Enabling tools to identify ligands for hormone nuclear receptors

• US 20060134670A1
• Filed: 11/18/2005
• Published: 06/22/2006
• Est. Priority Date: 11/19/2004
• Status: Abandoned Application

• Current Assignee: Acadia Pharmaceuticals Inc.
• Original Assignee: Acadia Pharmaceuticals Inc.
• Inventors: Piu, Fabrice
• Application Number:
  US11/283,329
• Publication Number:
  US 20060134670A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/00 Drugs for disorders of the endocrine system
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/6875 Nucleoproteins
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• CPC Class Codes: G01N 33/78 Thyroid gland hormones , e.g. T3, T4, TBH, TBG or their receptors

Abstract

A method and kit developed to identify substances that act as ligands, corepressors, coactivators, agonist and antagonists for cloned nuclear hormone receptors, as well as a test kit for use in the methods is provided herein. More specifically, the method involves expressing a nuclear hormone receptor, receptor heterodimer, and/or receptor homodimer, DNA encoding one or more signaling molecules and DNA encoding a marker, incubating the cells with a test substance, and identifying whether the test substance interacts with the receptor quantitatively or qualitatively by identifying the amount of marker and/or the proliferation of the cells.

Claims

1. A method of assessing the effect of a candidate compound on the activity of a nuclear receptor comprising:

obtaining a cell expressing one or more nuclear receptors and one or more helper proteins, wherein at least one of said nuclear receptor and said helper protein is expressed from a nucleic acid which has been introduced into said cell;

contacting said cell with said candidate compound; and

determining whether said candidate compound influences the activity of said nuclear hormone receptor.

2. The method of claim 1 wherein both said one or more nuclear receptor and said one or more helper protein are expressed from a nucleic acid which has been introduced into said cell.

3. The method of claim 1, wherein said one or more nuclear receptor and said one or more helper protein are expressed from the same nucleic acid which has been introduced into said cell.

4. The method of claim 1, wherein said one or more nuclear receptor is expressed from a first nucleic acid which has been introduced into said cell and said helper protein is expressed from a second nucleic acid which has been introduced into said cell.

5. The method of claim 1, wherein said determining step comprises comparing the activity of said nuclear hormone receptor in a first cell which expresses said nuclear receptor and said helper protein and which has been contacted with said candidate compound to the activity of said nuclear receptor in a second cell which expresses said nuclear receptor and said helper protein and which has not been contacted with said candidate compound, wherein said candidate compound is determined to influence the activity of said nuclear receptor if said activity of said nuclear receptor in said first cell is significantly different from the activity of said nuclear receptor in said second cell.

6. The method of claim 1, wherein said one or more nuclear receptors is encoded by a nucleic acid selected from the group consisting of SEQ ID NOs.:

1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, and 143 and said one or more helper proteins is encoded by a nucleic acid selected from the group consisting of 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, and 241.

7. The method of claim 1 wherein said one or more nuclear receptors is encoded by a nucleic acid comprising a nucleic acid having at least 70% nucleotide sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOS.:

1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, and 143 and said one or more helper proteins is encoded by a nucleic acid comprising a nucleic acid having at least 70% nucleotide sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOS.;

145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, and 241.

8. The method of claim 1, wherein said one or more nuclear receptors comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:

2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, and 144 and wherein said one or more helper proteins comprises an amino acid sequence selected from the group consisting of SEQ ID NOs;

146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, and 242.

9. The method of claim 1, wherein said one or more nuclear receptors comprises an amino acid sequence having at least 70% amino acid identify to an amino acid sequence selected from the group consisting of SEQ ID NOs:

2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, and 144 and wherein said one or more helper proteins comprises an amino acid sequence having at least 70% amino acid identify to an amino acid sequence selected from the group consisting of SEQ ID NOs;

146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, and 242.

