METHOD FOR IDENTIFYING CART RECEPTOR AND USES THEREOF

RELATED APPLICATION

[0001] This application claims priority of application Serial No. 60/693,660, filed June 24, 2005, incorporated by reference in its entirety

FILED OF THE INVENTION

[0002] This invention relates to methods for detemu ^' ning if a molecule interacts with a receptor. More particularly, the invention involves the identification of a receptor binding partner for "Cocaine and Amphetamine Regulated Transcript," or "CART" as it will be referred to hereafter.

BACKGROUND AND PRIOR ART

[0003] Cells recognize and respond to a great variety of extracellular stimuli using specific receptor molecules located on the cell surface. The largest class of cell surface receptors are the G-protein coupled receptors (GPCRs), an exceedingly diverse group of protein containing molecules, involved in the recognition of endogenous ligands such as hormones, neurotransmitters, peptides, glycoproteins, lipids, nucleotides and ions, as well as exogenous stimuli, including light, odors, pheromones and tastes Strader, et al., Ann. Rev. Biochem., 63:101-32(1994); Bockaert, et al., Embo J.. 18(7):1723-9(1999); Mombarets, et al., Science, 286(5440):707-711(1999). As a result of their central role in many signaling events, GPCRs are the targets of an increasingly large number of therapeutic agents (Howard, et al., Trends Pharmacol. ScI. 22(3): 132-40(2001), and mutations in GPCRs have been linked to numerous diseases and disorders. Spiegel, Ann- Rev. Phvsiol. 58:143-70(1996); Rana, et al., Ann. Rev. Pharmacol Toxicol, 41:593- 624(2001).

[0004] The discovery of candidate therapeutic agents that act on GPCR-mediated pathways requires the development of assays to monitor the activity of specific GPCR targets. Different GPCRs are coupled to distinct G-protein-regulated signal transduction pathways, and thus assays that measure G protein-regulated signaling pathways depend on knowledge of the G-protein specificity of the target receptor, or require engineering of the cellular system to force coupling of the target receptor to a measurable pathway. In contrast, one apparently common feature of GPCRs is that ligand binding triggers receptor desensitization, a process that is mediated by the recruitment of intracellular

arrestin proteins to the activated receptor. Thus, the ligand-induced activation or GPCRs may be assayed by monitoring the interaction of arrestin with the test GPCR. A major advantage of this approach is that no knowledge of G protein pathways is required.

[0005] As part of a broad effort to identify receptors that mediate the activity of heretofore "orphan" signaling factors and elucidate the biological function of these ligand-receptor pairs, cell-based assays have been developed to measure the ligand- mediated activation of a large fraction of the GPCRs found in the human genome. A panel of GPCR assays were screened for a receptor that is selectively activated by CART (Cocaine-and Amphetamine-Regulated Transcript), a neuroendocrine ligand for whom receptor had not been identified until now.

[0006] CART is a neuromodulatory peptide that is highly expressed in the hypothalamus and is believed to play a key role in appetite regulation and body weight homeostasis (reviewed in Hunter, et al., Trends Endocrin Metab., 15(9):454-9(2004); Hunter, et al., Curr Trends CNS Neurol Disord. 2(3):201 -5(2003)). Intracerebroventricular (ICV) injection of CART strongly suppresses feeding in rodents (Kristensen, et al., Nature, 393(6680):72~76(1998)), and targeted deletions of the CART gene result in increased body weight relative to wild type littermates Asnicar et al., Endocrinology, 142(10):4394-4400(2001); Wierup, et al., Regul Pept. 129(l-3):203- 11(2005). Expression of CART in the hypothalamic arcuate nucleus is regulated by the neuroendocrine factor leptin and is nearly absent in the obese ob/ob mutant mouse. Kristensen, et al., supra. Support for the involvement of CART in the regulation of body weight in humans comes from genetic studies that have shown association of mutations in CART with obesity del Giudice, et al., Diabetes, 50(9):2157-2160(2001); Yamada, et al., Obes Relat. Metab Disord, 26(l):132-6(2002). CART has also been implicated in neuronal reward pathways, addiction, stress, pancreatic islet function and bone remodeling. Elefeteriou, et al., Nature, 434(7032):514-20(2005); Dominguez, et al., Ann. N. Y. Acad. Sci, 1025:363-9(2004). As a large, unstable peptide, CART lacks therapeutic efficacy as an oral or IV drug, but small molecule drugs that mimic the action of CART could offer very valuable treatments for obesity, addiction, pathological stress conditions, defects in bone metabolism, cardiovascular and neuroendocrine diseases.

