Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1996-01-17
2001-07-03
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S007200, C435S069100, C435S069700, C435S254200, C435S254210, C536S023400, C536S023500, C530S350000, C530S300000
Reexamination Certificate
active
06255059
ABSTRACT:
BACKGROUND OF THE INVENTION
A common technique for cloning receptors is to use nucleic acid hybridization technology to identify receptors which are homologous to other, known receptors. For instance, originally the cloning of seven transmembrane domain G protein-coupled receptors (GCR) depended on the isolation and sequencing of the corresponding protein or the use of expression cloning techniques. However, when sequences for these receptors became available, it was apparent that there were significant sequence homologies between these receptors. This technology, since it does not require that the ligand of the receptor have been identified, has resulted in the cloning of a large number of “orphan receptors”, which have no known ligand and often whose biological function is obscure. Receptors of all types comprise this large family. Known orphan receptors include the nuclear receptors COUP-TF1/EAR3, COUP-TF2/ARP1, EAR-1, EAR-2, TR-2, PPAR1, HNF-4, ERR-1, ERR-2, NGFIB/Nur77, ELP/SF-1 and MPL (Parker et al, supra, and Power et al. (1992) TIBS 13:318-323). A large number of orphan receptors have been identified in the EPH family (Hirai et al (1987)
Science
238:1717-1720). HER3 and HER4 are orphan receptors in the epidermal growth factor receptor family (Plowman et al. (1993)
Proc. Natl. Acad. Sci. USA
90:1746-1750). ILA is a newly identified member of the human nerve growth factor/tumor necrosis factor receptor family (Schwarz et al. (1993)
Gene
134:295-298). IRRR is an orphan insulin receptor-related receptor which is a transmembrane tyrosine kinase (Shier et al. (1989)
J. Biol Chem
264:14606-14608). Several orphan tyrosine kinase receptors have been found in Drosophila (Perrimon (1994)
Curr. Opin. Cell Biol
. 6:260-266). The importance of identifying ligands for orphan receptors is clear; it opens up a wide area for research in the area of drug discovery.
One large subgroup of orphan receptors, as alluded to above, are found in the G protein coupled receptor family. Approximately 100 such receptors have been identified by function and these mediate transmembrane signaling from external stimuli (vision, taste and smell), endocrine function (pituitary and adrenal), exocrine function (pancreas), heart rate, lipolysis, and carbohydrate metabolism. Structural and genetic similarities suggest that G protein-coupled receptor superfamily can be subclassified into five distinct groups: (i) amine receptors (serotonin, adrenergic, etc.); (ii) small peptide hormone (somatostatin, TRH, etc.); (iii) large peptide hormone (LH-CG, FSH, etc.); (iv) secretin family; and (v) odorant receptors (Buck L. and Axel, R. (1991)
Cell
65:175-187), with orphan receptors apparently occurring in each of the sub-families.
Previous work describes the expression of recombinant mammalian G protein-coupled receptors as a means of studying receptor function as a means of identifying agonists and antagonists of those receptors. For example, the human muscarinic receptor (HM1) has been functionally expressed in mouse cells (Harpold et al. U.S. Pat. No. 5,401,629). The rat V1b vasopressin receptor has been found to stimulate phosphotidy.inositol hydrolysis and intracellular Ca2+ mobilization in Chinese hamster ovary cells upon agonist stimulation (Lolait et al. (1995)
Proc Natl. Acad Sci. USA
92:6783-6787). Likewise, the C5a receptor {to be completed} These types of ectopic expression studies have enabled researchers to study receptor signalling mechanisms and to perform mutagenisis studies which have been useful in identifying portions of receptors that are critical for ligand binding or signal transduction.
