System for detection of a functional interaction between a compound and a cellular signal transduction component
First Claim
1. A method for identifying a compound that modulates a heterologous G protein-coupled receptor in a yeast cell, said method comprising:
- providing a first recombinant 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;
providing a second recombinant yeast cell that does not comprise a heterologous G protein coupled receptor, but comprises a reporter gene, which produces a detectable signal upon activation of a second signal transduction pathway of said second yeast cell, in response to production of said signal molecule by said first yeast cell;
contacting said first yeast cell with a test compound;
contacting said second yeast cell with said signal molecule produced by said first yeast cell; and
detecting said detectable signal produced by said second yeast cell, thereby identifying a compound that modulates a heterologous G protein-coupled receptor in a yeast cell.
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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
23 Claims
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1. A method for identifying a compound that modulates a heterologous G protein-coupled receptor in a yeast cell, said method comprising:
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providing a first recombinant 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; providing a second recombinant yeast cell that does not comprise a heterologous G protein coupled receptor, but comprises a reporter gene, which produces a detectable signal upon activation of a second signal transduction pathway of said second yeast cell, in response to production of said signal molecule by said first yeast cell; contacting said first yeast cell with a test compound; contacting said second yeast cell with said signal molecule produced by said first yeast cell; and detecting said detectable signal produced by said second yeast cell, thereby identifying a compound that modulates a heterologous G protein-coupled receptor in a yeast cell. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method for detecting a heterologous gene product produced by a yeast cell comprising:
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causing a first recombinant yeast cell comprising a heterologous G protein coupled receptor to transmit a signal through a pheromone response signal transduction pathway of said first yeast cell, such that a heterologous gene product is produced by said first yeast cell upon transmission of said signal through said signal transduction pathway; and contacting said heterologous gene product with a second recombinant yeast cell that does not comprise a heterologous G protein coupled receptor, but comprises a reporter gene, which produces a detectable signal upon activation of a signal transduction pathway of said second yeast cell, detecting said detectable signal produced by said second yeast cell, thereby detecting said a heterologous gene product produced by said first yeast cell.
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23. A method for identifying a compound that modulates production of a heterologous gene product produced by a yeast cell, said method comprising:
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providing a first recombinant yeast cell, comprising a heterologous G protein coupled receptor that is functionally integrated into a pheromone response signal transduction pathway of said first yeast cell, wherein said first yeast cell produces a heterologous gene product upon activation or said signal transduction pathway; providing a second recombinant yeast cell that does not comprises a heterologous G protein coupled receptor, but comprises a reporter gene, which produces a detectable signal activation of a signal transduction pathway of said second yeast cell, contacting said first yeast cell with a test compound; contacting said second yeast cell with said heterologous gene product produced by said first yeast cell; and detecting said detectable signal expressed by said second yeast cell, thereby detecting a compound that modulates production of said heterologous gene product produced by said first yeast cell, contacting said first yeast cell with a test compound; contacting said second yeast cell with said heterologous gene product produced by said first yeast cell; and detecting said detectable signal expressed by said second yeast cell, thereby detecting a compound that modulates production of heterologous gene product produced by said first yeast cell.
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Specification