A novel GPER antagonist protects against the formation of estrogen-induced cholesterol gallstones in female mice

Article abstract of DOI:10.1194/jlr.RA119000592

Many clinical studies and epidemiological investigations have clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other estrogen therapies dramatically increase that risk. Further, anim

Full text of DOI:10.1194/jlr.RA119000592

A Novel GPER Antagonist Protects Against the Formation of Estrogen-Induced
Cholesterol Gallstones in Female Mice
Chelsea DeLeon,1 Helen H. Wang,2 Joseph Gunn,1 McKenna Wilhelm,1 Aidan Cole,1 Stacy
Arnett,3,# David Q.-H. Wang,2 Christopher K. Arnatt1
1Department of Chemistry, Saint Louis University, St. Louis, MO 63104, USA;
2Department of Medicine and Genetics, Division of Gastroenterology and Liver Diseases,
Marion Bessin Liver Research Center, Einstein-Mount Sinai Diabetes Research Center, Albert
Einstein College of Medicine, Bronx, NY 10461, USA; and
3Center for World Health and Medicine, Saint Louis University, St. Louis, MO 63104, USA
Address correspondence to:
Christopher K. Arnatt, Ph.D., Department of Chemistry, Saint Louis University, St. Louis, MO
63104, USA. Phone: (314) 977-8290, Fax: (314) 977-2521, Email: chris.arnatt@slu.edu
and
David Q.-H. Wang, M.D., Ph.D., Department of Medicine, Division of Hepatology, Marion
Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Phone: (718) 430-8865, Fax: (718) 430-8568, Email: david.wang@einsteinmed.org
#Current address: Center for Clinical Pharmacology, Washington University, St. Louis, MO
63110
1

Abbreviations: CSI, cholesterol saturation index; DMSO, dimethyl sulfoxide; ERE, estrogen
response elements; E2ꢀꢁꢂꢃȕ-estradiol; (5ꢀꢁHVWURJHQꢁUHFHSWRUꢄꢁ(5Įꢀꢁ(5ꢁVXEW\SHꢁĮꢄꢁ(5ȕꢀꢁ(5ꢁ
VXEW\SHꢁȕ; FBS, fetal bovine serum; GPER1, G protein-coupled estrogen receptor 1; GPR30, G
protein-coupled receptor 30; HTRF, homogeneous time-resolved fluorescence; OVX,
ovariectomized.
Keywords: bile; bile salts; cholesterol crystallization; Lith gene; mucin; G protein-coupled
estrogen receptor, GPER1, GPR30.
2

ABSTRACT
Many clinical studies and epidemiological investigations have clearly demonstrated that women
are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other
estrogen therapies dramatically increase that risk. Further animal studies revealed that estrogen
promotes cholesterol gallstone formation through the HVWURJHQꢁUHFHSWRUꢁĮꢁꢅ(5ĮꢆꢀꢁEXWꢁQRWꢁ(5ȕꢀꢁ
pathway. More importantly, some genetic and pathophysiological studies found that the G
protein-coupled estrogen receptor 1 (Gper1) is a new gallstone gene, Lith18, on chromosome 5
in mice DQGꢁSURGXFHVꢁDGGLWLRQDOꢁOLWKRJHQLFꢁDFWLRQVꢀꢁZRUNLQJꢁLQGHSHQGHQWO\ꢁRIꢁ(5ĮꢀꢁWRꢁmarkedly
increase cholelithogenesis in female mice. Based upon computational modeling of GPER, a
novel series of GPER-selective antagonists were designed, synthesized, and subsequently
assessed for their therapeutic effects via calcium mobilization, cyclic AMP, and (5ĮꢁDQGꢁ(5ȕꢁ
fluorescence polarization binding assays. From this series of compounds, one new compound,
CIMBA, exhibits superior antagonism and selectivity exclusively for GPER. Furthermore,
CIMBA UHGXFHVꢁWKHꢁIRUPDWLRQꢁRIꢁꢂꢃȕ-estradiol-induced gallstones in a dose-dependent manner
in ovariectomized mice fed a lithogenic diet for 8 weeks. At ꢇꢈꢁȝJꢉGD\ꢉNJꢁRIꢁCIMBA, no
gallstones are found in ovariectomized ERĮ (-/-) mice even WUHDWHGꢁZLWKꢁꢂꢃȕ-estradiol ꢅꢊꢁȝJꢉGD\ꢆꢁ
and fed the lithogenic diet for 8 weeks. In conclusion, CIMBA treatment protects against the
formation of estrogen-induced cholesterol gallstones by inhibiting the GPER signaling pathway
in female mice. This may provide a new effective therapy on cholesterol gallstone disease in
women.
3

INTRODUCTION
Prior to the identification of the G protein-coupled estrogen receptor 1 (GPER), also
known as the G protein-coupled receptor 30 (GPR30), the biological and physiological actions of
estrogens were considered to originate exclusively from the nuclear estrogen receptors (ER),
ERD and ERE (1-3). Upon activation of the classical ER, individual ER subunits dimerize and
bind to estrogen response elements (ERE) to directly regulate the expression of target genes (4-
7). While the ER is primarily responsible for the genomic signaling, rapid, non-genomic
signaling has been observed in response to estrogen. GPER was identified when there was the
absence of both ERD and ERE activation of mitogen-activated protein kinases 1 and 2 in
response to 17E-estradiol (E2) (8, 9). Since the identification of GPER as a novel ER, a variety of
pathways have been studied, including second messengers (e.g., Ca2+ and cAMP), tyrosine
kinase epidermal growth factor receptor, and protein/lipid kinases (e.g., protein kinase A, protein
kinase C, and Src family) (10-14). Despite the affinity of E2 for GPER, other estrogenic
compounds such as 17D-estradiol, estrone, and estriol show little affinity for the receptor (Figure
1) and other hormones such as progesterone, testosterone, and cortisol exhibit no significant
binding (15).
Several groups have identified GPER-selective agonists and antagonists (16-23). Using
virtual screening and molecular docking of E2-like compounds, Bologa and colleagues have
found a GPER-selective agonist, G-1, that exhibits a binding constant (Ki) of 11 nM with no
significant binding WRꢁ(5ĮꢁRUꢁ(5ȕꢁDWꢁFRQFHQWUDWLRQVꢁXSꢁWRꢁꢂꢁ—0 (Figure 1) (16). Subsequently,
two GPER-selective antagonists, G-15 and G-36, were identified based upon the same
dihydroquinoline scaffold (17, 18). Although G-15 possessed a relative strong binding constant
of 20 nM to GPER1, further studies revealed significant binding to the classical ER and
4