10. The method of claim 1 wherein the combination of said nucleic acid sequence encoding said one or more nuclear receptors and said one or more helper proteins are selected from the group consisting of:

SEQ ID NO;

5 and SEQ ID NOs;

161 or 213;

SEQ ID NO;

9 and SEQ ID NOs;

145, 147, 161, or 213;

SEQ ID NOs;

21, 23, 25, or 27 and SEQ ID NOs;

145, 147, or 213;

SEQ ID NO;

49 and SEQ ID NOs;

145, 147, 161, or 213;

SEQ ID NO;

45 and SEQ ID NOs;

145 or 213;

SEQ ID NO;

51 and SEQ ID NOs;

145 or 147;

SEQ ID NOs;

53, 55, or 57 and SEQ ID NOs;

145 or 147;

SEQ ID NO;

69 and SEQ ID NOs;

161 or 213;

SEQ ID NO;

93 and SEQ ID NOs;

145, 147, 161, or 213;

SEQ ID NO;

107 and SEQ IDNO;

161;

SEQ ID NO;

103 and SEQ ID NOs;

145, 147, or 161;

SEQ ID NO;

101 and SEQ ID NOs;

145 or 147;

SEQ ID NO;

143 and SEQ ID NOs;

145, 147 or 161;

SEQ ID NO;

29 and SEQ ID NO;

213;

SEQ ID NO;

43 and SEQ ID NO;

161;

SEQ ID NO;

61 and SEQ ID NOs;

145, 147, 161 or 213;

SEQ ID NO;

75 and SEQ ID NOs;

145 or 147;

SEQ ID NO;

79 and SEQ ID NO;

161;

SEQ ID NO;

87 and SEQ ID NO;

145, 147, or 161;

SEQ ID NO;

89 and SEQ ID NOs;

145, 147 or 161;

SEQ ID NO;

99 and SEQ ID NO;

161;

SEQ ID NOs;

123, 125, or 127 and SEQ ID NOs;

145, 147, 161, or 213; and

SEQ ID NO;

135 and SEQ ID NOs;

145, 147 or 213.

11. The method of claim 6 wherein the combination of the amino acid sequence of said one or more nuclear receptors and said one or more helper proteins are selected from the group consisting of:

SEQ ID NO;

6 and SEQ ID NOs;

162 or 214;

SEQ ID NO;

10 and SEQ ID NOs;

146, 148, 162, or 214;

SEQ ID NOs;

22, 24, 26 or 28 and SEQ ID NOs;

146, 148, or 214;

SEQ ID NO;

50 and SEQ ID NOs;

146, 148, 162, or 214;

SEQ ID NO;

46 and SEQ ID NOs;

146, 148 or 214;

SEQ ID NO;

52 and SEQ ID NOs;

146 or 148;

SEQ ID NOs;

54, 56, or 58 and SEQ ID NOs;

146 or 148;

SEQ ID NO;

70 and SEQ ID NOs;

162 or 214;

SEQ ID NO;

94 and SEQ ID NOs;

146, 148, 162, or 214;

SEQ ID NO;

108 and SEQ ID NO;

162;

SEQ ID NO;

104 and SEQ ID NOs;

146, 148, or 162;

SEQ ID NO;

102 and SEQ ID NOs;

146 or 148;

SEQ ID NO;

144 and SEQ ID NOs;

146, 148, or 162;

SEQ ID NO;

30 and SEQ ID NO;

214;

SEQ ID NO;

44 and SEQ ID NO;

162;

SEQ ID NO;

62 and SEQ ID NOs;

146, 148, 162, or 214;

SEQ ID NO;

76 and SEQ ID NOs;

146 or 148;

SEQ ID NO;

80 and SEQ ID NO;

162;

SEQ ID NO;

89 and SEQ ID NO;

146, 148, or 162;

SEQ ID NO;

90 and SEQ ID NOs;

146, 148, or 162;

SEQ ID NO;

100 and SEQ ID NO;

162;

SEQ ID NOs;

124, 126, or 128 and SEQ ID NOs;

146, 148, 162, or 214; and

SEQ ID NO;

136 and SEQ ID NOs;

146, 148 or 214.

12. The method of claim 1, wherein the combination of the nuclear receptor expressed by said cell and the helper protein expressed by said cell are selected from the group consisting of:

TR beta and DRIP 205 or ERK2;

RAR beta and SRC1, DRIP205 or ERK2;

PPAR gamma and SRC1 or ERK2;

FXR and SRC1, DRIP205 or ERK2;

LXR beta and SRC1 or ERK2;

VDR and SRC1;

PXR and SRC1;

RXR alpha and DRIP205 or ERK2;

ER beta and SRC1, DRIP205 or ERK2;

AR and DRIP205;

MR and SRC1 or DRIP205;

GR and SRC 1;

SHP and SRC1 or ERK2;

RevERb alpha and ERK2;

ROR gamma and DRIP205;

HNF4 alpha and SRC1, DRIP205 or ERK2;

TR2 alpha and SRC1;

TLX and ERK2;

COUP-TF beta and SRC1 or DRIP205;

EAR2 and SRC1, DRIP205 or ERK2;

ERR gamma and ERK2;

NOR-1 and SRC1, DRIP205 or ERK2; and

GCNF and SRC1 or ERK2.

