# Patent

## Title
Assays for nuclear hormone receptor binding

## Abstract

Methods and genetic constructs are provided for detecting the binding of nuclear hormone receptors to a coactivator/corepressor. The methods employ enzyme fragment complementation using fragments of β-galactosidase as the detection system. Cells are transformed to express the large fragment of β-galactosidase fused to a member of the complex with NHR for initiation of transcription and have it localized in the nucleus and to express the small fragment of β-galactosidase fused to the nuclear hormone receptor for binding to the member upon stimulation with a ligand.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. Provisional Patent Application No. 61/077,686, filed on Jul. 2, 2008, which is hereby incorporated by reference in its entirety.


STATEMENT OF GOVERNMENTAL SUPPORT
None.
REFERENCE TO SEQUENCE LISTING
Applicants assert that the paper copy of the Sequence Listing is identical to the Sequence Listing in computer readable form found on the accompanying computer file. Applicants hereby incorporate the contents of the sequence listing by reference in its entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The field of this invention is the determination of the effect of changes of the environment of a cell on the binding of nuclear hormone receptors to form transcriptional regulatory proteins.
2. Related Art
Nuclear hormone receptors (“NHRs”) are a significant group of proteins that act as transcription factors when bound to an agonist. The natural agonists are lipophilic compounds such as steroids. Nuclear hormone receptors, more commonly referred to as nuclear receptors, define a family of ligand activated transcription factors (Tenbaum et al, Int J Biochem Cell Biol, 29:1325-41 (1997); Willson et al, Mol Endocrinol, 16:1135-44 (2002)). Structurally, they are characterized by the presence of modular domains: a zinc-finger DNA binding domain, a ligand binding domain and two transcriptional activation domains AF-1 and AF-2, ligand-independent and ligand-dependent, respectively. Depending upon the nuclear receptor, monomers or dimers (homodimers or heterodimers with other nuclear receptors) constitute the functional effectors. This gene family regulates a wide variety of physiological functions and has thus a broad therapeutic potential ranging from metabolic, endocrinological diseases to neurological disorders, to cancer.
Nuclear receptors operate by recruiting an array of auxiliary polypeptides, denoted corepressors and coactivators (further described in Mol. Endocrinol. 1996 October; 10(10):1167-77, incorporated by reference to further define the properties of these putative coregulatory factors), and it is these auxiliary proteins that mediate the molecular events that result in transcriptional repression or activation. For most nuclear receptors, this recruitment event is initiated upon the binding of the nuclear receptor to a ligand. It can be envisioned that certain ligands can only trigger the recruitment of a particular set of coactivators or corepressors and thus promote very selective effects. Furthermore, phosphorylation/dephosphorylation events can also affect the activity of the nuclear receptor itself and/or the auxiliary proteins. Similarly, it is plausible to assume that certain ligands exclusively responsive to such modifications could be identified. Generally speaking, these selective modulators would be of tremendous interest from a therapeutic standpoint, exhibiting maximized therapeutic value and minimum adverse effects.
Nuclear hormone receptors have been divided into four types: (I) Exist in the cytosol and upon ligand binding dissociates from heat shock proteins, homodimerizes, translocates to the nucleus, and binds to hormone response elements (“HRE”) of the chromosome; (II) Exist in the nucleus, binds to the HRE as heterodimers (usually with RXR), complexed with corepressors, upon binding with ligand dissociates from corepressors and recruits coactivators; (III) Similar to Type 1 binding to DNA as a homodimer; (IV) Exists as monomers or dimers and upon ligand binding recruits coactivators. The heat shock protein family is further defined in J Biosci Bioeng. 2008 October; 106(4):324-36, incorporated herein by reference to further describe the heat shock protein family.
The first step in the characterization of ligand interaction with a cloned receptor is to express the receptor in a ligand sensitive form. While a few receptors can be expressed in easily manipulated model systems such as yeast and E. coli, the interactions of ligands with most receptors are influenced by post-translational modifications that are only present in mammalian cells. Moreover, many of these receptors require mammalian proteins to accurately transduce their biological effects. Thus for wide applicability, an assay system is best when it is based on cloned receptors expressed in a mammalian system.
Historically, the ability of ligands to interact with nuclear receptors has been evaluated by competition with a radiolabeled ligand for a binding site on the receptor. Such assays are popular because they involve relatively few steps. However binding assays have many limitations: (i) for many technical reasons, binding assays are performed in non-physiological conditions which can influence receptor pharmacology; (ii) agonists and antagonists cannot be reliably discriminated; (iii) only binding sites for which radiolabeled ligands are available can be studied; (iv) binding assays are not easily applicable to orphan receptors for which ligands haven't yet been identified; (v) purchase, handling and disposal of radioisotopes are major expenses; (vi) local governments are concerned about contamination; and (vii) the industry has primarily looked to assays where the result can be detected optically for high throughput screening.
In order to develop such modulating ligands, it is essential to have an effective screening assay. Such assays act to winnow the large number of compounds that are tested to provide a small class that is then tested in slower, less economical, screens. The initial screening should lend itself to robotics, have few false positives, require a minimum of steps in its protocols, and allow for sensitive determination. Over the years, a large number of systems, methods and reagents, have been developed for screening compounds for their effect on cellular proteins. One group of systems involves employing fused proteins, fusing the protein of interest to a detectable polyamide label.
In developing these assays, one cannot predict the effect of the fusion to the label on the functioning of the target protein. There can be disruption as to folding, interactions between the label and the target protein, effects on translocation and binding to other intracellular proteins, interference with the detection of the label, and the like. There is also the problem of sensitivity in that one should be able to detect at the concentration of the EC50, which is generally below about 1 μM. One of the attractive systems involves enzyme fragment complementation, generally employing fragments of β-galactosidase, where the fragments may complex independently to form an active enzyme or require being fused to auxiliary binding proteins which complex, bringing the fragments together to form an active enzyme. The degree to which the fragments independently complex without the presence of the auxiliary binding proteins can substantially affect the success of the assay. In the case of NHRs, how one organizes the interaction of the fusion protein, the complexing of the members of the NHR transcription factor, and the coactivator can be crucial in providing an effective assay.
There is a substantial need for the development of assays for screening ligands for nuclear hormone receptors that are accurate, utilize the full length protein, are applicable to orphan targets, are dependable, and for which the operators have familiarity and for which there is a substantial history of know-how and show-how for acceptance and adoption of the assays.
U.S. patent applications and patents of interest include 2007/0218456; 2006/0134670; 2006/0035813; 2005/0130232; 2003/0077664; and U.S. Pat. No. 5,846,711. Scientific articles of interest include: Beck, et al 2008 Anal Biochem 373, 263-71; and Le Guevel and Pakdel 2001 Biotechniques 30, 1000-4.
BRIEF SUMMARY OF THE INVENTION
Methods and reagents are capable of being used for high throughput screening employing enzyme fragment complementation with β-galactosidase as the enzyme. Genetic constructs are employed for a first fusion protein of the small β-galactosidase fragment with the nuclear hormone receptor (“NHR”) and for a second fusion protein of the large β-galactosidase fragment and a second molecule that forms a complex with the NHR (usually a coactivator) to form a transcriptionally active complex. The second moiety may be a second NHR member, heat shock protein or the coactivator/corepressor sequence that becomes localized to the nucleus. Mammalian cells are transformed with the constructs and the resulting mutant cells used in the assay protocol in the presence of a candidate ligand. The assays are found to be highly sensitive and capable of distinguishing ligand binding to the NHR receptor member.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a cartoon of the assay mechanism; it shows the NHR PathHunter assays using protein-protein interaction strategy. SRC is the steroid receptor coactivator. The triangle represents the agonist. The activated NHR translocates to the nucleus, as shown in the figure at 100. The activated NHR-PK interacts with SCR 1EA in the nucleus of the cell at 110 to generate enzyme complementation by combining ED and EA.
FIG. 2A is a graph of the results of the subject assay for ERRα (estrogen-related receptor α). It should be noted that ERRα is constitutively active and resides in the nucleus, so the assay is for an inverse agonist and is a loss of signal assay. It shows excellent performance for the CHO ERRα cell line. The data represents: stability to 20 passages; S/B=3 fold; EC50=300 nM; accurate pharmacology for inverse agonist. The ERRα is a constitutively active NHR and will reside in the nucleus under normal physiological conditions, making this a loss of signal assay. FIG. 2B is a photograph of a blot showing increased bands at the expected Mr of the expressed protein.
FIG. 3A is a graph of the results of the subject assay for THRα (thyroid hormone receptor α); it shows excellent performance for the THRα cell line. These data show: stability to passage 20; S/B=78 fold; EC50=15 nM and accurate pharmacology for the agonist. Shorter incubation times can be used. However, this results in higher EC50s and lower induction ratios. The cell conditioning involved first, the cells were grown in complete medium; second, they were washed in PBS twice; third, they were incubated in serum free medium overnight. FIG. 3B is a blot as in FIG. 2B.
FIG. 4A is a graph of the results of the subject assay for RARα (retinoic acid receptor α); it shows excellent performance for the CHO RARα cell line. These data show: stability to passage 20; S/B=3 fold; EC50=29 nM and accurate pharmacology for the agonist. The cell conditioning involved first, the cells were grown in complete medium; second, they were washed in PBS twice; third, they were incubated in serum free medium overnight. FIG. 4B is a blot as in FIG. 2B.
FIG. 5A is a graph of the results of the subject assay for RARβ (retinoic acid receptor β); it shows excellent performance for the CHO PGC1 RARβ cell line. These data show: stability to passage 20; S/B=3 fold; EC50=29 nM and accurate pharmacology for the agonist. The cell conditioning involved first, the cells were grown in complete medium; second, they were washed in PBS twice; third, they were incubated in serum free medium overnight. FIG. 5B is a blot as in FIG. 2B.



DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Ligands are capable of binding to NHRs and initiating a change in the binding of the NHR to other proteins, usually a second transcriptional co-activator. Generally, agonists will provide for recruitment of a second component, namely a coactivator peptide or another protein member of the transcription factor, e.g., a second NHR to form a homodimer or a different protein to form a heterodimer or binding of the agonist can release a co-repressor that is bound to the NHR. Alternatively, an antagonist will recruit a corepressor to the NHR to inhibit initiation of transcription, block binding of the agonist to the NHR or alter the dissociation of the corepressor. Candidate compounds are screened using enzyme fragment complementation as the system for detection. Genetic constructs are prepared encoding for fusion proteins under the regulatory control of transcriptional and translational genetic signals for expression of the fusion protein in a mammalian host cell. The cells are grown to a predetermined density with expression of the fusion proteins and then used in an assay medium with the candidate ligand. The turnover of the substrate by the functional enzyme resulting from the complexing of the fragments is determined optically. Further description of NHRs and their binding partners may be found at the dissertation, “Regulation of Nuclear Hormone Receptors by Corepressors and Coactivators: a Dissertation,” Xiaoyang Wu, University of Massachusetts Medical School, available on the internet at hypertext transfer protocol colon slash slash escholarship.umassmed. edu/gsbs_diss/106/, which is hereby incorporated by reference in its entirety. The genetic constructs are a first construct that fuses the NHR of interest to one of the fragments of β-galactosidase, conveniently the small fragment of β-galactosidase (“ED”, referred to as “PK” when a low affinity mutant of ED is employed). One may therefore have the wild-type sequence of the bacterial β-galactosidase, e.g., E. coli, or the enzyme from a different source or a functional mutant of the wild-type sequence. There are two possibilities: a short small fragment that weakly binds to the large fragment of β-galactosidase or a mutant that weakly binds to the large fragment of β-galactosidase; or longer small fragment that has a much higher affinity for the large fragment than the short small fragment. A mutant ED fragment referred to as PK has the point mutation H31R and is of 42 amino acids. By weakly binding is intended that when ED is co-expressed with the large fragment of β-galactosidase (“EA”) under assay conditions, the activity will be less than about 50% of the activity with a long small fragment of at least about 90 amino acids of the N-terminal portion of β-galactosidase in the absence of protein-protein interaction.
The second construct is a fusion protein of the large β-galactosidase fragment (“EA”) and a second component of the transcription factor that is recruited upon binding of a ligand. Conveniently, a steroid receptor coactivator/corepressor, usually coactivator, can be employed. Alternatively, one may use another polypeptide, such as a heat shock protein or the other member of the NHR dimer, which may include the NHR AF1 or a different protein, such as AF2, RXR, etc.
Either a short small fragment of about 36 to 60, usually 36 to 50 and more usually 36 to 45 amino acids of the N-proximal portion of β-galactosidase or a longer small fragment of at least 50, more usually at least about 60, and not more than about 110, usually not more than about 100 amino acids of the N-proximal portion of β-galactosidase is used. The short small fragment (or mutant) is desirable in providing lower background, but also results in lower signal. However, the background signal to noise ratio is usually better than the results with the long short fragment. The long short fragment is useful when the binding of the SRC to the NHR is weak, since the long short fragment has a higher affinity for the EA than the mutant ED and the overall binding between the two constructs will be increased. For the most part, the short small (mutant) fragment will be preferred.
Any NHR may be assayed whether located in the cytosol, nucleus or both. Until the NHR is activated it will not alter its association with the coactivator/corepressor to provide for formation of a functional β-galactosidase. NHRs include estrogen receptor-α and −β, progesterone receptor-α and -β, glucocorticoid receptor, mineralocorticoid receptor, thyroid hormone receptor-α and -β; retinoic acid receptor-α, -β and -γ; peroxisome proliferator-activated receptor-α, -β/δ, and γ; Rev-ErbAα and -β; RAR-related orphan receptor-α, -β and -γ; liver X receptor-α, and -β; farnesoid X receptor; vitamin D receptor; pregnane X receptor; constitutive androstane receptor; etc. Further descriptions of NHRs may be found for example in US 2005/0130232 A1, US 2004/0132038 A1, etc., which are hereby incorporated by reference in their entirety. NHR sequences may be found in NuReBase or at NURSA.org. Standard designations are used herein. For example, RARα is designated by the official symbol RARA; RARβ is designated by the official symbol RARβ; ERR α is designated by the official symbol ESRA; THRα is designated by the official symbol THRA; THRβ is designated by the official symbol THRB; PPARγ is designated by the official symbol PPARG; Retinoid X receptor γ (RXR) is designated by the official symbol RXRG; ESR1a designates Estrogen receptor (ESR1); ERa designates the a isoform and ERb the b isoform of the estrogen receptor; PRa designates progesterone receptor (Pgr) α; PRb designates progesterone receptor (Pgr) β; NURR1 designates Nur-related protein 1; PPARa designates peroxisome proliferator activated receptor alpha, PPARd designates peroxisome proliferator activated receptor delta; LXRa, and LXRb designate oxysterol receptor α and β, respectively.
All information on accessing and installing NUREBASE may be found at world wide web ens-lyon.fr/LBMC/laudet/nurebase/nurebase.html.
Illustrative of fusion of the small fragment of β-galactosidase, see, for example, U.S. Pat. No. 7,135,325, hereby incorporated by reference in its entirety.
The small fragment may be linked directly to a terminus, usually the N-terminus of the protein, or may be linked by up to about 20 amino acids. The amino acids for the linking group will be chosen to be non-interfering and will be primarily a matter of convenience, for example, in constructing the genetic sequence.
The large fragment will be fused to the other binding component of the NHR complex, usually at the N-terminus, conveniently the steroid receptor coactivator/corepressor, usually a coactivator (exemplified as “SRC”), or the other protein that forms the dimer. Known coactivators include C2-2, (see Thompson US 2003/0039980, hereby incorporated by reference in its entirety), TERHKILHRLLQEGST (SEQ ID NO:1), GPQTPQAQQKSLLQQLLTE (SEQ ID NO:2), TRAP220 (Thyroid Hormone Receptor-associated Protein, see J Biol Chem, Vol. 274, Issue 10, 6667-6677, Mar. 5, 1999, hereby incorporated by reference), NTKNHPMLMNLLKDNPAQD (SEQ ID NO:3), d-22, LPYEGSLLLKLLRAPVEEV (SEQ ID NO:4), EAEEPSLLKKLLAPANTQ (SEQ ID NO:5), and pgc1-α. In addition to the SRC (coactivator) there will also be nuclear localization and nuclear retention signals, so that the EA resides in the nucleus. Agonist binding to the first fusion protein will result in binding of the SRC to the NHR and concomitant complex formation to produce a functional β-galactosidase enzyme. Alternatively, antagonist binding will result in the recruitment of a corepressor or blockade of agonist binding.
For the preparation of the fusion protein and its expression construct, conventional splicing and insertion techniques are employed. The ED (PK) will usually be joined at its C-terminus. The ED (PK) will come from the N-terminus proximal region of the β-galactosidase enzyme.
The fusion proteins provide a functional protein that is soluble, does not aggregate so as to be unavailable for complexing, has substantially the natural folding, so as to be susceptible to binding to endogenous proteins that normally complex to the polypeptide fused to the ED, and will usually be able to perform substantially the same functions that such polypeptide performs. Therefore, the polypeptide is capable of acting as a surrogate for the natural protein to allow for measurements that are predictive of the activity of the natural protein.
The ED may be joined to the coding region in a variety of ways. For a cDNA gene, one may select a suitable restriction site for insertion of the sequence, where by using overhangs at the restriction site, the orientation is provided in the correct direction.
Various conventional ways for inserting encoding sequences into a gene can be employed. For expression constructs and decryptions of other conventional manipulative processes, See, e.g., Sambrook, Fritsch & Maniatis, “Molecular Cloning: A Laboratory Manual,” Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook et al., 1989”); “DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover ed. 1985); “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic Acid Hybridization” [B. D. Hames & S. J. Higgins EDs. (1985)]; “Transcription And Translation” [B. D. Hames & S. J. Higgins, EDs. (1984)]; “Animal Cell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning” (1984), which are incorporated by reference herein.
