A sphingosine 1-phosphate receptor 2 selective allosteric agonist

Article abstract of DOI:10.1016/j.bmc.2013.06.012

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.

Full text of DOI:10.1016/j.bmc.2013.06.012

Bioorganic & Medicinal Chemistry 21 (2013) 5373–5382
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A sphingosine 1-phosphate receptor 2 selective allosteric agonist q
Hideo Satsu a,f, Marie-Therese Schaeffer b, Miguel Guerrero c, Adrian Saldana d, Christina Eberhart d,
Peter Hodder d, Charmagne Cayanan b, Stephan Schürer e, Barun Bhhatarai e, Ed Roberts c, Hugh Rosen b,f
,
Steven J. Brown b,
⇑
a Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
b The Scripps Research Institute Molecular Screening Center, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA
c Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA
d Scripps Research Institute Molecular Screening Center, Lead Identification Division, Translational Research Institute, 130 Scripps Way, Jupiter, FL 33458, USA
e Center for Computational Science, Miller School of Medicine, University of Miami, FL 33136, USA
f Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for
investigating the multiple biological processes in which the S1PR2 receptor has been implicated.
Amongst these are NF-jB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here
Received 3 April 2013
Revised 29 May 2013
Accepted 6 June 2013
Available online 15 June 2013
we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We
employed high throughput screening to identify two compounds which activate the S1PR2 receptor.
SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and
CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not com-
petitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwit-
terionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not
activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that
CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling sug-
gests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is ener-
getically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
Keywords:
Allosteric
Signaling
GPCR
Sphingosine 1-phosphate
Molecular modeling
S1PR2
1. Introduction
K562 cells transiently transfected with S1pr2 cDNA, S1P increased
intracelluar calcium levels from intra- and extracellular reserves.3
Sphingolipids are an important family of bioactive molecules
with cell signaling properties. Sphingosine 1-phosphate (S1P) is a
pleiotropic lysophospholipid mediator present in plasma and is re-
leased in large amounts from activated platelets. S1P regulates var-
ious biological processes such as cell proliferation, migration,
survival, and differentiation. The five sphingosine-1-phosphate
receptors S1PR1 through S1PR5 mediate cellular functions upon
S1P binding.1 S1PR1, S1PR2 and S1PR3 are widely expressed on
various tissues and cell types, whereas the expression of S1PR4
and S1PR5 is prominent in cells of the immune and nervous sys-
tems, respectively.
S1PR2 is a high affinity subnanomolar receptor for S1P and has
been implicated in multiple biological functions, including Rho
activation, inhibition of Rac and cell migration, and in ‘feed for-
ward’ autocrine signaling in NF-jB survival signaling of tumor
cells.4,5 S1PR2 promiscuously couples to the heterotrimeric G pro-
1
teins Gq, Gs, Gi/o, and G12/13
.
Studies with genetic deletions can
provide insights into the physiologic functions of the targeted gene
product. Kono et al. reported that S1PR2 expression is essential for
proper functioning of the auditory and vestibular system.6 Skoura
et al. reported the essential role of S1PR2 in pathological angiogen-
esis of the mouse retina.7 About one half of S1pr2 gene null mice
develop clonal B-cell lymphomas by age 1.5–2 years.8 In addition
to these observations, it is further expected that S1PR2 exerts other
unknown physiological functions.
The S1pr2 gene was cloned from rat smooth muscle cDNA li-
brary as an orphan receptor homologous to the S1pr1 gene.2 In
S1P is a unique, amphiphilic GPCR ligand, consisting of a hydro-
philic, polar zwitterionic phosphoamine headgroup and a hydro-
phobic aliphatic straight chain C18 tail (Fig. 1A). The flexibility of
the acyl chain may allow binding to many diverse sites. Along with
genetic manipulations, chemical approaches provide novel insights
into the function of S1P receptors. In the case of S1PR1, SEW-2871
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
⇑
Corresponding author.
E-mail address: sbrown@scripps.edu (S.J. Brown).
0968-0896/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.012

5374
H. Satsu et al. / Bioorg. Med. Chem. 21 (2013) 5373–5382
2.3. Jump-In CHO S1PR2 wild type and head group triple mutant
cell lines
Multisite Gateway cloning was utilized to generate in-frame
S1pr2-eGFP expression constructs from pEnter-15-S1pr2 and pEN-
TER-52-eGFP. The S1pr2-eGFP fusion protein expression vector
was cloned into pDEST-CMV-JTI (Invitrogen). S1PR2 head group
binding side chains were identified by alignment with S1PR1.
These mutations were generated by overlapping oligonucleotide
PCR.14 The triple mutant S1PR2 (R108A, E109A and K269A) was
generated by overlapping oligonucleotide PCR mutagenesis. All
constructs were confirmed by DNA sequencing. These vectors were
transfected into CHO JumpIn cells (Invitrogen) and selected with
10 l
g/mL blasticidin as described.15 A homogenous pool of cells
was generated by FACS sorting of GFP positive cells.
Figure 1. Chemical structure of ligands used in this study. (A) Sphingosine-1-
phosphate, (B) XAX-162, and (C) CYM-5520.