Experiments have also been undertaken to express functional G protein coupled receptors in yeast cells. For example, U.S. Pat. No. 5,482,835 to King et al. describes a transformed yeast cell which is incapable of producing a yeast G protein &agr; subunit, but which has been engineered to produce both a mammalian G protein &agr;-subunit and a mammalian receptor which is “coupled to” (i.e., interacts with) the aforementioned mammalian G protein &agr;-subunit. Specifically, U.S. Pat. No. 5,482,835 reports expression of the human beta-2 adrenergic receptor (&bgr;2AR), a seven transmembrane receptor (STR), in yeast, under control of the GAL1 promoter, with the &bgr;2AR gene modified by replacing the first 63 base pairs of coding sequence with 11 base pairs of noncoding and 42 base pairs of coding sequence from the STE2 gene. (STE2 encodes the yeast &agr;-factor receptor). The Duke researchers found that the modified &bgr;2AR was functionally integrated into the membrane, as shown by studies of the ability of isolated membranes to interact properly with various known agonists and antagonists of &bgr;2AR. The ligand binding affinity for yeast-expressed &bgr;2AR was said to be nearly identical to that observed for naturally produced &bgr;2AR.
U.S. Pat. No. 5,482,835 describes co-expression of a rat G protein &agr;-subunit in the same cells, yeast strain 8C, which lacks the cognate yeast protein. Ligand binding resulted in G protein-mediated signal transduction. U.S. Pat. No. 5,482,835 teaches that these cells may be used in screening compounds for the ability to affect the rate of dissociation of G&agr; from G&bgr;&ggr; in a cell. For this purpose, the cell further contains a pheromone-responsive promoter (e.g. BAR1 or FUS1), linked to an indicator gene (e.g. HIS3 or LacZ). The cells are placed in multi-titer plates, and different compounds are placed in each well. The colonies are then scored for expression of the indicator gene.
SUMMARY OF THE INVENTION
The present invention relates to a rapid, reliable and effective assay for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a cellular receptor or ion channel. The subject assay enables rapid screening of large numbers of polypeptides in a library to identifying those polypeptides which agonize or antagonize receptor bioactivity. In general, the assay is characterized by the use of a library of recombinant cells, each cell of which include (i) a target receptor protein whose signal transduction activity can be modulated by interaction with an extracellular signal, the transduction activity being able to generate a detectable signal, and (ii) an expressible recombinant gene encoding an exogenous test polypeptide from a polypeptide library. By the use of a variegated gene library, the mixture of cells collectively express a variegated population of test polypeptides. In preferred embodiments, the polypeptide library includes at least 10
3
different polypeptides, though more preferably at least 10
5
, 10
6
, or 10
7
different (variegated) polypeptides. The polypeptide library can be generated as a random peptide library, as a semi-random peptide library (e.g., based on combinatorial mutagenesis of a known ligand), or as a cDNA library.
The ability of particular constituents of the peptide library to modulate the signal transduction activity of the target receptor can be scored for by detecting up or down-regulation of the detection signal. For example, second messenger generation via the receptor can be measured directly. Alternatively, the use of a reporter gene can provide a convenient readout. In any event, a statistically significant change in the detection signal can be used to facilitate isolation of those cells from the mixture which contain a nucleic acid encoding a test polypeptide which is an effector of the target receptor.
By this method, test polypeptides which induce receptor signaling can be identified. If the test polypeptide does not appear to directly induce the activity of the receptor protein, the assay may be repeated and modified by the introduction of a step in which the recombinant cell is first contacted with a known activator of the target receptor to induce the signal transduction pathways from the receptor. In one embodiment, the test polypeptide is assayed for its ability to antagonize, e.g., inhibit or block the activity of the activator
Broach James
Fowlkes Dana M.
Klein Christine A.
Manfredi John
Murphy Andrew J. M.
Cadus Pharmaceutical Corporation
DeConti, Jr., P Giulio A.
Kaufman Claire M.
Lahive & Cockfield LLP
Lauro, Esq. Peter C.
LandOfFree
Methods for identifying G protein coupled receptor effectors does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods for identifying G protein coupled receptor effectors, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods for identifying G protein coupled receptor effectors will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2517623