assay's sensitivity, as indicated in the
reduction of EC50 value of noradrenaline (Table 1). For all the
receptors tested, the sensitivity of Gα15/16-mediated
calcium assay was comparable with or sometimes more
sensitive than the traditional cAMP or
[35S]-GTPγS assays (Table 1). We also found cells stably transfected with DOR and
Gα15/16 gave higher and longer-sustained calcium signals
compared to transient transfected cells (supplement Figure
1S). This was likely due to the higher expression level of the
receptor and Gα15/16 protein in stably transfected cells
(supplement Figure 1S, 2S and Table 1S). For further
characterization of this calcium assay, stable cell lines were used.
Agonist and antagonist mode of the calcium assay
DOR was chosen as a model receptor to test the applicability of
this calcium assay. DOR is a Gαi/o-coupled receptor that
plays important roles in various diseases, but lacks
straightforward functional HTS assays. We tested a group of known
DOR ligands (including 3 agonists: DPDPE, deltorphin II,
and DADLE, and 2 antagonists: TIPP-ψ and naltrindole) on
cells that stably express DOR and Gα15/16 with 2 different
setups. In the antagonist testing mode (Figure 2A), test
compounds were pre-incubated with the cells for 15 min;
then calcium assay was initiated by the addition of agonist
DPDPE. In this setup, antagonists showed blocking effects
as anticipated, and agonists also blocked the DPDPE-induced
calcium response due to receptor desensitization during the
pre-incubation period[24,25]. In the agonist-testing mode
(Figure 2A), calcium assay was initiated by the direct
addition of test compounds. All agonists showed a robust
calcium-elevating effect, and antagonists did not cause any
changes. So in the later HTS campaign, all compounds were
tested in the antagonist mode in the primary screening to
reveal any compounds that might block (antagonist) or
desensitize (agonist) the receptor. The agonist mode was used
in the secondary screening to distinguish agonists from
antagonists.
The EC50 values of known agonists were generated with
the agonist mode of the calcium assay and the
IC50 of known antagonists with the antagonist mode. These values were
compared with those obtained with the traditional
[35S]-GTPγS binding assay (Table 2), and both assays showed similar
sensitivity.
Optimization and performance of the HTS assay
Various experimental conditions were tested to optimize the
assay for HTS. We found that cell density did not affect the
EC50 value of the agonist DPDPE, but the signal to
background (S/B) ratio of the calcium response reached a plateau
at a cell density of 30 000/well. The solvent used for
compounds, DMSO, did not affect the S/B ratio at
concentrations up to 1%, and hardly interfered with the
dose-response curves at concentrations up to 2% (Figure 3C,3D).
The final assay conditions for HTS were determined as
follows: the cell density was 30 000/well, the final
concentration of DMSO was 1%, and the DPDPE concentration was 10
nmol/L (approximately EC80).
The Z' value is a metric used to assess the robustness of
an assay for screening and is the normalized 3 standard
deviation window between the negative controls and positive
controls[26]. As shown in Figure 4A, the Z' value for the
assay was 0.64, and the S/B ratio was 18.86, indicating that
the system was adequately optimized for HTS. Furthermore,
to investigate reproducibility between duplicate plates, the
corresponding wells from 2 different 96-well plates were
treated with the same concentration of TIPP-ψ and then 10
nmol/L DPDPE. The data from the corresponding wells of
different plates were investigated with liner regression
analysis[27]. The correlation coefficient was 0.95, showing a high
degree of reproducibility between duplicate sample plates.
Results of HTS campaign Of the 48 000 compounds
initially screened, 273 hits (0.57%) showing greater than 70%
inhibition on 10 nmol/L DPDPE-induced calcium response
were discovered (Figure 5A). Secondary screening (single
compound per well) was done to further confirm the hits
(Figure 5B). Finally, 8 compounds displaying consistent
inhibitory effects from the secondary screening were picked
out and tested on other GPCR (CCR5 and CXCR4; data not
shown) for receptor specificity. Four compounds with
relatively high receptor specificity for DOR were further tested
to distinguish their agonist or antagonist nature (Figure
5C,5D). Three of the compounds (TZ-02, TZ-03, and TZ-04)
showed moderate to weak agonist properties, as they were
found to induce calcium response in DOR- and Gα15/16
co-expressing cells. One compound TZ-01 showed pure
antagonist property. The activities of these compounds were
further validated with [35S]-GTPγS binding assay (Table 3).
