US5401629A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Assay methods and compositions useful for measuring the transduction of an intracellular signal
Assignee: Merck and Co Inc
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Filing: 1990-08-07
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Granted: 1995-03-28
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Expires: 2010-08-07
Abstract
Abstract Recombinant cells are provided which are useful for assaying compounds for their agonist or antagonist activity with respect to ion channels and/or cell surface-localized receptors. In addition, assay methods employing the invention recombinant cells are provided.
Inventors
- 👤 Michael M. Harpold
- 👤 Paul Brust
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 09:34
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/68; G01N33/6872; C; C07; C07K; C07K14/00; C07K14/435; C07K14/705; C07K14/82; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6897; G01N33/94; G01N33/9406; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
248 words
A method for identifying compounds that modulate cell surface protein-mediated activity by detecting intracellular transduction of a signal generated upon interaction of the compound with the cell surface protein, comprising: comparing the amount of transcription of a reporter gene or the amount of reporter gene product expressed in a first recombinant cell in the presence of the compound with the amount of transcription or product in the absence of the compound, or with the amount of transcription or product in a second recombinant cell; and selecting compounds that change the amount of transcription of a reporter gene or the amount of reporter gene product expressed in the first recombinant cell in the presence of the compound compared to the amount of transcription or product in the absence of the compound, or compared to the amount of transcription or product in the second recombinant cell, wherein: the cell surface protein is a surface receptor or ion channel; the first recombinant cell contains a reporter gene Construct and expresses the cell surface protein; the second recombinant cell is identical to the first recombinant cell, except that it does not express the cell surface protein; and the reporter gene constructs contains: (a) a transcriptional control element that is responsive to the intracellular signal that is generated by the interaction of an agonist with the cell surface protein; and (b) a reporter gene that encodes a detectable transcriptional or translational product and that is in operative association with the transcriptional control element.
Claim 2
Independent Claim
248 words
A method for identifying compounds that modulate cell surface protein-mediated activity by detecting intracellular transduction of a signal generated upon interaction of the compound with the cell surface protein, comprising: comparing the amount of transcription of a reporter gene or the amount of reporter gene product expressed in a first recombinant cell in the presence of the compound with the amount of transcription or product in the absence of the compound, or with the amount of transcription or product in a second recombinant cell; and selecting compounds that change the amount of transcription of a reporter gene or the amount of reporter gene product expressed in the first recombinant cell in the presence of the compound compared to the amount of transcription or product in the absence of the compound, or compared to the amount of transcription or product in the second recombinant cell, wherein: the cell surface protein is a surface receptor or ion channel; the first recombinant cell contains a reporter gene Construct and expresses the cell surface protein; the second recombinant cell is identical to the first recombinant cell, except that it does not express the cell surface protein; and the reporter gene constructs contains: (a) a transcriptional control element that is responsive to the intracellular signal that is generated by the interaction of an agonist with the cell surface protein; and (b) a reporter gene that encodes a detectable transcriptional or translational product and that is in operative association with the transcriptional control element.
Claim 3
Dependent Claim
21 words
The method of claim 1, wherein said compound is an agonist or antagonist of said cell surface receptor or ion channel.
Claim 4
Dependent Claim
25 words
The method of claim 1, wherein the amount of transcription is determined by measuring the amount of mRNA that is transcribed from said reporter gene.
Claim 5
Dependent Claim
23 words
The method of claim 1, wherein the amount of transcription is measured by measuring the amount of reporter gene protein that is produced.
Claim 6
Dependent Claim
11 words
The method of claim 2, wherein said compound is an antagonist.
Claim 7
Dependent Claim
42 words
The method of claim 2, further comprising, prior to comparing the difference in the amount of transcription of the reporter gene, contacting the recombinant cell with an agonist that activates said cell surface protein, whereby transcription of said reporter gene is induced.
Claim 8
Dependent Claim
37 words
The method of claim 1, wherein said reporter gene construct includes a transcriptional regulatory region that includes a sequence of nucleotides that modulates transcription from said promoter in the absence of said cell surface protein or compound.
Claim 9
Dependent Claim
50 words
The method of claim 1, wherein the transcriptional control element includes a promoter selected from the group consisting of the c-fos gene promoter, the vasoactive intestinal peptide gene promoter, the somatostatin gene promoter, the proenkephalin gene promoter, the phosphoenolpyruvate carboxykinase gene promoter and the nerve growth factor-1 A gene promoter.
Claim 10
Dependent Claim
38 words
The method of claim 1, wherein the reporter gene is selected from the group consisting of the gene encoding bacterial chloramphenicol acetyltransferase, the gene encoding firefly luciferase, the gene encoding bacterial luciferase, and the gene encoding alkaline phosphatase.
Claim 11
Dependent Claim
38 words
The method of claim 1, wherein the transcriptional control region includes at least one regulatory element selected from the group consisting of serum responsive elements, cyclic adenosine monophosphate responsive elements, and elements responsive to intracellular calcium ion levels.
Claim 12
Dependent Claim
34 words
The method of claim 1, wherein the cell surface receptor is the Type 1 human muscarinic receptor, the promoter is the c-fos promoter and the reporter gene is the gene encoding bacterial chloramphenicol acetyltransferase.
Claim 13
Dependent Claim
40 words
The method of claim 1, wherein the cell surface receptor is selected from the group consisting of muscarinic receptors, neuronal nicotinic acetylcholine receptors, gamma aminobutyric acid receptors, glutamate receptors, adrenergic receptors, dopamine receptors, nerve growth factor receptors, and serotonin receptors.
Claim 14
Dependent Claim
21 words
The method of claim 1, wherein the ion channel is a calcium channel, potassium ion channel, or a sodium ion channel.
Claim 15
Dependent Claim
11 words
The method of claim 6, wherein said compound is an antagonist.
References Cited (Backward Citations)
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