WO2004060282A2

WO2004060282A2 - Uses of the snorf207 receptor

Info

Publication number: WO2004060282A2
Application number: PCT/US2003/039465
Authority: WO
Inventors: Liliana Delgado; Karim Berrada; Roman Artymyshyn
Original assignee: Synaptic Pharmaceutical Corporation
Priority date: 2002-12-19
Filing date: 2003-12-10
Publication date: 2004-07-22
Also published as: AU2003300867A8; WO2004060282A3; AU2003300867A1

Abstract

The invention provides methods of treating an abnormality that is linked to the activity of the mammalian SNORF207 receptor, as well as methods of determining binding of compounds to the mammalian SNORF207 receptor, methods of identifying agonists and antagonists of the SNORF207 receptor, and agonists and antagonists so identified.

Description

USES OF THE SNORF207 RECEPTOR

BACKGROUND OF THE INVENTION

This application claims priority of U.S. Serial No. 10/323,611, filed December 19, 2002, the entire contents of which are hereby incorporated by reference.

Throughout this application various publications are referred to by partial citations within parenthesis. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the invention pertains.

G-proteir- coupled receptors (GPCRs) represent a .a or class of cell surface receptors with which many biomclecules interact to mediate their effects. GPCRs are characterized by seven membrane- spanning domains interconnected with three intracellular loops and three extracellular loops . Binding of a bicmolecule tc its cognate G protein-coupled receptor typically triggers a change in cellular physiology resulting from activation, stabilization, or inactivation of the G proteins coupled to the receptor. This marks the beginning of a biochemical cascade that may result in the production of second messengers such as cA ? or the accumulation of intracellular calcium,. >.hιle primary structural motifs that characterize GPCRs can be recognized in the predicted amine acid sequence of a novel receptor, the endogenous cognate lιganα(s) cannot always oe inferred from, tne primary structure cr tr-e i-.-'.K. iπus, a nove_ receptor sequence is designated an orphan GPCR when it possesses structural motifs characteristic of GPCRs but lacks an identified cognate ligand. Identification of the endogenous ligand or other ligands that interact with an orphan receptor (i.e. "deorphanizing" ) greatly facilitates the assessment of both its physiological roles and therapeutic utility.

GPCR ligands comprise a diverse group of molecules that subserve or modulate communication between cells . They include, but are not limited to, neuropeptides , amino acids, biogenic amines, lipids and lipid metabolites, ether m.etabolic byproducts, and synthetic molecules. Determination of the endogenous (cognate) ligand acting through an orphan GPCR in vivo can be problematic. Endogenous biomolecules that may not normally interact with a given GPCR (due to differential localization or compartmentalization) may be shown to interact with the GPCR at high potency in vi zrc ; endogenous biomolecules other than the cognate ligand may activate or modulate the GPCR at reduced potencies; finally, GPCRs can be activated or mcdulateα by ligands ether tnan the cognate ligand or other endogenous biomolecules. The discovery of any ligand that activates or modulates an orphan GPCR through direct molecular interaction is extremely advantageous. For example, the identification of activating ligands which mimic tne effects of an endogenous ligand allows both the characterization of the receptor signal transduction pathway and the development of methods for screening for compounds (agonists cr antagonists) that activate or block receptor function. Such agonists or antagonists perm.it determination of the biochemical role of the receotor in normal and oatnological states, a a thus tne therapeutic pctentia-- of drugs that would act at the receptor. The use of such- agonists accelerates the discovery of an endogenous ligand through iterative structure/function analyses and datamming. Finally, the discovery of such agonists may further shed light on the physiological and biological function of a GPCR, leading to the discovery of new therapies to treat various human disorders. The present invention describes methods for identifying compounds that bind to, bind to and activate, cr bind to and inhibit the activation of the GPCR designated S CRF207. The present invention identifies SNORF207 agonists. Furthermore, the present invention describes the use of SXORF2C7 agonists and/or antagonists m the treatment of disorders, including, but not limited to, metabolic disorders, inflammation, and pain.

SUMMARY OF THE INVENT!UK

This invention provides a process for identifying a chemical compound which specifically binds to a mammalian S ORF207 receptor which comprises contacting cells containing DXA encoding, and expressing on their cell surface, the mammalian SNORF207 receptor, wherein such cells do not normally express the mammalian SNORF207 receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian SKORF207 receptor.

Furthermore, this invention provides a process for identifying a chemical compound which specifically binds to a mammalian SKORF207 receptor which comprises contacting a membrane preparation from ceils containing DNA encoding, and expressing en their cell surface, the mammalian SNORF207 receptor, wherein such cells do not normally express the mammalian SKORF207 receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian SNORF207 receptor.

Moreover, this invention provides a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian SXORF2C7 receptor which comprises separately contacting cells expressing en their cell surface the mammalian SNORF2C7 receptor, wherein such cells do not normally express the mammalian SNORF207 receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of such compounds to the receptor, and detecting specific binding of the chem.icai

- L - compound to tne mammalian receptor, a decrease in the binding of the second chemical com.pound to the mam-talian SKORF2C7 receptor in the presence of the chemical compound being tested indicating that such chemical compound binds to the m.ammalian SNORF207 receptor .

This invention also provides a process involving competitive binding for identifying a chemical compound which specifically binds to a miammalian SNORF207 receptor which comprises separately contacting a membrane preparation from cells expressing on their cell surface the mamrr.alian SNCRF207 receptor, wherein such cells do not norm.aliy express the mammalian SXORF207 receptor, with both the chemical com.pound and a second chemical com.pound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of such compounds to the receptor, and detecting specific binding of the chem.icai com.pound to the mamrr.alian SKORF207 receptor, a decrease m the binding of the second chemical compound to the m.ammalian SNORF207 receptor in the presence of the chemical compound being tested indicating that such chemical compound binds to the mammalian. SNORF207 receptor.

This invention further provides a method of screening a plurality of chemical compounds not known to bind to a m.ammalian SNORF2C7 receptor to identify a compound which specifically bir.αs to the mam alian SNORF207 receptor, which comprises (a) contacting cells transfected w th, and expressing, DNA encoding the mammalian SNORF2C7 receptor with a compound .<nown to bine specifically to the mammalian SKCRF207 receptor; (b) contacting the cells of step (a) with the plurality of compounds not known to bind specifically to the mammalian SNORF2U/ receptor, under conditions permitting binding of compounds known to bind to the mammalian SNORF2C7 receptor; (c) determining whether the binding of the compound known to bind to the mammalian SNORF207 receptor is reduced m the presence of the plurality of compounds, relative to the oinding of the com.pound in the aosence of the plurality of compounds; and if so (d) separately determining the binding to the mammalian SXORF207 receptor of each com.pound included in the plurality of compounds, so as to thereby identify any compound included therein which specifically binds to the m.ammalian SNORF207 receptor.

This invention still further provides a method of screening a plurality of chemical compounds not known to bind to a mamrr.alian SXORF207 receptor to identify a compound which specifically binds to the mammalian SNCRF207 receptor, which comprises (a) contacting a membrane preparation from ceils transfected with, and expressing, DNA encoding the mammalian SNORF207 receptor with the plurality of compounds not known to bind specifically to the m.ammalian 5XORF2C7 receptor under conditions permitting binding of compounds known to oind to the mammalia SNORF207 receptor; (b) determining whether the binding of a compound known to bind to the Lam.alian SXORF207 receptor is reduced m the presence of the plurality of compounds, relative to tne binding of the compound in the absence of the plurality of compounds; and if so (c) separately determining the binding to the mammalian SNORF207 receptor of each compound included in the plurality of compounds, so as to thereby identify any compound included therein which specifically binds to the mammalia SNORF207 receptor. This invention also provides a process ror determining whether a chemical compound is a m.ammalian SNORF207 receptor agonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian SNORF207 receptor with the compound under conditions permitting the activation of the mammalian SNORF207 receptor, and detecting any increase in mammalian SNCRF207 receptor activity, so as to thereby determine whether the compound is a mammalian SNORF207 receptor agonist.

This invention further provides a process for determining whether a chemical compound is a m.ammalian SNORF207 receptor antagonist which comprises contacting cells transfected with and expressing DXA encoding the mammalian SNORF207 receptor with the compound in the presence of a known mammalian SNORF207 receptor agonist, under conditions permitting the activation of the mammalian SNCRF2C7 receptor, and detecting any decrease in mammalian SNORF2C7 receptor activity, so as to thereby determine whether the compound is a mammalian SXORF207 receptor antagonist.

Moreover, this invention provides a process for determining whether a chemical compound specifically binds to and activates a mammalian SNORF207 receptor, which comprises contacting cells producing a second messenger response and expressing on their cell surface the mammalian SNORF2C7 receptor, wherein such cells do not normally express the mammalian SXORF2G7 receptor, with the chemical compound under conditions suitable for activation of the m.ammalian SXORF207 receptor, and measuring the second messenger response in the presence and in the absence of the chemical compound, a change m the second messenger response m the presence of the chem.ical compound indicating that the compound activates the mammalian SXORF207 receptor.

This invention further provides a process for determining whether a chemical compound specifically binds to and inhibits activation of a mammalian SNORF2C7 receptor, which comprises separately contacting cells producing a second messenger response and expressing on their cell surface the mammalian SNORF2C7 receptor, wherein such cells do not normally express the mammalian SNORF207 receptor, with both the chemical compound and a second chemical compound known to activate the mammalian SXORF207 receptor, and with only the second chemical compound, under conditions suitable for activation of the m.ammalian SNORF207 receptor, and measuring the second messenger response in the presence of only the second chemical compound and in the presence of both the second chem.ical compound and the cnemical compound, a smaller change in the second messenger response in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound indicating that the chemical compound inhibits activation of the m.ammalian SNORF207 receptor.

This invention provides a method of screening a plurality of chemical compounds net known to activate a mammalian SXORF207 receptor to identify a compound which activates the mammalian SNORF2C7 receptor which comprises: (a) contacting cells transfected with and expressing the mammalian SNORF207 receptor with the plurality of compounds not known to activate the mammalian SNORF207 receptor, under conditions permitting activation of the mammalia SKORF207 receptor; (b) determining whether the activity of the m.ammalian SNORF20/ receptor is increased in the presence of one or more of the compounds; and if so (c) separately determining whether the activation of the mammalian SXORF207 receptor is increased by any compound included in the plurality of compounds, so as to thereby identify each compound which activates the maiτt .aiian SNORF2C7 receptor.

This invention further provides a method of screening a plurality of chem.ical compounds not known to inhibit the activation of a rr.amm.alian SNORF207 receptor to identify a compound which inhibits the activation of the mammalian SXORF207 receptor, which comprises: (a) contacting cells transfected with and expressing the m.ammialian SNORF207 receptor with the plurality of compounds in the presence of a known m.ammalian SNORF207 receptor agonist, under conditions permitting activation of the -mammalian SNORF2C7 receptor; (b) determining whetner the extent or amount of activation of the mammalian SNORF207 receptor is reduced m the presence of one or more of the compounds, relative to the extent or amount of activation of the m.ammalian SNORF2C7 receptor in the absence of such one or m.ore compounds; and if so (c) separately determining whether each such compound inhibits activation of the mammalian SNORF207 receptor for each compound included in the plurality of compounds, so as to thereby identify any compound included in such plurality of compounds which inhibits the activation of the mammalian 3NCRF207 receptor.

This invention additionally provides a method of treating an abnormality m a subject wherein the aor.ormality is alleviated by increasing the activity of a mammalian SNORF207 receptor which comprises adminis ering to the subject a compound which is a m.ammialian S-*J*JRF_-U / receptor agonist m an amount effective to treat the abnormality.

This invention further provides a m.etnod of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a mammalian SXORF207 receptor which comprises administering to the subject a compound which is a mammalian SNORF207 receptor antagonist m an amount effective to treat the abnormality .

This invention provides an isolated nucleic acid encoding a mammalian SXORF2C7 receptor.

This invention further provides a purified mammalian SNORF207 receptor protein.

Furthermore, this invention provides a nucleic acid probe comprising at least 15 nucleotides, which prcbe specifically hybridizes with a nucleic acid encoding a m.ammalian SXCRF207 receptor, wnerem the probe has a sequence complementary to a unique sequence present within one of the two strands of the nucleic acid encoding the human SNORF207 receptor contained in plasmid MSP70-hSXORF2C7-f (ATCC Patent Deposit Designation PTA- 4790) .

This invention provides a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian SXORF207 receptor, wherein the probe has a sequence complementary to a unique sequence present within (a) the nucleic acid sequence shown in Figures 1A-13 (SEQ ID NO: 1) or (b) the reverse complement thereof. Furthermore, this invention provides a m.ethod for diagnosing a predisposition to a disorder associated with the activity of a specific mammalian allele which comprises: (a) obtaining DNA of subjects suffering from the disorder; (b) performing a restriction digest of the DNA with a panel of restriction enzymes; (c) electrophoretically separating the resulting DXA fragments on a sizing gel; (d) contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a mammalian SNORF207 receptor and labeled with a detectable marker; (e) detecting labeled bands which have hybridized to the DNA encoding a mammalian SNORF207 receptor of this invention to create a unique band pattern specific to the DNA of subjects suffering from the disorder; (f) repeating steps (a) -(e) with DNA obtained for diagnosis from, subjects not yet suffering from, the disorder; and (g) comparing the unique band pattern specific to the DXA of subjects suffering, from the disorder from step (e) with the band pattern from, step (f) for subjects not yet suffering from the disorder so as to determine whether tne patterns are the same or different and tnereoy diagnose predisposition to the disorder if tne patterns are the same .

This invention further provides a transgenic, nonhuman mammal comprising a homologous recombination knockout of the native mammalian SXORF207 receptor.

ι ι BRIEF DESCRIPTION OF THE FIGURES

Figures 1A-1B

Nucleotide sequence including the sequence encoding a human SNORF207 receptor (SEQ ID NO: 1) . The putative open reading fram.e is indicated by underlining the start (ATG) coder, (at positions 1-3) and the stop codon (at positions 1117-1119) . In addition, a partial 3' untranslated sequence is shown.

Figures 2A-23

Deduced amino acid sequence (SEQ ID NO: 2) of the human SNORF207 receptor encoded by the open reading frame indicated in the nucleotide sequence shewn in Figures 1A- 1B (SEQ ID NO: 1) . The seven putative transmembrane (TM) regions are underlined.

Figures 3A-3B

Exaunple 1 activates SNORF 207. CHO ceils were transiently transfected with human SXORF207 or with expression vector only (mock-transfected) , then prepared for assay by FLIPR^rϊ": as described in Materials and Methods. Intracellular calcium concentrations were m.onitored as relative fluorescence units (RFU) every 1.5 seconds. After β seconds, cells were exposed to various concentrations of Example 1 (DMSC = 1%), and fluorescence monitoring continued. A transient and saturable increase in RFU was observed within 20 seconds of exposure for hSXORF207-trar.sfected cells (Figure 3A) . The m.axim.al change in RFU was plotted vs. ; Example 1] to derive an Eπ*a_>::r.s::cRr2C _; of 1C,2C0 RFU and an EC₅₃ ( s.joRF-.T) of 1.6 x 10^"5 M (Figure 3B, wherein each point represents an average of 4 replicate measurements) . hSN0RF207-trar.sfected ceils were markedly more sensitive to Example 1 than mock-

- 17 - transfected cells, which yielded a only a small response (<50% of E_maX(_hs_NORF20 ) ) at the highest concentration tested (le^-4 M, Figure 3B) .

Figures 4A-4B

Example 2 activates SNORF 207. CHO ceils were transiently transfected with r.uman SNORF207 or with expression vector only (mock-transfected) , then prepared for assay by F1I?R^T:': as described in Materials and Methods. Intracellular calcium, concentrations were monitored as relative fluorescence units (RFU) every 1.5 seconds. After 6 seconds, cells were exposed to various concentrations of Example 2 (DMSO ■= 1%), and fluorescence monitoring continued. A transient and saturable increase in RFU was observed within 30 seconds of the experimental record for h.SNORF2 C7-transfected cells (Figure 4A) . The maxim.al cna ge m RFU was plotted vs. [Example 2] to derive an Ξ_.τaκ of 9600 RFU and an EC₅₀ of 4.1 x 1C^"6 M (Figure 43, wherein each point represents an average of 4 replicate measurements) . hSNORF207- transfected cells were markedly .cre sensitive to Example 2 than mock-transfected cells, which yielded a only a small response (<50 of E_π._a._:/_nS;;_CRF2C7; ) £~ the highest concentration tested (le^""' M, Figure 4B) .

