WO2001012838A2 - Compounds which modulate the activity of an lpa receptor - Google Patents

Abstract

The present invention provides novel methods for identifying and characterizing compounds which modulate the activity of an LPA receptor.

Description

DESCRIPTION

Compounds Which Modulate the Activity of an LPA Receptor

Technical Field

The present invention relates to compounds that modulate the activity of lysophosphatidic acids (LPA) , and the LPA receptor EDG-2 (gene sequence and amino acid sequence represented by SEQ ID NO 1) .

Background

Phospholipids . Phospholipids are a class of amphipathic phosphorous- containing lipids which are essential constituents of biological membranes. Various phospholipid preparations have been used for cooking, drug delivery (liposomes) , slow release delivery systems, carrier media for hydrophobic drugs, gene transfer and replacement therapy, sunscreens, emulsions, anti-foaming agents, replacement of damaged or absent pulmonary surfactants, detergents and membrane stabilization. Phosphatidic acid (PA) , phosphatidylinositol (PI) , lysophosphatidic acid, lysophosphatidylinositol (LPI) , and lysophosphatidylcholine (LPC) are found in a variety of plant and animal products. Lysophosphatidic acid analogs have been reported to have a variety of physiological activities including mitogenesis (i.e. prevention of hyperproliferative diseases), vasodilation, growth factor, wound healing and to be an anti-wrinkle agent. U.S. Patent Nos. 4,263,286; 4,746,652; 5,326,690; 5,480,877; 5,565,439; and 5,340,568. Lysophosphatidic acid is reviewed in detail by Moolenaar (1994) TICB 4:213-219; Eichholtz et al. (1990) Biochem . J. 291:677-680; and Moolenaar (1995) J. Biol . Chem . 270:12949-12952.

Previous studies have shown that lysophosphatidic acid, when bound to serum albumin, can activate membrane currents in Xenopus oocytes and induce neurite retraction in PC12 pheochromocytoma cells. Signal Transduction .

Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. Frequently, binding of a ligand to a cell-surface receptor represents the first step in a cascade of events that results in a cellular response. The ligands recognized by specific receptors include a diverse array of molecules such as peptides, deoxyribonucleotide triphosphates and phospholipids.

Research into phospholipid signaling is an area of intense scientific investigation, as more and more bioactive lipids are being identified and their actions characterized. One important addition to the growing list of lipid messengers is lysophosphatidic acid (l-acyl-2-hydroxy-sn- glycero-3-phosphate, LPA) , the simplest of all glycerophospholipids . While LPA has long been known as a precursor of phospholipid biosynthesis in both eukaryotic and prokaryotic cells, only recently has LPA emerged as an intercellular signaling molecule that is rapidly produced and released by activated cells, notably platelets, to influence target cells by acting on a specific cell-surface receptor. Moolenaar (1994) Trends Cell Biol . 4:213-219. Besides being synthesized and processed to more complex phospholipids in the endoplasmic reticulum, LPA can be generated through the hydrolysis of pre-existing phospholipids following cell activation. The best documented example concerns thrombin-activated platelets, where LPA production is followed by its extracellular release. Eichholtz et al . (1993) Biochem . J. 291:677-680.

Platelet LPA is formed, at least in large part, through phospholipase A₂ (PLA₂) -mediated deacylation of newly generated phosphatidic acid (PA) . Gerrard and Robinson

(1989) Biochim . Biophys . Acta 1001:282-285. Distinct PA- specific PLA₂ activity has been identified in platelets (Ca²⁺-dependent) and in rat brain (Ca²⁺-independent) , but little is known about its mode of regulation. Billah et al. (1981) J. Biol . Chem . 256:5399-5403; and Thompson and Clark (1995) Biochem . J. 306:305-309.

It remains to be examined at what stage of the platelet activation response LPA is produced and how it is released into the extracellular environment. Given the wide variety of LPA responsive cell types, LPA production and release are unlikely to be restricted to platelets. Indeed, there is preliminary evidence that growth factor-stimulated fibroblasts can also produce LPA. Fukami and Takenawa (1992) J. Biol . Chem . 267:10988-10993. Furthermore, LPA may be formed and released by injured cells, probably due to nonspecific activation of phospholipases . Many other cell systems remain to be examined for LPA production.

In freshly prepared mammalian serum, LPA concentrations are estimated to be in the range of approximately 2-20 μM, with oleoyl- and palmitoyl-LPA being the predominant species. Tokumura et al. (1994) Am . J. Physiol . 267:C204- C210; and Eichholtz et al. (1993) Biochem . J. 291:677-680. LPA is not detectable in platelet-poor plasma, whole blood, or cerebrospinal fluid. Tigyi and Miledi (1992) J. Biol . Chem . 267:21360-21367. In common with long chain fatty acids, LPA binds with high affinity to serum albumin at a molar ratio of about 3:1. Tigyi et al. (1991) J. Biol . Chem . 266:20602-20609; Thumser et al. (1994) Biochem . J. 301:801-806. It is notable that serum albumin contains several other, as yet unidentified lipids (methanol- extractable) with LPA-like biological activity. Tigyi and Miledi (1992) J. Biol . Chem . 267:21360-21367. This raises the interesting possibility that LPA may belong to a new family of phospholipid mediators showing overlapping biological activities and acting on distinct receptors; conceivably, the ether-linked phospholipid platelet- activating factor (PAF) and the mitogenic lipid sphingosine 1-phosphate may also belong to this putative family. Zhang et al. (1991) J. Cell Biol . 114:155-167.

The range of biological responses to LPA is quite diverse, ranging from induction of cell proliferation to stimulation of neurite retraction and even slimemold chemotaxis, and the body of knowledge continues to grow as more and more cellular systems are tested for LPA responsiveness. Jalink et al. (1993) Proc. Na tl . Acad. Sci . U. S. A. 90:1857-1861; Jalink et al. (1993) Cell Growth and Differ. 4:247-255; and Moolenaar (1995) Curr. Opin . Cell Biol . 7:203-210; Dyer et al. (1992) Molec . Brain Res . 14:293-301; Dyer et al . (1992) Molec. Brain Res . 14:302-309; Tigyi and Miledi (1992) J. Biol . Chem . 267:21360-21367. Although its precise physiological and pathological functions in vivo remain to be explored, LPA derived from platelets has all the hallmarks of an important mediator of wound healing and tissue regeneration. Thus, in addition to acting as an autocrine stimulator of platelet aggregation, LPA stimulates the growth of fibroblasts, vascular smooth muscle cells, endothelial cells, and keratinocytes.

Moolenaar (1994) Trends Cell Biol . 4:213-219; Jalink et al.

(1994) Biochim . Biophys . Acta 1198:185-196; Van Corven et al. (1989) Cell 59:45-54; Tigyi et al. (1994) Proc . Na tl . Acad. Sci . U. S . A . 91:1908-1912; Tokumura et al. (1994) Am . J. Physiol . 267 : C204-C210; and Piazza et al. (1995) Exp . Cell Res . 216:51-64. Intriguingly, it has been observed that LPA acts as an inhibitor of eukaryotic DNA polymerase α. Murakami-Murofushi et al. (1992) J. Biol . Chem . 267:21512-21517. LPA also exhibits anti-mitogenic activity toward myeloma cells, presumably through a distinct receptor subtype. Tigyi et al. (1994) Proc. Na tl . Acad. Sci . 91:1908- 1912; Murakami-Murofushi et al . (1993) Cell Structure and Function 18:363-370. In addition to stimulating cell growth and proliferation, LPA promotes cellular tension and cell- surface fibronectin binding, which are important events in wound repair and regeneration. Zhang et al. (1994) J. Cell Biol . 127:1447-1459; Kolodney et al. (1993) J. Biol . Chem . 268:23850-23855; and Lapetina et al. (1981) J. Biochem . 256:5037-5040. As a product of the blood-clotting process, LPA is a normal constituent of serum (but not platelet-poor plasma) , where it is present in an albumin-bound form at physiologically relevant concentrations. Tigyi and Miledi

(1992) J. Biol . Chem . 267:21360-21367; and Eichholtz et al.

(1993) Biochem . J. 291:677-680. Recently, anti-apoptotic activity has also been ascribed to LPA. PCT Application No. PCT/US94/13649. In this study, an actively proliferating cell line was rescued from serum withdrawal-induced apoptosis by LPA. In another study, evidence has been presented suggesting that LPA can suppress apoptosis in vitro as well as in ischemic organs such as heart and liver. Wu et al. (1996) Transplanta tion (in press) .

Apoptosis .

Apoptosis is a normal physiologic process that leads to individual cell death. This process of programmed cell death is involved in a variety of normal and pathogenic biological events and can be induced by a number of unrelated stimuli. Changes in the biological regulation of apoptosis also occur during aging and are responsible for many of the conditions and diseases related to aging. Recent studies of apoptosis have implied that a common metabolic pathway leading to cell death may be initiated by a wide variety of signals, including hormones, serum growth factor deprivation, chemotherapeutic agents, ionizing radiation, and infection by human immunodeficiency virus (HIV). Wyllie (1980) Nature 284:555-556; Kanter et al . (1984) Biochem . Biophys . Res . Commun . 118:392-399; Duke and Cohen (1986) Lymphokine Res . 5:289-299; Tomei et al. (1988) Biochem . Biophys . Res . Commun . 155:324-331; Kruman et al. (1991) J. Cell . Physiol . 148:267-273; Ameisen and Capron

(1991) Immunol . Today 12:102-105; and Sheppard and Ascher

(1992) J. AIDS 5:143-147. Agents that affect the biological control of apoptosis thus have therapeutic utility in numerous clinical indications. Cellular shrinkage, chromatin condensation, cytoplasmic blebbing, increased membrane permeability and interchromosomal DNA cleavage characterize Apoptotic cell death. Gerschenson et al . (1992) FASEB J. 6:2450-2455; and Cohen and Duke (1992) Ann . Rev. Immunol . 10:267-293. The blebs, small, membrane-encapsulated spheres that pinch off of the surface of apoptotic cells, may continue to produce superoxide radicals which damage surrounding cell tissue and may be involved in inflammatory processes.

While apoptosis is a normal cellular event, pathological conditions and a variety of injuries can also induce it. Apoptosis is involved in a wide variety of conditions, including, but not limited to, cardiovascular disease; cancer regression; immune disorders, including, but not limited to, systemic lupus erythematosus; viral diseases; anemia; neurological disorders; diabetes; hair loss; rejection of organ transplants; prostate hypertrophy; obesity; ocular disorders; stress; aging; and gastrointestinal disorders, including, but not limited to, diarrhea and dysentery. In the myocardium, apoptotic cell death follows ischemia and reperfusion.

In Alzheimer's disease, Parkinson's disease, Huntington's chorea, epilepsy, amyotrophic lateral sclerosis, stroke, ischemic heart disease, spinal cord injury and many viral infections, for example, abnormally high levels of cell death occur. In at least some of these diseases, there is evidence that the excessive cell death occurs through mechanisms consistent with apoptosis. Among these are 1) spinal cord injury, where the severing of axons deprives neurons of neurotrophic factors necessary to sustain cellular viability; 2) stroke, where after an initial phase of necrotic cell death due to ischemia, the rupture of dead cells releases excitatory neurotransmitters such as glutamate and oxygen free radicals that stimulate apoptosis in neighboring healthy neurons; and 3) HIV infection, which induces apoptosis of T-lymphocytes .

In contrast, the level of apoptosis is decreased in cancer cells, which allows the cancer cells to survive longer than their normal cell counterparts. As a result of the increased number of surviving cancer cells, the mass of a tumor can increase even if the doubling time of the cancer cells does not increase. Furthermore, the high level of expression in a cancer cell of the bcl-2 gene, which is involved in regulating apoptosis and, in some cases, necrotic cell death, renders the cancer cell relatively resistant to chemotherapeutic agents and to radiation therapy.

There is considerable evidence of plasma membrane receptors for LPA. LPA-binding proteins have been reported in mammalian tissues and labeled using a photoaffinity crosslinker derivative. Liliom et al. (1996) Am . J. Physiol . 270 : C772-C778 ; Thomson et al. (1994) Mol . Pharmacol . 45:718-723; and van der Bend et al . (1992) EMBO J. 11:2495-2501. In X. laevis oocytes, LPA elicits oscillatory CI^" currents. Tigyi and Miledi (1992) J. Biol .

Chem . 267:21360-21367. This current, like other effects of

LPA, is consistent with a plasma membrane receptor-mediated activation of G protein-linked signal transduction pathways.

In view of the potential physiological significance of LPA and its interaction with receptors in terms of wound healing, cell regeneration, cell proliferation and apoptosis, there is a need for compositions that modulate the acitivity of LPA and its interaction with receptors.