activation of ERE at concentrations above 100 nM (18). To address off-target binding and non-
selectivity, Dennis et al. made modifications to the dihydroquinoline scaffold. As a result, G-36
was discovered. In this compound, the addition of a bulky, lipophilic group led to decreased
binding to ER without significantly affecting activity at GPER (18). While decreased binding to
the classical ER was reduced at concentrations above 1 PM, the ERE activation was still weakly
present (18). These results show that the G-series GPER antagonists may be limited as GPER-
selective antagonists due to the presence of off-target effects. Nevertheless, commercial
availability of the G-series of ligands has made them valuable GPER chemical probes for
research.
It is well known that the annual medical cost for treating gallstone disease exceeded $6
billion in 2004 and even higher in 2019 in the USA (24). The burden of gallstone disease is
exacerbated by the fact that laparoscopic cholecystectomy remains the standard treatment for
symptomatic gallstones worldwide (25). In addition, clinical and epidemiological studies have
clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men,
and oral contraceptives or other estrogen therapies significantly increase that risk (26, 27).
Although (5ĮꢀꢁEXWꢁQRWꢁ(5ȕꢀ plays a key role in estrogen’s lithogenic effects (28), new evidence
has shown that GPER can SURGXFHꢁDGGLWLRQDOꢁOLWKRJHQLFꢁDFWLRQVꢀꢁZRUNLQJꢁLQGHSHQGHQWO\ꢁRIꢁ(5Į,
to promote gallstone formation in female mice (29, 30). More importantly, genetic analysis has
found that Gper1 is a new gallstone gene, Lith18, on chromosome 5 in mice (31). All these
studies strongly suggested that GPER could play a critical role in the formation of estrogen-
induced cholesterol gallstones (32).
To differentiate the GPER1-mediated effects from the (5Į-mediated events in estrogen-
induced cholesterol gallstones, we have created three knockout mouse lines: GPER1 (-/-ꢆꢀꢁ(5Įꢁꢅ-
5

/-), and GPER1(-/-ꢆꢉ(5Į(-/-) (29). Our results have shown that compared to ovariectomized
(OVX) wild-type mice, deletion of either ErĮ or Gper1 significantly reduced the prevalence of
estrogen-induced gallstones, but could not abolish it completely. Furthermore, no gallstones were
found in OVX GPER1(-/-ꢆꢉ(5Į(-/-) mice even treated with E2 at 6 μg/day and fed the lithogenic
diet for 8 weeks (29). These results clearly indicated that GPER plays an independent role from
(5Įꢁin the pathogenesis of cholesterol gallstone disease. Therefore, it is imperative to develop a
new, potent GPER-selective antagonist that could prevent estrogen-induced gallstones for
women.
Drug discovery for GPER ligands has been reliant upon the identification of individual
ligands via in silico molecular screening, and only several synthetic GPER modulating molecules
have been reported in the literature (16-23). However, there has been little effort from these
studies to understand the important binding interactions of ligands within the binding pocket of
GPER to aid in optimization. In this study, homology modeling was utilized to design and
synthesize a series of GPER-selective antagonists (33, 34). Using calcium mobilization and
cAMP assays, we have established the first series of compounds for exploring the importance of
binding interactions for GPER-selective antagonists. Additionally, our animal studies have
revealed that the lead compound discovered from this study can prevent the formation of E2-
induced cholesterol gallstones in OVX mice by inhibiting the GPER signaling pathway and may
provide an additional effective therapeutic option for gallstone disease in women and patients
exposed to high levels of estrogen.
6

MATERIALS AND METHODS
Chemistry
The compounds were synthesized according to the procedures shown in Figure 2.
Characteristics of the compounds were analyzed utilizing nuclear magnetic resonance (NMR)
and high-resolution mass spectroscopy. 1H and 13C NMR spectra were performed in chloroform-
D from Cambridge Isotope Laboratories (Andover, MA) on a 400 MHz Bruker AVANCE III.
Data was analyzed utilizing TopSpin 3.2 software. Experiments with high-resolution mass
spectroscopy were carried out using a MaXis plus electrospray-quadrupole time-of-flight mass
spectrometer (Bruker, Billerica, MA). Ionization was done by electrospray with the samples
infused into the instrument in ~5 μM acetonitrile/water/formic acid (50/50/0.1%) solutions at a
flow rate of 3 μl/minute. Nitrogen was used as nebulizing, drying, and collision gas. HPLC-
grade acetonitrile and water were purchased from Sigma-Aldrich (St. Louis, MO). CIMBA
exhibited a purity of greater than 95% as did all synthesized compounds. High-resolution mass
spectroscopy for CIMBA (C23H32NO+) was calculated as m/z = 338.2478 and found to be m/z =
338.2471.
Cell line and culture
The human promyelocytic leukemia (HL-60) cells, purchased from the American Type
Culture Collection (Manassas, VA), were cultivated in phenol red-free RPMI 1640 media
containing 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin, and 1% GlutaMax.
Three days prior to assays, the HL-60 cells were switched into the media containing 10%
charcoal-stripped FBS. Cells were passaged every 3 days and maintained at a cell concentration
below 1x106 to prevent differentiation. The cells were incubated at 37°C under 5% CO2.
7

