Methods of using the A form of the progesterone receptor to screen for antagonists of steroid intracellar receptor-mediated transcription

• US 5,506,102 A
• Filed: 10/28/1993
• Issued: 04/09/1996
• Est. Priority Date: 10/28/1993
• Status: Expired due to Term

• Current Assignee: Ligand Pharmaceuticals, Inc.
• Original Assignee: Ligand Pharmaceuticals, Inc.
• Inventors: McDonnell, Donald P.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/144,554
• Time in Patent Office: 894 Days
• Field of Search: 435/6, 435/8, 435/240.2, 435/69.1, 435/810, 935/33, 935/34, 935/36
• US Class Current: 435/6.17
• CPC Class Codes: C07K 14/721 Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: G01N 2333/723 Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
• CPC Class Codes: G01N 33/743 Steroid hormones
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Methods to screen for antagonists of steroid IR transcription as mediated by the PR-A isoform of the progesterone receptor are provided. Such cell-based methods include a cell in which PR-A is transcriptionally inactive containing asteroid IR other than PR, PR-A and a reporter vector that includes a promoter which is transcriptionally active in the presence of the steroid IR and an activator of said steroid IR, but is substantially transcriptionally inactive in the presence of PR-A. This cell is contacted with the activator of the steroid IR and a compound to be assayed, and the level of reporter product produced in the cell is measured and compared to the level of reporter product expressed in a similar cellular system absent PR-A as an indication of the potential antagonist activity of the compound being assayed. Also provided are an assay kit, a method of screening PR active compounds as ER antagonists and compounds that inhibit the transcriptional activity of asteroid IR in the presence of PR-A in a cellular context where PR-A is substantially transcriptionally inactive.

Claims

1. A method for screening for antagonists of steroid intracellular receptor-mediated transcription comprising:

(a) introducing a first vector encoding asteroid intracellular receptor other than progesterone receptor and a second vector encoding progesterone receptor A form along with a third reporter vector into a cell in which the progesterone receptor A form is a negligible activator of transcription, wherein the reporter vector includes a gene encoding a reporter product and a promoter which is transcriptionally active in the presence of the steroid intracellular receptor and an activator of said steroid intracellular receptor-mediated transcription, but is a negligible activator of transcription in the presence of the progesterone receptor A form;

(b) contacting the cell with the activator of steroid intracellular receptor-mediated transcription and a second compound; and

(c) comparing the level of reporter product expressed in the cell relative to the level of reporter product expressed in a second cell containing the steroid intracellular receptor and reporter vector, and contacted with the activator and second compound, as an indication of the potential antagonist activity of the second compound on steroid intracellular receptor-mediated transcription.

2. The method of claim 1, wherein the steroid intracellular receptor is selected from the group consisting of androgen receptor, glucocorticoid receptor, mineralocorticoid receptor and estrogen receptor.

3. The method of claim 1, wherein the first and second cells are CV-1 cells or HeLa cells.

4. The method of claim 3, wherein the reporter vector comprises a gene encoding luciferase and the MMTV promoter.

5. The method of claim 1, wherein the steroid intracellular receptor is glucocorticoid receptor and the activator of steroid intracellular receptor-mediate transcription comprises a glucocorticoid receptor agonist.

6. The method of claim 5, wherein the glucocorticoid receptor agonist is dexamethasone.

7. The method of claim 1, further comprising, contacting a third cell containing a progesterone receptor isoform and a reporter vector with the second compound, wherein the progesterone receptor isoform is transcriptionally active in the third cell, and comparing the level of reporter product expressed in the third cell relative to the level of reporter product expressed in the third cell absent the progesterone receptor isoform.

8. The method of claim 7, wherein the second compound comprises an agonist of progesterone receptor.

9. The method of claim 7, wherein the third cell is a HepG2 cell and the reporter vector comprises a gene encoding luciferase and the MMTV promoter.

10. The method of claim 7, further comprising, contacting the third cell containing the progesterone receptor isoform and the reporter vector with an activator of progesterone receptor-mediated transcription and the second compound, and comparing the level of reporter product expressed in the third cell relative to the level of reporter product expressed in the third cell in the absence of the second compound.

11. The method of claim 10, wherein the second compound comprises an antagonist of progesterone receptor.

12. The method of claim 1, further comprising, contacting a third cell containing a progesterone receptor isoform and a reporter vector with an activator of progesterone receptor-mediated transcription and the second compound, wherein the progesterone receptor isoform is transcriptionally active in the third cell, and comparing the level of reporter product expressed in the third cell relative to the level of reporter product expressed in the third cell in the absence of the second compound.

13. The method of claim 12, wherein the second compound comprises an antagonist of progesterone receptor.

14. A method for screening for antagonists of steroid intracellular receptor-mediated transcription comprising:

(a) providing a cell comprising asteroid intracellular receptor other than progesterone receptor, progesterone receptor A form and a reporter vector, wherein the progesterone receptor A form is a negligible activator of transcription in the cell, and wherein the reporter vector includes a gene encoding a reporter product and a promoter which is transcriptionally active in the presence of the steroid intracellular receptor and an activator of said steroid intracellular receptor-mediated transcription, but is a negligible activator of transcription in the presence of the progesterone receptor A form;

(b) contacting the cell with the activator of steroid intracellular receptor-mediated transcription and a second compound; and

(c) comparing the level of reporter product expressed in the cell relative to the level of reporter product expressed in a second cell containing the steroid intracellular receptor and reporter vector, and contacted with the activator and second compound, as an indication of the potential antagonist activity of the second compound on steroid intracellular receptor-mediated transcription.

15. An assay kit to screen for antagonists of steroid intracellular receptor-mediated transcription comprising a first cell containing asteroid intracellular receptor other than progesterone receptor, progesterone receptor A form, a reporter vector including a gene encoding a reporter product and a promoter which is transcriptionally active in the presence of the steroid intracellular receptor but is a negligible activator of transcription in the presence of the progesterone receptor A form, a second cell containing the steroid intracellular receptor and reporter vector, and an activator of steroid intracellular receptor-mediated transcription, wherein the progesterone receptor A form is a negligible activator of transcription in the first cell, and wherein the contacting of the first cell with the activator and a compound to be assayed yields a level of expressed reporter product that can be compared to the level of expressed reporter product in the second cell when contacted with the activator and second compound, as an indication of the potential antagonist activity of the compound to be assayed on steroid intracellular receptor-mediated transcription.

16. A method for screening progesterone receptor agonist or antagonist for estrogen receptor antagonist activity comprising:

(a) introducing a first vector encoding estrogen receptor and a second vector encoding progesterone receptor A form along with a third reporter vector into a cell in which the progesterone receptor A form is a negligible activator of transcription, wherein the reporter vector includes a gene encoding a reporter product and a promoter which is transcriptionally active in the presence estrogen receptor but is a negligible activator of transcription in the presence of the progesterone receptor A form;

(b) contacting the cell with an estrogen receptor agonist and a progesterone receptor agonist or antagonist; and

(c) comparing the level of reporter product expressed in the cell relative to the level of reporter product expressed in a second cell containing estrogen receptor and the reporter vector, and contacted with the estrogen receptor agonist and progesterone receptor agonist or antagonist, as an indication of the potential antagonist activity of the progesterone receptor agonist or antagonist on estrogen receptor-mediated transcription.

17. The method of claim 16, wherein the first and second cells are CV-1 cells.

18. The method of claim 17, wherein the reporter vector comprises a gene encoding luciferase and the ERE-MMTV promoter.

19. The method of claim 16, wherein the estrogen receptor agonist is 17-β

-estradiol.