Transformed cells are cloned that have various expression levels of the two fusion proteins. The best clone is then commonly chosen by lowest EC50 and best signal to background ratio. The cells are transformed by conventional methods. Methods include transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, using a viral vector, use of a carrier molecule, with a DNA vector transporter, and the like. For permanent insertion into the genome, various techniques are available for the insertion of the sequence in a homologous or non-homologous fashion. These techniques are well known. For random insertion, the introduction of the nucleic acid by any of the above methods will usually be sufficient. For homologous recombination, see, for example, U.S. Pat. Nos. 7,361,641, 5,578,461, 5,272,071 and PCT/US92/09627, incorporated by reference in their entirety and references cited therein, also hereby incorporated by reference.
Any eukaryotic cell may be employed, for the most part cell lines being employed. The cell lines will usually be mammalian, but for some purposes unicellular organisms or cells from non-vertebrates can be used. Mammalian cell lines include CHO, HeLa, HT1080, U2OS, MMTV, HepG2, HEK, and the like. Various vectors that are commercially available can be used successfully to introduce the two expression constructs into the eukaryotic cell. For an extensive description of cell lines, vectors, methods of genetic modification, and expression constructs, see published U.S. patent application serial no. 2003/0092070, Zhao, et al., May 15, 2003, paragraphs 00046-00066, which are specifically incorporated herein by reference.
Regulatory regions that may be used will be functional in the cell and may be obtained from cellular or viral genes. Illustrative regulatory regions include many promoters that are commercially available today. Expression of the fusion protein may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host or host cell selected for expression. Promoters which may be used to control fusion gene expression include, but are not limited to, the SV40 early promoter region (Benoist and Chambon, 1981, Nature, 290:304-310), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell, 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature, 296:39-42), etc., all incorporated herein by reference.
The screening method involves growing the cells in an appropriate medium and then washing the cells with an appropriate buffered aqueous solution, e.g., PBS, to remove any hormones. The cells are then incubated in low serum or serum-free medium, usually at least about 12 h, preferably at least about 24 h, where shorter times appear to degrade performance in some cases. Following the incubation, the cells are seeded in a medium in an appropriate environment in a small volume, followed by providing the desired stimulus, e.g., candidate compound, to provide the assay sample. The volume will generally not exceed about 250 μl, usually not more than about 200 μl, and generally be at least about 10 μl, more usually at least about 20 μl, where the volume of the candidate compound solution addition will generally dilute the cell medium less than about 1:1, usually not more than about 0.5:1. When the reagent is dry, there will be no dilution. After incubating the assay sample for sufficient time for the event of interest to occur, generally from about 1 h to about 0.5 day, a reagent solution for lysis of the cells and containing a detectable β-galactosidase substrate is added to the assay sample and one or more readings taken of the product from the substrate. The ratio of dilution will be not more than about 1:2, usually in the ratio of about 1:0.25 to 1:2, more usually 1:1 and as little at 1:0.25 or less. This dilution factor allows for reduced formation of complex during the reading period, while allowing for a robust signal, providing at least a three-fold, usually at least a 10-fold of ratio of signal to background during the period of the reading. One or more readings will be taken within 150 min, more usually within 120 min, preferably within about 60 min, and usually after about 10 min, more usually after about 15 min.
The primary interest is binding of the NHR to a second component, e.g., the coactivator/corepressor, involved in the ability of the NHR to initiate transcription. In this assay, little, if any, formation of the active enzyme occurs without there being binding of the two fusion proteins. The ultimate goal is to find ligands that can modulate cell metabolism/function through binding to the NHR.
The transformed cells to be used in the assay will be treated conventionally, generally being grown in a complete medium, washed twice with PBS and then incubated in serum-free medium overnight. The media will be conventional for the particular cells used; F-12 for CHO cells, modified Eagle's media for U20S cells, standard DMEM for HEK cells etc. The cells are then seeded in media containing fetal calf serum, conveniently media treated with charcoal/dextran to remove steroids that may be present in the fetal calf serum. The cells for use in the assay will be grown in accordance with the nature of the cells. For the most part, cells will be grown in wells in microtiter plates, the number of wells generally ranging from about 96 to 3456, generally being from 96 to 384 wells, or analyzed in suspension using capillary flow or flow cytometry, or they will be analyzed optically. The bottom will generally be clear, so that readings may be taken from the bottom of the wells, however opaque plates with instruments reading from the top can be used. The number of cells plated in a well will generally range from about 102 to 104 cells. The volume of the medium will usually be in the range of about 10 to 200 μl. The cells are then allowed to adhere overnight using conventional conditions of 37° C./5% CO2.
After sufficient time for the stimulation of the cells to take effect from the candidate ligand and the NHR protein, substrate is provided and the turnover of the substrate is determined, where the substrate provides a detectable product. If a substrate that can permeate the cell membrane and produce a product that is not leaky, a reagent solution is added for permeabilization or lysis of the cells. It is found that the formed enzyme complex is retained, the potential for new complex to form as a result of the permeabilizing of the cells is inhibited and the background from other than complex formed from the complexing of the fusion proteins as a result of binding of the second protein is minimal. In this way a robust response to the activity of the stimulation is achieved. No further additions are required. A conventional commercially available luminescent plate reader can be used effectively. The second protein component may be a kinase, preferably a protein kinase, such as protein kinase C. The protein kinase C family is further described in Science 1 Jul. 1988: Vol. 241. no. 4861, pp. 42-52 and incorporated herein by reference for further description of the kinase family.
The reagent solution provides for lysis of the cells and exposure of any complex formed in the nucleus to the assay medium. Any conventional lysis buffer may be employed that does not interfere with the β-galactosidase reaction with its substrate. Various ionic buffers, such as CHAPS, may be employed at 1-5%, generally not more than 3%, in a convenient buffer, such as PBS or HEPES, where numerous other substitutes are known in the field.
Also present will be a β-galactosidase substrate, desirably a luminescent reagent and optionally a signal enhancer. The luminescent reagent will be in large excess in relation to the maximum amount of β-galactosidase that is likely to be formed. Conveniently, a luminescent substrate is used, available as Galacton Star from ABI in conjunction with the Emerald II enhancer. Any equivalent luminescent or fluorescent substrate composition may be employed. The substrate will be present in about 1 to 10 weight percent, while the enhancer will be present in about 10 to 30 weight percent of the reagent solution. These amounts will vary depending upon the particular substrate composition employed. The reagent solution may be prepared as a 5-20× concentrate or higher for sale or the solids may be provided as powders and dissolved in water at the appropriate proportions.
Standards will usually be used, whereby the signal is related to the concentration of a known stimulator performed under the same conditions as the candidate compound. A graph can be prepared that shows the change in signal with the change in concentration of the standard compound. The assay is sensitive to EC50 s of not greater than 50 micromolar of candidate compound, generally sensitive to less than about 1 μM, in most cases sensitive to less than about 500 nM, frequently sensitive to less than 100 nM and can in many cases detect EC50 values of less than 5 nM. The S/B (signal/background) ratios are generally are at least about 3 fold and can be greater than about 50 fold.
For convenience kits can be provided. In the subject assays, the EA fusion protein may be provided as a construct for expression of EA to be introduced into the cell or cells may be provided that are appropriately modified to provide EA in the cell. Generally, the kits would include an insert with instructions for performing the assay. The instructions may be printed or electronic, e.g., a CD or floppy disk. The kits find use in marketing the product and encouraging the use of the assay for research and commercial settings.
The following examples are offered by way of illustration and not by way of limitation.
EXPERIMENTAL
The following is the full sequence (SEQ ID NO:6) for the plasmid introduced into the CHO cells for expression of the TRAP-220-EA-Nuc fusion protein.









