Discovery of Fluorescence Polarization Probe for the ELISA-Based Antagonist Screening of α1-Adrenergic Receptors

Article abstract of DOI:10.1021/acsmedchemlett.6b00048

High-throughput screening (HTS) of ligand library to find new active molecules for G protein-coupled receptors is still a major interest, as well as an actual challenge. Fluorescence polarization (FP) assay portrays an essential role in HTS; however, in many cases, it was restricted by the absence of FP probes, the narrow measurement window, and low signal-to-noise (S/N) ratio. Herein, based on the modification of our previous probe 1 (QFL), we discovered an FP probe 3 (QGGFL) for α₁-adrenergic receptors (α₁-ARs), which has satisfactory fluorescence intensity, specific binding ability to receptors, and suitable fluorescence properties that were compatible with the filters in the FP system. Meanwhile, an ELISA-like strategy was designed for FP-based HTS assay in which proteins were adhered into a solid phase to improve the measurement window and S/N ratio. With fluorescent antagonist QGGFL and the ELISA strategy, we succeeded in establishing the first competitive binding FP assay for α₁-AR antagonists as the alternative of the radioligand binding assay.

Full text of DOI:10.1021/acsmedchemlett.6b00048

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Technology Note
Discovery of fluorescence polarization probe for the ELISA-
based antagonist screening of #1-adrenergic receptors
Zhao Ma, Zhenzhen Liu, Tianyu Jiang, Tianchao Zhang, Huateng Zhang, Lupei Du, and Minyong Li
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00048 • Publication Date (Web): 26 Aug 2016
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Discovery of fluorescence polarization probe for the ELISA-based an-
tagonist screening of α1-adrenergic receptors
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Zhao Ma, Zhenzhen Liu, Tianyu Jiang, Tianchao Zhang, Huateng Zhang, Lupei Du and Minyong
Li*
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong
University, Jinan, Shandong 250012, China.
KEYWORDS.α1-adrenergic receptors, fluorescence polarization, ligandscreening, fluorescent probe, ELISA, GPCR.
(This article is dedicated to Professor Lin Xia on the occasion of her 80th birthday)
ABSTRACT: High-throughput screening (HTS) of ligand library to find new active molecules for GPCRs is still a major
interest, as well as an actual challenge. Fluorescence polarization (FP) assay portrays an essential role in HTS, however, in
many cases, it was restricted by the absence of FP probes, the narrow measurement window, and low signal-to-noise
(S/N) ratio. Herein, based on the modification of our previous probe 1 (QFL), we discovered an FP probe 3 (QGGFL) for
α1-adrenergic receptors (α1-ARs), which has satisfactory fluorescence intensity, specific binding ability to receptors and
suitable fluorescence properties to that were compatible with the filters in the FP system. Meanwhile, an “ELISA-like”
strategy was designed for FP-based HTS assay, in which proteins were adhered into a solid phase, to improve the meas-
urement window and S/N ratio. With fluorescent antagonist QGGFL and the ELISA strategy, we succeeded in establishing
the first competitive binding FP assay for α1-AR antagonists as the alternative of the radioligand binding assay.
G protein-coupled receptors (GPCRs) represent the
largest family of transmembrane proteins in the hu-
man genome that account for about 40% of therapeu-
tic agents in the prescription pharmaceutical indus-
try.1, 2 Unbiased identification of compounds interact-
ing with GPCRs is strongly dependent on high-
throughput screening (HTS) of chemical libraries.
Figure 1. Chemical structure, U-shaped preferential
Therefore, developing analytical methods to find new
conformation and structure modification strategy of
lead compounds targeting GPCRs is a great interest,
our previous fluorescent ligand 1 (QFL) of the α1-AR.
as well as an actual challenge.3 Currently, HTS ap-
proaches for GPCR ligands mainly involve in the lig-
and-receptor binding assay using radioactive or fluo-
rescent tracers.4 Radioligand binding is a well-
established tool for screening at GPCRs, but with
drawbacks of safety concerns and costly consuma-
bles.5 In parallel to the rapid development of fluores-
cent techniques, fluorescence assay is becoming a
multifaceted tool in life science.6 Among these tech-
niques, the nonradioactive, low-cost and self-
referencing FP assay using fluorescent ligands pro-
pose another credible alternative.7, 8 Polarization val-
ue is defined as the ratio of fluorescence intensities
parallel (I∥) and perpendicular (I⊥) with respect to
Fluorescent ligands of GPCRs sprang up in recent
decades, but their applications were restricted pri-
marily to receptor visualization and localization.11,
12Although fluorescent ligand-based assay has opened
a brand new standpoint in the fields of GPCRs, it still
requires further development.13 Due to the absence of
FP probes, the narrow measurement window, and
low signal-to-noise (S/N) ratio, FP assays have not
been extensively applied.13 The key issue in such a
matter is that the polarization is not totally or com-
pletely inexistent when fluorescent ligands bind to
GPCRs because there is not quite a difference on ro-
tation rate between the free fluorescent ligand and
ligand confined by the receptor.