Figures 5A-5D

Activation of calcium-activated chloride currents in Xenopus la evis oocytes expressing human 3NORF207 (hSNORF2C7) .

Ξlectrophysiological responses of a non-m; ected, voltage clamped oocyte (Figure 5A) vs. a voltage clamped oocyte injected wit.. cDNA encoding hSNORF207 (Figure 5B) to application of 50 uM Example 1. Each arrow indicates the bullet application of Example 1. (Representative of IU separate experiments.)

Electrophysiological responses of a non-injected, voltage clamped oocyte (Figure 50) vs. a voltage clamped oocyte injected with cDNA encoding hSNORF207 (Figure 5D) to application of 50 uM Example 2. Each arrow indicates the bullet application of Example 2. (Representative of 5 separate experiments.)

DETAILED DESCRIPTION OF THE INVENTION

This invention provides for a process for identifying a chemical com.pound which specifically binds to a m.ammalian SNORF207 receptor which comprises contacting cells transfected with DXA encoding, and expressing on their cell surface, the mammalian SNORF2C7 receptor, wherein such ceils prior to being transfected with sucn DNA do not normally express the mammalian SNORF2Q7 receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chem.ical compound to the mammalian SXORF207 receptor.

This invention further provides for a process for identifying a chemical compound which specifically binds to a mammalian SNCRF207 receptor which comprises contacting a membrane preparation from, cells transfected with DNA encoding and expressing on their cell surface the m.am.aliar. SNORF207 receptor, wherein such cells prior to being transfected with such DNA do not normally express the mammalian SNORF207 receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian SXORF207 receptor.

This invention still further provides a process involving competitive binding for identifying a chemical com.pound which specifically binds to a mammalian SNORF207 receptor which comprises separately contacting cells transfected with DNA encoding and expressing on their cell surface the mammalian SNORF207 receptor, wherein such cells prior to being transfected with such DNA do not normally express the mammalian SNCRF207 receptor, with both the chemical compound and a second chemical compound known to

_ - ς _ bind to tne receptor, and w th on.y tne seconα cnemicai compound, under conditions suitable for binding of such compounds to the receptor, and detecting specific^' binding of the chemical compound to the mammalian SNORF207 receptor, a decrease in the binding of the second chem.ical compound to the mammalian SXORF207 receptor in the presence of the chem.ical compound being tested indicating that such chemical compound binds to the mammalian SXORF207 receptor.

This invention provides a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian SNORF2C7 receptor which comprises separately contacting a membrane preparation from, cells transfected with DNA encoding ana expressing on their cell surface the mammalian. SNORF207 receptor, wherein such cells prior to oeing transfected with such DNA do not normally express the m.ammalian SNORF2C7 receptor, with both the chemical compound and a second chemical com.pound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of such compounds to the receptor, and detecting specific binding of the chemical compound to the mammalian SNORF2C7 receptor, a decrease in the binding of the second chemical compound to the mammalian SXORF2C7 receptor in the presence of the chemical compound being tested indicating that such chemical compound oinds to the mammalian SXORF207 receptor.

In an embodiment of the present invention, tne second chemical compound s 7- [ 2 - ( 2-chlorophenyl ) -4- (2- hyd o yphe yl )-l,3-dioxar.-cιs-5-yl]he -4z-enoic acid or n-linolecylglycine . In an embodiment, the mammalian SNORF20/ receptor is a human SNORF2C7 receptor. In another embodiment, the miammalian SXORF207 receptor has substantially the same amino acid sequence as the human SNORF207 receptor encoded by plasmid MSP70-hSNORF207-f (ATCC Patent Deposit Designation PTA-4790) .

In another embodiment, the mammalian SNORF207 receptor has substantially the same amino acid sequence as that shown in Figures 2A-2B (SEQ ID XO: 2) . In another embcdim.ent, the mammalian SXORF207 receptor has the amino acid sequence shown in Figures 2A-23 (SEQ ID NO: 2) .

In one embodiment, the compound is not previously known to bind to a mammalian SNORF2C7 receptor. In one embodiment, the cell is an insect cell. In one emocdiment, the cell is a mammalian, cell. In another embcdim.ent, the cell is r.or.r.euronai m origin. In another embodiment, the nonneuronai cell is a OOS-7 cell, a human embryonic kidney cell, a CHO cell, a NIH-3T3 cell, a mouse Yl cell, or a LM(tk-) cell. In another embodiment, the compound is a compound not previously known to bind to a m.ammalian SNORF207 receptor. This invention provides a compound identified by the preceding processes according to this invention.

Methods for preparing transfected cells and membrane preparations from, such cells are described hereinafter.

This invention provides for a m.etnod of screening a plurality of chemical compounds not known to bmo to a mammalian SNORF207 receptor to identify a compound which specifically binds to the m.ammialian SNORF207 receptor, which comprises (a) contacting cells transfected with, and expressing, D.\A encoding tne m.amm.a ian NUKt^./ receptor with a compound known to bind specifically to the m.ammialian SNORF207 receptor; (b) contacting the cells of step (a) with the plurality of compounds not known to bind specifically to the mammalian ΞNORF207 receptor, under conditions permitting binding of compounds known to bind to the m.amm.aiian SXORF207 receptor; (c) determining whether the binding of the compound known to bind to the mamrr.alian SXORF207 receptor is reduced in tne presence of the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so (d) separately determining the binding to the mammalian SNORF207 receptor of each compound included in the plurality of compounds, so as to thereby identify any compound included therein which specifically binds to the m.ammalian SNORF207 receptor.

This invention provides a method of screening a plurality of chemical compounds not known to bind to a m.ammalian SNORF207 receptor to identify a compound which specifically binds to the miammalian SNCRF2C7 receptor, which comprises (a) contacting a membrane preparation from cells transfected with, and expressing, DNA encoding the miammalian SNORF2C7 receptor with the plurality of compounds not known to bind specifically to tne mammalian SNCRF207 receptor under conditions permitting binding of compounds known to bind to the mammalian SNORF2C7 receptor; (b) determining whether the binding of a compound known to bind to the m.ammalian SNORF207 receptor is reduced in the presence of the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so (c) separately determining the binding to the mammalian SNORF2C7 receptor of each compound included in the plurality of compounds, so as to tnerepy identity any compound included therein which specifically binds to the mammalian SNORF2C7 receptor.

Furthermore, this invention provides a process for determining whether a chem.ical compound is a mammalian SNORF207 receptor agonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian SNORF207 receptor with the compound under conditions permitting the activation of the mamrr.alian SNORF207 receptor, and detecting any increase in mammalian SNORF2C7 receptor activity, so as to thereby determine whether the compound is a mammalian SNORF207 ^"receptor agonist.

This invention further provides a process for determining whether a chemical compound is a mammalian SNORF2C7 receptor agonist which comprises contacting cells transfected with and expressing DNA encoding the mamrr.alian SNORF207 receptor with the compound under conditions permitting the activation of the mammalian 3NORF207 receptor, and detecting any increase in mammalian SNORF207 receptor activity, so as to thereby determine whether the compound is a m.ammalian SNORF207 receptor agonist.

This invention also provides a process for determining whether a chemical compound is a m.ammalian SNORF C7 receptor antagonist which comprises contacting cells transfected with and expressing DXA encoding the mammalian SNORF207 receptor with the compound in the presence of a kr.owr. mammalian SXORF207 receptor agonist, under conditions permitting the activation of the mammalian SNORF207 receptor, and detecting any decrease in mammalian SNORF 0/ receptor activity, so as to tnerepy determine whetner the compound is a m.ammalian SNORF207 receptor antagonist.

This invention also provides a process for determining whether a chemical compound is a mammalian SNORF207 receptor antagonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian SNORF207 receptor with the compound in the presence of a known mammalian SNORF207 receptor agonist, under conditions permitting the activation of the mammalian SNORF207 receptor, and detecting any decrease m mammalian SNORF2C7 receptor activity, so as to thereby determine whether the compound is a mammalian SNORF207 receptor antagonist.

In an embodiment, the mammalian SNORF207 receptor is a human SNORF207 receptor. In another embodiment, the m.amm.aiian SXORF2C7 receptor has substantially the same amino acid sequence as the human SNORF2C7 receptor encoded by plasmid MSP70-hSXORF207-f (ATCC Patent Deposit Designation PTA-4790) .

In another embodi .ent, the m.amm.aiian SXORF207 receptor has substantially the same amino acid sequence as that shown m Figures 2A-23 (SEQ ID XC : 2). In another emαcdiment, the mammalian SXORF207 receptor has the amino acid sequence shewn in Figures 2A-23 (SEQ ID NO: 2) .

In one embodiment, tne compound s net previously known to bind to a m.amm.aiian SXORF207 receptor.^' In one embodiment, the cell is an insect cell. In one embodiment, the cell is a m.ammalian cell. In another embodim.ent, the cell is nonneuronal in origin. In another embodiment, the nonneuronal ce--_ is a iυ--/ ceι_, a nuiuaii embryonic kidney cell, a CHO cell, a NIH-3T3 ceil, a mouse Yl cell, cr a LM(tk-) cell, In another embodiment, the com.pound is a compound not previously known to bind to a m.ammalian SNORF207 receptor. This invention provides a compound identified by the preceding processes according to this invention.

Methods for preparing transfected ceils and membrane preparations from such cells are described hereinafter.

This invention still further provides a composition, for example a p.narmaceutical composition, which comprises an amount of a mammalian SNORF207 receptor agonist determined by a process according to this invention effective to increase activity of a mammalian SNORF207 receptor and a carrier, for example, a pharmaceutically acceptaole carrier. In one embodiment, the mammalian SNORF2C7 receptor agonist s not previously known.

Also, this invention provides a composition, for example a pharmaceutical composition, which comprises an amount of a mamrr.alian SNORF207 receptor antagonist determined by a process according to this invention effective to reduce activity of a mamm.alian SNORF207 receptor and a carrier, for example, a pharmaceutically acceptable carrier. Also, this invention provides a com.position, for example a pharmaceutical composition, which comprises an amount of a mammalian SXORF207 receptor antagonist determined by a process according to this invention effective to reduce activity of a m.ammalian SNORF2C7 receptor and a carrier, for example, a pharmaceutically acceptaole carrier. In one embodiment, tne mammalian ΛOΛ- u / receptor antagonist is not previously known. In an embodiment, the mammalian SNORF207 receptor antagonist is a human SNORF207 receptor antagonist.

This invention moreover provides a process for determining whether a chemical compound specifically binds to and activates a mammalian SNCRF207 receptor, which comprises contacting ceils producing a second messenger response and expressing on their cell surface the mammalian SNORF207 receptor, wherein such cells do not normally express the mammalian SNORF207 receptor, with the chemical com.pound under conditions suitable for activation of the mammalian SNORF2C7 receptor, and m.easuring the second messenger response in the presence and m the absence of the chemical compound, a change, e.g. an increase, m the second messenger response in the presence of the chemical compound indicating that the compound activates the mammalian SNORF207 receptor.

In one embodim.ent, the second messenger response comprises chloride channel activation and the change m second messenger is an increase in the level of chloride current. In another embodim.ent, the second messenger response comprises change in intracellular calcium levels and the change m second messenger is an increase in the measure of intracellular calcium. In another embodiment, the second messenger response comprises release of inositol phosphate and the change in second messenger is an increase in the level of inositol phosphate. In another embodiment, the second messenger response comprises release of arachidonic acid and the change m second messenger is an increase m the level of arachidonic acid. In yet another embodiment, the second messenger response comprises Gl-yb --iganα tunαing and tne change in second messenger is an increase in GTPγS ligand binding. In another embodiment, the second messenger response comprises activation of MAP kinase and the change m second messenger response is an increase in MAP kinase activation. In a further embodiment, the second messenger response comprises cAMP accumulation and the change in second messenger response is a reduction in cAMP accumulation.

This invention still further provides a process for determining whether a chemical compound specifically binds to and inhibits activation of a mamm.alian SNORF207 receptor, which comprises separately contacting cells producing a second messenger response and expressing on their cell surface the miammalian SNORF207 receptor, wherein such cells do not normally express the mamm.alian SNORF207 receptor, with both the chemical compound and a second chemical compound known to activate the miammalian SNORF207 receptor, and with only the second chemical compound, under conditions suitable for activation of the mammalian SNORF2C7 receptor, and measuring the second messenger response in the presence of only the second chemical compound and in the presence of both the second chemical compound and the chemical compound, a smaller change, e.g. increase, n tne second messenger response in the presence of both the chem.ical compound and the second chem.ical compound than in the presence of only the second chem.ical compound indicating that the chemical compound inhibits activation of the mammalian SNORF207 receptor .

In one embodiment of the present invention, the second chem.ical compound is 7- [2- ( 2-chlcropher.yl ) -4- (2- hydroxyphenyl) -1 , 3-cιoxan-cιs-o-y_ J r.ex-.z-er.oic acid or r.-linoleoylglycine .

In one embodiment, the second messenger response comprises chloride channel activation and the change in second messenger response is a smaller increase in the level of chloride current in the presence of both the chemical compound and the second chem.ical compound than in the presence of only the second chemical compound. In another embodiment, the second messenger response comprises change in intracellular calcium levels and the change in second messenger response is a smaller increase in the measure of intracellular calcium in the presence of both the chem.ical compound and the second chemical com.pound than in the presence of only the second chemical compound. In another embodiment, the second messenger response comprises release of inositol phosphate and the change in second messenger response is a smaller increase m the level of mcsitol phosphate m the presence of both the chem.ical compound and t e second chemical compound than in the presence of only the second chemical compound .

In one embodiment, the second messenger response comprises activation of MAP kinase and the change in second messenger response is a smaller increase in the level of MAP kinase activation in the presence of both the chem.ical compound and the second chem.ical compound than m the presence of only the second chem.ical compound. In another embodiment, the second messenger response comprises change in cAMP levels and the change m second messenger response is a smaller change in the level of cAMP m the presence of both the chemical compound and the second chemical compound than m the presence of only the second chemical compound. In another embodiment, the second messenger response comprises release of aracnidonic acid and the change in second messenger response is an increase m the level cf arachidonic acid levels m the presence of both the chemical compound and the second cnemical compound than in the presence of only the second chemical compound. In a further embodiment, the second messenger response comprises GTPγS ligand binding and the change in second messenger is a smaller increase in GTPyS ligand binding in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound.

In an embodiment, the mammalian SXORF207 receptor is a human SNORF207 receptor. In another embodiment, the mamm.alian 3NORF207 receptor has sudstantialiy the same am.mo acid sequence as the numan SNORF207 receptor encoded by plasmid MSP7 -hSXORF2 C7-f (ATCC Patent Deposit Designation PTA-4790) .

In another embodiment, the mammalian SNORF2C7 receptor has substantially the sam.e amino acid sequence as that shown in Figures 2A-2B (SEQ ID NO: 2) . In another embodiment, the mamm.alian SNORF207 receptor has the amino acid sequence sr.owr. in Figures 2A-2B (SEQ ID NO: 2) .

In one embcdim.ent, the compound is not previously known to bind to a m.ammalian SXORF207 receptor. In one embodiment, the cell is an insect cell. In one embodim.ent, tne cell is a m.amm.aiian cell. In another embodiment, the cell is nc r.euror.al in origin. In another embodiment, the nonneurcnal cell is a COS-7 cell, a human emcrycnic kidney cell, a CHO cell, a XIH-3T3 cell, a mouse Yl cell, or a LM(tk-) cell. In another embodiment, the compound is a compound not previously known to bind to a mammialian SNORF207 receptor. This invention provides a compound identified by the preceding processes according to this invention.

Methods for preparing transfected cells and membrane preparations from such cells are described hereinafter.

Further, this invention provides a compound determined by a process according to this invention and a composition, for example, a pharmaceutical composition, which comprises an amount of a mammalian SNORF207 receptor agonist determined to be such by a process according to this invention effective to increase activity of a mammalian SXORF207 receptor and a carrier, for example, a pharmaceutically acceptable carrier. In one embodiment, the mammalian SNORF2C7 receptor agonist is not previously known .

