Fatty alcohol phosphates are subtype-selective agonists and antagonists of lysophosphatidic acid receptors

Article abstract of DOI:10.1124/mol.63.5.1032

A more complete understanding of the physiological and pathological role of lysophosphatidic acid (LPA) requires receptor subtype-specific agonists and antagonists. Here, we report the synthesis and pharmacological characterization of fatty alcohol phosphates (FAP) containing saturated hydrocarbon chains from 4 to 22 carbons in length. Selection of FAP as the lead structure was based on computational modeling as a minimal structure that satisfies the two-point pharmacophore developed earlier for the interaction of LPA with its receptors. Decyl and dodecyl FAPs (FAP-10 and FAP-12) were specific agonists of LPA₂ (EC₅₀ = 3.7 ± 0.2 μM and 700 ± 22 nM, respectively), yet selective antagonists of LPA₃ (K_i = 90 nM for FAP-12) and FAP-12 was a weak antagonist of LPA₁. Neither LPA₁ nor LPA₃ receptors were activated by FAPs; in contrast, LPA₂ was activated by FAPs with carbon chains between 10 and 14. Computational modeling was used to evaluate the interaction between individual FAPs (8 to 18) with LPA₂ by docking each compound in the LPA binding site. FAP-12 displayed the lowest docked energy, consistent with its lower observed EC₅₀. The inhibitory effect of FAP showed a strong hydrocarbon chain length dependence with C12 being optimum in the Xenopus laevis oocytes and in LPA₃-expressing RH7777 cells. FAP-12 did not activate or interfere with several other G-protein-coupled receptors, including S1P-induced responses through S1P_1.2,3.5 receptors. These data suggest that FAPs are ligands of LPA receptors and that FAP-10 and FAP-12 are the first receptor subtype-specific agonists for LPA₂.

Full text of DOI:10.1124/mol.63.5.1032

0026-895X/03/6305-1032–1042$7.00
MOLECULAR PHARMACOLOGY
Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 63:1032–1042, 2003
Vol. 63, No. 5
2054/1060488
Printed in U.S.A.
Fatty Alcohol Phosphates are Subtype-Selective Agonists and
Antagonists of Lysophosphatidic Acid Receptors
TAMAS VIRAG, DON B. ELROD,1 KAROLY LILIOM, VINEET M. SARDAR, ABBY L. PARRILL, KAZUAKI YOKOYAMA,
GANGADHAR DURGAM, WENLIN DENG, DUANE D. MILLER, and GABOR TIGYI
Departments of Physiology (T.V., K.Y., W.D. G.T.) and Pharmaceutical Sciences (D.B.E., G.D., D.D.M.), University of Tennessee Health Science
Center, Memphis, Tennessee; Department of Chemistry and Computational Research on Materials Institute, University of Memphis, Memphis,
Tennessee (V.M.S., A.L.P.); and Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary (K.L.)
Received August 5, 2002; accepted February 7, 2003
This article is available online at http://molpharm.aspetjournals.org
ABSTRACT
A more complete understanding of the physiological and LPA2 was activated by FAPs with carbon chains between 10
pathological role of lysophosphatidic acid (LPA) requires recep- and 14. Computational modeling was used to evaluate the
tor subtype-specific agonists and antagonists. Here, we report interaction between individual FAPs (8 to 18) with LPA2 by
the synthesis and pharmacological characterization of fatty docking each compound in the LPA binding site. FAP-12 dis-
alcohol phosphates (FAP) containing saturated hydrocarbon played the lowest docked energy, consistent with its lower
chains from 4 to 22 carbons in length. Selection of FAP as the observed EC50. The inhibitory effect of FAP showed a strong
lead structure was based on computational modeling as a hydrocarbon chain length dependence with C12 being opti-
minimal structure that satisfies the two-point pharmacophore mum in the Xenopus laevis oocytes and in LPA3-expressing
developed earlier for the interaction of LPA with its receptors. RH7777 cells. FAP-12 did not activate or interfere with several
Decyl and dodecyl FAPs (FAP-10 and FAP-12) were specific other G-protein-coupled receptors, including S1P-induced re-
agonists of LPA2 (EC50 ϭ 3.7 Ϯ 0.2 ␮M and 700 Ϯ 22 nM, sponses through S1P1,2,3,5 receptors. These data suggest that
respectively), yet selective antagonists of LPA3 (Ki ϭ 90 nM for FAPs are ligands of LPA receptors and that FAP-10 and FAP-12
FAP-12) and FAP-12 was a weak antagonist of LPA1. Neither are the first receptor subtype-specific agonists for LPA2.
LPA1 nor LPA3 receptors were activated by FAPs; in contrast,
Lysophosphatidic acid (LPA) is a member of the phospho- 1-phosphate (S1P) (Chun et al., 2002). The S1P receptors
lipid growth factor (PLGF) family. PLGFs exert pleiotropic
biological effects, such as activating platelet aggregation and
affecting cell proliferation, apoptosis, migration, and cell
shape (for reviews, see Goetzl et al., 2000; Tigyi, 2001). LPA
elicits its biological effects through the activation of G pro-
tein-coupled receptors. In mammalian cells, three LPA-spe-
cific receptors have been identified, including LPA1 (EDG-2),
LPA2 (EDG-4) and LPA3 (EDG-7), all members of the endo-
thelial differentiation gene (EDG) family (for review, see
Contos et al., 2000). In addition to LPA1, the PSP24 receptor
was shown to elicit LPA-induced Ca2ϩ-dependent ClϪ-cur-
rents in Xenopus laevis oocytes (Guo et al., 1996; Fischer et
al., 1998; Kimura et al., 2001). Receptors LPA1–3 share 50 to
54% sequence identity (Chun et al., 2002). The EDG receptor
family also contains five other receptors, S1P1–5 (EDG-1, -5,
-3, -6, -8), specific for the sphingolipid PLGF sphingosine
S1P1–5 share 50% amino acid sequence identity and 35%
identity with the LPA receptors (Chun et al., 2002), suggest-
ing possible similarities in the characteristics of ligand rec-
ognition in these receptors. Most cells express a combination
of these receptors, making it difficult to dissect the biological
effects mediated by an individual receptor subtype. The need
to understand the biological function of PLGF receptors and
the desire to pharmacologically exploit the differences in
their ligand recognition requires the development of receptor
subtype-specific agonists and antagonists. Until recently, no
such compounds were available. Local and general anesthet-
ics have been reported to inhibit PLGF receptors in X. laevis
oocytes (Chan and Durieux, 1997; Tigyi et al., 1997). Like-
wise, N-acyl serine phosphoric acid and N-acyl tyrosine phos-
phoric acid were shown to inhibit LPA-induced platelet ag-
gregation, ClϪ-currents in X. laevis oocytes, and neutrophil
adhesion to vascular endothelial cells (Sugiura et al., 1994;
Liliom et al., 1996a; Hooks et al., 1998; Siess et al., 1999).
However, in MDA MB231 cells, N-acyl serine phosphoric acid
This work was supported in part by grants HL61469 and CA92160 from the
National Institutes of Health.
1 Current address: Lynntech, Inc., College Station, TX 77840.
ABBREVIATIONS: LPA, lysophosphatidic acid; EDG, endothelial differentiation gene; FAP, fatty alcohol phosphate; GPCR, G protein-coupled
receptor; GTP-␥-S, guanosine 5Ј-3-O-(thio)triphosphate; MS, mass spectrometry; LPP, lipid phosphate phosphatase; PLGF, phospholipid growth
factor; S1P, sphingosine 1-phosphate; RT-PCR, reverse transcription-polymerase chain reaction.
