This invention relates to the identification of lysophosphatidic acid (LPA) as a ligand for the G-protein coupled receptors OSGPR114 and OSGPR78, and is directed to in vitro methods for screening candidate drugs for their ability to modulate the activity of OSGPR114 or OSGPR78, and to methods of treating disease by administering to an individual a therapeutic amount of a modulator of OSGPR114 or OSGPR78.
G-protein coupled receptors (GPCRs) are a super-family of membrane receptors that mediate a wide variety of biological functions. Upon binding of extracellular ligands, GPCRs interact with a specific subset of heterotrimeric G proteins that can, in their activated forms, inhibit or activate various effector enzymes and/or ion channels. All GPCRs are predicted to share a common molecular architecture consisting of seven transmembrane helices linked by alternating intracellular and extracellular loops. The extracellular receptor surface has been shown to be involved in ligand binding whereas the intracellular portions are involved in G-protein recognition and activation. Different G-protein alpha-subunits, and beta-gamma subunit complexes, preferentially stimulate or inhibit particular effector molecules to modulate various biological functions in a cell. Typical effector molecules include adenylate cyclase, phospholipases C and A2, cGMP phosphodiesterase-γ, and potassium, sodium and calcium channels. Additional regulation of GPCR activity is thought to occur via receptor oligomerization and interaction with the protein β-arrestin (e.g. see Rocheville, M. et. al. (2000) Science 288:154-157; Gether, U. (2000) Endocrine Reviews 21:90-113; Luttrel, L. M. et. al. (1999) Science 283:655-661). G-protein coupled receptors are found ubiquitously in all cell types within mammalian organisms. Many therapeutic agents targeting GPCR receptors have been successfully introduced onto the market, thereby establishing their value as targets for drug discovery and development (e.g. Wise, A. et al. 2002, DDT, 7:235-246). Over 30% of clinically marketed drugs are active on GPCRs.
It has been estimated that for about 40% of GPCRs in the human genome (excluding sensory receptors) the ligand remains unknown. Such GPCRs are commonly referred to as “orphan” receptors. For example, the primary structures of a human isoform of the orphan GPCRs OSGPR114 and OSGPR78, or closely related GPCRs, have been described in recent patent applications (e.g. OSGPR114 in WO 01/90187, WO 01/87937, WO 01/42288, WO 01/77326, WO 01/48189, WO 01/31014, WO 01/04292, WO 01/02563, WO 00/23588, WO 00/31258, WO 00/50458 and EP 1090926 A1; and OSGPR78 in WO 96/30406). The DNA and amino acid sequences for OSGPR114 and OSGPR78 have also been described in the scientific literature. A chicken homolog of OSGPR78 was originally identified in chicken activated T-cells and named 6H1 by Kaplan (Kaplan et. al. (1993) J. Immunol. 151(2):628-636). Webb (Webb et. al. (1996) Biochem. Biophys. Res. Commun. 219(1):105-110) followed the disclosure of the sequence with a proposal that the receptor bound to ATP, and therefore named it P2Y5 as the fifth member of the purinergic GPCR P2Y family. However, subsequent studies (Li et. al. (1997) Biochem. Biophys. Res. Commun. 236(2):455-60); and experiments described herein) could not find evidence that the receptor was in fact activated by nucleotides, thus calling into question the classification as a P2Y receptor. The human OSGPR78 was sequenced earlier, while sequencing the complete genomic sequence of the human retinoblastoma susceptibility gene (Toguchida et al. (1993) Genomics 17: 535-543), but it was not appreciated until later that the human P2Y5 receptor is encoded in the intron 17 of the retinoblasoma gene (Herzog et al. 1996, Genome research 6: 858-61; Bohm et al. 1997, Genbank entry locus AAB62190, direct submission to Genbank). In the scientific literature, OSGPR114 was originally identified by White (White et. al. (2000) Nat. Genet. 26, 345-348) and named gpr92. Lee (Lee et. al. (2001) Gene 275(1):83-91) also disclosed the sequence.