[0007] Although CART has been very actively studied, drug development at this target has been impossible because the CART receptor is unknown. Since experimental evidence has supported the notion that activity of CART is mediated by an unkown G protein coupled receptor (GPCR), a panel of nearly 120 cell-based GPCR assays were

screened and a receptor known as GHSR (Growth Hormone Secretagogue Receptor) (tirst described by Howard, et al., Science, 273 (5277) :974-7(l 996), incorporated by reference in its entirety), was potently activated by CART. This receptor is also activated by a previously identified ligand, ghrelin [Kojitna, et al., Nature. 407(6762):656-60(1999)], and this receptor-ligand pair has been previously implicated in obesity and body weight regulation (see, for example, Hoist, et al., Trends Pharmacol Sci. 25(3): 113-7(2004)). The availability of a cell-based assay to measure CART activity provides a means of developing drugs that act like CART to suppress appetite, or to modulate any of CART's known activities in other therapeutic areas.

[0008] Details of the assay methodology have been described previously. See, e.g., U.S. Patent No. 7,049,076, referred to supra. Briefly, the ligand-mediated activation of a target GPCR is measured by monitoring the interaction of an arrestin with target GPCRs in vivo. The GPCR of interest is fused at its C-terminal end to a non-endogenous transcription factor via a protease cleavage sequence. This is expressed in a cell line containing a quantifiable reporter gene regulated by the tethered transcription factor, together with chimeric protein consisting of arrestin fused to the protease specific for the cleavage site above. The assay is then performed by adding a ligand to the growing cells for a defined period, and measuring the activity of the reporter gene. If the ligand binds to the target receptor, it stimulates the recruitment of the interacting arrestin fusion protein, which results in cleavage of the protease site and release of the transcription factor. The free transcription factor then enters the nucleus and stimulates expression of the reporter gene. Using this approach, quantification of the reporter gene activity affords a measurement of the degree of binding of the interacting arrestin protein to the test GPCR. This assay system has been validated for a diverse array of GPCRs, including receptors that couple to each of the major G protein pathways, and receptors activated by a variety of ligand types, such as hormones, neurotransmitters, peptides and chemokines.

BRIEF DESCRIPTION OF THE FIGURES

[0009] Figure 1 presents the data of Example 1, in dose response curve form, testing rat and human CART peptides, for binding to and activation of, GHSR.

[0010] Figure 2 depicts the results of the experiments discussed in Example 2, which describes competitive binding assays designed to determine if CART 55-102 alters the properties of GHSR.

[0011] Figure 3 presents the date of Example 3, in the form of dose response curves, where CART peptides were tested in functional assays, using the SRE reporter gene.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

[0012] To produce a cell based ("Tango" TM) assay of the human GHSR gene, a fusion construct was created, comprising DNA encoding human GHSR (366 amino acids), which can be found in Genbank under Accession Number NM_198407 (SEQ ID NO: 1), fused in frame to a DNA sequence encoding amino acids 3-335 of the tetracycline controlled transactivator tTA, described by Gossen, et al., Proc Natl Acad Sci USA, 89(12):5547-5551(1992), incorporated by reference. Inserted between these sequences is a DNA sequence encoding the amino acid sequence GSENLYFQL (SEQ ID NO: 2) which includes the low efficiency variant cleavage site for TEV NIa-Pro, ENLYFQL (amino acids 3-9 of SEQ ID NO: 2), described previously. The CMV promoter was placed upstream of the GHSR coding region, and a polyadenylation sequence was placed downstream of the tTA region. This construct is designated GHSR- L-tTA.

[0013] A second fusion construct was also produced containing a C-terminal "tail" sequence designed to enhance the affinity of the interacting arrestin fusion partner and the resulting response of the assay. This construct comprised a DNA sequence encoding the first 342 amino acids of human GHSR fused in frame to a DNA sequence encoding the C-terminal 25 amino acids of the human Arginine Vasopressin Receptor 2 (AVPR2) (Genbank Accession Number: NM_005314) (SEQ ID NO: 3), followed by the ENLYFQL cleavage site described supra, followed by the tTA transactivator sequence. The junction between the GHSR and AVPR2 tail sequences further contains an Xbal restriction site TCTAGA encoding the amino acids Ser-Arg (SR). This construct was designated GHSR-AVPR2ct-L-fTA.

[0014] Each of these receptor constructs was individually transfected into a HEK293 cell line harboring a tTA-dependent luciferase gene and a stable integration of a β-arrestin 2 (ARRB2) - TEV NIa protease fusion gene ("HTLA 5D4" cells described previously). These cells were transfected in a 10 cm cell culture dish with 0.5 μg receptor construct DNA and 7.5 μg carrier DNA using a lipid-based transfection reagent following

the vendor's specified instructions. Transfected cells were cultured for about 24 hours before cryopreservation.

