WO2005059550A1 - Adn codant snorf138 et ses utilisations - Google Patents
Adn codant snorf138 et ses utilisations Download PDFInfo
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- WO2005059550A1 WO2005059550A1 PCT/US2003/031659 US0331659W WO2005059550A1 WO 2005059550 A1 WO2005059550 A1 WO 2005059550A1 US 0331659 W US0331659 W US 0331659W WO 2005059550 A1 WO2005059550 A1 WO 2005059550A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
- G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- GPCRs G-protein coupled receptors
- GPCRs represent a major class of cell surface receptors with which many ligands interact to mediate their effects. GPCRs are characterized by seven membrane-spanning domains interconnected with three intracellular loops and three extracellular loops. Ligand binding to its cognate receptor results in the activation of the G proteins coupled to the receptor. This marks the beginning of a biochemical cascade that results in the production of second messengers such as cA P or accumulation of calcium in the cytosol . While structural motifs that characterize a GPCR can be recognized in the predicted amino acid sequence of a novel receptor, an endogenous ligand that activates the GPCR cannot always be predicted from its primary structure.
- a novel receptor sequence may be designated as a GPCR when it possesses the characteristic structural motif, even though an endogenous ligand for the receptor has not yet been defined.
- GPCRs can also be activated by ligands other than an endogenous ligand.
- Receptor 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, other metabolic byproducts, and synthetic molecules. Many of these ligands interact with specific cell surface receptors such as GPCRs which transduce signals from the extracellular space to the intracellular space (cytosol) of the cell.
- GPCRs cell surface receptors which transduce signals from the extracellular space to the intracellular space (cytosol) of the cell.
- Such agonists or antagonists permit determination the biochemical role of the receptor in normal and pathological states, and thus therapeutic potential of drugs that would act at the receptor. Finally, the use of such agonists accelerates the discovery of an endogenous ligand through iterative structure/function analyses and datamining.
- the present invention describes methods for identifying compounds that bind to, bind to and activate, or bind to and inhibit the activation of the GPCR designated SNORF138.
- the present invention identifies a- SNORF138 agonist.
- the present invention describes the use of SNORF138 agonists and/or antagonists in the treatment of disorders, including, but not limited to, immune disorders and inflammatory disease .
- This invention provides a process for identifying a chemical compound which specifically ⁇ binds to a mammalian SNORF138 receptor which comprises contacting cells containing DNA encoding, and expressing on their cell surface, the mammalian SNORF138 receptor, wherein such cells do not normally express the mammalian SMORF138 receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian SNORF138 receptor.
- this invention provides a process for identifying a chemical compound which specifically binds to a mammalian SNORF138 receptor which comprises contacting a membrane preparation from cells containing DMA encoding, and expressing on their cell surface, the mammalian SNORF138 receptor, wherein such cells do not normally express the mammalian SNORF138 receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian SNORF138 receptor.
- this invention provides a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian SNORF138 receptor which comprises separately contacting cells expressing on their cell surface the mammalian SNORF138 receptor, wherein such cells do not normally express the mammalian SNORF138 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 chemical compound to the mammalian SNORF138 receptor, a decrease in the binding of the second chemical compound to the mammalian SNORF138 receptor in the presence of the chemical compound being tested indicating that such chemical compound binds to the mammalian SNORF138 receptor.
- This invention also provides a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian SNORF138 receptor wbiich comprises separately contacting a membrane preparation from cells expressing on their cell surface the mammalian SNORF138 receptor, wherein such cells do not normally express the mammalian SNORF138 receptor, with both the chemical compound and a second c ⁇ iemical 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 mammalian SNORF138 receptor, a decrease in the binding of the second chemical compound to the mammalian SNORF138 receptor in the presence of the chemical compound being tested indicating that such chemical compound binds to the mammalian SNORF138 receptor.
- This invention further provides a method of screening a plurality of chemical compounds not known to bind to a mammalian SNORF138 receptor to identify a compound which specifically binds to the mammalian SNORF138 receptor, -which comprises (a) contacting cells transfected with, and expressing, DNA encoding the mammalian SNTORF138 receptor with a compound known to bind specifically to the mammalian SNORF138 receptor; (b) contacting the cells of step (a) with the plurality of compounds not known to bind specifically to the mammalian SNORF138 receptor, under conditions permitting binding of compounds known to bind to the mammalian SNORF138 receptor; (c) determining whether the binding of the compound known to bind to the mammalian SNORF138 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 (d) separately determining the binding to the mammalian SNORF138 receptor of each compound included in the plurality
- This invention still further provides a method of screening a plurality of chemical compounds not known to bind to a mammalian SNORF138 receptor to identify a compound which specifically binds to the mammalian ⁇ N0RF138 receptor, which comprises (a) contacting a membrane preparation from cells transfected with, and expressing, DNA encoding the mammalian SNORF138 receptor with the plurality of compounds not known to bind specifically to the mammalian SNORF138 receptor under conditions permitting binding of compounds known to bind to the mammalian SNORF138 receptor; (b) determining whether the binding of a compound known to bind to the mammalian SNORF138 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 SNORF138 receptor of each compound included in the plurality of compounds, so as to thereby identify any compound included therein which specifically binds to the mammalian SNORF
- This invention also provides a process for determining whether a chemical compound is a mammalian SNORF138 receptor agonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian SNORF138 receptor with the compound under conditions permitting the activation of the mammalian SNORF138 receptor, and detecting any increase in mammalian SNORF138 receptor activity, so as to thereby determine whether the compound is a mammalian SNORF138 receptor agonist.
- This invention further provides a process for determining whether a chemical compound is a mammalian SNORF138 receptor antagonist which comprises contacting cells transfected -with and expressing DNA encoding the mammalian SNORF138 receptor with the compound in the presence of a known mammalian SNORF138 receptor agonist, under conditions permitting the activation of the mammalian SNORF138 receptor, and detecting any decrease in mammalian SNORF138 receptor activity, so as to thereby determine whether the compound is a mammalian SNORF138 receptor antagonist.
- this invention provides a process for determining whether a chemical compound specifically binds to and activates a mammalian SNORF138 receptor, which comprises contacting cells producing a second messenger response and expressing on their cell surface the mammalian SNORF138 receptor, wherein such cells do not normally express the mammalian SNORF138 receptor, with the chemical compound under conditions suitable for activation of the mammalian SNORF138 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 in the presence of the chemical compound indicating that the compound activates the mammalian SNORF138.receptor.
- This invention further provides a process for determining whether a chemical compound specifically binds to and inhibits activation of a mammalian
- ⁇ NORF138 receptor which comprises separately contacting cells producing a second messenger response and expressing on their cell surface the mammalian SNORF138 receptor, wherein such cells do not normally express the mammalian SNORF138 receptor, with both the chemical compound and a second chemical compound known to activate the mammalian SNORF138 receptor, and with only the second chemical compound, under conditions suitable for activation of the mammalian SNORF138 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 chemical compound than in the presence of only the second chemical compound indicating that the chemical compound inhibits activation of the mammalian SNORF138 receptor.
- This invention provides a method of screening a plurality of chemical compounds not known to activate a mammalian SNORF138 receptor to identify a compound which activates the mammalian SNORF138 receptor which comprises: (a) contacting cells transfected with and expressing the mammalian SNORF138 receptor with the plurality of compounds not known to activate the mammalian SNORF138 receptor, under conditions permitting activation of the mammalian SNORF138 receptor; (b) determining whether the activity of the mammalian SNORF138 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 SNORF138 receptor is increased by any compound included in the plurality of compounds, so as to thereby identify each compound which activates the mammalian SNORF138 receptor.