Evaluation of GPER-associated calcium mobilization in HL-60 cells
The HL-60 cells were grown in 10% charcoal-stripped FBS, phenol red-free RPMI 72
hours before assays were performed. The cells were centrifuged and counted with a
hemocytometer. The assay required 100,000 cells per well, for a total number of cells on a 96-
well plate assay of about 1x107. HL-60 cells (1x107) were incubated in 50:1 HBSS/HEPES
containing 5 PM Indo-1 AM (Thermo Fisher Scientific, Waltham, MA) and 0.05% pluronic acid
for 0.5 hours at room temperature. Cells were spun down and washed with 50:1 HBSS/HEPES
and resuspended in media. Resuspension was placed on ice for no longer than 5 minutes. Cells
were loaded into the plate at 100,000 cells/well. For agonist, cells were immediately incubated
for 15 minutes at 37°C. Following the 15 minutes of incubation, FlexStation 3 Multimode Plate
Reader (Molecular Devices, Sunnyvale, CA) added the appropriate amount of agonist and was
read for 150 seconds at 37°C. For antagonists, once the cells were seeded, the antagonist was
added and incubated for 15 minutes at 37qC. After 15 minutes of equilibration, the
experimentally determined EC80 value of G-1 (3 PM) was added by the FlexStation and read for
150 secs at 37°C. Calcium mobilization was determined ratiometrically using Oex 350 nm and
O
em 405/490 nm, respectively.
Evaluation of cAMP response in HL-60 cells
A homogeneous time-resolved fluorescence (HTRF) components for cAMP was
purchased from CisBio (Bedford, MA). HL-60 cells were grown in 10% charcoal-stripped FBS,
phenol red-free RPMI media for 72 hours prior to assays. Cells were centrifuged and counted
with a hemocytometer. The total number of cells needed to complete the assay was determined
8

based upon 8,000 cells/well. The determined number of cells was diluted in 5:1 (5x) stimulation
buffer containing 500 PM of 3-isobutyl-1-methylxanthine (Sigma Aldrich, St. Louis, MO). To an
HTRF 96-well low volume white plate from CisBio (cat:62AMgPEB), 5 Pl of cold cell
suspension was added to each well, followed by 4 Pl of (2.5x) of agonist or stimulation buffer
(negative control and non-stimulated cells). Cells were covered with a clear plastic film and
incubated at 37°C for 15 minutes in an incubator. After 15 minutes, 1 Pl of 10 PM (10x)
forskolin was added to each well. The plate was once again sealed and incubated at 37°C for 15
minutes. Subsequently, 5 Pl of cAMP-d2 (acceptor) was added to all wells, including controls.
Conversely, 5 Pl of monoclonal anti-cAMP Eu3+ cryptate (donor) was added to only the wells
with a test compound and non-stimulated cells. Following the addition, the plate was sealed,
covered with aluminum foil, and incubated at room temperature for 30 minutes. After the allotted
time cells were read using Flexstation3 with Oex 314 nm and dual-emission wavelengths for the
acceptor and donor emission signals (Oem donor = 620 nm and Oem acceptor = 655 nm). A ratio of the
acceptor to the donor was calculated and utilized in the determination of EC50 value of G-1. For
an antagonism platform, the procedure was slightly altered. The EC80 value of G-1 was re-
determined based upon cAMP agonism results. To the plate, 2 Pl of (5x) antagonist were added,
followed by the addition of 5 Pl of the cold cell suspension to all wells, with top and bottom
wells being designated as G-1 control and IRUVNROLQꢁFRQWUROꢁꢅꢈꢋꢁȝ0ꢆꢀꢁrespectively. Cells and
antagonists were incubated for 15 minutes at 37°C. Then, 2 Pl of the (5x) EC80 of G-1 was added
to wells with the antagonist. For the G-1 control, 2 Pl of 5:1 stimulation buffer and 2 μl of the
(5x) EC80 G-1 were added. For the forskolin control, 4 Pl of 5:1 stimulation buffer and 1 PL of
(10x) forskolin were combined. For all other wells, following the addition of antagonists, cells,
and agonists, 1 Pl of (10x) forskolin were add for a final concentration of 1 PM. After the
9

addition of forskolin, cells were incubated at 37°C for 5 minutes before dyes and lysis buffer
were added. Cells were allowed to equilibrate for 30 minutes at room temperature before being
read at Oex 314 nm dual-emission wavelengths for the acceptor and donor emission signals (Oem
donor = 620 nm and Oem acceptor 655 = nm). A ratio of the acceptor to donor was calculated and
utilized to determine the IC50 values of tested antagonists. HTRF values were converted to %
cAMP using the manufacturer’s suggested protocol.
ERD and ERE fluorescence polarization assay
The ERD and ERE PolarScreen Competitor Assays were purchased from Invitrogen
(Carlsbad, CA). The concentrations of the ERD and ERE enzymes varied between the two
assays. A 4x sample of ERD was prepared for a final concentration of 75 nM. Conversely, a 4x
sample of ERE was prepared for a final concentration of 23 nM within the assay. For both ERD
and ERE, a 4x aliquot of Fluoromone was prepared for a final assay concentration of 4.5 nM.
Despite the differences in concentration of ERD and ERE, the binding assay protocol for the two
different receptors remained the same. Drug dilutions were prepared for a 2x dilution in the ER-
specific buffer that was provided within the assay kit. In addition to the drug dilutions, a 2x
aliquoWꢁRIꢁ)OXRURPRQHꢁZDVꢁSUHSDUHGꢁE\ꢁDGGLQJꢁꢂꢋꢁȝOꢁRIꢁꢌ[ꢁZLWKꢁꢂꢋꢁȝOꢁRIꢁ(5-specific buffer. A 2x
dilution of enzyme and fluorophore was achieved by adding equal portions of each to each other.
To a black 384-plate well plate, 10 ȝl of the compound was added to thHꢁZHOOVꢀꢁIROORZHGꢁE\ꢁꢂꢋꢁȝOꢁ
of the 2x mixture of enzyme and fluoromone. Two controls were included in the plate design. In
one control, enzyme and compound were omitted so that only ER-specific buffer and fluoromone
remained. A separate control contained the enzyme and fluoromone control. Once all
components were added, the plate was covered with a film and aluminum foil. The plate was
10