10
the plane-polarized excitation light.9, In general,
fast rotation of freely moving fluorophores will
scramble polarization, while constrained fluoro-
phores that bind to bulk receptors, will emit highly
polarized fluorescence.
α1-Adrenergic receptors(α1-ARs) are members of
GPCR superfamily that mediate many physiological
processes such as smooth muscle contraction, myo-
cardial inotropy and chronotropy, and hepatic glu-
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cose metabolism.14 As therapeutic targets, the antag- of α1-AR antagonists and could not become an FP probe.21
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onists of α1-AR play critical roles in the treatment of
hypertension, benign prostatic hyperplasia (BPH) and
lower urinary tract symptoms (LUTS).Moreover,
someα1-adrenoceptor antagonists could induce pros-
tate cancer cell apoptosis.15, 16 However, the screening
of α1-AR antagonists is heavily dependent on radiolig-
With the help of Chemdraw 3D software, we found QFL’s
essential conformation with a crowded U-shape (Figure 1).
In such a conformation, the quinazoline group comes
close to the fluorescein core, which may exert an influ-
ence the conjugate plane of fluorophores, and thereby the
quantum yield decreases. While the quinazoline moiety
represents an interesting pharmacophoric scaffold of an-
tagonist to develop FP agents that bind to the α1-AR re-
ceptor tightly, to circumvent the issue about fluorescence
intensity, two novel fluorescent ligands, QGFL and
QGGFL (2 and 3, Figure 1) were designed by introducing
the linker, γ-aminobutyric acid (GABA), to conjugate
pharmacophore and fluorophore. Although GABA isthe
inhibitory neurotransmitter targeted at GABA recep-
tors, the new compounds including GABA linker here
will show no agonism activity to GABA receptors, be-
cause the primary amino group at 4-position in GABA
18
and binding assay at present.17, Although several
fluorescent probes have been synthesized to study α1-
ARs in cells, a few of them are qualified for the high-
throughput screening of antagonists, and no FP-
based assay has been discovered or reported.18-22 In
this paper, through the structure modification of our
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previous fluorescent antagonist QFL (
designed and synthesized an excellent FP antagonist
probe QGGFL ( ) with maintained binding affinity
1
) of α1-AR21, we
3
and increased fluorescence quantum yield (~10-fold),
which could serve as an efficient toolkit to achieve an
FP-based HTS assay (Figure 1). To settle the matters
of narrow measurement window and low S/N ratio in
FP assay, an “ELISA-like” strategy was designed to
amplify FP effect.23, 24 This strategy is to adhere α1-AR
proteins to the solid phase and then to measure the
FP effect of fluorescent ligands binding to these pro-
teins. When bound to the confined solid-phase pro-
teins, the rotation of fluorescent ligands was con-
strained greatly, which would produce a considerable
polarization effect.
is necessary for its agonism activity27
. As the linker,
GABA could increase the flexibility of molecules and re-
duce the steric hindrance between quinazoline and fluo-
rescein groups. The target compounds were conveniently
synthesized as depicted in Scheme 1, in which piperazine-
substituted chlorine of 6 to afford compound 7, and then
the amide condensation and N-BOC deprotection reac-
tions were involved to introduce one or two GABA(s) to
the key intermediates 9 and 10. Finally, the key interme-
diate reacted with FITC to provide 2 (QGFL) and 3
(QGGFL), respectively.
The affinities of two target compounds
3
2 (QGFL)
and (QGGFL) were assessed routinely by competi-
tive binding assays on three α1-AR subtypes stably
expressed in CHO cells, using radiolabeled
[3H]prazosin, an established α1-AR competitive an-
tagonist. As expected, each compound revealed up to
nanomolar affinities for all α1-AR subtypes (Table 1).