[0015] To conduct the assay, cryopreserved, transiently transfected cells were thawed and plated in 96 well plates at a density of 10,000 cells per well in serum-free medium (SFM). After 5-6 hours of recovery, test ligands were added at various concentrations and the cells were incubated for a period of 8-16 hours. After the incubation period, cells were lysed and luciferase activity was assayed using a standard, commercially available luminescence assay.

[0016] Figure 1 shows dose-response curves for recombinant rat and human CART proteins representing the active C-terminal fragment (designated CART55-102) in assays with the GHSR-A VPR2ct-L-tTA receptor constructs. These results indicate that GHSR is activated by rat CART55-102 with an EC50 of 20OnM and by human CART with an EC50 of lμM. A similar cell-based assay for the human β2-adrenergic receptor (ADRB2) or of the related human motilin receptor (MNLR) showed no response to either recombinant CART protein.

EXAMPLE 2

[0017] This example shows that CART 55-102, upon binding to GHSR, alters the binding and signaling properties of human ghrelin, which is the orthosteric ligand for GHSR.

[0018] An assay was carried out, using COS-7 cells that had been transfected, in 10 cm cell culture dishes, using 24 μg of GHSR cDNA in vector pcDNA3, using lipofectamme, and art recognized methods. These transfected cells were then cultured, for 24 hours, before being cryopreserved, via standard methods.

[0019] Following cryopreservation, the cells were thawed, and plated in 24 well plates, which were covered with poly-D-lysine, using standard protocols. The plating was at a density of 4 x 10 ⁴ cells/well, with a goal of binding from 5-10% of the radioactive materials employed, as described infra.

[0020] One day after the plating of the cells described herein, competitive binding experiments were carried out. A fixed concentration of radiolabeled ghrelin ( ²⁵ I ghrelin, 0.16nm), was added, together with increasing concentrations of unlabeled, human ghrelin (hghrelin), or hCART 55-102. The assays were carried out for 1 hour, at 37°C, using 0.25 ml of 50 mM HEPES binding buffer, at pH7.4, which had been supplemented with 1 mM CaCl ₂ , 5 mM MgSO ₄ , 0.1% BSA, and 40 μg/ml bacitracin.

[0021] In order to determine non-specific binding, 1 μM ot unlabeled gnreπn was used.

[0022] Following the incubation, the COS-7 cells were washed, twice, in 0.5 ml of pre- warmed binding buffer, as described supra, and 1 ml of lysis buffer (IN NaOH, 0.2% SDS). Radioactively labeled ghrelin bound to the cells was counted using standard methods. Assays were carried out in triplicate.

[0023] In experiments not reported here, it had been determined that steady state binding of I ghrelin was achieved using these conditions.

[0024] The results of the assays, summarized in Figure 2, show that ghrelin, did displace the radiolabel, but hCART55-102 did not. Ghrelin displaced the radiolabel with an IC50 of about 8 nM, while hCART 55-102 not only did not displace the radiolabel, but when a concentration of 1 μM was used, the affinity of binding of ¹²⁵ I ghrelin increased by 25%.

[0025] These experiments suggest that CART 55-102 and ghrelin bind to different sites on the GHSR molecule. CART 55-102 modifies affinity and binding kinetics of ghrelin. This suggests that CART 55-102 is an "allosteric modifier" for the GHSR receptor, i.e., one which binds to a site on the receptor that is topographically distinct from the orthosteric binding site.

EXAMPLE 3

[0026] May, et al., Curr. Opin. Pharmacol.. 3:551-556(2003), suggest that allosteric modulators may modify receptor signaling, while having negligible impact on standard, radiolabel binding assays. Christopoulos, et al., Biochem. Soc. Trans., 32:873- 877(2004), suggest that functional assays are less biased than classical binding assays in detecting orthosteric ligands, as compared to allosteric ligands. In light of this background, the ability of CART peptides to stimulate GHSR was tested, using a functional assay, the SRE reporter gene assay. It has been proposed that transcriptional regulation through the SRE pathway is stimulated by various G protein systems, including Gαl3, Gαi, and Gβγ (Mao, et al., J. Biol. Chem., 273:27118-27123(1998); Gruijthuijsen, et al., J. Virol. 76:1328-1338(2002); and Niu, et al., Circ. Res.. 9:846-858.

[0027] The assay was carried out by transfecting HEK293 cells in 10 cm cell culture dishes, with 8 μg of SRE-LUC reporter plasmid, with or without 0.01 μg of GHSR cDNA in pcDNA3, using Fugene 6, and standard protocols. Transfected cells were cultured for 24 hours using the same medium as for COS-7, and then cryopreserved.