1032

FAPs Are LPA Agonists/Antagonists
1033
was shown to be a potent activator of LPA-like responses Chemical Synthesis of the FAP Compounds
(Hooks et al., 1998), although the receptor activated by this
All reagents and chromatography media were purchased from
Sigma-Aldrich Chemical Co. or Fisher Scientific (Pittsburgh, PA)
and were used without further purification. Thin-layer chromatog-
raphy was performed on 200-␮m alumina plates (Silica gel 60 Å;
E.M. Science, Hawthorne, NY). Flash chromatography was per-
formed on silica gel (60 Å, 200–425 mesh). Melting points were
determined on a Thomas-Hoover capillary melting point apparatus
and are uncorrected. 1H, 13C, and 31P nuclear magnetic resonance
spectra were obtained on an AX 300 spectrometer (Bruker, Billerica,
MA). Chemical shifts for 1H and 13C are reported as parts per million
relative to tetramethylsilane. Spectra for 31P are reported as parts
per million relative to 0.0485 M triphenylphosphate in CDCl3. Elec-
trospray ionization liquid chromatography/mass spectrometry was
performed on a Bruker Esquire LC/MS system.
compound was not identified. This same compound was a
weak agonist of LPA1 and LPA3 receptors heterologously
expressed in Jurkat T cells (An et al., 1998). Cyclic phospha-
tidic acid was also shown to inhibit LPA-induced platelet
aggregation (Gueguen et al., 1999); however, it is an LPA
receptor agonist in X. laevis oocytes (Liliom et al., 1996b;
Fischer et al., 1998) and in cells that heterologously express
EDG family LPA receptors (Bandoh et al., 2000).
Computational models for S1P1 (Parrill et al., 2000), LPA1,
and LPA2 (Wang et al., 2001; Sardar et al., 2002) confirmed
the importance of two specific interactions between each
receptor and its phospholipid ligand. One of these interac-
tions involves ion pairing between the phosphate group of
LPA and two positively charged conserved residues in the
third and seventh putative transmembrane segments of the
LPA receptor subfamily. A second interaction involves hydro-
phobic residues within the transmembrane segment of the
receptor and the hydrophobic tail of LPA (Fischer et al.,
2001). Based on these two points of interaction with the LPA
pharmacophore, we identified dioctylglycerol pyrophosphate
and dioctyl-phosphatidic acid as selective antagonists of
LPA1 and LPA3 receptors, with an order of magnitude higher
potency for LPA3 (Fischer et al., 2001). A stereoisomer of the
2-substituted N-acyl ethanolamide phosphate LPA analog
containing a bulky benzyl-4-oxybenzyl group was shown to be
a dual LPA1/LPA3 competitive antagonist with higher inhib-
itory potency for LPA1 (Heise et al., 2001).
Herein we report the synthesis and characterization of
fatty alcohol phosphates (FAPs) that lack a glycerol backbone
and therefore consist of only a polar phosphate head group
and a hydrophobic tail and represent a minimal structure
that satisfies the two-point pharmacophore introduced ear-
lier for LPA-like structures (Fischer et al., 2001, Sardar et
al., 2002). Pharmacological characterization of the ligand
properties of FAPs showed an exquisite dependence on the
length of the hydrocarbon chain. FAPs with 10, 12, and 14
carbons are agonists for LPA2 and antagonists for the LPA3
receptor. In contrast, other FAPs, with carbon chain lengths
of 4, 8, 16, 18, and 22, showed no agonist effect on any
mammalian LPA receptor. FAP-14 and FAP-18 were weak
antagonists of the LPA3 receptor, along with FAP-10.
Synthesis of Phosphoric Acid Dibenzyl Ester Alkyl Esters
(1–8a)
The synthesis of protected alkyl monophosphates (Fig. 1) was
performed according to the method of Bittman et al. (1996) except
that peracetic acid was used for the oxidation step instead of m-
chloroperoxybenzoic acid. Each anhydrous alcohol (1.0 mmol) and
365 mg (5.17 mmol) of 1H-tetrazole were dissolved in 34 ml of
anhydrous methylene chloride. A solution of 0.895 g (2.58 mmol) of
dibenzyl-N,N-diisopropyl phosphoramidite in 5 ml of anhydrous
methylene chloride was added under an argon atmosphere. The
reaction mixture was stirred at room temperature for 2 h and was
then cooled to Ϫ38°C in an isopropyl alcohol/dry ice bath. Peracetic
acid [0.815 g (3.43 mmol) of 32% acid] in 28 ml of anhydrous meth-
ylene chloride was added drop-wise, and the temperature of the
reaction mixture was raised to 0°C and stirred for 1 h. To the
reaction mixture, 200 ml of methylene chloride was added, and the
organic layer was washed with 10% sodium metabisulfite (2 ϫ 40
ml), saturated sodium bicarbonate (2 ϫ 40 ml), water (30 ml), and
brine (40 ml). The organic layer was dried with anhydrous sodium
sulfate, filtered, and concentrated under vacuum to dryness. The
resulting crude products were purified by silica gel chromatography
using hexane/ethyl acetate (1:1 for 1a and 7:3 for 2–8a) to elute the
desired product.
Spectral Characterization of Phosphoric Acid Dibenzyl
Ester Alkyl Esters (1–8a)
1a Isolated As a Clear Oil (309 mg) That Was Contaminated
with Excess Phosphorylating Reagent). 1H NMR (CDCl3) ␦ 0.88
(t,
J
ϭ
7.2 Hz, 3H, CH3), 1.34 (sextet,
J
ϭ
7.2 Hz, 2H,
OCH2CH2CH2CH3), 1.59 (quintet,
J
ϭ
6.6 Hz, 2H,
OCH2CH2CH2CH3), 3.99 (q, 6.6 Hz, 2H, OCH2CH2CH2CH3), 5.02 (d,
J ϭ 1.8 Hz, 2H, OCH2Ar), 5.05 (d, J ϭ 2.1 Hz, 2H, OCH2Ar), 7.35 (br
s, 10H, 2 ϫ ArH); 13C NMR (CDCl3) ␦ 13.55, 18.60, 32.16 (d, JC,P
6.8 Hz), 67.72 (d, JC,P ϭ 6.1 Hz), 69.13(d, JC,P ϭ 5.5 Hz), 127.90,
128.47, 128.55, 136.00 (d, JC,P ϭ 6.8 Hz); 31P NMR (CDCl3) ␦ 16.84;
MS: [M ϩ 23Na] at m/z 357.3.
ϭ
Materials and Methods
Materials
Lipids were purchased from Avanti Polar Lipids (Alabaster, AL);
other chemicals and fetal bovine serum were obtained from Sigma-
Aldrich Chemical Co. (St. Louis, MO). Cell culture medium (Dulbec-
co’s modified Eagle’s medium) and G418 were purchased from Cell-
gro (Herndon, VA). Fura-2 acetoxymethyl ester was from Molecular
Probes (Eugene, OR). Oocyte positive X. laevis frogs were from Xe-
nopus I (Dexter, MI). Collagenase A was purchased from Roche
Molecular Biochemicals (Mannheim, Germany). [35S]GTP-␥-S was
purchased from Amersham Biosciences (Uppsala, Sweden). FLAG
epitope-tagged cDNAs encoding human LPA2 and LPA3 receptors in
pCDNA3 plasmids (Invitrogen, Carlsbad, CA) were a gift from Dr.
Junken Aoki (University of Tokyo, Tokyo, Japan). S1P1 cDNA was a
gift from Dr. Timothy Hla (University of Connecticut, Storres, CT).
PCDNA3.1 expression vector containing S1P5 cDNA was a gift from
Dr. Kevin Lynch (University of Virginia, Charlottesville, VA).
Fig. 1. The synthesis of FAPs. 1, FAP-4, n ϭ 3; 2, FAP-8, n ϭ 7; 3,
FAP-10, n ϭ 9; 4, FAP-12, n ϭ 11; 5, FAP-14, n ϭ 13; 6, FAP-16, n ϭ 15;
7, FAP-18, n ϭ 17; 8, FAP-22, n ϭ 21.

1034
Virag et al.
2a Isolated As a Clear Oil (351 mg, 90% Yield). 1H NMR 30.18(d, JC,P ϭ 6.9 Hz), 31.93, 68.06 (d, JC,P ϭ 6.0 Hz), 69.13 (d, JC,P
(CDCl3) 0.88 (t,
6.9 Hz, 3H,CH3), 1.24 (br s, 10H, ϭ 5.6 Hz), 127.89, 128.47, 128.55, 135.98 (d, JC,P ϭ 6.9 Hz); 31P NMR
OCH2CH2(CH2)5CH3), 1.60 (quintet,
6.9 Hz, 2H, (CDCl3) ␦ 16.83; MS: [M ϩ 23Na]ϩ at m/z 609.3.
OCH2CH2(CH2)5CH3), 3.98 (q, 6.6 Hz, 6.9 Hz, 2H,
␦
J ϭ
J
ϭ
J
ϭ
OCH2CH2(CH2)5CH3), 5.02 (d, J ϭ 2.1 Hz, 2H, OCH2Ar), 5.05 (d, J ϭ
2.4 Hz, 2H OCH2Ar), 7.34 (br s, 10H, 2 ϫ ArH); 13C NMR (CDCl3) ␦
14.09, 22.62, 25.38, 29.06, 29.14, 30.17 (d, JC,P ϭ 6.9 Hz), 31.75, 68.05
(d, JC,P ϭ 6.2 Hz), 69.12 (d, JC,P ϭ 5.5 Hz), 127.90, 128.47, 128.56,
135.97 (d, JC,P ϭ 6.9 Hz); 31P NMR (CDCl3) ␦ 16.83; MS: [M ϩ 23Na]ϩ
at m/z 413.4.