- This invention further provides a method of screening a plurality of chemical compounds not known to inhibit the activation of a mammalian SNORF138 receptor to identify a compound which inhibits the activation of the mammalian SNORF138 receptor, which comprises: (a) contacting cells transfected with and expressing the mammalian SNORF138 receptor with the plurality of compounds in the presence of a known mammalian SNORF138 receptor agonist, under conditions permitting activation of the mammalian SNORF138 receptor; (b) determining whether the extent or amount of activation of the mammalian SN0RF138 receptor is reduced in the presence of one or more of the compounds, relative to the extent or amount of activation of the mammalian SNORF138 receptor in the absence of such one or more compounds; and if so (c) separately determining whether each such compound inhibits activation of the mammalian SNORF138 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 activ
- This invention additionally provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian SNORF138 receptor which comprises administering to the subject a compound which is a mammalian SNORF138 receptor agonist in an amount effective to treat the abnormality.
- This invention further provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a mammalian SNORF138 receptor which comprises administering to the subject a compound which is a mammalian SNORF138 receptor antagonist in an amount effective to treat the abnormality.
- This invention provides an isolated nucleic acid encoding a mammalian SNORF138 receptor.
- This invention further provides a purified mammalian SNORF138 receptor protein.
- this invention provides a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian SNORF138 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 human SNORF138 receptor contained in plasmid MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) .
- This invention provides a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian SNORF138 receptor, wherein the probe has a sequence complementary to a unique sequence present within (a) the nucleic acid sequence shown in Figures 1A-1B (SEQ ID NO: 1) or (b) the reverse complement thereof.
- this invention provides a method 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 DNA 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 SNORF138 receptor and labeled with a detectable marker; (e) detecting labeled bands which have hybridized to the DNA encoding a mammalian SNORF138 receptor of claim 1 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
- This invention further provides a transgenic, nonhuman mammal comprising a homologous recombination knockout of the native mammalian SNORF138 receptor.
- Nucleotide sequence including the sequence encoding a human SNORF138 receptor (SEQ ID NO : 1) .
- the putative open reading frame is indicated toy underlining the start (ATG) codon (at positions 1-3) and the stop codon (at positions 991-993).
- ATG start (ATG) codon
- stop codon at positions 991-993.
- a partial 3' untranslated sequence is shown.
- SNORF138 receptor encoded by the open reading frame indicated in the nucleotide sequence shown in Figures 1A-1B (SEQ ID NO: 1) .
- the seven pu ative transmembrane (TM) regions are underlined.
- This invention provides for a process for identifying a chemical compound which specifically binds to a mammalian SNORF138 receptor which comprises contacting cells transfected with DNA encoding, and expressing on their cell surface, the mammalian SNORF138 receptor, wherein such cells prior to being transfected with such DNA do not normally express the mammalian SNORF138 receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian SNORF138 receptor.
- This invention further provides for a process for identifying a .chemical compound which specifically binds to a mammalian SNORF138 receptor which comprises contacting a membrane preparation from cells transfected with DNA encoding and expressing on their cell surface the mammalian SNORF138 receptor, wherein such cells prior to being transfected with such DNA do not normally express the mammalian SNORF138 receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian SNORF138 receptor.
- This invention still further provides a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian SNORF138 receptor which comprises separately contacting cells transfected with DNA encoding and expressing on their cell surface the mammalian SNORF138 receptor, wherein such cells prior to being transfected with such DNA do not normally express the mammalian SNORF138 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 bindzLng of the chemical compound to the mammalian SNORF138 receptor, a decrease in the binding of the second chemicaZL compound to the mammalian SNORF138 receptor in the presence of the chemical compound being tested indicating that such chemical compound binds to the mammalian SNORF138 receptor.
- This invention provides a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian SNORF138 receptor which comprises separately contacting a membrane preparation from cells transfected with DNA. encoding and expressing on their cell surface the mammalian SNORF138 receptor, wherein such cells prior to being transfected with such DNA do not normally express the mammalian SNORF138 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 chemical compound to the mammalian SNORF138 receptor, a decrease in the binding of the second chemical compound to the mammalian SNORF138 receptor in the presence of the chemical compound being tested indicating that such chemical compound binds to the mammalian SNORF138 receptor.
- the second chemical compound is a glucocorticosteroic .
- trie second chemical compound is betamethasone valerate.
- the mammalian SNORF138 receptor is a human SNORF138 receptor.
- the mammalian SNORF138 receptor has substantially the same amino acid sequence as the human SN0RF138 receptor encoded by plasmid MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) .
- the mammalian SNORF138 receptor has substantially the same amino acid sequence as that shown in Figures 2A-2B (SEQ ID NO : 2) . In another embodiment, the mammalian SNORF138 receptor has the amino acid sequence shown in Figures 2A-2B (SEQ ID NO: 2) .
- the compound is not previously known to bind to a mammalian SNORF138 receptor .
- the cell is an insect cell.
- the cell is a mammalian cell.
- the cell is nonneuronal in origin.
- the nonneuronal cell is a COS -7 cell, a human embryonic kidney cell, a CHO cell, a NIH- 3T3 cell, a mouse Yl cell, or a LM(tk-) cell.
- the compound is a compound not previously known to bind to a mammalian SNORF138 receptor. This invention provides a compound identified by the preceding processes according to this invention.
- This invention provides for a method of screening a plurality of chemical compounds not known to bind to a mammalian SNORF138 receptor to identify a compound which specifically binds to the mammalian SNORF138 receptor, which comprises (a) contacting cells transfected with, and expressing, DNA encoding the mammalian SNORF138 receptor with a compound known to bind specifically to the mammalian SNORF138 receptor; (b) contacting the cells of step (a) with the plurality of compounds not known to bind specifically to the mammalian SNORF138 receptor, under conditions permitting binding of compounds known to bind to the mammalian SNORF138 receptor; (c) determining whether the binding of the compound known to bind to the mammalian SNORF138 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 (d) separately determining the binding to the mammalian SNORF138 receptor of each compound included in the plurality of compounds
- This invention provides a method of screening a plurality of chemical compounds not known to bind to a mammalian SNORF138 receptor to identify a compound which specifically binds to the mammalian SNORF138 receptor, which comprises (a) contacting a membrane preparation from cells transfected with, and expressing, DNA encoding the mammalian SNORF138 receptor with the plurality of compounds not known to bind specifically to the mammalian SNORF138 receptor under conditions permitting binding of compounds known to bind to the mammalian SNORF138 receptor; (b) determining whether the binding of a compound known to bind to the mammalian SNORF138 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 SNORF138 receptor of each compound included in the plurality of compounds, so as to thereby identify any compound included therein which specifically binds to the mammalian SNORF138 receptor.
- this invention provides a process for determining whether a chemical compound is a mammalian SNORF138 receptor agonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian SNORF138 receptor with the compound under conditions permitting the activation of the mammalian SNORF138 receptor, and detecting any increase in mammalian SNORF138 receptor activity, so as to thereby determine whether the compound is a mammalian SNORF138 receptor agonist.
- This invention further provides a process for determining whether a chemical compound is a mammalian SNORF138 receptor agonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian SNORF138 receptor with the compound under conditions permitting the activation of the mammalian SNORF138 receptor, and detecting any increase in mammalian SNORF138 receptor activity, so as to thereby determine whether the compound is a mammalian SNORF138 receptor agonist.
- This invention also provides a process for determining whether a chemical compound is a mammalian SNORF138 receptor antagonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian SNORF138 receptor with the compound in the presence of a known mammalian SNORF138 receptor agonist, under conditions permitting the activation of the mammalian SNORF138 receptor, and detecting any decrease in mammalian SNORF138 receptor activity, so as to thereby determine whether the compound is a mammalian SNORF138 receptor antagonist.