[0028] Following cryopreservation, cells were thawed, and plated in 96 well plates at a density of 1 x 10 ⁴ cells/well, in DMEM-F 12 serum free medium. After overnight recovery, test ligands (human ghrelin, human CART 55-102, and a mixture of hCART 55-102 and 1 nM human ghrelin), were added at varying concentrations, and incubated for 5 hours (37 ⁰ C).

[0029] Following incubation, cells were lysed, and luciferase activity was determined, using standard methods.

[0030] Figure 3 summarizes the results in the form of dose response curves. The control was cells transfected with SRE-LUC reporter plasmid, but no GHSR.

[0031] Study of these results via Figure 3 shows that human ghrelin dose dependently stimulated SRE activation (EC50 of 3.4 nM), while hCART 55-102 produced no effect. The combination of hCART 55-102 and human ghrelin, however, stimulated SRE activation (EC50 of 16.2 nM). CART 55-102 produced no activity in the SRE reporter gene assay, when GHSR was absent, indicating that CART stimulated SRE activity is GHSR receptor specific.

[0032] The identification of GHSR as a receptor for CART thus "deorphanizes" the ligand. As CART has a known function one aspect of the invention relates to any method for determining if a test compound, or compound of interests, serves as a potential CART inhibitor, or as a substance with more profound effect than CART. The assay, in its broadest aspect involves a comparison of the interaction of GHSR, or a CART binding fragment of GHSR, with GHSR in presence and absence of a test compound. Comparison of the activities of CART, with or without the test compound leads to a determination of the modulating, e.g., inhibiting or potentiating value, of the test compound or mixture. Hence, one can determine whether a particular test compound is, or if a mixture contains, an analogue, of CART. "Analogue" as used herein refers to a substance which possesses one or more of the properties possessed by CART. The property in question can be, e.g., the ability to bind to GHSR ₃ or the capability of initiating some downstream activity following binding, that the binding of CART to GHSR initiates also.

[0033] The GHSR may be present as an endogenously expressed molecule, or more preferably, in the context of a transformed or transfected cell. CART and test material may be admixed in a test assay, or may be tested separately.

[0034] The assays of the invention may be carried out extracellularly, or more preferably, on a cellular basis. Carrying out the assays using cells which express GHSR

is especially preferred because it permits determination of modulators via a downstream activity assay, which an extracellular assay does not allow.

[0035] The interaction of a compound with GHSR may be determined, for example, by comparing the binding of CART to GHSR, in the presence and absence of the test compound, where the GHSR or binding fragment is labeled, using any of the detectable labels known to the art.

[0036] Labeled molecules can be used, but are not necessary when a cell based assay is used. This is because the interaction of the molecules with GHSR leads to a chain of "downstream activities," any of which can be measured.

[0037] An especially preferred format for carrying out the assay is that described in U.S. Patent No. 7,049,075, referred to supra.

[0038] In the aforementioned, preferred method for determining if a test compound binds to GHSR, this comprises contacting the compound, either "neat" or in a mixture to a cell which has been transformed or transfected with (a) a nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes GHSR, (ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, and (iii) a nucleotide sequence which encodes a protein which activates a reporter gene in said cell, and (b) a nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes a test protein whose interaction with GHSR in the presence of said test compound is to be measured, and (ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for said cleavage site, and determining activity of said reporter gene as a determination of whether said compound modulates GHSR activity.

[0039] The protease or portion of a protease may be a tobacco etch virus nuclear inclusion A protease. The protein which activates said reporter gene may be a transcription factor, such as tTA or GAIA The test protein may be an inhibitory protein, such as an arrestin. The cell may be a eukaryote or a prokaryote. The reporter gene may be an exogenous gene, such as β-galactosidase or luciferase.

[0040] The nucleotide sequence encoding GHSR may be modified to increase interaction with the test protein. Such modifications include but are not limited to replacing all or part of the nucleotide sequence of the C-terminal region of said first test protein with a nucleotide sequence which encodes an amino acid sequence which has higher affinity for said second test protein than the original sequence. For example, the

c-termmai region may be replaced by a nucleotide sequence encoding the C-terminal region of AVPR2, AGTRLI, GRPR, F2RL1, CXCR2/IL-8b, CCR4, or GRPR.

[0041] The method may comprise contacting more than one test compound to a plurality of samples of cells, each of said samples being contacted by one or more of said test compounds, wherein each of said cell samples have been transformed or transfected with the aforementioned nucleic acid molecules, and determining activity of reporter genes in said plurality of said samples to determine if any of said test compounds modulate a specific, protein/protein interaction. The method may comprise contacting each of said samples with one test compound, each of which differs from all others, or comprise contacting each of said samples with a mixture of said test compounds.

