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Introduction
G-protein coupled receptors (GPCR) constitute one of the largest families of molecular targets applicable to drug
discovery in the human genome, and about 40% to 50% of currently marketed drugs are directed towards this class of cell surface
receptors[1,2]. Therefore, they have been the logical choice of numerous high-throughput screening (HTS) efforts to search
for new leads of potential therapeutic value.
MrgD and MrgA1 belong to a recently identified family of orphan GPCR genes, namely mas-related genes (Mrg). A
putative role for their protein products such as MrgD and MrgA1 receptors in nociception has been postulated based on
specific expression in sensory neurons of the dorsal root ganglion
(DRG)[3_5]. Studies published in 2004 suggest that they are
important regulators in pain perception and conduction, and may provide novel therapeutic opportunities for
analgesia[6_8]. Although b-alanine has been shown to stimulate the MrgD receptor in heterologous systems, the natural ligand remains
unknown at this time[9]. It was reported that the MrgA1 receptor could be activated by certain opioid-related peptides, but
its endogenous ligand(s) is still
illusive[3,4,10]. Meanwhile, high
EC50 values of some known modulators practically limit their
application in receptor-binding
assays[9]. Therefore, new and non-peptide small molecules would serve not only as potential
drug leads, but also as powerful molecular probes to elucidate the functions of the Mrg receptor family in pain perception.
It is known that both MrgD and MrgA1 receptors are coupled to the
G-protein a subunit, Gq. Upon activation,
intracellular calcium influx is
evoked[9,10], which can be measured using a calcium-sensitive dye and a fluorescence plate
reader[11]. In the present paper, we describe the development and validation of a homogeneous HTS assay based on the analysis of
calcium mobilization using cells expressing MrgD or MrgA1 receptors. Since the MrgD receptor is also coupled to Gi,
confirmed hits were further assessed for their ability to inhibit cAMP content in the MrgD receptor expressing cells.
Materials and methods
Reagents b-alanine was purchased from Shanghai Chemical Reagents Co, Ltd (Shanghai, China). FLIPR calcium 3 assay
kit and CatchPoint cyclic-AMP fluorescent assay kit were bought from Molecular Devices (Sunnyvale, CA, USA). Dynorphin
A (fragment 1_13), probenecid, 3-isobutyl-1-methylxanthine (IBMX) and forskolin were obtained from Sigma-Aldrich (St
Louis, MO, USA). Fetal bovine serum (FBS) was the product of Hyclone (Logan, UT, USA), cell culture medium F12 and
Dulbecco's Modified Eagle's Medium (DMEM) was from GIBCO BRL (Rockville, ML, USA), G418 was from Merck KGaA
(Darmstadt, Germany), EcoR I and Xho I were obtained from TaKaRa Biotechnology (Dalian) Co, Ltd (Dalian, China), and
Fugene 6 from Roche Diagnostics (Indianapolis, IN, USA).
Plasmid and stable cell lines
Full-length MrgD and MrgA1 genes were cloned by PCR using mouse genomic DNA as
templates. The PCR primers were as follows:
For the MrgD sense primer:
5'-ggggaattcatgaactccactcttgacagcag-cccagctcc
-3';
anti-sense primer:
5'-gggctcgaggaccccatcatta-gtacacgtggatggcgtctc-3'.
For the MrgA1 sense primer:
5'-ggggaattcatggacaataccatccctggagg-tatcaacatcac
-3';
anti-sense primer:
5'-gggctcgagtggctctgatttgc-ttcttgacatctccaccatg-3'.
PCR fragments were digested with
EcoR I and Xho I, and then cloned into pCMV-Tag2B (Strategene, La Jolla, CA, USA).
CHO-K1 (MrgD) or HEK293 (MrgA1) cells (ATCC,Manassas, VA, USA), grown to 50%_80% confluent in a 3.5 cm culture
dish and were transfected with the plasmids using a Fugene 6 (µL) plasmid (µg) ratio of 3:1. Four hundred (MrgA1) or 800
(MrgD) mg/L G418 was added 24 h afterwards, and individual clones were selected in 96-well clear culture plates (Corning,
Acton, MA, USA) with 400 or 800 mg/L G418 in DMEM (MrgA1) or F12 medium (MrgD) containing 10% FBS. Each clone was
characterized by the calcium mobilization assay. The stable cells established (MrgD-CHO and MrgA1-HEK293 cells) were
maintained in F12 or DMEM medium in the presence of 400 mg/L G418.
