ACCELERATED COMMUNICATION Characterization of a Receptor Subtype-Selective Lysophosphatidic Acid Mimetic

Despite an intriguing cell biology and the suggestion of a role in pathophysiological responses, the mechanism of action of such lipid phosphoric acid mediators as lysophosphatidic acid (LPA) remains obscure, in part because of an underdeveloped medicinal chemistry. We report now the agonist activity of a synthetic phospholipid in which the glycerol backbone of LPA is replaced by L-serine. Like LPA, the L-serine-based lipid mobilizes calcium and inhibits activation of adenylyl cyclase in the human breast cancer cell line MDA MB231. Treatment with LPA desensitizes MDA MB231 cells to subsequent application of the L-serine compound; when the order of application is reversed, however, the L-serine compound does not prevent calcium mobilization by LPA, which might indicate the existence of two LPA receptors in these cells. The analogous D-serine-based phospholipid was distinctly less potent than the L-isomer in those assays; this finding demonstrates stereoselectivity by an LPA receptor. Unlike LPA, the L-serine-based lipid does not evoke a chloride conductance in Xenopus laevis oocytes, but injection of poly(A) RNA from HEK 293 cells confers this phenotype on the oocyte. The latter result has practical importance in that it allows use of the frog oocyte for expression cloning of an LPA receptor DNA, an assay system made problematic by the oocyte’s strong endogenous response to LPA. The ability of LPA to mediate a wide range of responses in many cell types has been demonstrated repeatedly during the past decade. In a variety of cultured cells, these responses include calcium mobilization (Jalink et al., 1990), inhibition of adenylyl cyclase activation (van Corven et al., 1992), and formation of focal adhesions (Ridley and Hall, 1992). Less is known about LPA’s effect on tissues, but LPA has been shown to induce platelet aggregation (Watson et al., 1985), smooth muscle contraction (Tokumura et al., 1991; Tokumura et al., 1980) and skin thickening (Piazza et al., 1995). It is assumed widely that LPA signals cells at least in part through one or more G protein-coupled receptors. Although there exists a well-developed cell biology and a good understanding of intracellular signaling events in cultured cells after LPA activation, our understanding of LPA’s interaction with its receptor(s) is primitive. This is partly because of a lack of LPA-based medicinal chemistry, including LPA mimetics and receptor antagonists. Sugiura et al. (1994) synthesized a series of lipid phosphoric acids and investigated their effects on aggregation of human platelets. Among the compounds described in their seminal paper (Sugiura et al., 1994) were N-palmitoyl serine phosphoric acid (NASPA) and N-palmitoyl tyrosine phosphoric acid, both of which they found to act as insurmountable antagonists of LPA-induced platelet aggregation. Subsequently, Liliom et al. (1996a) synthesized the same compounds and demonstrated that both act as competitive antagonists of the chloride conductance elicited when LPA is applied to the surface of Xenopus laevis oocytes. They synthesized both stereoisomers and found them to be equipotent. In light of the blocking activity ascribed to these phosphoamino-acid-based compounds, we considered using them as This work was supported by grants from the National Institute of General Medical Sciences: R01-GM52722 (K.R.L., T.L.M.), R29-GM52387 (M.E.D.), and T32-GM08136, predoctoral traineeship (S.B.H.). C.H. is supported by Innovative Medizinische Forschung, HO-1–6-11/96–8. ABBREVIATIONS: LPA, lysophosphatidic acid; NOEPA, N-oleoyl ethyl phosphoric acid; NASPA, N-acyl serine phosphoric acid; TRAP, thrombin receptor activating peptide; HPLC, high performance liquid chromatography; BSA, bovine serum albumin; HEK, human embryonic kidney; ICl(Ca), calcium-induced chloride current; EDG-2, endothelin differentiation gene-2. 0026-895X/98/020188-07$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 53:188–194 (1998). 