Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Phosphorus containing other than solely as part of an...
Reexamination Certificate
2000-06-23
2002-04-30
Carr, Deborah D. (Department: 1621)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Phosphorus containing other than solely as part of an...
C514S143000, C554S078000
Reexamination Certificate
active
06380177
ABSTRACT:
FIELD OF INVENTION
Applicant has probed the Edg2 lysophosphatidic acid (LPA) receptor with a series of LPA analogs to determine receptor activation. The present invention is drawn to a series of LPA analogs which function as Edg2 receptor agonists, and methods of using such compounds to activate the Edg2 receptor of the surface of a cell. The compounds of the invention comprise a glycerol backbone with an ester-linked saturated or unsaturated alkyl group containing 15-17 carbons in addition to the carbonyl, substitutions of the hydroxyl group (—OH) at carbon two of the glycerol backbone, and optional replacement of the phosphate di-anion with either a hydroxyl group or a dimethylated phosphate. These LPA analogs may find uses in cancer and neurological disorders.
BACKGROUND
Lysophosphatidic acid (O-acyl-2-hydroxyl-sn-glycero-3-phosphate, LPA) is an intensely studied lipid phosphoric acid mediator of many well characterized and diverse biological responses. Although certain pathways in which LPA acts as a signal have been studied, specific LPA receptors are not well understood. One of the difficulties associated with studying a particular effect of LPA is that most cells are in some way responsive to LPA, thus assigning a specific effect with a specific receptor has been problematic. Three receptors for LPA have been described in the literature; PSP24, Edg2 (also referred to as vzg1, lp
A1
and Gpcr26) and Edg4. Edg2 and Edg4 are a part of a small gene family of related receptors that currently has five members. Edg1, Edg3 and Edg5/H218 have been reported to be receptors for sphingosine-1-phosphate. PSP24, an LPA receptor from
X. leavies
and mice shares only 10% identity with Edg2 and is not considered to be a part of the Edg family. The following references are specifically incorporated herein in their entirety. An, S., M. A. Dickens, T. Bleu, O. G. Hallmark and E. J. Goetzl (1997)
Biochem. Biophys. Res. Comm
. 231, 619-622
An, S., T. Bleu, O. G. Hallmark and E. J. Goetzl (1998) Characterization of a novel subtype of human G protein-coupled receptor for lysophosphatidic acid.
J. Biol. Chem
. 273, 7906-7910
Erickson, J. R., J. J. Wu, J. G. Goddard, G. Tigyi, K. Kawanishi, L. D. Tomei and M. C. Kiefer (1998) Edg-2/vzg-1 couples to the yeast pheromone response pathway selectively in response to lysophosphatidic acid
J. Biol. Chem
. 273, 1506-1510
Fischer, D. J., K. Liliom, Z. Guo, N. Nusser, T. Virag, K. Murakami-Murofushi, S. Kobayashi, J. R. Erickson, G. Sun, D. D. Miller and G. Tigyi. (1998) Naturally occurring analogs of lysophosphatidic acid elicit different cellular responses through selective activation of multiple receptor subtypes.
Mol. Pharmacol
. 54:979-988
Guo, Z., K. Liliom, D. J. Fischer, I. C. Bathurst, L. D. Tomei, M. C. Kiefer and G. Tigyi (1996) Molecular cloning of a high-affinity receptor for the growth factor-like lipid mediator lysophosphatidic acid from Xenopus oocytes
Proc. Natl. Acad. Sci. USA
93, 14367-14372
Hecht, J. H., J. A. Weiner, S. R. Post and J. Chun (1996) Ventricular zone gene-1 (vzg-1) encodes a lysophosphatidic acid receptor expressed in neurogenic regions of the developing cerebral cortex
J. Cell Biol
. 135, 1071-1083
Hopper, D. W., S. P. Ragan, S. B. Hooks, K. R. Lynch and T. L. Macdonald (1999) Structure-activity relationship of lysophosphatidic acid: Conformationally restricted backbone mimetics.
J. Med. Chem
. 42:963-970
Hooks, S. B., S. P. Ragan, D. W. Hopper, C. W. Honemann, M. E. Durieux, T. L. Macdonald and K. R. Lynch (1998) Characterization of a receptor subtype-selective lysophosphatidic acid mimetic.
