Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-03-12
2002-05-28
Kunz, Gary L. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S300000, C530S403000
Reexamination Certificate
active
06395878
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the cloning and expression of a novel human prostaglandin receptor. Methods of identifying compounds capable of both binding to and activating this receptor are also disclosed. As determined using the disclosed methods, the receptor exhibits EP
2
pharmacology.
BACKGROUND OF THE INVENTION
Prostaglandins are a group of hormone mediators derived from the metabolism of arachidonic acid via the cyclooxygenase enzymatic pathway. In the prostaglandin biosynthetic pathway, arachidonic acid is first converted to prostaglandin endoperoxide H2 (PGH
2
) by PGH
2
synthases followed by the cell-specific isomerization or reduction of PGH
2
to the active prostaglandins: PGD
2
, PGE
2
, PGF
2&agr;
, prostacyclin (PGI
2
) and thromboxane (TxA
2
). Following enzymatic conversion, the major biologically active prostaglandins exert their actions locally on the cells in which they were synthesized (autocrine) and/or on nearby cells (paracrine) through specific G protein-coupled receptors (Smith, (1992)
Am. J. Physiol.,
263: F181-F191) to either stimulate or inhibit the production of second messengers. Prostaglandins elicit a diverse spectrum of often opposing biological effects including muscle contraction and relaxation, potentiation and inhibition of platelet aggregation, and vasodilation and vasoconstriction. Prostaglandins also exhibit both pro-inflammatory and anti-inflammatory effects. They synergize with other pro-inflammatory mediators such as leukotrienes and bradykinins, but attenuate interleukin-1 (IL-1) production and inhibit various aspects of leukocyte function (Giles, (1990)
Trends Pharmacol. Sci.,
11:301-304).
Prostaglandin E
2
(PGE
2
) exhibits a broad range of actions in a number of tissues by binding to at least four EP receptor subtypes. It acts through pharmacologically distinct stimulatory (EP
2
) and inhibitory (EP
3
) receptor subtypes to stimulate and inhibit cyclic AMP (cAMP) formation, respectively (Sonnenburg, and Smith, (1988)
J. Biol. Chem.,
263: 6155-6160). PGE
2
also stimulates calcium release and protein kinase C activity in the rabbit kidney collecting tubule, most likely by binding to the EP
1
receptor subtype which is coupled to stimulation of phospholipase C (Hebert et al., (1990)
Am. J. Physiol.,
259: F318-F325). The EP
4
receptor is an additional subtype of PGE
2
-sensitive receptor that was recently identified based on agonist effects and blockade by the antagonist AH 23848B (Louttit et al., (1992)
The Eighth International Congress on Prostaglandins and Related Compounds,
Montreal, 258; Coleman et al., (1994)
Prostaglandins,
47:151-168). Other PGE
2
-sensitive receptors with distinct agonist pharmacology have been described (Milne et al., (1994)
Br. J. Pharmacol.,
111:79), but it is not clear whether they are different from the EP
4
receptor.
Analogs of PGE
2
that are therapeutically useful will elicit or block only a subset of its actions by acting on a single EP receptor subtype. Because prostaglandin receptors are present in tissues in low abundance, the discovery of such analogs is facilitated by the cloning of the receptors. Assigning cloned receptors to a corresponding pharmacologically defined binding site is an iterative process. Defining novel subtypes requires selective compounds, which may only be developed once the receptor is cloned.
Three human receptors that bind PGE
2
have been cloned. The EP
1
(Funk et al., (1993)
J. Biol. Chem.,
268: 26767-26772) and EP
3
(Regan et al., (1994)
Br. J. Phamacol.,
112:377-385) subtypes have been well characterized with subtype-selective compounds, but the pharmacology of the putative EP
2
receptor (An et al., (1993)
Biochem. Biophys. Res. Commun.,
197:263-270; Honda et al., (1993)
J. Biol. Chem.,
268:7759-7762) is not entirely consistent with the pharmacology derived from tissue models of the EP
2
receptor. In particular, the EP
2
-selective agonist butaprost, is inactive (Gardiner (1986)
Br. J. Pharmacol.,
87:45-56; Coleman, (1993) in
Eicosanoids and Other Bioactive Lipids in Cancer, Inflammation and Radiation Injury,
Nigan et al., eds., pp. 135-141). The pharmacology of this putative EP
2
clone is more similar to that of the EP
4
receptor, but it was named before the EP
4
receptor had been described.