Synthesis of Phosphoric Acid Mono Alkyl Esters (1–8b)
200 mg of 1–8a was dissolved in 30 ml of anhydrous methanol in
a pressure vessel (Fig. 1). The vessel was purged with argon and
ϳ200 mg of 10% Pd/C catalyst was added. The vessel was connected
to a hydrogenation apparatus and a hydrogen atmosphere of ϳ 50 psi
was maintained inside the reaction vessel at room temperature for
8 h. The reaction mixture was then filtered by vacuum through a pad
of methanol-washed celite. Solvent was evaporated under vacuum,
yielding the desired product.
3a Isolated As a Clear Oil (334 mg, 80% Yield). 1H NMR
(CDCl3)
OCH2CH2(CH2)7CH3), 1.58 (quintet,
OCH2CH2(CH2)7CH3), 3.98 (q,
␦
0.88 (t,
J
ϭ
6.9 Hz, 3H,CH3), 1.24 (br s, 14H,
6.9 Hz, 2H,
6.7 Hz, 2H,
J
ϭ
J
ϭ
OCH2CH2(CH2)7CH3), 5.02 (d, J ϭ 2.2 Hz, 2H, OCH2Ar), 5.04 (d, J ϭ
2.3 Hz, 2H OCH2Ar), 7.34 (br s, 10H, 2 ϫ ArH); 13C NMR (CDCl3) ␦
13.56, 22.13, 24.85, 28.57, 28.75, 28.95 (d, JC,P ϭ 1.6 Hz), 29.65 (d,
JC,P ϭ 6.9 Hz), 31.34, 67.52 (d, JC,P ϭ 6.1 Hz), 68.59 (d, JC,P ϭ 5.6
Hz), 126.40, 126.97, 127.35, 127.96 (d, JC,P ϭ 6.6 Hz), 135.47 (d, JC,P
ϭ 6.8 Hz); 31P NMR (CDCl3) ␦ 16.82; MS: [M ϩ 23Na]ϩ at m/z 441.4.
4a Isolated As a Clear Oil (361 mg, 81% Yield). 1H NMR
(CDCl3) ␦ 0.88 (t, J ϭ 7.2 Hz, 3H, CH3), 1.24 (br s, 18 H,
Spectral Characterization of Phosphoric Acid Mono alkyl
Esters (1–8b)
1b Isolated As a Yellow Oil (70 mg, 86% Yield). 1H NMR
(CDCl3/MeOH-d4) ␦ 0.95 (t, J ϭ 7.2 Hz, 3H, CH3), 1.43 (sextet, J ϭ
7.5 Hz, 2H, OCH2CH2CH2CH3), 1.66 (quintet,
J ϭ 6.9, 2H,
OCH2CH2CH2CH3), 3.99 (q, J ϭ 6.6 Hz, 2H, OCH2CH2CH2CH3); 13
C
NMR (CDCl3/MeOH-d4) ␦ 13.71, 19.02, 32.72 (d, JC,P ϭ 7.2 Hz), 66.86
(d, JC,P ϭ 5.5 Hz); 31P NMR (CDCl3/MeOH-d4) ␦ 18.84; MS: [M Ϫ H]Ϫ
at m/z 153.0.
OCH2CH2(CH2)9CH3), 1.60 (quintet,
OCH2CH2(CH2)9CH3), 3.98 (q,
J
ϭ
6.9 Hz, 2H,
6.9 Hz, 2H,
J
ϭ
OCH2CH2(CH2)9CH3), 5.02 (d, J ϭ 2.1 Hz, 2H, OCH2Ar), 5.05 (d, J ϭ
2.1 Hz, 2H, OCH2Ar), 7.34 (br s, 10H, 2 ϫ ArH); 13C NMR (CDCl3) ␦
14.13, 22.69, 25.38, 29.12, 29.35, 29.49, 29.56, 29.63, 30.18 (d, JC,P ϭ
7.0 Hz), 31.92, 68.05 (d, JC,P ϭ 6.1 Hz), 69.12 (d, JC,P ϭ 5.4 Hz),
127.89, 128.46, 128.55, 135.97 (d, JC,P ϭ 6.8 Hz); 31P NMR (CDCl3) ␦
16.84; MS: [M ϩ 23Na]ϩ at m/z 469.1.
2b Isolated As a White/Yellow Tacky Solid (100 mg, 93%
Yield). 1H NMR (CDCl3/MeOH-d4) ␦ 0.89 (t, J ϭ 6.9 Hz, 3H, CH3),
1.29 (br s, 10H, OCH2CH2(CH2)5CH3), 1.67 (quintet, J ϭ 6.9 Hz, 2H,
OCH2CH2(CH2)5CH3),
3.97
(q,
J
ϭ
6.6
Hz,
2H,
OCH2CH2(CH2)5CH3); 13C NMR (CDCl3/MeOH-d4) ␦ 14.18, 22.98,
25.89, 29.57, 29.58, 30.76 (d, JC,P ϭ 7.3 Hz), 32.18, 67.16 (d, JC,P
ϭ
5a Isolated As a Clear Oil (384 mg, 81% Yield). 1H NMR
(CDCl3) ␦ 0.88 (t, J ϭ 6.9 Hz, 3H, CH3), 1.27 (br s, 22 H,
5.2 Hz); 31P NMR (CDCl3/MeOH-d4) ␦ 20.55; MS: [M Ϫ H]Ϫ at m/z
209.1.
OCH2CH2(CH2)11CH3), 1.64 (quintet,
OCH2CH2(CH2)11CH3), 3.98 (q,
OCH2CH2(CH2)11CH3), 5.04 (d, J ϭ 2.1 Hz, 2H, OCH2Ar), 5.06 (d,
J ϭ 2.1 Hz, 2H, OCH2Ar), 7.34 (br s, 10H, 2 ϫ ArH); 13C NMR
(CDCl3) ␦ 13.55, 22.14, 24.85, 25.26, 28.57, 28.80, 28.98 (d, JC,P ϭ 5.2
Hz), 29.12 (m), 29.65 (d, JC,P ϭ 6.9 Hz), 31.38, 32.31, 62.41, 67.50 (d,
J
ϭ
6.8 Hz, 2H,
6.9 Hz, 2H,
3b Isolated As a White/Yellow Tacky Solid (102 mg, 90%
Yield). 1H NMR (CDCl3/MeOH-d4) ␦ 0.89 (t, J ϭ 6.9 Hz, 3H, CH3),
1.28 (br s, 14 H, OCH2CH2(CH2)7CH3), 1.67 (quintet, J ϭ 6.8 Hz, 2H,
J
ϭ
OCH2CH2(CH2)7CH3),
3.97
(q,
J
ϭ
6.9
Hz,
2H,
OCH2CH2(CH2)7CH3); 13C NMR (CDCl3/MeOH-d4) ␦ 12.83, 21.86,
24.79, 28.51 (d, JC,P ϭ 5.9 Hz), 28.79 (d, JC,P ϭ 1.3 Hz), 29.66 (d, JC,P
ϭ 7.2 Hz), 31.12, 65.97 (d, JC,P ϭ 5.6 Hz); 31P NMR (DMSO-d6) ␦
16.55; MS: [M Ϫ H]Ϫ at m/z 236.9.
J
C,P ϭ 6.1 Hz), 68.59 (d, JC,P ϭ 5.6 Hz), 127.34, 127.95 (d, JC,P ϭ 6.8
Hz), 135.48 (d, JC,P ϭ 6.8 Hz); 31P NMR (CDCl3) ␦ 16.85; MS: [M ϩ
23Na]ϩ at m/z 497.2.
