Systems and methods for detection of nuclear receptor function using reporter enzyme mutant complementation

Abstract

Systems and methods for detecting and assaying nuclear receptor activity and screening for nuclear receptor ligands, cofactors, and other compounds that interact with components of the nuclear receptor regulatory process are provided. The assay systems and methods employ complementary protein fused enzyme fragment technology to assay nuclear receptor activity. These methods have application for nuclear receptor ligand and cofactor screening. In particular, these techniques can be used to find ligands for orphan nuclear receptors and to determine the function of orphan nuclear receptors. In this manner, the methods of the invention can be used to find new drugs.

Description

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to methods of detecting nuclear receptor (NR) activity, and provides methods of assaying NR activity, methods for screening for NR ligands and cofactors (i.e., corepressors and coactivators), methods for screening natural and surrogate iigands for orphan NRs, and methods for screening compounds that interact with components of the NR regulatory process.

[0004] 2. Background of the Technology

[0005] Regulation of gene expression involves a large number of transcription factors with unique DNA-recognition properties. Many transcription factors belong to families of related proteins, the members of which bind to similar but distinct DNA sequences.

[0006] Nuclear receptors constitute a large family of ligand-activated transcription factors that interact with response elements within regulated genes. Nuclear receptors include receptors for steroid hormones, thyroid hormones, hormonal forms of vitamin A and D, peroxisomal activators, and ecdysone. Exemplary of steroid hormone receptors are estrogen receptor (ER) and progesterone receptor (PR). Other nuclear receptors include PPARγ (peroxisome proliferator activated receptor γ), RAR (retinoic acid receptor), RXR (retinoid X receptor), TR (thyroid hormone receptor) and VDR (vitamin D receptor).

[0007] Gene regulation by steroid hormones has been extensively studied. See, for example, Clever and Karlson, Exp. Cell Res., 20, 623 (1960). Additionally, steroid hormone receptors have been characterized and purified, hormonally regulated genes have been cloned, and hormone response sequences have been identified in the vicinity of genes regulated by steroid hormones. Further, dozens of regulatory elements for steroid hormones have been described, and the cDNAs for virtually all known hormone receptors have been cloned. See Evans, Science, 240, 889 (1988).

[0008] It has been widely proposed that steroid hormones mediate their biological responses by crossing the plasma membranes of cells and interacting with receptor proteins (i.e., steroid hormone or nuclear receptors) in the cytosol or nucleus of a cell to thereby form complexes. These ligand/receptor complexes then accumulate in the nucleus of cells where they bind to specific regulatory DNA sequences called hormone response elements (i.e., HREs). A dimer of the nuclear receptor/ligand complex is considered to bind to the appropriate response element, specifically to the core sequence of the response element. In this manner, the nuclear receptor/ligand complex can affect the transcription rate of dependent gene(s). Steroid hormone receptor/ligand complexes may also affect the stability of specific mRNAs.

[0009] Hormone response elements have been identified and characterized by several methods and have been shown to contain consensus sequences for the hormonal receptors. See, for example, Beato, Cell, 56, 335-344 (1989); Lopez de Haro, et al., FEBS Lett., 265, 20-22 (1990); and Baniahmad and Tsai, J. Cell Biochem, 51, 151-156 (1993).

[0010] Nuclear receptor complexes can bind to hormone response elements as homodimers and/or as heterodimers. Nuclear receptors that bind as homodimers include steroid receptors and retinoid X receptor. Nuclear receptors that bind as heterodimers include retinoic acid receptor, thyroid hormone receptor and vitamin D receptor. See for example, Moras, et al., “The Nuclear Receptor Ligand Bonding Domain: Structure and Function”, Current Opinion in Cell Biology, 10, 384-391 (1998).

[0011] Nuclear receptors may also regulate gene expression via association with histone acetyltransferase (HAT) or deacetyltransferase complexes. See, for example, Chen, et al., “Regulation of Hormone-Induced Histone Hyperacetylation and Gene Activation via Acetylation of an Acetylase”, Cell, Vol. 98 (11999). In particular, in the absence of their corresponding hormone, nuclear receptors are believed to repress the transcription of target genes via their association with corepressor complexes that contain histone deacetylase activity. Hormone binding is believed to trigger the release of these corepressors allowing for the subsequent association of an array of coactivators.

[0012] Various coactivators have been identified for nuclear receptors. These coactivators include p300/CBP, P/CAF (CBP associated factor), ACTR and SRC-1. These proteins have been demonstrated to interact with nuclear receptors and potentiate their transactivation activity. See Chen et al. (1999), supra. ACTR, for example, has been found to form a multimeric activation complex with P/CAF and CBP/p300. See Chen et al., “Nuclear Receptor Coactivator ACTR is a Novel Histone Acetyltransferase and Forms a Multimeric Activation Complex with P/CAF and CBP/p300”, Cell, Vol. 90, 569-580 (1997).

[0013] Various nuclear receptor bioassays are known. U.S. Pat. Nos. 5,071,773 and 5,298,429, for example, disclose bioassays for determining whether a protein suspected of being a hormone receptor has transcription-activating properties and for evaluating whether a compound is a functional ligand for receptor proteins. Additionally, U.S. Pat. No. 5,770,176 discloses a method of detecting the presence or absence of functional nuclear receptors in a cell or tissue sample comprising simultaneously binding the nuclear receptor under assay occupied by its ligand to its associated response element and to an anti-receptor antibody.