2.4. Glo-sensor cAMP transient transfection assay
The GloSensor vector (pGLoSensor-20FcAMP, Promega) was
transfected using Fugene HD into S1PR2-eGFP or S1PR2-TM-eGFP
Jump-In CHO cells. The following day, cells were harvested with
0.05% trypsin EDTA, resuspended to 500,000 cells/mL in CO2 inde-
pendent Media (Invitrogen) containing 2% charcol dextran stripped
serum (CDS, Invitrogen) and 20 lL of the cell suspension was
added to 384 well tissue culture treated white plates (Corning, part
number 3570). These plates were incubated overnight at 37 °C, 5%
CO2. 25 lL of CO2 independent media containing 2%CDS and 4%
GloSensor Reagent (Promega) were then added and the plates were
incubated for 2 h at room temperature. Antagonist (JTE-013) or
vehicle were added and incubated for 20 min followed by agonist
compounds or S1P. After 15 min, luminescence was read on a Per-
kin–Elmer Envision plate reader.
is well recognized as an S1PR1-agonist and demonstrates the
essential role of S1PR1 in lymphocyte trafficking. Recently a
S1PR1 subtype selective agonist provided insights into the pul-
monary response to viral infection.9 Further, Sanna et al. reported
that W146, a chiral S1PR1 antagonist, enhances capillary leakage
and restores lymphocyte egress in vivo.10 Therefore S1P receptor
subtype selective agonists and antagonists will be of broad utility
in understanding the regulatory mechanism of cell functions
in vitro and physiological phenomenon in vivo.
There are few existing chemical probes for S1PR2. JTE-013 has
been developed as an S1PR2 selective antagonist.11 However, to
our knowledge there is no known S1PR2 selective agonist. An
S1P analogue, DS-SG-44, (2S,3R)-2-amino-3-hydroxy-4-(4-octyl-
phenyl)butyl phosphoric acid), was reported to block isoprena-
line-mediated morphological changes in rat C6 glioma cells, and
was hypothesized to acted as an S1PR2 receptor agonist.12 How-
ever, no receptor pharmacology for DS-SG-44 is reported, and is
likely that it will be problematic for biological studies because of
solubility and metabolic liabilities.
We report here HTS-driven identification of novel chemical
scaffolds for S1PR2, chemical optimization and characterization
of receptor activation via binding to the hydrophobic portion of
the S1P biotopic, orthosteric binding site. Receptor binding and
activation were delineated by radioligand binding competition,
antagonist inhibition of CRE-reporter responses, cAMP biosensor
detection of activation of wild type and head group mutant
S1PR2 receptors, and molecular modeling studies.
2.5. 33P-S1P radioligand competition binding assay
Sphingosine, D
-erythro [33P] 1-phosphate was purchased from
American Radiolabeled Chemicals, Inc (St Louis). S1PR2-CRE bla
cells were seeded into wells of a 24 well plate at 200,000 cells in
1.0 mL growth media and the plate incubated overnight in an incu-
bator with 100% humidity, 5% CO2, 37 °C. The media was replaced
with 1% CDS serum media for 4 h prior to the assay. At 4 °C, the
media was removed and replaced with test compounds or vehicle
controls in binding buffer (20 mM Tris, pH 7.5, 100 mM NaCl,
15 mM NaF with freshly added 1 mM Na3VO4 and protease
inhibitors).
2.6. Compound synthesis and characterization
2. Materials and methods
2.1. Chemicals
2.6.1. (CYM- 5482)
1-(1-Benzyl-2,5-dimethyl-1H-pyrrol-3-yl)-2-chloroethanone
(50 mg, 0.191 mmol) in DMF were added sequentially DIPEA
(66 lL, 0.38 mmol) and succinamide (38 mg, 0.38 mmol). The reac-
S1P was purchased from Biomol (Plymouth Meeting, PA) and
dissolved in methanol (1 mM) and stored at À80 °C. Forskolin
was purchased from Sigma–Aldrich and stored as a 10 mM DMSO
solution at À35 °C. JTE-013 was purchased from Calbiochem (San
Diego, CA) and Cayman Chemical (Ann Arbor, MI) and stored as a
10 mM DMSO solution at À35 °C.
tion was stirred 40 min in the microwave at 130 °C. The mixture
was diluted in water and extracted with Ethyl acetate
(4 Â 50 mL). The combined organic phase was washed with brine
(2 Â 50 mL) and concentrated under reduced pressure. The mix-
ture was purified by column chromatography using DCM/MeOH
to yield 12 mg (0.036 mmol, 19%) of product as pale yellow
powder.
2.2. S1P reporter and counterscreen assays
1H NMR (400 MHz, CDCl3): d 7.34–7.25 (m, 3H), 6.87 (d,
J = 7.68 Hz, 2H), 6.34 (s, 1H), 5.05 (s, 2H), 4.73 (s, 2H), 2.84 (s,
4H), 2.45 (s, 3H), 2.14 (s, 3H); 13C NMR (125 MHz, CDCl3): d
186.46, 177.91, 137.72, 138.00, 137.20, 129.83, 129.64, 128.42,
126.35, 117.68, 107.64, 47.52, 46.36, 29.20, 13.08, 12.70. IR
(cmÀ1): 1702 s, 1663 s. MS (EI) m/z: 325 (M+H).
S1pr1 CRE-bla CHO, S1pr2 CRE-bla CHO, S1pr3-Ga16 NFAT-bla
CHO, S1pr4-TANGO, S1pr4-TANGO and the counterscreen CRE-bla
CHO reporter assays were performed as described.13 PubChem as-
says are listed in Table S1.