4b Isolated As a White Solid (112 mg, 94% Yield). 1H NMR
(CDCl3/MeOH-d4) ␦ 0.88 (t, J ϭ 6.6 Hz, 3H, CH3), 1.27 (br s, 18 H,
6a Isolated As a Clear Oil (427 mg, 85% Yield). 1H NMR
(CDCl3)
OCH2CH2(CH2)13CH3), 1.62 (quintet,
OCH2CH2(CH2)13CH3), 3.99 (q,
␦
0.88 (t,
J
ϭ
6.9 Hz, 3H, CH3), 1.28 (br s, 26H,
6.9 Hz, 2H,
6.9 Hz, 2H,
OCH2CH2(CH2)9CH3), 1.67 (quintet,
OCH2CH2(CH2)9CH3), 3.97 (q,
J
ϭ
6.6 Hz, 2H,
6.6 Hz, 2H,
J
ϭ
J
ϭ
OCH2CH2(CH2)9CH3); 13C NMR (CDCl3/MeOH-d4) ␦ 14.21, 22.98,
25.84, 29.57, 29.67, 29.89, 29.92, 29.96, 29.98, 30.69 (d, JC,P ϭ 7.4
Hz), 32.25, 67.22 (d, JC,P ϭ 5.7 Hz); 31P NMR (CDCl3/MeOH-d4) ␦
21.22; MS: [M Ϫ H]Ϫ at m/z 265.0.
J
ϭ
OCH2CH2(CH2)13CH3), 5.04 (d, J ϭ 2.1 Hz, 2H, OCH2Ar), 5.07 (d,
J ϭ 2.1 Hz, 2H, OCH2Ar), 7.35 (br s, 10H, 2 ϫ ArH); 13C NMR
(CDCl3) ␦ 13.57, 22.15, 24.85, 28.58, 28.83, 28.99 (d, JC,P ϭ 6.8 Hz),
29.17, 29.66 (d, JC,P ϭ 6.9 Hz), 31.39, 67.49 (d, JC,P ϭ 6.1 Hz), 68.59
(d, JC,P ϭ 5.6 Hz), 127.35, 127.95 (d, JC,P ϭ 6.6 Hz), 135.48 (d, JC,P ϭ
6.8 Hz); 31P NMR (CDCl3) ␦ 16.88; MS: [M ϩ 23Na]ϩat m/z 525.3.
7a Isolated As a Hygroscopic White Solid (474 mg, 89%
Yield), mp 32–33°C. 1H NMR (CDCl3) ␦ 0.88 (t, J ϭ 6.9 Hz, 3H,
CH3), 1.25 (br s, 30H, OCH2CH2(CH2)15CH3), 1.60 (quintet, J ϭ 6.9
5b Isolated As a White Solid (105 mg, 85% Yield), mp 58–
60°C. 1H NMR (CDCl3/MeOH-d4) ␦ 0.89 (t, J ϭ 6.9 Hz, 3H, CH3), 1.27
(br s, 22 H, OCH2CH2(CH2)11CH3), 1.64 (quintet, J ϭ 6.8 Hz, 2H,
OCH2CH2(CH2)11CH3),
3.96
(q,
J
ϭ
6.9
Hz,
2H,
OCH2CH2(CH2)11CH3); 13C NMR (CDCl3/MeOH-d4) ␦ 12.66, 21.82,
24.75, 28.48 (d, JC,P ϭ 8.5 Hz), 28.78 (d, JC,P ϭ 1.3 Hz), 28.85, 29.62
Hz, 2H, OCH2CH2(CH2)15CH3), 3.98 (q,
J ϭ 6.9 Hz, 2H, (d, J
C,P ϭ 7.2 Hz), 31.14, 65.88 (d, JC,P ϭ 5.7 Hz); 31P NMR (DMSO-
OCH2CH2(CH2)15CH3), 5.02 (d, J ϭ 2.1 Hz, 2H, OCH2Ar), 5.05 (d, d6) ␦ 16.51; MS: [M Ϫ H]Ϫ at m/z 293.0.
J ϭ 2.1 Hz, 2H, OCH2Ar), 7.34 (br s, 10H, 2 ϫ ArH); 13C NMR
6b Isolated As a White Solid (118 mg, 92% Yield), mp 71–
(CDCl3) ␦ 14.12, 22.70, 25.40, 29.13, 29.38, 29.51, 29.58, 29.68, 29.72, 72°C. 1H NMR (CDCl3/MeOH-d4) ␦ 0.89 (t, J ϭ 6.9 Hz, 3H, CH3), 1.28
30.20 (d, JC,P ϭ 6.9 Hz), 31.94, 68.06 (d, JC,P ϭ 6.1 Hz), 69.14 (d, JC,P (br s, 26 H, OCH2CH2(CH2)13CH3), 1.64 (quintet, J ϭ 6.8 Hz, 2H,
ϭ 5.4 Hz), 127.90, 128.47, 128.55, 136.00 (d, JC,P ϭ 6.8 Hz).; 31P NMR OCH2CH2(CH2)13CH3),
3.96
(q,
J
ϭ
6.9
Hz,
2H,
(CDCl3) ␦ 16.83; MS: [M ϩ 23Na]ϩat m/z 553.3.
OCH2CH2(CH2)13CH3); 13C NMR (CDCl3/MeOH-d4) ␦ 12.77, 21.85,
24.77, 28.48 (d, JC,P ϭ 8.5 Hz), 28.80 (d, JC,P ϭ 1.3 Hz), 28.88, 29.64
8a Isolated As a Hygroscopic White Solid (516 mg, 88%
Yield), mp 43.5–44.5°C. 1H NMR (CDCl3) ␦ 0.88 (t, J ϭ 6.9 Hz, 3H, (d, JC,P ϭ 7.3 Hz), 31.16, 65.94 (d, JC,P ϭ 5.7 Hz); 31P NMR (DMSO-
CH3), 1.25 (br s, 38H, OCH2CH2(CH2)19CH3), 1.60 (quintet, J ϭ 6.9 d6) ␦ 16.51; MS: [M Ϫ H]Ϫ at m/z 321.0.
Hz, 2H, OCH2CH2(CH2)19CH3), 3.98 (q,
OCH2CH2(CH2)19CH3), 5.02 (d, J ϭ 2.4 Hz, 2H, OCH2Ar), 5.05 (d, (CDCl3/MeOH-d4) ␦ 0.89 (t, J ϭ 6.9 Hz, 3H, CH3), 1.27 (br s, 30H,
J ϭ 2.4 Hz, 2H, , OCH2Ar), 7.35 (br s, 10H, 2 ϫ ArH); 13C NMR OCH2CH2(CH2)15CH3), 1.68 (quintet,
6.9 Hz, 2H,
(CDCl3) ␦ 14.13, 22.70, 25.39, 29.12, 29.37, 29.50, 29.57, 29.66, 29.71, OCH2CH2(CH2)15CH3); 3.98 (q, 6.9 Hz, 2H,
J
ϭ
6.6 Hz, 2H,
7b Isolated As a White Solid (104 mg, 79% Yield). 1H NMR
J
ϭ
J
ϭ

FAPs Are LPA Agonists/Antagonists
1035
OCH2CH2(CH2)15CH3); 13C NMR (CDCl3/MeOH-d4) ␦ 14.26, 23.14, were prepared fresh from methanolic stock solutions by diluting
26.01, 29.74, 29.84, 30.06, 30.09, 30.16, 30.87 (d, JC,P ϭ 7.2 Hz), them in perfusion buffer directly before application. LPA samples
32.42, 67.32 (d, JC,P ϭ 5.8 Hz); 31P NMR (CDCl3/MeOH-d4) ␦ 21.69; were prepared the same way from a bovine serum albumin-com-
MS: [M Ϫ H]Ϫ at m/z 349.1.
plexed stock solution.
8b Isolated As a White Solid (98 mg, 71% Yield). 1H NMR
(CDCl3/MeOH-d4) ␦ 0.88(t, J ϭ 6.9 Hz, 3H), 1.26 (br s, 38H,
Lipid Phosphate Phosphatase Assay
OCH2CH2(CH2)19CH3), 1.66 (quintet,
OCH2CH2(CH2)19CH3), 3.97 (q,
OCH2CH2(CH2)19CH3); 13C NMR (CDCl3/MeOH-d4) ␦ 14.22, 23.01,
J
ϭ
6.9 Hz, 2H,
6.6 Hz, 2H,
Lipid phosphate phosphatase (LPP) activity was measured as
described previously (Yokoyama et al., 2002). Competitive inhibition
of FAP-12 on LPP activity was analyzed according to the surface
dilution kinetics model (Carman et al., 1995; Dillon et al., 1997). A
100,000g pellet from mouse fibroblasts overexpressing mouse LPP1
was used as the enzyme source, and the reaction was performed in
the presence of 2 mol% ([lipid]/([lipid] ϩ [Triton X-100]) [32P]LPA (50
␮M, 2 ϫ 104 cpm/assay), 2.5 mM Triton X-100, and indicated amount
of competitors, including FAP-12. The released [32P]phosphate was
extracted and measured.