13. The method of claim 1, wherein said determining step comprises determining whether said compound influences the activity of said one or more nuclear receptors by evaluating a cellular parameter selected from the group consisting of morphology, phosphorylation, differentiation, apoptosis, process formation, motility, gene expression, expression of a cellular receptor, and a phenotypic change

14. The method of claim 1, further comprising introducing a nucleic acid comprising a promoter from which the level of transcription is responsive to activation of said nuclear receptor into said cell, said promoter being operably linked to a nucleic acid encoding a detectable product and determining whether said candidate compound influences the activity of said nuclear receptor by measuring the amount of said detectable product.

15. A method of identifying interaction between a nuclear receptor and one or more helper proteins, said method comprising:

co-transfecting a first cell culture with DNA encoding a nuclear receptor and DNA encoding a marker of cell amplification, along with DNA encoding one or more helper proteins;

incubating the cell culture with varying concentrations of a ligand which is an agonist or antagonist for said nuclear receptor for a period of time sufficient to permit cell amplification of said transfected cells in said first cell culture;

co-transfecting a second cell culture with DNA encoding a nuclear receptor and DNA encoding a marker of cell amplification;

incubating the cell culture with varying concentrations of a ligand which is an agonist or antagonist for said nuclear receptor for a period of time sufficient to permit cell amplification of said transfected cells in said second cell culture;

determining whether said one or more helper proteins interact with said nuclear receptor by comparing the level of amplification of transfected cells expressing said nuclear receptor and said one or more helper proteins to the level of amplification of cells which were transfected with DNA encoding said nuclear receptor but which were not transfected with DNA encoding said one or more helper proteins.

16. A method according to claim 15, wherein said one or more helper proteins is a coactivator.

17. A method according to claim 15, wherein said one or more helper proteins is a corepressor.

18. A method according to claim 15, wherein said one or more helper proteins is a kinase.

19. A method according to claim 15, wherein said one or more helper proteins is a signaling molecule.

20. A method according to claim 15, wherein said one or more helper proteins comprises at least two helper proteins selected from the group consisting of corepressors, and kinases, signaling molecules.

21. The method of claim 15, wherein said DNA encoding said nuclear receptor comprises a sequence selected from the group consisting of SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, and 143.

22. The method of claim 15, wherein said DNA encoding said nuclear receptor encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.:

2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, and 144.

23. The method of claim 15, wherein said DNA encoding said one or more helper proteins comprises a sequence selected from the group consisting of SEQ ID NOs.:

145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, and 241.

24. The method of claim 15, wherein said DNA encoding said one or more helper proteins encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.:

146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, and 242.

25. The method of claim 15, wherein said DNA encoding said nuclear receptor and said DNA encoding said marker of cell amplification are on the same vector.

26. The method of claim 15, wherein said DNA encoding said nuclear receptor and said DNA encoding said marker of cell amplification are on separate vectors.

27. A method of identifying a substance which is a ligand of a nuclear receptor, said method comprising:

incubating a cell culture which comprises a mixture of cells transfected with DNA encoding a nuclear receptor, DNA encoding a marker of cell amplification and DNA encoding one or more helper proteins and untransfected cells, with a test substance which is a potential agonist or antagonist for said nuclear receptor for a period of time sufficient to permit cell amplification of said transfected cells; and

determining any increase or decrease in cell amplification by measuring the level of the marker in said transfected cells.

28. A method of identifying a substance which is a selective modulator of a particular combination of a nuclear receptor and one or more helper proteins, said method comprising:

co-transfecting a first cell culture comprising cells of a first cell type with DNA encoding a nuclear receptor and DNA encoding a marker of cell amplification, along with DNA encoding said one or more helper proteins;

incubating the first cell culture with a test substance;

determining whether said test substance increases or decreases amplification of said transfected cells of said first cell type relative to untransfected cells of said first cell type;

co-transfecting a second cell culture comprising cells of a second cell type with DNA encoding said nuclear receptor and DNA encoding said marker of cell amplification, along with DNA encoding said one or more helper proteins;

incubating the second cell culture with said test substance;

determining whether said test substance increases or decreases amplification of said transfected cells of said second cell type relative to untransfected cells of said second cell type;

wherein said test substance is a selective modulator of said nuclear receptor if the effects of said test substance on said first cell type are opposite to the effects of said test substance on said second cell type.