[0014] There still exists a need, however, for bioassays which can be used to directly monitor protein-protein interactions between a nuclear receptor and a second protein such as a second nuclear receptor or a cofactor for the nuclear receptor. Such techniques would allow for the direct detection of protein-protein interactions in situ in a range of cell types and species. Such techniques could also be used to find ligands for orphan nuclear receptors by monitoring the interactions between an orphan nuclear receptor and a coactivator or a second nuclear receptor in the presence of a suspected ligand.

SUMMARY OF THE INVENTION

[0015] According to a first aspect of the invention, a method of detecting nuclear receptor interactions is provided. The method includes providing a cell that expresses a first nuclear receptor as a fusion protein to a first inactive mutant form of a reporter enzyme. The cell also expresses a protein partner as a fusion protein to a second inactive mutant form of the reporter enzyme. The first and second inactive mutant forms of the reporter enzyme can interact upon formation of a complex between the first nuclear receptor and the protein partner to form an active reporter enzyme. The method according to this aspect of the invention further includes determining the presence and/or amount of the reporter enzyme, wherein reporter enzyme activity in the cell indicates the formation of the complex between the nuclear receptor and the protein partner. The cell according to this aspect of the invention can also contain a hormone response element operatively linked to a reporter gene such that binding of the nuclear receptor/protein partner complex to the hormone response element results in expression of the reporter gene. The presence and/or amount of a surrogate reporter protein encodeu by the reporter gene can then be determined wherein the presence and/or amount of the surrogate reporter protein is an indication of the transcription-activating properties of the nuclear receptor/protein partner complex.

[0016] According to a second aspect of the invention, a DNA molecule comprising a sequence encoding a biologically active hybrid nuclear receptor is provided. The hybrid nuclear receptor comprises a nuclear receptor as a fusion protein to an inactive mutant form of a reporter enzyme.

[0017] According to a third aspect of the invention, a DNA molecule comprising a sequence encoding a biologically active hybrid of a nuclear receptor cofactor, wherein the hybrid cofactor comprises a nuclear receptor cofactor as a fusion protein to an inactive mutant form of a reporter enzyme is provided.

[0018] According to a fourth aspect of the invention, a cell is provided wherein the cell comprises a first DNA construct capable of directing the expression of a first biologically active hybrid nuclear receptor in a cell and a second DNA construct capable of directing the expression of a biologically active protein partner in a cell. The first DNA construct includes a first promoter operatively linked to a first DNA molecule, the first DNA molecule comprising a sequence encoding a first biologically active nuclear receptor as a fusion protein to a first inactive mutant form of a reporter enzyme. The second DNA construct includes a second promoter operatively linked to a second DNA molecule, the second DNA molecule comprising a sequence encoding a biologically active protein partner as a fusion protein to a second inactive mutant form of the reporter enzyme. The first and second inactive mutant forms of the reporter enzyme can interact upon formation of a complex between the first nuclear receptor and the protein partner to form an active reporter enzyme.

[0019] According to a fifth aspect of the invention, a solid support having deposited thereon a plurality of cells is provided wherein the cells express a first nuclear receptor as a fusion protein to a first inactive mutant form of a reporter enzyme and a protein partner as a fusion protein to a second inactive mutant form of the reporter enzyme. The first and second inactive mutant forms of the reporter enzyme can interact to form an active reporter enzyme upon the formation of a complex between the first nuclear receptor and the protein partner.

[0020] According to a sixth aspect of the invention, a method of detecting nuclear receptor interactions is provided. The method includes providing a cell that expresses a first nuclear receptor as a fusion protein to a first fragment of a reporter molecule and a protein partner as a fusion protein to a second fragment of the reporter molecule and determining the presence and/or amount of the reporter molecule. The first and second fragments of the reporter molecule independently have no reporter function. However, the first and second fragments of the reporter molecule can interact to restore reporter function upon formation of a complex between the first nuclear receptor and the protein partner. The presence of the reporter molecule indicates formation of a complex between the nuclear receptor and the protein partner. The reporter molecule can be an enzyme (e.g., a monomeric or multimeric enzyme), a fluorescent protein, a luminescent protein, or a phosphorescent protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The invention will be described with reference to the accompanying figures, wherein:

[0022] FIGS. 1A-1C illustrates a method of monitoring nuclear receptor dimerization according to the invention wherein a first nuclear receptor is fused to one complement enzyme fragment and a second NR is fused to a second complement enzyme fragment and wherein dimerization of the two NRs results in complementation of the enzyme fragments to produce an active enzyme complex;

[0023] FIGS. 2A-2C illustrate the use of complementation technology in the method of the invention wherein two inactive mutant reporter enzymes become active upon the interaction of a nuclear receptor fused to a first galactosidase fragment and a cofactor for the nuclear receptor fused to a second galactosidase fragment;

[0024] FIGS. 3A and 3B illustrate a method for determining ligands (e.g. ligand fishing) for orphan nuclear receptors byO-galactosidase mutant complementation wherein a test cell expressing two β-gal fusion proteins, (e.g., an orphan nuclear receptor fused to a first galactosidase fragment—NRorphan-Δα) and a known cofactor (e.g., ACTR fused to a second galactosidase fragment—ACTR-Δω), is subjected to treatments with samples containing ligands;

[0025] FIG. 4 illustrates Type I nuclear receptor complex formation (i.e., homodimerization) and gene expression in a cell wherein a ligand binds to the nuclear receptor in the cytoplasm activating the receptor for transport into the nucleus of the cell and binding to a hormone response element therein; and

[0026] FIG. 5 illustrates Type II nuclear receptor complex formation (i.e., heterodimerization) and gene expression in a cell wherein ligands for each of the nuclear receptors are transported into the nucleus of the cell and bind to each of the nuclear receptors in the cell nucleus before dimerization and bonding to a hormone response element therein.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention relates to protein-protein interaction assays for nuclear receptors. According to the invention, interactions between a nuclear receptor and a second protein can be monitored by reporter enzyme (e.g., β-galactosidase) complementation. The present invention provides a method to interrogate nuclear receptor function and pathways.