H. Satsu et al. / Bioorg. Med. Chem. 21 (2013) 5373–5382
5375
2.6.2. (CYM- 5477)
To stirred solution of potassium tert-butoxide (20 mg,
0.076 mmol) in THF at 0 °C was added slowly the 2-pyrrolidone
(12 L, 0.15 mmol) and the mixture was stirred at 0 °C for
20 min. The reaction was warmed to room temperature and stirred
for additional 20 min. The mixture was cooled to 0 °C followed by
addition of 1-(1-benzyl-2,5-dimethyl-1H-pyrrol-3-yl)-2-chloro-
ethanone (17 mg, 0.15 mmol), the reaction was stirred for 20 min
at 0 °C and overnight at room temperature. The mixture was di-
luted with water, and the product extracted with ethyl acetate.
The product was purified by column chromatography using
DCM/MeOH to yield 14 mg (0.045 mmol, 60%) as pale yellow
powder.
(s, 2H), 2.30 (s, 3H), 2.01 (s, 3H); 13C NMR (125 MHz, CDCl3):
d 187.25, 162.20, 138.89 (q, J3 = 5 Hz), 138.40, 137.28, 135.79 (q,
J3 = 5 Hz), 130.18, 129.95, 129.35, 128.30, 123.78 (q, J1 = 268 Hz),
121.50, 117.47, 109.62 (q, J2 = 35 Hz), 106.80, 55.50, 13.40, 13.10.
IR (cmÀ1): 1675 s, 1650 s, 1334 s. MS (EI) m/z: 375 (M+H).
a
l
2.6.7. (CYM-5520)
Product obtained in 27% yield.
1H NMR (400 MHz, CDCl3): d 7.75 (d, J = 2.4 Hz, 1H), 7.43 (dd,
J = 9.5, 2.4 Hz, 1H), 7.34–7.25 (m, 3H), 6.88 (d, J = 7.0 Hz, 2H),
6.61 (d, J = 9.5 Hz, 1H), 6.38 (s, 1H), 5.18 (s, 2H), 5.06 (s, 2H), 2.47
(s, 3H), 2.15 (s, 3H); 13C NMR (125 MHz, CDCl3): d 186.46,
161.28, 146.83, 139.55, 138.00, 136.49, 129.65, 129.40, 128.05,
125.89, 121.67, 117.05, 116.73, 107.13, 91.52, 55.22, 47.16, 12.64,
12.31. IR (cmÀ1): 2227 s, 1736 s, 1659s. MS (EI) m/z: 346 (M+H).
1H NMR (400 MHz, CDCl3): d 7.33–7.25 (m, 3H), 6.87 (d,
J = 7.28 Hz, 2H), 6.31 (s, 1H), 5.04 (s, 2H), 4.51 (s, 2H), 3.53 (t,
J = 7.08 Hz, 2H), 2.52 (t, J = 8.04 Hz, 2H), 2.47 (s, 3H), 2.13–2.06
(m, 5H); 13C NMR (125 MHz, CDCl3): d 189.89, 176.58, 136.82,
136.82, 129.32, 129.00, 127.90, 125.87, 117.80, 107.21, 50.29,
48.72, 47.00, 30.92, 18.37, 12.58, 12.20. IR (cmÀ1): 1740 s, 1663s.
MS (EI) m/z: 311 (M+H).
2.7. Computational studies
2.7.1. S1PR1/S1PR2 structure wild type (WT) and mutant models
The initial S1PR1 receptor structure was taken from the antag-
onist X-ray co-crystal structure (PDB code 3V2W).16 The structure
was prepared using the protein preparation workflow in Maestro
(Schrodinger Inc.) to assign hydrogens, optimize hydrogen bonds
and to perform constraint minimization (impref). The homology
model of S1PR2 was built using the Uniprot sequence S1PR2_Hu-
man (accession O95136) in Prime (Schrodinger Inc.). This initial
S1PR2 model was optimized using the same protein preparation
workflow above. Both the S1PR1 and the S1PR2 model with the
antagonist sphingolipid mimic ML5 ligand were then optimized
using a multi-step all-atom minimization and molecular dynamics
(MD) simulation implemented in the software package Desmond
(DE Shaw Research).17 Prior to the MD multi-step simulation, a
membrane bilayer model (POPC 300 K) was added to both the
S1PR1 and S1PR2 models. The system was set up using the OPLS-
AA force field, the TIP4P explicit solvent model in an orthorhombic
simulation box 10Å distance in all directions and adding counter
ions. Simulations were performed at 300 K and 1.01325 bar using
the NPT ensemble class. All other settings were default. The pro-
duction simulation time was 12 ns. Simulations were run on an
IBM E-server 1350 cluster (36 nodes of 8 Xeon 2.3 GHz cores and
12 GB of memory). Several later simulation frames were extracted
from the S1PR1 and S1PR2 simulations based on conformational
diversity, low (stable) RMSD, and a stable ML5 (ligand) pose with
maximum H-bonds. To avoid clashing side chains, constraint (im-
pref) minimization (in Maestro, Ref: Schrodinger Inc.) was per-
formed for the WT and mutant S1PR1 and S1PR2 receptor
structures. These structures were then used for further modeling.
2.6.3. (CYM- 5478)
To a stirred solution of 1-(1-benzyl-2,5-dimethyl-1H-pyrrol-3-
yl)-2-chloroethanone (50 mg, 0.191 mmol) in DMF (2.5 mL) were
added sequentially DIPEA (66 lL, 0.38 mmol) and 2-hydroxy-5-tri-
fluoromethyl pyridine (62 mg, 0.38 mmol). The reaction was stir-
red 48 h at 70 °C. The mixture was diluted in water and
extracted with ethyl acetate (4 Â 50 ml). The combined organic
phase was washed with brine (2 Â 50 mL) and concentrated under
reduced pressure. The mixture was purified by column chromatog-
raphy using DCM/MeOH to yield 20 mg (0.052 mmol, 27%) of prod-
uct as a pale yellow powder.