- This invention also provides a process for determining whether a chemical compound is a mammalian SNORF138 receptor antagonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian SNORF138 receptor with the compound in the presence of a known mammalian SNORF138 receptor agonist, under conditions permitting the activation of the mammalian SNORF138 receptor, and detecting any decrease in mammalian SNORF138 receptor activity, so as to thereby determine whether the compound is a mammalian SNORF138 receptor antagonist.
- the mammalian SNORF138 receptor is a human SNORF138 receptor.
- the mammalian SNORF138 receptor has substantially the same amino acid sequence as the human SNORF138 receptor , encoded by plasmid MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) .
- the mammalian SNORF138 receptor has substantially the same amino acid sequence as that shown in Figures 2A-2B (SEQ ID NO: 2) . In another embodiment, the mammalian SNORF138 receptor has the amino acid sequence shown in Figures 2A-2B (SEQ ID NO: 2) .
- the compound is not previously known to bind to a mammalian SNORF138 receptor.
- the cell is an insect cell.
- the cell is a mammalian cell.
- the cell is nonneuronal in origin.
- the nonneuronal cell is a COS-7 cell, a human embryonic kidney cell, a CHO cell, a NIH- 3T3 cell, a mouse Yl cell, or a LM(tk-) cell.
- the compound is a compound not previously known to bind to a mammalian SNORF138 receptor. This invention provides a compound identified by the preceding processes according to this invention.
- composition for example a pharmaceutical composition, which comprises an amount of a mammalian SNORF138 receptor agonist determined by a process according to this invention effective to increase activity of a mammalian SNORF138 receptor and a carrier, for example, a pharmaceutically acceptable carrier.
- a carrier for example, a pharmaceutically acceptable carrier.
- the mammalian SNORF138 receptor agonist is not previously known.
- this invention provides a composition, for example a pharmaceutical composition, which comprises an amount of a mammalian SNORF138 receptor antagonist determined by a process according to this invention effective to reduce activity of a mammalian SNORF138 receptor and a carrier, for example, a pharmaceutically acceptable carrier.
- a composition for example a pharmaceutical composition, which comprises an amount of a mammalian SNORF138 receptor antagonist determined by a process according to this invention effective to reduce activity of a mammalian SNORF138 receptor and a carrier, for example, a pharmaceutically acceptable carrier.
- the mammalian SNORF138 receptor antagonist is not previously known. In an embodiment, the mammalian SNORF138 receptor antagonist is a human SNORF138 receptor antagonist.
- This invention moreover provides a process for determining whether a chemical compound specifically binds to and activates a mammalian SNORF138 receptor, which comprises contacting cells producing a second messenger response and expressing on their cell surface the mammalian SNORF138 receptor, wherein such cells do not normally express the mammalian SNORF138 receptor, with the chemical compound under conditions suitable for activation of the mammalian SNORF138 receptor, and measuring the second messenger response in the presence and in the absence of the chemical compound, a change, e.g. an increase, in the second messenger response in the presence of the chemical compound indicating that the compound activates the mammalian SNORF138 receptor.
- a change e.g. an increase
- the second messenger response comprises chloride channel activation and the change in second messenger is an increase in the level of chloride current.
- the second messenger response comprises change in intracellular calcium levels and the change in second messenger is an increase in the measure of intracellular calcium.
- the second messenger response comprises release of inositol phosphate and the change in second messenger is an increase in the level of inositol phosphate.
- the second messenger response comprises release of arachidonic acid and the change in second messenger is an increase in the level of arachidonic acid.
- the second messenger response comprises GTP S ligand binding and the change in second messenger is an increase in GTP S ligand binding.
- the second messenger response comprises activation of MAP kinase and the change in second messenger response is an increase in MAP kinase activation.
- 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 mammalian SNORF138 receptor, which comprises separately contacting cells producing a second messenger response and expressing on their cell surface the mammalian SNORF138 receptor, wherein such cells do not normally express the mammalian SNORF138 receptor, with both the chemical compound and a second chemical compound known to activate the mammalian SNORF138 receptor, and with only the second chemical compound, under conditions suitable for activation of the mammalian SNORF138 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, 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 mammalian SNORF138 receptor.
- a smaller change e.g. increase
- the second chemical compound is a glucocorticosteroid. In an embodiment of the present invention, the second chemical compound is betamethasone valerate.
- 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 chemical compound than in the presence of only the second chemical compound.
- 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 chemical compound and the second chemical compound than in the presence of only the second chemical compound.
- the second messenger response comprises release of inositol phosphate and the change in second messenger response is a smaller increase in the level of inositol phosphate in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound.
- 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 chemical compound and the second chemical compound than in the presence of only the second chemical compound.
- the second messenger response comprises change in cAMP levels and the change in second messenger response is a smaller change in the level of cAMP in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound.
- the second messenger response comprises release of arachidonic acid and the change in second messenger response is an increase in the level of arachidonic acid levels in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound.
- the second messenger response comprises GTP S ligand binding and the change in second messenger is a smaller increase in GTP S 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.
- the mammalian SNORF138 receptor is a human SNORF138 receptor.
- the mammalian SNORF138 receptor has substantially the same amino acid sequence as the human SNORF138 receptor encoded by " plasmid MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) .
- the mammalian SNORF138 receptor has substantially the same amino acid sequence as that shown in Figures 2A-2B (SEQ ID NO: 2) .
- the mammalian SNORF138 receptor has the amino acid sequence shown in Figures 2A-2B (SEQ ID NO: 2) .
- the compound is not previously known to bind to a mammalian SNORF138 receptor.
- the cell is an insect cell.
- the cell is a mammalian cell.
- the cell is nonneuronal in origin.
- the nonneuronal cell is a COS-7 cell, a human embryonic kidney cell, a CHO cell, a NIH- 3T3 cell, a mouse Yl cell, or a LM(tk-) cell.
- the compound is a compound not previously known to bind to a mammalian SNORF138 receptor. This invention provides a compound identified by the preceding processes according to this invention.
- 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 SNORF138 receptor agonist determined to be such by a process according to this invention effective to increase activity of a mammalian SNORF138 receptor and a carrier, for example, a pharmaceutically acceptable carrier.
- a mammalian SNORF138 receptor agonist is not previously known.
- This invention also provides a composition, for example, a pharmaceutical composition, which comprises an amount of a mammalian SNORF138 antagonist determined to be such by a process according to this invention, effective to reduce activity of the mammalian SNORF138 receptor and a carrier, for example a pharmaceutically acceptable carrier.
- a carrier for example a pharmaceutically acceptable carrier.
- the mammalian SNORF138 antagonist is not previously known.
- This invention yet further provides a method of screening a plurality of chemical compounds not known to activate a mammalian SNORF138 receptor to identify a compound which activates the mammalian SNORF138 receptor which comprises: (a) contacting cells transfected with and expressing the mammalian SNORF138 receptor with the plurality of compounds not known to activate the mammalian SNORF138 receptor, under conditions permitting activation of the mammalian SNORF138 receptor; (b) determining whether the activity of the mammalian SNORF138 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 SNORF138 receptor is increased by any compound included in the plurality of compounds, so as to thereby identify each compound which activates the mammalian SNORF138 receptor.