Compounds TZ-02 and TZ-04 were proven to be agonists,
and compound TZ-01 was an antagonist. Compound TZ-03
was a partial agonist/antagonist. Its weak agonist activity
can only be detected in the sensitive calcium assay, but not
in the [35S]-GTPγS binding assay due to a limited assay
window. The strong antagonist property of TZ-03 was
confirmed by both assays.
Discussion
Considerable effort has been directed towards the
development of HTS platforms for the GPCR because these cell
surface receptors represent important drug
targets[28]. Detection methods have moved a long way from
membrane-based radioligand binding assay towards cell-based
functional assays. Most functional assays rely on the detection
of the changes of different downstream effectors induced
by receptor activation. Due to the versatility of
GPCR-induced intracellular changes, it is sometimes difficult to
handle and compare results from different assay systems.
Meanwhile, with the cloning of more and more orphan GPCR,
their implications as potential drug targets require vigorous
validation. Little knowledge exists today regarding their
native ligands and coupling mechanisms, and this makes HTS
assay development extremely difficult. Thus, a universal
HTS approach for GPCR ligand screening would be highly
valuable.
Calcium mobilization assay with fluorescent dyes is a
highly sensitive and easy-to-handle method that has been
widely applied to study ligand or voltage-gated ion
channels and GPCR coupled to the
Gαq-protein[29]. It is critical to provide the receptors with a universal and efficient calcium
signal transducer if this method is to be used to search
modulators for various GPCR. It has been reported that the
Gαq-protein with the last 5 amino acids exchanged with
Gαs- or Gαi-proteins (designated as Gqs5 and
Gqi5[30]) can couple to GPCR that originally coupled to
Gαs or Gαi, and induce calcium mobilization upon stimulation. In the present study,
we tested the versatility of another Gαq subfamily protein
Gα15/16.
A panel of 9 GPCR that originally coupled to different
types of G-proteins was studied. These included 5
Gαi/o-coupled (DOR, CB1, CB2, CCR5, and CXCR4), 3
Gαs-coupled (α2bAR, β2AR, and DRD5) and 1 Gαq-coupled
(α1aAR) receptors. With the exception of α1aAR, which originally
coupled to Gαq, and α2bAR, which was reported to
modulate plasma membrane calcium
channels[23], other receptors could not elicit measurable calcium responses upon
stimulation when they were expressed alone in CHO-K1 or HEK293
cells. After co-expression with Gα15/16, all receptors were
coupled to the calcium mobilization pathway, and the
EC50 values of the ligands measured with this assay were in close
agreement or more sensitive than other reported methods.
We further characterize the Gα15/16-mediated calcium
assay on DOR. DOR has been heavily studied in the past for
its roles in pain and drug
addiction[31,32]. It has been a focus of attention again recently due to its involvement in
neurode-generative and autoimmune
diseases[16,18]. Traditional HTS methods for searching DOR ligands include radioligand
binding, [35S]-GTPγS binding, and cAMP assay. Both
[35S]-GTPγS binding and cAMP assay can only be used to search
agonists or antagonists in a single HTS run. Radioligand
binding is the only way to find both agonists and
antagonists simultaneously, even though the separation of
agonists from antagonists needs a secondary functional assay.
We found the calcium assay to be very efficient in detecting
both agonists and antagonists if the testing compounds were
pre-incubated with the cells before the addition of agonist
DPDPE. In this experimental setup, agonists can also block
the DPDPE-induced calcium response due to receptor
desensitization during the pre-incubation period. Thus, any
compounds that reduce the DPDPE-elicited calcium signal
could be a potential DOR modulator. Later on, the agonist
or antagonist nature of the compound can be simply
distinguished by direct application of the compound to the cells
to see whether it can induce calcium change or not.
Various assay parameters were optimized to improve the
assay window and stability. The Z' factor is a useful tool for
evaluating bioassay qualities[26]. In general, a Z' value above
0.5 suggests that an assay is robust enough for HTS. The
calcium mobilization system described herein displayed a Z'
value of 0.64, which indicated that the assay
was of a high-quality nature. This assay was applied to a large-scale
screening of a compound library consisting of 48 000 synthetic
compounds. Four compounds with novel structures and
relatively high receptor specificity were sorted out and
further validated with a traditional
[35S]-GTPγS binding assay. Two of these compounds were found to be agonists and 1 to
be antagonist. The other was a partial agonist/antagonist
that displayed very weak agonist, but strong antagonist
activity.
In summary, a universal, cell-based, Gα15/16-mediated
calcium assay was developed and validated for the
identification of compounds that modulate DOR activity. Its
application may be expanded to other GPCR and even orphan
receptors.
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