J
ϭ
25.87, 29.61, 29.71, 29.93, 29.97, 30.04, 30.73 (d, J
ϭ 7.4 Hz),
C,P
32.29, 67.27 (d, JC,P ϭ 5.6 Hz); 31P NMR (CDCl3/MeOH-d4) ␦ 20.66;
MS: [M Ϫ H]Ϫ at m/z 405.1.
Molecular Modeling of the LPA2 Receptor
The inactive and active models of LPA2 were previously developed
in our research group (Sardar et al., 2002). Briefly, the inactive
model of LPA2 was developed by homology modeling using the MOE
(molecular operating environment) program (version 2002:01; Chem-
ical Computing Group, Montreal, ON, Canada) and was based on the
bovine rhodopsin crystal structure (Palczewski et al., 2000). The
active model of the LPA2 receptor was developed via homology mod-
eling using MOE and was based on the validated model of S1P1
(Parrill et al., 2000). Autodock 3.0 (Morris et al., 1996, 1998) was
used to calculate the docked energies for FAP-12 with both the
inactive and active model of LPA2. Automated docking using the
Lamarckian genetic algorithm (Morris et al., 1998) was applied to
generate 100 complexes of FAP with the LPA2 receptor (inactive and
active forms) and LPA with the LPA2 receptor (active form), to
evaluate the binding region of the ligand. The best complex for each
receptor, in terms of energy and binding position of the ligand, was
energy minimized with the MMFF94 force field (Halgren, 1996) to a
root-mean-square gradient of 0.01 kcal/mol/Å to allow the receptor to
adapt to the presence of the ligand. The ligands were then removed
from the minimized receptor-ligand complexes and 100 additional
complexes were generated with Autodock to re-examine the binding
energy after allowing the receptor to acclimatize to the ligand.
Autodock 3.0 allows full flexibility of ligand torsion angles but does
not provide opportunity for protein side chains to adapt to the pres-
ence of a ligand. Thus, the best protein-ligand complex found in an
initial docking run was geometry optimized to allow protein side
chains to optimize in the presence of the ligand. The ligand was then
removed and docked back into the protein to obtain a final docked
energy that better reflects the induced fit that occurs when protein-
ligand binding occurs. If a conformation similar to the one before
minimization was not obtained, the docking run was repeated. When
the preminimized conformation was located, the complex with the
lowest docked energy was chosen as the best complex.
DNA Fragmentation Assay
The topoisomerase inhibitor camptothecin induces DNA fragmen-
tation and apoptosis in rat IEC-6 intestinal epithelial cells that can
be prevented by LPA (Deng et al., 2002). To assess the agonist
properties of FAP-12, IEC-6 cells were exposed to 20 ␮M camptoth-
ecin for 6 h with or without FAP-12 (10 ␮M) or LPA (10 ␮M)
pretreatment (15 min before camptothecin) and DNA fragmentation
was measured using an enzyme-linked immunosorbent assay
method with the cell death detection enzyme-linked immunosorbent
assay kit from Roche (Indianapolis, IN). Briefly, cells were harvested
and lysed in DNA lysis buffer for 30 min and centrifuged at 200 rpm
for 10 min. An aliquot of the supernatant was incubated with anti-
histone-biotin plus anti-DNA peroxidase conjugated antibody in 96-
well streptavidin-coated plates on a shaker for 2 h. After washing
with the incubation buffer, 100 ␮l of substrate buffer was added to
each well and incubated for an additional 5 to 10 min. DNA absor-
bance was read at 405 nm in a microplate reader. Duplicates of the
samples were used to quantify protein using the bicinchoninic acid
assay kit from Pierce (Rockford, IL). DNA fragmentation was ex-
pressed as absorbance units per microgram of protein per minute.
RT-PCR Analysis of LPA Receptor Expression
To assess the expression of LPA receptor subtypes in N1E-115
cells, RT-PCR analysis was performed using a primer set and PCR
protocol described in previous publications (Tigyi et al., 1999; Fischer
et al., 2001).
Data Analysis
The significance of differences between the groups was deter-
mined using the Student’s t test. Values were considered signifi-
cantly different at p Ͻ 0.05. The antagonists’ binding constant, Ki,
Cells and Cell Culture
was estimated by the method of Cheng (2002), as follows: Ki ϭ IC50/(1
nH
ϩ ([LPA]/EC50
) ), where IC50 is the half-effective concentration of
Oocytes were harvested and treated as described earlier (Tigyi et
al., 1999). RH7777 cells, stably expressing human LPA2 receptors,
were provided by Dr. Kevin Lynch (University of Virginia, Char-
lottesville, VA). All cell lines were maintained in Dulbecco’s modified
Eagle’s medium, containing 10% fetal bovine serum and 2 mM glu-
tamine, containing 250 ␮g/ml G418 for the stable transfectants.
RH7777 cells stably expressing LPA1 and LPA3 receptors have been
generated by our group and characterized elsewhere (Fischer et al.,
2001). N1E-115 and IEC-6 cells were purchased from American Type
Culture Collection (Manassas, VA).
the inhibitor, EC50 is the half-effective concentration of the agonist
(LPA), nH is the slope function (Hill- or cooperativity-exponent) in
the logistic function that describes the agonist’s activation curve, and
[LPA] is the concentration of LPA against which the antagonist was
being tested.
Results
The synthetic pathway used to generate FAPs is shown in
Fig. 1. Molecular structures were verified by NMR and mass
spectrometry, and all spectral data were consistent with the
assigned structures (see Materials and Methods).
FAPs with hydrocarbon chain lengths of 4, 8, 12, 18, and 22
were tested for their ability to enhance or inhibit LPA-in-
duced ClϪ currents in X. laevis oocytes. None of the FAP
compounds activated ClϪ currents in the oocytes when ap-
Cellular Assays
Electrophysiological recording from X. laevis oocytes was done
using the standard two-electrode voltage clamp technique (Tigyi et
al., 1999). Monitoring of intracellular Ca2ϩ changes using Fura-2
acetoxymethyl ester fluorescent indicator (Tigyi et al., 1999) and
ligand-induced [35S]GTP-␥-S binding assays (Parrill et al., 2000) was
performed as described in our previous publications. FAP samples plied up to 10 ␮M (data not shown). In contrast, all FAPs

1036
Virag et al.
inhibited LPA-induced ClϪ currents with varying potency. high- and low-affinity binding sites (Guo et al., 1996; Liliom
Dose-inhibition measurements revealed a strong correlation et al., 1996b). When the LPA activation curve was measured
between chain length and inhibitory action (Fig. 2A). The in the presence of FAP-12, it could be fitted by a simple
lowest IC50 was observed using FAP-12 and FAP-8 (IC50
ϭ
Langmuir isotherm, suggestive of only one binding site (Fig.
2.4 Ϯ 1 and 6 Ϯ 1 nM, respectively, against 5 nM oleoyl-LPA). 2B). The activation curves measured in the absence and
FAPs with shorter or longer hydrocarbon chains than 12 presence of the inhibitor run parallel at high LPA concentra-
were less effective in inhibiting the LPA responses.
When coapplied with LPA, FAP-12 shifted the LPA dose- affinity LPA binding site.