This invention also provides a composition, for example, a pharmaceutical composition, which comprises an amount of a m mm.alian SNORF207 antagonist determined to be such by a process according to this invention, effective to reduce activity of the mammalian SXORF207 receptor and a carrier, for example a pharmaceutically acceptable carrier. In one embodiment, the mammalian SNOR.F2C7 antagonist is not previously known.

This invention yet further provides a m.etnod of screening a plurality of chemical compounds not known to activate a m.amm.aiian SNORF2C7 receptor to identify a compound which activates the m.amm.aiian SNORF207 receptor which comprises: (a) contacting cells transfected with and expressing the mammalian SNO t-.u/ receptor witn tne plurality of compounds not known to activate the mammalian SNORF207 receptor, under conditions permitting activation of the mammalian SXORF207 receptor; (b) determining whether the activity of the mammalian SNORF207 receptor is increased in the presence of one or more of the compounds; and if so (c) separately determining whether the activation of the mammalian SXORF207 receptor is increased by any compound included in the plurality of compounds, so as to thereby identify each compound whic activates the mammalian SNORF2C7 recepto .

This invention provides a method of screening a plurality cf chem.ical compounds not known to inhibit the activation of a mammalian SNORF207 receptor to identify a compound which inhibits the activation of the mammalian SNORF207 receptor, which comprises: (a) contacting cells transfected with and expressing the m.ammalian SNORF2C7 receptor with the plurality of compounds in the presence of a known mamm.alian SNORF207 receptor agonist, under conditions permitting activation of the mammalian SNORF2C7 receptor; (b) determining whether the extent cr amount of activation of tne mamm.alian SXORF207 receptor is reduced in the presence of one cr m.ore of the compounds, relative to t e extent cr amount of activation of the mammalian SNORF2C7 receptor m the absence of such one or m.ore compounds; and if so (c) separately deter .ining whether each such compound inhibits activation of the mamm.alian SXORF207 receptor for each compound included in the plurality of compounds, so as to thereby identify any compound included in such plurality of compounds which inhibits the activation cf the m.ammalian SNORF207 receptor. In an embodiment, the mammialian SNORF207 receptor is a human SNORF2C7 receptor. In another em.bodiment, the mammalian SXORF2C7 receptor has substantially the same amino acid sequence as the human SXORF207 receptor encoded by plasmid MSP70-hSNORF207-f (ATCC Patent Deposit Designation PTA-4790) .

In another embodiment, the mammalian SNORF207 receptor has substantially the same amino acid sequence as that shown in Figures 2A-2B (SEQ ID NO: 2) . In another em.bodiment, the mammalian SNORF207 receptor has the amino acid sequence shown n Figures 2A-2B (SEQ ID NO: 2) .

In one embodiment, the compound is not previously known to bind to a mammalian SXORF207 receptor. In one embodiment, the cell is an insect cell. In one embodiment, the cell is a mamm.alian cell. In another embodiment, the ceil is nonneuronal in origin. In another embodiment, the nonneuronal cell is a CC3-7 cell, a human embryonic kidney cell, a CHO cell, a XIH-3T3 cell, a mouse Yl cell, or a IM(tk-) cell. In another em.bodiment, the compound is a compound not previously known to bind to a mammalian SNORF207 receptor. This invention provides a compound identified by the preceding processes according to this invention.

This invention also provides a composition, for example, a pharmaceutical composition, comprising a compound identified by a method according to this invention in an amount effective to increase mammalian SXORF2C7 receptor activity and a carrier, tor example, a pharmaceutically acceptable carrier.

This invention still further provides a composition, for example, a pharmaceutical composition, comprising a compound identified by a method according to this invention m an amount effective to decrease mammalian SNORF207 receptor activity and a carrier, for example, a pharmaceutically acceptable carrier.

Furthermore, this invention provides a m.etnod of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian SNORF207 receptor which comprises administering to the subject a compound which is a mammalian SXORF207 receptor agonist in an amount effective to treat the abnormality.

This invention additionally provides a m.ethod cf treating an abnormality in a suoject wherein the abnormality is alleviated by decreasing the activity of a mammalian SNORF207 receptor which comprises administering to the subject a compound which is a mammalian SNORF207 receptor antagonist in an amount effective to treat the abnormality .

In some embodiments, the abnormality is diabetes, hypercholesterolemia , dyslipidem.ia, obesity, artherosclercsis , thrombosis, chronic and acute inflammation, pulmonary disorders, bronchial asthma, allergy, intraocular pressure, pain, neuropathic pain, inflammatory pain, visceral pain, trigem.inal neuralgia, orcfacial pain, irritable bowel syndrome, 3arrett's esophagus, pain associated with cancer, diabetes, diabetic neuropathies, migraine, skin sensitivity associated with migraine, aiiooynia, neuroma, nerve compression, pair, due to damage of the spinal cord or brain, glaucoma, visual impairment, dementia, dyslexia, movement disorders, dyskinesia, tremor, Parkinson's, anxiety, chorea, epilepsy, dry-eye disorders, cystic fibrosis, hyperactive bladder, or urinary retention.

In one embodiment, the mamm.alian SXORF207 receptor is a human SNORF207 receptor.

This invention also provides a process for making a composition of matter which specifically binds to a mammalian SNORF207 receptor which comprises identifying a chemical compound using a process in accordance with this invention and then synthesizing the chemical compound or a novel structural and functional analog or homolog thereof .

This invention further provides a process for preparing a composition, for example a pharmaceutical composition which comprises admixing a carrier, for example, a pharmaceutically acceptable carrier, and a therapeutically effective amount of a cnem.ical compound identified by a process in accordance with this invention or a novel structural and functional analog cr nomoiog thereof .

This invention further provides a process for preparing a composition, for example a pharmaceutical com.position which comprises identifying a chem.ical compound by a process m accordance with this invention or a novel structural and functional analog cr homolog thereof, recovering the chemical compound free of any receptor, and then admixing a carrier, for example, a

- "3 ^ - pharmaceutically acceptable carrier, and a therapeutically effective amount of the chemical compound.

In one embodiment, the mammalian SNORF207 receptor is a human SNORF207 receptor.

This invention provides a pharmaceutical composition made by com.bining a therapeutically effective am.ount of the compound cf this invention and a pharmaceutically acceptable carrier.

This invention provides a process for making a pharmaceutical composition comprising combining a therapeutically effective amount of the compound of this invention and a pharm.aceutically acceptable carrier.

This invention further provides a pharmaceutical composition comprising a therapeutically effective amount of the compound of the invention and a pharm.aceutically acceptable carrier. In one embodiment, the amount of the compound is an am.ount from about 0.01 .g to about 800 mg . In another em.Dodim.ent, the am.ount of the com.pound is an am.ount from about 0.01 m.g to about 500 mg . In another embodiment, the am.ount cf the compound is an am.ount from about C.01 mg to about 250 mg . In another embodiment, the amount cf the compound is an am.ount from, about 0.1 mg to about 60 m.g. In another embodiment, the amount of the compound is an amount from, about 1 m.g to about 2 C mg . In a further embodiment, the carrier is a liquid and the composition is a solution. In another embodiment, the carrier is a solid and the composition is a powder or tablet. In a further embodiment, the carrier is a gel and the composition is a capsule or suppository. In the present invention the term "pharmaceutically acceptable carrier" is any pharmaceutical carrier known to those of ordinary skill in the art as useful in formulating pharmaceutical compositions.

In an embodiment of the present invention, the pharmaceutical carrier may be a liquid and the pharmaceutical composition would be in the form of a solution. In another embodiment, the pharmaceutically acceptable carrier is a solid and the composition is in the form of a powder or tablet. In a further embodiment, the pharmaceutical carrier is a gel and the composition is in the form of a suppository or cream. In a further em.bodiment the compound may be formulated as a part of a pharmaceutically acceptable transdermal patch. In yet a further embodiment, the compound may be delivered to the subject by means of a spray or inhalant.

A solid carrier can include one or more substances which may also act as endogenous carriers (e.g. nutrient or micronutrient carriers), flavoring agents, lubricants, solubilizers , suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it car. also be an encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary compression properties in suitable proportions and compacted m tne shape and size desired. The powders and tablets preferably contain up to 99% of the active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, poiyvinylpyrro idine, --ow me--tιr.g waxes and ion excnange resins .

Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient can be dissolved or suspended m a pharm.aceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmoregulators , Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferaoly sodium carboxymethyl cellulose solution), alcohols *, including monchydric alcohols and pclyhydπc alconols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arach s oil) . For parenteral administration, the carrier can also be an oily ester such as ethyl oieate or isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form, compositions for parenteral administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellen .

Liquid p.narmaceutical compositions which are sterile solutions or suspensions can oe utilized by for example, intramuscular, intrathecal, epidural, mtraperitoneal cr subcutaneous injection. Sterile solutions can also be aαm.inistered intravenously. The compounds m.ay be prepared as a sterile solid composition which may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.. Carriers are intended to include necessary and inert binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, dyes, and coatings.

In the subject invention a "therapeutically effective amount" is any amount cf a compound which, when administered to a subject suffering from a disease against which the compounds are effective, causes reduction, remission, or regression of the disease. In the subject applicaticn, a "subject" is a vertebrate, a mammal, or a human.

The compound can be administered orally in the form, cf a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline cr glucose to make the solution isctonic), bile salts, acacia, gelatin, sorbitan m.onoieate, pclysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.

The compound can also be administered orally either in liquid or solid composition form.. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid form.s, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

Optimal dosages to be administered may be determined by those skilled in the art, and w ll vary with the particular compound in use, the strength of the preparation, the mode of administration, and the advancement cf the disease condition. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time cf administration.

This invention provides a recombinant nucleic acid comprising a nucleic acid encoding a mammalian SXORF207 receptor, wherein the mamm.alian receptor-encoding nucleic acid hybridizes under high stringency conditions to a nucleic acid encoding a human SXORF207 receptor. This invention provides a recombinant nucleic acid comprising a nucleic acid encoding a m.ammalian SNORF207 receptor, wherein the receptor has a sequence identical to the sequence of the human SNORF207 receptor encoded by plasmid MSP70-hSXORF207-f (ATCC Patent Deposit Designation FTA-479C) .

This invention further provides a recombinant nucleic acid comprising a nucleic acid encoding a human SNORF2C7 receptor, wherein the human SXORF2C7 receptor comprises an a .ino acid sequence identical to the sequence of the human SNORF207 receptor as indicated in Figures IA-13 (SEQ ID NO: 1) . In one embodim.ent, the num.an SNORF207 receptor is encoded by the nucleotide sequence beginning at the start codo at positions 1-3 and ending at the stop codon at positions 1117-1119 as indicated in Figures 1 -1B (SEQ ID NO: 1) .

The plasmid MS?70-hSNORF207-f was deposited on November 7, 2002, with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Virginia 20110- 2209, U.S.A. under the provisions of the Budapest Treaty for the International Recognition cf the Deposit of Microorganisms tor tne Purposes or tater.t procedure and was accorded ATCC Patent Deposit Designation PTA-4790.

This invention contemplates recombinant nucleic acids which comprise nucleic acids encoding naturally occurring allelic variants of the mamm.alian SXORF207 receptors described above. This invention also contemplates recombinant nucleic acids which comprise nucleic acids encoding variants of the mammalian SNORF207 receptor that result from. single nucleotide polymorphisms (SNPs), deletions of one or more nucleotides, insertions of one or more nucleotides, inversions, etc.

Hybridization methods are well known to those of skill in the art. For purposes of this invention, hybridization under high stringency conditions means hybridization performed at 40^CC in a hybridization buffer containing 50% formamide, 5X SSC, 7 mM Tris, IX Denhardt's, 25ug/ml salmon sperm DXA; wash at 50^CC in 0. IX SSC, 0.1%SDS.

Throughout this application, the following standard abbreviations are used tc indicate specific nucleotide bases :

A = adenine

G = guanine

C = cytosme

T = thymme

M = adenine or cytosine

R = adenine or guanine

7. = adenine or thymine

S = cytosme cr guanine

Y = cytosine or thymine K = guanine or thymine

V = adenine, cytosine, or guanine (not thymine) H = adenine, cytos e, or thymine inot cytosme)

B = cytosine, guanine, or thymine (not adenine)

N = adenine, cytosme, guanine, or thymine (or other modified base such as i osine)

I = inosine

Furthermore, the term "agonist" is used throughout this application to indicate any peptide or non-peptidyl compound which increases the activity of any of the polypeptides of the subject invention. The term "antagonist" is used throughout this application to indicate any peptide or non-peptidyl compound which decreases the activity of any of the polypeptides of the subject invention.

Furthermore, as used herein, the phrase "pharmaceutically acceptable carrier" means any cf the standard pharmaceutically acceptaole carriers. Examples include, but are not limited to, phosphate buffered saline, physiological saline, water, and emulsions, such as oil/water em.ulsions.

It is possible that the mammalian SNORF207 receptor gene contains intror.s and furthermore, the possibility exists that additional introns could exist in coding or non- coding regions. In addition, spliced form, (s) of mRNA m.ay encode additional am.ino acids either upstream, of the currently defined starting methionine or within the ceding region. Further, the existence and use cf alternative exons is possible, whereby the RNA may encode different am.ino acids within tne region comprising the exon. In addition, single amine acid substitutions m.ay arise via the mechanism, of RNA editing such that the amine acid sequence of the expressed protein is different than that encoded by tne original gene. (Burns, C.M. et al., 1997; Chu, et al . , 1996) . Such variants may exhibit pharmacological properties differing from the polypeptide encoded by the original gene.

This invention provides splice variants of the m.ammalian SXORF207 receptor disclosed herein. This invention further provides for alternate translation initiation sites and alternately spliced or edited variants of nucleic acids encoding the mammalian SXORF207 receptor of this invention.

The nucleic acids of the subject invention also include nucleic acid analogs of the human SXCRF207 receptor gene, wherein the human SNORF207 receptor gene comprises the nucleic acid sequence shown in Figures 1A-1B (SEQ ID NO: 1) or contained in plasmid MSP70-hSNORF207-f (ATCC Patent Deposit Designation PTA-4790) . Nucleic acid analogs of the human SNORF2C7 receptor genes differ from, the human SXORF207 receptor genes described herein m terms of the identity or location of one or m.ore nucleic acid bases (deletion analogs containing less than ail of the nucleic acid bases shown in Figures 1A-1B or contained in plasmid MSP70-hSXCRF207-f , substitution analogs wherein one or mere nucleic acid bases shown in Figures 1A-13 or contained in plasmid MSP70-hSNORF207-f (ATCC Patent Deposit Designation PTA-4790), are replaced by other nucleic acid bases, and addition analogs, wherein one or more nucleic acid bases are added to a terminal or medial portion of the nucleic acid sequence) and which enccde proteins which share some cr all of the properties of the proteins encoded by the nucleic acid sequences shown in Figures 1A-13 or contained plasmid MSP70-hSNORF2C7-f (ATCC Patent Deposit Designation FTA-4790) . In one embodiment of the present invention, the nucleic acid analog encodes a protein which has an amino acid sequence identical to that shown in Figures 2A-2B or encoded by the nucleic acid sequence contained in plasmid MSP7C-hSNORF207-f (ATCC Patent Deposit Designation PTA- 4790) . In another embodiment, the nucleic acid analog encodes a protein having an amine acid sequence which differs from the amino acid sequences shown in Figures 2A-2B or encoded by the nucleic acid contained in plasm.id M5P7C-hSXORF207-f (ATCC Patent Deposit Designation PTA- 4 90) .

In a further embodiment, the protein encoded by the nucleic acid analog has a function which is the same as the function of the receptor proteins having the amino acid sequence shown m Figures 2A-23. In another embodiment, the function cf the protein encoded by the nucleic acid analog differs from the function of the receptor protein having the amino acid sequence shown in Figures 2A-2B. In another embodiment, the variation in the nucleic aciα sequence occurs within the transmembrar.e (TM) region of the protein. In a further embodim.ent, the variation in the nucleic acid sequence occurs outside cf the TM region.

In another embodiment, the nucleic acid analog encodes a mammalian SNORF2C7 receptor which has above 75% amino acid identity, preferably above 85% amino acid identity, more preferably above 95% am.ino acid identity to the SNORF207 receptor encoded by the plasmid MS?70-hSNORF207- f (ATCC Patent Deposit Designation PTA-4790) . This invention provides tne aoove-oescπoed isolated nucleic acid, wherein the nucleic acid is DNA. In an embodiment, the DXA is cDNA. In another embodiment, the DXA is genomic DNA. In still another embodiment, the nucleic acid is RXA. Methods for production and manipulation of nucleic acid molecules are well known in the art.