incubated for 2 hours at room temperature before being read with Oex 485 nm and Oem 535 nm,
respectively.
Animal and diet
Although it has been found that inbred AKR/J mice are a gallstone-resistant strain, they
are still susceptible to E2-induced cholesterol gallstone formation (28). Although AKR/J mice
have an intact expression of the Gper1, (UĮ, and (Uȕ genes, mRNA levels of (Uȕ in the liver are
almost undetectable under normal physiological conditions. Hepatic expression of (Uȕ is 50-fold
lower than that of (UĮ even under the stimulation of E2. In addition, we have established
EUHHGLQJꢁFRORQLHVꢁRIꢁ(5Įꢁꢅꢍꢉ-) mice on an AKR/J genetic background in-KRXVHꢎꢁ(5Įꢁꢅꢍꢉ-)
heterozygotes were also fertile and showed no obvious phenotypes in association with the
disrupted (UĮ JHQRW\SHꢎꢁ$ꢁFURVVꢁEHWZHHQꢁKHWHUR]\JRXVꢁ(5Įꢁꢅꢍꢉ-) mice produced the live birth of
QRUPDOꢁOLWWHUꢁVL]HVꢁRIꢁKRPR]\JRXVꢁ(5Į (-/-) mice. Mice were maintained in a temperature-
controlled room (22±1°C) with a 12-hour day cycle (lights on 0600 h - 1800 h) and were
provided free access to water and normal mouse chow containing trace cholesterol (<0.02%)
(Lab Rodent Diet, St. Louis, MO). To exclude possible interindividual differences in endogenous
estrogen concentrations, all female mice, at the age of 4 weeks, were ovariectomized (OVX). At
8 weeks of age, these mice were implanted subcutaneously with pellets (Innovative Research of
$PHULFDꢀꢁ6DUDVRWDꢀꢁ)/ꢆꢁUHOHDVLQJꢁꢂꢃȕ-estradiol (E2) at 6 μg/day for 8 weeks. As reported (28),
plasma estradiol concentrations were significantly increased to 81±21 pg/ml in OVX mice
treated with E2 at 6 μg/day compared to wild-type mice (26±11 pg/ml) receiving neither surgery
nor E2 treatment. To study the role of the GPER-selective antagonist CIMBA in the prevention
of E2-induced cholesterol gallstones, the mice, at 8 weeks old, were injected intraperitoneally
11

with CIMBA DWꢁꢋꢀꢁꢂꢊꢀꢁRUꢁꢇꢈꢁȝJꢉGD\ꢉNJꢀꢁDVꢁZHOOꢁDVꢁIHGꢁWKHꢁOLWKRJHQLFꢁGLet containing 1%
cholesterol, 15% butter fat, and 0.5% cholic acid for 8 weeks. All procedures were in accordance
with current NIH guidelines and were approved by the Institutional Animal Care and Use
Committees of Albert Einstein College of Medicine (Bronx, NY) and Saint Louis University (St.
Louis, MO).
Gallstone studies
After 8 weeks of feeding the lithogenic diet, mice were fasted overnight but had free
access to water. After anesthetization with pentobarbital, cholecystectomy was performed during
laparotomy. The entire gallbladder bile was studied by polarizing light microscopy without a
cover slip and then with a cover slip using phase contrast optics for the presence of mucin gels,
liquid crystals, cholesterol monohydrate crystals, sandy stones, and real gallstones according to
previously established criteria (35). The images of cholesterol monohydrate crystals and
gallstones were analyzed by a Carl Zeiss Imaging System with an AxioVision Rel 4.6 software
(Carl Zeiss Microimaging GmbH, Göttingen, Germany). Under the circumstances, gallstones
exhibit rounded contours and black centers from light scattering/absorption, and gallstones were
counted at x100 or x200 magnification. The prevalence rate of gallstones is determined by the
number of mice per group with evident stones. After microscopic studies, gallbladder bile was
collected, frozen and stored at -20°C for lipid studies.
Biliary lipid analysis
Cholesterol, phospholipid, and bile salt concentrations in pooled gallbladder bile (n=10
mice per group) were determined according to previously published methods (36). Cholesterol
12

saturation index (CSI) of pooled gallbladder bile was calculated from critical tables (37) that was
established for taurocholate, the predominant bile salts in the bile of mice on the lithogenic diet
(38). Relative lipid composition of pooled gallbladder bile was plotted on condensed phase
diagrams. For phase analysis, the phase limits of the micellar zones and the crystallization
pathways were extrapolated from model bile systems developed for taurocholate at 37°C and at a
total lipid concentration of ~10 g/dl (39).
Quantitative real-time PCR assay
Total RNA was extracted from fresh liver tissues of mice (n=4 per group) according to
our published methods (29). Primer Express Software (Applied Biosystems, Foster City, CA)
was used to design the primers based on sequence data available from GenBank. Quantitative
real-time PCR assays of the hepatic Gper1, (UĮ, and (Uȕ genes were performed in triplicate
according to previously established methods (29). The sequences of the primers for these genes
have been reported (29). Relative mRNA levels were calculated using the threshold cycle of an
unknown sample against a standard curve with known copy numbers. To obtain a normalized
target value, the target amount was divided by the endogenous reference amount of mouse ȕ-
Actin as internal control.
Liver compound accumulation study
After liver samples were collected from mouse gallstone experiments, they were
immediately frozen and stored at -80°C until analysis. Liver tissues were weighed and stored in
Eppendorf tubes. Corresponding liver tissues were used for preparing standard curves in a tissue
matrix. To each tissue sample or standard, the appropriate volume of cold PBS was added to
13