[0028] Only a fraction of nuclear receptors have been identified and even fewer have been associated with ligands (i.e., hormones). A ligand or hormone can be considered to be any small molecule that can bind to the receptor and affect its function. The means by which the identified orphan nuclear receptors and newly discovered orphan nuclear receptors will be associated with their cognate ligands and physiological functions represents a major challenge to biological and biomedical research.

[0029] The identification of an orphan nuclear receptor typically requires an individualized assay and a guess as to the function of the nuclear receptor. The present invention, however, involves the interrogation of nuclear receptor function by monitoring the activation of the receptor using activation dependent protein-protein interactions between the nuclear receptor and a second protein. The second protein can be a second nuclear receptor or a cofactor.

[0030] According to the invention, the specific protein-protein interactions can be measured using mutant enzyme complementation technology as described below. This assay system can eliminate guessing at the function of the nuclear receptor because it can be performed either with or without prior knowledge of other signaling events. Further, the assay system according to the invention is relatively sensitive and easy to perform. The assay system may also be applicable to nuclear receptors generically because many of the nuclear receptors are activated by a common mechanism. These generic mechanisms can be used, for example, to develop assays for orphan nuclear receptors.

[0031] According to the present invention, enzyme complementation technology can be used to monitor interactions between nuclear receptors and other proteins. Enzyme complementation technology is disclosed in U.S. application Ser. Nos. 09/654,499 (hereinafter the '499 application), filed Sep. 1, 2000, pending, and U.S. Pat. No. 6,342,345, issued Jan. 29, 2002 (hereinafter the '345 patent), both of which are incorporated herein by reference in their entirety.

[0032] As described in the '499 application and the '345 patent, enzyme complementation technology involves the use of two inactive enzyme (e.g., β-galactosidase) mutants, each of which is fused with one of two interacting target protein pairs. The formation of an active enzyme (e.g., β-galactosidase) complex is driven by the interaction of the target proteins. When the proteins of interest do not interact, the reporter enzyme remains inactive. When the proteins of interest do interact (e.g., when the proteins bind or dimerize), the reporter enzyme mutants come together and form an active enzyme.

[0033] Complementation techniques other than mutant enzyme complementation can also be used according to the invention. For example, protein fragment complementation assays can be used to measure the interactions between nuclear receptors and other proteins according to the invention. Protein fragment complementation assays are disclosed in U.S. Pat. Nos. 6,270,964; 6,294,330; and 6,428,951. Each of these patents is incorporated herein by reference in its entirety. Any of the protein fragment complementation assays disclosed in the aforementioned patents can be used to detect interactions between nuclear receptors and other proteins according to the invention.

[0034] According to a first aspect of the invention, a method of detecting nuclear receptor interactions using mutant enzyme complementation is provided. The method includes steps of: providing a cell that expresses a first nuclear receptor as a fusion protein to a first inactive mutant form of a reporter enzyme and a protein partner as a fusion protein to a second inactive mutant form of the reporter enzyme; and determining the presence and/or amount of the reporter enzyme. The first and second inactive mutant forms of the reporter enzyme can interact upon formation of a complex between the first nuclear receptor and the protein partner to form an active reporter enzyme. Reporter enzyme activity therefore indicates formation of a complex between the nuclear receptor and the protein partner. The protein partner according to the invention can be a wildtype protein or a mutant protein or any other regulatory protein, either known or unknown. For example, the protein partner can be a cofactor for the first nuclear receptor or a second nuclear receptor which can be the same or different than the first nuclear receptor. The co-factor can be a corepressor or a coactivator for the nuclear receptor. The cell can be a mammalian cell, a nematode cell, a yeast cell, a bacteria cell, or an insect cell.

[0035] According to a further aspect of the invention, a method of detecting nuclear receptor interactions is provided using protein fragment complementation. The method according to this aspect of the invention includes providing a cell that expresses a first nuclear receptor as a fusion protein to a first fragment of a reporter molecule and a protein partner as a fusion protein to a second fragment of the reporter molecule and determining the presence and/or amount of the reporter molecule. The first and second fragments of the reporter molecule independently have no reporter function. However, the first and second fragments of the reporter molecule can interact to restore reporter function upon formation of a complex between the first nuclear receptor and the protein partner. The presence of the reporter molecule indicates formation of a complex between the nuclear receptor and the protein partner. The reporter molecule can be an enzyme (e.g., a monomeric or multimeric enzyme), a fluorescent protein, a luminescent protein, or a phosphorescent protein.

[0036] According to a further aspect of the invention, interactions between nuclear receptors and protein partners can be detected and quantitated in vivo (i.e., within living cells) using the methods of the present invention. Vital enzyme substrates (e.g., vital β-gal substrates) which can be used in living cells are disclosed in U.S. Pat. No. 6,342,345. Any of these vital substrates can be used according to the invention for in vivo detection.