[0042] In each case, reference is then made to the same assay, carried out with CART. Exemplary of the type of molecule which can be tested are, e.g., cyclic forms of CART, modified forms of CART with added materials at the C- and/or N-termini, moficiations to side chains, larger peptides containing the CART structure and so forth.

[0043] In another embodiment, there is provided a recombinant cell, transformed or transfected with (a) a nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes GHSR, (ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, and (iii) a nucleotide sequence which encodes a protein which activates a reporter gene in said cell, and (b) a nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes a test protein whose interaction with GHSR in the presence of said test compound is to be measured, and (ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for said cleavage site.

[0044] One or both of said nucleic acid molecules may be stably incorporated into the genome of said cell. The cell also may have been transformed or transfected with said reporter gene.

[0045] The protease or portion of a protease may be a tobacco etch virus nuclear inclusion A protease. The protein which activates said reporter gene may be a transcription factor, such as tTA or GAL4. The second protein may be an inhibitory protein. The cell may be a eukaryote or a prokaryote. The reporter gene may be an exogenous gene, such as β-galactosidase or luciferase. The nucleotide sequence encoding GHSR may be modified to increase interaction with said test protein, such as by replacing all or part of the nucleotide sequence of the C- terminal region of GHSR with a nucleotide sequence which encodes an amino acid sequence which has higher affinity for said test protein than the original sequence. The C-terminal region may be replaced by a

nucleotide sequence encoding the C-terminal region of AVJfK2, AUi KLi, UKFK, F2RL1, CXCR2/IL-8B, CCR4, or GRPR.

[0046] In still yet another embodiment, there is provided an isolated nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes GHSR (ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, and (iii) a nucleotide sequence which encodes a protein which activates a reporter gene in said cell. The protease or portion of a protease may be a tobacco etch virus nuclear inclusion A protease. The protein which activates said reporter gene may be a transcription factor, such as tTA or GAL4. As above, the invention is not to be viewed as limited to these specific embodiments.

[0047] In still a further embodiment, there is provided an expression vector comprising an isolated nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes GHSR (ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, and (iii) a nucleotide sequence which encodes a protein which activates a reporter gene in said cell, and further being operably linked to a promoter.

[0048] In still yet a further embodiment, there is provided an isolated nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes a test protein whose interaction with GHSR in the presence of a test compound is to be measured, and (ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for said cleavage site. The test protein may be an inhibitory protein, such as an arrestin.

[0049] Also provided is an expression vector comprising an isolated nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes a test protein whose interaction with GHSR in the presence of a test compound is to be measured, and (ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for said cleavage site, said nucleic acid further being operably linked to a promoter.

[0050] An additional embodiment comprises a fusion protein produced by expression of: an isolated nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes a test protein (ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, and (iii) a nucleotide sequence which encodes a protein which activates a reporter gene in said cell, and further being operably linked to a promoter; or an isolated nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes a test protein whose interaction with GHSR in the

presence of a test compound is to be measured, and (ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for said cleavage site.

[0051] In yet another embodiment, there is provided a test kit useful for determining if a test compound modulates GHSR activity comprising a separate portion of each of (a) a nucleic acid molecule which comprises, a nucleotide sequence which encodes GHSR (i) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, (ii) a nucleotide sequence which encodes a protein which activates a reporter gene in said cell, and (b) a nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes test protein whose interaction with GHSR in the presence of said test compound is to be measured, (ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for said cleavage site, and container means for holding each of (a) and (b) separately from each other.

[0052] The protease or portion of a protease may be tobacco etch virus nuclear inclusion A protease. The protein which activates said reporter gene may be a transcription factor, such as tTA or GAL4. The test protein may be an inhibitory protein, such as an arrestin. The kit may further comprise a separate portion of an isolated nucleic acid molecule which encodes a reporter gene. The reporter gene may encode β- galactosidase or luciferase. The nucleotide sequence encoding GHSR may be modified to increase interaction with said second test protein, such as by replacing all or part of the nucleotide sequence of the C-terminal region of said first test protein with a nucleotide sequence which encodes an amino acid sequence which has higher affinity for said second test protein than the original sequence. The nucleotide sequence of said C- terminal region may be replaced by a nucleotide sequence encoding the C-terminal region of AVPR2, AGTRLI, GRPR, F2RL1, CXCR2/IL-8B, CCR4, or GRPR.

[0053] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0054] These, and other, embodiments of the invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following description, while indicating various embodiments of the invention and numerous specific

details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions and/or rearrangements may be made within the scope of the invention without departing from the spirit thereof, and the invention includes all such substitutions, modifications, additions and/or rearrangements.