    1
ACTAGTTAGC TAACTAGCTC TGTATCTGGC GGACCCGTGG TGGAACTGAC



 


   51
GAGTTCTGAA CACCCGGCCG CAACCCTGGG AGACGTCCCA GGGACTTTGG


 


  101
GGGCCGTTTT TGTGGCCCGA CCTGAGGAAG GGAGTCGATG TGGAATCCGA


 


  151
CCCCGTCAGG ATATGTGGTT CTGGTAGGAG ACGAGAACCT AAAACAGTTC


 


  201
CCGCCTCCGT CTGAATTTTT GCTTTCGGTT TGGAACCGAA GCCGCGCGTC


 


  251
TTGTCTGCTG CAGCGCTGCA GCATCGTTCT GTGTTGTCTC TGTCTGACTG


 


  301
TGTTTCTGTA TTTGTCTGAA AATTAGGGCC AGACTGTTAC CACTCCCTTA


 


  351
AGTTTGACCT TAGGTCACTG GAAAGATGTC GAGCGGATCG CTCACAACCA


 


  401
GTCGGTAGAT GTCAAGAAGA GACGTTGGGT TACCTTCTGC TCTGCAGAAT


 


  451
GGCCAACCTT TAACGTCGGA TGGCCGCGAG ACGGCACCTT TAACCGAGAC


 


  501
CTCATCACCC AGGTTAAGAT CAAGGTCTTT TCACCTGGCC CGCATGGACA


 


  551
CCCAGACCAG GTCCCCTACA TCGTGACCTG GGAAGCCTTG GCTTTTGACC


 


  601
CCCCTCCCTG GGTCAAGCCC TTTGTACACC CTAAGCCTCC GCCTCCTCTT


 


  651
CCTCCATCCG CCCCGTCTCT CCCCCTTGAA CCTCCTCGTT CGACCCCGCC


 


  701
TCGATCCTCC CTTTATCCAG CCCTCACTCC TTCTCTAGGC GCCGGAATTC


 


  751
CGATCTGATC AGCTTGCCAC AACCCGTACC AAAGATGGAT AGATCCGGAA


 


  801
AGCCTGAACT CACCGCGACG TCTGTCGAGA AGTTTCTGAT CGAAAAGTTC


 


  851
GACAGCGTCT CCGACCTGAT GCAGCTCTCG GAGGGCGAAG AATCTCGTGC


 