The present invention addresses this need. Herein are described novel compositions and methods of using these compositions to modulate the activity of LPA and similar proteins, and to modulate the interaction of LPA with its various receptors .

All references cited herein are incorporated by reference in their entirety.

Summary Of The Invention

This invention relates to therapeutically effective compositions of matter, which have been found to modulate the activity of LPA. Also encompassed by the present invention are methods of using these compositions to modulate the activity of LPA and LPA receptors, and to modulate the interaction of LPA with its various receptors, are provided.

The present invention encompasses methods of modulating the biological activity of a lysophosphatidic acid or a lysophosphatidic acid receptor, comprising introducing to said lysophosphatidic acid, or its receptor, a composition comprising one of the following structures:

R,—O-R, or

or

or

OR*

or

OR_{4 Q}

R₃O _P — R, — O^R₂

OR₄

wherein Ri is selected from the group comprising a substituted or unsubstituted , saturated or unsaturated, straight or branched-chain alkyl group having from 1 to about 4 carbon atoms, or a cyclic ether; R₂ is a substituted or unsubstituted, saturated or unsaturated, straight or branched-chain alkyl having from about 7 to about 15 carbon atoms; and R₃ and R are independently H or a substituted or unsubstituted, saturated or unsaturated, straight or branched-chain alkyl having from 1 to about 3 carbon atoms, or a salt thereof. In a specific embodiment, the composition is a lysophosphatidic acid having a fatty acid chain of from approximately 10 to approximately 14 carbon atoms . The present invention also encompasses a variety of novel compositions, including: 1-O-Decyl-rac-glycerol;

3-O-Tetradecyl-rac-glycero-l-phosphate; 1-O-Decyl-glycidol; 1-O-Tetradecyl-glycidol ; 1-O-Oleyl-glycidol;

3-O-Decyl-rac-glycero-l-phosphate; 3-O-Oleyl-rac-glycero-l-phosphate; 3-Hydroxypropyl decyl ether; 3-Hydroxypropyl decyl ether; and 3-Decyloxypropyl-l-phosρhate.

Brief Description of the Drawings

Figure 1 shows a schematic of the yeast pheromone- inducible MAP Kinase cascade. Components of this pathway ( SST2 and FAR1 ) that were genetically inactivated by mutation are identified by underlines.

Figure 2 is a graph depicting EDG-2-mediated stimulation of FUSl::lacZ. Yeast cells carrying the galactose-inducible edg-2 gene were grown in SC media containing either 2% galactose (filled bars) or 2% glucose (hatched bars) for seven hours in the presence of lysophosphatidic acid (LPA) or on galactose in the absence of LPA (open bars) . After seven hours, the cells were assayed for β-galactosidase (lacZ) activity.

Figures 3A and 3B are graphs depicting the stimulation by LPA of FUSl::lacZ activity in cells expressing EDG-2 in a time and dose dependent manner, respectively. 3A: Yeast cells carrying edg-2 (■) or empty vector (♦) were grown in synthetic complete media (S.C.) + 2% galactose for the indicated time prior to assaying β-galactosidase activity. 3B, Yeast cells carrying edg-2 were grown for seven hours at the indicated dose of LPA.

Figure 4 is a graph depicting the specific activation by LPA, but not other related lysophospholipids or Sphingosine-1-phosphate (SPP) , of FUSl::lacZ. Yeast cells carrying edg-2 were grown in S.C. + 2% galactose in the presenceof LPA (■) , LPC (♦) , LPE (•) , LPG (A), LPS (D) or Sph-l-P (O) at the indicated concentrations for seven hours. All lyso-glycerophospholipid were resuspended in BBS/EDTA + lmg/ml fatty acid free bovine serum albumin to enhance solubility. Figure 5 is a graph depicting the specific activation of FUSl::lacZ by LPA, but not Diacyl-glycerophospholipids . Yeast cells were cultured in S.C. + 2% galactose in the presence of PA (■) , PC (♦) , PE (•) , PG (A) PS (D) or LPA (O) at the indicated concentration for seven hours. All diacyl-glycerophospholipid were resuspended in BBS/EDTA + lmg/ml fatty acid free bovine serum albumin to enhance solubility.

Figure 6 is a graph depicting the effect of the fatty acid side-chain of LPA on activation of FUSl::lacZ. Yeast cells expressing EDG-2 were cultured in S.C. + 2% galactose in the presence of 18:1 oleoyl LPA (♦) , 18:0 steroyl LPA (•) , 16:0 palmitoyl LPA (A), 14:0 LPA (O) , 24:1 LPA (■) , 10:0 LPA (□) , and 6:0 LPA (hollow diamond), at the indicated concentration for seven hours. The numerical representation refers to the chain length and degree of saturation. All forms of LPA were resuspended in BBS/EDTA + lmg/ml fatty acid free bovine serum albumin to enhance solubility.

Figure 7 is a graph depicting the activation of FUSl::lacZ by LPA presented either as a liposomal formulation or as freely soluble LPA. Yeast cells carrying EDG-2 were culture in S.C. + 2% galactose in the presence of freely soluble LPA (■) , LPA + PC liposomes (♦) , PC alone (O), LPA + PG liposomes (A) or PG alone (D) . Note that the concentration of lipid reflects only the LPA component of the liposome.

Figures 8, 9 and 10 are graphs depicting the activities of various analogs of lysophosphatidic acid and compounds otherwise related to lysophosphatidic acid. Compound numbers and detailed structures are set forth in Example 6. Modes For Carrying Out The Invention

Herein are described novel compositions and methods of using these compositions to modulate the activity of LPA and similar proteins, and to modulate the interaction of LPA with its various receptors.

A. Compounds of the Present Invention. 1. Lysophosphatidic acids .

"LPA" is defined herein as a lysophosphatidic acid or analog thereof. Although phospholipid structures are well defined, they can vary with respect to lipid chain length and saturation. Typically, LPA has the following structures but can include other structures known in the art provided they are effective in producing therapeutic response.

LPA has the following general structure:

O OH O

R- ■C-O-CH,-CH-CH, -O—P- OH

LPA is an acid in which only one of the hydroxyl groups of the glycerol is esterified to a fatty acid. Generically, LPA is a phosphatidic acid in which the sn-2 position of the glycerol moiety is not esterified and the sn-3 position is bound to the 0-P0₃H₂ group, or, in the case of the salt, one or more hydrogen atoms are replaced, for example with Na⁺. The sn-1 position contains an acyl ester of fatty acids. While natural LPAs occur with the phospho- moiety at the sn- 3 position, synthetic LPAs can have alternative stereochemistry with, e.g., the phospho- group at the sn-1 or sn-2 positions. The present invention encompasses these and other sterioisomers and positional isomers of LPA. As used herein, LPA includes compounds having any one of a variety of fatty acids esterified at the #1 carbon position. Examples include compounds wherein the fatty acid ester is lauryl, myristyl, palmityl, stearyl, palmitoleyl, oleyl or linoleyl. For a representative example of suitable phospholipids see any chemical catalog of a phospholipid supplier, for instance, the (1994) Avanti Polar Lipids catalog, particularly pages 14 and 21. R can be an unsubstituted or substituted, saturated or unsaturated, straight or branched chain alkyl having from 11 to about 24 carbon atoms. Substitutions include, but are not limited to, halogen, hydroxy, phenyl, amino or acylamino. A wide variety of LPAs are known in the art and many of these can be purchased from commercial sources such as Avanti Polar Lipids Inc. (Alabaster, AL) , or they can be synthesized by methods known in the art.

The term "UB" is used in reference to the various structures herein to describe the number of unsaturated carbon atoms in R. For example, if R is 18 and UB is 1, R contains 18 carbon atoms, with 1 unsaturated bond. Some LPAs are also referred to herein as R.-UB-LPA (i.e. 18:1-LPA, wherein R is 18 carbon atoms with 1 unsaturated bond) . The term "LPA" also encompasses salts of the phospholipids encompassed by the present invention, which include, but are not limited to, the free acid form, alkali metal salts, such as sodium and potassium; alkaline earth metal salts, such as calcium and magnesium; non-toxic heavy metal salts; ammonium salts; trialkylammonium salts, such as trimethyl-ammonium and triethyla monium; and alkoxyammonium salts, such as triethanolammonium, tri (2-hydroxyethyl) ammonium, and trometha ine (tris (hydroxymethyl) aminomethane) . Particularly preferred are sodium and ammonium salts. Also preferred are pharmaceutically acceptable salts.

LPA has also been shown to have some inhibitory effect on neuronal growth and/or axon stimulation. In methods of the present invention, the LPA modulators disclosed herein can be used to interfere with LPA' s biological effects on neurons, for a variety of therapeutic purposes, including, without limitation, the treatment of diseases characterized by slowed growth or repair of neuronal cells, neurodegenerative diseases, and acute neuron damage. Methods of treatment of such neuronal diseases encompass the direct or indirect presentation of the LPA modulators of the present invention to the vicinity of the diseased neurons.

2. LPA Modulators .

It has been discovered that a subset of analogs of 18:1 LPA can act as modulators of the activity of 18:1 LPA, and other of its analogs, as well as modulators of LPA receptor activity and the interaction between 18:1 LPA and its receptors. This class of compounds generally has a) an approximately 6 to approximately 16 carbon atom chain, which may be saturated or unsaturated, branched or unbranched and substituted or unsubstituted, and b) an ether, ester or reverse ester at one end of the chain. In one embodiment, the In a preferred embodiment, the ester or ether linkage connects the carbon chain to a glycerol moiety, which can also optionally have an 0-P0₃H₂ addition, as in, for example, 10:1 LPA. In an alternative embodiment, the carbon chain is connected by the ester or ether linkage to a cyclic ether, such as, without limitation, an epoxide moiety. In a specific embodiment, the LPA modulators of the present invention have one of the following general structures:

or

O

II or

III or

OR₄

IV or

or

OR*

VI

wherein Ri is selected from the group comprising H, HP0₄, a substituted or unsubstituted , saturated or unsaturated, straight or branched-chain alkyl having from 1 to about 4 carbon atoms, or a cyclic ether, and R₂ is a substituted or unsubstituted, saturated or unsaturated, straight or branched-chain alkyl having from about 6 to about 16 carbon atoms, preferably from about 7 to about 15 carbon atoms, more preferably from about 10 to about 14 carbon atoms, and R₃ and R₄ are independently H or a substituted or unsubstituted, saturated or unsaturated, straight or branched-chain alkyl having from 1 to about 3 carbon atoms. Additionally, any oxygen atom on the phosphate group of structures IV - VI can be substituted with a sulfer atom, to make a phosphothionate moiety.

3. Obtaining compounds of the present invention .

The phospholipids can be obtained from any source including, but not limited to, commercial, isolated from a variety of different plants (including plant organs) and animals or created synthetically. Preferably the plants are in the soybean family, but the phospholipids can be isolated from other plants including, but not limited to, those in the leguminosae (beans and peas etc.). The phospholipids can also be isolated from partially purified plant extracts including, but not limited to, soy molasses, lecithin (fluid, deoiled or other forms) , partially purified protein concentrates, partially purified protein hydrolysates, defatted soy flakes, refined soy oils, soy grits, soy flours and other soy fractions from which lipids can be extracted. It is within the skill of one in the art, utilizing the methods described herein, to determine whether the phospholipids of the present invention can be isolated from a particular species of plant, plant extract or organ within a plant. In addition, U.S. Patent No. 3,365,440 describes extraction of lipids from soybeans. U.S. Patent Nos. 5,567,425; 5,602,885; 5,624,675; 5,635,186; 5,635,187 have further general descriptions of a variety of techniques useful for the present invention.

The phospholipids can be obtained from plant sources by any method known in the art provided it results in purification of at least one of the phospholipids of the invention. A variety of methods are known in the art for purifying and analyzing phospholipids from plant sources. For review, see Bligh and Dyer (1959) Can . J. Biochem . Physiol . 37:911-917; Patton et al. (1982) J. Lipid Res . 23:190-196; Jungalwala (1985) Recent Developments in Techniques for Phospholipid Analysis, in Phospholipids in Nervous Tissues (ed. Eichberg) John Wiley and Sons, pp. 1- 44; Hamilton et al . (1992) in the series, A Practical Approach (Rickwood et al . eds . ) IRL Press at Oxford University Press; and Kates (1986) Techniques of Lipidology: Isolation, Analysis and Identification in Laboratory Techniques in Biochemistry and Molecular Biology (Burdon et al. eds.) Elsevier.

Phospholipids can also be derived from animal sources. Preferably, the animal is a mammal. Even more preferably, the phospholipids are derived from liver cells. Such phospholipids are commercially available or can be purified from animal tissue by methods known in the art, for instance from animal and egg lecithin or from the compositions described in WO 95/15173. Phospholipids in general, and LPAs in particular, can also be derived from blood.