- This invention provides a method of screening a plurality of chemical compounds not known to inhibit the activation of a mammalian SNORF138 receptor to identify a compound which inhibits the activation of the mammalian SNORF138 receptor, which comprises: (a) contacting cells transfected with and expressing the mammalian SNORF138 receptor with the plurality of compounds in the presence of a known mammalian SNORF138 receptor agonist, under conditions permitting activation of the mammalian SNORF138 receptor; (b) determining whether the extent or amount of activation of the mammalian SNORF138 receptor is reduced in the presence of one or more of the compounds, relative to the extent or amount of activation of the mammalian SNORF138 receptor in the absence of such one or more compounds; and if so (c) separately determining whether each such compound inhibits activation of the mammalian SNORF138 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 SNORF138 receptor is a human SNORF138 receptor.
- the mammalian SNORF138 receptor has substantially the same amino acid sequence as the human SNORF138 receptor encoded by plasmid MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) .
- the mammalian SNORF138 receptor has substantially the same amino acid sequence as that shown in Figures 2A-2B (SEQ ID NO: 2) . In another embodiment, the mammalian SNORF138 receptor has the amino acid sequence shown in Figures 2A-2B (SEQ ID NO: 2) .
- the compound is not previously known to bind to a mammalian SNORF138 receptor.
- the cell is an insect cell.
- the cell is a mammalian cell.
- the cell is nonneuronal in origin.
- the nonneuronal cell is a COS -7 cell, a human embryonic kidney cell, a CHO cell, a NIH-
- the compound is a compound not previously known to bind to a mammalian SNORF138 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.
- composition for example, a pharmaceutical composition, comprising a compound identified by a method according to this invention in an amount effective to increase mammalian SNORF138 receptor activity and a carrier, for example, a pharmaceutically acceptable carrier.
- a pharmaceutical composition comprising a compound identified by a method according to this invention in an amount effective to increase mammalian SNORF138 receptor activity and a carrier, for example, a pharmaceutically acceptable carrier.
- composition for example, a pharmaceutical composition, comprising a compound identified by a method according to this invention in an amount effective to decrease mammalian SNORF138 receptor activity and a carrier, for example, a pharmaceutically acceptable carrier.
- a pharmaceutical composition comprising a compound identified by a method according to this invention in an amount effective to decrease mammalian SNORF138 receptor activity and a carrier, for example, a pharmaceutically acceptable carrier.
- this invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian SNORF138 receptor which comprises administering to the subject a compound which is a mammalian SNORF138 receptor agonist in an amount effective to treat the abnormality.
- This invention additionally provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a mammalian SNORF138 receptor which comprises administering to the subject a compound which is a mammalian SNORF138 receptor antagonist in an amount effective to treat the abnormality.
- the abnormality is a regulation of a steroid hormone disorder, an epinephrine release disorder, a gastrointestinal disorder, a cardiovascular disorder, an electrolyte balance disorder, hypertension, diabetes, a respiratory disorder, asthma, a reproductive function disorder, an immune disorder, such as psoriasis, an endocrine disorder, a musculoskeletal disorder, a neuroendocrine disorder, a cognitive disorder, a memory disorder, so atosensory and neurotransmission disorders, metabolic disorders, a motor coordination disorder, a sensory integration disorder, a motor integration disorder, a dopaminergic function disorder, an appetite disorder, such as anorexia or obesity, a sensory transmission disorder, drug addiction, an olfaction disorder, an autonomic nervous system disorder, pain, neuropsychiatric disorders, affective disorder, migraine, circadian disorders, visual disorders, urinary disorders, blood coagulation-related disorders, developmental disorders, or ischemia-reperfusion injury-related diseases.
- an immune disorder such as psorias
- the mammalian SNORF138 receptor is a human SNORF138 receptor.
- This invention also provides a process for making a composition of matter which specifically binds to a mammalian SNORF138 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 chemical compound identified by a process in accordance with this invention 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 identifying a chemical compound by a process in accordance with this invention or a novel structural and functional analog or homolog thereof, recovering the chemical compound free of any receptor, and then admixing a carrier, for example, a pharmaceutically acceptable carrier, and a therapeutically effective amount of the chemical compound.
- the mammalian SNORF138 receptor is a human SNORF138 receptor.
- This invention provides a pharmaceutical composition made by combining a therapeutically effective amount of the compound of 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 pharmaceutically acceptable carrier .
- This invention further provides a pharmaceutical composition
- a pharmaceutical composition comprising a therapeutically effective amount of the compound of the invention and a pharmaceutically acceptable carrier.
- the amount of the compound is an amount from about 0.01 mg to about 800 mg.
- the amount of the compound is an amount from about 0.01 mg to about 500 mg.
- the amount of the compound is an amount from about 0.01 mg to about 250 mg.
- the amount of the compound is an amount from about 0.1 mg to about 60 mg .
- the amount of the compound is an amount from about 1 mg to about 20 mg .
- the carrier is a liquid and the composition is a solution.
- the carrier is a solid and the composition is a powder or tablet.
- the carrier is a gel and the composition is a capsule or suppository.
- pharmaceutically acceptable carrier is any pharmaceutical carrier known to those of ordinary skill in the art as useful in formulating pharmaceutical compositions.
- the pharmaceutical carrier may be a liquid and the pharmaceutical composition would be in the form of a solution.
- the pharmaceutically acceptable carrier is a solid and the composition is in the form of a powder or tablet .
- the pharmaceutical carrier is a gel and the composition is in the form of a suppository or cream.
- the compound may be formulated as a part of a pharmaceutically acceptable transdermal patch.
- 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 can also be an encapsulating material.
- the carrier is a finely divided solid which is in admixture with the finely divided active ingredient.
- the active ingredient is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the 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, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
- Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
- the active ingredient can be dissolved or suspended in a pharmaceutically 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 preferably sodium carboxymethyl cellulose solution
- alcohols including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil)
- oils e.g. fractionated coconut oil and arachis oil
- the carrier can also be an oily ester such as ethyl oleate 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 propellent.
- Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utilized by for example, intramuscular, intrathecal, epidural , intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously.
- the compounds may 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.
- a “therapeutically effective amount” is any amount of 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.
- a “subject” is a vertebrate, a mammal, or a human.
- the compound can be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic) , bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
- solutes or suspending agents for example, enough saline or glucose to make the solution isotonic
- bile salts for example, enough saline or glucose to make the solution isotonic
- acacia gelatin
- sorbitan monoleate oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide
- compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, 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 will vary with the particular compound in use, the strength of the preparation, the mode of administration, and the advancement of 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 of administration.
- This invention provides a recombinant nucleic acid comprising a nucleic acid encoding a mammalian SNORF138 receptor, wherein the mammalian receptor-encoding nucleic acid hybridizes under high stringency conditions to a nucleic acid encoding a human SNORF138 receptor.
- This invention provides a recombinant nucleic acid comprising a nucleic acid encoding a mammalian SNORF138 receptor, wherein the receptor has a sequence identical to the sequence of the human SNORF138 receptor encoded by plasmid MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) .
- This invention further provides a recombinant nucleic acid comprising a nucleic acid encoding a human SNORF138 receptor, wherein the human SNORF138 receptor comprises an amino acid sequence identical to the sequence of the human SNORF138 receptor as indicated in Figures 1A-1B (SEQ ID NO: 1) .
- the human SNORF138 receptor is encoded by the nucleotide sequence beginning at the start codon at positions 1-3 and ending at the stop codon at positions 991-993 as indicated in Figures 1A-1B (SEQ ID NO: 1) .
- the plasmid MSP70-hSNORF138-f was deposited on September 25, 2002, with the American Type Culture Collection (ATCC), 10801 University Boulevard., Manassas, Virginia 20110-2209, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and was accorded ATCC Patent Deposit Designation PTA-4725.