  901
TTTCAGCTTC GATGTAGGAG GGCGTGGATA TGTCCTGCGG GTAAATAGCT


 


  951
GCGCCGATGG TTTCTACAAA GATCGTTATG TTTATCGGCA CTTTGCATCG


 


 1001
GCCGCGCTCC CGATTCCGGA AGTGCTTGAC ATTGGGGAAT TCAGCGAGAG


 


 1051
CCTGACCTAT TGCATCTCCC GCCGTGCACA GGGTGTCACG TTGCAAGACC


 


 1101
TGCCTGAAAC CGAACTGCCC GCTGTTCTGC AGCCGGTCGC GGAGGCCATG


 


 1151
GATGCGATCG CTGCGGCCGA TCTTAGCCAG ACGAGCGGGT TCGGCCCATT


 


 1201
CGGACCGCAA GGAATCGGTC AATACACTAC ATGGCGTGAT TTCATATGCG


 


 1251
CGATTGCTGA TCCCCATGTG TATCACTGGC AAACTGTGAT GGACGACACC


 


 1301
GTCAGTGCGT CCGTCGCGCA GGCTCTCGAT GAGCTGATGC TTTGGGCCGA


 


 1351
GGACTGCCCC GAAGTCCGGC ACCTCGTGCA CGCGGATTTC GGCTCCAACA


 


 1401
ATGTCCTGAC GGACAATGGC CGCATAACAG CGGTCATTGA CTGGAGCGAG


 


 1451
GCGATGTTCG GGGATTCCCA ATACGAGGTC GCCAACATCT TCTTCTGGAG


 


 1501
GCCGTGGTTG GCTTGTATGG AGCAGCAGAC GCGCTACTTC GAGCGGAGGC


 


 1551
ATCCGGAGCT TGCAGGATCG CCGCGGCTCC GGGCGTATAT GCTCCGCATT


 


 1601
GGTCTTGACC AACTCTATCA GAGCTTGGTT GACGGCAATT TCGATGATGC


 


 1651
AGCTTGGGCG CAGGGTCGAT GCGACGCAAT CGTCCGATCC GGAGCCGGGA


 


 1701
CTGTCGGGCG TACACAAATC GCCCGCAGAA GCGCGGCCGT CTGGACCGAT


 


 1751
GGCTGTGTAG AAGTACTCGC CGATAGTGGA AACCGACGCC CCAGCACTCG


 


 1801
TCCGAGGGCA AAGGAATAGA GTAGATGCCG ACCGAACAAG AGCTGATTTC


 


 1851
GAGAACGCCT CAGCCAGCAA CTCGCGCGAG CCTAGCAAGG CAAATGCGAG


 


 1901
AGAACGGCCT TACGCTTGGT GGCACAGTTC TCGTCCACAG TTCGCTAAGC


 


 1951
TCGCTCGGCT GGGTCGCGGG AGGGCCGGTC GCAGTGATTC AGGCCCTTCT


 


 2001
GGATTGTGTT GGTCCCCAGG GCACGATTGT CATGCCCACG CACTCGGGTG


 


 2051
ATCTGACTGA TCCCGCAGAT TGGAGATCGC CGCCCGTGCC TGCCGATTGG


 


 2101
GTGCAGATCT AGAGCTCGCT GATCCGGCCA TTAGCCATAT TATTCATTGG


 


 2151
TTATATAGCA TAAATCAATA TTGGCTATTG GCCATTGCAT ACGTTGTATC


 


 2201
CATATCATAA TATGTACATT TATATTGGCT CATGTCCAAC ATTACCGCCA


 


 2251
TGTTGACATT GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC


 


 2301
ATTAGTTCAT AGCCCATATA TGGAGTTCCG CGTTACATAA CTTACGGTAA


 


 2351
ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA


 


 2401
ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA


 


 2451
ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT


 


 2501
ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC


 


 2551
GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA


 


 2601
GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC


 


 2651
AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT


 


 2701
CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG


 


 2751
GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG


 


 2801
GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCT CTGGCTAACT


 


 2851
AGAGAACCCA CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG


 


 2901
GGAGACCCAA GCTGGCTAGC ACCATGCATA ACACAAAGAA TCACCCTATG


 


 2951
CTCATGAACC TGCTGAAGGA CAATCCTGCC CAGGACACCG GTTCAAAGCT


 


 3001
TGGTACCATG GATCCTCGCG CCTCCTCCAA TTCACTGGCC GTCGTTTTAC


 


 3051
AAGAGGCCCG CACCGATCGC CCTTCCCAAC AGTTGCGCAG CCTGAATGGC


 


 3101
GAATGGCGCT TTGCCTGGTT TCCGGCACCA GAAGCGGTGC CGGAAAGCTG


 


 3151
GCTGGAGTGC GATCTTCCTG AGGCCGATAC TGTCGTCGTC CCCTCAAACT


 


 3201
GGCAGATGCA CGGTTACGAT GCGCCCATCT ACACCAACGT GACCTATCCC


 


 3251
ATTACGGTCA ATCCGCCGTT TGTTCCCACG GAGAATCCGA CGGGTTGTTA


 


 3301
CTCGCTCACA TTTAATGTTG ATGAAAGCTG GCTACAGGAA GGCCAGACGC


 


 3351
GAATTATTTT TGATGGCGTT AACTCGGCGT TTCATCTGTG GTGCAACGGG


 


 3401
CGCTGGGTCG GTTACGGCCA GGACAGTCGT TTGCCGTCTG AATTTGACCT


 


 3451
GAGCGCATTT TTACGCGCCG GAGAAAACCG CCTCGCGGTG ATGGTGCTGC


 


 3501
GCTGGAGTGA CGGCAGTTAT CTGGAAGATC AGGATATGTG GCGGATGAGC


 


 3551
GGCATTTTCC GTGACGTCTC GTTGCTGCAT AAACCGACTA CACAAATCAG


 


 3601
CGATTTCCAT GTTGCCACTC GCTTTAATGA TGATTTCAGC CGCGCTGTAC


 


 3651
TGGAGGCTGA AGTTCAGATG TGCGGCGAGT TGCGTGACTA CCTACGGGTA


 


 3701
ACAGTTTCTT TATGGCAGGG TGAAACGCAG GTCGCCAGCG GCACCGCGCC


 


 3751
TTTCGGCGGT GAAATTATCG ATGAGCGTGG TGGTTATGCC GATCGCGTCA


 


 3801
CACTACGTCT GAACGTCGAA AACCCGAAAC TGTGGAGCGC CGAAATCCCG


 


 3851
AATCTCTATC GTGCGGTGGT TGAACTGCAC ACCGCCGACG GCACGCTGAT


 


 3901
TGAAGCAGAA GCCTGCGATG TCGGTTTCCG CGAGGTGCGG ATTGAAAATG


 


 3951
GTCTGCTGCT GCTGAACGGC AAGCCGTTGC TGATTCGAGG CGTTAACCGT


 


 4001
CACGAGCATC ATCCTCTGCA TGGTCAGGTC ATGGATGAGC AGACGATGGT


 


 4051
GCAGGATATC CTGCTGATGA AGCAGAACAA CTTTAACGCC GTGCGCTGTT


 


