Identification of ligands by selective amplification of cells transfected with a 5HT2A receptor
First Claim
1. A method of determining whether a substance is a ligand of a constitutively active wild-type 5HT2A receptor, wherein said 5HT2A receptor has a basal response, said method comprising:
- obtaining a test cell culture which comprises a first population of cells and a second population of cells, wherein DNA coding for said 5HT2A receptor and also DNA encoding a marker indicative of the extent of cellular proliferation has been introduced into said first population of cells such that the amount of said marker produced by said first population of cells is influenced by the degree to which said 5HT2A receptor is active, and wherein the amount of marker produced by said second population of cells is not influenced by the degree to which said 5HT2A receptor is active or wherein said second population of cells does not comprise DNA coding for said marker;
contacting said test cell culture with a test substance which is a potential ligand of said 5HT2A receptor;
obtaining a control cell culture, said control cell culture comprising cells comprising said DNA coding for said 5HT2A receptor and said DNA coding for said marker, wherein said control cell culture has not been contacted with said test substance;
determining whether the extent of proliferation of said first population of cells in said test cell culture is significantly greater or significantly less than the extent of proliferation of said cells in said control cell culture by comparing the amount of marker activity produced by said first population of cells in said test culture to the amount of marker activity produced by said cells in said control culture, wherein said test substance is an agonist of said 5HT2A receptor if the extent of proliferation of said first population of cells in said test culture is significantly greater than the extent of proliferation of said cells in said control culture and wherein said test substance is an antagonist or inverse agonist of said 5HT2A receptor if the extent of proliferation of said first population of cells in said test culture is significantly less than the extent of proliferation of said cells in said control culture.
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Abstract
The invention is directed to a method for identifying substances acting as ligands for transfected receptors by using transfected markers to measure receptor/ligand interactions. The present invention also relates to a method of identifying compounds which act as inverse agonists of the 5-HT2A receptor, the method comprising contacting a constitutively active 5-HT2A receptor with at least one test compound and determining any decrease in the amount of basal activity of the receptor so as to identify a test compound which is an inverse agonist of the 5-HT2A receptor. Such inverse agonists may be used in the treatment of schizophrenia and related psychoses.
9 Citations
65 Claims
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1. A method of determining whether a substance is a ligand of a constitutively active wild-type 5HT2A receptor, wherein said 5HT2A receptor has a basal response, said method comprising:
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obtaining a test cell culture which comprises a first population of cells and a second population of cells, wherein DNA coding for said 5HT2A receptor and also DNA encoding a marker indicative of the extent of cellular proliferation has been introduced into said first population of cells such that the amount of said marker produced by said first population of cells is influenced by the degree to which said 5HT2A receptor is active, and wherein the amount of marker produced by said second population of cells is not influenced by the degree to which said 5HT2A receptor is active or wherein said second population of cells does not comprise DNA coding for said marker; contacting said test cell culture with a test substance which is a potential ligand of said 5HT2A receptor; obtaining a control cell culture, said control cell culture comprising cells comprising said DNA coding for said 5HT2A receptor and said DNA coding for said marker, wherein said control cell culture has not been contacted with said test substance; determining whether the extent of proliferation of said first population of cells in said test cell culture is significantly greater or significantly less than the extent of proliferation of said cells in said control cell culture by comparing the amount of marker activity produced by said first population of cells in said test culture to the amount of marker activity produced by said cells in said control culture, wherein said test substance is an agonist of said 5HT2A receptor if the extent of proliferation of said first population of cells in said test culture is significantly greater than the extent of proliferation of said cells in said control culture and wherein said test substance is an antagonist or inverse agonist of said 5HT2A receptor if the extent of proliferation of said first population of cells in said test culture is significantly less than the extent of proliferation of said cells in said control culture. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method of determining whether a substance is a ligand of a constitutively active wild-type 5HT2A receptor, wherein said 5HT2A receptor has a basal response, said method comprising,