[0037] Interactions between nuclear receptors and protein partners can also be detected and quantitated in vitro according to the invention. For example, according to one embodiment of the invention, the method as set forth above can further include lysing the cells and incubating the cell lysate with a substrate which emits a detectable signal after cleavage by the reporter enzyme. The substrate can be a chemiluminescent, colorimetric or fluorescent substrate. According to a preferred embodiment of the invention, the substrate is a chemiluminescent 1,2-dioxetane substrate.

[0038] According to a further aspect of the invention, the cell can also contain a hormone response element operatively linked to a reporter gene such that binding of a nuclear receptor complex to the hormone response element results in expression of the reporter gene. According to this aspect of the invention, the method further includes a step of determining the presence and/or amount of a surrogate reporter protein encoded by the reporter gene. The presence and/or amount of the surrogate reporter protein is therefore an indication of the transcription-activating properties of the nuclear receptor/protein partner complex. The surrogate reporter protein can be a surrogate reporter enzyme (e.g., luciferase) which is different than the reporter enzyme. The method according to this aspect of the invention can further include steps of lysing the cells and incubating the cell lysate with first and second substrates wherein the first substrate emits a first detectable signal after cleavage by the reporter enzyme and the second substrate emits a second detectable signal different than the first detectable signal after cleavage by the surrogate reporter enzyme. The first and second substrates can be chemiluminescent or fluorescent substrates.

[0039] According to a further aspect of the invention, the method can also include a step of exposing the cell to a compound comprising one or more ligands wherein increased reporter enzyme activity indicates agonist activity of the compound and decreased reporter enzyme activity indicates inverse agonist or antagonist activity of the compound. According to this aspect of the invention, the first nuclear receptor can be an orphan nuclear receptor.

[0040] According to a further aspect of the invention, a DNA molecule comprising a sequence encoding a biologically active hybrid nuclear receptor is provided wherein the hybrid nuclear receptor comprises a nuclear receptor as a fusion protein to an inactive mutant form of a reporter enzyme. The inactive mutant form of the reporter enzyme can be a β-galactosidase mutant. A DNA construct capable of directing the expression of the biologically active hybrid nuclear receptor in a cell is also provided. The DNA construct comprises a promoter operatively linked to a DNA molecule as set forth above. The inactive mutant form of the reporter enzyme in the DNA construct can be a β-galactosidase mutant. A cell comprising a DNA construct as set forth above is also provided.

[0041] According to a further aspect of the invention, a DNA molecule comprising a sequence encoding a biologically active hybrid of a nuclear receptor cofactor is provided wherein the hybrid cofactor comprises a nuclear receptor cofactor as a fusion protein to an inactive mutant form of a reporter enzyme. The inactive mutant form of the reporter enzyme can be β-galactosidase mutant. A DNA construct capable of directing the expression of a biologically active hybrid nuclear receptor cofactor in a cell is also provided. The DNA construct comprises a promoter operatively linked to a DNA molecule as set forth above. The inactive mutant form of the reporter enzyme in the DNA construct can be a β-galactosidase mutant. A cell comprising a DNA construct as set forth above is also provided. According to a further embodiment of the invention, the nuclear receptor cofactor can be ACTR.

[0042] According to a further aspect of the invention, a cell comprising a first DNA construct capable of directing the expression of a first biologically active hybrid nuclear receptor in a cell and a second DNA construct capable of directing the expression of a biologically active protein partner in a cell is provided. The first DNA construct comprises a first promoter operatively linked to a first DNA molecule comprising a sequence encoding a first biologically active nuclear receptor as a fusion protein to a first inactive mutant form of a reporter enzyme. The second DNA construct comprises a second promoter operatively linked to a second DNA molecule comprising a sequence encoding a biologically active protein partner as a fusion protein to a second inactive mutant form of the reporter enzyme. The first and second inactive mutant forms of the reporter enzyme can interact upon formation of a complex between the first nuclear receptor and the protein partner to form an active reporter enzyme. The protein partner can be a cofactor for the first nuclear receptor or a second nuclear receptor which can be the same as or different than the first nuclear receptor.

[0043] According to a further aspect of the invention, a solid support having a plurality of cells deposited thereon is provided. According to this aspect of the invention, the cells express a first nuclear receptor as a fusion protein to a first inactive mutant form of a reporter enzyme and a protein partner as a fusion protein to a second inactive mutant form of the reporter enzyme. The first and second inactive mutant forms of the reporter enzyme can interact to form an active reporter enzyme upon the formation of a complex between the first nuclear receptor and the protein partner. According to this aspect of the invention, the cells can further comprise an enzyme substrate comprising an enzyme-labile group which, upon cleavage by the reporter enzyme, emits a detectable signal. The signal can be a calorimetric, fluorescent or chemiluminescent signal. The solid support can be made of glass, plastic, ceramic, semiconductor, silica, fiber optic, diamond, and bio-compatible materials (e.g., bio-compatible monomers or polymers).

[0044] According to a preferred embodiment of the invention, a first inactive β-galactosidase mutant is fused to a first nuclear receptor and a second β-galactosidase mutant is fused to second protein (i.e., protein partner). The second protein can interact with the first nuclear receptor. The first and second inactive β-galactosidase mutants can form an active enzyme when the first nuclear receptor and the second protein interact to form a complex. The second protein can be a second nuclear receptor (which may be the same or different than the first nuclear receptor) or a cofactor for the first nuclear receptor.

