Compositions and methods for detecting ligand-dependent nuclear receptor and coactivator interactions

• US 6,410,245 B1
• Filed: 04/01/1998
• Issued: 06/25/2002
• Est. Priority Date: 04/01/1998
• Status: Expired due to Fees

• Current Assignee: Affymax Incorporated
• Original Assignee: Affymax Incorporated
• Inventors: Northrop, Jeffrey P., Hart, Charles P., Schatz, Peter J.
• Primary Examiner(s): CELSA, BENNETT M
• Application Number:
  US09/053,611
• Time in Patent Office: 1,546 Days
• Field of Search: 436/518, 424/141.1, 536/23.5, 536/23.1, 435/7.1, 435/7.2, 435/69.1
• US Class Current: 506/9
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways
• CPC Class Codes: G01N 33/5047 Cells of the immune system
• CPC Class Codes: G01N 33/5067 Liver cells
• CPC Class Codes: G01N 33/5073 Stem cells
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• CPC Class Codes: G01N 33/6875 Nucleoproteins

Abstract

The invention provides methods for identifying agents that are ligands for nuclear receptors. The methods include conducting multiplexed assays utilizing positive hybrid systems, reverse hybrid systems, direct interaction assays and other assays to screen for ligands having activity with a receptor of interest. The methods can be performed in various multiplexing formats to produce a profile that can be used to categorize a test ligand relative to known agonists and antagonists.

Claims

1. A method for identifying nuclear receptor ligands comprising performing at least two different types of assays in multiplex format, said assays for identifying a ligand and/or quantifying the efficacy and/or potency of said ligand as an antagonist or agonist for a nuclear receptor and selected from the group of assays consisting of the following assay types (1) a positive hybrid system:

coactivator assay, (2) a positive hybrid system;

corepressor assay, (3) a reverse hybrid system;

coactivator assay and (4) a reverse hybrid system;

corepressor assay,

wherein

(a) the positive hybrid system;

coactivator assay comprises

(i) contacting a first fusion protein, a second fusion protein, a first test compound and a first reporter construct that comprises a first regulatory sequence responsive to a first transcription factor and a first reporter sequence operably linked thereto, and wherein

the first fusion protein comprises a ligand binding domain (LBD) of the nuclear receptor linked to a transcriptional activation domain (AD) of the first transcription factor and the second fusion protein comprises a domain from a nuclear receptor coactivator protein (CA) and is linked to a DNA binding domain (DBD) of the first transcription factor, or

the first and second fusion protein are as described in section (a)(i), except that the LBD of the first fusion protein is fused to the DBD of the first transcription factor and the CA domain of the second fusion protein is fused to the AD of the first transcription factor, whereby

in the presence of agonist the first and second fusion proteins interact via the LBD and the CA domain to form a first reconstituted transcription factor that binds to the first regulatory sequence to activate transcription of the first reporter sequence; and

(ii) determining whether expression of the first reporter sequence is activated, activation being an indication that the first test agent is a ligand for the nuclear receptor and is an agonist rather than an antagonist;

(b) the positive hybrid system;

corepressor assay comprises

(i) contacting a third fusion protein, a fourth fusion protein, a second test agent, and a second reporter construct that comprises a second regulatory sequence responsive to a second transcription factor and a second reporter sequence operably linked thereto, and wherein

the third fusion protein comprises the LBD linked to an AD from a second transcription factor and the fourth fusion protein comprises a domain from a nuclear corepressor protein (CR) linked to a DBD of the second transcription factor, or

the third and fourth fusion protein are as described in section (b) (i), except that the LBD of the third fusion protein is linked to the DBD of the second transcription factor and the CR domain of the fourth fusion protein is linked to the AD of the second transcription factor, whereby

in the presence of antagonist the third and fourth fusion proteins interact via the LBD and the CR domain to form a second reconstituted transcription factor that binds to the second regulatory sequence to activate transcription of the second reporter sequence; and

(ii) determining whether expression of the second reporter sequence is activated, activation being an indication that the second test agent is a ligand for the nuclear receptor and is an antagonist rather than an agonist;