achieve a tissue concentration of 200 mg/ml. Stainless steel beads (2-3 mm) were added to the
tubes that were then placed in a bead beater for 1-2 minutes. Tissue samples and standards (100
μl) were then added to a 96-well plate. Standards (100 μl) were also added to a separate 96-well
SODWHꢎꢁ7RꢁHDFKꢁWLVVXHꢁZHOOꢀꢁꢌꢋꢋꢁȝOꢁRIꢁFROGꢁDFHWRQLWULOHꢁFRQWDLQLQJꢁꢂꢋꢋꢁQJꢉPOꢁLQWHUQDOꢁVWDQGDUGꢁ
enalapril was added. Plates were vortexed for 5 minutes at 4°C, then centrifuged at 3,200 rpm at
4°C for 10 minutes. The supernatant (400 μl) was transferred to a second 96-well plate, and
evaporated to dryness under nitrogen, as well as reconstituted with 100 μl of 0.1% formic acid in
water/acetonitrile (9:1, vol/vol), and vortexed for 5 minutes. After that, the samples were briefly
centrifuged and submitted for LC/MS analysis. Compound concentrations were determined on a
Sciex API-4000 LC/MS system in positive electrospray mode. Analyses were eluted from an
Amour C18 reverse phase column (2.1x30 mm, 5 μm) using a 0.1% formic acid (aqueous) to
100% acetonitrile gradient mobile phase system at a flow rate of 0.35 ml/minute. Peak areas for
the specific mass transitions were integrated using Analyst 1.5.1 software. Peak area ratios of
analyses to the internal standard were plotted against concentration with a 1/x-weighted linear
regression to determine compound concentration.
Statistical methods
All data are expressed as means ± SD. Statistically significant differences among groups
of E2-treated OVX mice fed the lithogenic diet and administrated with various doses of CIMBA
were assessed by Student’s t-test, or Chi-square tests, or by Mann-Whitney U-test. The 50%
excitatory (EC50) and 50% inhibitory concentrations (IC50) were determined by nonlinear
regression analysis using GraphPad Prism, version 5.02 (GraphPad, La Jolla, CA). All ANOVA
14

analyses were also run using the same statistical software with Tukey’s post hoc analysis for
multiple comparisons. Statistical significance was defined as a two-tailed probability of <0.05.
15

RESULTS
Design and synthesis of the GPER-selective antagonists, as well as inhibition of calcium
mobilization and cAMP Gi/o signaling by the GPER antagonists 5–25
Previous docking studies revealed the potential importance of hydrogen bonding to
N3107.40 (Ballesteros-Weinstein numbering) and E2756.52ꢀꢁʌ-ʌꢁVWDFNLQJꢁZLWKꢁ)ꢈꢋꢏꢁꢅH[WUDFHOOXODUꢁ
loop 2, EL2) and H3077.37, and various hydrophobic interactions with an extensive hydrophobic
pocket (33). For this work, a scaffold was designed, as shown in Figure 2A (34). The synthetic
route that was utilized in the synthesis of the GPER-selective antagonists is summarized in
Figure 2B.
To assess the therapeutic efficacy of the synthesized GPER-selective antagonists, the
human promyelocytic leukemia (HL-60) cell line was utilized because it displays high
expression of GPER1 in addition to both ERD and ERE. Thus, using the HL-60 cells, off-target
signaling was analyzed with a fluorescence polarization assay that will be discussed later. Prior
to running antagonism assays, compounds 5–25 were tested for GPER agonism in a similar
fashion as G-1, and no compounds VKRZHGꢁDJRQLVPꢁEHORZꢁꢂꢋꢁȝ0. Within the antagonism assay,
nearly every compound exhibited an IC50 value in the high nanomolar range (Table 1). For
comparison purposes, we determined that within the assay, the IC50 values of G-15 and G-36
were 1,550 ± 170 nM and 1,350 ± 220 nM, respectively. The previously reported calcium
mobilization IC50 values of G-15 and G-36 varied from the determined values within this study.
For G-15, the reported IC50 value was 185 nM, whereas the G-36 exhibited slightly greater
potency with an IC50 value of 165 nM (18). Variation in the reported and observed IC50 values
for G-15 and G-36 may exist due to differences in antagonism assay methodology between the
study that reported these values and our study. For antagonism assays, Dennis et al. utilized 200
16

nM of G-1 to antagonize against G-15 and G-36 because there was a similar amount of calcium
mobilization in SKBr3 cells for both E2 and G-1 at this concentration (18). Standard
methodology suggests that the standard method for performing an antagonism assay requires the
EC80/90 value of the agonist to create the necessary signal window for detecting inhibitory
response (40, 41). While the EC50 value of G-1 has been reported in the literature, based upon
this methodology it is unclear whether 200 nM truly represents the EC50/80 value of G-1. In the
HL-60 cell line, the EC80 value was determined (data not shown) in a dose-dependent manner
DQGꢁH[LVWHGꢁDWꢁDSSUR[LPDWHO\ꢁꢇꢁȝ0ꢎꢁ8VLQJꢁꢇꢁȝ0ꢁDVꢁWKHꢁ(&ꢏꢋꢁYDOXHꢁRIꢁ*-1, there was an
approximate 10-fold magnitude difference in the IC50 values obtained in this study as compared
to the literature for selective GPER antagonists, G-15 and G-36. This observed difference was
approximately the same fold magnitude between the different values of G-1 (200 nM in the
previous study (18) YVꢎꢁꢇꢁȝM in this study) used to agonize the cells in the individual studies.
Differences amongst the determined EC50 values may also exist due to the difference in
expression levels of endogenous GPER between the cell lines.
The results from the calcium mobilization data revealed several key interactions of
antagonists with GPER. In this series of compounds (5–25), the R1 substitution greatly impacted
GPER antagonism. Larger hydrophobic groups (isopropyl and tert-butyl) were well tolerated at
R1 position, whereas the methyl-substituted compounds (12–18) exhibited the lowest antagonism
activity. For the R1 substitution, isopropyl derivatives (5–11) had lower IC50 values than the tert-
butyl derivatives (19–25). Overall, this suggests that small molecules may be limited in size due
to restriction within the GPER binding pocket. Changes in activity observed by various R2
substitutions were less straightforward. While mono-substitution appears to be favored over 3-4-
disubstitution (11, 18, and 25), there was an insufficient number of compounds to verify whether
17