[0045] FIGS. 1A-1C illustrate the use of β-galactosidase complementation technology according to the invention wherein two inactive β-galactosidase mutants 2, 4 (e.g., Δα and Δω) become active 16 when the protein fusion partners of the two inactive β-galactosidase mutants 6, 8 interact to form a dimer 14. As shown in FIGS. 1A-1C, these protein fusion partners 6, 8 are first and second nuclear receptors (e.g., NR1 and NR2) each of which are activated by their respective ligands 10, 12. FIG. 1A shows protein fusion partners 6, 8 (i.e., the nuclear receptors) before ligand activation, FIG. 1B shows the nuclear receptors 6, 8 associated with their respective ligands 10, 12 and FIG. 1C shows receptors 6, 8 after formation of the dimer 14. According to the invention, the active β-galactosidase 16 resulting from the interaction of the two protein fusion partners 6, 8 can cleave an enzyme labile group on a chemiluminescent substrate to produce light.

[0046] The first and second nuclear receptors 6, 8 shown in FIGS. 1A-1C can be the same, in which case homo-dimer formation can be monitored. Alternatively, the first and second nuclear receptors 6, 8 can be different, in which case heterodimer formation can be monitored. As shown in FIGS. 1B and 1C, each of the nuclear receptors 6, 8 are associated with ligands 10, 12 respectively. Nuclear receptor dimer formation, which is illustrated in FIG. 1C, may be ligand activated in which case increased chemiluminescence can be observed if a ligand for the nuclear receptor is present. As indicated in FIGS. 1A-1C, agonist activity 15 promotes dimer formation whereas antagonist or inverse agonist activity 17 promotes monomer formation. According to the invention, the agonist or antagonist activity of various compounds on nuclear receptor complex formation can be monitored.

[0047] FIGS. 2A-2C illustrate the use of complementation technology according to the invention wherein two inactive β-galactosidase mutants 2, 20 (e.g., Δα and Δω) become active 24 when the protein fusion partners (i.e., a nuclear receptor and a co-factor) of the two inactive β-galactosidase mutants 6, 18 interact to form a dimer 22. In FIGS. 2A-2C, a nuclear receptor 6 is shown as a fusion protein with a first β-galactosidase mutant 2 (e.g., Δα), and a cofactor 18 (e.g., ACTR) is shown as a fusion protein with a second β-galactosidase mutant 20 (e.g., Δω). FIG. 2A shows the nuclear receptor 6 before activation by ligand 10, FIG. 2B shows the ligand activated receptor 6, and FIG. 2C shows the nuclear receptor-cofactor complex 22.

[0048] After enzyme complementation, enzyme activity according to the invention can be measured by enzyme activity assays according to techniques known in the art. Increased β-galactosidase activity can be an indication that the receptor and the cofactor have interacted to form a complex.

[0049] Nuclear receptor assays according to the invention can also be used to determine ligands for orphan nuclear receptors. This process is commonly referred to as a “de-orphaning” or a “ligand fishing” process. A procedure of this type is illustrated in FIGS. 3A and 3B wherein a P-galactosidase fusion protein 30 of an orphan receptor 6 (e.g., NRo-Δα) is co-expressed in a test cell 32 with a fusion protein 34 of a known cofactor 18 (e.g., ACTR-Δω) for the nuclear receptor. In FIGS. 3A and 3B, the β-galactosidase mutants fused to orphan receptor 6 and cofactor 18 are denoted by reference numerals 2 and 18 respectively.

[0050] As shown in FIG. 3B, when test cell 32 is subjected to compounds containing various ligands 36, including the ligand 38 for the receptor 6, ligand activation of the nuclear receptor 6 can result in the formation of a receptor-cofactor complex 39. According to the invention, formation of complex 39 can produce an increase in β-galactosidase 40 activity which can be used to indicate that the compound contains either a natural or surrogate ligand for the nuclear receptor.

[0051] The technique illustrated in FIGS. 3A and 3B can also be practiced with a second nuclear receptor capable of dimerizing with the orphan nuclear receptor rather than with a cofactor for the orphan nuclear receptor.

[0052] The above assay technique can be used to find and develop potential drugs for orphan nuclear receptors. For example, increased β-galactosidase activity in the test cell after treatment with a compound can indicate agonist activity of the compound. Alternatively, decreased β-galactosidase activity in the test cell can indicate antagonist activity or inverse agonist activity of the compound.

[0053] Also according to the invention, the complementation technology for measuring nuclear receptor complex formation can be combined with the use of a surrogate reporter (e.g., luciferase) for monitoring the effects of nuclear receptor complex formation on gene expression. Reporter gene assays are widely used in the art to measure the activity of a gene's promoter. This technique takes advantage of molecular biology techniques in which heterologous genes under the control of any promoter are introduced into the genome of a mammalian cell. See, for example, Gorman, et al., Mol. Cell Biol. 2, 1044-1051 (1982); and Alam et al., Anal. Biochem. 188, 245-254 (1990). Activation of the promoter induces the expression of the reporter gene. By design, the reporter gene codes for a reporter protein that can easily be detected and measured. The reporter protein is typically a reporter enzyme that can convert a substrate (e.g., a fluorescent or a chemiluminescent substrate) into a product. This conversion can be conveniently followed by direct optical measurement and can therefore allow for the quantification of the amount of reporter enzyme activity produced.

[0054] Gene expression according to the invention can also be monitored using other assay technology as known in the art. For example, mRNA can be quantified using a reverse transcription polymerase chain reaction assay. An assay of this type is the “TaqMan” assay which utilizes the 5′ nuclease activity of the DNA polymerase to hydrolyze a hybridization probe bound to its target amplicon. “TaqMan” is a registered trademark of Hoffman-La-Roche, Inc. The “TaqMan” assay and other methods of quantifying mRNA are reviewed in Bustin, Absolute Quantification of mRNA using Real Time Reverse Transcription Polymerase Chain Reaction Assays, J. of Mol. Endocrinology, 25, 169-193 (2000).