1H NMR (400 MHz, CDCl3): d 7.63 (br s, 1H), 7.48 (dd, J = 9.5,
2.4 Hz, 1H), 7.34–7.26 (m, 3H), 6.89 (d, J = 7.2 Hz, 2H), 6.66 (d,
J = 9.5 Hz, 1H), 6.40 (s, 1H), 5.18 (s, 2H), 5.06 (s, 2H), 2.48 (s, 3H),
2.16 (s, 3H); 13C NMR (125 MHz, CDCl3): d 187.07, 162.26, 138.86
(q, J3 = 5 Hz), 137.71, 136.59, 135.77 (q, J3 = 2 Hz), 129.47, 129.37,
128.00, 125.89, 123.77 (q, J1 = 268 Hz), 121.48, 117.25, 109.65 (q,
J2 = 35 Hz), 107.17, 55.44, 47.11, 12.62, 12.27. IR (cmÀ1): 1740 s,
1675 s, 1330 s. MS (EI) m/z: 389 (M+H).
2.6.4. (CYM- 5491)
Product obtained in 32% yield.
1H NMR (400 MHz, CDCl3): d 7.39–7.24 (m, 4H), 6.89 (d,
J = 8.16 Hz, 2H), 6.88 (s, 1H), 6.40 (s, 1H), 6.33 (d, J = 8.6 Hz, 1H),
5.19 (s, 2H), 5.06 (s, 2H), 2.47 (s, 3H), 2.15 (s, 3H); 13C NMR
(125 MHz, CDCl3):
d
187.28, 161.81, 141.58 (q, J2 = 34 Hz),
140.77, 137.72, 136.59, 129.56, 129.44, 128.00, 125.89, 122.53 (q,
J1 = 273 Hz), 118.57 (q, J3 = 4 Hz), 117.32, 107.19, 101.12 (q,
J3 = 3 Hz), 55.26, 47.11, 12.62, 12.26. IR (cmÀ1): 1679 s, 1609 s,
1167 s, 1133 s. MS (EI) m/z: 321 (M+H).
2.7.2. Ligand receptor binding models
Using the optimized S1PR1 and S1PR2 WT and mutant receptor
models, we generated initial binding poses for the ligands CYM-
5520 and S1P as follows. Ligands were prepared using ligprep
Schrodinger Inc.) to generate ionization states (pH = 7) and stereo-
isomers resulting in a single representation for both S1P and CYM-
5520. Ligands were initially docked into the receptor structures
using the Induced Fit Docking (IFD) (Ref: Schrodinger Inc.) protocol
with default settings. The IFD protocol includes a constraint recep-
tor minimization step followed by initial flexible Glide docking of
the ligand using a softened potential to generate an ensemble of
poses. For each pose, the nearby receptor structure is then refined
using Prime. Each ligand is then re-docked (using Glide) into its
corresponding optimized low-energy receptor structure and
ranked by Glide score. For S1P, we required two hydrogen bond
interactions of polar receptor side chains known to interact with
S1P (R120/108, E121/109, R292/K269; S1PR1/S1PR2). For CYM-
5520 no constrains were used. The best pose with highest IFD score
2.6.5. (CYM-5481)
Product obtained in 46% yield.
1H NMR (400 MHz, CDCl3): d 7.39–7.22 (m, 5H), 6.89 (d,
J = 7.08 Hz, 2H), 6.61 (d, J = 9.08 Hz, 1H), 6.43 (s, 1H), 6.20 (td,
J = 7.9, 1.2, 1H), 5.18 (s, 2H), 5.06 (s, 2H), 2.47 (s, 3H), 2.15 (s,
3H); 13C NMR (125 MHz, CDCl3):
d 188.80, 163.46, 140.63,
139.54, 137.86, 137.21, 129.82, 129.63, 128.42, 126.39, 121.64,
118.09, 107.80, 106.46, 55.64, 47.54, 13.10, 12.72. IR (cmÀ1):
1740 s, 1655 s. MS (EI) m/z: 321 (M+H).
2.6.6. (CYM- 5473)
Product obtained in 34% yield.
1H NMR (400 MHz, CDCl3): d 7.66 (br s, 1H), 7.54–7.47 (m, 4H),
7.17 (d, J = 7.8 Hz, 2H), 6.66 (d, J = 8.6 Hz, 1H), 6.44 (s, 1H), 5.19

5376
H. Satsu et al. / Bioorg. Med. Chem. 21 (2013) 5373–5382
obtained for each ligand was again subjected to MD simulation
(3–5 ns production runs) for further optimization of the protein li-
gand complex. The MD protocol includes a multi-step procedure of
minimizations and short MD runs followed by the production MD
simulation. The same parameter and settings as described above
were used. Poses were stable during the production MD runs.
The final frames of these simulations were then used for docking
of ligands after constraint (impref) minimization (Maestro,
Schrodinger Inc.). Ligands were re-docked using Glide SP and XP
with default potential and other settings. The best pose of the li-
gand was selected based on the Glide scores, known interactions
(e.g., head group) and visual inspection. MM-GB/SA implemented
in Prime was performed to calculate the relative binding free ener-
gies for the studied ligands. Receptor flexibility cutoff was set to 4 Å
around the ligand. 2D ligand-receptor interaction diagrams were
generated in Maestro and 3D plots were produced using PyMol.
