Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-06-12
2003-04-29
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S007100, C435S007200, C435S069100, C435S069700, C435S254200, C435S091500, C435S023000, C435S023000, C435S091500, C435S350000, C435S300100
Reexamination Certificate
active
06555325
ABSTRACT:
FIELD OF THE INVENTION
The invention is related to the detection of the transduction of an extracellular signal by an intracellular signal transduction pathway. In particular, the invention relates to methods and compositions useful for identifying a test compound as an agonist or antagonist of a cellular receptor.
BACKGROUND OF THE INVENTION
The identification of biological activity in new molecules has historically been accomplished through the use of in vitro assays or whole animals. Intact biological entities, either cells or whole organisms, have been used to screen for anti-bacterial, anti-fungal, anti-parasitic and anti-viral agents in vitro. Cultured mammalian cells have also been used in screens designed to detect potential therapeutic compounds. A variety of bioassay endpoints have been exploited in cell screens including the stimulation of growth or differentiation of cells, changes in cell motility, the production of particular metabolites, the expression of specific proteins within cells, altered protein function, and altered conductance properties. Cytotoxic compounds used in cancer chemotherapy have been identified through their ability to inhibit the growth of tumor cells in vitro and in vivo. In addition to cultures of dispersed cells, whole tissues have served in bioassays, as in those based on the contractility of muscle.
In vitro testing is a preferred methodology in that it permits the design of high-throughput screens: small quantities of large numbers of compounds can be tested in a short period of time and at low expense. Optimally, animals are reserved for the latter stages of compound evaluation and are not used in the discovery phase, inasmuch as the use of whole animals is labor-intensive and extremely expensive.
The search for agonists and antagonists of cellular receptors has been an intense area of research aimed at drug discovery because of the elegant specificity of these molecular targets. Drug screening has been carried out using whole cells expressing functional receptors and, recently, binding assays employing membrane fractions or purified receptors have been designed to screen compound libraries for competitive ligands.
G protein-coupled receptors (GPCRs) are a particularly important category of cell surface receptors. The medical importance of these receptors is evidenced by the fact that more than 60% of all commercially available prescription drugs work by interacting with known GPCRs. Hundreds, if not thousands, of receptors convey messages through heterotrimeric G proteins, of which at least 17 distinct forms have been isolated. Most G protein-coupled receptors are comprised of a single protein chain that is threaded through the plasma membrane seven times. Such receptors are often referred to as seven-transmembrane receptors (STRs). More than a hundred different GPCRs have been found, including many distinct receptors that bind the same ligand, and there are likely many more GPCRs awaiting discovery. The development of new drug discovery assays to identify novel modulators of GPCRs would be of tremendous benefit.
The heterologous expression of recombinant mammalian G protein-coupled receptors in mammalian cells which do not normally express those receptors has been described as a means of studying receptor function for the purpose 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 phosphotidylinositol hydrolysis and intracellular Ca
2+
mobilization in Chinese hamster ovary cells upon agonist stimulation (Lolait et al. (1995)
Proc. Natl. Acad Sci.
USA 92:6783-6787). These types of ectopic expression studies have enabled researchers to study receptor signaling mechanisms and to perform mutagenesis 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.) It was 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 also describes co-expression of a rat G protein &agr;-subunit in 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 further 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. DNA vectors and host yeast cells for use in the method are also disclosed (see U.S. Pat. No. 5,739,029).
U.S. Pat. No. 5,789,184 describes yeast cells engineered to express a heterologous kinase as a yeast pheromone system protein surrogate, and a heterologous polypeptide. The yeast cells are used in assays to screen for peptides that modulate the activity of non-yeast surrogates.
U.S. Pat. No. 5,879,591 describes yeast cells engineered to express a heterologous protein (e.g., a farnesyl transferease) which functions as a surrogate for, and performs the function of, a yeast pheromone system protein, as well as a heterologous polypeptide. The yeast cells are useful in screening assays to identify polypeptides which modulate the interaction of the surrogate with the yeast pheromone system.
U.S. Ser. No. 08/322,137 describes yeast cells engineered to express both a surrogate, e.g. a G protein-coupled receptor, of a pheromone pathway component and a potential peptide modulator of the surrogate. This is performed in such a manner that inhibition or antagonism of the surrogate by the peptide modulator affects a screenable or selectable trait of the yeast cell. Also included are mechanisms by which the signal-to-noise ratio of the system may be improved. The yeast cells are useful in assays to screen for peptides that modulate the activity of endogenous and heterologous yeast pheromone system surrogates.
Published PCT international application WO 98/13513 describes methods for identifying modulators of heterologous receptors expressed in yeast. Modulators are identified by detecting an alteration in a signal produced by an endogenous yeast signaling pathway.
Published PCT international application WO 99/18211 describes novel yeast cells which express a heterologous G protein coupled receptor and mutant and/or chimeric G protein subunit molecules which serve to functionally integrate the heterologous receptor into the pheromone signaling pathway of the yeast cell.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a novel, rapid, reproducible, robust assay system for screening and
Cadus Technologies, Inc.
DeConti, Jr. Giulio A.
Kemmerer Elizabeth
Lahive & Cockfield LLP
Lauro Peter C.
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