(c) the reverse hybrid system;

coactivator assay comprises

(i) contacting a fifth fusion protein, a sixth fusion protein, a third test agent, a third reporter construct encoding a third reporter, and a first relay construct responsive to a third transcription factor and encoding a first relay product that inhibits expression of the third reporter; and

wherein

the fifth fusion protein comprises the LBD linked to an AD from the third transcription factor and the sixth fusion protein comprises the CA domain linked to the DBD of the third transcription factor, or

the fifth and sixth fusion protein are as described in section (c)(i), except that the LBD of the fifth fusion protein is linked to the DBD of the third transcription factor and the CA domain of the sixth fusion protein is linked to the AD from the third transcription factor, whereby

in the presence of antagonist, interaction between the fifth and sixth fusion protein via the LBD and the CA domain to form a third reconstituted transcription factor is inhibited, thereby inhibiting expression of the first relay product and thus activating expression of the third reporter; and

(ii) determining whether expression of the third reporter is activated, activation being an indication that the third test agent is a ligand for the nuclear receptor and is an antagonist rather than an agonist; and

(d) the reverse hybrid system;

corepressor assay comprises

(i) contacting a seventh fusion protein, an eight fusion protein, a fourth test agent, a fourth reporter construct encoding a fourth reporter, and a second relay construct responsive to a fourth transcription factor and encoding a second relay product that inhibits expression of the fourth reporter; and

wherein

the seventh fusion protein comprises the LBD linked to an AD from the fourth transcription factor and the eighth fusion protein comprises the CR domain linked to a DBD of the fourth transcription factor, or

the seventh and eighth fusion protein are as described in section (d)(i), except that the LBD of the seventh fusion domain is linked to the DBD of the fourth transcription factor and the CR domain of the eighth fusion protein is linked to the AD from the fourth transcription factor, whereby

in the presence of agonist, interaction between the seventh and eighth fusion protein via the LBD and the CR domain to form a fourth reconstituted transcription factor is inhibited, thereby inhibiting expression of the second relay product and activating expression of the fourth reporter; and

(ii) determining whether expression of the fourth reporter is activated, activation being an indication that the fourth test agent is a ligand for the nuclear receptor and is an agonist rather than an antagonist.

2. The method of claim 1, wherein said at least two assays are selected from the group consisting of assay type (1), (3) and (4).

3. The method of claim 1, wherein said at least two assays are selected from the group of assays consisting of assay type (1), (2) and (4).

4. The method of claim 1, wherein said at least two assays are selected from the group of assays consisting of assay type (2), (3) and (4).

5. A method for identifying nuclear receptor ligands comprising performing at least two assays in multiplex format, said assays for identifying a ligand and/or quantifying the efficacy and/or potency of said ligand as an antagonist or agonist of a nuclear receptor and selected from the group of assays consisting of the following assay types (1) a positive hybrid system:

coactivator assay, (2) a positive hybrid system;

corepressor assay, (3) a reverse hybrid system;

coactivator assay, (4) a reverse hybrid system;

corepressor assay, (5) a direct interaction assay and (6) another assay for identifying and/or quantifying the efficacy and/or potency of nuclear receptor ligands, and wherein at least one of said assays is of assay type (2), (3) or (4) and assays (1), (2), (3) and (4) are performed as described in claim 1.

6. The method of claim 5, wherein at least one of said assays is of assay type (2) or (4).

7. A method for identifying nuclear receptor ligands comprising performing at least three assays in multiplex format, said assays for identifying a ligand and/or quantifying the efficacy and/or potency of said ligand as an antagonist or agonist of a nuclear receptor and selected from the group of assays consisting of the following assay types (1) a positive hybrid system:

coactivator assay, (2) a positive hybrid system;

corepressor assay, (3) a reverse hybrid system;

coactivator assay, (4) a reverse hybrid system;

corepressor assay and (5) a direct interaction assay, wherein of said at least three assays, at least three are of different assay types, and wherein assays (1), (2), (3) and (4) are performed as described in claim 1.