To evaluate the hydrophobic interaction of ligand and receptor,
the structural interaction fingerprint (SiFT) of the binding was cal-
culated post docking. The resulting fingerprint was visualized in a
matrix as heat map, sorted by similarity and clustered considering
only the hydrophobic amino acid residues. The presence of interac-
tion is shown as red and the absence green.
at 5 lmol concentration. Compounds that were confirmed active in
the primary assay and inactive in the counterscreen were pur-
chased as powders for further testing. From the Maybridge screen
only one compound, XAX-164 (Table 1), was active in the S1PR2
CRE-bla assay and inactive in the counterscreen assay. Dose re-
sponse from a powder sample determined an EC50 of 1.3 lM in
the S1PR2 CRE-bla reporter assay and absence of activity in the
CRE-bla parental cell line counterscreen assay.
In the MLSCN S1PR2 uHTS campaign (deposited in PubChem
AID 729), 96,881 compounds were tested in 1,536 well format at
5 lmol. The 61 (of 64) available compounds with activation greater
than 50% were tested in dose response against the S1PR2 Agonist
(PubChem AID 854) and parental cell line counterscreen (PubChem
AID 843) dose response assays. Only 2 compounds were active in
the S1PR2 dose response assay, and inactive in the CRE-bla dose re-
sponse counterscreen. The MLSMR compound MLS000049871 was
inactive in the CRE-bla counterscreen, but was active in PubChem
AID 662, an assay designed to find CRE activators, and was there-
fore not considered a viable lead. We resynthesized the remaining
compound, CYM-5482 (1) (Table 1) and confirmed S1PR2 agonist
activity with an EC50 of 1.0 lM.
3.2. Synthesis of 1, 10 and analogs
3. Results
The structural integrity and biological activity of the original hit
(1) were confirmed by re-synthesizing the title compound
(Scheme 1). Reaction of commercially available chloroketone I
with succinamide using DIPEA as a base provided the hit 1. Simi-
larly, condensation of chloroketone I with 2-pyrrolidone using KOt-
Bu as base furnished derivative 9. The synthesis of 1 and 9 is shown
in Scheme 1.
Condensation of 2-pyridone derivatives III–VI with chloroke-
tones I and II, using DIPEA as base, provided the final products
10–14 (Scheme 2).
3.1. S1PR2 HTS and uHTS screening
We developed a HTS compatible assay for S1PR2 with beta-lac-
tamase reporter readout. In order to identify the most appropriate
reporter for detecting S1PR2 activation we compared the signal
generated by transiently transfecting NFAT-bla or CRE-bla CHO
cells with either pcDNA3.1 human S1pr2 or LacZ constructs. Stim-
ulation with 1 lmol S1P resulted in a reproducible 1.4-fold in-
crease in the blue/green readout for the S1PR2 transfected cells
and no increase for the control transfections. A stable cell line
expressing S1PR2 in CRE-bla CHO was generated and conditions
for HTS and ultra HTS (uHTS) optimized.18 Two complementary
screening sets were probed, the Maybridge HitFinder and NIH
MLSMR library. The Maybridge S1PR2 agonist screen was run in
3.3. Structure–activity analysis of analogues
Six compounds similar to XAX-164 were purchased to explore
what functional groups were required for S1PR2 activation (Ta-
ble 2). The three compounds with 4-substituted phenyl groups in
the R1 region are active. The methyl substitutedXAX-162 (EC50
0.55 lmol) and bromide 4 (EC50 0.82 lmol derivatives are mod-
estly more potent than XAX-164. Substitution of the phenyl moiety
384 well format, with 10 lmol test compound. The screen had
acceptable assay statistics (Z’ave = 0.63; S/B 6.7). For the May-
bridge screen a cutoff of greater than 40% of control was selected
and 57 compounds were cherry picked and retested in the primary
and parental CRE-bla cell line counterscreening assays in triplicate
with 6-(trifluoromethyl)pyridin-2-yl (5) or cyclohexy (6) results in
Table 1
Confirmed S1PR2 agonists from the Maybridge and MLMSR screens
Name
Structure
IC50 S1PR2 (
lM)
IC50 CRE-bla counterscreen
Not active
O
N
O
N
1 (CYM-5482)
1.0
O
S
S
O
N+
O-
S
2 (XAX-164)
1.3
Not active
Cl

H. Satsu et al. / Bioorg. Med. Chem. 21 (2013) 5373–5382
5377
Scheme 1. Reagents and conditions: (i) I (1 equiv), succinamide (2 equiv), DIPEA (2 equiv), DMF, mw, 130 °C, 40 min, 19%; (ii) I (1 equiv), 2-pyrrolidone (2 equiv), KOtBu
(0.5 equiv), THF, 0 °C to rt, overnight, 60%.
Scheme 2. Reagents and conditions: (i) I or II (1 equiv), III–VI (2 equiv), DIPEA (2 equiv), DMF, 70 °C, 48 h (27–46%).