[0055] An aspect of the present invention relates to methods for determining if a substance of interest binds to GHSR and shares properties with CART, or interferes therewith. The methodology involves cotransforming or cotransfecting a cell, which may be prokaryotic or eukaryotic, with two constructs. The first construct includes, a sequence encoding (i) GHSR, such as a transmembrane receptor, (ii) a cleavage site for a protease, and (iii) a sequence encoding a protein which activates a reporter gene. The second construct includes, (i) a sequence which encodes a test protein whose interaction with GHSR is measured and/or determined, and (ii) a nucleotide sequence which encodes a protease or a portion of a protease sufficient to act on the cleavage site that is part of the first construct, In especially preferred embodiments, these constructs become stably integrated into the cells.

Expression Constructs and Transformation

[0056] The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be "exogenous," which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (see, for example, Maniatis, et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor, 1990) and Ausubel, et al., 1994, Current Protocols In Molecular Biology (John Wiley & Sons, 1996), both incorporated herein by reference).

[0057] The term "expression vector" refers to any type of genetic construct comprising a nucleic acid coding for a RNA capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "control sequences," which

refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleotide-sequences that serve other functions as well and are described infra.

[0058] In certain embodiments, a plasmid vector is contemplated for use in cloning and gene transfer. In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. In a non-limiting example, E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, for example, promoters which can be used by the microbial organism for expression of its own proteins.

[0059] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, the phage lambda GEM™"11 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as, for example, E. coli LE392.

[0060] Bacterial host cells, for example, E. coli, comprising the expression vector, are grown in any of a number of suitable media, for example, LB. The expression of the recombinant protein in certain vectors may be induced, as would be understood by those of skill in the art, by contacting a host cell with an agent specific for certain promoters, e.g., by adding IPTG to the media or by switching incubation to a higher temperature. After culturing the bacteria for a further period, generally of between 2 and 24 h, the cells are collected by centrifugation and washed to remove residual media.

[0061] Many prokaryotic vectors can also be used to transform eukaryotic host cells. However, it may be desirable to select vectors that have been modified for the specific purpose of expressing proteins in eukaryotic host cells. Expression systems have been designed for regulated and/or high level expression in such cells. For example, the insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patents 5,871,986 and

4,879,236, both herein incorporated by reference, and which can be bought, tor example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH®.

[0062] Other examples of expression systems include STRATAGENE®'S COMPLETE CONTROL Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E.coli expression system. Another example of an inducible expression system is available from

INVITROGEN®, which carries the T-REX™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter.

INVITROGEN® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

Regulatory Signals

[0063] The construct may contain additional 5' and/or 3' elements, such as promoters, poly A sequences, and so forth. The elements may be derived from the host cell, i.e., homologous to the host, or they may be derived from distinct source, i.e., heterologous.

[0064] A "promoter" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors, to initiate the specific transcription a nucleic acid sequence. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.

[0065] A promoter generally comprises a sequence that functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as, for example, the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of

initiation. Additional promoter elements regulate the frequency ot transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. To bring a coding sequence "under the control of a promoter, one positions the 5 end of the transcription initiation site of the transcriptional reading frame "downstream" of (i.e., 3 of) the chosen promoter. The "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.

[0066] The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0067] A promoter may be one naturally associated with a nucleic acid molecule, as may be obtained by isolating the 5 non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid molecule, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid molecule in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid molecule in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. For example, promoters that are most commonly used in recombinant DNA construction include the -lactamase (penicillinase), lactose and tryptophan (tip) promoter systems. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology,

including PCR™, in connection with the compositions disclosed herein (see U.S. Patents Nos. 4,683,202 and 5,928,906, each incorporated herein by reference). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0068] Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, (see, for example Sambrook, et al, 1989, incorporated herein by reference). The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

[0069] Additionally any promoter/enhancer combination (as per, for example, the Eukaryotic Promoter Data Base EPDB, www.epd.isb-sib.ch/) could also be used to drive expression. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

[0070] A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in- frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

[0071] In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, Nature.

334:320-325(1988)). IRES elements from two members of the picornavirus lamily (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, supra), as well an IRES from a mammalian message (Macejak and Sarnow, Nature. 353:90-94(1991)). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patent Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).

Other Vector Sequence Elements

[0072] Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector (see, for example, Carbonelli, et al, FEMS Microbiol. Lett., 172(1):75-82(1999), Levenson, et al, Hum. Gene Ther,, 9(8):1233-1236(1998), and Cocea, Biotechniques, 23(5):814-816(1997)), incorporated herein by reference.) "Restriction enzyme digestion" refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. "Ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.

