Method of identifying compounds that modulate interaction of androgen receptor with beta-catenin

• US 20060269950A1
• Filed: 05/17/2006
• Published: 11/30/2006
• Est. Priority Date: 05/19/2005
• Status: Abandoned Application

• Current Assignee: Wyeth LLC (Pfizer Inc.)
• Original Assignee: Wyeth (Pfizer Inc.)
• Inventors: Berrodin, Thomas J., Kilbourne, Edward Judson
• Application Number:
  US11/435,947
• Publication Number:
  US 20060269950A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Q 2565/201 Two hybrid system
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways
• CPC Class Codes: G01N 33/743 Steroid hormones

Abstract

Methods for determining if test compounds are able to modulate the interaction between androgen receptor and β-catenin are disclosed. Methods for the determining whether a test compound selectively modulates an androgen receptor signaling pathway over a β-catenin-Wnt signaling pathway or a β-catenin-Wnt signaling pathway over an androgen receptor signaling pathway are also disclosed.

Claims

1. A method of determining if a test compound is able to modulate the interaction between androgen receptor and β

-catenin comprising the steps of;

(a) providing a cell comprising;

(i) a DNA sequence encoding a hybrid protein comprising a DNA binding domain fused to the NH₃-terminal region of β

-catenin,

(ii) a DNA sequence comprising an upstream activation sequence corresponding to said DNA binding domain operably linked to and controlling transcription of a reporter gene, and

(iii) a DNA sequence encoding androgen receptor protein,

(b) introducing the test compound to the cell, optionally in the presence of androgen; and

(c) measuring the expression of the reporter gene,

wherein an increase or decrease of expression by the reporter gene indicates that the test molecule is able to modulate the interaction between androgen receptor and β

-catenin.

2. The method of claim 1, wherein the DNA binding domain of (i) comprises a GAL-4 DNA binding domain.

3. The method of claim 1, wherein the upstream activation sequence operably linked to a reporter gene of (ii) comprises GALA UAS.

4. The method of claim 3, wherein there are one or more copies of the GALA UAS sequence operably linked to the reporter gene.

5. The method of claim 1, wherein the reporter gene is luciferase.

6. The method of claim 1, wherein the NH₃-terminal region of β

-catenin of (i) comprises amino acids 2 through 424 of human β

-catenin.

7. The method of claim 1, wherein the NH₃-terminal region of β

-catenin of (i) comprises a nucleotide sequence encoding an amino acid sequence having at least 65% identity with amino acids 2424 of SEQ ID NO;

1.

8. The method of claim 7, wherein the NH₃-terminal region of β

-catenin of (i) comprises a nucleotide sequence encoding an amino acid sequence having at least 75% identity with amino acids 2-424 of SEQ ID NO;

1.

9. The method of claim 8, wherein the NH₃-terminal region of β

-catenin of (i) comprises a nucleotide sequence encoding an amino acid sequence having at least 85% identity with amino acids 2424 of SEQ ID NO;

1.

10. The method of claim 9, wherein the NH₃-terminal region of β

-catenin of (i) comprises a nucleotide sequence encoding an amino acid sequence having at least 95% identity with amino acids 2424 of SEQ ID NO;

1.

11. The method of claim 1, wherein the NH₃-terminal region of β

-catenin of (i) comprises a nucleotide sequence which hybridizes with a nucleotide sequence encoding amino acids 2424 of SEQ ID NO;

1 under the following conditions;

6×

SSC at 45°

C. and washed at least once with 0.2×

SSC, 0.1% SDS at 50°

C.

12. The method of claim 11, wherein the NH₃-terminal region of β

-catenin of (i) comprises a nucleotide sequence which hybridizes with a nucleotide sequence encoding amino acids 2424 of SEQ ID NO;

1 under the following conditions;

6×

SSC at 45°

C. and washed at least once with 0.2×

SSC, 0.1% SDS at 55°

C.

13. The method of claim 12, wherein the NH₃-terminal region of β

-catenin of (i) comprises a nucleotide sequence which hybridizes with a nucleotide sequence encoding amino acids 2-424 of SEQ ID NO;

1 under the following conditions;

6×

SSC at 45°

C. and washed at least once with 0.2×

SSC, 0.1% SDS at 65°

C.

14. The method of claim 1, wherein said cell is a eukaryotic cell.

15. The method of claim 14, wherein said cell is a mammalian cell.

16. The method of claim 1, wherein the modulation of expression of the reporter gene is a decrease in expression.

17. The method of claim 1, wherein said androgen at step (b) is DHT.

18. The method of claim 1, wherein the DNA binding domain of (i) comprises a GAL-4 DNA binding domain;

wherein the NH₃-terminal region of β

-catenin of (i) comprises amino acids 2 through 424 of human β

-catenin;

wherein there are more than one copy of the upstream activation sequence GAL-4 UAS operably linked to the reporter gene;

wherein the reporter gene is luciferase;

wherein the androgen at step (b) is DHT; and

wherein the modulation is a decrease in expression.