This result also indicated that the new compounds
could serve as antagonists of α1-AR. It should be not-
ed that our previous research indicated clearly that
the large moiety was permitted to conjugate to the
N4 position of 2-piperazine group in the quinazoline
pharmacophore without losing much binding affinity,
but bulk fluorescein was not under consideration for
selection.21 These results revealed that introducing a
linker between fluorescein and quinazoline groups
was beneficial for affinity improvement. Moreover,
Scheme 1.Synthesis of fluorescent ligands 2and 3.
To achieve the FP-based ligand binding assay, the first
thing we need to do is to afford an excellent FP probe
with bright fluorescence and high binding affinity to re-
ceptors. In particular, the fluorescence intensity must be
strong enough in order to increase S/N ratio and measure
up to the microplate reader. Among masses of fluoro-
phores, fluorescein performs brightly as the signal emitter
in FP assay by virtue of its high quantum yield.25, 26 How-
ever, in our previous research on α1-AR, the reported fluo-
rescent quinazoline-fluorescein conjugate QFL (1，Figure
1), possessing nanomolar binding affinity to α1-ARs,
showed very low quantum yield (Table 1).21As a result, this
compound failed to be employed in quantitive screening
compound
3
higher affinity than compound
(QGGFL) with the longer linker showed
(QGFL).
2
A summary of spectral properties for all com-
pounds is shown in Table 1as well. Compared to the
reported compound 1 (QFL), the newly synthesized
fluorescent ligands
2 (QGFL) and 3 (QGGFL) dis-
played about 15 nm red-shift in wavelength. Signifi-
cantly, the major improvement in optical properties
among them was fluorescence quantum yield and the
quantum yield was positively associated with the
length of the linker. In brief, compound
3
(QGGFL)
2
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ACS Medicinal Chemistry Letters
with two GABAs as the linker displayed about 10-fold
quantum yield (27.2%) than compound (QFL) with-
out linker (2.6%), and compound (QGFL) with one
At present up to, we have gotten an excellent fluo-
rescent ligand (QGGFL) with improved fluores-
1
2
3
4
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7
8
1
3
2
cence intensity, the specific binding ability to α1-AR
and suitable fluorescent properties that were compat-
ible with the filters in our FP system. In order to iden-
tify whether the probe QGGFL may serve as an FP
probe or if it could be qualified for the FP-based lig-
and screening of α1-AR, polarization at 520 nm of
QGGFL with various concentrations of α1-AR proteins
was measured firstly using the 96-well or 384-well
no-binding flat bottom black plate (Corning, USA) in
classical FP assay. However, as shown in Figure S4Aa,
the polarization values seemed no correlation with
the amount of proteins. This set of irregular data in-
cubated that the measurement window was too nar-
row for FP assay. To amplify the polarization signal,
the ELISA strategy was surrogated (Figure S4B), in
which the enzyme or antigen was attached to the sol-
id-phase surface.23, 24 By adhering to stationary phase,
the constrained proteins could slow the spin of fluo-
rescent ligands down, and thus, the fluorescent lig-
and would produce the obvious polarization effect.
To achieve this strategy, 384-well flat bottom black
plate with high protein-binding force (Fluotrac 600,
Greiner) was employed in 1 h incubation at room
temperature. By contrast with the irregular data in
Figure S4Aa, three nice saturation curves in Figure
S4Ba showed that addition of 16 nM QGGFL into in-
creasing concentrations of three α1-ARproteins gave a
GABA linker showed 6-fold yield (17.1%).
Table 1. Characterizationof fluorescent ligands in
spectroscopy and binding affinity to α1-ARs.
Kia/nM
λex/ λem/
nm
Φ/
%
Compd
nm
α1A
1.3
4.7
α1B
α1D
9
Phentolamine
QFL21
2
3
Atropine
--
--
--
2.6
7.7 13.8
7.3 20.7
17.1 33.5 8.1 31.3
2.7 23.8
NA NA NA
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485
500
500
--
517
530
530 27.2 4.0
-- --
a Ki was calculated from IC50 using the Cheng-Prusoff
equation (Equation S3).
An essential requirement of a fluorescent ligand of
GPCR is to visualize the target protein. As an im-
proved fluorescent ligand, compound 3 (QGGFL)
shows the reasonable receptor affinity and appropri-
ate quantum yield, especially the latter. Next, we es-
tablished the transfected HEK293A cells transiently
with α1B-AR and determined the ability of QGGFL to
bind to such intact cells expressing α1-AR in culture.