[0073] Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression (see, for example, Chandler, et al., 1997, herein incorporated by reference).

[0074] The vectors or constructs of the present invention will generally comprise at least one termination signal. A "termination signal" or "terminator" comprises a DNA sequence involved in specific termination of an RNA transcript by an RNA polymerase.

Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.

[0075] In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 adenosine residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message. The terminator and/or polyadenylation site elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

[0076] Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not being limited to, for example, the termination sequences of genes, such as the bovine growth hormone terminator, viral termination sequences, such as the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as an untranslatable/untranscribable sequence due to a sequence truncation.

[0077] In expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, both of which are convenient, readily available, and known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.

[0078] In order to propagate a vector in a host cell, it may contain one or more origins of replication (often termed "on"), sites ₄ which are specific nucleotide sequences at which replication is initiated. Alternatively, an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

Transformation Methodology

[0079] Suitable methods for nucleic acid delivery for use with the current invention are believed to include virtually any method by which a nucleic acid molecule (e.g., DNA) can be introduced into a cell as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection (Wilson, et al, Science. 244:1344-1346(1989), Nabel et al., Science. 244:1342-1344(1989), by injection (U.S. Patent Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harlan and Weintraub, J. Cell Biol. 101(3):1094-1099(1985); U.S. Patent No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference; Tur-Kaspa, et al., MoI. Cell Biol.. 6:716-718(1986); Potter, et al., Proc. Natl. Acad. Sci. USA. 81:7161-7165(1984); by calcium phosphate precipitation (Graham and Van Der Eb, Virology. 52:456-467(1973); Chen and Okayama, MoI. Cell Biol.. 7(8):2745-2752(1987); Rippe, et al., MoI. Cell Biol.. 10:689- 695(1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, MoI. Cell Biol. 5:1188-190(1985); by direct sonic loading (Fechheimer, et al., Proc. Natl. Acad. Sci. USA. 89(17):8463-8467(1987); by liposome mediated transfection (Nicolau and Sene, Biochem. & Biophvs. Acta.. 721:185-190(1982); Fraley, et al., Proc. Natl. Acad. Sci. USA. 76:3348-3352(1979); Nicolau, et al., Meth. Enzvm.. 149:157-176(1987); Wong, et al., Gene. 10:879-894(1980); Kaneda, et al., Science. 243:375-378(1989); Kato, et al., J. Biol. Chem., 266:3361-3364(1991) and receptor-mediated transfection (Wu and Wu, J. Biol. Chem.. 262:4429-4432(1987); Wu and Wu, 1988); by PEG-mediated transformation of protoplasts (Omirulleh, et al., Plant MoI. Biol.. 21 (3):415-428(1987); U.S. Patent Nos. 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation/inhibition-mediated DNA uptake (Potrykus, et al., MoI. Gen. Genet., 199(2): 169-177(1985), and any combination of such methods.

Components of the Assay System Host Cells

[0080] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to

deliberate or inadvertent mutations. JL ne ήost cells generally will have been engineered to express a screenable or selectable marker which is activated by the transcription factor that is part of a fusion protein, along with GPRl .

[0081] In the context of expressing a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. When host cells are "transfected" or "transformed" with nucleic acid molecules, they are referred to as "engineered" or "recombinant" cells or host cells, e.g., a cell into which an exogenous nucleic acid sequence, such as, for example, a vector, has been introduced. Therefore, recombinant cells are distinguishable from naturally-occurring cells which do not contain a recombinantly introduced nucleic acid.

[0082] Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Cell types available for vector replication and/or expressioninclude, but are not limited to, bacteria, such as E. coli (e.g., E. coli strain RRl, E. coli Lε392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325), DH5, JM109, and KC8, bacilli such as Bacillus subtilis; and other enterobacteriaceae such as Salmonella typhimurium, Serratia marcescens, various Pseudomonas specie, as well as a number of commercially available bacterial hosts such as SURE® Competent

Cells and SOLOPACK Gold Cells (STRATAGENE®, La Jolla). In certain embodiments, bacterial cells such as E. coli LE392 are particularly contemplated as host cells for phage viruses.

[0083] Examples of eukaryotic host cells for replication and/or expression of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.

Test Protein

[0084] The present invention contemplates the use of GHSR and a test protein. The GHSR and test protein, will exist as fusions proteins with GHSR fused to a transcription factor, and the test protein fused to a protease that recognizes a cleavage site in the first fusion protein, cleavage of which releases the transcription factor. The only requirements for the test proteins/fusions are (a) that the first construct be such that the test protein cannot localize to the nucleus prior to cleavage, and (b) that the protease must remain active following both fusion to the test protein and binding of the GHSR to the second test protein.