The deduced protein sequences of the cloned receptors indicate that all are members of the G protein-linked receptor superfamily, having seven putative membrane-spanning hydrophobic domains. The proteins share significant amino acid sequence similarity with other members of this family including the thromboxane (TP) receptor (Hirata et al., (1991)
Nature
349: 617-620), rhodopsin and the adrenergic receptors.
The cloning of EP
2
and/or additional EP receptors will facilitate identification of prostaglandins which can modulate specific effects elicited by this receptor. Since these effects will differ from those activated by other EP receptors, such compounds will have therapeutic utility.
SUMMARY OF THE INVENTION
One embodiment of the present invention is an isolated DNA molecule encoding a novel mammalian prostaglandin EP receptor, herein called HP4 (Human Placental clone Number 4). Preferably, the DNA molecule is human; most preferably, the DNA molecule has the nucleotide sequence shown in SEQ ID NO: 3. According to another aspect of the invention, there is provided an isolated DNA molecule having at least 18 consecutive nucleotides of the DNA molecule encoding the HP4 receptor. In accordance with another aspect of the invention, there is provided an isolated amino acid sequence derived from the HP4 receptor DNA sequence. Preferably, the amino acid sequence is human; most preferably it is SEQ ID NO:4. Advantageously, there is also provided a recombinant construct comprising the HP4 receptor DNA sequence operably linked to a heterologous promoter. In another aspect of this preferred embodiment, there is provided an isolated antibody having binding affinity for the isolated HP4 receptor amino acid sequence. Preferably, the antibody is monoclonal.
Another embodiment of the invention is a method of screening compounds for binding to the prostaglandin HP4 receptor comprising:
transfecting cells with a DNA molecule encoding an HP4 receptor, wherein the DNA molecule is operably linked to a promoter in an expression vector;
culturing the cells to express the HP4 receptor;
incubating at least the cell membranes of the cells in the presence of a labeled compound to be tested for binding affinity to the HP4 receptor; and
measuring the amount of label bound to the cell membranes, wherein an increased amount of the label associated with the cell membranes indicates that the compound binds to the receptor.
Preferably, the cells are mammalian; most preferably, they are COS-7 cells. In another aspect of this preferred embodiment, the HP4 receptor is human. Preferably, it is encoded by the polynucleotide of SEQ ID NO:3. Advantageously, the expression vector is mammalian; most preferably, it is pBC12BI. In accordance with this aspect of the invention, the label is radioactive, calorimetric or fluorimetric.
In accordance with another aspect of the invention, there is provided a method of determining the ability of a compound to inhibit ligand binding to the prostaglandin HP4 receptor, comprising:
transfecting cells with a DNA molecule encoding a prostaglandin HP4 receptor, wherein the DNA molecule is operably linked to a promoter in an expression vector;
culturing the cells to express the HP4 receptor;
incubating at least the cell membranes of the cultured cells in the presence of a labeled ligand having binding affinity for the receptor and a test compound; and
determining the level of binding of the ligand to the prostaglandin HP4 receptor in the presence of the compound, wherein a lower level of ligand binding in the presence of the compound indicates that the compound binds to the receptor.
Preferably, the cells are mammalian; most preferably, they are COS-7 cells and the HP4 receptor is human. In another aspect of the invention,
Gil Daniel W.
Regan John W.
Woodward David F.
Allergan Sales Inc.
Baran Robert J.
Fisher Carlos A.
Kunz Gary L.
Landsman Robert S.
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