 4101
CGCATTATCC GAACCATCCG CTGTGGTACA CGCTGTGCGA CCGCTACGGC


 


 4151
CTGTATGTGG TGGATGAAGC CAATATTGAA ACCCACGGCA TGGTGCCAAT


 


 4201
GAATCGTCTG ACCGATGATC CGCGCTGGCT ACCGGCGATG AGCGAACGCG


 


 4251
TAACGCGAAT GGTGCAGCGC GATCGTAATC ACCCGAGTGT GATCATCTGG


 


 4301
TCGCTGGGGA ATGAATCAGG CCACGGCGCT AATCACGACG CGCTGTATCG


 


 4351
CTGGATCAAA TCTGTCGATC CTTCCCGCCC GGTGCAGTAT GAAGGCGGCG


 


 4401
GAGCCGACAC CACGGCCACC GATATTATTT GCCCGATGTA CGCGCGCGTG


 


 4451
GATGAAGACC AGCCCTTCCC GGCTGTGCCG AAATGGTCCA TCAAAAAATG


 


 4501
GCTTTCGCTA CCTGGAGAGA CGCGCCCGCT GATCCTTTGC GAATACGCCC


 


 4551
ACGCGATGGG TAACAGTCTT GGCGGTTTCG CTAAATACTG GCAGGCGTTT


 


 4601
CGTCAGTATC CCCGTTTACA GGGCGGCTTC GTCTGGGACT GGGTGGATCA


 


 4651
GTCGCTGATT AAATATGATG AAAACGGCAA CCCGTGGTCG GCTTACGGCG


 


 4701
GTGATTTTGG CGATACGCCG AACGATCGCC AGTTCTGTAT GAACGGTCTG


 


 4751
GTCTTTGCCG ACCGCACGCC GCATCCAGCG CTGACGGAAG CAAAACACCA


 


 4801
GCAGCAGTTT TTCCAGTTCC GTTTATCCGG GCAAACCATC GAAGTGACCA


 


 4851
GCGAATACCT GTTCCGTCAT AGCGATAACG AGCTCCTGCA CTGGATGGTG


 


 4901
GCGCTGGATG GTAAGCCGCT GGCAAGCGGT GAAGTGCCTC TGGATGTCGC


 


 4951
TCCACAAGGT AAACAGTTGA TTGAACTGCC TGAACTACCG CAGCCGGAGA


 


 5001
GCGCCGGGCA ACTCTGGCTC ACAGTACGCG TAGTGCAACC GAACGCGACC


 


 5051
GCATGGTCAG AAGCCGGCCA CATCAGCGCC TGGCAGCAGT GGCGTCTGGC


 


 5101
GGAAAACCTC AGTGTGACGC TCCCCGCCGC GTCCCACGCC ATCCCGCATC


 


 5151
TGACCACCAG CGAAATGGAT TTTTGCATCA AGCTGGGTAA TAAGCGTTGG


 


 5201
CAATTTAACC GCCAGTCAGG CTTTCTTTCA CAGATGTGGA TTGGCGATAA


 


 5251
AAAACAACTG CTGACGCCGC TGCGCGATCA GTTCACCCGT GCACCGCTGG


 


 5301
ATAACGACAT TGGCGTAAGT GAAGCGACCC GCATTGACCC TAACGCCTGG


 


 5351
GTCGAACGCT GGAAGGCGGC GGGCCATTAC CAGGCCGAAG CAGCGTTGTT


 


 5401
GCAGTGCACG GCAGATACAC TTGCTGACGC GGTGCTGATT ACGACCGCTC


 


 5451
ACGCGTGGCA GCATCAGGGG AAAACCTTAT TTATCAGCCG GAAAACCTAC


 


 5501
CGGATTGATG GTAGTGGTCA AATGGCGATT ACCGTTGATG TTGAAGTGGC


 


 5551
GAGCGATACA CCGCATCCGG CGCGGATTGG CCTGAACTGC CAGCTGGCGC


 


 5601
AGGTAGCAGA GCGGGTAAAC TGGCTCGGAT TAGGGCCGCA AGAAAACTAT


 


 5651
CCCGACCGCC TTACTGCCGC CTGTTTTGAC CGCTGGGATC TGCCATTGTC


 


 5701
AGACATGTAT ACCCCGTACG TCTTCCCGAG CGAAAACGGT CTGCGCTGCG


 


 5751
GGACGCGCGA ATTGAATTAT GGCCCACACC AGTGGCGCGG CGACTTCCAG


 


 5801
TTCAACATCA GCCGCTACAG TCAACAGCAA CTGATGGAAA CCAGCCATCG


 


 5851
CCATCTGCTG CACGCGGAAG AAGGCACATG GCTGAATATC GACGGTTTCC


 


 5901
ATATGGGGAT TGGTGGAGAC GACTCCTGGA GCCCGTCAGT ATCGGCGGAA


 


 5951
TTACAGCTGA GCGCCGGTCG CTACCATTAC CAGTTGGTCT GGTGTCAAAA


 


 6001
AGCGGCCGCA GATCCAAAAA AGAAGAGAAA GGTAGATCCA AAAAAGAAGA


 


 6051
GAAAGGTAGA TCCAAAAAAG AAGAGAAAGG TAGATACGGC CCTCGAGCCT


 


 6101
CCCCCAGTGT CCAAGAGGGA ATCCAAATCC AGGTCGCGAT CGAAGAGTCC


 


 6151
CCCCAAGTCT CCTGAAGAGG AAGGAGCGGT GTCCTCTTCT AGGGGTCTTT


 


 6201
CCCCTCTCGC CAAAGGAATG CAAGGTCTGT TGAATGTCGT GAAGGAAGCA


 


 6251
GTTCCTCTGG AAGCTTCTTG AAGACAAACA ACGTCTGTAG CGACCCTTTG


 


 6301
CAGGCAGCGG AACCCCCCAC CTGGCGACAG GTGCCTCTGC GGCCAAAAGC


 


 6351
CACCCTTAAT GGCCTAACAT CGATAAAATA AAAGATTTTA TTTAGTCTCC


 


 6401
AGAAAAAGGG GGGAATGAAA GACCCCACCT GTAGGTTTGG CAAGCTAGCT


 


 6451
TAAGTAACGC CATTTTGCAA GGCATGGAAA AATACATAAC TGAGAATAGA


 


 6501
GAAGTTCAGA TCAAGGTCAG GAACAGATGG AACAGCTGAA TATGGGCCAA


 


 6551
ACAGGATATC TGTGGTAAGC AGTTCCTGCC CCGGCTCAGG GCCAAGAACA


 


 6601
GATGGAACAG CTGAATATGG GCCAAACAGG ATATCTGTGG TAAGCAGTTC


 


 6651
CTGCCCCGGC TCAGGGCCAA GAACAGATGG TCCCCAGATG CGGTCCAGCC


 


 6701
CTCAGCAGTT TCTAGAGAAC CATCAGATGT TTCCAGGGTG CCCCAAGGAC


 