The phospholipids of the invention can also be synthesized by methods known in the art. Suitable semi- synthetic phospholipids and their synthesis are described in Kates, Techniques of Lipidology (1972) . A synthesis of lysophosphatidic acid is described in W. Stoffel and G.D. Wolf, Chemische Synthese von 1-0- [3H] Palmitoyl-L-glycerin-3-phosphate (L-3-

Lysophosphatidsaure) , Chem. Ber., 347 (1966) 94-101.

The synthesis of various cyclic phosphate LPAs is described in A.J. Slotboom, et al., Synthesis of Lysophosphoglycerides, Chem. Phys. Lipids, 1 (1967) 317-336; PCT Publication No. WO 92/21323; and US Patent 5,565,439.

The synthesis of a phosphonate analog of 1-0-hexadecyl- 2-O-methyl-glycero-phosphate is described in Z. Li, et al., Phosphonate isosteres of phospholipids, Tetrahedron Lett., 34 (1993) 3539-3542.

Procedures for synthesis of functionalized glycerol ether derivatives which can be used in the synthesis of compounds suitable for use in the present invention are described in K. Agarwal, et al. Synthesis of carbamyl and ether analogs of phosphatidylcholines, Chem. Phys. Lipids, 39 (1984) 169-177, and H. Eibl and P. Woolley, A general synthetic method for enantiomerically pure ester and ether lysophospholipids, Chem. Phys. Lipids, 47 (1988) 63-68.

The preparation of l-0-benzyl-2-deoxy-2-bromo glycerol, a starting material for the synthesis of 2-bromo LPA Compound 37, is described in W.L.F. Armarego, B.A. Milloy and W. Pendergast, A highly stereospecific synthesis of (R) - and (S)-[2-2Hl]glycine, J.C.S. Perkin I, (1976) 2229-2237.

The synthesis of 2-deoxy-2-bromo-phosphatidylcholine is described in C.J. Lacey and L.M. Loew, Phospholipid synthesis based on new sequential phosphate and carboxylate ester bond formation steps, J. Org. Chem. , 48 (1983) 5214- 5221.

The synthesis of bisphosphatidic acid and its conversion to bis-lysophosphatidic acid using phospholipase A2 from pig pancreas is described in Q. Quan Dang, et al., Synthesis and identification of bis (diacylglycero) phosphoric acid and bis (monoacylglycero) phosphoric acid, Lipids, 17 (1982) 798-802, and Q. Quan Dang and L. Douste-Blazy, Synthesis and stereochemical study of some biologically relevant phosphoglycerides: dicarboxylic phosphatidyl cholines and bis (diacylglycero) phosphoric acids, Phosphorus and Sulfur, 18 (1983) 377-380.

A method for the preparation of lysophosphatidic acid or lysophosphatidates by reacting glycidyl esters with anhydrous phosphoric acid is described in U.S. Patent 3,423,440.

A synthesis of lysothiophosphatidic acid is described in N.V. Heeb and K.P. Nambiar, Synthesis of (R) - lysothiophosphatidic acid and (R) -thiophosphatidic acid, Tetrahedron Lett. , 34 (1993) 6193-6196.

The preparation of LPA amide analogs and 2-deoxy LPA plus various derivatives is described in D.W. Hopper, et al., Facile synthesis of lysophospholipids containing unsaturated fatty acid chains, Tetrahedron Lett., 37 (1996) 7871-7874; and K.R. Lynch, et al., Structure/activity relationships in lysophosphatidic acid: the 2-hydroxyl moiety, Mol. Pharmacol., 52 (1997) 75-81. The following papers describe synthetic routes which can be used for the synthesis of additional LPA analogs: M.

Fuji, et al., A stereoselective and highly practical synthesis of cytosolic phospholipase A2 substrate, 2-S- arachidonoyl-l-O-hexadecyl-sn-thioglycero-3-O- phosphocholine, J. Org. Chem., 62 (1997) 6804-6809;

(Strategy for the preparation of 2-thioglycero phosphocholines and guidance for the synthesis of 2-deoxy-2- thiol LPA); and A. Markowska, et al., Etheranaloge der Thio- und Dithiophospholipide mit C-S-P-Bindung, Liebigs Ann. Chem. , (1993) 1327-1329 (synthesis of l-O-hexadecyl-2-O- methyl-3-thioglycero-3-phosphocholine and l-S-hexadecyl-2-O- methyl-1, 3-dithioglycero-3-phosphocholine, guidance for the preparation of LPA analogs containing sulfur linked phosphates) .

Various degrees of purity of the phospholipids can be used. Purity can be assayed by any method known in the art such as two dimensional TLC or HPLC and assayed for total lipids, phospholipids or phosphate. Various suitable methods are outlined in Kates (1972) . Preferably, the phospholipids must be of sufficient purity such that, when mixed at a total concentration of about 10 mg/mL, the mixture can be sonicated as described below to provide a relatively translucent solution. Preferably, the phospholipids are at least 90% pure, more preferably, they are at least 95% pure and, most preferably, they are at least 99% pure.

B . LPA Receptors .

The edg-2 gene product, a lysophosphatidic acid (LPA) receptor, also reported as vzg-1, couples to the yeast heterotrimeric G-protein and activates a MAP kinase cascade- dependent reporter. The response to LPA can be quantitated by using a reporter gene, including, without limitation, the lacZ gene or the luc gene fused to the FUS1 promoter, a mating pheromone-inducible gene promoter, the HIS3 gene, or any other gene that can genetically compliment an auxotropic growth mutation. The yeast strain used is able to grow in the presence of activated G-protein due to a mutation in the FAR1 gene. This mutation has the phenotypic effect of uncoupling G-protein/map kinase activation from cell cycle arrest.

The following definitions are for the purpose of clarifying the terms used herein, and are not meant to be limiting.

Vectors useful for practicing the present invention include plasmids, viruses (including phage) , and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination) . The vector may replicate and function independently of the host genome, as in the case of a plasmid, or may integrate into the genome itself, as in the case of an integratable DNA fragment. Suitable vectors will contain replicon and control sequences that are derived from species compatible with the intended expression host. For example, a promoter operable in a host cell is one which binds the RNA polymerase of that cell, and a ribosomal binding site operable in a host cell is one which binds the endogenous ribosomes of that cell.

DNA regions are "operably" associated when they are functionally related to each other. For example: a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.

Heterologous DNA sequences are expressed in a host by means of an expression vector. An expression vector is a replicable DNA construct in which a DNA sequence encoding the heterologous DNA sequence is operably linked to suitable control sequences capable of effecting the expression of a protein or protein subunit coded for by the heterologous DNA sequence in the intended host. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and (optionally) sequences which control the termination of transcription and translation.

Transformed host cells of the present invention are cells which have been transformed or transfected with the vectors constructed using recombinant DNA techniques and express the protein or protein subunit coded for by the heterologous DNA sequences.

A variety of yeast cultures, and suitable expression vectors for transforming yeast cells, are known. See, e.g., U.S. Pat. No. 4,745,057; U.S. Pat. No. 4,797,359; U.S. Pat. No. 4,615,974; U.S. Pat. No. 4,880,734; U.S. Pat. No. 4,711,844; and U.S. Pat. No. 4,865,989. Saccharomyces cerevisiae is the most commonly used among the yeast, although a number of other strains are commonly available.

See, e.g., U.S. Pat. No. 4,806,472 (Kluveromyces lactis and expression vectors therefor); U.S. Pat. No. 4,855,231

( Pichia pastoris and expression vectors therefor) . Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence

(ARS) , a promoter, DNA encoding the heterologous DNA sequences, sequence^'s for polyadenylation and transcription termination, and a selection gene. An exemplary plasmid is

YRp7, (Stinchcomb et al., Nature 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschemper et al., Gene 10, 157 (1980)). This plasmid contains the trpl gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85, 12 (1977)). The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include, but are not limited to, the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al . , J. Adv. Enzyme Reg. 7, 149 (1968) and Holland et al., Biochemistry 17, 4900 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPO Publn. No. 73,657. Other promoters, which have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned metallothionein and glyceraldehyde-3- phosphate dehydrogenase, as well as enzymes responsible for maltose and galactose utilization.

In constructing suitable expression plasmids, the termination sequences associated with these genes may also be ligated into the expression vector 3' of the heterologous coding sequences to provide polyadenylation and termination of the mRNA.

A "reporter gene" is a gene that is operably linked to control sequences for expression of a gene of interest, and that encodes a gene product that can be detected for the purpose of evaluating expression of the associated gene of interest.

The "active state" of a receptor is the state at which the ligand that stimulates the receptor can bind to activate a signaling pathway controlled by the receptor.

An "LPA agonist" is defined herein as a composition that is capable of combining with the active state of an LPA receptor to up-regulate a signaling pathway controlled by an LPA receptor. An "LPA antagonist" is defined herein as a composition that is capable of combining with the LPA receptor in either the active or inactive state, thereby impeding the biological action of LPA. An "inverse agonist" of LPA is defined herein as a composition that is capable of combining with the inactive state of an LPA receptor to down-regulate a signaling pathway controlled by an LPA receptor.

A composition that "modulates" LPA activity or the interaction of LPA with an LPA receptor is defined herein as a composition that changes LPA activity or interaction with its receptor by either increasing, decreasing, or stabilizing such activity or interaction.

As used herein, the terms "purified" or "isolated" are intended to refer to a molecule used in the present invention in an enriched or pure form obtainable from a natural source or by means of genetic engineering or synthetic chemistry. The purified protein, DNA or RNA of the invention may be useful in ways that the protein, DNA and RNA as they naturally occur are not, such as identification of compounds selectively modulating the expression or the activity of the EDG-2 of the invention.

The isolated polypeptide and polypeptide fragments of EDG-2 means EDG-2 which is free of one or more components of its natural environment. Purified EDG-2 includes purified EDG-2 in recombinant cell culture. The enriched form of the receptor refers to a preparation containing said receptor in a concentration higher than natural, or in a cell where it is not found under native conditions e.g., a cellular membrane fraction comprising said receptor. If the receptor is in a pure form it is substantially free from other macromolecules, particularly from naturally occurring proteinaceous contamination. If desired, the receptor may be solubilized. Preferably, the receptor of the invention is in an active state meaning that it has both ligand binding and signal transduction activity. Receptor activity is measured according to methods known in the art, e.g., using a binding assay or a functional assay, e.g., an assay as described below.

The invention is further intended to encompass variants of the receptor of the invention. For example, a variant of the EDG-2 receptor of the invention is a functional equivalent of said receptor. A functional equivalent is a protein displaying a physiological profile essentially identical to the profile characteristic of the EDG-2 having the amino acid sequence set forth in SEQ ID NO:l. The physiological profile in vitro and in vivo includes receptor effector function, electrophysiological and pharmacological properties, e.g., selective interaction with agonists or antagonists. Exemplary functional equivalents may be amino acid mutants including those having amino acid deletions, substitutions or insertions, and glycosylation variants. Functional equivalents may also include EDG-2 from a different organism. The present invention also encompasses methods for comparing the agonist profile of other EDG-2 related receptors such as EDG-1 (Lee, M.-J., et al . (1996) J. Biol . Chem . 271 (19), 11272-11279; Hla, T., and Maciag, T. (1990) J. Biol . Chem . 265 (16), 9308-9313), EDG-3

(Yamaguchi, F. , et al . (1996) Biochem . Biophys . Res . Comm .

227, 608-614) and H218 (Okazaki, H., et al . (1993) Biochem .

Biophys . Res . Com . 190, 1104-1109; MacLennan, A. J. , et al.

(1994) Mol . Cell . Neurosci . 5, 201-209) as well as the Xenopus high-affinity LPA receptor, PSP-24 (Guo, Z., et al . (1996) Proc . Na tl . Acad. Sci . USA 93 , 14367-14372).

Covalent derivatives include, for example, aliphatic esters or amides of a receptor carboxyl group, O-acyl derivatives of hydroxyl group containing residues and N-acyl derivative of amino group containing residues. Such derivatives can be prepared by linkage of functionalities to reactable groups that are found in the side chains and at the N- and C-terminus of the receptor protein. Polypeptides of this invention may be modified post-translationally (e.g., acetylation or phosphorylation) .

The invention also encompasses methods wherein EDG-2 is conjugated to a label capable of producing a detectable signal or other functional moieties. Suitable labels include, but are not limited to, radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent dyes, chemiluminescent dyes, bioluminescent compounds and magnetic particles. A protein for use in the invention is obtainable from a natural source, e.g., by isolation, by chemical synthesis or by recombinant techniques.