the 3- or 4-position confers favorable binding. The larger R2 substituents such as naphthyl or
biphenyl (5, 6, 12, 13, 19, and 20) had mixed tolerances based upon the R1 substitution.
Differences in these observations may exist due to the size of the GPER binding pocket or the
existence of different binding modes. The 4-Cl derivatives (10, 17, and 24) were unaffected by
the R1 substitution, whereas the other small substituents were all affected by the R1 substitution.
The two compounds with the lowest IC50 values, 8 (R1 = isopropyl, R2 = 4-OMe) and 21 (R1 =
tert-butyl, R2 = 4-CH3), had different substitution patterns and illustrated the potential for further
optimization in future studies. These results were further verified in a secondary HTRF cAMP
assay (Figure 3A). In this assay, HTRF was inversely proportional to cellular levels of cAMP.
Accordingly, it was observed that G-1 increased HTRF signal, which indicated a decrease in the
level of cAMP. Blocking GPER with an antagonist exhibited a decrease in HTFR signal, which
corresponded to an increase in cAMP levels. Presented data shows % cAMP, which was
calculated using the manufacturer’s suggested protocol. Cellular response to G-1 was reversed
with treatment of pertussis-toxin (1 ȝM), suggesting that G-1 signals through the Gi/o pathway
(Figure 3A). Results with G-36, 8, and 21 show that there was a return to basal levels of cAMP
by blocking the activation of the receptor with the experimentally determined cAMP EC80 value
of GPER activation with agonist, G-1 (EC80= 3 ȝM) (Figure 3B).
Selectivity of 8 and 21 for GPER over the classical ER
Due to the existence of multiple targets for estrogenic compounds, the selectivity of 8 and
21 for GPER over the classical ER was established. Unlike E2, there was no appreciable binding
for ERD observed by G-1, G-36, or 8 at any tested concentrations (Figure 4A). At the highest
(10 ȝM) concentration evaluated, 21 showed a low level of binding that was significantly
18

different from G-1, G-36, and 8. For this reason, 21 may be limited in effectiveness as a GPER
antagonist due to the potential of off-WDUJHWꢁHIIHFWVꢎꢁ6WXGLHVꢁRIꢁ(5ȕꢁELQGLQJꢁUHYHDOHGꢁWKDWꢁDWꢁKLJKꢁ
ꢅꢂꢋꢁȝ0ꢆꢁFRQFHQWUDWLRQVꢀꢁRQO\ꢁ*-1 and G-36 exhibited binding (Figure 4B). In contrast to the G-
series, both 8 and 21 VKRZHGꢁDꢁODFNꢁRIꢁELQGLQJꢁDWꢁWKHꢁꢂꢋꢁȝ0ꢁFRQFHQWUDWLRQꢎꢁ2YHUDOOꢀꢁELQGLQJꢁ
studies revealed that both 8 and 21 GRꢁQRWꢁH[KLELWꢁVLJQLILFDQWꢁELQGLQJꢁWRꢁHLWKHUꢁ(5ĮꢁRUꢁ(5ȕꢁDWꢁ
concentrations belRZꢁꢂꢋꢁȝ0ꢎꢁ6LQFHꢁ8 displayed greater selectivity at high concentrations
compared to 21, we have established 8 as the lead compound within this series. Henceforth, the
compound 8 (2-Cyclohexyl-4-Isopropyl-N-(4-MethoxyBenzyl)Aniline) is referred to as
CIMBA.
Prevention of estrogen-induced gallstones by CIMBA in OVX mice
Although inbred AKR/J mice have been found to be a gallstone-resistant strain, they are
still susceptible to the formation of E2-induced cholesterol gallstones (28). Notably, the inbred
AKR/J strain expresses Gper1, ErĮ, and Erȕ in the liver. To further explore whether GPER-
selective antagonists play a key role in preventing the formation of estrogen-induced gallstones,
CIMBA was first studied in OVX AKR/J mice fed a lithogenic diet and treated with exogenous
E2 DWꢁꢊꢁȝJꢉGD\ꢁIRUꢁꢏꢁZHHNV. Figure 5A shows that without treatment of CIMBA (i.e., at 0
ȝJꢉGD\ꢉNJꢆꢀꢁ100% of E2-treated OVX mice developed gallstones in response to being fed the
lithogenic diet for 8 weeks. However, gallstone prevalence was significantly reduced from 80%
to 40% in E2-treated OVX mice receiving CIMBA from 16 to 32 ȝg/day/kg for 8 weeks. Figure
5B displays representative photomicrographs of amorphous mucin gel, liquid crystals,
cholesterol monohydrate crystals, and gallstones in these mice, as observed by phase contrast
and polarizing light microscopy. As shown in Table 2, the highest mole percent cholesterol and
19

cholesterol saturation index (CSI) value in pooled gallbladder bile were found in E2-treated OVX
mice receiving no CIMBA. In contrast, the mole percent of cholesterol in gallbladder bile was
gradually reduced with an increase in doses of CIMBA. Thus, CSI values of pooled gallbladder
bile were dramatically decreased from 1.61 to 1.23 by CIMBA (Table 2), which is consistent
with a dose-dependent reduction in gallstone prevalence in E2-treated OVX mice receiving
various doses of CIMBA (Figure 5A). Figure 5C shows that without CIMBA treatment, the
relative lipid composition of pooled gallbladder bile from E2-treated OVX mice fed the
lithogenic diet for 8 weeks is located in the central three-phase area denoted Region C on a
taurocholate-rich bile phase diagram, in which the bile is composed mainly of solid cholesterol
monohydrate crystals, liquid crystals, and saturated micelles (39). With an increase in doses of
CIMBA, the relative lipid composition of pooled gallbladder bile progressively shifts downward
and to the left of the phase diagram. These alterations are caused by a dramatic reduction in
cholesterol content, a relative decrease in phospholipid content, and a relative increase in bile salt
content (Table 2).
In addition, utilizing pooled liver tissues harvested from the mice used in the above
gallstone studies, we found that CIMBA was absorbed into the liver at both doses as determined
by HPLC/MS. When the doses of CIMBA WUHDWPHQWꢁZHUHꢁDXJPHQWHGꢁIURPꢁꢂꢊꢁȝJꢉGD\ꢉNJꢁWRꢁꢇꢈꢁ
ȝg/day/kg for 8 weeks, the concentrations of CIMBA in the liver were increased from 5 ng/g
liver tissue to 25 ng/g liver tissue in OVX mice. Taken together, these results are consistent with
a dose-dependent reduction in gallstone prevalence in E2-treated OVX mice receiving CIMBA
from 0 to 32 ȝg/day/kg (Figure 5A).
Figure 5D exhibits the effect of E2 and CIMBA on the expression of Gper1, ErĮ, and
Erȕ in the liver. Compared to control OVX mice receiving neither E2 nor CIMBA, hepatic
20