Calcium mobilization assay
MrgD-CHO cells or MrgA1-HEK293 cells were detached and plated onto 96-well plates at a
density of 30 000 cells for MrgD-CHO cells and 20 000 cells for MrgA1-HEK293 cells (100 µL/well) and incubated overnight.
After changing to 80 µL of serum-free medium, the cells were loaded with 80 µL of the calcium 3 assay dye supplemented with
2.5 mmol/L probenecid and incubated at 37 °C for 60 min. A baseline fluorescence signal was measured for the first 17 s, after
which test compounds prepared as a 5×stock in Hank's balanced salt solution buffer (supplied with the assay kit) were added
to the plate through an automated pipetter (40 µL/well) equipped within FlexStation
II384 (Molecular Devices, USA). The intracellular calcium influx was analyzed by the same instrument with an excitation wavelength of 485 nm and emission
wavelength of 525 nm, and relative fluorescence signal measured at 1.6 s intervals for
150_300 s.
cAMP assay MrgD-CHO cells were detached and plated onto 96-well plates at a density of
30 000 cells (100 µL/well) and incubated overnight. After 1 wash step with Krebs-Ringer Bicarbonate Buffer (KRBG) containing 10 mmol/L glucose , the
cells were pre-incubated in KRBG with 500 µmol/L IBMX for 10 min, and then stimulated with test compounds in the presence
of 1.0 µmol/L forskolin for 15 min. Forskolin alone was used as maximal signal control and the blank well as the negative
control (background). They were lysed thereafter and quantified for cAMP content using a
CatchPoint cyclic-AMP fluorescent assay kit according to manufac-turer's instruction. Briefly, 20 µL cell lysate or standard cAMP was added to the 384-well
plate pre-coated with goat anti-rabbit IgG, and then 20 µL rabbit anti-cAMP antibody followed by 20 µL horseradish
peroxidase-labeled cAMP. After 2 h incubation, the plate was washed with 80 µL washing buffer 4 times before the addition of 50
µL substrate solution. Fluorescence intensity was recorded by FlexStation
II384 with an excitation wavelength of 530 nm and
emission wavelength of 590 nm.
HTS campaign The compound library used for the screening of MrgD receptor agonists consisted of 8000 pure synthetic
compounds and extracts from natural products. A 10-compound pool per well mix dissolved in 100% dimethyl-sulphoxide
(DMSO) solution was applied to the primary screening, with an average final concentration of 1.7 µmol/L of each compound.
This matrix system was known to maximize the advantage of HTS and allowed duplicate screening of each
compound[12]. In each of the 96-well plate, 16 wells were used either as the
positive control (b-alanine or dynorphin A) or as the negative
control (assay buffer), and samples showing greater than 20% agonist activities compared to the maximum response elicited
by the positive control were considered `hits'.
Data analysis Data were analyzed using GraphPad Prism software (San Diego, CA, USA). Non-linear regression analyses
were performed to generate dose-response curves to calculate
EC50 values. Time-course study results from the calcium
mobilization assay were expressed as relative fluorescence unit (RFU), and all other calcium influx measurements were
represented as the peak RFU per well. Values presented are mean±SEM of at least 3 independent experi-ments. Percentage
responses of hits were estimated based on the response to 1.0 mmol/L
b-alanine or 60 µmol/L dynorphin A, which was defined
as 100%. Z' factors were calculated as described by Zhang
et al[13].
Results
Assay validation In the present study, we first assessed the kinetics of the signal generated in the assay system.