The invention further provides a method for expressing EDG-2 in host cells. Suitable host cells producing the receptor of the invention are multiplied in vi tro or in vivo . Preferably, the host cells are transformed

(transfected) with a hybrid vector comprising an expression cassette comprising a promoter and an edg-2 DNA sequence. Subsequent to expression of the edg-2 gene under control of the promoter, EDG-2 may be recovered. Recovery comprises, e.g., isolating the host cells comprising the receptor, e.g., from the culture broth.

The term "functional" or "biologically active", when used herein as a modifier of EDG-2, refers to a polypeptide that is able to produce one or more of the functional characteristics exhibited by native EDG-2. In one embodiment, functional means capable of binding a EDG-2 ligand. In another embodiment, functional means that a signal is transduced as a consequence of binding of a ligand.

Suitable host cells include eukaryotic cells, e.g., animal cells, plant cells and fungi, and prokaryotic cells, such as Gram-positive and Gram-negative bacteria, e.g., Escheria coli .

Isolated polynucleotides (or nucleic acids) encoding a polypeptide substantially identical to EDG-2, or any fragment thereof, is designated edg-2. The term polynucleotide as used herein, may be DNA or RNA, either coding or noncoding strands, edg-2 cDNA, genomic DNA and synthetic or semi-synthetic DNAs and RNAs.

The invention includes modifications to edg-2 DNA such as deletions, substitutions and additions particularly in the non-coding regions of genomic DNA. Such changes are useful to facilitate cloning and modify gene expression in methods of the present invention. Various substitutions can be made within the coding region that either do not alter the amino acid residues encoded or result in conservatively substituted amino acid residues. Nucleotide substitutions that do not alter the amino acid residues encoded are useful for optimizing gene expression in different systems. Suitable substitutions are known to those of skill in the art and are made, for instance, to reflect preferred codon usage in the particular expression systems. The invention encompasses methods using functionally equivalent variants and derivatives of edg-2 that may enhance, decrease or not significantly affect the properties of EDG-2. For instance, changes in the DNA sequence that do not change the encoded amino acid sequence, as well as those that result in conservative substitutions of amino acid residues, one or a few amino acid deletions or additions, and substitution of amino acid residues by amino acid analogs are those which will not significantly affect its properties, such as its ability to bind to LPA or analogs thereof.

EDG-2 is preferably incorporated into a vector (a virus, phage, or plasmid) which can be introduced by transfection or infection into a cell. The vector preferably includes one or more expression control sequences, in which case the cell transfected by the vector is capable of expressing the polypeptide. By "isolated DNA" is meant a single- or double-stranded DNA that is free of the genes which, in the naturally-occurring genome of the animal from which the isolated DNA is derived, flank the edg-2 gene. The term therefore includes, for example, either or both strands of a edg-2 cDNA or an allelic variant thereof; a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryotic or eukaryotic cell; or a genomic DNA fragment ( e . g. , produced by PCR or restriction endonuclease treatment of human or other genomic DNA) . The term also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

Stringent conditions for both DNA/DNA and DNA/RNA hybridization assays are as described by Sambrook et al . , Molecular Cloning, A Labora tory Manual , 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, herein incorporated by reference. For example, see page 7.52 of Sambrook et al .

Given the guidance of the present invention, the nucleic acids used in the invention are obtainable according to the methods well known in the art.

For example, a DNA used in the invention is obtainable by chemical synthesis, by recombinant DNA technology or by PCR. Preparation by recombinant DNA technology may involve screening a suitable cDNA or genomic library. A suitable method for preparing a DNA or of the invention may, e.g., comprise the synthesis of a number of oligonucleotides, their amplification by PCR methods, and their splicing to give the desired DNA sequence. Suitable libraries are commercially available or can be prepared from individual tissues or cell lines.

For an individual receptor related to EDG-2, the expression pattern in different tissues may vary. Thus, in order to isolate cDNA encoding a particular EDG-2-related receptor, it is advantageous to screen libraries prepared from different suitable tissues or cells. As a screening probe, there may be employed a DNA or RNA comprising substantially the entire coding region of edg-2 or a suitable oligonucleotide probe based on said DNA. A suitable oligonucleotide probe (for screening involving hybridization) is a single stranded DNA or RNA that has a sequence of nucleotides that includes at least 14 contiguous bases that are the same as (or complementary to) any 14 or more contiguous bases set forth in SEQ ID NO:l. The probe may be labeled with a suitable chemical moiety for ready detection. The nucleic acid sequences selected as probes should be of sufficient length and be sufficiently unambiguous so that false positive results are minimized.

Preferred regions from which to construct probes include 5' and/or 3' coding sequences, sequences predicted to encode ligand binding sites, and the like. For example, either the full-length cDNA clone disclosed herein or fragments thereof can be used as probes. Preferably, nucleic acid probes of the invention are labeled with suitable label means for ready detection upon hybridization. For example, a suitable label means is a radiolabel. The preferred method of labeling a DNA fragment is by incorporating ³²P-labeled -dATP with the Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art. Oligonucleotides are usually end-labeled with ³²P-labeled -ATP and polynucleotide kinase. However, other methods (e.g., non-radioactive) may also be used to label the fragment or oligonucleotide, including, e.g., enzyme labeling and biotinylation.

After screening the library, e.g., with a portion of DNA including substantially the entire edg-2 gene or a suitable oligonucleotide based on a portion of said DNA, positive clones are identified by detecting a hybridization signal; the identified clones are characterized by restriction enzyme mapping and/or DNA sequence analysis, and then examined, e.g., by comparison with the sequences set forth herein, to ascertain whether they include a full length edg-2 gene (i.e., if they include translation initiation and termination codons) . If the selected clones are incomplete, they may be used to rescreen the same or a different library to obtain overlapping clones. If the library is genomic, then the overlapping clones may include exons and introns. If the library is a cDNA library, then the overlapping clones will include an open reading frame. In both instances, complete clones can be identified by comparison with the DNAs and deduced amino acid sequences provided herein. It is envisaged that the nucleic acid of the invention can be readily modified by nucleotide substitution, nucleotide deletion, nucleotide insertion or inversion of a nucleotide stretch, and any combination thereof. Such modified sequences can be used to produce a mutant EDG-2 which differs from the receptors found in nature. Mutagenesis may be predetermined (site-specific) or random. A mutation that is not a silent mutation should not place sequences out of reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structures such as loops or hairpins.

The edg-2 cDNA or genomic DNA can be incorporated into vectors for transfection of a host cell. Furthermore, the invention concerns a recombinant DNA which is a hybrid vector comprising at least one of the above mentioned genes.

The hybrid vectors of the invention comprise an origin of replication or an autonomously replicating sequence, one or more dominant marker sequences and, optionally, expression control sequences, signal sequences and additional restriction endonuclease sites.

Preferably, the hybrid vector of the invention comprises an above described nucleic acid insert operably linked to an expression control sequence, in particular those described hereinafter. Vectors typically perform two functions in collaboration with compatible host cells. One function is to facilitate the cloning of the edg-2 gene, i.e., to produce useable quantities of the nucleic acid (cloning vectors) . The other function is to provide for replication and expression of the gene constructs in a suitable host, either by maintenance as an extrachromosomal element or by integration into the host chromosome (expression vectors) . A cloning vector comprises the DNAs as described above, an origin of replication or an autonomously replicating sequence, selectable marker sequences, and optionally, signal sequences and additional restriction sites. An expression vector additionally comprises expression control sequences essential for the transcription and translation of the edg-2 gene. Thus, an expression vector refers to a recombinant DNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into a suitable host cell, results in expression of the cloned DNA. Suitable expression vectors are well known in the art and include those that are replicable in eukaryotic and/or prokaryotic cells.

Most expression vectors are capable of replication in at least one class of organisms but can be transfected into another organism for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even though it is not capable of replicating independently of the host cell chromosome. DNA may also be amplified by insertion into the host genome. However, the recovery of the genomic edg-2 gene is more complex than that of exogenously replicated vector because restriction enzyme digestion is required to excise the gene. DNA can be amplified by PCR and directly transfected into the host cells without any replication component.

Advantageously, expression and cloning vectors contain a selection gene also referred to as selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g., ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media.

Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the edg-2 gene, such as dihydrofolate reductase (DHFR, methotrexate resistance) , thymidine kinase, or genes conferring resistance to G418 or hygromycin. The mammalian cell transfectants are placed under selection pressure in which only those transfectants that are uniquely adapted to survive are those which have taken up and are expressing the marker.

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the edg-2 gene. Suitable promoters may be inducible or constitutive. The promoters are operably linked to the edg-2 gene by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Both the native edg-2 promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of edg-2. However, heterologous promoters are preferred, because they generally allow for greater transcription and higher yields of EDG-2 as compared to native edg-2 promoter.

Promoters suitable for use with prokaryotic hosts include, for example, the -lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published thereby enabling the skilled worker to ligate them to the edg-2 gene using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Dalgarno sequence operably linked to the edg-2 gene.

The various DNA segments of the vector DNA are operably linked, i.e., they are contiguous and placed in a functional relationship to each other. Construction of vectors according to the invention employs conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a manner known in the art. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing edg-2 expression and function are known to those skilled in the art. Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis) , in si tu hybridization, using an appropriately labeled probe based on a sequence provided herein, binding assays, immunodetection and functional assays .

Suitable methods for manipulation of polynucleotides include those described in a variety of references, including, but not limited to, Molecular Cloning: A Labora tory Manual , 2nd Ed., Vol. 1-3, eds. Sambrook et al . Cold Spring Harbor Laboratory Press (1989) ; and Current Protocols in Molecular Biology, eds. Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987) and periodic updates. Those skilled in the art will readily envisage how these methods may be modified, if desired.

The invention further provides host cells capable of producing edg-2 and heterologous (foreign) polynucleotides encoding said receptor. The nucleic acids of the invention can be expressed in a wide variety of host cells, e.g., those mentioned above, that are transformed or transfected with an appropriate expression vector. EDG-2 (or a portion thereof) may also be expressed as a fusion protein. Recombinant cells can then be cultured under conditions whereby the protein (s) encoded by edg-2 is (are) expressed.

Suitable prokaryotes include eubacteria, such as Gram- negative or Gram-positive organisms, such as E. coli , e.g., E. coli K-12 strains, DH5 and HB101, or bacilli. Further host cells suitable for edg-2-encoding vectors include eukaryotic microbes such as filamentous fungi or in a preferred embodiment, yeast, e.g., Saccharomyces cerevisiae.

G proteins are comprised of three subunits: a guanyl- nucleotide binding α subunit; a β subunit; and a γ subunit. G proteins cycle between two forms, depending on whether GDP or GTP is bound thereto. When GDP is bound the G_αβγ protein exists as an inactive heterotrimer, the G_αpγ complex. When GTP is bound the subunit dissociates, leaving a G_βγ complex. Importantly, when a G_oβγ complex operatively associates with an activated G protein coupled receptor in a cell membrane, the rate of exchange of GTP for bound GDP is increased and, hence, the rate of dissociation of the bound the α subunit from the G_βγ complex increases. This fundamental scheme of events forms the basis for a multiplicity of different cell signaling phenomena.

At least four G protein-mediated signaling pathways have been identified in the action of LPA. These are:

1) stimulation of phospholipase C and phospholipase D;

2) inhibition of adenylyl cyclase; 3) activation of Ras and the downstream Raf/MAP kinase pathway; and 4) tyrosine phosphorylation of focal adhesion proteins in concert with remodeling of the actin cytoskeleton in a Rho-dependent manner.

GTP-binding proteins fall into two broad classes of regulatory proteins; the heterotrimeric G-proteins, and small GTPases. The alpha subunit of heterotrimeric G- proteins (Gα) and the small GTPases, as typified by the proto-oncogene Ras, share certain structural homology, and cycle between an active GTP-bound state and an inactive GDP- bound state. When stimulated by an appropriate signal, G- proteins and small GTPases become activated by the binding of GTP and physically interact with effector molecules to transduce the signal to the cell. In the case of G- proteins, binding of GTP to the α subunit causes the low molecular weight Gα to dissociate from the Gβγ dimer where either Gα or Gβγ can act as the signal transducer. An intrinsic GTPase activity hydrolyses GTP to GDP and thus attenuates the signal. Ancillary proteins collectively known as exchange factors are responsible for replacing GDP for GTP and reactivating the GTP-binding protein. Heterotrimeric G-protein coupled receptors are a special class of receptors. It is estimated that G-protein coupled receptors comprise 0.1% of the human genome (including olfactory and visual receptors) which could place the number of different receptors in the thousands. The common structural feature of these receptors are seven hydrophilic membrane spanning domains. Based on the three dimensional model of bacterial rhodopsin, it is predicted that the receptors would form a barrel shaped structure with the ligand binding domains being the extracellular loops and/or the transmembrane domains.