expression of Gper1 was significantly increased in OVX mice treated with E2 DWꢁꢊꢁȝJꢉGD\.
However, expression of Gper1 was significantly reduced by CIMBA in a dose-dependent
manner. Of note, expression of ErĮ was significantly increased in three groups of OVX mice
treated with E2, regardless of whether mice received varying doses of CIMBA. As expected,
expression of Erȕ was slightly increased in all mice because its expression was approximately
50-fold lower compared to ErĮ in the mouse liver (28).
To investigate whether CIMBA protects against E2-induced gallstone formation through
WKHꢁ*3(5ꢁSDWKZD\ꢀꢁZHꢁVWXGLHGꢁ29;ꢁ(5Įꢁꢅ-/-) mice fed the lithogenic diet and treated with
exogenous E2 DWꢁꢊꢁȝJꢉGD\ꢁIRUꢁꢏꢁZHHNVꢎꢁ&RQVLVWHQWꢁZLWKꢁRXUꢁSXEOLVKHGꢁUHVXOWV (30), at 8 weeks on
the lithogenic diet, 30% of 29;ꢁ(5Įꢁꢅ-/-) mice receiving no CIMBA ꢅLꢎHꢎꢀꢁꢋꢁȝJꢉGD\ꢉNJꢆ suffered
from E2-induced gallstones (Figure 6A). However, under 8-week treatment of CIMBA at 32
ȝJꢉGD\ꢉNJꢀꢁQRꢁJDOOVWRQHVꢁZHUHꢁGHWHFWHGꢁLQꢁ29;ꢁ(5Įꢁꢅ-/-) mice, and only 40% of these mice
formed mucin gel, liquid crystals, and cholesterol monohydrate crystals (Figure 6B). As shown
in Table 2, the CSI value of pooled gallbladder bile was 0.84 LQꢁ29;ꢁ(5Įꢁꢅ-/-) mice treated
with CIMBA DWꢁꢇꢈꢁȝJꢉGD\ꢉNJ was markedly lower compared to that (CSI = 1.28ꢆꢁLQꢁ29;ꢁ(5Įꢁꢅ-
/-) mice receiving no CIMBA. These results indicate that pooled gallbladder bile is unsaturated
with cholesterol after 8-week treatment of CIMBA LQꢁ29;ꢁ(5Įꢁꢅ-/-) mice.
21

DISCUSSION
Despite the implication of estrogen and GPER in a variety of health and disease states,
there has been limited clinical success with currently available GPER ligands. Although the G-
series (G-1, G-15, and G-36) has become the standard for studies pertaining to GPER, the series
may be limited by off-target effects and weak solubility in water or oil. A particular disadvantage
of the G-series is the presence of ERE activation at high concentrations (18). To improve the
solubility and address promiscuous binding of the G-series, we have rationally and successfully
synthesized a new series of the GPER-selective antagonists that provide the first evidence for
key binding interactions within the binding pocket of GPER.
Previously, our group explored binding interactions in a series of indole-thiazole
derivatives that exhibited agonism at GPER (42). Molecular homology modeling found that the
indole-thiazole derivatives exhibited S-S stacking similar to G-1 of the aromatic groups with
F20645.49 and F20845.51 within the binding pocket of GPER (33, 34, 42). These results suggested
that the tetrahydroquinoline is not required for GPER activity. Prior modifications of the G-series
focused on site-selective alteration on the tetrahydroquinoline structure, but have not focused on
additional substitution patterns. To explore the binding modes and interactions that are important
for antagonism, the indole-thiazole scaffold has been revised. In the revised scaffold, the amide
bond was replaced with an amine, and the heteroatoms of the indole-thiazole were changed to
substituted benzene ring systems. We hypothesized that removal of the heteroatoms in the
indole-thiazole would alter the pharmacological activity of the derivatives from agonists to
antagonists. The structure of the G-series is similar in rigidity to estrogen and other steroids,
which may contribute to weak solubility. Additionally, the lack of flexibility may reduce the
22

ability of GPER ligands to effectively probe the binding pocket. To address the concerns of the
solubility and probing ability, the scaffold included more rotatable bonds than the G-series.
Using the hypothesized scaffold, we developed a simplistic route to synthesize the initial
set of compounds (5–25) with varying substituents. The results of calcium mobilization studies
show that the majority of the compounds in the series have the ability to antagonize the EC80 of
the known GPER-selective agonist, G-1. Among these new series of compounds, CIMBA (8)
and 21 display superior antagonism of the G-1 signaling activity in the HL-60 cell line. These
two molecules have different substitutions at both the R1 and R2 positions of our scaffold,
CIMBA (R1 = isopropyl and R2 = 4-OMe) and 21 (R1 = tert-butyl and R2 = 4-CH3). At the R1
substitution, it appears that GPER prefers bulkier hydrophobic groups, and varying the bulkiness
of that groups influences the substitution pattern that is tolerated at the R2 substitution. At the R2
position, smaller electron donating groups are favored when R1 is either an isopropyl or tert-
butyl group. The electron donating nature of these two groups most likely contributes to the
electron-rich aromatic ring, which influences either aryl–aryl or aryl–cation interactions (43).
Trends in efficacy and potency were validated by evaluating cAMP. Overall, the findings from
the current studies provide some critical insights into the binding interactions that can be utilized
in the development of new ligands for modulating the GPER activity.
The selectivity studies show that CIMBA and 21 VSHFLILFDOO\ꢁELQGꢁ*3(5ꢁRYHUꢁ(5ĮꢁRUꢁ
(5ȕꢁEHORZꢁꢂꢋ PM. While CIMBA GRHVꢁQRWꢁELQGꢁWRꢁHLWKHUꢁ(5ĮꢁRUꢁ(5ȕꢀꢁ21 displays binding to
(5ĮꢁDWꢁꢂꢋꢁPM. These results are not surprising because the molecular modeling studies on GPER
and the classical ER from our group and others are in agreement that estrogenic compounds bind
within a similar binding pocket as each other (33, 34, 44-47). Due to the proposed similarity
between the binding pockets for these three receptors, it is not surprising to observe off-target
23