 6751
CTGAAATGAC CCTGTGCCTT ATTTGAACTA ACCAATCAGT TCGCTTCTCG


 


 6801
CTTCTGTTCG CGCGCTTCTG CTCCCCGAGC TCAATAAAAG AGCCCACAAC


 


 6851
CCCTCACTCG GGGCGCCAGT CCTCCGATTG ACTGAGTCGC CCGGGTACCC


 


 6901
GTGTATCCAA TAAACCCTCT TGCAGTTGCA TCCGACTTGT GGTCTCGCTG


 


 6951
TTCCTTGGGA GGGTCTCCTC TGAGTGATTG ACTACCCGTC AGCGGGGGTC


 


 7001
TTTCATTTGG GGGCTCGTCC GGGATCGGGA GACCCCTGCC CAGGGACCAC


 


 7051
CGACCCACCA CCGGGAGGTA AGCTGGCTGC CTCGCGCGTT TCGGTGATGA


 


 7101
CGGTGAAAAC CTCTGACACA TGCAGCTCCC GGAGACGGTC ACAGCTTGTC


 


 7151
TGTAAGCGGA TGCCGGGAGC AGACAAGCCC GTCAGGGCGC GTCAGCGGGT


 


 7201
GTTGGCGGGT GTCGGGGCGC AGCCATGACC CAGTCACGTA GCGATAGCGG


 


 7251
AGTGTATACT GGCTTAACTA TGCGGCATCA GAGCAGATTG TACTGAGAGT


 


 7301
GCACCATATG CGGTGTGAAA TACCGCACAG ATGCGTAAGG AGAAAATACC


 


 7351
GCATCAGGCG CTCTTCCGCT TCCTCGCTCA CTGACTCGCT GCGCTCGGTC


 


 7401
GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG TAATACGGTT


 


 7451
ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC


 


 7501
AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT


 


 7551
AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG


 


 7601
GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA


 


 7651
GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG


 


 7701
TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA GCTCACGCTG


 


 7751
TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC


 


 7801
ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT


 


 7851
CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC


 


 7901
TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT


 


 7951
TGAAGTGGTG GCCTAACTAC GGCTACACTA GAAGGACAGT ATTTGGTATC


 


 8001
TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG


 


 8051
ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC


 


 8101
AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT


 


 8151
TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT


 


 8201
GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA


 


 8251
AATGAAGTTT TAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC


 


 8301
AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA TCTGTCTATT


 


 8351
TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC


 


 8401
GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA


 


 8451
CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC


 


 8501
CGAGCGCAGA AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA


 


 8551
ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC


 


 8601
AACGTTGTTG CCATTGCTGC AGGCATCGTG GTGTCACGCT CGTCGTTTGG


 


 8651
TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT


 


 8701
CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT


 


 8751
GTCAGAAGTA AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT


 


 8801
GCATAATTCT CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG


 


 8851
GTGAGTACTC AACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT


 


 8901
TGCTCTTGCC CGGCGTCAAC ACGGGATAAT ACCGCGCCAC ATAGCAGAAC


 


 8951
TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA


 


 9001
GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC


 


 9051
AACTGATCTT CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA


 


 9101
AACAGGAAGG CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT


 


 9151
GTTGAATACT CATACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG


 


 9201
GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA


 


 9251
ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTCT


 


 9301
AAGAAACCAT TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG


 


 9351
AGGCCCTTTC GTCTTCAAGA ATTCATACCA GATCACCGAA AACTGTCCTC


 


 9401
CAAATGTGTC CCCCTCACAC TCCCAAATTC GCGGGCTTCT GCCTCTTAGA


 


 9451
CCACTCTACC CTATTCCCCA CACTCACCGG AGCCAAAGCC GCGGCCCTTC


 


 9501
CGTTTCTTTG CTTTTGAAAG ACCCCACCCG TAGGTGGCAA GCTAGCTTAA


 


 9551
GTAACGCCAC TTTGCAAGGC ATGGAAAAAT ACATAACTGA GAATAGAAAA


 


 9601
GTTCAGATCA AGGTCAGGAA CAAAGAAACA GCTGAATACC AAACAGGATA


 


 9651
TCTGTGGTAA GCGGTTCCTG CCCCGGCTCA GGGCCAAGAA CAGATGAGAC


 


 9701
AGCTGAGTGA TGGGCCAAAC AGGATATCTG TGGTAAGCAG TTCCTGCCCC


 


 9751
GGCTCGGGGC CAAGAACAGA TGGTCCCCAG ATGCGGTCCA GCCCTCAGCA


 


 9801
GTTTCTAGTG AATCATCAGA TGTTTCCAGG GTGCCCCAAG GACCTGAAAA


 


 9851
TGACCCTGTA CCTTATTTGA ACTAACCAAT CAGTTCGCTT CTCGCTTCTG


 


 9901
TTCGCGCGCT TCCGCTCTCC GAGCTCAATA AAAGAGCCCA CAACCCCTCA


 


 9951
CTCGGCGCGC CAGTCTTCCG ATAGACTGCG TCGCCCGGGT ACCCGTATTC


 


10001
CCAATAAAGC CTCTTGCTGT TTGCATCCGA ATCGTGGTCT CGCTGTTCCT


 


10051
TGGGAGGGTC TCCTCTGAGT GATTGACTAC CCACGACGGG GGTCTTTCAT


 


10101
TTGGGGGCTC GTCCGGGATT TGGAGACCCC TGCCCAGGGA CCACCGACCC


 


10151
ACCACCGGGA GGTAAGCTGG CCAGCAACTT ATCTGTGTCT GTCCGATTGT


 


10201
CTAGTGTCTA TGTTTGATGT TATGCGCCTG CGTCTGT.






The genes for the NHRs may be obtained from any convenient source: commercial supplier; RT_PCR from mRNA isolated in accordance with conventional procedures using known sequences as probes; PCR from genomic DNA using primers from known sequences. The genes are PCR amplified to remove the stop codon at the 3′ end. The genes are then digested with restriction enzymes where the restriction site is included with the primer sequences. These products are then purified in conventional ways and then ligated into a commercial vector into which the ED or EA has been inserted. Separating the ED and the EA from the gene is a linker that provides flexibility to the fusion proteins to enhance complementation. This linker is not required for activity. The transcriptional regulatory region is generally present in commercial vectors, such as the 5′ LTR of the virus used for the vector. Alternatively, the CMV promoter may be used. The resulting vector is then introduced into the host cell by liposome mediated transfection or retroviral infection with Moloney murine leukemia virus vector and packaging cell lines. The resulting virus is then used for viral infection. The vectors also include selection genes, such as hygromycin resistance and cells into which the construct is integrated are selected in a conventional selection medium. The surviving cells are then screened in an agonist dose response assay using adherent cells and the Path-Hunter® Detection Kit reagents in white-walled microplates.
The following TABLE 1 provides exemplary protocols:









TABLE 1






Volumes
Volumes


Steps
(384-well full volume)
(96-well full volume)







Step 1:
Add 25 μL of cells in each
Add 100 μL of cells in each


Plate Cells
well at a preferred density of
well at a preferred density of



5,000-10,000 cells per well.
20,000-30,000 cells per well.