Time-course experiments showed that agonist stimulation with 1.0 mmol/L
b-alanine produced a significant increase in
fluorescence intensity relative to the background (non
b-alanine stimulated) and the peak RFU appeared around 70 s followed by a
gradual signal decay to the baseline (Figure 1A). As for the MrgA1 receptor, a similar time-course curve was observed with
60 µmol/L dynorphin A (Figure 1B). Meanwhile,
b-alanine and dynorphin A each did not induce any increase in fluorescence
intensity in the blank CHO or HEK293 cells (data not shown). The effects of cell seeding density on the assay signal and
background are shown in Figure 2. A density of 30
000 cells per well for MrgD-CHO and 20 000 cells per well for
MrgA1-HEK293 generated the best signal to background (S/B) ratios (58.1 and 50.3). These numbers were selected for respective
HTS campaign. DMSO, at concentrations of up to 0.5%, did not affect the assay performance (data not shown). Under these
optimized conditions, the determined
EC50 values of the known MrgD receptor ligand
b-alanine and the MrgA1 receptor agonist dynorphin A were 8.6 µmol/L and 1.1 µmol/L, respectively (Figures 3A, 3B), consistent with those reported in the
literature[3,9].
Assay performance In order to apply this assay system to HTS format, both signal intensity evoked by 1.0 mmol/L
b-alanine and background noise without alanine were extensively studied. As shown in Figure 4, the Z' value for the calcium
mobilization assay was 0.65 with an S/B ratio of 54.0, and the coefficient of variation (CV) value for the signal was 10.7%. In
the case of the MrgA1 receptor, the Z' factor, S/B ratio, and CV value for the signal were 0.5%, 73.6%, and
15.8%, respectively (Figure 4B). These parameters indicate that the system was adequately optimized for HTS.
Identification of MrgD receptor agonists
Of the 8000 samples initially screened for potential MrgD receptor agonists, 31
initial hits (0.39%) showing greater than 20% agonistic
activities relative to b-alanine were discovered, as shown in the scatter
plot of the actual readouts (Figure 5). The HTS campaign resulted in a Z' value of 0.6 that conferred the assay quality
requirement of above 0.5[13]. Secondary screening with a single compound per well confirmed that 14 of the above 31 hits
displayed consistent agonistic properties. Follow-up experiments using CHO cells lacking the MrgD receptor revealed that
only 2 of the above 14 hits elicited calcium influx through an MrgD receptor-mediated mechanism. They were further studied
in a functional assay where intracellular cAMP levels were measured following MrgD activation. When validating the assay,
b-alanine did not exhibit any effect on the cAMP levels in the blank CHO cells (data not shown). As displayed in Figure 6, like
b-alanine, both hits (121145 and 011531) induced calcium influxes and cAMP responses in CHO cells expressing MrgD, thus
verifying their agonist status.
Identification of MrgA1 receptor agonists
Based on the above HTS experience, we applied the same method to screen
MrgA1 receptor agonists. Briefly, HEK293 cells expressing MrgA1 were used to optimize assay conditions. As a result, a cell
density of 20 000 per well and DMSO tolerance of 0.5% were selected for the HTS campaign. The assay parameters studied
were comparable to that of MrgD (Table 1), suggesting its suitability. Following a major HTS campaign on
16 000 synthetic compounds or natural product extracts, 48 initial hits (0.30%) showing greater than 20% agonistic activities
relative to dynorphin A were discovered (Figure 7). Follow-up screening with blank HEK293 cells confirmed that 2 hits
(077802 and 078728) consistently displayed MrgA1 agonist effects on calcium mobilization (Figure 8). These, together with
the 2 MrgD agonists above, were further examined by reciprocal calcium mobilization assay to check their specificities for
MrgD vs MrgA1 receptors. No cross-reactivity was noted between the 2 sets of hits (Table 2).
Discussion
Opioids have been used clinically as effective analgesics for many pain conditions, but their application is limited by
considerable central nervous system (CNS) mediated side-effects. Therefore, the development of peripheral analgesics that
do not cross-react with central opioid receptors is of common interest for many
researchers[16]. The restricted expression of
MrgD and MrgA1 in sensory neurons of DRG offers a new strategy in the study of peripherally active analgesics to mitigate
or avoid undesirable CNS side-effects such as mental impairment and drug dependence. This is the rationale behind the
studies presented in this paper.