In confocal imaging study, the α1B-AR transfected
HEK293A cells could be “dyed” by strong green fluo-
rescence when incubated with 300nM of compound
3
(QGGFL) for 20 min, and the strong fluorescence
would weaken while a co-incubation of QGGFL (300
nM) and an excess of prazosin (3 μM) was carried
out(Figure S3). Meanwhile, the untransfected
HEK293A cells were also employed as a negative con-
trol, in which very weak fluorescence was observed.
This negative result indicated that non-specific bind-
ing is absent between QGGFL and cell components.
The untransfected HEK293A cells also express some
β-ARs natively, so these imaging results also indicat-
ed that QGGFL showed no interactions with the
aminergic β-ARs. As is known, the α1B-AR subtype
mainly distributes on cytomembrane.28 In our confo-
cal imaging, most cells with highlighted membrane
by QGGFL came into view, indicative of a cell surface
localization for α1B-AR, which was highly consistent
with the conclusion from Piascik and declared that
saturable increase in FP. We also measured the Z
'
factor in both the traditional assay and the ELISA-like
assay (Figure S4c), which is a statistical benchmark to
judge whether the response in a particular assay is
large enough to warrant further attention.30, 31
The Z' factors calculated for our ELISA-based FP as-
say are 0.84, 0.77 and 0.70 for α1A-, α1B-, α1D-AR pro-
teins, respectively (Figure S4Bc), and in the tradition-
al FP assay, the Z' factors are -0.49, -0.64 and -0.56,
respectively (Figure S4Ac). A Z' factor greater than 0.5
means the assay is considered acceptable for a good
HTS, while that less than 0 confirms there is too
much overlap between the positive and negative con-
trols for the assay to be used. In addition, when treat-
ed with excess membrane proteins (0.4 mg/mL), the
FP value displays a dose-dependent enhancement,
which yields Kd value of 0.56 ± 0.14, 1.21 ± 0.49 and
0.64 ± 0.30 nM for α1A-, α1B- and α1D-AR, respectively
(Figure 2). The results above indicates that com-
the probe
3
(QGGFL) could recognize α1-
adrenoceptors specifically.28 In addition, to check the
specificity or selectivity of QGGFL, we measured its
activities to three kinases (BMX, KIT and ALK) be-
cause quinazoline may cause the inhibitory activity
against kinases.29 As shown in Figure S5, we found all
pound3 (QGGFL) is qualified to become a FP probe in
the evolutionary ELISA-based FP assay. The specific
interaction between QGGFL and α1-AR proteins
reached equilibrium in 60 min and was saturable.
Therefore, the FITC-derivative compound 3 (QGGFL)
is suitable for the measurement of binding affinity of
ligands.
the GI50 values were greater than 10
μM. Moreover,
QGGFL also showed low affinities to other adrenergic
receptors (α2- and β1-ARs). As a result, we can con-
clude that this fluorescent antagonist QGGFL with
quinazoline core has few off-target activities.
We further investigated whether this “ELISA-like”
FP strategy could be used in HTS forα1-AR antago-
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nists. Competitive binding experiments were carried plify the FP signal and increase the measurement
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out in the presence of 16 nM QGGFL and α1-AR mem-
brane proteins (625 ng of α1A-AR and α1B-AR, 1.25 μg of
α1D-AR, respectively, Table S1). As known competitive
antagonists, prazosin, doxazosin, and phentolamine
could reduce FP in a concentration-dependent man-
ner, which gave three anticipated sigmoidal response
window and S/N ratio. Based on the FP probe QGGFL
and the ELISA strategy, we established a safe and af-
fordable FP-based screening method for α1-ARs for
the first time, which could provide the convincing
data of binding affinity to three α1-AR subtypes of
known antagonists, such as prazosin, doxazosin, and
phentolamine, with the classical radioligand binding
assay. To the best of our knowledge, this research is
the first report that the ELISA strategy is applied in a
small-molecule probe-based FP assay. It is our expec-
tation that this strategy would find broad applica-
tions in the FP-based HTS for other target proteins to
help addressing their signal issue.
curves (Figure 2) and convincing IC50 and Ki values to
three α1-AR subtypes with the similar values acquired
from radioligand binding assay (Table S2). The differ-
ence of Ki values in the two assays may be caused by
the different systems: the unbound ligands in radi-
oligand assay were washed away while the FP values
were recorded in the homogeneous reaction system.
