Patent US5436128A successfully refreshed from Google Patents.
US5436128A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Assay methods and compositions for detecting and evaluating the intracellular transduction of an extracellular signal
Assignee: Merck and Co Inc
•
Filing: 1993-01-27
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Granted: 1995-07-25
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Expires: 2013-01-27
Abstract
Abstract Transcription based assays that identify extracellular signals that modulate the activity of cell surface proteins are provided. Extracellular signals are identified by measuring the amount of transcription of a reporter gene in a recombinant cell that expresses the cell surface protein and contains DNA encoding the reporter gene under the transcriptional control of a promoter that is regulated, directly or indirectly, by the cell surface protein. The assays, provide a means for identifying potential pharmaceutical compounds that can be used to treat disease by virtue of their agonistic or antagonistic effects on the cell surface protein. Recombinant cells that express cell surface receptors and that contain reporter gene constructs that include transcriptional regulatory elements that are responsive to the activity of the cell surface receptors are also provided.
Inventors
- 👤 Michael M. Harpold
- 👤 Paul Brust
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 20:37
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: 51
Claim 1
Independent Claim
101 words
A method for identifying extracellular signals that modulate cell surface protein-mediated activity, comprising: comparing the difference in the amount of transcription of a reporter gens in a recombinant cell in the presence of the signal with the amount of transcription in the absence of said signal, or with the amount of transcription in the absence of the cell surface protein, wherein: the recombinant cell contains a reporter gens construct and expresses the cell surface protein; and transcription of the reporter gens is under the control of at least one transcriptional control element whose activity is regulated by the cell surface protein.
Claim 2
Independent Claim
101 words
A method for identifying extracellular signals that modulate cell surface protein-mediated activity, comprising: comparing the difference in the amount of transcription of a reporter gens in a recombinant cell in the presence of the signal with the amount of transcription in the absence of said signal, or with the amount of transcription in the absence of the cell surface protein, wherein: the recombinant cell contains a reporter gens construct and expresses the cell surface protein; and transcription of the reporter gens is under the control of at least one transcriptional control element whose activity is regulated by the cell surface protein.
Claim 3
Dependent Claim
16 words
The method of claim 1, wherein said protein is a cell surface receptor or ion channel.
Claim 4
Dependent Claim
21 words
The method of claim 2, wherein said signal is an agonist or antagonist of said cell surface receptor or ion channel.
Claim 5
Dependent Claim
18 words
The method of claim 3, wherein the concentration of said agonist or antagonist is less than 100 μM.
Claim 6
Dependent Claim
18 words
The method of claim 3, wherein the concentration of said agonist or antagonist is less than 10 μM.
Claim 7
Dependent Claim
18 words
The method of claim 3, wherein the concentration of said agonist or antagonist is less than 1 μM.
Claim 8
Dependent Claim
18 words
The method of claim 3, wherein the concentration of said agonist or antagonist is less than 0.1 μM.
Claim 9
Dependent Claim
18 words
The method of claim 3, wherein the concentration of said agonist or antagonist is less than 10 nM.
Claim 10
Dependent Claim
18 words
The method of claim 3, wherein the concentration of said agonist or antagonist is less than 1 nM.
Claim 11
Dependent Claim
18 words
The method of claim 3, wherein the concentration of said agonist or antagonist is less than 0.1 nM.
Claim 12
Dependent Claim
18 words
The method of claim 3, wherein the concentration of said agonist or antagonist is less than 0.01 nM.
Claim 13
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 14
Dependent Claim
23 words
The method of claim 1, wherein the amount of transcription is determined by measuring the amount of reporter gene protein that is produced.
Claim 15
Dependent Claim
11 words
The method of claim 3, wherein said signal is an antagonist.
Claim 16
Dependent Claim
45 words
The method of claim 14, further comprising, prior to or simultaneously with 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 17
Dependent Claim
35 words
The method of claim 1, wherein said reporter gene construct includes transcriptional regulatory sequences of nucleotides that modulate transcription from said transcriptional control element in the absence of said cell surface protein or extracellular signal.
Claim 18
Dependent Claim
49 words
The method of claim 16, wherein said transcriptional regulatory sequence is the c-fos gene intragenic regulatory element, whereby the level of transcription of said reporter gene in the absence of said cell surface protein or extracellular signal is less than in the absence of the the intragenic regulatory element.