- ATCC American Type Culture Collection
- This invention also contemplates recombinant nucleic acids which comprise nucleic acids encoding naturally occurring allelic variants of the mammalian SNORF138 receptors described above.
- hybridization under high stringency conditions means hybridization performed at 40°C in a hybridization buffer containing 50% formamide, 5X SSC, 7 mM Tris, IX Denhardt ' s , 25 g/ml salmon sperm DNA; wash at 50°C in 0.IX SSC, 0.1%SDS.
- N adenine, cytosine, guanine, or thymine (or other modified base such as inosine)
- I inosine
- the term "agonist" is used throughout this application to indicate any peptide or non-peptidyl compound
- 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.
- pharmaceutically acceptable carrier means any of the standard pharmaceutically acceptable carriers . Examples include, but are not limited to, phosphate buffered saline, physiological saline, water, and emulsions, such as oil/water emulsions.
- the mammalian SNORF138 receptor gene contains introns and furthermore, the possibility exists that additional introns could exist in coding or non-coding regions.
- spliced form(s) of mRNA may encode additional amino acids either upstream of the currently defined starting methionine or within the coding region.
- the existence and use of alternative exons is possible, whereby the mRNA may encode different amino acids within the region comprising the exon .
- single amino acid substitutions may arise via the mechanism of RNA editing such that the amino acid sequence of the expressed protein is different than that encoded by the original gene. (Burns, CM. 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 mammalian SNORF138 receptor disclosed herein. This invention further provides for alternate translation initiation sites and alternately spliced or edited variants of nucleic acids encoding the mammalian SNORF138 receptor of this invention.
- the nucleic acids of the subject invention also include nucleic acid analogs of the human SNORF138 receptor gene, wherein the human SNORF138 receptor gene comprises the nucleic acid sequence shown in Figures 1A-1B (SEQ ID NO: 1) or contained in plasmid MSP70- hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) .
- Nucleic acid analogs of the human SNORF138 receptor genes differ from the human SNORF138 receptor genes described herein in terms of the identity or location of one or more nucleic acid bases (deletion analogs containing less than all of the nucleic acid bases shown in Figures 1A-1B or contained in plasmid MSP70- hSNORF138-f, substitution analogs wherein one or more nucleic acid bases shown in Figures 1A-1B or contained in plasmid MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) , 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 encode proteins which share some or all of the properties of the proteins encoded by the nucleic acid sequences shown in Figures 1A-1B or contained in plasmid MSP70- hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) .
- 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 MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) .
- the nucleic acid analog encodes a protein having an amino acid sequence which differs from the amino acid sequences shown in Figures 2A-2B or encoded by the nucleic acid contained in plasmid MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) . See, for example, PCT International Publication No. WO 01/98330 or Genembl Database Accession No. AC0121016.
- 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 in Figures 2A-2B. In another embodiment, the function of 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 acid sequence occurs within the trans embrane (TM) region of the protein. In a further embodiment, the variation in the nucleic acid sequence occurs outside of the TM region.
- TM trans embrane
- the nucleic acid analog encodes a mammalian SNORE138 receptor which has above 75% amino acid identity, preferably above 85% amino acid identity, more preferably above 95% amino acid identity to the SNORF138 receptor encoded by the plasmid MSP70- h.SNORF138-f (ATCC Patent Deposit Designation PTA-4725) .
- nucleic acid is DNA.
- the DNA is cDNA.
- the DNA is genomic DNA.
- nucleic acid is RNA. Methods for production and manipulation of nucleic acid molecules are well known in the art .
- nucleic acid which is degenerate with respect to the DNA encoding any of the polypeptides described herein.
- the nucleic acid comprises a nucleotide sequence which is degenerate with respect to the nucleotide sequence shown in Figures 1A-1B (SEQ ID NO: 1) or the nucleotide sequence contained in the plasmid MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) , that is, a nucleotide sequence which is translated into the same amino acid sequence.
- This invention also encompasses DNAs and cDNAs which encode amino acid sequences which differ from those of the polypeptides of this invention, but which should not produce phenotypic changes.
- this invention also encompasses DNAs, cDNAs, and RNAs which hybridize to the DNA, cDNA, and RNA of the subject invention. Hybridization methods are well known to those of skill in the art.
- nucleic acids of the subject invention also include nucleic acid molecules coding 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 less than all of the 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 amino acid residues is added to a terminal or medial portion of the polypeptides) and which share some or all properties of naturally-occurring forms.
- These molecules include: the incorporation of 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 DNA sequences that facilitate construction of readily expressed vectors.
- the creation of polypeptide analogs is well known to those of skill in the art (Spurney, R. F. et al . (1997); Fong, T.M. et al . (1995); Underwood, ' D.J. et al . (1994); Graziano, M.P. et al . (1996); Guan X.M. et al. (1995) ) .
- modified polypeptides of this invention may be transfected into cells either transiently or stably using methods well-known in the art, examples of which are disclosed herein.
- This invention also provides for binding- assays using the modified 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 herein.
- nucleic acids described and claimed herein are useful for the information which they provide concerning the amino acid sequence of the polypeptide and as products for the large scale synthesis of the polypeptides by a variety of recombinant techniques.
- the nucleic acid molecule is useful for generating new cloning and expression vectors, transformed and transfected prokaryotic and eukaryotic host cells, 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 acid encoding species homologs of the SNORF138 receptor encoded by the nucleic acid sequence shown in Figures 1A-1B (SEQ ID NO: 1) or encoded by the plasmid MSP70- hSN0RF138-f (ATCC Patent Deposit Designation PTA-4725) .
- the nucleic acid encodes a mammalian SNORF138 receptor homolog which has substantially the same amino acid sequence as does the SNORF138 receptor encoded by the plasmid MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) . Examples of methods for isolating and purifying species homologs are described elsewhere (e.g., U.S. Patent No. 5,602,024, W094/14957, W097/26853, WO98/15570) .
- the nucleic acid encodes a mammalian SNORF138 receptor homolog which has above 75% amino acid identity to the SNORF138 receptor encoded by the plasmid MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725); preferably above 85% amino acid identity to the SNORF138 receptor encoded by the plasmid MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) ; most preferably above 95% amino acid identity to the SNORF138 receptor encoded by the plasmid MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) .
- the mammalian SNORF138 receptor homolog has above 70% nucleic acid identity to the SNORF138 receptor gene contained in plasmid MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725); preferably above 80% nucleic acid identity to the SNORF138 receptor gene contained in the plasmid MSP70-hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) ; more preferably above 90% nucleic acid identity to the SNORF138 receptor gene contained in the plasmid MSP70-hSNORF138 -f (ATCC Patent Deposit Designation PTA-4725) .
- nucleic acid encoding a mammalian SNORF138 receptor.
- the nucleic acid is DNA.
- the DNA is cDNA.
- the DNA is genomic DNA.
- the nucleic acid is RNA.
- the mammalian SNORF138 receptor is a human SNORF138 receptor.
- the human SNORF138 receptor has an amino acid sequence identical to that encoded by the plasmid MSP70- hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) .
- the human SNORF138 receptor has an amino acid sequence identical to the amino acid sequence shown in Figures 2A-2B (SEQ ID NO: 2) .
- This invention provides a purified mammalian SNORF138 receptor protein.
- the SNORF138 receptor protein is a human SNORF138 receptor protein.
- This invention provides a vector comprising the nucleic acid of this invention.
- This invention further provides a vector adapted for expression in 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 to permit expression thereof, wherein the cell is a bacterial, amphibian, yeast, insect or mammalian cell.
- the vector is a baculovirus .
- the vector is a plasmid.
- This invention provides a plasmid designated MSP70- hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) .
- 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.