Table 2
SAR by purchase for the Maybridge Hit XAX-164
O
O-
N+
S
R1
S
S
Name
R1
EC50 S1PR2 (lM)
Max activity (% of 1 lM S1P)
2 (XAX-164)
3 (XAX-162)
4 (XAX-166)
5 (XAX-159)
6 (XAX-160)
4-Chlorophenyl
4-Methylphenyl
4-Bromophenyl
6-(Trifluoromethyl)pyridin-2-yl
Cyclohexyl
1.3
80%
130%
80%
30%
20%
0.55
0.82
>10
>10
O
O-
N+
S
Cl
Name
R2
R2
EC50 S1PR2 (lM)
Max activity (% of 1 lM S1P)
7 (BTB06747)
8 (CD05115)
Carboxyl
Cl
>10
>10
0%
0%
loss of activity. Replacement of the 1,3-dithiolane group (R2) in 2
with either carboxyl (7) or chloride (8) results in a loss of S1PR2
activation. GPCR ligands for adrenergic and 5-HT receptors con-
taining 1,3-dithiolanes have been reported.19
58 compounds structurally related to CYM-5482 were pur-
chased and evaluated in the S1PR2 and counterscreen dose re-
sponse assays (PubChem AIDs 872 and 874). Several compounds
were active and are shown in Table S3 along with some of the
inactive analogues. Interestingly, amongst the commercial analogs
the 5-(trifluoromethyl)pyridin-2-one (10) was found slightly more
potent than 1. Based upon these results, we then synthesized se-
lected compounds (Table 3) in order to improve potency while

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H. Satsu et al. / Bioorg. Med. Chem. 21 (2013) 5373–5382
Table 3
Synthesized SAR compounds
Name
Structure
EC50 S1PR2 (
l
M)
Max Activity (% of 1 lM S1P)
O
N
O
N
1 (CYM-5482)a
1.03
95%
O
O
O
O
N
N
9 (CYM-5477)
10 (CYM-5478)
>10
35%
N
O
0.78
100%
N
CF3
O
O
O
N
N
11 (CYM-5491)
12 (CYM-5481)
>10
>10
9%
CF3
N
O
10%
N
O
O
N
13 (CYM-5473)
>10
0%
N
CF3
O
O
N
14 (CYM-5520)
0.48
100%
N
CN
a
Resynthesized original hit.
maintaining selectivity. First we removed the labile functional
group from 1, however the pyrrolidone (9) is inactive. Next we ex-
plored the SAR around 10. The 4-(trifluoromethyl)pyridin-2-one
(11), the unsubstituted pyridine-2-one (12) and the N-phenyl
(13) are completely inactive. Interestingly, changing the trifluoro-
methyl for a cyanide group (14, CYM-5520) leads to a 1.6-fold in-
crease in potency and inactivity in the CHO CRE-bla counterscreen.
either S1P or CYM-5520 against several concentrations of
JTE-013 in the S1PR2 CRE-bla reporter assay (Fig. 2). The S1P
concentration response curve shifts to the right in response to
JTE-013, consistent with competitive antagonism. Linear
regression of the Schild plot yields a Ki of 20 nM. With increasing
JTE-013, the CYM-5520 dose–response curves both shift to the
right and the magnitude of the response is diminished. Both
XAX-164 and XAX-162 have a similar response as CYM-5520 in
the presence of increasing concentrations of JTE-013 (Supplemen-
tary Fig. S1). This type of agonist inhibition is best described with
the noncompetitive binding model.21
3.4. Compound selectivity
CYM-5520 and XAX-162 are highly selective for S1PR2 and
were inactive in the S1PR1, S1PR3, S1PR4 and S1PR5 agonist
assays (Supplementary Table S4). We further characterized
CYM-5520 in the PanLabs HitProfiling Screen of 29 receptors and
transporters confirming selectivity for S1PR2 over other molecular
targets with no assay inhibition greater than 20% (Supplementary
Table S3).
3.6. Radioligand binding competition to S1PR2
The competitive binding of the ligands used in this study for the
S1PR2 receptor were evaluated with radiolabeled S1P. Both S1P
and JTE-013 demonstrate dose-dependent inhibition of binding
of 33P-S1P to S1PR2. The unlabeled S1P displaces the radiolabeled
S1P with an IC50 of 25 nM. JTE-013 has an IC50 of 53 nM in this as-
say. In contrast, CYM-5520 does not block radiolabeled S1P binding
(Fig. 4). CYM-5520 was tested at a smaller range of higher concen-
trations because, in pilot experiments, we observed that it was not
3.5. Competition with the S1PR2 antagonist JTE-013
JTE-013 is an antagonist of S1PR2.20 To compare S1P and CYM-
5520 binding to S1PR2 we evaluated dose–response curves of

H. Satsu et al. / Bioorg. Med. Chem. 21 (2013) 5373–5382
5379
Figure 2. JTE-013 inhibition of agonists in the S1PR2-CRE-bla assay. Dose response of S1P (A) or CYM-5520 (B) on S1PR2- Cre-bla CHO cells was challenged with increasing
concentrations of JTE-013 (d, DMSO control; j, 10 nM JTE-013; N, 30 nM JTE-013; ., 90 nM JTE-013).
Figure 3. The effect of S1P and CYM-5520 on intracellular cAMP levels in S1PR2 wild type- and triple mutant-expressed cells. Dose response of S1P (d) or CYM-5520 (j) on
intracellular content of cAMP in wild type- (A) and triple mutant- (B) S1PR2 expressed CHO cells was measured with luciferase activity.
competitive with S1P binding. These results demonstrate that
CYM-5520 is not competitive with S1P.