8. A method for identifying a candidate pharmaceutical agent from a library of test agents, wherein the candidate pharmaceutical agent has a desired biological effect profile and potentially affects binding between a nuclear receptor and a ligand, comprising:

performing at least three assays with each of a plurality of test agents from said library to obtain for each test agent measurements of a plurality of biological effects as detected as a ligand-induced conformational change or a binding interaction change as determined by said plurality of assays, said assays being selected from the group consisting of the following assay types (1) a positive hybrid system;

coactivator assay, (2) a positive hybrid system;

corepressor assay, (3) a reverse hybrid system;

coactivator assay, (4) a reverse hybrid system;

corepressor assay and (5) a direct interaction assay, wherein of said at least three assays, at least three are of different assay types;

assigning a separate score value for each of said biological effects based upon the detection or nondetection of a ligand-induced conformational change or binding interaction change in each of said plurality of assays to generate a score matrix for each of said test agents; and

comparing said score matrix for each of said test agents to an equivalent score matrix for one or more predetermined agonist(s) and/or antagonist(s) to obtain an indication of the value of the test agent as a candidate pharmaceutical agent; and

wherein

(a) the positive hybrid system;

coactivator assay comprises

(i) contacting a first fusion protein, a second fusion protein, one of the test agents and a first reporter construct that comprises a first regulatory sequence responsive to a first transcription factor and a first reporter sequence operably linked thereto, and wherein

the first fusion protein comprises a ligand binding domain (LBD) of the nuclear receptor linked to a transcriptional activation domain (AD) of the first transcription factor and the second fusion protein comprises a domain from a nuclear receptor coactivator protein (CA) and is linked to a DNA binding domain (DBD) of the first transcription factor, or

the first and second fusion protein are as described in section (a)(i), except that the LBD of the first fusion protein is fused to the DBD of the first transcription factor and the CA domain of the second fusion protein is fused to the AD of the first transcription factor, whereby

in the presence of agonist the first and second fusion proteins interact via the LBD and the CA domain to form a first reconstituted transcription factor that binds to the first regulatory sequence to activate transcription of the first reporter sequence; and

(ii) determining whether expression of the first reporter sequence is activated, activation being an indication that the test agent is a ligand for the nuclear receptor and is an agonist rather than an antagonist;

(b) the positive hybrid system;

corepressor assay comprises

(i) contacting a third fusion protein, a fourth fusion protein, one of the test agents, and a second reporter construct that comprises a second regulatory sequence responsive to a second transcription factor and a second reporter sequence operably linked thereto, and wherein

the third fusion protein comprises the LBD linked to an AD from a second transcription factor and the fourth fusion protein comprises a domain from a nuclear corepressor protein (CR) linked to a DBD of the second transcription factor, or

the third and fourth fusion protein are as described in section (b)(i), except that the LBD of the third fusion protein is linked to the DBD of the second transcription factor and the CR domain of the fourth fusion protein is linked to the AD of the second transcription factor, whereby

in the presence of antagonist the third and fourth fusion proteins interact via the LBD and the CR domain to form a second reconstituted transcription factor that binds to the second regulatory sequence to activate transcription of the second reporter sequence; and

(ii) determining whether expression of the second reporter sequence is activated, activation being an indication that the test agent is a ligand for the nuclear receptor and is an antagonist rather than an agonist;

(c) the reverse hybrid system;

coactivator assay comprises

(i) contacting a fifth fusion protein, a sixth fusion protein, one of the test agents, a third reporter construct encoding a third reporter, and a first relay construct responsive to a third transcription factor and encoding a first relay product that inhibits expression of the third reporter; and

wherein

the fifth fusion protein comprises the LBD linked to an AD from the third transcription factor and the sixth fusion protein comprises the CA domain linked to the DBD of the third transcription factor, or

the fifth and sixth fusion protein are as described in section (c)(i), except that the LBD of the fifth fusion protein is linked to the DBD of the third transcription factor and the CA domain of the sixth fusion protein is linked to the AD from the third transcription factor, whereby

in the presence of antagonist, interaction between the fifth and sixth fusion protein via the LBD and the CA domain to form a third reconstituted transcription factor is inhibited, thereby inhibiting expression of the first relay product and thus activating expression of the third reporter; and

(ii) determining whether expression of the third reporter is activated, activation being an indication that the test agent is a ligand for the nuclear receptor and is an antagonist rather than an agonist;