- a vector or plasmid with untranslated sequences of varying lengths may express differing amounts of the polypeptide depending upon the host cell used.
- the vector or plasmid comprises the coding sequence of the polypeptide and the regulatory elements necessary for expression in the host cell.
- the cell is a non-mammalian cell.
- the non- mammalian cell is a Xenopus oocyte cell or a Xenopus melanophore cell.
- the cell is a mammalian cell.
- the cell is a COS-7 cell, a human embryonic kidney cell, a HEK293 cell, a NIH-3T3 cell, a LM(tk-) cell, a mouse Yl cell, or a CHO cell.
- the cell is an insect cell.
- the insect cell is an Sf9 cell, an Sf21 cell or a Trichoplusia ni 5B-4 cell.
- This invention provides a membrane preparation isolated from the cell in accordance with this invention.
- this invention provides for a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian SNORF138 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 SNORF138 receptor contained in plasmid MSP70 -hSNORF138-f (ATCC Patent Deposit Designation PTA-4725) .
- This invention further provides a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian SN0RF138 receptor, wherein the probe has a sequence complementary to a unique sequence present within (a) the nucleic acid sequence shown in Figures 1A-1B (SEQ ID WO: 1) or (b) the reverse complement to (a) .
- the nucleic acid is DNA.
- the nucleic acid is RNA.
- the phrase "specifically hybridizing” means the ability of a nucleic acid molecule to 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.
- nucleic acids further enables elucidation of possible receptor diversity and of the existence of multiple subtypes within a family of receptors of which SNORF138 is a member.
- the receptors of this invention will serve as a valuable tool for designing drugs for treating various pathophysiological conditions such as chronic and acute inflammation, arthritis, autoimmune diseases, transplant rejection, graft vs. host disease, bacterial, fungal, protozoan and viral infections, septicemia, AIDS, pain, psychotic and neurological disorders, including anxiety, depression, schizophrenia, dementia, mental retardation, memory loss, epilepsy, neuromotor disorders, locomotor disorders, respiratory disorders, asthma, eating/body weight disorders including obesity, bulimia, diabetes, anorexia, nausea, hypertension, hypotension, vascular and cardiovascular disorders, ischemia, stroke, cancers, ulcers, urinary retention, sexual /reproductive disorders, circadian rhythm disorders, renal disorders, bone diseases including osteoporosis, benign prostatic hypertrophy, gastrointestinal disorders, nasal congestion, dermatological disorders such as psoriasis, allergies, Parkinson's disease, Alzheimer's disease, acute heart failure, angina disorders, delirium, dyskinesias such as Huntington
- transfected cells may also be used to test compounds and screen co ⁇ npound libraries to obtain compounds which bind to the SNORF138 receptor, as well as compounds which activate or inhibit activation of functional responses in such cells, and therefore are likely to do so in vivo .
- This invention provides an antibody capable of binding to a mammalian SNORF138 receptor encoded by a nucleic acid encoding a mammalian SNORF138 receptor.
- the mammalian SNORF138 receptor is a human SNORF138 receptor.
- This invention also provides an agent capable of competitively inhibiting the binding of the antibody to a mammalian SNORF138 receptor.
- the antibody is a monoclonal antibody or antisera.
- This invention provides for an antisense oligonucleotide having a sequence capable of specifically hybridizing to RNA encoding a mammalian SNORF138 receptor, so as to prevent translation of such RNA.
- This invention further provides for an antisense oligonucleotide having a sequence capable of specifically hybridizing to genomic DNA encoding a mammalian SNORF138 receptor, so as to prevent transcription of such genomic DNA.
- the oligonucleotide comprises chemically modified nucleotides or nucleotide analogues.
- This invention still further provides a pharmaceutical composition
- a pharmaceutical composition comprising (a) an amount of an oligonucleotide in accordance with this invention capable of passing through a cell membrane and effective to reduce expression of a mammalian SNORF138 receptor and (b) a pharmaceutically acceptable carrier capable of passing through the cell membrane.
- the oligonucleotide is coupled to a substance which inactivates mRNA.
- the substance which inactivates mRNA is a ribozyme.
- the pharmaceutically acceptable carrier comprises a structure which binds to a mammalian SNORF138 receptor on a cell capable of being taken up by the cells after binding to the structure.
- the pharmaceutically acceptable carrier is capable of binding to a mammalian SNORF138 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 SNORF138 receptor and a pharmaceutically acceptable carrier.
- This invention further provides a transgenic, nonhuman mammal expressing DNA encoding a mammalian SNORF138 receptor in accordance with this invention.
- This invention provides a transgenic, nonhuman mammal comprising a homologous recombination knockout of a native mammalian SNORF138 receptor.
- This invention further provides a transgenic, nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a mammalian SNORF138 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 SNORF138 receptor so as to thereby reduce translation or such mRNA and expression of such receptor.
- the DNA encoding the mammalian SNORF138 receptor additionally comprises an inducible promoter. In another embodiment, the DNA encoding the mammalian SNORF138 receptor additionally comprises tissue specific regulatory elements. In another embodiment, the transgenic, nonhuman mammal is a mouse .
- This invention also provides a method of detecting expression of a mammalian SNORF138 receptor by detecting the presence of mRNA coding for the mammalian SNORF138 receptor which comprises obtaining total mRNA 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 SNORF138 receptor by the cell.
- This invention further provides for a method of detecting the presence of a mammalian SNORF138 receptor on the surface of a cell which comprises contacting the cell 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 cell, and thereby detecting the presence of the mammalian SNORF138 receptor on the surface of the cell.
- This invention still further provides a method of determining the physiological effects of varying levels of activity of a mammalian SNORF138 receptor which comprises producing a transgenic, nonhuman mammal in accordance with this invention whose levels of mammalian SNORF138 receptor activity are varied by use of an inducible promoter which regulates mammalian SNORF138 receptor expression.
- This invention additionally provides a method of determining the physiological effects of varying levels of activity of a mammalian SNORF138 receptor which comprises producing a panel of transgenic, nonhuman mammals in accordance with this invention each expressing a different amount of a mammalian SNORF138 receptor .
- this invention provides method for identifying an antagonist capable of alleviating an abnormality wherein the abnormality is alleviated by decreasing the activity of a mammalian SNORF138 receptor comprising administering a compound to a transgenic, nonhuman mammal according to this invention, and determining whether the compound alleviates any physiological and/or behavioral abnormality displayed by the transgenic, nonhuman mammal as a result of overactivity of a mammalian SNORF138 receptor, the alleviation of such an abnormality identifying the compound as an antagonist.
- the mammalian SNORF138 receptor is a human SNORF138 receptor.
- the 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 to 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 SNORF138 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.
- this invention provides a method for identifying an agonist capable of alleviating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian SNORF138 receptor comprising administering a compound to a transgenic, nonhuman mammal according to this invention, and determining whether the compound alleviates any physiological and/or behavioral abnormality displayed by the transgenic, nonhuman mammal, the alleviation of such an abnormality identifying the compound as an agonist .
- the mammalian SNORF138 receptor is a human SNORF138 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.
- this invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian SNORF138 receptor which comprises administering to the subject an effective amount of the pharmaceutical composition of this invention so as to thereby treat the abnormality.
- this invention provides a method 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 DNA 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 SNORF138 receptor and labeled with a detectable marker; (e) detecting labeled bands which have hybridized to the DNA encoding a mammalian SNORF138 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 is a disorder associated with the activity of a specific mammalian allele is diagnosed.
- This invention also provides a method of preparing a purified mammalian SNORF138 receptor according to this invention which comprises: (a) culturing cells which express the mammalian SNORF138 receptor; (b) recovering the mammalian SNORF138 receptor from the cells; and (c) purifying the mammalian SNORF138 receptor so recovered.