[0055] FIGS. 4 and 5 illustrate how both nuclear receptor complex formation (e.g., dimerization) and the downstream effects of complex formation on gene expression can be monitored in a recombinant cell line. In FIGS. 4 and 5, a β-galactosidase fusion protein of a nuclear receptor is co-expressed in a test cell with a fusion protein of a second receptor. In FIG. 4, the second receptor is the same as the first receptor whereas, in FIG. 5, the second receptor is different than the first receptor. The test cell can also contain a DNA sequence encoding a hormone response element operatively linked to a reporter gene. DNA sequences of this type are described in U.S. Pat. Nos. 5,071,773 and 5,298,429, which are hereby incorporated by reference in their entirety.

[0056] FIG. 4 illustrates dimerization and gene expression for Type I nuclear receptors. It is known that Type I ligands (e.g., cortisol, testosterone, etc.) and estrogen (i.e., estradiol) bind to their corresponding nuclear receptors in the cytosol. As shown in FIG. 4, a ligand 42 is transferred 44 through the plasma membrane 45 and into the cytoplasm 47 of a cell 41. As shown in FIG. 4, the cell 41 contains a nuclear receptor 50 associated with a heat shock protein 48. Once ligand 42 binds 46, 48 to nuclear receptor 50, nuclear receptor 50 dissociates 52 from heat shock protein 49 and becomes “activated” for binding to a hormone response element. The activated receptor can then move 54, 56 into the nucleus 43 of the cell 41. Once inside the nucleus 43, the activated receptor either dimerizes then binds 54, 57 or binds sequentially 56 to the corresponding hormone response element (HRE) 52. As a result, the transcription 58 of the particular DNA to which the dimer has bound can be regulated. The transcribed messenger RNA in the nucleus 43 can then move 59 into the cytosol 47 where it can be translated on ribosomes into a protein.

[0057] According to the invention, the protein-protein interactions 54, 56 can be used to produce a first signal 51 and gene expression 58, 59 can be used to produce a second signal 53. The second signal 53 can be generated from the expression of a reporter gene. In this manner, the nuclear receptor interactions and the downstream effects of these interactions on gene expression can be monitored.

[0058] FIG. 5 illustrates dimerization and gene expression for Type II nuclear receptors. Ligands for Type II nuclear receptors include vitamin D, thyroid hormones (T3) and retinoids (Vitamin A). Activation for a Type II receptor can lead to either homo- or hetero-dimerization (which is shown in FIG. 5) and then DNA binding. In the case of the retinoic acid receptor (RAR), for example, the DNA response element binds two receptors of the same type (homodimer) in the presence of all-trans retinoic acid ligand and represses DNA transcription. In the presence of T3 (e.g., triiodiothyronine), however, one receptor for T3 exchanges with one receptor of bound RAR to produce an RAR/T3 receptor heterodimer. This heterodimer turns on the DNA transcription. The effect an activated receptor has on its target DNA can be dependent upon a variety of factors including the relationship of its hormone response elements to other DNA elements as well as the transcription factors for that particular DNA.

[0059] As shown in FIG. 5, ligands 62, 66 move 61, 65 through plasma membrane 71 and cytoplasm 72 and into nucleus 74 of a cell 60 without binding any receptors. Once in the nucleus, ligands 62, 66 can activate receptors 64, 68. The ligand activated receptors can then either bind sequentially 77, 79 to the hormone response element 70 or dimerize first 73, 75 and then bind in dimer form 81 to hormone response element 70. As a result, the transcription of messenger RNA (i.e., mRNA) can be effected. As shown in FIG. 5, the transcribed messenger RNA in the nucleus can then move 85 into the cytosol 72 where it can be translated on ribosomes into a protein. According to the invention, the protein-protein interactions 73, 75 or 77, 79 can be used to produce a first signal 76 and gene expression 83, 85 can be used to produce a second signal 78. In this manner, both nuclear receptor interactions and the downstream effects of these interactions on gene expression can be monitored.

[0060] According to the invention, the transcribed mRNA can be assayed using various techniques known in the art. In this manner, the effects of protein-protein interactions on gene expression can be monitored. Alternatively, a reporter gene (e.g., luciferase) linked to a promoter (e.g., the regulatory sequences of a particular hormone response element) can be used to determine the effects of protein-protein interactions on gene expression. In this manner, the influence of protein-protein interactions on expression of the reporter gene can be related to expression of the gene of interest.

[0061] FIGS. 4 and 5 are merely intended to illustrate various factors that may be present and which may interact within the cell. These figures and the discussion thereof are not intended to limit the invention in any way.

[0062] According to the invention, β-galactosidase activity resulting from protein-protein interactions can be detected using an assay system wherein cell lysis is combined with chemiluminescent detection of β-galactosidase reporter enzyme activity. The assay system according to the invention can, for example, employ a chemiluminescent substrate such as a 1,2-dioxetane compound having an enzyme labile substituent and one or more stabilizing groups. Chemiluminescent substrates of this type are disclosed, for example, in U.S. Pat. Nos. 5,851,771; 5,538,847; 5,326,882; 5,145,772; 4,978,614 and 4,931,569, the contents of each of which are incorporated herein by reference in their entirety. Any of the chemiluminescent substrates disclosed in the aforementioned references can be used in assays according to the invention. Other chemiluminescent substrates can also be used. Any substrate which emits light upon enzyme cleavage can be used according to the invention. According to a preferred embodiment of the invention, the chemiluminescent substrate is a 1,2-dioxetane having an adamantyl stabilizing group. A material of this type is available under the trademark Galacton-Star®, which is a registered trademark of Applera Corporation or its subsidiaries.