To characterize the effects of the FAP compounds in a
tions. Consequently, FAP-12 seems to inhibit only the high-
response curve to the right, suggesting a competitive mech-
anism for the antagonist activity (Fig. 2B). In X. laevis oo- mammalian system, we chose the RH7777 rat hepatoma cell
cytes the LPA dose-response curve has been shown to be line, which does not respond to LPA or S1P in a variety of
biphasic and has been attributed to the presence of both cellular assays, including Ca2ϩ mobilization, and does not
express any of the known LPA and S1P receptors as moni-
tored by RT-PCR (Zhang et al., 1999). In cells stably express-
ing LPA1, FAP-12 weakly inhibited LPA-induced Ca2ϩ-mo-
bilization (Fig. 3). In contrast, FAP-12 alone did not elicit
intracellular Ca2ϩ transients when applied up to 10 ␮M (data
not shown). Surprisingly, LPA2-expressing cells showed a
dose-dependent increase in intracellular Ca2ϩ mobilization
in response to FAP-10 and FAP-12, with EC50 values of 3.7 Ϯ
0.2 ␮M and 700 Ϯ 22 nM, respectively (Fig. 4A). FAP-14 was
found to be a weak agonist, when applied at 10 ␮M. In
contrast, no other FAP analog elicited Ca2ϩ mobilization, or
inhibited the LPA response when applied in concentrations
up to 10 ␮M. The maximal response to FAP-12 was 50% of
that elicited by LPA, suggesting that FAP-12 was a partial
agonist of the LPA2 receptor. When a concentration of 10 ␮M
was applied only FAP-10, FAP-12 and FAP-14 showed sig-
nificant agonist activity (Fig. 4B) with FAP-10 being the most
efficacious.
Computational docking studies were used to evaluate the
Fig. 2. Pharmacological characterization of the FAPs on LPA-elicited ClϪ
currents in X. laevis oocytes. A, 5 nM LPA 18:1 mixed with increasing
concentrations of FAP-4 (f), -8 (E), -12 (Œ), -18 (F), or -22 (‚) was
superfused over oocytes and peak ClϪ current amplitudes were mea-
sured. 100% represents the peak amplitude of the ClϪ current activated
by 5 nM LPA, and 0% corresponds to baseline holding current. The bar
graph represents the IC50 values for each FAP compound. IC50 values
were 423 Ϯ 150, 6 Ϯ 1, 2.4 Ϯ 1, 26 Ϯ 12, and 32 Ϯ 16 nM for FAP-4, -8,
-12, -18, and -22, respectively. Data points represent the average of at
least three measurements Ϯ S.D. B, effect of FAP-12 on the LPA dose-
response curve in X. laevis oocytes. Oocytes were treated with increasing
concentrations of LPA (18:1) (f) alone, or mixed with 30 nM FAP-12 (F).
100% represents the peak amplitude for the maximal LPA-activated ClϪ
current, and 0% corresponds to baseline holding current. EC50 values
were 30 Ϯ 8 and 300 Ϯ 40 nM for LPA and LPA ϩ 30 nM FAP-12,
respectively. Data points represent the average values of at least three
experiments Ϯ S.D.
Fig. 3. Inhibition of LPA-induced Ca2ϩ mobilization by FAP-12 in
RH7777 cells expressing LPA1 receptors. Cells were exposed to 250 nM
LPA (18:1) mixed with increasing concentrations of FAP-12. Peak areas
of Ca2ϩ responses were measured. 100% represents the peak area of the
Ca2ϩ transient elicited by 250 nM LPA, and 0% corresponds to baseline
fluorescence. Ca2ϩ mobilization was measured as the ratio of fluorescence
values at 340 and 380 nm. Data points represent the average from at
least three experiments Ϯ S.D. Inset, representative time-courses for
Ca2ϩ transients induced by 250 nM LPA alone or by coapplication of 250
nM LPA with 10 ␮M FAP-12. R is the ratio of fluorescence measured at
340 and 380 nm.

FAPs Are LPA Agonists/Antagonists
1037
interactions of LPA and FAP with LPA2. When LPA (18:1) (18:1) was in the transmembrane domain with the phosphate
was docked against the active LPA2 model, the lowest docked group of LPA forming ion pairs with arginine 107 and lysine
free energy was Ϫ15.08 kcal/mol. The binding pocket of LPA 278 (Fig. 5A) and the 2-hydroxyl group of LPA hydrogen
bonding with glutamine 108.
FAP-12 was docked against both the active and inactive
models of LPA2. The lowest docked energy obtained upon
evaluation of FAP-12 with the inactive form of LPA2 was
Ϫ9.98 kcal/mol. A more favorable docked energy, Ϫ12.44
kcal/mol, was obtained when FAP-12 was docked against the
active form of LPA2, consistent with the agonist effect ob-
served experimentally. The binding pocket of FAP-12 was
located in the transmembrane domain and ion pairs were
observed between the phosphate and two cationic residues,
arginine 107 and lysine 278 of LPA2 (Fig. 5B). These inter-
actions are consistent with our previous studies on the bind-
ing of LPA to the LPA receptors (Wang et al., 2001; Sardar et
al., 2002).
Docking studies between FAP-8, FAP-10 (Fig. 5C), FAP-14,
and FAP-18 were performed using the active LPA2 model,
with resulting docked energies of Ϫ9.17, Ϫ10.46, Ϫ12.06, and
Ϫ10.84 kcal/mol, respectively. The binding mode observed for
these structures was similar to that observed for FAP-12. All
four of these compounds had lower (less favorable) docked
energies than FAP-12, as expected based on the experimental
observation that FAP-12 had the lowest EC50. However, the
relative energies did not correlate further with the experi-
mentally observed receptor activation. Several factors prob-
ably contribute to this discrepancy. First, energies from dock-
ing studies are most reflective of binding affinity rather than
Fig. 4. A, dose-dependent activation of LPA2 by FAPs. Increasing con-
centrations of LPA (f), FAP-10 (ꢀ), or FAP-12 (F) were applied, and the
peak areas of Ca2ϩ responses were measured in RH7777 cells stably
expressing LPA2. The EC50 values for FAP-10 and FAP-12 were 700 Ϯ 22
nM and 3.7 Ϯ 0.2 ␮M, respectively. 100% represents the peak area for
maximal LPA-activated Ca2ϩ mobilization, and 0% corresponds to base-
line. Data points represent the average of at least three measurements Ϯ
S.D. Inset, comparison of time courses of maximal Ca2ϩ responses to LPA
(18:1), FAP-10, and FAP-12 in LPA2-expressing RH7777 cells. LPA, FAP-
10, or FAP-12 (30 ␮M) was applied. R is the ratio of fluorescence mea-
sured at 340 and 380 nm. B, chain length dependence of FAP agonist
activity in RH7777 cells expressing LPA2. FAP compounds were applied
at 10 ␮M, and peak areas of Ca2ϩ responses were measured. Data points
represent the average of at least three measurements Ϯ S.D. Inset,
comparison of time courses of Ca2ϩ transients elicited by 10 ␮M FAP-8,
-10, -12, and -14. R is the ratio of fluorescence measured at 340 and 380
nm.
Fig. 5. Model of the LPA2 receptor docked with LPA (18:1) (A), FAP-12
(B), and FAP-10 (C). LPA2 is shown as a ribbon model (side view, top)
with the ligands LPA (18:1) (blue), FAP-12 (green), and FAP-10 (yellow)
as space-filling models. Residues R 107 (magenta), Q 108 (teal), and K
278 (orange) are also shown as space-filling models. Close-up view (top
view, bottom) of critical interactions between LPA2 residues and the
phosphate group of the three ligands is shown.

1038
Virag et al.
receptor activation. Second, binding is governed by the free
energy change that occurs when a ligand moves from one
environment (often aqueous solution) to another (a protein
binding site). The docked energies represent only the enthal-
pic part of the free energy. The entropic part of the free
energy, which consists of changes in conformational freedom
and solvation effects, may be an important contributor to the
differences in FAP compound activity.
RH7777 cells stably expressing the LPA3 receptor did not
respond to any of the FAPs by Ca2ϩ mobilization when ap-
plied up to 10 ␮M (data not shown). However, FAP-12 dose
dependently inhibited LPA-induced Ca2ϩ mobilization with a
Ki of 90 nM (Fig. 6A). Coapplication of 300 nM FAP-12 with
LPA shifted the dose-response curve of LPA to the right,
increasing the EC50 value from 300 Ϯ 14 to 1000 Ϯ 30 nM
and suggesting a competitive type of inhibition (Fig. 6B).
All FAPs were tested for their ability to inhibit LPA-elic-
ited Ca2ϩ-mobilization in cells expressing LPA3. Applied at a
concentration of 200 nM, FAP-12 was the most effective (60%
inhibition), followed by FAP-10, -14, and -18 (ϳ25% inhibi-
tion) (Fig. 6C). FAP-4, -8, -16, and -22 did not inhibit the LPA
response significantly at this concentration.