No information outwith nucleic acid and amino acid sequence homologies and expression patterns for OSGPR114 or OSGPR78 have been described in the scientific literature, e.g. no activating ligand has been identified. Similarly, in the above patent applications OSGPR114 and OSGPR78 are described as tools for identifying drugs for the treatment of a variety of pathophysiological conditions. However, none of these applications identify a function for either of these GPCRs, or describes a ligand that binds to either receptor and modulates its activity.
It has been previously shown that LPA has the ability to modulate cell motility and growth and to stimulate tumor growth; to modulate the development and regulation of the cardiovascular system including a contribution to artherosclerosis and a role in wound healing and tissue regeneration; to regulate the differentiation of multiple cell types including the induction of differentiation of preadipocytes into adipocytes; and to have influence over the physiology and pathophysiology of the reproductive tracts of males and of females. Prior to the work described herein, the effects of LPA in these and other systems were thought to be exclusively the result of interaction with the LPA receptors LPA-1, -2 and -3, previously known as Edg-2, -4 and -7 (Chun, J., et al. (2002) Pharmacol. Reviews 54:265-269).
LPA is one of the simplest phospholipids found in nature and consists of a glycerol moiety with a fatty acid backbone at the sn1 (or sn2) position, a phosphate group at the sn3 position and a hydroxyl at the sn2 (or sn1) site. It has been shown that endogenous LPA species can contain multiple fatty acids. These fatty acids may vary in their chain length, the amount of unsaturation/saturation and may consist of an acyl or alkyl linkage. It has been shown that in some cases, the predominant species of LPA may vary between tissues and/or cell types and is influenced by the available precursor lipids within a particular cell or tissue. The biosynthetic pathways and metabolic pathways of LPA may also vary between cells and are only moderately well characterized. Intracellular and extracelullar synthetic and degradative pathways for LPA are also different, as are the physiological roles of LPA on either side of the cell membrane.
Amongst the multiple known biological roles of LPA, much of the scientific literature attention has been focused on the ability of LPA to act as a proliferative signal to cells of multiple origins, especially malignant cells. In addition to acting as a growth stimulator to cancer cells, LPA has been demonstrated by multiple studies to act as a motility factor and an angiogenic factor in carcinogenesis and cancer progression. It has been shown to increase the invasive capacity of cancer cells, and to have an important role in increasing the metastatic potential of tumors. It is therefore known that LPA is strongly implicated in controlling and contributing towards virtually all aspects of malignant disease. One of the original studies on the role of LPA in cancer identified “ovarian cancer activating factor” as LPA (Xu et. al. 1995 Clin. Cancer Res. 1(10): 1223-32). Additionally, increased tumor production of LPA has been observed and the enzyme responsible for this shown to be upregulated in multiple cancers. Further validation of the role of LPA in cancer disease is shown by the fact that induction or expression of enzymes that degrade LPA do not only prevent the activity of LPA in disease progression in vitro, but also dramatically reduce tumor growth in vivo (Tanyi et. al. 2003 Cancer Res. 63(5):1073-1082). Additionally, LPA levels in the blood, and in ascites, have been shown to be significantly higher in patients with ovarian cancer than in patients who do not have ovarian cancer. The degree of this elevation in blood LPA has been correlated with tumor malignancy. As well as inducing the growth of ovarian cancer cells, LPA also increases their motility and invasiveness and at concentrations present in ascites, prevents cisplatin-induced apoptosis. In summary, there is an extensive body of public literature that conclusively demonstrates that LPA signaling is aberrant in multiple cancers. Cancer types that have been implicated as involving dysregulated LPA signaling, in addition to ovarian cancer, include cancers of the lung, prostate, pancreas, colon, breast, esophagus, kidney and stomach, and glioma, lymphoma, leukemia and melanoma.