[0063] Light output resulting from enzymatic activity according to the invention can be measured in a luminometer to thereby provide a measure of nuclear receptor complex formation as well as the effects of complex formation on gene expression. For example, β-galactosidase reporter enzyme activity can provide a measure of nuclear receptor complex formation whereas the enzyme activity of a surrogate reporter (e.g., luciferase) can provide a measure of gene expression. Light output according to the invention can also be measured using a scintillation counter or any other known light measuring device.

[0064] An assay according to the invention can be performed by incubating the cell lysate with a reaction buffer containing the chemiluminescent substrate and, optionally, a chemiluminescent substrate enhancer. Any known chemiluminescent substrate enhancer can be used according to the invention. Incubation can be conducted until maximum light emission is reached at which point light output can be measured. An assay system of this type is available under the trademark Galacto-Star™, which is a trademark of Applera Corporation or its subsidiaries. Other assay systems and techniques, however, can also be used according to the invention.

[0065] As set forth above, the invention is achieved in part by using protein/protein interaction screening to map signaling pathways. This technology can be used with known and unknown nuclear receptors having diverse functions including orphan nuclear receptors wherein the natural ligand to the receptor has not been identified.

[0066] Use of galactosidase complementation technology provides many benefits to the nuclear receptor screening process, including the ability to monitor protein interactions in any sub-cellular compartment membrane (e.g., cytosol or nucleus). Moreover, the present invention provides nuclear receptor binding assays that can be achieved directly within the cellular environment in a rapid, non-radioactive assay format. The assay techniques of the present invention can therefore provide a more physiologically relevant model without the need for protein overexpression. The assay system of the invention can also provide a cell-based method for interrogating nuclear receptor pathways which is amenable to high-throughput screening (HTS).

[0067] The present invention has numerous additional advantages. First, it is applicable to a variety of cells including mammalian cells, nematode cells, yeast cells, bacterial cells and insect cells. Second, it can detect interactions in the cytosol or nucleus of a cell. Also, it does not rely on indirect read-outs such as transcriptional activation but, rather, allows interactions between a nuclear receptor and a second protein to be directly monitored. The present invention can thus provide assays with a high degree of physiological relevance.

[0068] The methods of the present invention can be used to determine the effects of mutations on the interactions between a nuclear receptor and a second protein. For example, the effect of single nucleotide polymorphisms (i.e., SNPs), such as coding SNPs, on interactions between nuclear receptors and other proteins can be determined according to the invention. Coding SNPs are SNPs which alter the sequence of the protein encoded by the altered or mutated gene. According to the invention, interactions between SNPs of nuclear receptors and protein partners (e.g., cofactors) can be determined. Alternatively, interactions between nuclear receptors and SNPs of protein partners (e.g., cofactors) can also be determined according to the invention. Additionally, the effect of various mutations of nuclear receptors and/or protein partners (e.g., cofactors) on the interaction between the two proteins can be determined according to the invention.

[0069] The assays of this invention, and their application and preparation have been described both generically and by specific example. The examples, however, are not intended to be limiting. Other embodiments will occur to those of ordinary skill in the art without the exercise of inventive faculty. Such modifications remain within the scope of the invention.

Claims

1. A method of detecting nuclear receptor interactions, the method comprising: providing a cell that expresses: a first nuclear receptor as a fusion protein to a first inactive mutant form of a reporter enzyme; and a protein partner as a fusion protein to a second inactive mutant form of the reporter enzyme, wherein the first and second inactive mutant forms of the reporter enzyme interact upon formation of a complex between the first nuclear receptor and the protein partner to form an active reporter enzyme; and determining the presence and/or amount of the active reporter enzyme; wherein reporter enzyme activity indicates formation of a complex between the nuclear receptor and the protein partner.

2. The method of claim 1, wherein the protein partner is a cofactor for the first nuclear receptor.

3. The method of claim 2, wherein the cofactor is a corepressor or a coactivator.

4. The method of claim 2, wherein the cofactor is a wildtype protein or a mutant protein.

5. The method of claim 2, wherein reporter enzyme activity is detected in a living cell.

6. The method of claim 1, wherein the protein partner is a second nuclear receptor.

7. The method of claim 6, wherein the second nuclear receptor is the same as the first nuclear receptor.

8. The method of claim 6, wherein the second nuclear receptor is different than the first nuclear receptor.

9. The method of claim 1, wherein the cell also contains a hormone response element operatively linked to a reporter gene such that binding of the nuclear receptor/protein partner complex to the hormone response element results in expression of the reporter gene, the method further comprising: determining the presence and/or amount of a surrogate reporter protein encoded by the reporter gene; wherein the presence and/or amount of the surrogate reporter protein is an indication of the transcription-activating properties of the nuclear receptor/protein partner complex.

10. The method of claim 9, wherein the surrogate reporter protein is a surrogate reporter enzyme which is different than the reporter enzyme.

11. The method of claim 10, wherein the surrogate reporter enzyme is luciferase.

12. The method of claim 1, further comprising: exposing the cell to a compound comprising one or more ligands; wherein increased reporter enzyme activity indicates agonist activity of the compound and decreased reporter enzyme activity indicates inverse agonist or antagonist activity of the compound.