[0085] With respect to the first construct, either GHSR as a whole and described supra, or portions of GHSR which function in the same manner as the full length first test protein may be used.

Reporters

[0086] The protein which activates a reporter gene may be any protein having an impact on a gene, expression or lack thereof which leads to a detectable signal. Typical protein reporters include enzymes such as chloramphenicol acetyl transferase (CAT), - glucuronidase (GUS) or -galactosidase. Also contemplated are fluorescent and chemilluminescent proteins such as green fluorescent protein, red fluorescent protein, cyan fluorescent protein luciferase, beta lactamase, and alkaline phosphatase.

Transcriptions Factors and Repressors

[0087] In accordance with the present invention, transcription factors are used to activate expression of a reporter gene in an engineered host cell. Transcription factors are typically classified according to the structure of their DNA-binding domain, which are generally (a) zinc fingers, (b) helix-turn-helix, (c) leucine zipper, (d) helix-loop-helix, or (e) high mobility groups. The activator domains of transcription factors interact with the components of the transcriptional apparatus (RNA polymerase) and with other regulatory proteins, thereby affecting the efficiency of DNA binding.

[0088] The Rel/Nuclear Factor kB (NF-kB) and Activating Protein-1 (AP-I) are among the most studied transcription factor families. They have been identified as important components of signal transduction pathways leading to pathological outcomes such as inflammation and tumorogenesis. Other transcription factor families include the heat shock/E2F family, POU family and the ATF family. Particular transcription factors,

such as tTA and GAL4, are contemplated for use in accordance with the present invention.

[0089] Though transcription factors are one class of molecules that can be used, the assays may be modified to accept the use of transcriptional repressor molecules, where the measurable signal is downregulation of a signal generator, or even cell death.

Proteases and Cleavage Sites

[0090] Proteases are well characterized enzymes that cleave other proteins at a particular site. One family, the Ser/Thr proteases, cleave at serine and threonine residues. Other proteases include cysteine or thiol proteases, aspartic proteases, metalloproteinases, aminopeptidases, di & tripeptidases, carboxypeptidases, and peptidyl peptidases. The choice of these is left to the skilled artisan and certainly need not be limited to the molecules described herein. It is well known that enzymes have catalytic domains and these can be used in place of full length proteases. Such are encompassed by the invention as well. A specific embodiment is the tobacco etch virus nuclear inclusion A protease, or an active portion thereof. Other specific cleavage sites for proteases may also be used, as will be clear to the skilled artisan.

Modification of GHSR

[0091] GHSR coding sequences may be modified to enhance their binding to their interacting protein, in this assay. For example, it is known that certain GPCRs bind arrestins more stably or with greater affinity upon ligand stimulation and this enhanced interaction is mediated by discrete domains, e.g., clusters of serine and threonine residues in the C-terminal tail (Oakley, et al., J. Biol. Chem.. 274:32248-32257(1999) and Oakley, et al., J. Biol. Chem., 276:19452-19460(2001). Using this as an example, it is clear that the GPRl encoding sequence itself may be modified, so as to increase the affinity of the membrane bound protein, such as the receptor, with the protein to which it binds. Exemplary of such modifications are modifications of the C-terminal region of the membrane bound protein, e.g., receptor, such as those described supra, which involve replacing a portion of it with a corresponding region of another receptor, which has higher affinity for the binding protein, but does not impact the receptor function.

[0092] In addition, the test protein may be modified to enhance its interaction with the GHSR. For example, the assay may incorporate point mutants, truncations or other variants of the second test protein, e.g., arrestin that are known to bind agonist-

occupied GPCRs more stably or in a phosphorylation-independent manner (Kovoor, et al, J. Biol. Chem., 274:6831-6834(1999).

Assay Formats

[0093] As discussed above, the present invention, in one embodiment, offers a straightforward way to assess the interaction of GHSR and a test protein, thereby determining the activity of a test compound. A first construct, as described supra, comprises a sequence encoding a GHSR, concatenated to a sequence encoding a cleavage site for a protease or protease portion, which is itself concatenated to a sequence encoding a reporter gene activator. By "concatenated" is meant that the sequences described are fused to produce a single, intact open reading frame, which may be translated into a single polypeptide which contains all the elements. These may, but need not be, separated by additional nucleotide sequences which may or may not encode additional proteins or peptides. A second construct inserted into the recombinant cells is also as described supra, i.e., it contains both a sequence encoding a second protein, and the protease or protease portion. Together, these elements constitute the basic assay format when combined with a candidate agent whose effect on target protein interaction is sought.

[0094] Other features of the invention will be clear to the skilled artisan and need not be reiterated here.