This invention further provides a nucleic acid which is degenerate with respect to the DNA encoding any of the polypeptides described herein. In an embodiment, the nucleic acid comprises a nucleotide sequence which is degenerate with respect to the nucleotide sequence shown m Figures 1A-13 (SEQ ID NO: 1) cr the nucleotide sequence contained in the plasmid MSP7 C-hSNORF207-f (ATCC Patent Deposit Designation PTA-4790), that is, a nucleotide sequence which is translated into the same amino acid sequence.

This invention also encompasses DNAs and cDNAs which encode ammo acid sequences which differ from those of the polypeptides of this invention, but which should not produce pher.otypic changes.

Alternately, this invention also encompasses DNAs, cDNAs, and RNAs whicn hybridize to the DNA, cDNA, and RXA of the subject invention. Hybridization methods are well .<nown tc these of skill m the art.

The nucleic acids of the subject invention also include nucleic acid molecules ceding for polypeptide analogs, fragments or derivatives of antigenic polypeptides which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (deletion analogs containing _-ess than al-- or tne residues specified for the protein, substitution analogs wherein one or more residues specified are replaced by other residues and addition analogs wherein one or more am.ino acio residues is added to a terminal or medial portion of the polypeptides) and which share some or ail properties of naturally-occurring orms. These molecules include: the incorporation cf codons "preferred" for expression by selected non-mammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DXA sequences that facilitate construction of readily expressed vectors. The creation of polypeptide analogs s well known to those cf skill in the art (Spurney, R. F. et al . (1997); For.g, T.M. et al . (1995); Underwood, O X . et al . (1994); Graziano, M.P. et al. (1996); Guan X . M . et al . (1995)) .

The modified polypeptides of this invention may be transfected into cells either transiently or staoly using methods well-known in the art, examples of which are disclosed herein. This invention also provides for binding assays using the m.odified polypeptides, in which the polypeptide is expressed either transiently or in stable cell lines . This invention further provides a compound identified using a modified polypeptide in a binding assay such as the binding assays described he ein.

The nucleic acids described and claim.ed herein are useful for the information which they provide concerning the amino acid sequence cf the polypeptide and as products for the large scale synthesis of the polypeptides by a variety cf recombinant techniques. The nucleic acid molecule is usefu-- for generating new cloning and expression vectors, transformed and transfected prokaryotic and eukaryotic host ceils, and new and useful methods for cultured growth of such host cells capable of expression of the polypeptide and related products.

This invention also provides an isolated nucleic ac d encoding species homologs of the SXCRF207 receptor encoded by the nucleic acid sequence shown m Figures 1A- 13 (SΞQ ID NO: 1) or encoded by the plasm.id MSP70- hSNORF207-f (ATCC Patent Deposit Designation PTA-4790) . In one embodiment, the nucleic acid encodes a m.ammalian SNORF207 receptor homolog which has substantially the same amino acid sequence as does the SNORF207 receptor encoded by the plasmid MSP70-hSNCRF2C7-f (ATCC Patent Deposit Designation PTA-4790) .

Examples of m.ethods for isolating and purifying species homologs are described elsewhere (e.g., U.S. Patent No. 5,602,024, W094/14957, W097/26853 , 7.C98 /15570 ) .

In another embodiment, the nucleic acid encodes a mammalian SNORF207 receptor homolog which has above 75% amino acid identity to the SNORF2C7 receptor encoded by the plasmid MSP70-hSXORF207-f (ATCC Patent Deposit Designation PTA-4790); preferably above 85% ammo acid identity to the 3XORF2C7 receptor encoded by tne plasmid MS?70-hSNORF207-f (ATCC Patent Deposit Designation PTA- 4790); most preferably above 95% amino acid identity to tne SNORF207 receptor encoded by the plasmid MSP70- hSXORF207-f (ATCC Patent Deposit Designation PTA-4790) . In another em.bodiment, the miammalian SNORF207 receptor homolog has above 70% nucleic acid identity to the SNORF2C7 receptor gene contained in plasmid MSP70- hSNCRF207-f (ATCC Patent Deposit Designation P'lA-*. / y u ) ; preferably above 80% nucleic acid identity to the SNORF207 receptor gene contained m the plasmid MSP70- hSNORF207-f (ATCC Patent Deposit Designation PTA-4790); iore preferably above 90% nucleic acid identity to the SNORF207 receptor gene contained in the plasmid MSP70- hSNORF207-f (ATCC Patent Deposit Designation PTA-4790) .

This invention provides an isolated nucleic acid encoding a mammalian SNORF207 receptor. In one embodiment, the nucleic acid is DNA. In another embodiment, the DNA is cDXA. In another embodim.ent, the DXA is genomic DXA. In another embodiment, the nucleic acid is RNA.

In one embodiment, the mammalian SNORF207 receptor is a human SNORF2C7 receptor. In another embodiment, the human SNORF207 receptor has an amino acid sequence identical tc that encoded by the plasm.id MSP70-hSNORF207- f (ATCC Patent Deposit Designation PTA-4790) . In another embodiment, the human SNCRF207 receptor has an amino acid sequence identical to the amino acid sequence shewn in Figures 2A-23 (SEQ ID NO: 2) .

This invention provides a purified mamm.alian SNORF207 receptor protein. In one embodiment, the SNORF207 receptor protein is a human SXORF207 receptor protein.

This invention provides a vector comprising the nucleic acid of this invention. This invention further provides a vector adapted for expression m a cell which comprises the regulatory elements necessary for expression of the nucleic acid in the cell operatively linked to the nucleic acid encoding the receptor so as tc permit expression thereof, wherem the cell is a bacterial, ampnipian, yeast, insect or mamma±iαii ce-- . n one embodiment, the vector is a baculovirus. In another embodiment, the vector is a plasmid.

This invention provides a plasmid designated MSP70- hSNORF207-f (ATCC Patent Deposit Designation PTA-4790) .

This invention further provides for any vector or plasmid which comprises modified untranslated sequences, which are beneficial for expression in desired host cells or for use in binding or functional assays. For example, a vector or plasmid with untranslated sequences of varying lengths may express differing amounts of the polypeptide depending upon the host cell used. In an em.bodim.ent , the vector or plasmid comprises the coding sequence of the polypeptide and the regulatory elements necessary for expression in the host cell.

This invention provides for a cell comprising tne vector of this invention. In one embodim.ent, the cell is a ncn- namm liar. ceil. In one embodim.ent, the non-mammalian cell is a Xenopus oocyte cell or a Xencpus melanophore cell. In another em.bodiment, the cell is a miammalian cell. In another embodiment, the cell is a 00S-7 cell, a human embryonic kidney ceil, a HEK293 cell, a XIH-3T3 cell, a LM(tk-) cell, a mouse Yl cell, or a CHO cell. In another embodiment, the ceil is an insect cell. In another embodiment, the insect cell is an Sf9 cell, an Sf21 cell cr a Trichoplusia ni 5B-4 cell.

This invention provides a membrane preparation isolated from the cell in accordance with this invention.

- c c Furthermore, this invention provides tor a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian SNORF207 receptor, wherein the probe has a sequence complementary to a unique sequence present within one of the two strands of the nucleic acid encoding the mammalian SNORF207 receptor contained m plasm.id MSF70-hSNORF207-f (ATCC Patent Deposit Designation PTA-4790) .

This invention further provides a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mamm.alian SNORF207 receptor, wherein the probe has a sequence complementary to a unique sequence present within (a) the nucleic acid sequence shown in Figures 1A- 13 (SEQ ID NO: 1) or (b) the reverse complement to (a) . In one em.bodiment, the nucleic acid is DNA. In another embodim.ent, the nucleic acid is RNA.

As used herein, the phrase "specifically hybridizing" means tne ability of a nucleic acid molecule tc recognize a nucleic acid sequence complementary to its own and to form, double-helical segments through hydrogen bonding between complementary base pairs.

The nucleic acids of this invention may be used as probes to obtain homologous nucleic acids from, other species and to detect the existence of nucleic acids having complementary sequences in samples.

The nucleic acids may also be used to express the receptors they encode in transfected cells.

Δ ^■--. The use of a constitutively active receptor encoded by SNORF207 either occurring naturally without further modification or after appropriate point mutations, deletions cr the like, allows screening for antagonists and in vivo use of such antagonists to attribute a role to receptor SXORF207 without prior knowledge of the endogenous ligand.

Use of the nucleic acids further enables elucidation of possible receptor diversity and of the existence of multiple subtypes within a family of receptors of which SNORF207 is a member.

It is contemplated that the receptors of this invention will serve as a valuable tool for designing drugs for treating various pathophysiological conditions such as diabetes, hypercholesterolemia, dyslipidemia, obesity, artherosclerosis , thrombosis, cr.rcnic and acute inflammation, pulmonary disorders, bronchial asthma, allergy, intraocular pressure, pain, neuropathic pain, inflammatory pain, visceral pain, trigem.inal neuralgia, orcfacial pain, irritable bowel syndrome, Barrett's esophagus, pain associated with cancer, diabetes, diabetic neuropathies, m.igraine, skin sensitivity associated with m.igraine, allodynia, neuroma, nerve compression, pain due to damage of the spinal cord or brain, glaucom.a, visual impairment, dementia, dyslexia, movement disorders, dyskmesia, tremor, Parkinson's, anxiety, chorea, epilepsy, dry-eye disorders, cystic fibrosis, hyperactive bladder, urinary retention, among ethers, and diagnostic assays for such conditions.

Methods of transfectir.g cells e.g. mamm.alian cells, with such nucleic acid to obtain cells which the receptor i s expre s sed or. the surf ace o r the ce -L l are well known in the art . ( See , f or example , U . S . Patent Nos . 5 , 053 , 337

5, 155, 218 5,360,735; 5,472,866; 5,476,782; 5,516,653 5,545, 549 5,556,753; 5,595,880; 5,602,024; 5,639,652 5,552,113 5,661,024; 5,766,879; 5,786,155; and 5,786, 157, the disclosures of which are hereby incorporated by reference in their entireties into this application . )

Such transfected cells may also be used to test compounds and screen compound libraries to obtain compounds which bind to the SNCRF207 receptor, as well as compounds which activate or inhibit activation of functional responses in such cells, and therefore are likely to do so in vi vo . (See, for example, U.S. Patent Xos . 5,053,337; 5,155,21

5,360,735 5,472,866; 5,476,782; 5,516,653; 5,545,549 5 , 556, 753 5,595,880; 5,602,024; 5,639,652; 5,652,113 5,661,02 5,766,379; 5,766,155; and 5,786,157, the disclosures of which are hereby incorporated by reference in their entireties into this application.)

This invention provides an antibody capable of binding to a m.amm.aiian SXORF207 receptor encoded by a nucleic acid encoding a mammalian SXORF207 receptor. In an embodiment of the present invention, the mammalian SNORF2C7 receptor is a human SNORF207 receptor.

This invention also provides an agent capable of competitively inhibiting the binding of the antibody to a mammalian SXORF207 receptor. In one embodim.ent, the antibody is a monoclonal antibody or antisera.

Methods .repar g anc p--oy ar.tiser.se oligonucieotides , an: .bodies, nucleic ac d probes and transgenic animals directed to tne 3NOR-!--u/ receptor are well known in the art. (See, for example, U.S. Patent Ncs. 5,053,337; 5,155,218; 5,360,735; 5,472,866; 5,476,782; 5,516,653; 5,545,549; 5,556,753; 5,595,880; 5,602,024; 5,639,652; 5,652,113; 5,661,024; 5,766,879; 5,786,155; and 5,786,157, the disclosures of which are hereby incorporated by reference in their entireties into this application.)

This invention provides for an antisense oligonucleotide having a sequence capable of specifically hybridizing to RNA encoding a mammalian SNORF207 receptor, so as to prevent translation of such RNA. This invention further provides for an antisense oligonucleotide having a sequence capable cf specifically hybridizing to genomic DXA encoding a mammalian SNORF207 receptor, so as to prevent transcription of such genomic DXA. In one embodiment, the oligonucleotide comprises chemically modified nucleotides or nucleotide analogues .

This invention still further provides a pharmaceutical composition comprising (a) an am.ount of an oligonucleotide in accordance with this invention capable of passing through a cell membrane and effective to reduce expression cf a mammalian SNORF2C7 receptor and (b) a pharmaceutically acceptable carrier capable of passing through the cell membrane.

In one embodiment, the oligonucleotide is coupled to a substance which inactivates mRNA. In another embodiment, the substance which inactivates mRNA is a ribozyme. In another embodiment, the pharmaceutically acceptable carrier comprises a structure which binds to a m.amm.aiian SNCRF207 receptor on a cell capable of being taken up by the cells after binding to the structure. In another embodiment, the pharmaceutically acceptable carrier is capable of binding to a mammalian SNORF207 receptor which is specific for a selected cell type.

This invention also provides a pharmaceutical composition which comprises an amount of an antibody in accordance with this invention effective to block binding of a ligand to a human SXORF207 receptor and a pharmaceutically acceptable carrier.

This invention further provides a transgenic, nor.hurr.ar. mammal expressing DNA encoding a m.ammalian SNORF2C7 receptor m accordance with this invention. This invention provides a transgenic, nonhuman mammal comprising a homologous recombination knockout of a native mammalian SNORF207 receptor. This invention further provides a transgenic, nonhuman mammal whose genome com.prises antisense DXA complementary to DNA encoding a mammalian SNORF207 receptor in accordance with this invention so placed within such genome as to be transcribed into antisense mRNA which is complementary and hybridizes with mRNA encoding the mammalian SXORF207 receptor so as tc thereby reαuce translation or such mRNA and expression of such receptor. In one embodiment, the DNA encoding the mammalian SNORF2C7 receptor additionally comprises an mducible promoter. In another embodiment, the DNA encoding the mammalian SNORF2C7 receptor additionally comprises tissue specific regulatory elements. In another em.bodiment, the transgenic, nonhuman mammal is a mouse.

This invention also provides a method of detecting expression of a mammalian SNORF207 receptor by detecting the presence of mRNA ceding for the m.ammalian SNORF20 / receptor which comprises obtaining total RNA from, the cell and contacting the mRNA so obtained with a nucleic acid probe according to this invention under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the mammalian SNORF2C7 receptor by the ceil.

This invention further provides for a method of detecting the presence of a m.ammalian SNORF207 receptor on the surface of a cell which comprises contacting the ceil with an antibody according to this invention under conditions permitting binding of the antibody to the receptor, detecting the presence of the antibody bound to the ceil, and thereby detecting the presence of the mamm.alian SXORF207 receptor on the surface of the cell.

This invention still further provides a method of determining the physiological effects of varying levels cf activity of a mammalian SNORF207 receptor which comprises producing a transgenic, nonhuman mammal in accordance with this invention whose levels of mammalian SNORF207 receptor activity are varied by use of an inducible promoter which regulates m.ammalian SNORF207 receptor expression.

This invention additionally proviαes a method of determining the physiological effects of varying levels of activity of a mammalian SNORF207 receptor which comprises producing a panel of transgenic, nonhuman mammals in accordance with this invention each expressing a different am.ount of a m.ammalian SNORF207 receptor. Moreover, this invention provides method for identifying an antagonist capable of alleviating an abnorm.ality wherein the abnormality is alleviated by decreasing the activity cf a mamm.alian SNORF207 receptor comprising administering a compound tc a transgenic, nonhuman mammal according to this invention, and determining whether the ccmpound alleviates any physiological and/or behavioral abnorm.ality displayed by the transgenic, nonhuman mammal as a result of overactivity of a mammalian SNORF207 receptor, the alleviation of such an abnormality identifying the compound as an antagonist. In an em.bodiment, the mamm.alian SNORF207 receptor is a human SXORF2C7 receptor.

^^' invention also provides an antagonist identified by the preceding method according to this invention. This invention further provides a composition, e.g. a pharmaceutical composition comprising an antagonist according tc this invention and a carrier, e.g. a pharmaceutically acceptable carrier.