188 at A PE T Jornals on M ay 9, 2017 m oharm .aspeurnals.org D ow nladed from lead structures that might, through subsequent synthetic manipulation, be optimized to develop a high affinity LPA receptor antagonist for a mammalian LPA receptor. Concentrating on the serine-based compound, we duplicated readily the synthetic route and antagonist activity on frog oocytes reported by Liliom et al. (1996a) and Bittman et al. (1996). To our surprise, however, we found that L-NASPA is a potent agonist on cultured mammalian cells with regard to both calcium mobilization and inhibition of adenylyl cyclase activation, whereas D-NASPA is less potent in these assays. With this article, we present the results of our characterization of these compounds. Materials and Methods Compound synthesis and purification. Synthesis of the Dand Lisomers of NASPA was conducted exactly as described by Bittman et al. (1996), except that we substituted 30% hydrogen peroxide (aqueous) for 3-chloroperoxybenzoic acid in oxidizing the phosphite intermediate. All reactions were carried out under inert atmosphere, all solvents were purified by filtration through alumina (activity I) and the reaction products were purified on 230–400 mesh silica gel. The purities of both Dand L-NASPA were determined using normal phase HPLC with an analytical Microsorb-MV column (4.6 3 250 mm; Rainin/Varian Instruments, Walnut Creek, CA) packed with 5-mm silica (1000-nm pore size). A Waters two-pump gradient system was used with a flow rate of 1 ml/min. The column effluent was monitored with an evaporative light scattering detector (Alltech Model 500; Alltech Associates, Inc., Deerfield, IL). The solvent system consisted of solvent A [composed of HCCl3/MeOH/NH4OH (30%) in a ratio of 80:19.5:0.5] and solvent B [composed of HCCl3/MeOH/ H2O/NH4OH (30%) in a ratio of 34.5:55:10:0.5]. The samples were prepared for HPLC in 1:1 chloroform/methanol at 5 mM concentrations. Injections of 10 ml were made with a U6K injector (Waters, Milford, MA) and eluted over an isocratic gradient 90:10 A/B over 5 min and a linear gradient to 30:70 A/B over 10 min followed by isocratic application of 30:70 A/B for 30 min. The detector evaporator tube was set to 92°, the exhaust tube to 52°, and the nitrogen gas flow was set to 2.45 standard liters per minute. The retention times of both the pure Dand L-NASPAs were 33 min under these conditions. Compound preparation. Compounds were quantified by colorimetric phosphate analysis (Kingsley and Feigenson, 1979) and freed of organic solvents in an evacuated microcentrifuge (SpeedVac; Savant Instruments, Farmingdale, NY). The compounds were dissolved in water containing 0.1% fatty acid-free BSA and serially diluted in the same solvent. To manipulate solutions containing large concentrations ($ 100 mM) of compounds, we used water containing 1.0% fatty acid-free BSA. Cell culture. Lipid phosphoric acids were tested on the human breast cancer cell line MDA MB231. This cell line was chosen to test compounds generated in our synthetic program because it is both highly and reproducibly responsive to LPA; also, these cells are simple to grow. The cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and were passaged after trypsinization every 5–7 days. Their growth medium was changed every third day. Our compounds were tested as well, albeit less extensively, on the adenovirus-transformed HEK 293 cell line and on the LPA-nonresponsive human myeloid cell line, K562. These cells were cultured in 1:1 Dulbecco’s modified Eagle’s medium/Ham’s F12 medium and RPMI 1640 medium, respectively. The growth media for all cell lines were supplemented with 10% fetal bovine serum and the cultures were kept at 37° in a humidified atmosphere containing 5% CO2. Calcium and cAMP assays. Calcium mobilization and cAMP assays were performed as we described previously (Lynch et al., 1997). Briefly, intracellular calcium fluxes were measured on cell populations (2–4 3 10 cells) that had been loaded with the calcium sensitive fluorophore INDO-1. Responses, which were measured in a temperature controlled fluorimeter (Aminco SLM 8000C; SLM Instruments, Urbana, IL), are reported as the fraction of the maximal response (i.e., response to 75 mM digitonin). The BSA vehicle was determined to have no response. Assays of adenylyl cyclase activity were conducted on populations of 5 3 10 cells stimulated with 10 mM forskolin in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. cAMP was measured by automated radioimmu-