System for detection of a functional interaction between a compound and a cellular signal transduction component
First Claim
1. A kit for screening of test compounds that modulate a heterologous G protein-coupled receptor in a yeast cell, said kit comprising:
- a first yeast cell which comprises a heterologous G protein-coupled receptor that is functionally integrated into a first pheromone response signal transduction pathway of said first yeast cell, wherein a signal molecule is produced by said first yeast cell upon activation of said first signal transduction pathway; and
a second yeast cell which is responsive to production of said signal molecule by said first yeast cell, such that a detectable signal is generated by said second yeast cell in response to production of said signal molecule by said first yeast cell.
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Accused Products
Abstract
The present invention makes available a rapid, reproducible, robust assay system for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a cellular protein, e.g., a receptor or ion channel. The subject assay enables rapid screening of large numbers of compounds to identify those which act as an agonist or antagonist to the bioactivity of the cellular protein. In this system, the first cell is treated with a compound, and functional interaction of this compound with a cellular receptor yields a secreted signal. A second cell, bearing a receptor for this secreted signal, makes use of an indicator gene in a signaling pathway coupled to this second receptor. The subject assays include methods of identifying compounds which specifically modulate, for example, heterologous receptors coupled to the pheromone response pathway in yeast. The subject assays are particularly amenable to the identification of specific agonists and antagonists of G protein-coupled receptors.
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Citations
11 Claims
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1. A kit for screening of test compounds that modulate a heterologous G protein-coupled receptor in a yeast cell, said kit comprising:
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a first yeast cell which comprises a heterologous G protein-coupled receptor that is functionally integrated into a first pheromone response signal transduction pathway of said first yeast cell, wherein a signal molecule is produced by said first yeast cell upon activation of said first signal transduction pathway; and
a second yeast cell which is responsive to production of said signal molecule by said first yeast cell, such that a detectable signal is generated by said second yeast cell in response to production of said signal molecule by said first yeast cell. - View Dependent Claims (2, 8, 9)
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- 3. A recombinant yeast cell comprising a surrogate of a pheromone response signal transduction pathway of said recombinant yeast cell, wherein a signal molecule is produced by said surrogate upon activation of said pheromone response signal transduction pathway, and wherein production of said signal molecule is capable of detection by a second yeast cell comprising means for generating a detectable signal, such that a detectable signal is generated by said second yeast cell in response to stimulation of said second yeast cell by said signal molecule.
- 6. A mixture of recombinant yeast cells comprising a first recombinant yeast cell and a second recombinant yeast cell, wherein said first cell comprises a surrogate of a pheromone response signal transduction pathway of said yeast cell, such that a signal molecule is produced by said surrogate upon activation of said pheromone response signal transduction pathway, and wherein said second yeast cell comprises means for generating a detectable signal such that a detectable signal is generated by said second yeast cell in response to stimulation of said second yeast cell by said signal molecule.
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7. A mixture of recombinant cells comprising a first recombinant yeast cell and a second recombinant yeast cell, wherein said first yeast cell comprises a heterologous receptor that is functionally integrated into a pheromone response signal transduction pathway of said first yeast cell, and produces a signal molecule upon activation of said pheromone response signal transduction pathway, and wherein said second yeast cell comprises means for generating a detectable signal, and is responsive to the signal molecule produced by said first yeast cell, such that a detectable signal is generated by said second yeast cell in response to production of said signal molecule by said first yeast cell.
Specification