To determine whether the effects of FAPs were selective to
LPA receptors, we examined the most potent analog, FAP-12,
on responses elicited by various mammalian G-protein cou-
pled receptors. In oocytes expressing poly(A)ϩ mRNA from
rat brain, responses elicited by serotonin (10 ␮M), kainate
(100 ␮M), and glutamate (10 ␮M), which are mediated
through the inositol trisphosphate-Ca2ϩ pathway, were
tested for interference by FAPs. In the presence of 10 ␮M
FAP-12, these responses relative to control were 103 Ϯ 8,
95 Ϯ 12, and 106 Ϯ 7% (n ϭ 3), respectively. The lack of
inhibition indicates that FAP-12 did not inhibit these neuro-
transmitter receptors or the second-messenger systems me-
diating Ca2ϩ-activated ClϪ currents. Additionally, in
RH7777 cells, FAP-12 (10 ␮M) did not affect ATP-induced (1
␮M) Ca2ϩ mobilization (101 Ϯ 10% of ATP alone control, n ϭ
3) indicating that in this mammalian cell line, just as in X.
laevis oocytes, the compound did not interfere with ligand-
induced Ca2ϩ transients.
The effects of FAP-12 were also tested on S1P receptors.
GTP-␥-S loading assays were performed on RH7777 cells
stably expressing S1P1. FAP-12, when coapplied with S1P
(300 nM) up to 10 ␮M, neither enhanced nor inhibited the
Fig. 6. A, FAP-12 inhibits LPA (18:1)-induced Ca2ϩ mobilization in
RH7777 cells stably expressing LPA3 in a dose-dependent manner. LPA
(250 nM; 18:1) was mixed with increasing concentrations of FAP-12. The
Ki value was 90 nM. Data points represent the average of at least three
measurements Ϯ S.D. Inset, time courses of Ca2ϩ mobilization after
application of 250 nM LPA alone or together with 1 ␮M FAP-12. R is the
ratio of fluorescence measured at 340 and 380 nm. B, FAP-12 shifts the
LPA dose-response curve to the right. Cells were exposed to increasing
concentrations of LPA (f) or mixed with 300 nM FAP-12 (F). EC50 values
were 300 Ϯ 14 nM for LPA and 1000 Ϯ 30 nM for LPA ϩ 300 nM FAP-12.
Data points represent the average of peak areas from at least three
measurements Ϯ S.D. Inset, time courses of Ca2ϩ mobilization after
application of 1 ␮M LPA alone or together with 300 nM FAP-12. R is the
ratio of fluorescence measured at 340 and 380 nm. C, the relationship
between FAP chain length and inhibition of LPA-elicited Ca2ϩ mobiliza-
tion through LPA3 in RH7777 cells. Each FAP compound (200 nM) was
mixed with 250 nM LPA (18:1), and the elicited Ca2ϩ peak areas were
measured. Data points represent the average of at least three measure-
ments Ϯ S.D. Inset, time courses of Ca2ϩ mobilization after coapplication
of 250 nM LPA with 200 nM FAP-4, -10, and -12. R is the ratio of
fluorescence measured at 340 and 380 nm.

FAPs Are LPA Agonists/Antagonists
1039
TABLE 1
Effect of FAP-12 on S1P receptors
FAP-12 Alone
S1P ϩ FAP-12
FAP-12
Response
S1P
FAP-12
Response
␮M
% of vehicle
␮M
␮M
% of S1P
S1P1
S1P2
S1P3
S1P5
10
10
10
10
95 Ϯ 5a
100 Ϯ 0b
100 Ϯ 0b
90 Ϯ 5a
0.3
0.3
1
10
1
97 Ϯ 4a
99 Ϯ 11b
104 Ϯ 13b
101 Ϯ 2a
1
0.3
10
a Results were obtained using ͓35S͔GTP-␥ -S binding assay.
b Results were obtained using intracellular Ca2ϩ-monitoring.
substantial S1P-induced GTP loading. FAP-12 alone did not et al., 2001; Sardar et al., 2002). Based on computational
induce GTP loading in these cells (data not shown). Similar modeling of the ligand-receptor interactions, we deduced and
results were obtained with RH7777 cells transiently trans- partially validated a model for receptor activation. The model
fected with S1P5 (Table 1). S1P elicits dose-dependent Ca2ϩ assigned distinct functions to the polar headgroup and the
transients in RH7777 cells expressing S1P2 or S1P3. FAP-12 hydrophobic tail within the LPA pharmacophore. In this
did not induce Ca2ϩ mobilization in RH7777 cells transiently model, the phosphate headgroup interacts with two posi-
transfected with S1P2 or S1P3, when applied in concentra- tively charged conserved residues in the third and seventh
tions as high as 10 ␮M, nor did it affect S1P-elicited Ca2ϩ transmembrane helices (Wang et al., 2001), whereas the
transients (Table 1). We could not test FAP-12 on S1P4 re- hydrocarbon tail interacts with hydrophobic side-chains of
ceptors, because, in our hands, this receptor did not respond amino acid residues lining the interhelical pocket (Sardar et
to S1P in the GTP-␥-S loading or Ca2ϩ-mobilization assays. al., 2002). These hydrophobic interactions are predicted to be
To test whether FAP-12 interacts with LPP, a key enzyme necessary for activation of the receptor (Fischer et al., 2001;
of LPA degradation, enzymatic activity was measured in the Wang et al., 2001). Specific recognition of S1P versus LPA is
absence or presence of FAP-12. LPP activity decreased to 73, achieved through hydrogen bonding between the hydroxyl
58, and 39% of control, when FAP-12 was added in 2-, 4-, and group of LPA and a glutamine or by ion pairing between the
8-fold excess, respectively (data not shown). This result sug- amino group of S1P and a glutamate in the third transmem-
gests that FAP12 also interferes with the LPP-mediated deg- brane helix conserved in the corresponding receptor subfam-
radative pathway of LPA.
ilies (Wang et al., 2001). The two-point pharmacophore is
To confirm the agonist properties of FAP-12 and FAP-10 on consistent with the experimentally established structure-ac-
LPA2, these compounds were tested in N1E-115 neuroblas- tivity relationships of LPA (Lynch and Macdonald, 2002;
toma cells for Ca2ϩ mobilization effect (Fig. 7A). N1E-115 Sardar et al., 2002).
cells express only LPA2 receptor transcripts (Fig. 7B) and
Our group (Fischer et al., 2001) and Dr. Lynch’s group
respond to LPA with a transient elevation in [Ca2ϩ]i. FAP-12 (Heise et al., 2001) have reported on LPA antagonists that
and FAP-10 both elicited Ca2ϩ transients in these neuroblas- show receptor subtype selectivity. A systematic screening of
toma cells, confirming their agonist properties in a cell line 2-OH-substituted N-acyl ethanolamide phosphates led to the
that endogenously expresses only the LPA2 receptor subtype. discovery of a benzyl-4-oxybenzyl derivative of ethanolamine
To further characterize the agonist properties of FAP-12 (Fig. phosphate, of which the S-enantiomer exhibited selective
7C), we examined its effect in an apoptosis protection assay antagonism of LPA1 over LPA3, whereas it did not affect the
using IEC-6 cells. These cells express predominantly LPA2 LPA2 receptor (Heise et al., 2001). We found that dioctyl
along with lesser amounts of LPA1 (Deng et al., 2002). In phosphatidic acid and dioctylglycerol pyrophosphate were
these cells, FAP-12 applied at 10 ␮M elicited a significant weak antagonists of LPA1 and strong antagonists for LPA3,
decrease in camptothecin-induced DNA fragmentation that whereas long-chain analogs (18:1) were not inhibitors of LPA
was comparable with the effect of LPA (10 ␮M). The results receptors (Fischer et al., 2001). It is important to note that
obtained from N1E-115 and IEC-6 cells lend strong support short-chain LPA (8:0) was neither an agonist nor an antag-
to the LPA-like agonist properties of FAPs in cell lines that onist in this system (Fischer et al., 2001). Both dioctylglyc-
express the LPA2 receptor subtype.