binding by some GPER ligands at high concentrations (16-18). In our binding studies, we found
that G-36 exhibits DQꢁLQFUHDVLQJꢁWUHQGꢁLQꢁELQGLQJꢁDWꢁ(5Į. This is the first study to suggest
potential off-target effects of G-36.
The results from this study show several key advantages of CIMBA over the G-series
antagonists, G-15 and G-36. One advantage of CIMBA is the removal of the tetrahydroquinoline
moiety. This could reduce potential off-target binding since these moieties are pan-assay
interference compounds which generally lead to off-target effect, confusing structure-activity
relationship of ligands, and poor downstream data (48, 49). Moreover, removing the
tetrahydroquinoline moiety allows for less molecular rigidity and a greater ability to probe the
GPER binding pocket. An additional advantage of CIMBA over G-15 and G-36 is that at high
concentrations, CIMBA does not bind to either (5ĮꢁRUꢁ(5ȕ, thereby preventing induction of any
ER-dependent ERE activation. Compared to the G-series antagonists, CIMBA also appears to be
a more potent inhibitor of the calcium release induced by G-1. The increase in potency may be
attributed to the increase in flexibility and the ability to probe the binding pocket of the receptor.
Furthermore, CIMBA does not contain chiral centers that are present in G-1. The lack of chiral
centers in CIMBA prevents a racemic mixture of both active and inactive stereoisomers,
allowing for ease of synthesis and more accurate determination of potency. Lastly, CIMBA is
more soluble than both G-15 and G-36 in 1% dimethyl sulfoxide (DMSO) solutions. In a
preliminary study, we found that both G-15 and G-36 dissolve sparingly in water, oil, or
alcohols. To be appropriately dissolved, G-15 and G-36 must be dissolved in a high
concentration of DMSO. The restriction in solvents has limited the use of G-15 and G-36 in
animal studies due to the significant cytotoxicity of DMSO. In contrast, CIMBA in the range of
24

pharmacological dosages can be dissolved in alcohol first and then oil so that it is convenient to
be used for animal experiments through intramuscular, subcutaneous, or intraperitoneal injection.
As found by clinical and epidemiological studies, the prevalence of cholesterol gallstone
disease in women is twice that of men (24), and oral contraceptives or other estrogen therapies
significantly increase that risk (26). Although the classical ERĮꢁSOD\VꢁDꢁcritical role in estrogen-
induced lithogenic effects, genetic studies have found that Gper1 is a new gallstone gene, Lith18,
in mice (31) and is also involved in estrogen-dependent lithogenic pathways (29, 50). In a mouse
model of E2-induced gallstones, CIMBA reduces gallstone formation in a dose-dependent
manner in OVX mice. Of note, at the highest ꢅꢇꢈꢁȝJꢉGD\ꢉNJꢆꢁconcentration, CIMBA does not
completely inhibit gallstone formation in OVX mice. One explanation for this observation is that
even higher doses of CIMBA are needed to fully prevent E2-induced gallstone formation.
However, the most likely explanation is that since CIMBA does not bind to the classical ERs,
(5ĮꢁLVꢁVWLOOꢁEHLQJꢁDFWLYDWHGꢁE\ꢁ(2, thus leading to gallstone formation (29). 8VLQJꢁ29;ꢁ(5Įꢁꢅ-/-)
mice, we further found that CIMBA protects against the formation of E2-induced gallstones by
inhibiting the Gper1 activity in the liver. The current results are consistent with the findings
reported in the literature (27), supporting the novel concept that GPER is involved in E2-
dependent lithogenic actions, working independently of ERĮꢎ These results also support the
QRWLRQꢁWKDWꢁERWKꢁ*3(5ꢁDQGꢁ(5ĮꢁFDQꢁZRUNꢁWKURXJKꢁGLIIHUHQWꢁSDWKZD\VꢁRQꢁKHSDWLFꢁFKROHVWHUROꢁDQGꢁ
bile salt metabolism, as well as biliary lipid secretion and cholesterol crystallization to promote
E2-induced cholesterol gallstones (29).
Taken together, the discovery of the potent GPER-selective antagonist, CIMBA, may
provide a novel and alternative strategy for the prevention of cholesterol gallstones in women
and, particularly for subjects who have to expose to high levels of estrogen. CIMBA is a
25

precedent for GPER antagonists to reduce GPER mRNA levels, as we found in the liver.
Furthermore, CIMBA and the other compounds from this study also offer new pharmacological
tools to evaluate the functions of GPER while exploring the ligand binding domain, which may
greatly aid in the development of an orally administered, liver-specific, GPER-selective
antagonist for the prevention of cholesterol gallstone disease in a subgroup of women at high
risk.
26

Acknowledgments: This work was supported in part by the start-up funds from Saint Louis
University (to CKA), as well as by research grants DK101793, DK106249, DK114516, and
AA025737 (to DQ-HW), as well as P30 DK041296 (to Marion Bessin Liver Research Center),
all from the National Institutes of Health (US Public Health Service).
Conflicts of interest: There is no conflict of interest for all authors.
27

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