29. A method of identifying a substance which is a selective modulator of a particular combination of a nuclear receptor and one or more helper proteins, said method comprising:

co-transfecting a first cell culture with DNA encoding a nuclear receptor and DNA encoding a marker of cell amplification, along with DNA encoding one or more first helper proteins;

incubating the first cell culture with a test substance;

determining whether said test substance increases or decreases amplification of said transfected cells in said first cell culture relative to untransfected cells;

co-transfecting a second cell culture with DNA encoding said nuclear receptor and DNA encoding a marker of cell amplification, along with DNA encoding a second one or more helper proteins, wherein said second one or more helper proteins are distinct from said first one or more helper proteins;

incubating the second cell culture with said test substance;

determining whether said test substance increases or decreases amplification of said transfected cells in said second cell culture relative to untransfected cells;

wherein said test substance is a selective modulator of said nuclear receptor if the effects of said test substance on said first cell culture are opposite to the effects of said test substance on said second cell culture.

30. A method of identifying a substance which is a selective modulator of a nuclear receptor comprising:

co-transfecting a first cell culture comprising cells of a first cell type with DNA encoding a nuclear receptor and DNA encoding a marker of cell amplification;

incubating the first cell culture with a test substance;

determining whether said test substance increases or decreases amplification of said transfected cells of said first cell type relative to untransfected cells of said first cell type;

co-transfecting a second cell culture comprising cells of a second cell type with DNA encoding said nuclear receptor and DNA encoding said marker of cell amplification;

incubating the second cell culture with said test substance;

determining whether said test substance increases or decreases amplification of said transfected cells of said second cell type relative to untransfected cells of said second cell type;

wherein said test substance is a selective modulator of said nuclear receptor if the effects of said test substance on said first cell type are opposite to the effects of said test substance on said second cell type.

31. A method of detecting a substance which is a ligand of two nuclear receptors comprising:

incubating a cell culture which comprises a mixture of cells transfected with DNA encoding a first nuclear receptor, DNA encoding a second nuclear receptor, and DNA encoding a marker of cell amplification with a test substance which is a potential agonist or antagonist for said nuclear receptor for a period of time sufficient to permit cell amplification of said transfected cells; and

determining any increase or decrease in cell amplification by measuring the level of the marker of cell amplification in said transfected cells.

32. A method of detecting a substance which is a selective modulator of a particular combination of two nuclear receptors and one or more helper proteins comprising:

co-transfecting a first cell culture comprising cells of a first cell type with DNA encoding a first nuclear receptor, DNA encoding a second nuclear receptor, DNA encoding one or more helper proteins, and DNA encoding a marker of cell amplification;

incubating the first cell culture with a test substance;

determining whether said test substance increases or decreases amplification of said transfected cells in said first cell culture relative to untransfected cells in said first cell culture;

co-transfecting a second cell culture comprising cells of a second cell type with DNA encoding said first nuclear receptor, DNA encoding said second nuclear receptor and DNA encoding a marker of cell amplification;

incubating the second cell culture with said test substance; and

determining whether said test substance increases or decreases amplification of said transfected cells of said second cell type relative to untransfected cells of said second cell type;

wherein said test substance is a selective modulator of said nuclear receptor if the effects of said test substance on said first cell type are opposite to the effects of said test substance on said second cell type.

33. A method of identifying a substance which is a selective modulator of a particular combination of two nuclear receptors and one or more helper proteins comprising:

co-transfecting a first cell culture with DNA encoding a first nuclear receptor, DNA encoding a second nuclear receptor and DNA encoding a marker of cell amplification, along with DNA encoding one or more first helper proteins;

incubating the first cell culture with a test substance;

determining whether said test substance increases or decreases amplification of said transfected cells in said first cell culture relative to untransfected cells;

co-transfecting a second cell culture with DNA encoding said first nuclear receptor, DNA encoding said second nuclear receptor and DNA encoding a marker of cell amplification, along with DNA encoding a second one or more helper proteins, wherein said second one or more helper proteins are distinct from said first one or more helper proteins;

incubating the second cell culture with said test substance;

determining whether said test substance increases or decreases amplification of said transfected cells in said second cell culture relative to untransfected cells;

wherein said test substance is a selective modulator of said nuclear receptor if the effects of said test substance on said first cell culture are opposite to the effects of said test substance on said second cell culture.