This invention provides a method of treating an abnormality in a subject wherein, the abnormality is alleviated by decreasing the activity of a mammalian SNORF2C7 receptor which comprises administering to the subject an effective amount of the pharmaceutical composition according to this invention so as to thereby treat the abnormality.

In addition, this invention provides a m.etnod for identifying an agonist capable of alleviating an abnormality m a subject wherein the abnormality is alleviated by increasing the activity of a mammalian SNORF2C7 receptor comprising adm.mistering a com.pound tc a transgenic, nonhuman mammal according tc this invention, and determining whether the compound alleviates any physiological and/or behavioral abnormality displayed by the transgenic, nonhum.ar. mammal, the alleviation of such an abnormality identifying the compound as an agonist. In an embodiment, the mammalian SNORF207 receptor is a human SNORF2C7 receptor. This invention provides an agonist identified by the preceding method according to this invention. This invention provides a composition, e.g. a pharmaceutical composition comprising an agonist identified by a method according to this invention and a carrier, e.g. a pharmaceutically acceptable carrier.

Moreover, this invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity cf a mammalian SNCRF2C7 receptor which comprises administering to the subject an effective am.ount cf the pharmaceutical composition of this invention so as to thereby treat the abnormality .

Yet further, this invention provides a method for diagnosing a predisposition to a disorder associated with the activity cf a specific mammalian aliele which comprises: (a) obtaining DNA of subjects suffering from the disorder; (b) performing a restriction digest of the DNA with a panel of restriction enzymes; (c) electrophoretically separating the resulting DXA fragments or. a sizing gel; (d) contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a mamm.alian SNORF207 receptor and labeled with, a detectable marker; c; 2 - (e) detecting labeled bands which have hybridized to the DXA encoding a mammalian SXORF207 receptor to create a unique band pattern specific to the DNA of subjects suffering from the disorder; (f) repeating steps (a) -(e) with DNA obtained for diagnosis from, subjects not yet suffering from, the disorder; and (g) comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step (e) with the band pattern from step (f) for subjects not yet suffering frcm. the disorder so as to determine whether the patterns are the same or different and thereby diagnose predisposition tc the disorder if the patterns are the same.

In one embodiment, the disorder is a disorder associated with the activity cf a specific mammalian aliele is diagnosed .

This invention also provides a method of preparing a purified mammalian SXORF207 receptor according to this invention which comprises: (a) culturing cells which express the mammalian SNORF2C7 receptor; (b) recovering the ma.mmalian SNORF207 receptor from, the cells; and (c) purifying the m.ammalian SNCRF207 receptor so recovered.

This invention further provides a method of preparing a purified mammalian 3XCRF207 receptor according to th s invention which comprises: (a) inserting a nucleic acid encoding the m.amm.aiian SNORF207 receptor into a suitable expression vector; (b) introducing the resulting vector into a suitable host cell; (c) placing the resulting host cell suitable condition permitting the production of the m.ammalian SNORF207 receptor; (d) recovering the m.amm.aiian SNORF207 receptor so produced; and optionally (e) isolating and/or purifying the mamm.alian SXORF207 receptor so recovered.

Once the gene for a targeted receptor subtype is cloned, it is placed into a recipient cell which then expresses the targeted receptor subtype on its surface. This cell, which expresses a single population of the targeted human receptor subtype, is then propagated resulting in the establishment of a ceil line. This cell line, which constitutes a drug discovery system, is used in two different types of assays: binding assays and functional assays. In binding assays, the affinity of a compound for both the receptor subtype that is the target of a particular drug discovery program, and other receptor subtypes that could be associated with side effects are measured. These measurements enable one to predict the potency of a compound, as well as the degree of selectivity that the compound has for the targeted receptor subtype over other receptor subtypes. The data obtained from binding assays also enable chemists to design compounds toward or away from one or more of the relevant subtypes, as appropriate, for optimal therapeutic efficacy. In functional assays, the nature of the response of the receptor subtype to the compound is determined. Data from, the functional assays show whether the compound is acting to inhibit or enhance the activity of the receptor subtype, thus enabling pharmacologists to evaluate compounds rapidly at their ultimate human receptor subtypes targets permitting chemists tc rationally design drugs that will be more effective and have fewer or substantially less severe side effects than existing drugs.

5 - Approaches to designing and synthesizing receptor subtype-selective compounds are well known and include traditional medicinal chemistry and the newer technology of combinatorial chemistry, both of which are supported by computer-assisted molecular modeling. With such approaches, chemists and pharmacologists use their knowledge of the structures of the targeted receptor subtype and compounds determined to bind and/or activate or inhibit activation of the receptor subtype to design and synthesize structures that will have activity at these receptor subtypes.

Combinatorial chemistry involves autom.ated synthesis of a variety cf novel compounds by assembling them using different combinations of chemical building blocks. The use of combinatorial chemistry greatly accelerates the process of generating compounds. The resulting arrays of compounds are called libraries and are used to screen for compounds ("lead compounds") that demonstrate a sufficient level of activity at receptors of interest. Using combinatorial chemistry it is possible to synthesize "focused" libraries of compounds anticipated to be highly biased toward the receptor target of interest .

Once lead compounds are identified, whether through the use of combinatorial chemistry or traditional medicinal chemistry or otherwise, a variety of r.omologs and analogs are prepared tc facilitate an understanding of the relationship between chemical structure and biclogical or functional activity. These studies define structure activity relationships which are then used to design drugs with improved potency, selectivity and pharmacokinetic properties. Combinatorial chemistry is also used to rapidly generate a variety of structures for lead optimization. Traditional medicinal chemistry, which involves the synthesis of compounds one at a time, is also used for further refinement and to generate compounds not accessible by automated techniques. Once such drugs are defined the production is scaled up using standard chem.ical manufacturing methodologies utilized throughout the pharmaceutical and chemistry industry.

This invention will be better understood from. the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

o EXPERIMENTAL DETAILS

Materials and Methods

Isolation cf the human SNORF2C7 receptor

A novel GPCR fragment was identified in the Ensembl Genscar. database by dataminir.g with the MOTIFSEARCH algorithm (GCG Wisconsin Package Version 10.3, Accelrys Inc., San Diego, CA) using a profile created with MEME (GCG Wisconsin Package Version 10.3, Accelrys Inc., San Diego, CA) built using a subset of human GPCR sequences. The receptor was subsequently named "SNORF207". BLAST analysis (GCG Wisconsin Package Version 10.3, Accelrys Inc., San Diego, CA) cf the GenEmbl sequence database using the SNORF207 GPCR fragment revealed an exact match to a human putative GPCR termed GPR92 (locus HSA272207; corresponding Gen3ank Accession No. AJ272207) . The full length DNA encoding the SNORF2C7 protein was generated by Blue Heron Biotechnology, using their proprietary gene synthesis m.etnod GeneMaker--^: (Blue Heron Biotechnology, 22310 20^t- Avenue, Suite 100, Bothell, WA 98021), and subclor.ee into the Synaptic vector MSP70. The resultant plasmid designated KI369 was used for further analysis.

Isolation of SNORF2C7 receptors

A nucleic acid sequence encoding a SXORF207 receptor from human or other species may also be isolated using standard molecular biology techniques and approaches such as those described below:

Approach ^■*? 1 : A genomic library (e.g., cosm.id, phage, PI, 3A0, YAC) generated from, the species of interest may be screened with a ^j2P-labeled oligonucleotide probe corresponding to a fragment of the human SXORF207 receptor whose sequence is snown m -igures ±Α- lϋ to isolate a genomic clone. The full-length sequence m.ay be obtained by sequencing this genomic clone. If one or more intrcns are present in the gene, the full-length introniess gene may be obtained from. cDNA using standard molecular biology techniques. For example, a forward PCR primer designed in the 5 ' UT and a reverse PCR primer designed in the 3'UT may be used to amplify a full- length, introniess receptor from cDXA. Standard m.olecular biology techniques could be used to subclor.e this gene into a mammalian expression vector.

Approach ?2: Standard m.olecular biology techniques m.ay be used tc screen commercial cDNA phage libraries of the species of interest by hybridization under reduced stringency with a ³²P-labeled oligonucleotide probe corresponding to a fragment of the sequences shown m Figures 1A-1B. One m.ay isolate a full-length SNORF2C7 receptor by obtaining a plaque purified clone from, the lambda libraries and then subjecting the clone to direct DNA sequencing. Alternatively, standard m.olecular biology techniques could be used to screen cDNA plasmid libraries by PCR amplification of library pools using primers designed against a partial species homolog sequence. A full-length clone m.ay be isolated by Southern hybridization of colony lifts cf positive pools with a ²^?-oligor.ucleonde probe.

Approach ³ⁱ3: 3' and 5' RACE may be utilized to generate PCR products from. cDNA derived from the species cf interest expressing SNORF207 which contain the additional sequence of SNORF207. These RACE PCR products may then be sequenced to determine the additional sequence. This new seσuence is then used to design a forward PCR primer m the 5 ' UT and a reverse primer in the 3 ' UT . These pπm.ers are then used to amplify a full-length SNORF207 clone from. cD A.

Examples of non-human species include, but are not limited to, rat, mouse, dog, monkey, hamster and guinea pig .

Host cells

A broad variety of host cells can be used to study heterologously expressed proteins. These ceils include but are net lim.ited to mammalian cell lines such as : COS- 7, CHO, LM(tk^"), HEK293 cells, etc.; insect cell lines such as: Sf9, Sf21, Trichcplusia r.i 53-4, etc.; amphibian cells sucn as Xenopus oocytes; assorted yeast strains; assorted bacterial cell strains; and others. Culture conditions fcr each cf these ceil types is specific ana is known to those familiar with the art.

C03-7 cells are grown on 150 mm plates in DMEM with supplemients (Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 m.M glutamine, 100 units/ml penicillin 100 μg/ml streptomycin) at 37°C, 5% CO^ . Stock plates of COS-7 cells are trypsinized and split 1:6 every 3-4 days. HEK cells (?eak^a?lc cells^τ:*, Edge BioSystems, Gaithersburg, MD) are maintained in DMEM growth medium supplemented with 10% bovine calf serum, 1% L-glutam.ine , 50 ug/m.l genta ycin at 37°C, 5% C0₂.

Transient expression

DNA encoding proteins to be studied can be transiently expressed m a variety of mammalian, insect, amphibian, yeast, bacterial and other cells lines by several trans fection methods including cut not limited tc; calcium phosphate-mediated, DEAL-eextran mediated; liposomal-mediated, viral-mediated, electroporation- mediated, and microinj ection delivery. Each of these methods may require optimization of assorted experimental parameters depending on the DXA, cell line, and the type of assay tc be subsequently employed.

A typical protocol for the DEAΞ-dextran method as applied to COS-7 and HEK293 cells is described as follows. Ceils to be used for transfecticn are split 24 hours prior to the transfection to provide flasks which are 70-80% confluent at the tim.e of transfection. Briefly, 8 μg of receptor DXA plus 8 ug of any additional DXA needed (e.g. G_α protein expression vector, reporter construct, antibiotic resistance marker, mock vector, etc.) are added to 9 m.l of complete DMEM plus DEAΞ-dextran mixture (10 mg/ml m PBS). Cells plated into a T225 flask (sub- confluent) are washed once with PBS and the DNA mixture is added to each flask. The cells are allowed tc mcubate for 30 minutes at 37°C, 5% C0₂. Following the incubation, 36 ml of complete DMEM with 80 μM chloroquine is added to each flask and allowed tc incubate an additional 3 hours. The medium is then aspirated and 24 ml of ccmplete medium containing 10% DMSO for exactly 2 minutes and then aspirated. The cells are then washed 2 times with PBS and 30 m.l of complete DMEM addeα to each flask. The cells are then allowed to mcubate over night. The next day the cells are harvested by trypsinization and reseeded into 96 well plates.

Alternatively, HEK cells may be transfected with the calcium phosphate m.etnod according tc Jordan, et al . (1996) . Stable expression

Heterologous DNA can be stably incorporated into host cells, causing the cell to perpetually express a foreign protein. Methods for the delivery of the DNA into the cell are similar to those described above for transient expression but require the cc-transfection of an ancillary gene to confer drug resistance or. the targeted host cell. The ensuing drug resistance can be exploited to select and maintain cells that have taken up the DNA. An assortment cf resistance genes are available including but net restricted to neomycin, kanam.ycm, and hygromycin. For purposes of studies concerning the receptor of this invention, stable expression of a heterologous receptor protein is typically carrier out in, mammalian cells including but not necessarily restricted to, CHO, HEK293, LM(tk-), etc. In addition native cell lines that naturally carry and express the nucleic acid sequences for the receptor may be used without the need to engineer the receptor com.plement.

Mem.brane preparations

Cell rr.emDrar.es expressing the receptor protein of this invention are useful fcr certain types of assays including but not restricted tc liganα binding assays, GT?-γ-S binding assays, and others. The specifics of preparing such cell membranes m.ay m some cases be determined by the nature cf the ensuing assay but typically involve harvesting whole cells and disrupting the cell pellet by sonication in ice cold buffer (e.g. 20 mM Tris HCl, mM EDTA, pH 7.4 at 4° 0) . The resulting crude cell lysate is cleared of cell debris by low speed centrifugation at 200xg for 5 m.in at 4° C. The cleared supernatant is then cer.trifuged at 40,000xg for 20 min at 4° C, and the resulting mem.brane pellet is v.'ashed by suspending in ice cold buffer and repeating the high speed centrifugation step. The final v.'ashed membrane pellet is resuspended in assay buffer. Protein concentrations are determined by the method of Bradford (1976) using bovine serum albumin as a standard. The membranes may be used imm.ediately or frozen for later use .

Radiolabeled ligand binding assays

Cells expressing the receptor cf this invention m.ay be used to screen for ligands for said receptor. The same assays may be used to identify agonists or antagonists of the receptor that may be employed for a variety of therapeutic purposes .

Radioligand binding assays are performed by diluting m.embranes prepared from cells expressing the receptor in 50 mM Tris buffer (pH = 7.4 at 0^CC) containing 0.1% bovine serum album.in (Sig .a), aprotinin (0.CC5 mg/ml, 3oehringer Mannheim.) and bestatin (C.l mM, Sigma) as protease inhibitors. The final protein concentration in the assay can be 12 - 40 ug/ml.

Membranes are then incubated with radiolabeled ligand either in the presence cr absence of competing ligands on ice for 60 mm in a total volume of 250 ul m 95 well microtiter plates. The bound ligand is then separated from, free ligands by filtration through GF/3 filters presoaked in 0.5% polyethyieneimine (PΞI), using a Tom.tec (Wallac) vacuum filtration device. After addition cf Ready Safe (Ξeckman) scintillation fluid, bound radioactivity is quantitated using a Trilux (Wallac) scintillation counter (approximately 40% counting efficiency of bound counts) . Alternatively, t may be preferable to collect bound ligand and then separate ligand from, receptor using procedures well known in the art. Data is fit to r.on-Imear curves using GraphPad Prism.

In this manner, agonist or antagonist compounds that bind to the receptor may be identified as they inhibit the binding of the radiolabeled ligand to the m.embrane protein of cells expressing the said receptor. Nonspecific binding is defined as the amount of radioactivity remaining after incubation of membrane protein in the presence of 100 r.M of the unlabeled peptide corresponding to the radioligand used. In equilibrium saturation binding assays m.embrane preparations or intact cells transfected with the receptor are incubated in the presence of increasing concentrations of the labeled ccmpound to determine the binding affinity cf the labeled ligand. The binding affinities of unlabeled compounds may be determined in equilibrium, competition binding assays, using a fixed concentration of labeled compound in the presence of varying concentrations of the displacing ligands .

Functional assays

Cells expressing the receptor DXA of this invention may be used to screen for ligands to said receptor using functional assays. Once a ligand is identified the same assays m.ay be used to identify agonists or antagonists of the receptor that may be employed for a variety of therapeutic purposes. It is well known to those in the art that the over-expression of a G-protεm coupled receptor can result in the constitutive activation of intracellular signaling pathways. In the same manner, over-expression of the receptors of the present invention in any cell line as described above, can result m the activation cf the functional responses described below, and any of the assays herein descriced can be used tc screen for agonist, partial agonist, inverse agonist and antagonist ligands of the SXORF207 receptor.