Recently, three putative receptors for LPA have been identified suggesting that functionally different LPA receptors may exist that dictate the particular cellular response of LPA. Hecht, J. H., et al. (1996) J. Cell. Biol. 135(4), 1071-1083; Macrae, A. D., et al . (1996) Mol. Brain. Res. 42, 245-254; An, S., et al. (1997) Biochm. Biophys. Res. Com. 231, 619-622; Guo, Z., et al . (1996) Proc. Natl. Acad. Sci. USA 93, 14367-14372; An, et al., J. Biol. Chem. (1998) . Most cell types respond to LPA making it difficult to characterize the receptor dependency of a particular response to LPA since the response cannot be solely attributed to a single LPA receptor. In particular, it is difficult to assess ligand binding specificity of an LPA receptor without a naive cell line because other lipid receptors may exist with overlapping ligand specificity. Therefor, the yeast Saccharomyces cerevisiae was used to study the human LPA receptor EDG-2 (or Vzg-1) . S. cerevisiae contain no endogenous LPA receptors and is therefore a potentially useful organism in which to functionally express LPA receptors and analyze their ligand specificity. Other mammalian receptors have been functionally expressed in Saccharomyces including the sommatostatin receptor. (Price, L. A., et al. (1995) Mol. Cell. Biol. 15(11), 6188-6195), the A2a adenosine receptor (Price, L. A., et al. (1996) Mol. Pharmacol. 50(4), 829-837) and the β2-adrenergic receptor (King, K., et al., (1990) Science 250, 121-123) . Figure 1 shows a detailed schematic of the yeast pheromone-inducible MAP Kinase cascade. Saccharomyces contains a single heterotrimeric G-protein that is activated by mating factor binding to a specific receptor. Blumer, K. J., and Thorner, J. (1990) Proc. Natl. Acad. Sci. USA 87, 4363-4367. Upon stimulation by an occupied receptor, the α subunit of the heterotrimeric G protein (Gα, the GPA1 gene product (Dietzel, C, and Kurjan, J. (1987) Cell 50, 1001- 1010; Miyajima, I., et al. (1987) Cell 50, 1011-1019) becomes bound to GTP and dissociates from the βγ dimer. In yeast, it is the βγ dimer that transduces the signal to Stell (the MEKK equivalent (Lange-Carter, C. A., et al . (1993) Science 260, 315-319)) and Ste7 (the MEK equivalent (Neiman, A. M., and Herskowitz, I. (1994) Proc . Natl . Acad. Sci . USA 91, 3398-3402)). The active GTP-bound version of Gα is inactivated by hydrolysis of GTP to GDP at which time, Gα can re-associate with Gβγ and attenuates the signal (Blinder, D., and Jenness, D. D. (1989) Mol . Cell . Biol . 9, 3720-3726; Cole, G., (1990) Mol . Cell . Biol . 10 (510-517); Dietzel, C, and Kurjan, J. (1987) Cell 50, 1001-1010; Miyajima, I., et al. (1987) Cell 50, 1011-1019). Like the mammalian MAP kinase, the yeast MAP kinases Fusl and Kssl activate a transcriptional activator, the STE12 gene product (Elion, E. A., et al. (1994) Mol . Biol . Cell 4, 495-510). Activated Stel2 in turn activates the transcription of several mating- inducible genes such as FUS1 (Elion, E. A., et al. (1991) Cold Spring Harbor Symp. Quant . Biol . 56, 41-49; Peter, M., et al. (1993) Cell 73, 747-760). To study the EDG-2 receptor using the yeast pheromone response pathway system, a strain carrying a mutation in the FAR1 gene was used. This mutation has the effect of uncoupling the MAP kinase cascade from cell cycle arrest allowing the yeast to continue growing during MAP kinase activation (Peter, M., et al. (1993) Cell 73, 747-760; Peter, M. , and Herskowitz, I. (1994) Science 265, 1228-1231). Secondly, a mutationally inactivated SST2 gene was created to increase the sensitivity of the strain to G-protein activation. The SST2 gene encodes a GTPase activating protein (GAP) for the Gα subunit (the GPA1 gene product) (Dohlman, H. G., et al. (1996) Mol . Cell . Biol . 16(9), 5194-5209). By inactivating the SST2 gene product, Gα remains in the GTP-bound state longer and thus increases the steady-state concentration of the signal transducing βγ dimer. Finally, to quantify the response, the bacterial lacZ gene was fused to the mating inducible FUS1 promoter to create a reporter gene.

The ubiquitous presence of the response elicited by LPA in almost every cell line tested, combined with the amphiphilic character of LPA that makes radioligand binding assays extremely difficult, has presented considerable difficulties in the molecular cloning of its receptors.

The advantage of a yeast system is that yeast contain few G-protein coupled receptors and it is therefor a simple task to show that the response of the EDG-2 receptor to a particular phospholipid is dependent on the expression of the receptor since it is expressed from a galactose inducible promoter. This is in contrast to mammalian cells in which identity and distribution of LPA and other glycerophospholipids receptors is unclear. The results show that EDG-2 specifically responds to LPA. EDG-2 does not respond to other lysophospholipids or to diacyl- glycerophospholipids, in particular Phosphatidic acid (PA) or to the related lipid messenger sphingosine-1-phosphate (SPP) .

Higher eukaryotic cells include insect, amphibian and vertebrate cells, or mammalian cells. The methods for expressing proteins of interest in Sf9 cells are known in the art and are described in, for example Current Protocols in Molecular Biology, Eds. Ausubel et al . , Greene Publishing and Wiley-Interscience: New York (1987) and references therein. In recent years, propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. The host cells referred to in this application comprise cells in in vi tro culture as well as cells that are within a host animal. Host cells are transfected or transformed with the above-captioned expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Heterologous DNA may be introduced into host cells by any method known in the art, such as transfection with a vector encoding a heterologous DNA by the calcium phosphate coprecipitation technique, by electroporation or by lipofectin-mediated transfection. Numerous methods of transfection are known to the skilled worker in the field. Successful transfection is generally recognized when any indication of the operation of this vector occurs in the host cell. Transformation is achieved using standard techniques appropriate to the particular host cells used.

Incorporation of cloned DNA into a suitable expression vector, transfection of eukaryotic cells with a plasmid vector or a combination of plasmid vectors, each encoding one or more distinct genes or with linear DNA, and selection of transfected cells are well known in the art (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Labora tory Manual, Second Edition, Cold Spring Harbor Laboratory Press) .

Transfected or transformed cells are cultured using media and culturing methods known in the art, preferably under conditions whereby edg-2 is expressed. Suitable culturing media are either commercially available or readily prepared.

The edg-2 gene is ligated into a vector, and introduced into suitable host cells to produce transformed cell lines that express edg-2. The resulting cell line can then be produced in amounts sufficient for reproducible qualitative and quantitative analysis of the effects of a receptor- specific agonist, antagonist or allosteric modulator. The transfected cells can then be employed in a drug screening assay provided hereinafter. Such drugs are useful in diseases associated with pathogenesis of LPA. Particularly useful for assessing the specific interaction of compounds with EDG-2 are stably transfected cell lines expressing EDG- 2. Cells expressing EDG-2 polypeptides are useful for identifying substances that bind to EDG-2. Identification of substances that bind to EDG-2 may be achieved by assessing the ability of a test compound to inhibit the binding of labeled ligand or analog thereof. Another method for identification of such substances involves assessing the ability of a test compound to inhibit specific antibody binding to EDG-2.

Cells expressing EDG-2 polypeptides are also useful for elucidating the signal transduction pathways to which EDG-2 is coupled. By "signal transduction pathway" is meant the sequence of events that involves the transmission of a message from a cell-surface receptor to the cytoplasm. The signal will ultimately cause the cell to perform a particular function.

Thus, host cells expressing EDG-2 are also useful for drug screening and it is an object of the present invention to provide a method for identifying a compound or signal which modulates the activity of EDG-2. The method includes exposing cells containing heterologous edg-2, wherein said cells produce functional EDG-2, to at least one compound or signal whose ability to modulate the activity of said EDG-2 is sought to be determined. The cells are then monitored for changes caused by the modulation. Such an assay enables the identification of agonists, antagonists and allosteric modulators of EDG-2.

In a further aspect, the invention relates to an assay for identifying compounds that modulate the activity of EDG- 2. The assay comprises the steps of: - contacting cells expressing an active EDG and containing heterologous edg with at least one compound to be tested for its ability to modulate the activity of said receptor, and analyzing cells for a difference in second messenger level or receptor activity. Additionally, to determine the specificity of the compound for a particular member of the EDG family of receptors, the assay can further comprise the steps of: contacting cells expressing one of the other EDG receptors, or the PSP-24 receptor and containing heterologous edg with the same compound tested above, and analyzing cells for a difference in second messenger level or receptor activity.

If the compound only effects receptor activity in one EDG family member, it is more specific, and may be preferable for certain therapeutic purposes, for example, to reduce the likelyhood of undesired biological effects.

In particular, the invention covers an assay for identifying compounds that modulate the activity of EDG-2, said assay comprising:

Contacting cells expressing active EDG-2 and containing heterologous edg-2 with at least one compound to be tested for its ability to modulate the activity of said receptor, and - Monitoring said cells for a resulting change in second messenger activity.

The results obtained in these assays are compared to an assay suitable as a negative control.

Assay methods generally require comparison to various controls. A change in receptor activity or in second messenger level is "induced" by a test compound if such an effect does not occur in the absence of the test compound. An effect of a test compound on the receptor of the invention is "mediated" by the receptor if this effect is not observed in cells that do not express the receptor or express decreased amounts of the receptor.

As used herein, a compound or signal that modulates the activity of EDG-2 refers to a compound or signal that alters the response pathway mediated by EDG-2 within a cell (as compared to the absence or decreased amount of said EDG-2) . A response pathway is activated by an extracellular stimulus, resulting in a change in second messenger concentration or enzyme activity, or resulting in a change of the activity of a membrane-bound protein, such as a receptor or ion channel. A variety of response pathways can be utilized, including but not limited to, the adenylate cyclase response pathway, the phospholipase C/intracellular calcium ion response pathway or a response pathway involving activation of Ras or Rho .

Apoptosis represents another important response pathway that may be modulated by EDG-2 agonists or antagonists. Suitable indications for therapeutic use of EDG-2 agonists or antagonists that result in modulation of apoptotic pathways include, but are not limited to, ischemic heart disease, tumors, viral diseases such as HIV infection, neurodegenerative disorders, inflammatory bowel disease, hair loss, and rejection of organ transplants.

Thus EDG-2 expressing cells may be employed for the identification of compounds, particularly low molecular weight molecules capable of acting as LPA agonists or antagonists. Within the context of the present invention, an agonist refers to a molecule that is capable of interacting with EDG-2, thus mimicking the action of LPA. In particular, an LPA agonist is characterized by its ability to interact with the EDG-2 of the invention, and thereby increasing or decreasing the stimulation of a response pathway within a cell. For example, an agonist increases or decreases a measurable parameter within the host cell, such as the concentration of a second messenger or modulation of apoptosis.

By contrast, in situations where it is desirable to decrease the activity of EDG-2, antagonists are useful. Liliom et al. (1996) Molec . Pharmacol . 50:616-623; Bittman et al. (1996) J. Lipid Res . 37:391-398. Within the context of the present invention, an antagonist refers to a molecule that is capable of interacting with EDG-2, but which does not stimulate a response pathway within a cell. In particular, LPA antagonists are generally identified by their ability to interact with EDG-2, and thereby reduce the ability of the natural ligand to stimulate a response pathway within a cell, e.g., by interfering with the binding of LPA to EDG-2 or by inhibiting other cellular functions required for the activity of EDG-2. For example, in a suitable assay, e.g., an assay involving suitable eukaryotic cells expressing EDG-2, a LPA antagonist is capable of modulating the activity of EDG-2 such that the ability of the natural ligand to activate the map kinase pathway is reduced. Yet another alternative to achieve an antagonistic effect is to rely on overexpression of antisense edg-2 RNA. Preferred is an agonist or antagonist selectively acting on EDG-2.

An allosteric modulator of EDG-2 interacts with the receptor protein at another site than that recognized by LPA, thus acting as agonist or antagonist. Therefore, the screening assays described herein are also useful for detecting an allosteric modulator of a receptor of the invention. For example, an allosteric modulator, acting as an agonist, may enhance the specific interaction between EDG-2 and LPA. For instance, if an allosteric modulator acts as an antagonist, it may interact with the receptor protein in such a way that binding of the agonist is functionally less effective. Examples include local anesthetics such as procaine, lidocaine, dibucaine and tetracaine.

An in vi tro assay for a LPA agonist or antagonist may require that EDG-2 is produced in sufficient amounts in a functional form using recombinant DNA methods. An assay is then designed to measure a functional property of EDG-2, e.g., interaction with LPA. Production of EDG-2 is regarded as occurring in sufficient amounts if activity of the receptor results in a measurable response.