FP was almost enacted on the muscarinic receptor
antagonist atropine, which could serve as the nega-
tive group for atropine do not bind to α1-AR. Compar-
ing with the radioligand competitive binding assay,
the safer fluorescent ligands were employed and few-
er membrane proteins (>10 fold) were consumed in
the FP binding assay (Table S1). According to these
results, we also concluded that the new fluorescent
antagonist QGGFL could serve as a competitive an-
tagonist.
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ASSOCIATED CONTENT
Supporting Information
Full experimental procedures, analytical and spectral charac-
terization data of all compounds, and results of cell imaging
and FP assay. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Minyong
Li,
Tel./fax:
+86ꢀ531ꢀ8838ꢀ2076;
E-mail:
mli@sdu.edu.cn
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
ACKNOWLEDGMENT
The present work was supported by grants from the Qilu
Young Professorship of Shandong University, the Interna-
tional Postdoctoral Exchange Fellowship Program, the Fok
Ying Tong Education Foundation (No. 122036), the Program
for Changjiang Scholars and Innovative Research Team in
University (No. IRT13028), the Major Project of Science and
Technology of Shandong Province (No. 2015ZDJS04001), the
Figure 2. Saturation of increasing concentrations of
compound 3 (QGGFL) binding to α1A-AR (A), α1B-AR
(B) and α1D-AR (C) proteins (Final con. 0.4 mg/mL).
Competition curves of prazosin, doxazosin, phen-
tolamine and atropine to three α1-AR subtype, includ-
ing α1A-AR (D), α1B-AR (E) and α1D-AR (F) using the
established “ELISA-like” FP assay.
Shandong Key Research
& Development Project (No.
2015GSF118166) and the Fundamental Research Funds of
Shandong University (No. 2014JC008). Our cell imaging work
was performed at the Microscopy Characterization Facility,
Shandong University.
In conclusion, developing analytical methods to
identify compounds interacting with GPCRs or to
find new drugs targeting GPCRs is drawing more and
more attentions from scientists and researchers. Alt-
hough FP assay can portray important roles in the
HTS of the chemical library for GPCRs, it is restricted
in many cases due to the lack of FP probes, the nar-
row measurement window, and low S/N ratio. To
overcome these limitations in α1-ARs, we firstly dis-
covered and synthesized an ideal FP probe QGGFL,
ABBREVIATIONS
GPCRs,
G
protein-coupled receptors; HTS, high-
throughput screening; FP, fluorescence polarization; S/N,
signal-to-noise;α1-ARs, α1-adrenergic receptors; BPH, be-
nign prostatic hyperplasia; LUTS, lower urinary tract
symptoms; GABA, γ-aminobutyric acid; ELISA, enzyme
linked immunosorbent assay, EDCI, 1-Ethyl-3-(3-
compound 3, for α1-ARs, which has improved quan-
tum yield, high receptor affinity and the ability of
receptor imaging specifically in living cells. Moreover,
an “ELISA-like” strategy was designed, in which pro-
teins are confined to the solid phase, in order to am-
dimethylaminopropyl)carbodiimide
HOBT, 1-hydroxybenzotriazole; FITC, fluorescein isothio-
cyanate; TFA, trifluoroacetic acid; BMX, bone marrow X
hydrochloride;
4
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17 Nagatomo, T.; Tsuchihashi, H.; Sasaki, S.; Nakagawa, Y.;
kinase; ALK, anaplastic lymphoma kinase; KIT, v-kit Har-
1
2
3
4
5
6
7
8
Nakahara, H.; Imai, S. Displacement by α1ꢀadrenergic agonists
and antagonists of Hꢀprazosin bound to the α1ꢀadrenoceptors
of the dog aorta and the rat brain. Jpn. J. Pharmacol. 1985, 37,
181ꢀ187.
dy-Zuckerman 4 feline sarcoma viral oncogene homologue.
3
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As an important HTS method, fluorescence polarization assay is restricted in many cases due to the
lack of FP probes, the narrow measurement window, and low S/N ratio. For the case of α1ꢀARs, we
firstly designed an ideal FP probe QGGFL with improved quantum yield, high affinity and high speciꢀ
ficity to α1ꢀARs. Then an “ELISAꢀlike” strategy was adopted to amplify the FP signal and increase the
measurement window and S/N ratio. Finally, based on the FP probe and the ELISA strategy, we estabꢀ
lished a safe and affordable FPꢀbased screening method for α1ꢀARantagonists for the first time.
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