13. The method of claim 12, wherein the first nuclear receptor is an orphan nuclear receptor.

14. The method of claim 1, further comprising: lysing the cells; and incubating the cell lysate with a substrate, wherein the substrate emits a detectable signal after cleavage by the reporter enzyme.

15. The method of claim 14, wherein the substrate is a chemiluminescent or fluorescent substrate.

16. The method of claim 14, wherein the substrate is a chemiluminescent 1,2-dioxetane substrate.

17. The method of claim 10, further comprising: lysing the cells; and incubating the cell lysate with first and second substrates, wherein the first substrate emits a first detectable signal after cleavage by the reporter enzyme and wherein the second substrate emits a second detectable signal different than the first detectable signal after cleavage by the surrogate reporter enzyme.

18. The method of claim 17, wherein the first and second substrates are independently selected from the group consisting of chemiluminescent and fluorescent substrates.

19. The method of claim 1, wherein the cell is selected from the group consisting of mammalian cells, nematode cells, insect cells, yeast cells and bacteria cells.

20. A DNA molecule comprising a sequence encoding a biologically active hybrid nuclear receptor, wherein the hybrid nuclear receptor comprises a nuclear receptor as a fusion protein to an inactive mutant form of a reporter enzyme.

21. The DNA molecule of claim 20, wherein the inactive mutant form of the reporter enzyme is a β-galactosidase mutant.

22. A DNA construct capable of directing the expression of a biologically active hybrid nuclear receptor in a cell, the DNA construct comprising the following operatively linked elements: a promoter; and the DNA molecule of claim 20.

23. The DNA construct of claim 22, wherein the inactive mutant form of the reporter enzyme is a β-galactosidase mutant.

24. A cell comprising the DNA construct of claim 22.

25. A cell comprising the DNA construct of claim 23.

26. A DNA molecule comprising a sequence encoding a biologically active hybrid of a nuclear receptor cofactor as a fusion protein to an inactive mutant form of a reporter enzyme.

27. The DNA molecule of claim 26, wherein the inactive mutant form of the reporter enzyme is a β-galactosidase mutant.

28. A DNA construct capable of directing the expression of a biologically active hybrid of a nuclear receptor cofactor in a cell, comprising the following operatively linked elements: a promoter; and the DNA molecule of claim 26.

29. The DNA construct of claim 28, wherein the inactive mutant form of the reporter enzyme is a β-galactosidase mutant.

30. A cell comprising the DNA construct of claim 28.

31. A cell comprising the DNA construct of claim 29.

32. The DNA molecule of claim 26, wherein the nuclear receptor cofactor is ACTR.

33. A cell comprising: a first DNA construct capable of directing the expression of a first biologically active hybrid nuclear receptor in a cell, the first DNA construct comprising a first promoter operatively linked to a first DNA molecule, the first DNA molecule comprising a sequence encoding a first biologically active nuclear receptor as a fusion protein to a first inactive mutant form of a reporter enzyme; and a second DNA construct capable of directing the expression of a biologically active protein partner in a cell, the second DNA construct comprising a second promoter operatively linked to a second DNA molecule, the second DNA molecule comprising a sequence encoding a biologically active protein partner as a fusion protein to a second inactive mutant form of the reporter enzyme; wherein the first and second inactive mutant forms of the reporter enzyme interact upon formation of a complex between the first nuclear receptor and the protein partner to form an active reporter enzyme.

34. The cell of claim 33, wherein the protein partner is a cofactor for the first nuclear receptor.

35. The cell of claim 33, wherein the protein partner is a second nuclear receptor.

36. The cell of claim 35, wherein the second nuclear receptor is the same as the first nuclear receptor.

37. The cell of claim 35, wherein the second nuclear receptor is different than the first nuclear receptor.

38. A solid support having deposited thereon a plurality of cells, wherein the cells express: a first nuclear receptor as a fusion protein to a first inactive mutant form of a reporter enzyme; and a protein partner as a fusion protein to a second inactive mutant form of the reporter enzyme; wherein first and second inactive mutant forms of the reporter enzyme interact to form an active reporter enzyme upon the formation of a complex between the first nuclear receptor and the protein partner.

39. A solid support according to claim 38, wherein the cells comprise an enzyme substrate comprising an enzyme-labile chemical group which, upon cleavage by the reporter enzyme, releases a product measurable by colorimetry, fluorescence or chemiluminescence.

40. A solid support according to claim 38, wherein the solid support is made of a material selected from the group consisting of glass, plastic, ceramic, semiconductor, silica, fiber optic, diamond, bio-compatible monomers and biocompatible polymer materials.

41. A method of detecting nuclear receptor interactions, the method comprising: providing a cell that expresses: a first nuclear receptor as a fusion protein to a first fragment of a reporter molecule; and a protein partner as a fusion protein to a second fragment of the reporter molecule, wherein the first and second fragments of the reporter molecule independently have no reporter function and wherein the first and second fragments of the reporter molecule interact to restore reporter function upon formation of a complex between the first nuclear receptor and the protein partner; and determining the presence and/or amount of the reporter molecule; wherein the presence of the reporter molecule indicates formation of a complex between the nuclear receptor and the protein partner.

42. The method of claim 41, wherein the reporter molecule is selected from the group consisting of a monomeric enzyme, a multimeric enzyme, a fluorescent protein, a luminescent protein, and a phosphorescent protein.