- This invention further provides a method of preparing a purified mammalian SNORF138 receptor according to this invention which comprises: (a) inserting a nucleic acid encoding the mammalian SNORF138 receptor into a suitable expression vector; (b) introducing the resulting vector into a suitable host cell; (c) placing the resulting host cell in suitable condition permitting the production of the mammalian SNORFI38 receptor; (d) recovering the mammalian SNORFI38 receptor so produced; and optionally (e) isolating and/or purifying the mammalian SNORF138 receptor so recovered.
- the gene for a targeted receptor subtype is cloned, it is placed into a recipient cell which thxen 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 cell 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.
- bindrng assays enable chemists to design compounds toward or away from one or more of the relevant subtypes, as appropriate, for optimal therapeutic efficacy.
- 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 to rationally design drugs that will be more effective and have fewer or substantially less severe side effects than existing drugs.
- Combinatorial chemistry involves automated synthesis of a variety of 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 .
- the primers synthesized were: TB1 : 5'- GTTCAAGTCCAGGTCGACACTGC (SEQ ID NO: 3) TB2 : 5'- GGCACAGGCTGGAGCCATGCTG (SEQ ID NO : 4)
- a 1375 bp cDNA was isolated from a human kidney cDNA library by PCR screening/sib selection with TB1/TB2. (The human kidney cDNA library was constructed in a plasmid vector.) Sequence analysis revealed a 993 bp open reading frame that could encode a 330 amino acid protein with predicted transmembrane domains and sequence motifs characteristic of the rhodopsin GPCR superfamily. The putative coding region was amplified by PCR using primers KS2588 and KS2589 designed from the cDNA clone and subcloned into a mammalian expression vector.
- PCR primers were: KS2588: 5 ' -ACGCCCAACAGCACTGGCGAGG (SEQ ID NO: 5) KS2589: 5'-CAGCTGAAGGGATCCCCTTTAGTTCAAGTCCAGGTCGAC (SEQ ID NO: 6)
- the primer KS2589 was designed to incorporate a BamH I restriction enzyme site into the 3' end of the amplified GPCR coding region.
- the PCR-amplified fragment was digested with BamH I restriction enzyme prior to ligation into the expression vector MSP 70.
- the resulting plasmid was designated K1335.
- a nucleic acid sequence encoding a SNORF138 receptor from other species may be isolated using standard molecular biology techniques and approaches such as those described below:
- a genomic library (e.g., cosmid, phage, PI, BAC, YAC) generated from the species of interest may be screened with a 32 P-labeled oligonucleotide probe corresponding to a fragment of the human SNORF138 receptor whose sequence is shown in Figures 1A-1B to isolate a genomic clone.
- the full-length sequence may be obtained by sequencing this genomic clone. If one or more introns are present in the gene, the full-length intronless gene may be obtained from cDNA using standard molecular biology techniques.
- 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, intronless receptor from cDNA.
- Standard molecular biology techniques could be used to subclone this gene into a mammalian expression vector.
- Standard molecular biology techniques may be used to screen commercial cDNA phage libraries of the species of interest by hybridization under reduced stringency with a 32 P- labeled oligonucleotide probe corresponding to a fragment of the sequences shown in Figures 1A-1B.
- One may isolate a full-length SNORF138 receptor by obtaining a plaque purified clone from the lambda libraries and then subjecting the clone to direct DNA sequencing.
- standard molecular 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 may be isolated by Southern hybridization of colony lifts of positive pools with a 32 P-oligonucleotide probe.
- RACE 3' and 5' RACE may be utilized to generate PCR products from cDNA derived from the species of interest expressing SNORF138 which contain the additional sequence of SNORF138. These RACE PCR products may then be sequenced to determine the additional sequence. This new sequence is then used to design a forward PCR primer in the 5'UT and a reverse primer in the 3 ' UT . These primers are then used to amplify a full-length SNORF138 clone from cDNA.
- Examples of other species include, but are not limited to, rat, mouse, dog, monkey, hamster and guinea pig.
- a broad variety of host cells can be used to study heterologously expressed proteins. These cells include but are not limited to mammalian cell lines such as: COS-7, CHO, LM(tk " ), HEK293 cells, etc.; insect cell lines such as: Sf9, Sf21, Trichoplusia ni 5B-4, etc.; amphibian cells such as Xenopus oocytes; assorted yeast strains; assorted bacterial cell strains; and others. Culture conditions for each of these cell types is specific and is known to those familiar with the art.
- COS-7 cells are grown on 150 mm plates in DMEM with supplements (Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin 100 g/ml streptomycin) at 37°C, 5% C0 2 .
- Stock plates of COS-7 cells are trypsinized and split 1:6 every 3-4 days.
- HEK cells (Peak Rapi cellsTM, Edge BioSystems, Gaithersburg, MD) are maintained in DMEM growth medium supplemented with 10% bovine calf serum, 1% L-glutamine, 50 g/ml gentamycin at 37°C, 5% C0 2 .
- DNA encoding proteins to be studied can be transiently expressed in a variety of mammalian, insect, amphibian, yeast, bacterial and other cells lines by several transfection methods including but not limited to; calcium phosphate-mediated, DEAE-dextran mediated; liposomal -mediated, viral-mediated, electroporation- mediated, and microinjection delivery. Each of these methods may require optimization of assorted experimental parameters depending on the DNA, cell line, and the type of assay to be subsequently employed.
- a typical protocol for the DEAE-dextran method as applied to COS-7 and HEK293 cells is described as follows.
- Cells to be used for transfection are split 24 hours prior to the transfection to provide flasks which are 70-80% confluent at the time of trans ection.
- 8 g of receptor DNA plus 8 g of any additional DNA needed e.g. G protein expression vector, reporter construct, antibiotic resistance marker, mock vector, etc.
- 9 ml of complete DMEM plus DEAE-dextran mixture (10 mg/ml in PBS) .
- Cells plated into a T225 flask (sub-confluent) are washed once with PBS and the DNA mixture is added to each flask.
- HEK cells are allowed to incubate for 30 minutes at 37°C, 5% C0 2 . Following the incubation, 36 ml of complete DMEM with 80 M chloroquine is added to each flask and allowed to incubate an additional 3 hours . The medium is then aspirated and 24 ml of complete medium containing 10% DMSO for exactly 2 minutes and then aspirated. The cells are then washed 2 times with PBS and 30 ml of complete DMEM added to each flask. The cells are then allowed to incubate 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 method according to Jordan, et al . (1996) .
- 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 co-transfection of an ancillary gene to confer drug resistance on the targeted host cell. The ensuing drug resistance can be exploited to select and maintain cells that have taken up the DNA.
- An assortment of resistance genes are available including but not restricted to neomycin, kanamycin, and hygromycin.
- 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.
- 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 complement.
- Cell membranes expressing the receptor protein of this invention are useful for certain types of assays including but not restricted to ligand binding assays, GTP- -S binding assays, and others.
- the specifics of preparing such cell membranes may in some cases be determined by the nature of 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 HC1 , mM EDTA, pH 7.4 at 4° C) .
- the resulting crude cell lysate is cleared of cell debris by low speed centrifugation at 200xg for 5 min at 4° C.
- the cleared supernatant is then centrifuged at 40,000xg for 20 min at 4° C, and the resulting membrane pellet is washed by suspending in ice cold buffer and repeating the high speed centrifugation step. The final washed 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 immediately or frozen for later use.
- Cells expressing the receptor of this invention may 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.
- the final protein concentration in the assay can be 12 - 40 g/ml.