(d) the reverse hybrid system;

corepressor assay comprises

(i) contacting a seventh fusion protein, an eight fusion protein, one of the test agents, a fourth reporter construct encoding a fourth reporter, and a second relay construct responsive to a fourth transcription factor and encoding a second relay product that inhibits expression of the fourth reporter; and

wherein

the seventh fusion protein comprises the LBD linked to an AD from the fourth transcription factor and the eighth fusion protein comprises the CR domain linked to a DBD of the fourth transcription factor, or

the seventh and eighth fusion protein are as described in section (d)(i), except that the LBD of the seventh fusion domain is linked to the DBD of the fourth transcription factor and the CR domain of the eighth fusion protein is linked to the AD from the fourth transcription factor, whereby

in the presence of agonist, interaction between the seventh and eighth fusion protein via the LBD and the CR domain to form a fourth reconstituted transcription factor is inhibited, thereby inhibiting expression of the second relay product and activating expression of the fourth reporter; and

(ii) determining whether expression of the fourth reporter is activated, activation being an indication that the test agent is a ligand for the nuclear receptor and is an agonist rather than an antagonist; and

(e) the direct interaction assay comprises

(i) contacting a ligand binding domain from a nuclear receptor and a coactivation domain or corepressor domain in the presence of one of the test agents; and

(ii) detecting binding between the nuclear receptor and the coactivation or corepressor domains.

9. The method of claim 8, wherein said plurality of assays are greater than 2 and less than 10 billion in number.

10. The method of claim 9, wherein said plurality of assays are at least 5 in number.

11. The method of claim 8, wherein said score value is a binary value.

12. The method of claim 8, wherein said score value is a quantitative value.

13. The method of claim 8, wherein said test agent is a mixture of test agents.

14. The method of claim 8, wherein the score matrix and the equivalent score matrix are compared by rank ordering of matrix similarity.

15. The method of claim 8, wherein the score matrix and the equivalent score matrix are compared by electronic computation using a trained neural network implementation.

16. A method for identifying a candidate pharmaceutical agent from a library of test agents, wherein the candidate pharmaceutical agent has a desired biological effect profile and potentially affects binding between a nuclear receptor and a ligand, comprising:

(a) performing a plurality of assays with each of a plurality of test agents from said library to obtain for each test agent measurements of a plurality of biological effects as detected as a ligand-induced conformational change or a binding interaction change as determined by said plurality of assays, said plurality of assays being selected from the group consisting of the following assay types (1) a positive hybrid system;

coactivator assay, (2) a positive hybrid system;

corepressor assay, (3) a reverse hybrid system;

coactivator assay, and (4) a reverse hybrid system;

corepressor assay, wherein at least two of the plurality of assays are of different assay types;

(b) assigning a separate score value for each of said biological effects based upon the detection or nondetection of a ligand-induced conformational change or binding interaction change in each of said plurality of assays to generate a score matrix for each of said test agents; and

(c) comparing said score matrix for each of said test agents to an equivalent score matrix for one or more predetermined agonist(s) and/or antagonist(s) to obtain an indication of the value of the test agent as a candidate pharmaceutical agent; and

wherein assays (1), (2), (3) and (4) are performed as described in claim 8.

17. A method for identifying a candidate pharmaceutical agent from a library of test agents, wherein the candidate pharmaceutical agent has a desired biological effect profile and potentially affects binding between a nuclear receptor and a ligand, comprising:

(a) performing a plurality of assays with each of a plurality of test agents from said library to obtain for each test agent measurements of a plurality of biological effects as detected as a ligand-induced conformational change or a binding interaction change as determined by said plurality of assays, said plurality of assays being selected from the group consisting of the following assay types (1) a positive hybrid system;

coactivator assay, (2) a positive hybrid system;

corepressor assay, (3) a reverse hybrid system;

coactivator assay, (4) a reverse hybrid system;

corepressor assay, (5) a direct interaction assay and (6) another assay for identifying and/or quantifying the efficacy and/or potency of nuclear receptor ligands, and wherein at least one of said assays is of assay type (2), (3) or (4);

(b) assigning a separate score value for each of said biological effects based upon the detection or nondetection of a ligand-induced conformational change or binding interaction change in each of said plurality of assays to generate a score matrix for each of said test agents; and

(c) comparing said score matrix for each of said test agents to an equivalent score matrix for one or more predetermined agonist(s) and/or antagonist(s) to obtain an indication of the value of the test agent as a candidate pharmaceutical agent; and

wherein assays (1), (2), (3), (4) and (5) are performed as described in claim 8.