3.8. Molecular modeling
3.8.1. Computational modeling building
3.7. CYM-5520 is an agonist for both wild type and headgroup
mutant S1PR2 receptor
Because CYM-5520 is an allosteric agonist we modeled ligand
binding and examined the binding pocket in detail to better under-
stand how our observations could be explained. The questions
examined were whether CYM-5520 and S1P co-binding in the opti-
mized model was reasonable, and what hydrophobic contacts are
important for CYM-5520 binding. A homology model was pro-
duced for S1PR2 by using the S1PR1 structure as obtained from
PDB (3V2W, resolution 3.35 Å) and the Uniprot sequence of
S1PR2_Human (accession O95136). The primary aligment of the
amino acids sequences used to build the model is in the Supple-
mentary Figure S3. The alignment score of S1PR1-S1PR2 was
0.016 and RMSD (C-alpha atoms of the aligned chains) was
0.625. RMSD is explained by the identity of two proteins of 51%
and a 3% gap in the structures. The S1PR2 receptor model showed
that disulfide bonds in the extracellular loops EL2 and EL3 were
aligned with the corresponding S1PR1 disulfide bonds. Impor-
tantly, the S1P binding region of S1PR2 was well aligned with
S1PR1 (compare Supplementary data) with a RMSD (alpha-C) of
0.322. The receptor structures were optimized by minimization
and molecular dynamics. Optimized structures were used further
for docking studies.
In S1PR1, the side chains from Arginine 120, Glutamic acid 121
and Arginine 292 form salt bonds with the phosphate and ammo-
nium moieties of the zwitterionic S1P headgroup. These interac-
tions are required for receptor binding of S1P to the receptor.22
Homologous polar residues responsible for S1P headgroup binding
in S1PR2 were identified by alignment with the S1PR1 amino acid
sequence. The residues in S1PR2 are Arginine 108, Glutamic acid
109 and Lysine 269. A S1pr2 cDNA was generated by overlapping
PCR mutagenesis in which all three of these residues were mutated
to alanine to generate the triple mutant (TM) construct. Stable cell
lines with a single copy of either wild type (WT) or TM S1PR2-GFP
fusion proteins were generated by targeted, single site integration.
The WT S1PR2-GFP and TM S1PR2-GFP cell lines were then used to
evaluate the ligand-stimulated cAMP response. We measured
intracellular cAMP with a genetically encoded biosensor, based
upon a single chain luciferase-cAMP fusion construct, which upon
binding cAMP changes conformation to higher luciferase activity.23
S1P (EC50 10 nM) and CYM-5520 (EC50 1.6 lM) are full agonists for
wild type S1PR2 (Fig. 3A). Stimulation of cells expressing the triple
mutant S1PR2 with S1P did not elicit a rise in luciferase activity,
3.8.2. Docking study of S1PR2 with S1P and CYM-5520
Ligand-receptor binding models were generated as described in
Section 2. The final ligand–receptor complexes were ranked by
whereas the CYM-5520 was an agonist with an EC50 of 1.5
(Fig. 3B).
lM

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H. Satsu et al. / Bioorg. Med. Chem. 21 (2013) 5373–5382
Figure 4. Radiolabeled 33P-S1P competition binding study. The graph shows the
average of replicate samples. Results are representative of 3 independent exper-
iments which was only tested one time. S1P (j), JTE-013 (d), CYM-5520 ( )
N
glide score, emodel, and MM GB/SA energies, based on known
interactions. The energetically most favorable and comparable
poses represent possible binding modes in the S1PR1 and S1PR2
receptors. The docking scores are reported in Supplementary mod-
eling Table S5. In S1PR2, the S1P head group interacts with R108
and E109 while the hydrocarbon alkyl tail interacts in the hydro-
phobic pocket.(Fig. 5A) CYM-5520 sits in the hydrophobic pocket
close to F250 (Fig. 5B) with no apparent head group interaction.
In the presence of S1P, CYM-5520 moves away from F86 closer
to the hydrophobic region formed by F250 and W256. Co-docking
of S1P and CYM-5520 suggested that the receptor pocket could
accommodate CYM-5520 in the hydrophobic pocket close to
W246 and F250 while the S1P tail adjusts in the space around it
(Fig. 5C). Molecular dynamics simulations did not show any change
in the binding pattern. The corresponding interaction diagrams for
individual and co-docked poses showing all of the residues in close
contact with the ligands are shown in Figure 6.
The SiFT analysis of the CYM-5520 ligand over several valid
poses of S1PR2 post docking suggests important amino-acid resi-
dues that take part in hydrophobic interactions. The majority of
interactions shown in the heat map matrix are conserved across
the different poses. The SiFT analysis complements the ligand
interaction map shown for CYM-5520 (Fig. 6B).
4. Discussion and conclusions
Chemical probes that elucidate interactions within and outside
of the orthosteric binding pockets of GPCRs provide important in-
sights both into mechanisms and potentially into interactions that
make the pocket more pharmaceutically tractable. Detailed analy-
sis of the recent liganded S1PR1 crystal structure24 has provided
insights into the S1PR1–S1PR5 ligand specificity. In the S1P recep-
tor family the orthosteric ligand binding pocket is highly con-
served, but residues that form binding pockets for allosteric
ligands are more diverse.25 Defining the orthosteric pocket of
S1PR1 defines the gatekeeper residues for selectivity of pharmaco-
logical ligands between receptors, and these side-chain protrusions
into the pocket define binding pocket shape and impact on the var-
iable efficiency of small molecule discovery between subtypes.13
For example, L276 in S1PR1 and its replacement by F263 in
S1PR3 provided both loss of function mutations L276F for
S1PR1,26 while that same mutation induced a gain of function for
selective ligands of S1PR3 to bind S1PR1.13 The selectivity of fingo-
limod-phosphate for S1PR1, S1PR3, S1PR4 andS1PR5 while having
no activity of S1PR227 can be explained by the steric interference of
the F274 aromatic side-chain in S1PR2 precluding the binding of
fingolimod while that residue is a conserved leucine in the remain-
ing four receptors. Notably, a close contact between CYM-5520 and
Figure 5. S1PR2 structure model. The ligands S1P (Panel A) and CYM-5520 (Panel
B) are shown docked in the ligand binding pocket. S1P and CYM-5520 are co-docked
in Panel C.