A wide spectrum of assays can be employed to screen for the presence cf receptor SXORF207 ligands. These assays range from traditional measurements of total inositol phosphate accumulation, cAMP levels, intracellular calcium. mobilization, and potassium currents, for example; tc systems measuring these same second messengers but which have been modified or adapted to be of higher throughput, m.ore generic and m.ore sensitive; to ceil based assays reporting m.ore general cellular events resulting from receptor activation such as metabolic changes, differentiation, cell division/proliferation. Description of several such assays follow.

Cyclic AMP (cAMP) assay

The receptor-mediated stimulation or inhibition of cyclic AMP (cAMP) formation may be assayed in cells expressing the receptor. COS-7 cells are transiently transfected with the receptor gene using the DEAE-dextran m.etnod and plated in 96-well plates. 48 hours after transfection, cells are washed twice with Dulbecco's phosphate buffered saline (PBS) supplemented with 10 mM HEPES, 10 mM glucose and 5 m.M theophyllme and are incubated in the same buffer for 20 mm at 37CC, m 5% C0₂. Test compounds are added and cells are incubated for an additional 10 min at 37^~C. The medium is then aspirated and tne reaction stopped by the addition cf 100 mM HCl. The plates are stored at -20ι_C for 2-5 days. For cAMP measurement, plates are thawed and tne cAMP content in each well is measured by cAMP Scintillation Proximity Assay (Amiersnam Pharmacia Biotech) . Radioactivity is quantified using m.icrobeta Trilux counter (Wallac) .

Arachidonic acid release assay

Cells expressing the receptor are seeded into 96 well plates or other vessels and crown for 3 days medium with supplements. ^JH-arachιdonic acid (specific activity = 0.75 μCi/ .l) is delivered as a IOC μL aliquot to each well and samples are incubated at 37° C, 5% C0₂ for 18 hours . The labeled cells are washed three times with medium. The wells are then filled with medium and the assay is initiated with the addition of test compounds or buffer in a total volume of 250 μL . Cells are incubated for 30 min at 37°C, 5% C0₂. Superr.atants are transferred tc a microtiter plate and evaporated to dryness at 75°C in a vacuum oven. Samples are then dissolved and resuspended m 25 μL distilled water. Scintillar.t (300 μL) is added to each well and samples are counted for ^JH in a Trilux plate reader. Data are analyzed using nonlinear regression and statistical techniques available in the GraphPAD Prism package (San Diego, CA) .

Intracellular calcium mobilization assays

In the present invention, CHO cells transiently transfected with hSXCRF2C7 or with expression vector only (mock-transfected) were seeded at a density of 10,000 cells per well into black walled clear-base 384-well plates coated with pcly-D-Lysme (Sector. Dickinson, USA) . The cells were cultured for 24 hrs after seeding m HAM' s F-12 mediu containing 1.5 % BCS, 2% L-glucamine, and 1% penicillin/streptomycin, 37°C in 5% C0₂. For measurements of intracellular free calcium, concentration ( [Ca^2*]_!) , the culture medium was replaced with a freshly prepared loading buffer. The loading buffer contains IX Hand's Buffered Saline Solution (HBSS, Gibco) , 20 .M HEPES

(Sigma), C.1% 3SA (Sigma), 1.5 μM Fluo-4-AM (Molecular Probes), and 2.5 mM freshly prepared probenecid (Sigma) . The plates were incubated for 1 hour at 37°C /5% CO₂, then washed three times with washing buffer (similar to loading buffer but without Fluo-4-AM) . Test compounds were diluted in DMSC/washing buffer to a 4X concentration

(DMSO = 4%) ana transferred into a 384-weli plate (clear V-bottom, Nunc) . Cells and compounds were then placed into a fluorescence imaging plate reader (FLIPR^τ;;, Molecular Devices) . Resting cell calcium was monitored using Fluo-4, a fluorescent calcium indicator dye. Fluo-4 was excited at wavelength = 488 nm wavelength by an argon ion laser; emission was detected at wavelengths 510-570 nm (Sullivan, E., et al, 1999) . Test compounds were then delivered to the plate as a 1:4 dilution (final DMSO = 1%) . Relative fluorescence units (RFU) were recorded and changes from resting levels (delta RFU) were used to construct concentration-response curves. Data were analyzed using FLIPR"^": software for primary data processing and GraphPad Prism, for nonlinear regression

( 4-parameter logistic) .

In another m.etnod, the intracellular free calcium concentration may be measured by microspectrofluorimetry using the fluorescent indicator dye Fura-2 /AM (Bush et al., 1991) . Cells expressing the receptor are seeded onto a 35mm culture dish containing a glass coversl p insert and allowed tc adhere overnight. Cells are then washed with H3S and leaded with 100 μL cf Fura-2 /AM (10 μM) for 20 to ^X mm. After washing with KBS to remove the Fura- 2/AM solution, cells are equilibrated in KBS for 10 to 20 min. Cells are then visualized under the 40X objective of a Leitz Fluovert FS microscope and r-luorescence emission is determined at 510 nM with excitation wavelengths alternating between 340 nM and 380 nM . Raw fluorescence data are converted to Ca²⁺ concentrations using standard Ca^2~ concentration curves and software analysis techniques .

In still other methods, [Ca^""]_: can also be performed on a 96-well format and with alternative Ca^^τ-sensitive indicators. Preferred examples of these are: aequorin, Fluo-2, Fluo-3, Fluo-5, Calcium Green-1, Oregon Green, and 488 BAPTA. After activation of the receptors with agonist ligands, the emission elicited by the change cf intracellular 0a'^τ concentration can be measured by a lummo etεr, or a fluorescence i .ager; a preferred example cf this is the fluorescence imager plate reader (FLIPR™, Molecular Devices) .

This m.etnod described above provides a strategy for identifying receptor agonists. Antagonists are identified similarly except that cells are pre-incubated with potential antagonist compounds before application of the agonist. An antagonist is identified by its ability to inhibit the agonist-evoked signal.

GTPyS functional assay

Membranes from. cells expressing the receptcr are suspended m assay buffer (e.g., 50 .M Tris, 100 m.M NaCl, 5 mM MgCl?, 10 μM GDP, pH 7.4) with or without protease inhibitors (e.g., 0.1% bacitracir.) . Mem.brar.es are incubated on ice for 20 minutes, transferred to a 96-well Millipore microtiter GF/C filter plate and m.ixed with GT?γ³⁵S (e.g., 250,000 cpm/sample, specific activity -1000 Ci/mmol) plus or minus unlabeled GT?γS (final concentration = 100 uM) . .ma- m.embrane protein concentration ~90 μg/ml. Samples are incubated in the presence or absence of test compounds for 30 min. at room temperature, then filtered en a Miliipore vacuum m.anifold and washed three times with cold (4^CC) assay buffer. Samples collected in the filter plate are treated with scintillant and counted for ^J~S in a Trilux (Wallac) liquid scintillation counter. It is expected that optimal results are obtained when the receptor membrane preparation is derived from an appropriately engineered heterologous expression system., i.e., an expression system resulting in high levels of expression of the receptor and/or expressing G-proteins having high turnover rates (for the exchange of GD? for GT?) . GTPγS assays are well-known to those skilled m the art, and it is contemplated that variations on the m.etnod described above, such as are described by Tiar. et al. (1994) or Lazarer.o and Birdsall (1993), m.ay be used.

Microphysiometric assay

Because cellular m.etabolism is intricately involved in a bread range of cellular events (including receptor activation of multiple messenger pathways), the use of microphysiometric measurements cf cell m.etabolism. can in principle provide a generic assay of cellular activity arising from the activation of any orphan receptor regardless of the specifics of the receptor' s signaling pathway .

General guidelines for transient receptor expression, cell preparation and microphysiometric recording are described elsewhere (Salon, J.A. and Owicki, J.A., 1996) . Typically cells expressing receptors are harvested and seeded at 3 x 10⁵ cells per micrephysiometer capsule in complete media 24 hours prior to an experiment. The media is replaced with serum free media 16 hours prior to recording to minimize non-specific metabolic stimulation by assorted and ill-defined serum factors. On the day of the experiment the cell capsules are transferred to the microphysiom.eter and allowed to equilibrate in recording media (low buffer RPMI 1640, no bicarbonate, no serum (Molecular Devices Corporation, Sunnyvale, CA) containing 0.1% fatty acid free BSA) , during which a baseline measurement of basal metabolic activity is established.

A standard recording protocol specifies a 100 ul/mir. flow rate, with a 2 min total pump cycle which includes a 30 sec flow interruption during which the acidification rate measurement is taken. Ligand challenges involve a 1 min 20 sec exposure to the sample just prior to the first post challenge rate measurement being taken, followed by two additional pump cycles for a total of 5 mm. 20 sec sample exposure. Typically, drugs in a primary screen are presented to the cells at 10 μM final concentration.

Fellow up experiments to examine dose-dependency of active compounds are then dene by sequentially challenging the cells with a drug concentration range that exceeds the amount needed tc generate responses ranging fro threshold to maximal levels. Ligand samples are then washed out and the acidification rates reported are expressed as a percentage increase of the peak response over the baseline rate observed just prior to challenge .

MAP kinase assay

MA? kinase (mitogen activated kinase) may be monitored tc evaluate receptor activation. MA? kinase is activated by multiple pathways in the ceil. A primary mode or activation involves the ras/raf/MEK/MAP kinase pathway. Growth factor (tyrosine kinase) receptors feed into this pathway via SHC/Grb-2/SOS/ras . Gi coupled receptors are alsc knovm to activate ras and subsequently produce an activation of MA? kinase. Receptors that activate phosphclipase C (such as Gq/Gll-coupled) produce diacylglycerol (DAG) as a consequence of phosphatidyl inositol hydrolysis. DAG activates protein kinase C which in turn phosp oryiates MAP kinase.

MAP kinase activation can be detected by several approaches . One approach is based on an evaluation of the phosphorylation state, either unphosphorylated (inactive) or phosphorylated (active). "^^' e phosphorylated protein has a slower mobility in SDS-PAGE and can therefore be compared with the unstimulated protein using Western blotting. Alternatively, antibodies specific for the phosphorylated protein are available (New England Biolabs) which can be used to detect an increase in the phosphorylated kinase. In either method, ceils are stimulated with the test compound and then extracted with Laemmli buffer. The soluble fraction is applied to an SDS-PAGE gel and proteins are transferred electrophoretically to nitrocellulose or Immcbilon. Immuncreactive bands are detected by standard Western blotting technique. Visible or chem.ilum.inescent signals are recorded or. film and may be quantified by densito etry .

Another approach is based on evaluation of the MA? kinase activity via a phosphorylation assay. Cells are stimulated with the test ccmpound and a soluble extract is prepared. The extract is incubated at 30°C for 10 min with gamma-^""r'-A-. f , an ήif reyeiie_c^*.ι_ι..ιu ays -.em, ctii a. specific substrate for MAP kinase such as phosphorylated heat and acid stable protein regulated by insulin, or PHAS-I. The reaction is terminated by the addition of H₃PO₄ and samples are transferred to ice. An aliquot is spotted onto Whatman P81 chromatography paper, which retains the phosphorylated protein. The chromatography paper is washed and counted for ^~2? in a liquid scintillation counter. Alternatively, the cell extract is incubated with gamma-³²P-ATP, an ATP regenerating system, and biotinylated myelm basic protein bound by streptavidm tc a filter support. The m.yelin basic protein is a substrate for activated MAP kinase. The phosphorylation reaction is carried cut for 10 min at 30°C. The extract can then by aspirated through the filter, which retains the phosphorylated myeiir. basic protein. The filter is washed and counted for ^J"P by liquid scintillation counting.

Cell proliferation assay

Receptor activation of the orphan receptor may lead to a mitogenic or proliferative response which can be monitored via "Η-thymidme uptake. When cultured cells are incubated with ³H-thymidir.e , the thymidine translocates into the nuclei where it is phosphorylated to thymidine triphosphate. The nucleotide triphosphate is then incorporated into the cellular DNA at a rate that is proportional to the rate of cell growth. Typically, cells are grown m culture for 1-3 days. Ceils are forced into quiescence by the removal of serum for 24 hrs . A mitogenic agent is then added to the media. 24 hrs later, the cells are incubated with ³H-thymidir.e at specific activities ranging from 1 tc 10 uCi/m.l for 2-6 hrs. Harvesting procedures may involve trypsinizaticn and trapping of ce-- s py nitration ever *J£ n-Lters WILI: or v/ithout a prior incubation m TCA to extract soluble thymidine. The filters are processed with scintilϊant and counted for ³H by liquid scintillation counting. Alternatively, adherent cells are fixed in MeOH or TCA, washed in water, and soiubiiized in 0.05% deoxychclate/C .1 N NaOH. "^^' e soluble extract is transferred to scintillation vials and counted for ³K by liquid scintillation counting.

Alternatively, cell proliferation can be assayed by measuring the expression of an endogenous or heterologous gene product, expressed by the cell line used to transfect the orphan receptor, which can be detected by methods such as, but not limited to, florescence intensity, enzymatic activity, immur-oreaccivity , DNA hybridization, polymerase chain reaction, etc.

Promiscuous second messenger assays

It is net possible to predict, a priori and based solely upon tne GPCR sequence, which of the cell's many- different signaling pathways any given receptor will naturally use. It is possible, however, to coax receptors of different functional classes to signal through a preselected pathway through the use of promiscuous G_a subunits. For example, by providing a cell based receptor assay system, with an endoger.ously supplied promiscuous G_Q subur.it such as G_κι₅ or G_a_s or a chimeric G_α subunit such as Gcq_z, a GPCR, which might normally prefer to couple through a specific signaling pathway (e.g., G_s, G_ , G_ς, Go, etc.), can be made to couple through the pathway defined by the promiscuous G_a suour.it and upon agonist activation produce the second messenger associated with that subunit' s pathway. In the case of G_3l5, G_Qlg and/or this would involve activation ot the G_q pathway ana production of the second messenger IPs. Through the use of similar strategies and tools, it is possible tc bias receptor signaling through pathways producing other second messengers such as Ca^*+, cAM?, and K^τ currents, for example (Milligan and Rees, 1999) .

It follows that the promiscuous interaction of the exoger.ously supplied G_a subunit w th the receptor alleviates the need to carry out a different assay for each possible signaling pathway ana increases the chances of detecting a functional signal upon receptor activation .

Methods for recording currents in Xenopus oocytes Heterologous expression of GPCRs in Xenopus oocytes has been widely used to determine the identity of signaling pathways activated by agonist stimulation. In tne present invention, activation of the phosphclipase C (PLC) pathway was assayed by applying a test compound in ND96 solution to oocytes previously injected with cDNA for the human SNORF207 receptor and observing inward currents at a holding potential of approximately -80 mV . The appearance of currents that reverse at -25 mV and display ether properties of the Ca^+~-activated CI^" channel is indicative cf receptor-activation of PLC and release of IP₃ and intracellular Ca^""*. Such activation is exhibited by GPCRs that couple to G_σ or Gn.

Oocytes were harvested from. Xenopus ia evis as previously described (Quick and Lester, 1994; Sm.ith et al . , 1997). Oocytes were injected with a cocktail of T3-dπven hSNCRF2C7 cDNA, T3-RXA polymerase and NTP, directly into the cytoplasm, as previously described (Geib et al., 2001) . After 2 days of incubation at 15°C, dual electrode voltage clamp was performed using 3 M KCl-filled glass microelectrodes with resistances of 1-2 MΩ . Unless otherwise specified, oocytes were vcitage-clamped at a holding potential of -80 mV . During recordings, oocytes were bathed in continuously flowing (1-3 m.l/min) medium containing 96 m.M XaCi, 2 mM KOI, 1.8 m.M CaCl₂, and 5 M HEPES, pH 7.5 (XD96 Buffer) . Drugs were applied by local perfusion from a 10 mil glass capillary tube at a distance of 0.5 mm. from the oocyte.

Alternatively, activation of the G_Ξ-ceupled AC pathway is assayed by applying test compound in ND96 solution to oocytes expressing the subject receptor and the cystic fibrosis transmembrane conductance regulator (CFTR) . Changes of inward CFTR currents at a holding potential of -80 mV are observed. CFTR channel activity is heavily regulated by the cytosolic levels of cyclic AMP which can be increased by activation of G_≤-coupled adenylyl cyclase (AC) .