The molecular mechanisms behind the involvement of LPA in cancer are the subject of multiple reviews (e.g. Fang et. al. (2000) Ann. N.Y. Acad. Sci. 905:188-208; Fujita et. al. (2003) Cancer Letts 192:161-9; Erickson et. al. (2001) Prostaglandins and other Lipid Mediators 64: 63-81; Daaka (2002) Biochim. Biophys. Acta 1582: 265-269; Fukushima et. al. (2001) Ann. Rev. Pharm. Toxicol. 41:507-34). Although LPA is known to act as both an intracellular and extracellular signaling moiety, most studies investigating the role of LPA in cancer have focused on its role as an autocrine and paracrine growth factor, predominantly stimulating the growth of cancer cells and tumors. Such extracellular signaling pathways have also been shown to be intimately involved in increases in cancer cell motility, invasiveness, angiogenesis and metastasis resulting from LPA administration to cancer cells. The extracellular signaling of LPA is known to be transduced to the cell interior via LPA specific GPCRs. Such receptors have been described in the literature, and there are three that have been well characterized to date, LPA1, LPA2 and LPA3. They were previously named Edg2, Edg4 and Edg7 due to their high homology with other phospholipid receptors Edg 1, 3, 5, 6 and 8, (which are now known as Sphingosine 1-phosphate (SIP) receptors 1 to 5). Edg is an acronym for “endothelial differentiation gene”. Very recently a fourth LPA receptor, p2y9/GPR23, has also been reported (Noguchi et. al. (2003) J. Biol. Chem. April 30, electronic manuscript M302648200 ahead of print). p2y9/GPR23 is only distantly related to LPA1, LPA2 and LPA3. Additionally, activation of GPCRs by LPA is also known to transactivate other growth factors involved in cancer development, such as the epidermal growth factor receptor and the platelet-derived growth factor receptor (for review see Wu and Cunnick (2002) Biochim. Biophys. Acta. 1582(1-3): 100-106). The effects of LPA on mitogenesis and survival likely involve activation of ERK and transactivation of other growth factor receptors, whereas the effects on motility and invasion probably occur through Rho-based signaling, probably via coupling of the receptor to Galpha 12/13. The effects on angiogenesis probably occur through the induction of proangiogenic factors such as VEGF.
Despite the considerable body of literature in this area, a complete understanding of LPA action in modulating cell proliferation and tumor growth at the molecular level has not yet been achieved. Consequently, without a full understanding of its mechanism of action, there are considerable problems associated with developing compounds that antagonize such effects of LPA. To help alleviate this problem, the surprising discovery described herein that certain LPA compounds are ligands of OSGPR114 and OSGPR78, and therefore that the latter are novel members of the family of LPA-activated GPCRs, suggests that the effect of LPA as a physiological modulator of various physiological systems is not limited to interaction with the previously known LPA receptors. Thus, this discovery provides an additional mechanism of action for LPA, additional targets for therapeutic modulation, and thus a basis for further assay and drug development. Several compounds are already under development as modulators of the activity of other LPA receptors (e.g. EP 1258484 A1, WO 02/29001, US 2003/0027800 A1 and U.S. Pat. No. 6,380,177; Fischer, D. J., et al. (2001) Mol Pharmacol. 60(4):776-84; Hasegawa, Y., et al. (2003) J. Biol. Chem. 278(14):11962-9; Heise, C. E., et al. (2001) Mol. Pharmacol. 60(6):1173-80; Hooks, S. B., et al. (2001) J Biol. Chem. 276(7):4611-21; Hopper, D. W., et al. (1999) J. Med. Chem. 42(6):963-70; Tigyi, G. (2001) Mol Pharmacol. 60(6): 1161-4; Yokoyama, K., et al. (2002). Biochim. Biophys. Acta 1582(1-3): 295-308; Gueguen, G., et al. (1999) Biochemistry 38(26): 8440-50; Lynch, K. R. and T. L. Macdonald (2002). Biochim. Biophys. Acta 1582(1-3): 289-94; Sardar, V. M., et al. (2002) Biochim. Biophys. Acta 1582: 309-307 and Virag, T., et al. (2003) Mol Pharmacol. 63(5):1032-42). Some of these compounds have been found to have selective activity on one or more LPA receptors, while others have equivalent activity on all LPA receptors tested.
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