Claim 19
Dependent Claim
39 words
The method of claim 1, wherein the cell surface protein is a cell surface receptor selected from: muscarinic receptors, neuronal nicotinic acetylcholine receptors, gamma-aminobutyric acid receptors, glutamate receptors, adrenergic receptors, dopamine receptors, nerve growth factor receptors, or serotonin receptors.
Claim 20
Dependent Claim
26 words
The method of claim 1, wherein the cell surface protein is an ion channel selected from: calcium ion channels, potassium ion channels, or sodrum ion channels.
Claim 21
Dependent Claim
45 words
The method of claim 1, wherein the transcriptional control element is a promoter selected from: the c-fos gene promoter, the vasoactive intestinal peptide gene promoter, the somatostatin gene promoter, the proenkephalin promoter, the phosphoenolpyruvate carboxykinase gene promoter or the nerve growth factor-1 A gene promoter.
Claim 22
Dependent Claim
38 words
The method of claim 1, wherein the reporter gene is selected from: the gene encoding bacterial chloramphenicol acetyltransferase, the gene encoding firefly luciferase, the gene encoding bacterial luciferase, the gene encoding β-galactosidase or the gene encoding alkaline phosphatase.
Claim 23
Dependent Claim
40 words
The method of claim 1, wherein the gene construct includes at least one transcriptional regulatory sequence of nucleotides selected from the group consisting of serum responsive elements, cyclic adenosine monophosphate responsive elements, and elements responsive to intracellular calcium ion levels.
Claim 24
Dependent Claim
34 words
The method of claim 18, wherein said receptor is the Type 1 human muscarinic receptor, said transcriptional control element is the c-fos promoter and said reporter gene is the gene encoding bacterial chloramphenicol acetyltransferase.
Claim 25
Dependent Claim
46 words
A recombinant cell, comprising: DNA that encodes a heterologous cell surface protein whose activity is modulated by extracellular signals; and a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional control element that is regulated by said cell surface protein.
Claim 26
Dependent Claim
46 words
A recombinant cell, comprising: DNA that encodes a heterologous cell surface protein whose activity is modulated by extracellular signals; and a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional control element that is regulated by said cell surface protein.
Claim 27
Dependent Claim
29 words
The cell of claim 24, wherein said cell is a PC12 cell or a COS-7 cell that has been modified by introduction of said DNA and reporter gene construct.
Claim 28
Dependent Claim
33 words
The cell of claim 25, wherein said reporter gene construct includes transcriptional regulatory sequences of nucleotides that modulate transcription from said promoter in the absence of said cell surface protein or extracellular signal.
Claim 29
Dependent Claim
38 words
The cell of claim 26, wherein said transcriptional regulatory sequence is the c-fos gene intragenic regulatory element, which is inserted downstream, with respect to the direction of transcription, from the transcription binding and initiation site of said promoter.
Claim 30
Dependent Claim
14 words
The cell of claim 27, wherein said reporter gene construct is FC4XP1FIRE or FC4XP2FIRE.
Claim 31
Dependent Claim
74 words
The cell of claim 27, wherein said reporter gene is fused to a sufficient portion of the c-fos gene first exon and intragenic region to reduce the level of transcription of said reporter gene in the absence of said cell surface protein or signal to levels that are substantially undetectable; and said c-fos gene intragenic regulatory element is inserted in reading frame at the 3' end of the first exon of said c-fos gene.
Claim 32
Dependent Claim
36 words
The recombinant cell of claim 24, wherein said cell surface proteins are receptors selected from: muscarinic receptors, neuronal nicotinic acetylcholine receptors, gamma-aminobutyric acid receptors, glutamate receptors, adrenergic receptors,dopamine receptors, nerve growth factor receptors, or serotonin receptors.
Claim 33
Dependent Claim
26 words
The recombinant cell of claim 24, wherein said cell surface proteins are ion channels selected from: calcium ion channels, potassium ion channels, or sodium ion channels.
Claim 34
Dependent Claim
44 words
The recombinant cell of claim 24, wherein said transcriptional control element is selected from: the c-fos gene promoter, the vasoactive intestinal peptide gene promoter, the somatostatin gene promoter, the proenkephalin promoter, the phosphoenolpyruvate carboxykinase gene promoter, or the nerve growth factor-1 A gene promoter.