Mol. Pharmacol
. 53, 188-194
Ishii, I., T. Izumi, H. Tsukamoto, H. Umeyama, M. Ui and T. Shimizu (1997)
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Jalink, K, T. Hengeveld, S. Mulder, F. R. Postma, M.-F. Simon, H. Chap, G. A. van der Marel, J. H. van Boom, W. J. van Blitterswijk and W. H. Moolenaar (1995)
Biochem. J.
307, 609-616
Lee, M.-J., M. Evans and T. Hla (1996)
J. Biol. Chem
. 271, 11272-11279
Liliom, K., R. Bitttnan, B. Swords and G. Tigyi (1996) N-palmitoyl-serine and N-palmitoyl-tyrosine phosphoric acids are selective competitive antagonists of the lysophosphatidic acid receptors.
Mol. Pharmacol
. 50:616-623
Lynch, K. R., D. W. Hopper, S. J. Carlisle, J. G. Catalano, M. Zhang and T. L. Macdonald (1997) Structure/activity relationships in lysophosphatidic acid: The 2-hydroxyl moiety.
Mol. Pharmacol
. 52, 75-81
MacLennan, A. J., C. S. Browe, A. A. Gaskin, D. C. Lado and G. Shaw (1994)
Mol. Cell. Neurosci
. 5, 201-209
Macrae, A. D., R. T. Premont, M. Jaber, A. S. Peterson, R. J. Lefkowtiz (1996)
Mol. Brain Res
. 42, 245-254
Okazaki, H. N. Ishizaka, T. Sakurai, K. Kurokawa, K. Goto, M. Kumada and Y. Takuwa (1993)
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227, 608-614
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although certain structure/activity relationships of LPA have been observed, the effects cannot be attributed to a particular or a single receptor because of the possibility that a particular analog could be signaling through different receptors to achieve the same phenotypic effect leading to conflicting results concerning the specific structure/activity relationship of LPA. Indeed, given that there are at least three LPA receptors present in mammalian cells and given the wide range of phenotypic responses to LPA, caution must be exercised when making such analysis.
A yeast expressing the Edg2 receptor is one way to avoid such problems of multiple receptors. Edg2 has been functionally expressed in the yeast
Saccharomyces cerevisiae
and provides a facile system with which to analyze this particular receptor. Edg2 couples to the yeast pheromone response pathway selectively in response to exogenously added LPA and activates two easily assayed reporter genes, lacZ and HIS3, that are fused to the pheromone-inducible FUS1 promoter. Using the FUS1::lacZ reporter, Edg2 in yeast has been shown to selectively respond to LPA and not other glycerophospholipids. Although the yeast assay is relatively insensitive to LPA compared with mammalian signaling events (the yeast assay has an E.C.
50
of 20 &mgr;M), yeast offers the advantage of specificity; the induction of FUS1::lacZ by LPA is absolutely dependent on the Edg2 receptor being expressed.
SUMMARY OF INVENTION
Using a series of 17 LPA analogs that alter the length and saturation of the fatty acid side chain, the linkage between the fatty acid, the glycerol backbone, and the 2-hydroxyl moiety and the phosphate moiety the Edg2 receptor is analyzed pursuant to this invention and the molecular determinants of LPA in Edg2 receptor activation in yeast is established. Therefore, as a preliminary study to the analysis of the Edg2 receptor in mammalian cells, we established the molecular determinants of LPA in Edg2 receptor activation in yeast.
Thus, a primary aspect of this invention are compounds which act as Edg2 agonists. These compounds of the present invention have the formula:
wherein, X is hydroxyl,
Z is hydrogen, bromine, chlorine, fluorine, iodine, or OR
2
, wherein R
2
is an unsaturated alkyl containing 1-3 carbons;
and wherein R
1
is a saturated or unsaturated alkyl chain containing 15-17 carbons.
In preferred compounds of the present invention, X is phosphate. Also, in preferred compounds of the present invention, R
1
is a saturated or unsaturated alkyl chain containing 17 carbons. In other preferred embodiments, Z is hydrogen, bromine, or oxymethyl. Particular preferred embodiments of this aspect of the present invention include 1-(3-propyl oleoate) phosphate, 3-O-oleoyl-2-O-methyl-rac-glycero-1-phosphate, 1-O-oleoyl-2-O-methyl-rac-glycerol, 3-O-oleoyl-2-deoxy-2-bromo-rac-glycero-1-phosphate, and 1-O-oleoyl-2-deoxy-2-bromo-rac-glycerol.
Another aspect of the
Atairgin Technologies, Inc.
Carr Deborah D.
Lyon & Lyon LLP
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