19. The method of claim 1, wherein the NH₃-terminal region of β

-catenin comprises armadillo repeats 1-12.

20. The method of claim 1, wherein the NH₃-terminal region of β

-catenin comprises armadillo repeats 1-7.

21. The method of claim 1, wherein the NH₃-terminal region of β

-catenin comprises armadillo repeats 1-6.

22. A method of determining if a test compound is able to modulate the interaction between androgen receptor and β

-catenin comprising the steps of;

(a) providing a cell comprising;

(i) a DNA sequence encoding a hybrid protein comprising a DNA binding domain fused to β

-catenin,

(ii) a DNA sequence comprising an upstream activation sequence corresponding to said DNA binding domain operably linked to and controlling transcription of a reporter gene, and

(iii) a DNA sequence encoding androgen receptor protein,

(b) introducing the test compound to the cell, optionally in the presence of androgen; and

(c) measuring the expression of the reporter gene,

wherein an increase or decrease of expression by the reporter gene indicates that the test molecule is able to modulate the interaction between androgen receptor and β

-catenin.

23. A method of determining if a test compound selectively modulates the β

-catenin-Wnt signaling pathway over an androgen receptor signaling pathway comprising;

(a) identifying a test compound which increases or decreases the expression of a gene by inhibiting the AR mediated interaction with β

-catenin, wherein the test compound removes androgen-liganded AR repression on Wnt signaling without repressing androgen-AR mediated transcription; and

(b) assaying the test compound of (a) to determine whether the test compound increases or decreases the expression of a gene through a β

-catenin independent androgen receptor signaling pathway;

whereby the test compound of (a) selectively modulates the β

-catenin-Wnt signaling pathway by inhibiting the ability androgen-liganded AR to interact with β

-catenin if the test compound fails to increase or decrease the expression of a gene through an androgen receptor signaling pathway.

24. The method of claim 23, wherein step (a) comprises the steps of

(A) providing a cell comprising:

(i) a DNA sequence encoding a hybrid protein comprising a DNA binding domain fused to β

-catenin;

(ii) a DNA sequence comprising an upstream activation sequence corresponding to said DNA binding domain operably linked to and controlling transcription of a reporter gene; and

(iii) a DNA sequence encoding androgen receptor protein;

(B) introducing the test compound to the cell, optionally in the presence of androgen; and

(C) measuring the expression of the reporter gene,

wherein an increase or decrease of expression by the reporter gene indicates that the test molecule is able to modulate the interaction between androgen receptor and β

-catenin.

25. The method of claim 24, wherein the DNA sequence of (i) encodes a hybrid protein comprising a DNA binding domain fused to the NH₃-terminal region of β

-catenin.

26. A method of determining if a test compound selectively modulates an androgen receptor signaling pathway over a β

-catenin-Wnt signaling pathway comprising;

(a) identifying a test compound which increases or decreases the expression of a gene through an androgen receptor signaling pathway; and

(b) assaying the test compound of (a) to determine whether the test compound increases or decreases the ability of androgen-liganded AR or non-liganded AR to inhibit β

-catenin/Wnt signaling;

whereby the test compound of (a) selectively modulates an androgen receptor signaling pathway without removing androgen-liganded AR repression of Wnt signaling or does not promote the interaction between AR and β

-catenin in the absence of an AR agonist resulting in the test compound having no activity in increasing or decreasing the expression of a gene regulated by β

-catenin-Wnt signaling pathway.

27. The method of claim 26, wherein step (b) comprises the steps of

(A) providing a cell comprising:

(i) a DNA sequence encoding a hybrid protein comprising a DNA binding domain fused to β

-catenin;

(ii) a DNA sequence comprising an upstream activation sequence corresponding to said DNA binding domain operably linked to and controlling transcription of a reporter gene; and

(iv) a DNA sequence encoding androgen receptor protein;

(B) introducing the test compound of (a) to the cell, optionally in the presence of androgen; and

(C) measuring the expression of the reporter gene,

wherein an increase or decrease of expression by the reporter gene indicates that the test molecule is able to modulate the interaction between androgen receptor and β

-catenin.

28. The method of claim 27, wherein the DNA sequence of (i) encodes a hybrid protein comprising a DNA binding domain fused to the NH₃-terminal region of β

-catenin.