Claim 35
Dependent Claim
39 words
The recombinant cell of claim 24, wherein said reporter gene is selected from: the gene encoding bacterial chloramphenicol acetyltransferase, the gene encoding firefly luciferase, the gene encoding bacterial luciferase, the gene encoding β-galactosidase or the gene encoding alkaline phosphatase.
Claim 36
Dependent Claim
35 words
The recombinant cell of claim 30, wherein said receptor is Type 1 human muscarinic receptor, said transcriptional control element is the c-fos gene promoter and said reporter gene is the gene encoding bacterial chloramphenicol acetyltransferase.
Claim 37
Dependent Claim
29 words
The method of claim 1, wherein said cell surface protein is an ion channel and prior to comparing the difference in the amount of transcription the cell is depolarized.
Claim 38
Dependent Claim
22 words
The method claim 1, wherein said cell surface protein is an ion channel and said signal causes depolarization of the cell membrane.
Claim 39
Independent Claim
351 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 a 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 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 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 construct 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. Transcription based assays that identify extracellular signals that modulate the activity of cell surface proteins are provided. Extracellular signals are identified by measuring the amount of transcription of a reporter gene in a recombinant cell that expresses the cell surface protein and contains DNA encoding the reporter gene under the transcriptional control of a promoter that is regulated, directly or indirectly, by the cell surface protein. The assays, provide a means for identifying potential pharmaceutical compounds that can be used to treat disease by virtue of their agonistic or antagonistic effects on the cell surface protein. Recombinant cells that express cell surface receptors and that contain reporter gene constructs that include transcriptional regulatory elements that are responsive to the activity of the cell surface receptors are also provided.
Claim 40
Independent Claim
351 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 a 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 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 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 construct 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. Transcription based assays that identify extracellular signals that modulate the activity of cell surface proteins are provided. Extracellular signals are identified by measuring the amount of transcription of a reporter gene in a recombinant cell that expresses the cell surface protein and contains DNA encoding the reporter gene under the transcriptional control of a promoter that is regulated, directly or indirectly, by the cell surface protein. The assays, provide a means for identifying potential pharmaceutical compounds that can be used to treat disease by virtue of their agonistic or antagonistic effects on the cell surface protein. Recombinant cells that express cell surface receptors and that contain reporter gene constructs that include transcriptional regulatory elements that are responsive to the activity of the cell surface receptors are also provided.
Claim 41
Dependent Claim
21 words
The method of claim 37, wherein the compound is an agonist or antagonist of said cell surface receptor or ion channel.
Claim 42
Dependent Claim
25 words
The method of claim 37, wherein the amount of transcription is determined by measuring the amount of mRNA that is transcribed from said reporter gene.
Claim 43
Dependent Claim
23 words
The method of claim 37, wherein the amount of transcription is determined by measuring the amount of reporter gene protein that is produced.
Claim 44
Dependent Claim
45 words
The method of claim 37, further comprising, prior to or simultaneously with 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 45
Dependent Claim
11 words
The method of claim 41, wherein said compound is an antagonist.
Claim 46
Dependent Claim
11 words
The method of claim 15, wherein said compound is an antagonist.
Claim 47
Dependent Claim
18 words
The method of claim 1, wherein transcription is assessed by measuring the activity of the reporter gene protein.
Claim 48
Dependent Claim
21 words
The method of claim 1, wherein the cell is preincubated with the extracellular signal prior to comparing the difference in transcription.
Claim 49
Dependent Claim
19 words
The method of claim 45, wherein the cell is contacted with an agonist when comparing the difference in transcription.
Claim 50
Dependent Claim
20 words
The method of claim 37, wherein the cell is preincubated with the compound prior to comparing the difference in transcription.
Claim 51
Dependent Claim
19 words
The method of claim 47, wherein the cell is contacted with an agonist when comparing the difference in transcription.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1998-07-24 | 1999-01-23 | 1999-07-25 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2002-07-24 | 2003-01-23 | 2003-07-25 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2006-07-24 | 2007-01-23 | 2007-07-25 | $7700.00 | Window Closed / Lapsed |