Measurement cf inwardly rectifying K^~ (potassium) channel (GIRK) activity m.ay be monitored in oocytes that have beer, co-mj ected with m.RXA or cDXA encoding the mamm.alian receptor plus GIRK subunits . GIRK gene products co- assemble to form, a G-protein activated potassium channel known to be activated (i.e., stimulated) by a number of GPCRs that couple tc G^ or G_c (Kubo et al., 1993; Dascai et al.,1993) . Oocytes expressing the mammalian receptor plus the GIRK subunits are tested for test compound responsitivity by measuring K^~ currents in elevated K^"' solution containing 49 mM K^'" . Inositol phosphate assay

Human SNORF207 receptor-mediated activation of the inositol phosphate (IP) second messenger pathways may be assessed by radiometric measurement of IP products.

For example, in a 96 well microplate format assay, COS-7 cells expressing the receptor of interest are plated at a density of 70,000 cells per well ana allowed to incubate for 24 hours. The cells are then labeled with 0.5 μCi

[ ^JH] myo-inositol overnight at 37°C, 5% C0₂. Immediately before the assay, the medium is removed and replaced with 180 μL cf Phosphate-Buffered Saline (PBS) containing 10 m.M LiCi . The plates are then incubated for 20 min at 37°C, 5% CC₂. Following the incubation, the cells are challenged with agonist (20 μl/well; lOx concentration) for 30 m.in at 37°C. The challenge is terminated by the addition of 100 uL of 5% v/v trichloroacetic acid, followed by incubation at 4^{^}0 for greater than 30 minutes. Total IPs are isolated from the lysate by ior. exchange chromatography. Briefly, the lysed contents of the wells are transferred to a Multiscreen HV filter plate

(Millipore) containing Dowex AG1-X8 (200-400 mesh, formate form) . The filter plates are prepared by adding 100 μL cf Dowex AG1-X8 suspension (50% v/v, water: resin) to each well. 1^'r.e filter plates are placed on a vacuum, manifold to wasr. or elute the resir. bed. Each well is first v.'ashed 2 times with 2CC ul of 5 M myo-inositcl . Total [^JK] inositol phosphates are eluted with 75 μl of 1.2M amm.onium formate/0. IM fcrmic acid solution into 96- well plates. 200 uL of scintillation cocktail is added to each well, and the radioactivity is determined by liquid scintillation counting.

Generation of baculcvirus The coding region cf DNA encoding the human receptor disclosed herein may be subcloned into pBlueBacIII into existing restriction sites or sites engineered into sequences 5' and 3' to the coding region of the polypeptides. To generate baculovirus, 0.5 μg of viral DXA (BaculoGold) and 3 μg of DXA construct encoding a polypeptide may be co-transfected into 2 x 10^s Spodoptera frugiperda insect Sf9 ceils by the calcium phosphate co- precipitation method, as outlined by Pharmingen (in "Baculovirus Expression Vector System: Procedures and Methods Manual") . The cells then are incubated for 5 days at 27°C.

The supernatant of the co-transfection plate may be collected by centrifugation and the recor.D r.ant virus plaque purified. The procedure to infect ceils with virus, to prepare stocks of virus and to titer the virus stocks are as described m Pharm.ingen ' s manual.

Localization of RXA coding for human SNORF207 receptor

Quantitative PCR using a fluoroger.ic probe with real time detection : Quantitative PCR using fiucrogenic probes used to characterize the distribution of SXORF207 RXA. This assay utilizes two oligonucleotides for conventional PCR amplification and a third specific oligonucleotide probe that is labeled with a reporter at the 5' end and a quencher at the 3' end of the oligonucleotide. In the instant invention, FAM ( 6-carbcxyfiuorescein) was used as the reporter, and Black Hole Quencher-l^τ,: (3H1) (Bicsearch) was used as the quencher. As amplification progresses, the labeled oligonucleotide probe hybridizes to the gene sequence between the two oligonucleotides useo for amplification. The nuclease activity of Ts q thermostable DNA polymerase is utilized to cleave the labeled probe. This separates the quencher from the reporter and generates a fluorescent signal that is directly proportional to the amount cf amplicon generated. This labeled probe confers a high degree of specificity. Non-specific amplification is not detected as the labeled probe does net hypπdize and as a consequence is not cleaved. All experiments were conducted in a FE7700 Sequence Detection System. (PE Biosyste s, Foster City, CA) .

Quantitative RT-PCR: Quantitative RT-PCR was used for the detection of SNORF207 RNA.

For use as a template in quantitative PCR reactions, cDNA was synthesized by reverse transcription from, total human RNA. Reverse transcription by SuperScriptll RNAse H^~ and (GibcoBRL/life Technologies) was primed using random hexa ers . Parallel reactions included ³²P labeled dCTP to allow quantification of the cDNA. Following reverse transcription, cDXA was phenol/chloroform extracted and precipitated. Incorporation of ^J^P dCTP was assessed after precipitation with tnchloroacetic acid and the amount of cDNA synthesized was calculated.

Primers were designed to amplify a region of human SNORF207. Primers with the following sequences were used:

Forward primer: hSX207-240F

5' -CTACTACGCACTGCACCACTGG -3' (SEQ ID NO: 7)

Reverse primer: hSN207-358R

5' -AGCGGTCCACGTTGATGAG -3' (SEQ ID XO : 8) Fluorogemc oligonucleotide probe : r.SN 2 C 7 - 2 65T

5 ' f tS- Ai^ -TTCCCCGACCTCCTGTGCCAGA - ( BHQ-1 ) 3 ' ( SEQ I D NO :

9 )

Using these primer pairs, ampliccn length is 119 bp for human SNORF207. Each PCR reaction contained 3.0 ng cDNA. Oligonucleotide concentrations were: 500 r.M of forward and reverse primers, and 200 nM of flucrogenic probe. PCR reactions were carried out in 50 ml volum.es using TaqMan universal PCR master mix (PE Applied Biosystems) . Buffer for RT-PCR reactions contained a fluor used as a passive reference (ROX: Perkin Elmer proprietary passive reference I) . All reagents for PCR (except cDNA and oligonucleotide primers) were obtained from Perkin Elmer (Fester City, CA) . Reactions were carried in a PE7700 sequence detection system. (PE Applied Biosystems) using the following thermal cycler profile: 50 °C 2 min., 95 °C 10 min., followed by 40 cycles of: 95 °C, 15 sec, 60 °C 1 mm .

Standard curves for quantification of human SNCRF2C7 were constructed using genomic DNA. Negative controls consisted of mRNA blanks, as well as primer and mRNA blanks. To confirm, that the mRNA was not contaminated with genomic DNA, PCR reactions were carried out without reverse transcription using Taq DXA polymerase. Integrity of RXA was assessed by amplification of RNA coding for cyclophilin or glyceraldehyde 3-phosphate dehydroger.ase (GAPDH) . Following reverse transcription and PCR amplification, data was analyzed using PE Biosystems sequence detection software. The fluorescent signal from each well was normalized using an internal passive reference, and data was titted a standard curve tc obtain relative quantities cf SNORF207 expression.

Localization of human SNORF207 in diabetic tissue: To determine if the level of SNORF207 transcripts is altered in diabetes, quantitative PCR was performed on cDNA synthesized from total RNA extracted from human skeletal muscle and peri-renal adipose tissue. Tissue samples were obtained from, diabetic donors that were insulin dependant for a minimum cf five years. Control samples were obtained from normal donors (perirenal adipose), or purchased (skeletal muscle; Clontech) . Tissue was homogenized in TRIZOL ( Ir.vitrogen) , extracted with chloroform, alcohol precipitated and quantified. To assess integrity of the isolated RNA, a small fraction of the RNA was size fractionated using an Agilent BioAnalyzer. Contaminating DXA was removed from the samples using DXAse. Reverse transcription was carried out using Superscript II (Invitrcgen) . PCR reactions were carried out in 50 μl volumes using Taq DNA polym.erase (Perkin Elm.er) . The primer, probe and PCR conditions used were the same as previously described.

Each human 3NORF207 PCR reaction contained cDNA from. either a diabetic or normal donor pool. Expression levels assayed m cDNA from individual diseased donors was compareα to cDNA from a pool of normal donors . For perirenal adipose cDNA, the normal pool contained cDNA synthesized twc donors, and skeletal muscle pool contained cDNA from eight donors. To equalize the am.ount of diabetic and normal cDNA in each reaction the amounts of cDNA normalized to cyclophilin. Each cDNA was assayed for cyclophilin m increasing quantities ranging from an estimated 0.25ng to 3.0ng. The cycle threshold values of tne Ul duti - i -. a .n — c o Λ e -. e ^■-- iii . - — ^■= -_. -. ■- . _ -_ _-• •= -. _ -• -- -. -- -. -- normal samples. The amount of diabetic cDNA that had the same cycle threshold as the normal cDNA was then used in the quantitative PCR assay. Oligonucleotide concentrations were: 500 nM of forward and reverse primers, and 50 nM of fluorogenic probe. The amplification reagent m each reaction was IX TaqMan Universal PCR Master Mix which contains AmpliTaq Gold DNA Polymerase, AmpErase UNG, dNTPS with dUTP, passive reference, and optimiized buffer components (Perkin Elmer proprietary enzymes and buffers) .

Following PCR amplification, data was analyzed using Perkin Elmer sequence detection software. The fluorescent signal from each well was normalized using an internal passive reference, and data from diabetic tissue was compared to the normal, Data was again normalized to a measurement of cyclophilin i each assay plate.

Chemical compounds

The TUPAC chemical name for Example 1 is 7-[2-(2- CHLOROPHENYL) -4- (2-HYDROXYPHENYL) -1, 3-DIOXAN-CIS-5- YL] HEX-4Z-ENOIC ACID; CAS Number : 117621-64-4; Synonym : ICI 192,605. Example 1 can be purchased for research use as a white solid from 3I0M0L Research Laboratories Inc. (5120 Butler Pike, Plymouth Meeting, PA 19462-1202, USA) or other commercial vendors . The structure for Example 1 is as follows :

Example 1 is described as a potent and selective orally active thromboxane A2 antagonist and has known therapeutic uses as brcnchoconstrictive agent, (al Jarad, et al, . 1994; Descombes, et al, 1993; Kawikova et al, 1995) .

The chem.ical name for Example 2 is X-LINOLEOYLGLYCINE . Example 2 can be purchased for research use as a white, waxy solid from BIOMOL Research Laboratories Inc. (5120 Butler Pike, Plymouth Meeting, PA 19462-1202, USA) or other commercial vendors . The structure for Example 2 is as follows :

Example 2 is classified as a bioactive lipid, possibly an endogenous iir.olenic acid m.etabolite and 18 : 2-anandamide metabolite. Example 2 has known therapeutic uses as an ar.ti-infla matcry agent (Burstein, et al, 20CC) . RESULTS

Isolation cf a full-length human SNORF2C7 receptor The full-length coding sequence of human SNORF207 was generated by Blue Heron Biotechnology, using their proprietary gene synthesis technology GeneMaker"^t-: (Blue Heron Technology, 22310 20^th Avenue, Suite ICC, Bothell, WA 98021) and cloned into Synaptic vector MSP7C. The published f ll-length r.SXORF20^~ sequence consists of a 1119 bp nucleotide coding region which can encode a 372 amino acid protein.

The predicted hSXORF207 amino acid sequence contains seven putative transmembrane domains and sequence motifs characteristic of the rhedopsin GPCR superfamily. Searches cf sequence databases using BLAST analysis (GCG Wisconsin Package Version 10.3, Accelrys Inc., San Diego, CA) shew that the most closely related human sequences (35-36% overall am.ino acid identity) are the orphan receptor termed GPR35, and the purinergic receptors P2Y5 and ?2Y9.

increase intracellular re.ease

CHO cells were transiently transfected with human SXORF207 cr vector DXA (mock-transfected) . Cells were then prepared for compound screening by FLI?R"'^': as described hereir.above in Materials and Methods. By this strategy, two molecules with agonist activity at hSNORF207 were discovered. Example 1 selectively activates r.SNORF207-trar.sfected cells with an E^_* = 11,200 RFU and EC ₀ = 1.6 x 10^"6 M (Figure 3B) . Example 2 selectively activates hSNCRF207-transfected ceils with Ξ_max = 9800 RFU and ΞC_5C = 4.1 x iC^~6 M (Figure 43) . The increase m calcium mobilization in CHO cells indicates that hSXORF207 is capable of coupling to a _q/_^::-n e signal transduction pathway.

Activation cf calcium-activated CI^" currents in hSNORF207- expressing Xenopus oocytes

In noninjected Xenopus oocytes, no significant changes in Ca^τ+-activated chloride currents were observed upon a bullet application of 50 uM Example 1 (n=5) cr 50 μM Example 2 (n=5) (see Figure 5A and Figure 5C) . However, application of 50 μM Example 1 elicited a significant increase of chloride currents (324 nA) in oocytes expressing hSXORF207 (n=10, see Figure 53) . Application of 50 μM Example 2 also elicited a significant increase of chloride currents (233 r.A) m oocytes expressing hSXORF207 (n=5, see Figure 5D) . The results indicate that Example 1 and Example 2 stimulate PLC and release of I?₃ and intracellular Ca^*"* via activation of the SNORF207 receptor .

Detection of mRNA coding for human SXORF207 receptor: mRNA was isolated from multiple tissues (Table 1) and assayed as described.

SNORF207 is expressed in moderate leλfels m all CNS region assayed. Highest levels are found m the midbram, hypothalamus and hippocampus. Tne high levels found m the midbrain are important as this region contains dopaminergic, and some serotonergic neurons. This situates SXORF207 to be active in tne modulation of dopaminergic and serotonergic systems and the multiple systems and behavior that they influence. Additionally, the substantia nigra and red nucleus are both located in the midbram and if SNORF2C7 is present in these two regions, it may have a significant impact or. Parkinson's disease or other movement αisuxuei*.. --.xpxebbiui- υ-. SNORF207 RNA m the hippocampal formation and amygdala support the hypothesis that SNORF207 is involved in the modulation of learning and m.e .ory. It may also have a role in the regulation of fear, mood, and may provide a target for the treatment of depression, anxiety, phobias and mood disorders . Other regions of the CNS containing SNORF207 include the hypothalamus which implies a role in feeding, endocrine regulation or circadian rhythms. It is also notable that moderate expression of SNORF207 in the dorsal root ganglia supports a role in the modulation of pain. The broad distribution of SNORF207 RNA throughout the CNS implies a modulatory role in multiple systems within the CNS .

In peripheral tissue, most tissues assayed expressed measurable SNORF207 mRNA levels. The peripheral tissues expressing the highest levels of SNORF2C7 m.RNA are spleen and small intestine. Expression of hig.n levels in the spleen implies a role in modulating immune function, and expression in the small intestine indicates a role in absorption or endocrine regulation of gastrointestinal function .

In addition to the potential therapeutic applications identified in Table 1, the localization data for RXA encoding the human SNORF2C7 receptor indicates that the DNA encoding the human SNORF207 receptor can be used to predict the likelihood that a tissue sample of unknown tissue origin is of midbram origin with respect to a given individual. In addition, with respect tc a given individual, one could determine whether a giver, tissue sample cf unknown, origin is of midbram origin as opposed to having the origin of another tissue, e.g. the kidney, pancreas, or skeletal muscle, bucr. determinations ay jje used for various purposes including but not limited to the detection, of tumor metastasis.

Another application is to use hum.an SNORF207 to identify or screen for antagonists or agonists that function in certain localized organs. This is especially useful for screening antagonists or agonists that have strong affinity for the SNORF207 receptor. Further is to provide targeted therapy at certain localized organs . Using antagonists and agonists that have strong affinity for SNORF207 or anti-SNORF207 antibodies, a person skilled in the art m.ay design a therapeutic treatment, that targets a particular tissue with relatively high SNORF207 expression level.

.aoie --

Distribution of RXA coding for human SNORF207 receptor using qRT-PCR

RXA encoding SXORF207 is expressed as copies/ng cDXA

hypotr.a-L.amus 3591 Appetite/ obesity, neuroendocrine reσulation kidney cortex 110 Electrolyte balance, hypertension kidney medulla .89 Electrolyte balance, hypertension

,ιver 32; Diabe :es

-unσ 537 Respiratory disorders, asthma medull; 37< Sensory transmission midbrain Regulation of dopaminergic and serotonergic systems, Parkinson's disease, m.ovement disorders pancreas Diabetes, endocrine disorders pituitary (whole) 200 Endocrine/ neuroendocrine regulation p--acenta Reproductive function por.tine reticular 184! Regulation of formation scm tosensory , meter, visual, auditory, auter.om.ic and affective processes prostate 408 Urinary dysfunction and male sexual disfunction putamen 1573 Modulation of dopaminergic function

;keietal m.uscle 16 Musculoskeleta. disorders

Regulation of SNORF207 RNA in diabetes.