H. Satsu et al. / Bioorg. Med. Chem. 21 (2013) 5373–5382
5381
Gly
94
Glu
A
B
Ser
93
109
Thr
97
Pro
184
Phe
113
Tyr
18
Leu
183
Asn
89
Asn
89
Phe
274
Arg
108
Phe
86
Ala
112
Ser
93
Glu
109
Ser
277
Phe
198
Pro
184
Leu
116
Ala
112
Val
Val
182
Ala
249
182
Leu
253
Val
Trp
246
Leu
183
195
Phe
86
Phe
113
Leu
253
Leu
116
Phe
250
Phe
274
Phe
250
Trp
246
Ala
249
Tyr
186
Ser
277
Val
195
Val
191
Phe
198
Val
194
Tyr
190
Tyr
18
Thr
97
Gly
94
C
D
Arg
108
Thr
181
Glu
109
Pro
184
Pro
184
Glu
Ser
93
Phe
113
109
Tyr
Leu
183
Asn
89
Leu
183
186
Ala
112
Ser
93
Val
182
Ala
112
Tyr
190
Val
191
Phe
113
Phe
274
Phe
198
Leu
253
Val
182
Leu
116
Val
194
Val
195
Leu
253
Phe
86
Ser
Phe
86
Val
Ala
249
Phe
274
Leu
195
Phe
198
277
116
Phe
250
Phe
250
Ser
277
Ile
252
Trp
246
Ala
249
Trp
246
Figure 6. Ligand interaction diagrams in the S1PR2 wild type pocket for (A) S1P, (B) CYM-5520, or in the S1PR2 co-docked WT pocket for (C) S1P and (D) CYM-5520. Amino
acid type (sphere color): red–acidic (charged negative), purple–basic (charged positive), green–hydrophobic, turquoise–polar and gray–other (Gly). Ligand exposure: yellow–
ligand atom exposed to solvent, Interaction type: solid pink–H-bond to protein backbone and dotted pink–H-bond to protein site chain.
F274 is predicted in the S1PR2 model (Fig. 6B). This residue is leu-
cine in the other S1P receptors and thus this interaction may be
important for the specificity of CYM-5520 binding to S1PR2. The
significant difference between S1PR1 and S1PR4 with regard to
subtype selectivity is M124 in S1PR1 (Leucine in S1PR4). In con-
trast S1PR1 and S1PR5 have no obvious gatekeeper residue differ-
ences and this is reflected in the parallel structure–activity
relationships seen between these two receptor subtypes. The
importance of screening is to define diverse chemical scaffolds that
are not limited by the very difficult physical properties of the phys-
iological lysophospholipid ligands.1 The hydrophobic, strong zwit-
terions, and their synthetic analogs28–32 are very sparingly soluble,
do not cross biological membranes, are not orally bioavailable and
require some significant stabilization of the phosphate ester bond
to limit excessive lability in vivo.1,33 Furthermore, the complete
conservation of the zwitterionic headgroup interactions
(E121R122) that provide >3log of potency27 for ligand binding, se-
lect for lysophospholipid-like scaffolds that discriminate poorly
between receptor subtypes. Only the addition of aromatic systems
in place of the long acyl chain, and especially with the elaboration
of heterocycles provides a useful basis for the identification of
probes of the binding pocket that are truly selective and spe-
cific.13,34 Reaching beyond the orthosteric interactions enhance
the possibilities of finding truly novel chemical space.
High throughput screening of two diversity libraries identified
novel S1PR2 agonists. The diaryl-oxadiazole chemical space activ-
ity ‘hot spot’ identified in the comparison of the S1PR1 and S1PR3
HTS campaigns does not extend to these S1PR2 agonists, suggest-
ing that the receptor family has significant diversity in ligand bind-
ing. We made analogues leading to CYM-5520 because this series
was considered more amenable to chemical optimization, in part
due to the nitro group in XAX-164. CYM-5520 lacks any functional
group with any similarity to the zwitterionic head group of S1P. For
these two reasons we focused these ligand-receptor binding stud-
ies to the CYM-5520/S1PR2 ligand/receptor system. Noncompeti-
tive antagonist inhibition, functional response with triple mutant
S1PR2, and absence of radioligand binding completion revealed a
consistent scenario in which CYM-5520 is an allosteric agonist that
can co-bind in the S1PR2 receptor with S1P. Since S1PR2 selective
agonists are not currently available, these compounds may serve as

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H. Satsu et al. / Bioorg. Med. Chem. 21 (2013) 5373–5382
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Acknowledgments
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This work was supported by research Grants from the National
Institute of Health, Molecular Library Probe Production Center
Grant U54 MH084512 (E.R., H.R. P.H.), and a Grant-in-Aid for
Young Scientists (A-20688005) from the Ministry of Education,
Culture, Sports, Science, and Technology (MEXT) of Japan (H.S.)
We thank Jill Ferguson for her help with editing.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.06.012.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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