obtaining a test cell culture which comprises a first population of cells and a second population of cells, wherein DNA coding for said 5HT2A receptor and also DNA encoding a marker indicative of the extent of cellular proliferation has been introduced into said first population of cells such that the amount of said marker produced by said first population of cells is influenced by the degree to which said 5HT2A receptor is active, and wherein the amount of said marker produced by said second population of cells is not influenced by the degree to which said 5HT2A receptor is active or wherein said second population of cells does not comprise DNA coding for said marker; -
contacting said test cell culture with a test substance which is a potential ligand of said 5HT2A receptor; and determining whether the test substance confers a competitive advantage or a competitive disadvantage on the cells in said first population of said test culture relative to the cells in said second population of said test culture by measuring the amount of said marker produced by said test culture. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
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25. A method of determining whether a substance is a ligand of a constitutively active wild-type 5HT2A receptor, wherein said 5HT2A receptor has a basal receptor response, said method comprising:
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obtaining a test cell culture which comprises a first population of cells and a second population of cells, wherein DNA coding for said 5HT2A receptor and also DNA encoding a marker indicative of the extent of cellular proliferation has been introduced into said first population of cells such that the amount of said marker produced by said first population of cells is influenced by the degree to which said 5HT2A receptor is active, wherein the transcription of said DNA coding for said marker is not under the control of said 5HT2A receptor, and wherein the amount of marker in said second population of cells is not influenced by the degree to which said 5HT2A receptor is active or wherein said second population of cells does not comprise said DNA coding for said marker; contacting said test cell culture with a test substance which is a potential ligand of said 5HT2A receptor; obtaining a control cell culture, said control cell culture comprising cells comprising said DNA coding for said 5HT2A receptor and said DNA coding for said marker, wherein said control cell culture has not been contacted with said test substance; determining whether the extent of proliferation of said first population of cells in said test cell culture is significantly greater or significantly less than the extent of proliferation of said cells in said control cell culture by comparing the amount of marker activity produced by said first population of cells in said test culture to the amount of marker activity produced by said cells in said control culture, wherein said test substance is an agonist of said 5HT2A receptor if the extent of proliferation of said first population of cells in said test culture is significantly greater than the extent of proliferation of said cells in said control culture and wherein said test substance is an antagonist or inverse agonist of said 5HT2A receptor if the extent of proliferation of said first population of cells in said test culture is significantly less than the extent of proliferation of said cells in said control culture. - View Dependent Claims (26, 27, 28, 29, 30, 31)
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32. A method of determining whether a substance is a ligand of a constitutively active wild-type 5HT2A receptor, wherein said 5HT2A receptor has a basal response, said method comprising:
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obtaining a test cell culture which comprises a first population of cells and a second population of cells, wherein DNA coding for a plurality of receptors and also DNA encoding a marker indicative of the extent of cellular proliferation has been introduced into said first population of cells such that the amount of said marker produced by said first population of cells is influenced by the degree to which said plurality of receptors are active, and wherein the amount of said marker produced by said second population of cells is not influenced by the degree to which said plurality of receptors are active or wherein said second population of cells does not comprise said DNA coding for said marker, wherein at least one of said plurality of receptors is a constitutively active wild-type 5HT2A receptor, wherein said 5HT2A receptor has a basal or constitutive receptor response; contacting said test cell culture with a test substance which is a potential ligand of said 5HT2A receptor; obtaining a control cell culture, said control cell culture comprising cells comprising said DNA coding for said plurality of receptors and said DNA coding for said marker, wherein said control cell culture has not been contacted with said test substance and wherein at least one of said plurality of receptors is a constitutive active wild type 5HT2A receptor; determining whether the extent of proliferation of cells in said first population of cells in said test cell culture is significantly greater or significantly less than the extent of proliferation of said cells in said control cell culture by comparing the amount of marker activity produced by said first population of cells in said test culture to the amount of marker