For example, mammalian cells (available, e.g., from the American Tissue Type Culture Collection) are grown in appropriate culture medium. An EDG-2 expression plasmid is transiently transfected into the cells, e.g., by calcium- phosphate precipitation. Ausubel, F.M. et al. (1993). Cell lines stably expressing EDG-2 may be generated, e.g., by lipofectin-mediated transfection with EDG-2 expression plasmids and a plasmid comprising a selectable marker gene. Southern and Berg (1982) J. Mol. Appl . Genet. 1:327-341. Cells surviving the selection are isolated and grown in the selection medium. Resistant clonal cell lines are analyzed, e.g., for immunoreactivity with EDG-2-specific antibodies or by assays for EDG-2 functional responses following agonist addition. Cells producing EDG-2 are used in a method for detecting compounds binding to EDG-2 or in a method for identifying a LPA agonist or antagonist.

Compound bound to the target EDG-2 may modulate functional properties of EDG-2 and may thereby be identified as a LPA agonist or antagonist in a functional assay. Functional assays are used to detect a change in the functional activity of EDG-2, for instance, as a result of the interaction of the compound to be tested with EDG-2. A functional response is a change (difference) in the concentration of a relevant second messenger influenced by the receptor of the invention within cells expressing functional EDG-2 (as compared to a negative control) . Those of skill in the art can readily identify an assay suitable for detecting a change in the level of an intracellular second messenger indicative of the expression of active EDG- 2 (functional assay) . Examples include cAMP assays (see, e.g., Nakajima et al. (1992) J. Biol . Chem . 247 :2437-2442) ; Tigyi et al. (1996) J. Neurochem . 66:549-558) measuring changes in inositol 1, 4, 5-triphosphate levels (Tigyi et al. (1996) J. Neurochem . 66:537-548), measuring CI^" ion efflux (Postma et al. (1996) EMBO J. 15:63-72; Watsky (1995) Am. J. Physiol. 269:C1385-C1393) , or measuring changes in intracellular Ca²⁺ levels (Tigyi et al. (1996) J. Neurochem . 66:537-548) .

More specifically, according to the invention, a method for detecting a LPA agonist comprises the steps of

(a) exposing a compound to EDG-2 coupled to a response pathway, under conditions and for a time sufficient to allow interaction of the compound with EDG-2 and an associated response through the pathway, and (b) detecting an increase or decrease in the stimulation of the response pathway resulting from the interaction of the compound with EDG-2, relative to the absence of the tested compound and therefrom determining the presence of a LPA agonist.

A method for identifying a LPA antagonist comprises the steps of (a) exposing a compound in the presence of a known LPA agonist to EDG-2 coupled to a response pathway, under conditions and for a time sufficient to allow interaction of the agonist with the receptor and an associated response through the pathway, and (b) detecting an inhibition of the stimulation of the response pathway induced by the agonist, said inhibition resulting from the interaction of the compound with EDG-2, relative to the stimulation of the response pathway by the LPA agonist alone and determining therefrom the presence of a LPA antagonist. Inhibition may be detected if the test compound competes with the LPA agonist for EDG-2. Compounds which may be screened utilizing such a method include, but are not limited to, blocking antibodies specifically binding to EDG-2. Furthermore, such an assay is useful for the screening for compounds interacting with LPA. In this case, the agonistic effect is neutralized or reduced, e.g., by binding of the test compound to the agonist, thus affecting agonist interaction with the receptor. Examples are soluble EDG-2 fragments comprising part or all of the ligand binding domain.

Preferentially, interaction of an agonist or antagonist with EDG-2 denotes binding of the agonist or antagonist to said EDG-2.

As employed herein, conditions and times sufficient for interaction of an LPA agonist or antagonist candidate with the receptor will vary with the source of the receptor, however, conditions generally suitable for binding occur between about 4°C and about 40°C, preferably between about 4°C and about 37 °C, in a buffer solution between 0 and 2 M NaCl, preferably between 0 and 0.9 M NaCl, with 0.1 M NaCl being particularly preferred, and within a pH range of between 5 and 9, preferably between 6.5 and 8. Sufficient time for the binding and response will generally be between about 1 ms and about 24 h after exposure.

Within one embodiment of the present invention, the response pathway is a membrane-bound Map Kinase pathway, and, for an agonist, the step of detecting comprises measuring a reduction or increase, preferably a reduction, in lacZ production by the membrane-bound response pathway, relative to the lacZ production in the relevant control setup. For the purpose of the present invention, it is preferred that the reduction or increase in lacZ production be equivalent or greater than the reduction or increase induced by LPA applied at a concentration corresponding to its IC₅o value. For an antagonist, the step of detecting comprises measuring in the presence of the antagonist a smaller LPA-induced decrease or increase in lacZ production by the membrane-bound response pathway, as compared to the lacZ production in the absence of the antagonist. The measurement of lacZ may be performed after cell destruction or by a lacZ sensitive molecular probe loaded into the cell.

Yeast contains multiple MAP kinase cascades that are functionally analogous to the Mitogen Activated Protein Kinase (MAPK) cascade in mammalian systems (Brewster, J. L., et al. (1993) Science 259, 1760-1763; Irie, K., et al . (1993) Mol . Cell . Biol . 13, 3076-3083; Neiman, A. M., and Herskowitz, I. (1993) Trends Genet . 9, 390-394; Chang, E. C, et al. (1994) Cell 79, 131-141). A schematic of the S . cerevisiae pheromone response pathway and the relevant genetic components are shown in Figure 1. The parental yeast strain, SY2069, contains the FUS1 promoter fused to lacZ and HIS3 integrated into different chromosomal loci and carries the farl-bad allele. The FAR1 gene product is required for cell cycle arrest following exposure to mating pheromone (see Figure 1) . By deleting this gene, the cells are able to grow in the presence of MAP kinase activation. In addition, a null mutation in the SST2 gene was created because it has been previously reported that the Sommatostatin receptor can efficiently couple to the endogenous yeast heterotrimeric G-protein after mutationally inactivating the SST2 gene (Price, L. A., Kajkowski, E. M. , Hadock, J. R., Ozenberger, B. A., and Pausch, M. H. (1995) Mol . Cell . Biol . 15 (11), 6188-6195). SST2 encodes a GTPase activating protein (GAP) for the GPAl gene product, the Gα subunit required for mating pheromone signal transduction. The effect of inactivating SST2 is that Gpal remains in the GTP-bound state longer and thus permits signaling through the βγ dimer to proceed at a higher rate, resulting in a higher signal from the receptor.

Further cell-based screening assays can be designed, e.g., by constructing cell lines in which the expression of a reporter protein, i.e., an easily assayable protein, such as β-galactosidase, chloramphenicol acetyltransferase (CAT) or luciferase, is dependent on the function of EDG-2. The resulting DNA construct comprising the enzyme DNA is stably transfected into a host cell. The host cell is then transfected with a second DNA construct containing edg-2 gene operably linked to additional DNA segments necessary for the expression of the receptor.

Thus, the edg-2 gene is useful for a variety of studies. These include, but are not limited to, testing LPA analogs for agonistic/antagonistic activity; dissecting the molecular signal transduction mechanism, analyzing receptor- ligand interactions by site-directed mutagenesis; determining the levels and distribution of the receptor; cloning related receptors; and determining the mechanism of tissue-specific response to LPA.

The following examples are provided to illustrate but not limit the invention.

Experimental The following abbreviations, used in the experimental section and throughout the specification, are herein set forth : LPA (lysophosphatidic acid) , LPE ( lysophosphatidlylethanolamine ) , LPS

( lysophosphatidlyserine) , LPC (lysophosphatidlycholine) , LPG ( lysophosphatidlyglycerol ) , PA (phosphatidic acid) , PE (phosphatidylethanolamine) , PS (phosphatidylserine) , PC (phosphatidylcholine) , PG (phosphatidylglycerol ) , Sph-l-P ( shingosine-1-phosphate) , BBS (Bicarbonate buffered saline ) .

Example 1

Construction Of The EDG-2 Expression Plasmid And Expression In Yeast

SY2069 (Ma ta , farl -bad3 , HIS3 : :pFUSl : :HIS3 , mfa2- Δl : :pFUSl : : lacZ , ura3 , leu2, adel , arg4 , trpl ) was used to derive an sst2^' strain for subsequent studies. SST2 was disrupted using pBC14 (Dohlman, H. G., et al. (1996) Mol . Cell . Biol . 16(9), 5194-5209). pBC14 was digested with Ncol and transformed into SY2069 by lithium acetate using the URA3 gene for selection. Ura⁺ colonies were grown in non- selective media (YEPD) and plated onto media containing 5- Flouro-orotic Acid (5-FOA, Sigma) . The resultant 5-FOA resistant isolates were tested for the supersensitive phenotype by assaying lacZ activity in response to α-factor (data not shown) . One sst2 strain was named JEY5 and used in all subsequent studies. Yeast were grown in SC + 2% Galactose or 2% Glucose media lacking Uracil. The EDG-2 coding region was amplified by RT-PCR using Pfu DNA polymerase under conditions described by the supplier (Stratagene) . The template for RT-PCR was cDNA (5ng) that was reverse transcribed from human fetal brain total RNA (Clontech) using Superscript II Reverse Transcriptase as described by the supplier (Gibco BRL) . lμM each of the following primers, FP; 5'- GCGATAGGATCCATCATGGCTGCCATCTCTACTTC-3' (SEQ ID NO. 2) and RP; 5'-GCGATACTCGAGCTAAACCACAGAGTGATCATTGC-3' (SEQ ID NO. 3) , were used for RT- PCR. Oligonucleotide Synthesis and DNA Sequencing-PCR primers and DNA sequencing primers were synthesized by the phosphoramidite method with an Applied Biosystems model 394 synthesizer, purified by polyacrylamide gel electrophoresis and desalted on Sep-Pak Cj.8 cartridges (Waters Associates, Milford, MA) . The edg-2 cDNA was sequenced in pYEUra3 by the dideoxy chain termination method using the T7 Sequenase7-deaza-dGTP sequencing kit as described by the supplier (Amersham Life Science) . The primers were designed based on the human edg-2 cDNA sequence submitted to Genbank by Zondag and Moolenaar (accession no. Y09479) and included restriction site extensions for subcloning into the pYEUra3 vector (Stratagene) . This placed the cDNA under the control of a galactose-inducible promoter (UASgal). The resulting plasmid was used to transform JEY5 by the lithium acetate method.

Expression of the EDG-2 to test the effects of the SST2 gene product on the edg-2 response to LPA, JEY5 (sst2Δ) expressing the EDG-2 receptor was compared to the parental SY2069 strain (SST2⁺) . Figure 2 shows that the SST2⁺ strain was unresponsive to LPA whereas the sst2^" derivative was activated by 200μM LPA. As a control, JEY5+EDG-2 was assayed in 2% glucose such that the GALl promoter would be repressed and thus not expressing the edg-2 gene (glucose repression is described in detail by Johnston, M. , and Carlson, M. (1992) in The molecular and cellular biology of the yeast Saccharomyces : gene expression (Broach, J. R. , Pringle, J. R. , and Jones, E. W. , eds), pp. 193-281, Cold Spring Harbor Laborartory Press, Plainview, N.Y.). lacZ assays in response to phospholipids: JEY5 + pJE15 was grown on SC media containing either 2% galactose or 2% glucose lacking uracil to an approximate optical density of 0.1-0.5 prior to the addition of lipid or α- factor. LPA and other glycerophospholipids (Avanti Polar Lipids, Alabaster, AL) were dissolved in chloroform and dried down under vacuum immediately prior to experiments and resuspended in BBS/EDTA (50mM NH₄HC0₃, 104mM NaCl, 250mM EDTA"2Na) at lmg/ml with sonication until the solution was clear. Sphingosine-1- phosphate (Matreya) was resuspended in ethanol/water (9:1) pH 3.0 immediately prior to use. Fatty Acid Free Bovine Serum Albumin was obtained from Sigma (St. Louis, MO) and used at 0. lmg/ml in BBS/EDTA. Cells were allowed to grow for the indicated time (7 hours for^' dose response experiments) prior to assaying. lOOμl of yeast culture were then added to 900μl assay buffer (per liter: 60 mM Na₂HP0₄, 40mM NaH₂P0₄, lOmM KCl, 0.1 mM MgS0₄, pH 7.0 plus 2.7ml β- mercaptoethanol per liter) plus 50μl 0.1%SDS + three drops Chloroform. Cells were vortexed for 10 seconds and incubated for 5 minutes at 28°C. 200μL of 4mg/ml o- nitrophenol-β-D-galactopyranoside (ONPG, Sigma) were added and the reaction was incubated 30 minutes at 28°C. The assay was stopped by the addition of 500μl 1M Na₂C0₃. Color development was measured at A_{4 0} and normalized to Agrjo- Units were expressed as Miller Units. Figure 2 shows that the induction of lacZ activity is dependent on 1) the pYEUra3-Edg2 plasmid being present in the yeast cell, 2) the yeast cell containing the pYEUra3- Edg2 plasmid being grown on the sugar galactose such that the UASgal promoter which drives the expression of the Edg2 gene is induced and 3) lysophosphatidic acid being present.