erol pyrophosphate and (S)-2-benzyl-4-oxybenzyl-N-acyl eth-
anolamide phosphate have a negatively charged phosphate
head group. They also have two hydrocarbon side chains,
unlike the physiological ligand, LPA. Therefore, both the
Discussion
The phospholipid growth factors LPA and S1P are involved length and size of the hydrophobic tail seems to affect the
in numerous physiological and pathological processes, in- ability to activate or inhibit LPA receptors. Based on the
cluding regulation of cell proliferation and differentiation, two-point pharmacophore, we hypothesized that modifica-
apoptosis, Ca2ϩ-homeostasis (Goetzl et al., 2000; Tigyi, 2001) tions within the hydrophobic tail of the ligand might have
and tumor cell invasion (Umezu-Goto et al., 2002), and ath- profound effects in determining agonist or antagonist behav-
erosclerosis (Siess et al., 1999). Given that most cells express ior and therefore might allow identification of molecules with
multiple PLGF receptor subtypes, the lack of subtype-specific selective antagonistic properties.
agonists and antagonists for LPA receptors remains a limit-
Our earlier work (Bittman et al., 1996; Liliom et al., 1996),
ing factor for the PLGF field. In an effort toward the rational as well as of that of others (Hooks et al., 1998; Heise et al.,
design of such ligands, structural models of the PLGF recep- 2001), has shown that the glycerol backbone can be replaced
tors have been developed recently (Parrill et al., 2000; Wang by serine, tyrosine, or ethanolamine but maintain the LPA

1040
Virag et al.
synthesized a series of FAP molecules with carbon chain
lengths from 4 to 22 and characterized their effects on indi-
vidual LPA and S1P receptor subtypes.
Pharmacological analysis of the FAP series supports our
hypothesis that the ionic headgroup attached to a hydropho-
bic tail are sufficient to make them ligands for all three LPA
receptors, although with markedly different efficacies, poten-
cies, and selectivities. FAP-10 and FAP-12 were specific ago-
nists for LPA2 with EC50 values of 3.7 Ϯ 0.2 ␮M and 700 Ϯ
22 nM, respectively, and specific antagonists for LPA3.
FAP-12 also weakly inhibited LPA1. The decyl and dodecyl
chain seem to be unique, which suggests that LPA2 differs
from the other two receptors in that it is activated by these
relatively short-chain analogs. FAP molecules with carbon
chain lengths shorter than 10 or longer than 14 did not affect
LPA2 and were weaker inhibitors of LPA3 than FAP-12.
Thus, the present results emphasize that acyl chain length
plays a significant role in the ligand properties of LPA-like
pharmacophores and establishes a distinguishing role in li-
gand recognition between the different LPA receptor sub-
types. Furthermore, FAPs lack the glycerol backbone; thus,
the present findings provide compelling evidence that it is
not required for ligand activity. These observations are in
complete agreement with earlier structure-activity studies in
which LPA receptors showed a high degree of tolerance to
analogs with serine, tyrosine, or ethanolamine in place of the
glycerol backbone (Sugiura et al., 1994; Jalink et al., 1995;
Lynch et al., 1997; Hooks et al., 1998).
X. laevis oocytes express three pharmacologically distin-
guishable receptors for LPA (Liliom et al., 1996b; Fischer et
al., 1998). This heterogeneity is reflected in the dose-re-
sponse curve, which is best described by assuming multiple
sites with different high and low affinity for LPA (Guo et al.,
1996; Liliom et al., 1996b). Our experiments indicate that
FAPs block only the high-affinity site (Fig. 2B). Thus far, two
high-affinity LPA sites have been identified in oocytes: the
PSP24 (Guo et al., 1996) and LPA1 receptors (Kimura et al.,
2001). Which of these is inhibited by FAPs remains unclear.
The human ortholog of the LPA1 receptor was only weakly
inhibited by FAP-12 in concentrations in the high micromo-
lar range (Fig. 3). The highly conserved nature of mamma-
lian and X. laevis LPA1 tends to argue against the hypothesis
that the inhibitor would target LPA1. On the other hand,
overexpression of X. laevis LPA1 in X. laevis oocytes aug-
mented the endogenous LPA response (Kimura et al., 2001),
whereas the human ortholog did not (Yokoyama et al., 2002).
Interestingly, the chain length-dependence of the inhibitory
potencies of FAPs in X. laevis oocytes (Fig. 2A) and in LPA3-
Fig. 7. Agonist properties of FAPs in cells that endogenously express expressing RH7777 cells (Fig. 6C) is very similar despite the
LPA2. A, time course of a representative experiment monitoring intracel-
consensus that oocytes do not express an LPA3 receptor sub-
type (Kimura et al., 2001).
lular Ca2ϩ concentration in N1E-115 cells. FAP-10 (30 ␮M), -12 (10 ␮M),
and LPA 18:1 (5 ␮M) were applied consecutively to N1E-115 cells and the
resulting changes in [Ca2ϩ
] were recorded. B, RT-PCR study of LPA
FAPs did not activate or inhibit S1P receptors, suggesting
that this cluster within the EDG receptor family is funda-
mentally different from the LPA receptor cluster despite
several similarities in ligand recognition. Based on earlier
structure-activity measurements, a free amino group on the
sphingoid backbone seems to be necessary for ligand recog-
nition of the S1P receptors (Van Brocklyn et al., 1998). This
i
receptor transcripts in N1E-115 cells. M, molecular weight marker; 1,
LPA1; 2, LPA2; 3, LPA3; 4, negative control (no template); 5, beta actin. C,
FAP-12 mimics the antiapoptotic effect of LPA in IEC-6 cells. Camptoth-
ecin (20 ␮M) induces apoptosis accompanied by DNA fragmentation
within 6 h. LPA (10 ␮M) or FAP-12 (10 ␮M) both significantly reduced
camptothecin-induced DNA fragmentation (mean Ϯ S.E.M., n ϭ 3).
mimetic effect of these lipid phosphate analogs. Fatty alcohol group is necessary for ligand binding (Parrill et al., 2000) and
phosphates provide the simplest set of easy to synthesize provides selectivity for these receptors to distinguish S1P
analogs required to delineate the minimal structural require- from LPA (Wang et al., 2001). The absence of this free amino
ment for a ligand using this hypothesis. For this reason we group in the FAP structure might explain the lack of effect of

FAPs Are LPA Agonists/Antagonists
1041
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these molecules on S1P receptors. The agonist and antago-
nist effects of FAP-12 seem to be specific for LPA receptors
because it did not modify the function of other heterologously
expressed GPCRs in the X. laevis bioassay or RH7777 cells.
The agonist properties of FAP-12 were confirmed in N1E-115
and IEC-6 cells that endogenously express LPA2 receptor
subtype (Fig. 7). Thus, the present data identify FAP-12 as
the first LPA2-selective agonist. Computational docking
studies of FAP-8, -10, -12, -14, -18, and LPA against the
active form of LPA2 suggest that the greatest affinity for all
structures occurs in an overlapping region in the receptor.
The energies observed for FAP-12 binding to the active and
inactive models of the LPA2 receptor, Ϫ12.44 kcal/mol versus
Ϫ9.98 kcal/mol, are consistent with the experimental obser-
vation that FAP-12 is a partial agonist and thus interacts
more strongly with the active conformation of the LPA2 re-
ceptor. The lowest docked energy was observed for FAP-12
binding to the active model, a finding consistent with the
observed lowest EC50 value (700 Ϯ 22 nM) for this structure.
Thus, the present results provide new refinements to our
computational models.
FAP-12 inhibits LPA degradation by LPP, indicating that
the simplified structure of the FAP molecule is also recog-
nized by a key enzyme in the established degradation path-
way of LPA. This observation suggests that FAP-12 will have
at least three molecular targets: the LPA3 and LPA2 recep-
tors and LPP enzymes.
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palmitoyl-tyrosine phosphoric acids are selective competitive antagonists of the
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The two-point pharmacophore for LPA receptor activa-
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models of the PLGF receptors, makes the rational design of
receptor subtype-selective agonists and antagonists possi-
ble, opening an important new avenue of research in the
PLGF field. This approach identified FAPs as selective
inhibitors of LPA3 and the first subtype-specific agonists of
LPA2 receptors. Although the compounds identified here
are not themselves likely to be useful clinically, they could
serve as lead compounds for further development. More-
over, the differential effects of the acyl chain length deriv-
atives of FAPs point to an important concept in designing
subtype-selective reagents.
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Address correspondence to: Gabor Tigyi, M.D., Ph.D., Department of Phys-
iology, The University of Tennessee Health Science Center, 894 Union Avenue,
Memphis, TN 38163. E-mail: gtigyi@physio1.utmem.edu
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