Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-06-03
2001-06-05
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007210, C435S007720, C435S069100, C530S350000, C536S023100, C536S023500
Reexamination Certificate
active
06242199
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to an assay which involves identification of cell membrane receptors, specifically growth hormone secretagogoue receptors (GHSRs). By varying the protocol, receptor ligands can be identified, or the presence of a GHSR can be identified.
BACKGROUND OF THE INVENTION
Growth hormone (GH) is an anabolic hormone capable of promoting linear growth, weight gain and whole body nitrogen retention. Classically, GH is thought to be released primarily from the somatotroph cells of the anterior pituitary under the coordinate regulation of two hypothalamic hormones, growth hormone releasing factor (GHRF or GRF) and somatostatin. Both GHRF stimulation and somatostatin inhibition of the release of GH occurs by the specific engagement of receptors on the cell membrane of the somatotroph.
Recent evidence has been mounting which suggests that GH release is also stimulated by a group of short peptides termed the growth hormone releasing peptides (GHRP; GHRP-6, GHRP-2 [hexarelin]) These peptides are described, for example, in U.S. Pat. No. 4,411,890, PCT Patent Pub. No. WO 89/07110, PCT Patent Pub. No. WO 89/07111, PCT Patent Pub. No. WO 93/04081, and
J. Endocrinol Invest.,
15(Suppl 4), 45 (1992). These peptides function by selectively binding to a distinct somatotroph cell membrane receptor, the growth hormone secretagogue receptor (GHSR). A medicinal chemical approach has resulted in the design of several classes of orally-active, low molecular weight, non-peptidyl compounds which bind specifically to this receptor and result in the pulsatile release of GH. Such compounds possessing growth hormone secretagogue activity are disclosed, for example, in the following: U.S. Pat. No. 3,239,345; U.S. Pat. No. 4,036,979; U.S. Pat. No. 4,411,890; U.S. Pat. No. 5,206,235; U.S. Pat. No. 5,283,241; U.S. Pat. No. 5,284,841; U.S. Pat. No. 5,310,737; U.S. Pat. No. 5,317,017; U.S. Pat. No. 5,374,721; U.S. Pat. No. 5,430,144; U.S. Pat. No. 5,434,261; U.S. Pat. No. 5,438,136; U.S. Pat. No. 5,494,919; U.S. Pat. No. 5,494,920; U.S. Pat. No. 5,492,916; EPO Patent Pub. No. 0,144,230; EPO Patent Pub. No. 0,513,974; PCT Patent Pub. No. WO 94/07486; PCT Patent Pub. No. WO 94/08583; PCT Patent Pub. No. WO 94/11012; PCT Patent Pub. No. WO 94/13696; PCT Patent Pub. No. WO 94/19367; PCT Patent Pub. No. WO 95/03289; PCT Patent Pub. No. WO 95/03290; PCT Patent Pub. No. WO 95/09633; PCT Patent Pub. No. WO 95/11029; PCT Patent Pub. No. WO 95/12598; PCT Patent Pub. No. WO 95/13069; PCT Patent Pub. No. WO 95/14666; PCT Patent Pub. No. WO 95/16675; PCT Patent Pub. No. WO 95/16692; PCT Patent Pub. No. WO 95/17422; PCT Patent Pub. No. WO 95/17423; PCT Patent Pub. No. WO 95/34311; PCT Patent Pub. No. WO 96/02530;
Science,
260, 1640-1643 (Jun. 11, 1993);
Ann. Rep. Med. Chem.,
28, 177-186 (1993);
Bioorg. Med. Chem. Ltrs.,
4(22), 2709-2714 (1994); and
Proc. Natl. Acad. Sci. USA
92, 7001-7005 (July 1995).
The use of such orally-active agents which stimulate the pulsatile release of GH would be a significant advance in the treatment of growth hormone deficiency in children and adults as well as provide substantial benefit under circumstances where the anabolic effects of GH might be exploited clinically (e.g. post-hip fracture rehabilitation, the frail elderly and in post-operative recovery patients).
Cell membrane receptors which are of low abundance on the cells can be difficult to isolate, clone and characterize. In the past, assays to identify a receptor in a mammalian cell or frog oocyte generally have depended on either: 1) directly detecting a receptor-ligand interaction, such as by binding of a radiolabeled ligand; or 2) indirectly detecting receptor-ligand binding by detecting either an intracellular event (such as calcium mobilization, or the identification of, for instance a calcium activated current) or an extracellular event (such as hormone secretion), that is the consequence of the ligand binding to its receptor. Most cloned receptors, which have been isolated using a functional expression assay have relied on immortalized cell lines or tumor derived tissues which are enriched for the receptor of interest.
There are numerous receptors which cannot be readily identified using these types of assays, due to: 1) a paucity of biochemical information about the protein; 2) the low abundance of receptors present on the cell; and/or 3) the lack of a cell line or tumor material expressing the receptor. It would be desirable to develop an assay which can be used to identify and characterize cell receptors not amenable to study by conventional means.
DETAILED DESCRIPTION OF THE INVENTION
This invention relates to an assay method to determine the presence of a nucleic acid which encodes a G protein-linked cell membrane receptor comprising:
a) introducing at least one nucleic acid suspected of encoding a G protein cell membrane receptor into a cell;
b) introducing a G-protein subunit into the cell;
c) introducing a detector molecule or a nucleic acid encoding a detector molecule into the cell, wherein the detector molecule responds directly or indirectly to a G-protein receptor-ligand binding event;
d) contacting the cell with a receptor ligand; and
e) determining whether the oligonucleotide encoded a receptor by monitoring the detector molecule.
In one preferred embodiment the cell does not naturally express the receptor on its cell membrane. In other preferred embodiments of the assay, the receptor is a member of the growth hormone secretagogue family of receptors, such as a growth hormone secretagogue receptor (GHSR) or a growth hormone secretagogue related receptor (GHSRR). Thus, another aspect of this invention is an assay method to determine the presence of a nucleic acid which encodes a member of the growth hormone secretagogue receptor family comprising:
a) introducing at least one nucleic acid suspected of encoding a GHSR or GHSRR into a cell which does not naturally express the receptor on its cell membrane;
b) introducing a G-protein subunit into the cell;
c) introducing a detector molecule or a nucleic acid encoding a detector molecule into the cell, wherein the detector molecule is directly or indirectly responsive to a GHSR-ligand or GHSRR-ligand binding event;
d) contacting the cell with a growth hormone secretagogue; and
e) determining whether the nucleic acid encodes a receptor by monitoring the detector molecule.
A further embodiment of this invention is an assay to determine the presence of a growth hormone secretagogue. Thus, this invention also comprises a method to determine the presence of a growth hormone secretagogue comprising:
a) introducing a nucleic acid which encodes a growth hormone secretagogue receptor into a cell under conditions so that growth hormone secretagogue receptor is expressed;
b) introducing a G-protein subunit into the cell;
c) introducing a detector molecule or a nucleic acid encoding a detector molecule into the cell, wherein the detector molecule is directly or indirectly responsive to a GHSR-ligand binding event;
d) contacting the cell with a compound suspected of being a growth hormone secretagogue; and
e) determining whether the compound is a growth hormone secretagogue by monitoring the detector molecule.
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patent: 5
Feighner Scott D.
Howard Andrew D.
Pai Lee-Yuh
Pong Sheng-Shung
Van Der Ploeg Leonardus H. T.
Cocuzzo Anna L.
Kaufman Claire M.
Kemmerer Elizabeth
Merck & Co. , Inc.
Tribble Jack L.
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