Cell-based assays can offer a wealth of information relative to the functionality of a test compound, and thus are widely
employed in the drug discovery process. As far as measuring intracellular calcium influx is concerned, cells are loaded with
the acetoxymethyl ester of Fluo-3 or Fluo-4 in a typical assay format, and unincorporated dye is removed by repeated
washing. Obviously, this non-homogeneous approach is not sufficiently robust for HTS. Fluorometric imaging plate reader
(FLIPR) calcium 3 is a newly developed fluorescent dye (calcium indicator) that uses quenching technology to reduce
background fluorescence, thereby providing a homogenous
solution[14]. A major practical benefit of this method is that it
eliminates cell-washing steps, resulting in easier automation and faster throughput. As demonstrated in the present study,
compounds were screened at a rate of 20 s per well; a minimum of 4000 compounds could be screen-ed each day in a 96-well
plate format (1 compound per well). This advantage could be further explored by utilizing high-density plates (eg 384-well
plate).
As Mrg receptors are still classified as orphan GPCR, their implication as potential drug targets requires vigorous
validation. Little knowledge exists today regarding the native ligands, which makes assay development extremely difficult.
Apart from discovering relevant modulators to alleviate pain symptoms, confirmed hits may well serve as molecular probes
to the understanding of Mrg receptor func-tions. Conceivably, exogenous ligands would be more easily screened in a format
in which the target is expressed and regulated in the most physiologically relevant manner. When an artificial system such
as an engineered cell line is employed, many factors must be considered in order to maximize the S/B ratio. As shown in Figure
2, varying cell seeding density influences the signal and background of the cellular reaction. We chose
20 000 or 30 000 cells/well in the HTS setting that resulted in an optimal S/B ratio. In addition, DMSO tolerance can limit the maximum concentration
of the compound to be assayed[15]. In our experience, 0.5% DMSO did not disturb the assay performance and a final
compound concentration of 17 µmol/L was achieved.
The Z' factor is a useful tool for evaluating bioassay qualities. In general, a Z' value above 0.5 suggests that an assay is
robust enough for HTS. The calcium mobilization system described herein consistently displayed a Z' value between 0.50
and 0.65. This, and in conjunction with other parameters, such as S/B ratio as well as CV values, indicates that the assay is
of a high quality nature.
False positives are a major problem for any type of HTS assay. In the calcium mobilization analysis, this translates into
issues such as auto-fluorescence of test compounds, calcium ionophores and chemicals that permeabilize the cell
membrane[11]. In our case, the majority of the non-specific compounds were discarded after running a secondary screening using cells
lacking MrgD or MrgA1 receptors. The bioactivities of the confirmed hits for MrgD discovered in the calcium mobilization
assay were supported by a decrease in the intracellular cAMP level, an indicator of Gi activation, and strikingly similar
potencies determined by these 2 different functional assays.
Because the MrgA1 receptor is only coupled to Gq, but not to the Gi or Gs signaling
pathway[10], cAMP measurements are not applicable in terms of functionality verification. Since the homology in DNA sequences of the 2 Mrg receptor genes is
only 37.9%[3_5], it is not surprising that the hits for the MrgD receptor did not cross-react with the MrgA1 receptor or vice
versa. This feature is of importance in the development of subtype-selective Mrg receptor modulators for therapeutic use. In
addition, the structural diversity of the 4 confirmed hits (Table 2) will certainly offer a good starting point for medicinal
chemistry efforts as well as an in-depth structure-activity relationship exploration.
In summary, we have successfully developed and evaluated a homogeneous calcium mobilization assay that can be
applied to HTS for small molecule MrgD or MrgA1 receptor agonists. The hits identified by the system could be functionally
verified by another assay based on a known signal transduction pathway. This approach is straightforward, robust and
amenable to automation, with a minimum screening throughput of 4000 compounds per day. It may also be applied to other
GPCR that involve calcium signaling upon stimulation.
Acknowledgements
The authors are indebted to Dr Xin XIE (The National Center for Drug Screening, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai) for technical advice and to Dr Dale E MAIS (Ligand Pharmaceuticals, Inc, USA) for
critical review of the manuscript.
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