activity produced by said cells in said control culture, wherein said test substance is an agonist of said 5HT2A receptor if the extent of proliferation of cells in said first population of cells in said test culture which comprise DNA coding for said 5HT2A receptor is significantly greater than the extent of proliferation of said cells in said control culture which comprise DNA coding for said 5HT2A receptor and wherein said test substance is an antagonist or inverse agonist of said 5HT2A receptor if the extent of proliferation of cells in said first population of cells in said test culture which comprise DNA coding for said 5HT2A receptor is significantly less than the extent of proliferation of cells in said control culture which comprise DNA coding for said 5HT2A receptor. - View Dependent Claims (33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
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46. A method of determining whether a substance is a ligand of a constitutively active wild-type 5HT2A receptor, wherein said 5HT2A receptor has a basal response, said method comprising,
obtaining a test cell culture which comprises a first population of cells and a second population of cells, wherein DNA coding for a plurality of receptors and also DNA encoding a marker indicative of the extent of cellular proliferation has been introduced into said first population of cells such that the amount of said marker produced by said first population of cells is influenced by the degree to which said plurality of receptors are active, and wherein the amount of said marker in said second population of cells is not influenced by the degree to which said plurality of receptors are active or wherein said second population of cells does not comprise said DNA coding for said marker wherein at least one of said plurality of receptors is a constitutively active wild-type 5HT2A receptor, wherein said 5HT2A receptor has a basal response; -
contacting said test cell culture with a test substance which is a potential ligand of said 5HT2A receptor; and determining whether the test substance confers a competitive advantage or a competitive disadvantage on cells in said first population of said test culture which express said 5HT2A receptor relative to the cells in said second population of said test culture which comprise said 5HT2A receptor by measuring the amount of said marker produced by said test culture. - View Dependent Claims (47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)
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59. A method of determining whether a substance is a ligand of a constitutively active wild-type 5HT2A receptor, wherein said 5HT2A receptor has a basal response, said method comprising:
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obtaining a test cell culture which comprises a first population of cells and a second population of cells, wherein DNA coding for a plurality of receptors and also DNA encoding a marker indicative of the extent of cellular proliferation has been introduced into said first population of cells such that the amount of said marker produced by said first population of cells is influenced by the degree to which said plurality of receptors are active, wherein the transcription of said DNA coding for said marker is not under the control of any of said plurality of receptors, and wherein the amount of said marker produced by said second population of cells is not influenced by the degree to which said plurality of receptors are active or wherein said second population of cells does not comprise said DNA coding for said marker, wherein at least one of said plurality of receptors is a 5HT2A receptor; contacting said test cell culture with a test substance which is a potential ligand of said 5HT2A receptor; obtaining a control cell culture, said control cell culture comprising cells comprising said DNA coding for said plurality of receptors and said DNA coding for said marker, wherein said control cell culture has not been contacted with said test substance and wherein at least one of said plurality of receptors is a constitutively active wild type 5HT2A receptor; determining whether the extent of proliferation of said first population of cells in said test cell culture is significantly greater or significantly less than the extent of proliferation of said cells in said control cell culture by comparing the amount of marker activity produced by said first population of cells in said test culture which comprise DNA coding for said 5HT2A receptor to the amount of marker activity produced by said cells in said control culture which comprise DNA coding for said 5HT2A receptor, wherein said test substance is an agonist of said 5HT2A receptor if the extent of proliferation of said first population of cells in said test culture which comprise DNA coding for said 5HT2A receptor is significantly greater than the extent of proliferation of said cells in said control culture which comprise DNA coding for said 5HT2A receptor and wherein said test substance is an antagonist or inverse agonist of said 5HT2A receptor if the extent of proliferation of said first population of cells in said test culture which comprise DNA coding for said 5HT2A receptor is significantly less than the extent of proliferation of said cells in said control culture which comprise DNA coding for said 5HT2A receptor. - View Dependent Claims (60, 61, 62, 63, 64, 65)
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Specification