To further characterize the LPA response to EDG-2, the time and dose dependency of LPA activation was tested. As seen in figure 3A, LPA results in a time-dependent increase in lacZ activity as compared to vector control with a maximal four-fold stimulation of activity at 7 hours. The dose response of EDG-2 to LPA is shown in Figure 3B

(E.C.50=20μM-30μM) . LPA concentrations above 600 μM could not be tested due to toxicity. However, the dose response curve can been seen to plateau suggesting that maximal activity has been reached. This toxicity was seen in other glycerophospholipids tested at 200μM (see below) . However, the response to LPA was significantly above the vector control suggesting that EDG-2 confers LPA responsiveness upon yeast. LPA resuspended in BBS/EDTA + fatty acid free albumin showed the same activity as did freely soluble LPA (data not shown and see below) . The results support that the expression of EDG-2 in yeast faithfully reconstitutes many of the key properties of an LPA receptor.

Example 2 EDG-2 responds selectively to LPA and not to other lysophospholipids or to corresponding diacyl- glycerophospholipids

Yeast does not have endogenous receptors for glycerphospholipids such as LPA. Therefor, yeast represented an excellent naϊve system to evaluate the agonist binding specificity of EDG-2.

Lysophosphatidylethanolamine (LPE) , -serine (LPS) , -glycerol

(LPG) and -choline (LPC) and Sphingosine-1-phosphate (SPP) were tested over the same dose range as LPA. The results are consistent with EDG-2 being a functional, specific LPA receptor. As seen in Figure 4, no other lyso-glycerolphospholipids or Sphingosine-1-phosphate activated EDG-2 as well as LPA at concentrations up to 200μM, the highest concentration tested due to toxicity. The results of a similar experiment testing the effects of the diacyl-glycerophospholipids are seen in Figure 5. In this experiment, no diacyl-glycerophospholipid significantly activated EDG-2 except phosphatidic acid (PA) . However, the activity of PA may be due to contaminating LPA as determined by HPLC (data not shown) .

Example 3

Acyl-chain length dependency of EDG-2 activity by LPA

Due to the apparent specificity of EDG-2 for LPA, the dependency of the acyl-chain length of the LPA molecule on EDG-2 activation was also determined. Six forms of LPA were tested: 24:1, 18:1 (Oleoyl) , 18:0 (Stearoyl), 16:0 (Palmitoyl), 14:0 (Myristoyl) and 6:0 (Caproyl) . The experiment was carried out as described in Example 2, above, using the four forms of LPA. Figure 6 shows that those LPA molecules containing 16 or 18 carbons activated EDG-2. This experiment was repeated with the LPA analogs suspended in 0.1 mg/ml fatty acid free BSA with similar results (data not shown) .

Example 4

LPA in a liposomal formulation is effective in EDG-2 activation

To test the effects of presenting LPA to the yeast cells as a liposome rather than as freely soluble LPA or bound to albumin, liposomes were formed with either phosphatidylcholine or phosphatidylglycerol . To formulate the liposomes, LPA and dioleoyl-phosphatidylcholine or 1- palmitoyl-2-oleoyl-phosphatidylglycerol were mixed at a weight ratio of 1:9 (LPA: PC or PG) in Chloroform solution, dried under vacuum (Savant) and resuspended in BBS/EDTA to a total lipid concentration of lOmg/ml (LPA concentration = lmg/ml) . The resultant opaque suspension was sonicated until the solution was clear (approximately 10 minutes, Lab Supplies Co., Hicksville, NY). The liposome size ranged from 50nm to 80nm as determined on a Coulter N4 Plus Particle Sizer. (Coulter) .

Figure 7 shows that LPA-containing liposomes showed equivalent activity towards EDG-2 as did freely soluble LPA. Phosphatidylcholine and phosphatidylglycerol as liposome preparations gave no activity on their own (Figure 4) . These results support that the form in which LPA is presented does not significantly effect its ability to activate EDG-2; albumin-bound, freely soluble and liposomal formulations of LPA all activated EDG-2 with equal efficacy.

Example 5

In the process of evaluating various analogs of LPA for their effect on the expression of edg-2 assay, as described in Example 3, it was discovered that some LPA analogs compete with LPA to interact with the edg-2 receptor, thereby acting as inverse agonists of LPA. To test the activity of LPA analogs, 100 μM 18:1 acyl- LPA was added to the yeast cultures described in Example 1, along with a dose range of the lipid being testing for modulating activity (0.02 to 200μM) . Yeast cultures were assayed for lacZ activity according to the protocol described in Example 2, above. All lipids tested were dried under vacuum and resuspended in BBS/EDTA at 20 mM prior to use except for LPA analogs that did not contain a phosphate group. These analogs were store in 100% DMSO and were added directly to the yeast cultures.

Results of these tests appear in Figures 8 - 10

14:0 LPA (ether)

18:1 LPA (ether)

6:0 LPA (ester)

10:0 LPA (ester)

14:0 LPA (ester)

As is clear from the figures, carbon chains having between 6 and 16 carbons, linked via an ester or an ether, to a substituted or unsubstituted glycerol moiety, display antagonistic or inverse agonistic activity in the presence of 18:1 LPA.

As set forth in Table 1, below, blocked phosphate substituted, as well as cyclic ether substituted compounds also have antagonistic or inverse agonistic activity against LPA or its receptor. TABLE 1

Example 6 Structure and Synthesis of Various Analogs of Lysophosphatidic Acid

The following example sets forth the synthetic methodology and analytical data used in the construction and characterization of several lysophosphatidic acid analogs and derivatives. In addition experimental procedures and analytical data has been provided for intermediates used in the construction of these LPAs.

Contained at the end of the experimental procedures and analytical data are reaction schemes which show the synthetic routes used in compound construction. The following nomenclature and abbreviations are used in the naming of the compounds:

Bn benzyl

BSA bis (trimethylsilyl) acetamide t-BuOOH tert . -butylhydroperoxide CNE cyanoethyl DMAP N,N-dimethylaminopyridine DMF N,N-dimethylformamide lie L-isoleucine Me methyl

Mel methyl iodide MeOH methanol sat. saturated

TBAF tetra-butylammonium fluoride TBS tert . -butyldimethylsilyl TBTU 0-benzotriazol-l-yl-N,N,N' ,N' - tetramethyluronium tetrafluoroborate

TEA triethylamine

THF tetrahydrofuran

TLC thin layer chromatography

TMSBr trimethylsilyl bromide

Tr trityl

Ts tosyl

Val L-valine

Z benzyloxycarbonyl

1-O-Decyl-rac-glycerol (Compound 1)

To a stirring mixture of NaH (0.36 g, 15.1 mmol) in dry DMF (15 ml) under N₂ was added solketal (0.94 ml, 7.6 mmol) dropwise over a 30 min period. After 30 min, Nal (0.30 g, 2.0 mmol) was added followed by the addition of 1- chlorodecane (1.34 g, 7.6 mmol) and stirring was continued at 50 °C for 16 hours. The reaction mixture was diluted with diethyl ether (50 mL) and washed with H₂0 (2 x 50 mL) , dried (MgS0 ) and concentrated to give a crude oil (1.48 g) . The crude oil (1.06 g, 3.9 mmol) was treated with 1/1 2M HCl/THF

(14 mL, v/v) for 2 hours, and the resulting solution was concentrated and redissolved in ethyl acetate (80 ml), washed with H₂0 (2 x 20 mL) , dried (MgS0₄) and concentrated to give a yellow oil. The oil was subjected to silica-gel column chromatography [eluent: ethyl acetate/hexane, 50/50, v/v] to give the title diol (Compound 1) (58 mg, 65%) as a colourless oil.

IH NMR (360 MHz; CDC13) : d 0.87 (3 H, t, J 7 Hz, Me), 1.26 (14 H, br s, 7 x CH2), 1.53-1.59 (2 H, m, b-H2), 3.41- 3.55 (4 H, m, a-H2 and 1-H2 or 1-H2), 3.64 (1 H, dd, J 11.5 and 5 Hz, 1-H or 3-H) , 3.72 (1 H, dd, J 11.5 and 4 Hz, 1-H or 3-H) and 3.83-3.88 (1 H, m, 2-H) ; ESI-MS (m/z, +ve) : 231 (MH+, 100%) .

1-O-Tetradecyl-rac-glycerol (Compound 2) (OD60/100/1) To a mixture of solketal (5.0 mL, 40 mmol), 1- chlorotetradecane (10.9 mL, 40 mmol) and a catalytic amount of Nal in DMF (200 mL) was added NaH (3.1 g, 80 mmol) and the reaction was stirred for 16 hours at 50 °C. After removal of the solvent in vacuo, the residue was redissolved in ethyl acetate (150 mL) , washed with H₂0 (3 x 50 mL) , dried (MgS0₄) and evaporated to dryness. The obtained oil was treated with 1/1 2M HCl/THF (150 mL, v/v) for 16 hours. The resulting mixture was concentrated and redissolved in ethyl acetate (150 mL) , washed with H₂0 (3 x 50 mL) , dried (MgS0₄) and evaporated to dryness. The crude product was purified by silica-gel column chromatography [eluent: CH₂C1₂—methyl acetate/CH₂Cl₂, 50/50, v/v] to yield diol Compound 2 (8.4 g, 73%) as a white solid.

IH NMR (360 MHz; CDC13): d 0.87 (3 H, t, J 7 Hz, Me), 1.25 (22 H, br s, 11 x CH2) , 1.57 (2 H, quintet, J 7 Hz, b- H2), 2.38 and 2.77 (each 1 H, br s, 2 x OH), 3.46 (2 H, dt, J 6.5 and 2 Hz, 1-H2 or a-H2) , 3.49-3.55 (2 H, m, a-H2 or 1- H2), 3.64 (1 H, dd, J 11.5 and 5 Hz, 3-H), 3.71 (1 H, dd, J 11.5 and 4 Hz, 3-H) and 3.85-3.86 (1 H, m, 2-H) .

3-O-Tetradecyl-rac-glycero-l-phosphate (Compound 4) To a solution of diol 2 (1.0 g, 3.5 mmol) and dibenzyl N,N- diisopropylphosphoramidite (1.16 mL, 3.5 mmol) in dry CH₃CN (30 mL) was added lH-tetrazole (0.12 g, 1.7 mmol). After stirring for 2 hours, t-BuOOH (2 mL) was added and after another 30 min the mixture was concentrated. The residue was purified by silica-gel column chromatography [eluent: hexane/ethyl acetate, 66/33, v/v) to give dibenzyl 3-0- tetradecyl-rac-glycero-1-phosphate (0.27 g, 14%) as an oil. Subsequent hydrogenolysis in MeOH (30 L) in the presence of 10% Pd/C (0.5 g) , followed by filtration over Celite and evaporation to dryness, afforded the title phosphate (Compound 4) (0.18 g, 99%) as a glass.

IH NMR (360 MHz; CDC13) : d 0.87 (3 H, t, J 6.5 Hz, Me), 1.25 (22 H, br s, 11 x CH2), 1.55 (2 H, br s, b-H2), 3.45 (4 H, br s, a-H2 and 3-H2) and 4.05-4.14 (3 H, m, 2-H and 1- H2); 31P NMR(146 MHz; CDC13) : d 1.11; ESI-MS (m/z, +ve) : 367 (M-H+, 100%) . 1-O-Decyl-glycidol (Compound 6) To a stirring solution of glycidyl tosylate (2.0 g, 8.8 mmol) and decyl alcohol (1.34 mL, 7.0 mmol) in dry CH₂C1₂ (40 mL) under N2 was added a BF3.0Et₂ solution (-2.2 mL, -10% in CH2C12). After 48 hours, TLC analysis showed the reaction to be complete and the solvent was removed in vacuo to give 1-O-decyl-rac- glycero-3-p-toluenesulfonate (Compound 5) as an oil.

Crude Compound 5 was taken up in 50% aqueous MeOH (30 mL) , NaOH (1.4 g, 35.0 mmol) was added and the mixture was left to stir for 16 hours. TLC analysis showed the reaction to be complete and the mixture was concentrated to remove MeOH. The remaining aqueous phase was extracted with diethyl ether (2 x 40 mL) and the combined organic phases were washed with H₂0 (30 mL) , sat. NaHC0₃ (30 mL) , dried (MgS0) and concentrated. The residue was subjected to silica-gel column chromatography [eluent: ethyl acetate/hexane, 91/9, v/v] to give epoxide Compound 6 (0.93 g, 62%) as a colourless oil.