Cells should be seeded
Cells should be seeded in



In media* + 1.5%
media* + 1.5% Charcoal/



Charcoal/Dextran treated serum
Dextran treated serum


Step 2:
Dissolve compounds**, add 1-5
Dissolve compounds, add 5-10


Treat Cells*
μl per well, and incubate for
μl per well, and incubate for



3 hrs @ 37° C.*
3 hrs @ 37° C.*


Step 3:
Add 12 μl of detection
Add 50 μl of detection


Add Detection
reagents*** per well.
reagents per well.


Reagents


Step 4:
Samples can be read on any
Samples can be read on any


Read Samples
standard luminescence plate
standard luminescence plate



reader after 60 minutes of
reader after 60 minutes of



incubation.
incubation.





Note:


Dissolve compound according to manufacturer's instructions and without exceeding 1% organic solvent such as DMSO or EtOH in the assay medium.


CHO:


*F12 + 10% FBS, 1X P/S/G and 250 mg/mL Hygromycin and 600 mg/mL G418 Cells are maintained at ~70% confluence, passed two to three times a week.


**Candidate compound is dissolved in a minimum amount of solvent, e.g., ethanol or DMSO


***Detection reagent is prepared by combining PathHunter TM Cell Assay Buffer (DiscoveRx, Fremont, CA), Galacton Star ® and Emerald II ® reagent to 25 μl of cells + compound at a ratio of 19:1:5.






The results of the assays are set forth in the figures and are further tabulated below in TABLE 2. All of the cells were CHO cells and were stable through 20 passages.











TABLE 2





Figure
NHR
S/B
EC50 nM
Ligand



















2
ERRα
3
300
XCT790*


3
THRα
78
15
T3


4
RARα
3
29
9-cis-RA


5
RARβ
3
29
9-cis-RA





*CAS registry 725247-18-7






It is evident from the above results that the subject assay provides for a sensitive, accurate technique for measuring modulation of activation of NHRs in response to candidate compounds. The assay is easy to perform, using conventional equipment, can be used for rapidly screening numerous candidates for their activity and is readily adaptable to any of the known or orphan NHRs. The assay has a novel format for pathway profiling. The assays have a reduced time relative to reporter genes, there being no requirement for transcriptional activity. There is no requirement for target over expression. The method is readily adaptable to automated screening employing robotics. NHR activation is detected directly without imaging.
Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims. All patents and publications referred to in the specification are incorporated by reference as if fully set forth therein.

## Claims

1. A method for screening a candidate compound for modulating the binding of a nuclear hormone receptor to a second protein component that binds to the nuclear hormone receptor to form a complex to initiate transcription, comprising:
(a) using a cell in an assay medium transformed with first and second genetic constructs having regulatory sequences functional in said cell for expression of said constructs,
(b) said method employing first and second fragments of β-galactosidase which when complexed form an active enzyme, one of said fragments being an enzyme donor (“ED”) and the other of said fragments being an enzyme acceptor (“EA”) that is larger than said ED,
(c) said first genetic construct encoding a first fusion protein of a first fragment of said β-galactosidase fused to said nuclear hormone receptor to serve as a surrogate for said nuclear hormone receptor;
(d) said second genetic construct encoding a second fusion protein of said second fragment of β-galactosidase fused to said second protein component, wherein said second protein component is selected from the group consisting of (i) coactivator, (ii) corepressor, (iii) member of a homodimer, and (iv) member of a heterodimer, of said nuclear hormone receptor;
(e) incubating said cell in said assay medium with a candidate compound for sufficient time for any binding between said first fusion protein fused to a nuclear hormone receptor and said second fusion protein, fused to said second enzyme fragment, that binds to the nuclear hormone receptor to form a complex to initiate transcription, to occur, said binding causing formation of active β-galactosidase;
(f) adding a β-galactosidase substrate that provides for a signal that is detectable; and
(g) determining said signal as a measure of the modulation resulting from said binding by said candidate compound, wherein binding results in a signal and a candidate compound modulating binding modulates said signal.

2. The method according to claim 1, wherein said ED fragment is a low affinity β galactosidase fragment from about 36 to 50 amino acids long.

3. The method according to claim 1, wherein a lysing reagent is added after said incubating.

4. The method according to claim 1, wherein said ED is fused to said nuclear hormone receptor.

5. The method according to claim 1, wherein said nuclear hormone receptor is selected from the group consisting of estrogen-related receptor α (ERRα), thyroid hormone receptor α (THRα), thyroid hormone receptor β (THRβ), retinoic acid receptor α (RARα), retinoic acid receptor β (RARβ), one of peroxisome proliferator-activated receptor γ (PPARγ) or retinoid X receptor γ (RXRγ), estrogen receptor 1a (ESR1a), the a isoform of the estrogen receptor (Era), the b isoform of the estrogen receptor (ERb), progesterone receptor α (PRa), progesterone receptor β (PRb), Nur-related protein 1 (NURR1), peroxisome proliferator-activated receptor alpha (PPARa), peroxisome proliferator-activated receptor delta (PPARd), oxysteroal receptor α (LXRa) and oxysterol receptor β (LXRb).

6. The method according to claim 1, wherein said cells are CHO or HepG2.

7. The method according to claim 1, wherein said determining is capable of determining an EC50 of less than about 1 μM.

8. The method according to claim 1, wherein a ratio of said signal to background is not less than 2 fold.

9. A method for screening candidate compounds for modulating the binding of a nuclear hormone receptor to a coactivator,
(a) using a cell in an assay medium transformed with first and second genetic constructs having regulatory sequences functional in said cell for expression of said constructs,
(b) said method employing a small (“ED”) and a large (“EA”) fragment of β-galactosidase which when complexed form an active enzyme,
(c) said first genetic construct encoding a fusion protein of said ED fused to said nuclear hormone receptor to serve as a surrogate for said nuclear hormone receptor;
(d) said second genetic construct encoding said coactivator fused to said EA;
(e) incubating said cells in said assay medium with a candidate compound for sufficient time for any binding between said coactivator fused to said EA and said fusion protein of ED fused to said nuclear hormone receptor to occur;
(f) adding a reagent solution and substrate that provides for a detectable signal to said cells to lyse said cells; and
(g) determining said detectable signal as a measure of the modulation resulting from said binding by said candidate compound, wherein binding results in a signal and a candidate compound modulating said binding modulates said signal.

10. The method according to claim 1, wherein said nuclear hormone receptor is selected from the group consisting of ERRα, THRα, RARα and RARβ.

11. The method according to claim 1 where said second protein component is steroid receptor coactivator-1 (SRC-1) or thyroid hormone receptor-associated protein (TRAP-220).

12. The method of claim 1 wherein said second genetic construct resides in a nucleus in said cell and said binding occurs in the nucleus.

13. The method of claim 1 wherein said second genetic construct further encodes nuclear localization and nuclear retention signals, whereby said second fusion protein is localized to the cell's nucleus.

14. The method of claim 2 wherein said ED comprises not more than 100 amino acids of the N-proximal portion of β-galactosidase.