The expression of SNORF207 was assayed in perirenal adipose tissue (5 donors) and skeletal m.uscle (7 donors) obtained from diabetic doors . All donors were insulin dependant for a minimum of five years . The levels of SNORF207 in diabetic donors was compared to the levels detected in a pool of normal controls .

In the 5 peri-renal adipose samples assayed, SNORF207 is up-regulated, approximately 3-10 fold. Skeletal muscle from. diabetic donors shows an inconsistent pattern indicating no net change in SXORF207 RNA extracted from, skeletal muscle.

These data indicate that SNORF2C7 is up-regulated in diabetes, and that this up-regulatior. is tissue specific. Its up-regulafion in peri renal adipose from diabetic

83 donors, combined with its expression proti.e indicate that the expression levels of this gene are altered in the disease state and compounds acting en this receptor m.ay provide a m.ethod for changing the clinical course of diabetes .

DISCUSSION

These data provide the first evidence for pharmacological characterization of SNORF207. Together with the expression pattern of SNORF207 mRNA, the data suggest a broad range of potential therapeutic applications related to regulation of SNORF207 activity (using molecules that bind to, agonize or antagonize SXORF207 activity) . Specific applications are outlined below. These applications are further supported by data from gene deletion studies in which case the SXORF207 receptor (otherwise known as 3ACH GPCR) was deleted from the mouse genome and phenotypic changes were reported (see PCT International Publication Xo. WO 02/38607 A2 ) .

Two molecules identified as agonists of SNORF 207 include 7- { 2- (2-chlorophenyi-4- ( 2-hydroxyphenyl ) -1, 3-dioxan-cis- 5-YL] hex-4Z-enoic acid (a.k.a. ICI 192,605) and N- iinolecylglycir.e . Structures are shown herir.above as Example 1 and Example 2. ICI 192,605 is a structural analog of particular importance for its greater potency as a SNORF207 agonist. N-linoleoylglycme , a possible metabolite of linolenic acid or anandamide, is of particular interest for its known biological property as an ar.ti-mflam.atory agent (Burstem et al . , 2000) . These agonist molecules may be used to activate SNORF207 and downstream consequences in biological m.odels predictive cf therapeutic utility. These agonist molecules may also be used to search for SNORF207 antagonists with therapeutic utility.

A rcle for SNORF2C7 and corresponding ligands in the treatment of pat ophysiology is supported by SNORF207 mRNA localization data. High levels of SNORF207 mRNA were identified in the central nervous system. (brain and soinal cord) . Specific regions of interest include amygdala, cerebral cortex, cerebellum, hippocampus, hypothalamus, medulla, midbrain, pontine reticular formation, putamen and lumbar spinal cord. Based on these data SNORF207 is likely to be involved in several disorders including affective (i.e. depression, anxiety), cognitive, seizure, learning, memory, vigilance, movement, balance, autonomic, metabolic (obesity, diabetes), neuroendocrine, sensory integration, nociceptive and pain disorders. SXORF207 mRNA was also identified in peripheral tissues. Specific regions of interest include dorsal root ganglia, thyroid, adrenal gland, sto .ach, small intestine, colon, liver, lung, spleen, heart and kidney (cortex and medulla) . Low but detectable levels were also found in adipose tissue, pancreas and skeletal muscle. Based on these data SNORF207 is likely to be involved in several disorders including metabolic (such as obesity, diabetes, dyslipidemia) , hypercholesterolemia, thermo-reguiation, depression, stress, fatigue, inflammatory, immune, gastrointestinal motility and secretion (including irritable bowel, inflammatory bowel, Crohn' s disease), immune, cardiovascular (hypertension), epithelial secretion (such as cystic fibrosis) and pain (including visceral pain) and sensory processing. The finding that SNORF207 rr.RNA is upregulated m perirenal fat from, diabetic patients further supports a role for this receptor and its ligands in the treatment and management of diabetes .

A role for SNORF207 and regulation thereof in the treatment of pathophysiclogy is further supported by localization data from PCT International Publication No. WO 02/38607 A2 , in which case SNORF207 mRNA was localized in m.ouse tissues by multiple techniques (lacZ reporter staining, Northern blot analysis and RT-PCR) . SXORF207 mRNA expression was m.apped to the following regions: brain (cerebellum, spinal column, substantia gelatinosa, dorsal root ganglia (A-delta and C fiber class), trigemir.al ganglion, trigeminal nucleus, cranial nerve 8 (with 2 m.ajor sets of afferent fibers conducting impulses from, inner ear to brain), eye (cells of conjunctiva), urinary bladder, gall bladder, tongue, skin (particularly around hair follicles and in the nasal region) , pleura and surface of lungs, salivary glands, regions of submaxiliary salivary glands, gut (esophagus, stomach, vilius of s .ail intestine, colon and rectum (crypts) and fat and pericardium, surrounding the heart. From this expression pattern, it was deduced that SNORF207 is likely to play a role in the pathophysiclegy and treatment of several disorders, including trigemir.al neuralgia, orcfacial pain, pain associated with toothache, migraine, irritable bowel syndrome, 3arrett' s esophagus, glaucoma, pain associated with cancer, diabetic neuropathies, Herpes infections, HIV infections, m.igraine and skin sensitivity associated with migraine, allodyna. toothache, neuroma (caused by amputation, nerve transaction or trauma), nerve compression (caused by tumors, entrapment or crush), pair, due to damage of spinal cord or brain, dementia, dyslexia, dyskinesis, tremor, Parkinson's, benign essential tremor, chorea, epilepsy cr ballisumus (for example occurring through stroke, trauma, degeneration or malignancy), dry-eye disorders, cystic fibrosis, hyperactive bladder, hypercholesterolemia, dyslipidemia and obesity (see PCT WO 02/38607 A2) . SNORF207 deletion studies in mice provide yet another body of evidence supporting a role for SXORF207 and regulation thereof in the treatment of pathophysiology

(see PCT WO 02/38607 A2 ) . Consistent with SNORF207 expression in the cranial nerve number 8

(vestibulecochlear) , some SNORF207 -/- mice have difficulty performing a wire balance maneuver test. Seme SNCRF207 -/- mice in this paradigm also display retrcpulsion (backwards walking) , as though compensating from a hallucinatory experience cf sliding downward. Thus a SNORF207 may be useful in the treatment cf vertigo and balance disorders related tc sensitivity or dysfunction of the inner ear.

In a model of spatial and cognitive processing, SXCRF207 -/- mice display impaired response to a visual "cliff"¹ edge, thereby suggesting defects in visual processing, anxiety and mobility. The mice are placed on a black-and- white checkered surface which adjoins a Perspe.x board, through which a 0.5 meter drop-off tc a lower surface may be viewed and perceived as though from the standpoint of a cliff. The "cliff" effect is accentuated by the high contrast pattern of the black-and-white checkered surface. Mice are placed at the boundary cf the "cliff" and Perspex board. The latency to m.ove and the direction of the movement (toward or away from Perspex) are coth scored. SNORF207 -/- mice display a "freezing" behavior (i.e. a longer latency to move) but tend to perform, like wild type counterparts m directional movement (favoring the direction away from. Perspex) . Retropulsion is accentuated in this m.cdel, as though compensating from a hallucinatory experience of falling downward. (Heterozygous mutants display defects in both latency and directional movement; one possibility is that the heterozygotes navigate m.ore by vibrissae than by visual cues.) These data provide further support for SNORF207 agonists m the treatment of vertigo and balance disorders. SNORF207 agonists m.ay also be useful for the treatment of motion sickness and symptoms thereof

(including headache and nausea). SNORF207 agonists m.ay also be useful for the treatment of anxiety in general, or as it relates to balance disorders in particular. SNORF2C7 agonists may also be useful for treating disorders of visual processing. SNORF207 agonists may also be useful for treating disorders of movement

(dyskinesis ) .

Consistent with SNORF207 expression in the cereoellun, SNORF207 -/- mice display improved motor coordination and balance when tested on a rotorod. Outbred (but not mored) SNORF207 -/- mice also display improved learning ability upon repeat testing. While this may seem, contradictory in light of the spatial and balance impairments described above, one possibility is that compensatory mechanisms for dealing with the perception of apparent movement (such as retropulsion) may favor appropriate responses m conditions of actual movement. Thus a SNORF207 agonist or antagonist may be useful for the treatment cf disorders related to motor and reflex learning skills.

Consistent with SNORF207 expression m sensory nerves, spinal cord ana tr.alamus, SNORF207 -/- mice display defects in sensory processing related to external stimuli and pain. SNORF207 -/- mice are less sensitive than wild type counterparts to paw pressure, as measured py latency to withdraw from a sharpened dowel rod pressed against the paw. SNORF207 -/- mice are also less sensitive to thermal pain, as measured by latency in the tail flick model. SNORF207 -/- mutants are also less sensitive to a noxious substance (formalin) when injected into a hind paw, as measured by the time spent licking and biting the affected paw. SNORF2C7 -/- mice are also less sensitive tc mechanical stimulation of a paw conveyed through a wire mesh platform, as indicated by the minimum gauge Von Frey hair (wire) required to elicit a withdrawal response. Thus SNORF207 antagonists may be useful for the treatment of pain and related sensory disorders.

Consistent with SNORF2C7 expression in the bladder, SNORF207 -/- m.ice displayed abnorm.al bladder m.otility. This was expressed as hypoactive bladder, with less frequent urination but larger voiding volumes. The data may be explained by defect in smooth muscle function and/or sensory processing. Thus SNORF207 agonists may be useful for the treatment of urinary hypomotility and retention. SXORF207 antagonists m.ay be useful for the treatment of urinary stress or urge incontinence. SXORF207 agonists or antagonists m.ay be useful for the treatment of erectile dysfunction and the control of motor fibers in the prostate.

Consistent with SXORF207 expression in liver, gallbladder and fat, SXORF207 -/- mice displayed increased levels of circulating cholesterol.

REFERENCES

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Claims

WHAT IS CLAIMED:

1. A process for identifying a chemical compound which specifically binds to a human SNORF207 receptor which com.prises contacting cells transfected with DNA. encoding, and expressing on their ceil surface, the human SXORF2C7 receptor, wherein such cells prior tc being transfected do not normally express the human SNORF207 receptor, or a membrane preparation of such cells, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the hum.an SNORF207 receptor.

2. A process involving competitive binding for identifying a chemical compound which specifically binds to a human SNORF207 receptor which comprises separately contacting cells expressing on their cell surface the human SNORF207 receptor, wherein such cells dc not normally express the human SNORF207 receptor, or a* membrane preparation of sucn cells, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of such compounds to the receptor, and detecting specific binding of the chemical compound to the human SXORF207 receptor, a decrease in the binding of the second chemical compound to the human SXORF2C7 receptor in the presence of the chemical com.pound being tested indicating that such chem.ical compound binds to the human SNORF207 receptor.

3. A method of screening a plurality of chemical compounds not known to bind to a human SNORF207 receptor to identify a compound which specifically binds to the human SNORF207 receptor, which comprises :

(a) contacting cells transfected with, and expressing, DXA encoding the hum.an SXORF207 receptor, or a membrane preparation of such cells, with a compound known to bind specifically to the human SNORF207 receptor;

(b) contacting the cells of step (a) with the plurality cf compounds net known to bind specifically to the human SXORF207 receptor, under conditions permitting binding cf compounds known tc bind to the human 3NORF207 receptor;

(c) determining whether the binding of the compound known to bind to the human SXORF2C7 receptor is reduced in the presence of the plurality of compounds, relative to the binding of the compound m the absence cf the plurality of compounds; and if so

(d) separately determining the binding to the human SXORF2C7 receptor of each compound included m the plurality of compounds, so as to thereby identify any compound included therein which specifically binds to the hum.an SXORF207 receptor .

4. A process for determining whether a chemical compound is a hum.an SNORF207 receptor agonist which comprises contacting cells transfected with and expressing DXA encoding the human SXORF207 receptor, with the compound under conditions permitting the activation of the human SXORF207 receptor, and detecting any increase m human SXORF207 receptor activity, so as to thereby determine whether the compound is a human SNORF207 receptor agonist.

5. A process for determining whether a chemical compound is a human SNORF207 receptor antagonist which comprises contacting cells transfected with and expressing DNA encoding the human SNORF207 receptor, with the compound in the presence of a known human SNORF207 receptor agonist, under conditions permitting the activation of the human SNORF207 receptor, and detecting any decrease in human SNORF2C7 receptor activity, so as to thereby determine whether the compound is a human SNORF207 receptor antagonist.

6. A process for determining whether a chem.ical compound specifically binds to and activates a human SNORF2C7 receptor, which comprises contacting cells, producing a second messenger response and expressing on their cell surface the hum.an SNORF207 receptor, wherein such cells do net normally express the human SNORF207 receptor, with the chem.ical com.pound under conditions suitable for activation of the human SXORF207 receptor, and measuring the second messenger response in the presence and in the absence of the chemical compound, a change in the second messenger response m the presence of the chemical compound indicating that the compound activates the hum.an 3NORF207 receptor.

7. A process for determining whether a chemical compound specifically binds to and inhibits activation of a human SNORF207 receptor, which comprises separately contacting cells producing a second messenger response and expressing on their cell surface the human SNORF207 receptor, wherein such cells do not normally express the human SNORF207 receptor, with both the chemical compound and a second chem.ical compound known to activate the human SNORF207 receptor, and with only the second chemical compound, under conditions suitable for activation of the human SNORF207 receptor, and measuring the second messenger response in the presence of only the second chemical compound and in the presence of both the second chemical compound and the chemical compound, a smaller change in the second messenger response in the presence of both the chemical compound and the second chem.ical compound than in the presence of only the second chemical compound indicating that tne cnemicai compound inhibits activation of the human SNORF207 receptor .

8. A method of screening a plurality of chem.ical compounds not known to activate a human SNORF207 receptor to identify a compound which activates the human SNORF2C7 receptor which comprises:

(a) contacting cells transfected with and expressing the human SNORF2C7 receptor with the plurality of compounds not known tc activate the human SNORF207 receptor, under conditions permitting activation of the human SNORF2C7 receptor;

(b) determining whether the activity of the human SNORF207 receptor is increased m the presence of cne or more of the compounds;- and if so (c) separately determining whether the activation cf the human SNORF207 receptor is increased by any compound included in the plurality of compounds, so as to thereby identify each compound which activates the human SNORF207 receptor .

9. A method of screening a plurality of chemical compounds not known to inhibit the activation of a human SNORF207 receptor to identify a compound which inhibits the activation of the human SNORF207 receptor, which comprises: a) contacting cells transfected with and expressing the human SNORF207 receptor with the plurality of compounds in the presence of a known human SNORF207 receptor agonist, under conditions permitting activation of the human SNORF207 receptor; b) determining whether the extent cr am.ount of activation of the human SXORF207 receptor is reduced in the presence of one or more of the compounds, relative to the extent or amount of activation of the human SXORF2C7 receptor in the absence of such one or mere compounds; and if so c) separately determining whether each such compound inhibits activation of the human SNORF207 receptor for each compound included in the plurality cf compounds, so as to thereby identify any compound included in such plurality of compounds which inhibits the activation of the human SXORF207 receptor.

.04

10. A process for preparing a pharmaceutical composition which comprises admixing a carrier, and a therapeutically effective amount of a chemical compound identified by the process any one of claims

4, 6 or 8.

11. A process for preparing a pharmaceutical com.position which comprises admixing a carrier, and a therapeutically effective amount of a chemical compound identified by the process any one cf claims

5, 7 or 9.

12. A method of treating an abnormality m a subject wherein the abnormality is alleviated by increasing the activity of a human SNORF2C7 receptor which comprises administering to the subject a com.pound which is a human SNORF207 receptor agonist in an amount effective to treat the abnorm.ality.

13. A method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a human SNORF2C7 receptor which comprises administering to the subject a compound which is a human SNORF207 receptor antagonist in an amount effective to treat the abnormality.

14. The method of claim 12 or 13, wherein the abnormality is diabetes .