IH NMR (360 MHz; CDC13) : d 0.87 (3 H, t, J 7 Hz, Me), 1.26 (14 H, br s, 7 x CH2) , 1.58 (2 H, m, b-H2) , 2.61 (1 H, dd, J 5 and 2.5 Hz, 3-H), 2.80 (1 H, t, J 4.5 Hz, 3-H), 3.13-3.17 (1 H, m, 2-H), 3.38 (1 H, dd, J 11.5 and 6 Hz, 1- H) , 3.42-3.54 (2 H, m, a-H2) and 3.70 (1 H, dd, J 11.5 and 3 Hz, 1-H) . 1-O-Tetradecyl-glycidol (Compound 8) To a solution of glycidyl tosylate (2.0 g, 8.8 mmol) and 1-tetradecanol (1.5 g, 7.0 mmol) in dry CH₂C1₂ (40 mL) under N₂ was added a BF₃.OEt₂ solution (-2.2 L, -10% in CH₂C1₂) . After 48 hours, TLC analysis showed the reaction to be complete and the solvent was removed in vacuo to give 1-O-tetradecyl-rac- glycero-3-p-toluenesulfonate (Compound 7) as an oil.

Crude Compound 7 was taken up in 50% aq. MeOH (30 mL) , NaOH (1.4 g, 35.0 mmol) was added and the mixture was left to stir for 16 hours. TLC analysis showed the reaction to be complete and the mixture was concentrated to remove MeOH. The remaining aqueous phase was extracted with diethyl ether (2 x 40 mL) and the combined organic phases were washed with H₂0 (30 mL) , sat. NaHC0₃ (30 mL) , dried (MgS0₄) and evaporated to dryness. The residue was subjected to silica- gel column chromatography [eluent: ethyl acetate/hexane, 91/9, v/v] to give epoxide Compound 8 (1.57 g, 83%) as a colourless oil.

IH NMR (360 MHz; CDC13) : d 0.88 (3 H, t, J 7 Hz, Me), 1.25 (22 H, m, 11 x CH2), 1.57 (2 H, m, b-H2), 2.61 (1 H, dd, J 5.5 and 3 Hz, 3-H), 2.80 (1 H, t, J 4.5 Hz, 3-H), 3.13-3.17 (1 H, m, 2-H), 3.38 (1 H, dd, J 11.5 and 6 Hz, 1- H) , 3.42-3.54 (1 H, m, a-H2) and 3.70 (1 H, dd, J 11.5 and 6 Hz, 1-H) .

1-O-Oleyl-glycidol (Compound 10) To a solution of glycidyl tosylate (2.0 g, 8.8 mmol) and oleyl alcohol (1.88 mL, 7.0 mmol) in dry CH₂C1₂ (40 mL) under N₂ was added a BF3.0Et₂ solution (-2.2 mL, -10% in CH₂C1₂) . After 48 hours, TLC analysis showed the reaction to be complete and the solvent was removed in vacuo to give 1-O-oleyl-rac-glycero- 3-p-toluenesulfonate (Compound 9) as an oil.

Crude Compound 9 was taken up in 50% aq. MeOH (30 mL) to which NaOH (1.4 g, 35.0 mmol) was added. After 16 hours, TLC analysis showed the reaction to be complete and the mixture was concentrated to remove MeOH. The remaining aqueous phase was extracted with diethyl ether (2 x 40 mL) and the combined organic phases were washed with H₂0 (30 mL) , sat. NaHC0₃ (30 mL) , dried (MgS0₄) and concentrated. The residual oil was purified by silica-gel column chromatography [eluent: ethyl acetate/hexane, 91/9, v/v] to furnish epoxide Compound 10 (1.65 g, 72%) as a colourless oil. IH NMR (360 MHz; CDC13) : d 0.88 (3 H, t, J 6.5 Hz, Me), 1.28 (22 H, apparent br d, separation 9 Hz, -(CH2)5- and - (CH2)6-), 1.58 (2 H, br s, b-H2), 2.01 (4 H, apparent br d, separation 4.5 Hz, -CH2CH=CHCH2-) , 2.61 (1 H, br s, 3-H), 2.80 (1 H, br t, J 4 Hz, 3-H), 3.15 (1 H, br s, 2-H), 3.38 (1 H, dd, J 11.5 and 6 Hz, 1-H), 3.45-3.54 (2 H, m, a-H2), 3.69-3.72 (1 H, m, 1-H) and 5.34 (2 H, br s, -CH2CH=CHCH2-) . 3-O-Decyl-rac-glycero-l-phosphate (Compound 11) A mixture of 98% phosphoric acid (0.18 g, 1.9mol) and decyl glycidol (Compound 6) (0.4 g, 1.9 mmol) in dry CH₂C1₂ was refluxed for 2 hours until TLC analysis showed the reaction to be complete. Then, the reaction mixture was concentrated to afford phosphate Compound 11 (0.56 g, 78%) as a colourless oil.

IH NMR (360 MHz; CDC13) : d 0.87 (3 H, t, J 6.5 Hz, Me), 1.25 (14 H, br s, 7 x CH2) , 1.56 (2 H, br s, b-H2), 3.44- 3.53 (4 H, m, a-H2 and 3-H2) and 3.61-4.13 (3 H, m, 2-H and 1-H2); 31P NMR (146 MHz; CDC13) : d 1.41; ESI-MS (m/z, -ve) : 311 (M-H+, 100%) .

3-O-Oleyl-rac-glycero-l-phosphate (Compound 12) A mixture of 98% phosphoric acid (0.18 g, 1.9 mol) and oleyl glycidol (Compound 10) (0.4 g, 1.2 mmol) in dry CH₂C1₂ was refluxed for 16 hours. TLC analysis showed the reaction to be complete and the mixture was concentrated to give the title phosphate (Compound 12) (0.501 g, 96%) as a colourless oil. IH NMR (360 MHz; CDC13) : d 0.87 (3 H, t J 6.5 Hz, Me),

1.27 (22 H, apparent br d, separation 6 Hz, -(CH2)5- and -

(CH2)6-), 1.55 (2 H, br s, b-H2), 1.98-2.03 (4 H, m, -

CH2CH=CHCH2-) , 3.44-3.51 (4 H, m, 3-H2 and a-H2), 3.53-4.12

(3 H, m, 2-H and 1-H2) , 5.30-5.38 (2 H, , -CH2CH=CHCH2-) and 6.43 (2 H, br s, 2 x OH); 31P NMR (146 MHz; CDC13) : d 1.68; ESI-MS (m/z, -ve) : 421 (M-H+, 100%).

3-Hydroxypropyl decyl ether (Compound 17) To a mixture of NaH (2.6 g, 70 mmol) and anhydrous Nal (9.9 g, 70 mmol) in dry DMF (80 mL) under N₂ was added dropwise a solution of 1,3-propanediol (4.75 mL, 70 mmol) in DMF (20 mL) . The mixture was stirred until hydrogen evolution had ceased, decyl chloride (1.34 g, 7.6 mmol) was added and stirring was continued at 50 °C for 18 hours. Then, the reaction mixture was poured into H₂0 (300 mL) and extracted with diethyl ether (3 x 250 mL) . The organic extracts were combined, washed with sat. NaCl (100 mL) , dried (MgS0 ) and evaporated to dryness. The residue was subjected to silica-gel column chromatography [eluent: ethyl acetate/hexane, 30/70, v/v] to furnish decyl ether Compound 17 as a colourless oil.

^XH NMR (360 MHz; CDC1₃) : δ 0.87 (3 H, t, J 7 Hz, Me), 1.25 (14 H, br s, 7 x CH₂) , 1.55 (2 H, quintet, J 7 Hz, β- H₂), 1.82 (2 H, quintet, J 5.5 Hz, 2-H₂) , 2.62 (1 H, br s, OH), 3.41 (2 H, t, J 7.5 Hz, α-H₂) , 3.60 (2 H, t, J 5.5 Hz, 1-H₂), and 3.77 (2 H, t, J 5.5 Hz, 3-H₂) ; ESI-MS (m/z, +ve) : 217 (Mi⁺, 100%) . 3-Decyloxypropyl-l-phosphate (Compound 19) To a solution of alcohol Compound 17 (0.5 g, 2.3 mmol) in dry CH₂C1₂ (20 mL) was added TEA (0.48 mL, 3.5 mmol) followed by the addition of trimethyl phosphite (0.41 mL, 3.5 mmol). After 90 min, the reaction was cooled to -40 °C, pyridinium tribromide (0.89 g, 2.8 mmol) was added and the reaction was allowed to warm to 20 °C overnight. The mixture was subsequently quenched with sat. KHS0₄ (30 mL) and extracted with ethyl acetate (2 x 50 mL) . The organic extracts were combined, washed with sat. NaCl (30 mL) , dried (MgS0₄) and concentrated to give phosphate triester Compound 18. H NMR (360 MHz; CDC1₃) : δ 0.87 (3 H, t, J 6.5 Hz, Me), 1.25 (14 H, br s, 7 x CH₂) , 1.50-1.56 (2 H, m, β-H₂) , 1.93 (2 H, quintet, J 6 Hz, 2-H₂) , 3.39 (2 H, t, J 6.5 Hz, α-H₂ or 3- H₂) , 3.49 (2 H, t, J 6 Hz, α-H₂ or 3-H₂) , 3.74 and 3.77 (each 3 H, s, 2 x OMe) and 4.14 (2 H, quartet, J 6.5 Hz, 1-H₂) .

To a stirring solution of phosphate triester Compound 18 (0.25 g, 0.72 mmol) in dry CH₂C1₂ (5 mL) was added BSA (0.25 mL, 1.0 mmol) followed by the addition of TMSBr (0.3 mL, 2.3 mmol). After 15 min, TLC analysis showed complete consumption of the starting material and the reaction was quenched with 1/1 MeOH/H₂0 (2 mL, v/v) for 15 min, followed by the addition of sat. KHS0₄ (5 mL) . The reaction mixture was subsequently extracted with ethyl acetate (2 x 30 mL) , and the combined organic extracts were dried (MgS0₄) and concentrated to give the title phosphate (Compound 19) as an oil . ^λ NMR (360 MHz; CDC1₃) : δ 0.88 (3 H, t, J 7 Hz, Me),

1.27 (14 H, br s, 7 x CH₂) , 1.55-1.58 (2 H, m, β-CH₂) , 1.95

(2 H, br s, 2-H₂), 3.42-3.48 (4 H, m, α-H₂ and 3-H₂) , 4.10 (2

H, br s, 1-H₂) and 6.35 (2 H, br s, 2 x OH); ESI-MS {m/z, - ve):295 ( -H⁺, 100%). Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention, which is delineated by the appended claims.

Claims

Claims

1. A method of modulating the biological activity of a lysophosphatidic acid or a lysophosphatidic acid receptor, comprising introducing to said lysophosphatidic acid, or its receptor, a composition comprising one of the following structures :

R_! — α_R₂

or

O

or

or

OR₄

or

or OR*

wherein Ri is selected from the group comprising a substituted or unsubstituted, saturated or unsaturated, straight or branched-chain alkyl group having from 1 to about 4 carbon atoms, or a cyclic ether; R₂ is a substituted or unsubstituted, saturated or unsaturated, straight or branched-chain alkyl having from about 7 to about 15 carbon atoms; and R₃ and R are independently H or a substituted or unsubstituted, saturated or unsaturated, straight or branched-chain alkyl having from 1 to about 3 carbon atoms, or a salt thereof.

2. The method of claim 1, wherein said composition is a lysophosphatidic acid having a fatty acid chain of from approximately 10 to approximately 14 carbon atoms.

3. The method of claim 2, wherein said composition has the following structure

OR₄

or a pharmaceutically acceptable salt thereof.

4. A composition comprising: 1-O-Decyl-rac-glycerol ,

5. A composition comprising: 3-O-Tetradecyl-rac- glycero-1-phosphate .

6. A composition comprising: 1-O-Decyl-glycidol .

7. A composition comprising: 1-O-Tetradecyl-glycidol ,

8. A composition comprising: 1-O-Oleyl-glycidol .

9. A composition comprising: 3-O-Decyl-rac-glycero-l- phosphate.

10. A composition comprising: 3-O-Oleyl-rac-glycero-l- phosphate.

11. A composition comprising: 3-Hydroxypropyl decyl ether.

12. A composition comprising: 3-Hydroxypropyl decyl ether.

13. A composition comprising: 3-Decyloxypropyl-l- phosphate.