- Membranes are then incubated with radiolabeled ligand either in the presence or absence of competing ligands on ice for 60 min in a total volume of 250 1 in 96 well microtiter plates.
- the bound ligand is then separated from free ligands by filtration through GF/B filters presoaked in 0.5% polyethyleneimine (PEI), using a Tomtec (Wallac) vacuum filtration device. After addition of Ready Safe (Beckman) scintillation fluid, bound radioactivity is quantitated using a Trilux (Wallac) scintillation counter (approximately 40% counting efficiency of bound counts) .
- agonist or antagonist compounds that bind to the receptor may be identified as they inhibit the binding of the radiolabeled ligand to the membrane 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 nM of the unlabeled peptide corresponding to the radioligand used.
- membrane preparations or intact cells transfected with the receptor are incubated in the presence of increasing concentrations of the labeled compound to determine the binding affinity of 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.
- Cells expressing the receptor DNA of this invention may be used to screen for ligands to said receptor using functional assays. Once a ligand is identified the same assays may be used to identify agonist ' s 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- protein 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 in the activation of the functional responses described below, and any of the assays herein described can be used to screen for agonist, partial agonist, inverse agonist and antagonist ligands of the SNORF138 receptor.
- a wide spectrum of assays can be employed to screen for the presence of receptor SNORF138 ligands. These assays range from traditional measurements of total inositol phosphate accumulation, cAMP levels, intracellular calcium mobilization, and potassium currents, for example; to systems measuring these same second messengers but which have been modified or adapted to be of higher throughput , more generic and more sensitive,- to cell based assays reporting more general cellular events resulting from receptor activation such as metabolic changes, differentiation, cell division/proliferation. Description of several such assays follow.
- human embryonic kidney cells (Peak Rapid cellsTM, Edge BioSystems, Gaithersburg, MD) were trypsinized and transfected with human SNORF138 and G z DNA (16 and 4 g per T150 flask, respectively) using the calcium-phosphate transfection method (Jordan, et al, 1996) . Mock transfection was also performed with vector DNA plus G z DNA for comparison. Five hours after plating the cells, the medium was removed and replaced with fresh medium. The following day, the cells were trypsinized and seeded onto poly D-lysine coated Costar 96-well plates (55,000 cells / well), and incubated overnight.
- HBS HEPES-buffered saline
- HEPES 20 mM
- NaCl 150 mM
- CaCl 2 1 mM
- MgCl 2 1 mM
- KC1 5 mM
- glucose 10 M
- BSA 0.2%)
- theophylline 0.9 gm/liter
- test compounds are added and cells are incubated for 10 min at 37°C.
- cAMP inhibition assays test compounds are added and cells are incubated for 5 min at 37°C, after which forskolin (10 M) is added to each well and cells are incubated for additional 5 min at 37°C. The medium is then decanted and the reaction stopped by the addition of 100 mM HC1.
- COS-7 cells can be transiently transfected with the receptor gene using the DEAE- dextran method 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 mM theophylline and are incubated in the same buffer for 20 min at 37°C, in 5% C0 2 . Test compounds are added and cells are incubated for an additional 10 min at 37°C. The medium is then aspirated and the reaction stopped by the addition of 100 mM HC1. The plates are stored at -20°C for 2-5 days.
- PBS Dulbecco's phosphate buffered saline
- Test compounds are added and cells are incubated for an additional 10 min at 37°C.
- the medium is then aspirated and the reaction stopped by the addition of 100 mM HC1.
- the plates are stored at -20°
- cAMP Scintillation Proximity Assay Amersham Pharmacia Biotech
- Radioactivity is quantified using microbeta Trilux counter (Wallac) .
- Cells expressing the receptor are seeded into 96 well plates or other vessels and grown for 3 days in medium with supplements.
- 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 L. Cells are incubated for 30 min at 37°C, 5% C0 2 .
- Supernatants are transferred to a microtiter plate and evaporated to dryness at 75°C in a vacuum oven.
- Intracellular calcium mobilization assays The intracellular free calcium (Ca 2+ ) 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 coverslip insert and allowed to adhere overnight. Cells are then washed with HBS and loaded with 100 L of Fura-2/AM (10 M) for 20 to 40 min. After washing with HBS to remove the Fura-2/AM solution, cells are equilibrated in HBS for 10 to 20 min.
- Cells are then visualized under the 40X objective of a Leitz Fluovert FS microscope and fluorescence emission is determined at 510 nM with excitation wavelengths alternating between 340 nM and 380 nM.
- Raw fluorescence data are converted to Ca 2+ concentrations using standard Ca 2+ concentration curves and software analysis techniques.
- the measurement of intracellular Ca 2+ can also be performed on a 96-well (or higher) format and with alternative Ca 2+ -sensitive indicators, preferred examples of these are: aequorin, Fluo-3, Fluo-4, Fluo-5, Calcium Green-1, Oregon Green, and 488 BAPTA.
- aequorin Fluo-3, Fluo-4, Fluo-5, Calcium Green-1, Oregon Green, and 488 BAPTA.
- the emission elicited by the change of intracellular Ca 2+ concentration can be measured by a luminometer, or a fluorescence imager; a preferred example of this is the fluorescence imager plate reader (FLIPRTM, Molecular Devices) .
- Cells expressing the receptor of interest are plated into clear, flat-bottom, black-walled 96-well plates (Costar) at a density of 80,000-150,000 cells per well and allowed to incubate for 48 hr at 5% C0 2 , 37°C.
- the growth medium is aspirated and 100 L of loading medium containing Fluo-3 dye is added to each well.
- the loading medium contains: 20 mM HEPES (Sigma), 0.1% BSA (Sigma), dye/pluronic acid mixture (e.g. 1 mM Fluo-3/AM (Molecular Probes) and 10% pluronic acid (Molecular Probes) mixed immediately before use), and 2.5 mM probenecid (Sigma) (prepared fresh) .
- the cells are allowed to incubate for about 1 hour at 5% C0 2 , 37°C.
- the compounds of interest are diluted in wash buffer (Hank's BSS (without phenol red), 20 mM HEPES, 2.5 mM probenecid) to a 4X final concentration and aliquoted into a clear v-bottom plate (Nunc) . Following the dye incubation, the cells are washed 4 times to remove excess dye using a Denley plate washer. 100 L final volume of wash buffer is then added to each cell well. Compounds are added to the cell plates and responses are measured using the FLIPRTM instrument. The data are then collected and analyzed using the FLIPRTM software and Graphpad Prism.
- Antagonist ligands are identified by the inhibition of the signal elicited by agonist ligands.
- Membranes from cells expressing the receptor are suspended in assay buffer (e.g., 50 mM Tris, 100 mM NaCl, 5 mM MgCl 2 , 10 M GDP, pH 7.4) with or without protease inhibitors (e.g., 0.1% bacitracin) .
- Assay buffer e.g., 50 mM Tris, 100 mM NaCl, 5 mM MgCl 2 , 10 M GDP, pH 7.4
- protease inhibitors e.g. 0.1% bacitracin
- GTP S assays are well-known to those skilled in the art, and it is contemplated that variations on the method described above, such as are described by Tian et al . (1994) or Lazareno and Birdsall (1993), may be used.
- microphysiometric measurements of cell metabolism 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) .
- cells expressing receptors are harvested and seeded at 3 x 10 5 cells per microphysiometer 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.
- the cell capsules are transferred to the microphysiometer 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.
- recording media low buffer RPMI 1640, no bicarbonate, no serum (Molecular Devices Corporation, Sunnyvale, CA) containing 0.1% fatty acid free BSA
- a standard recording protocol specifies a 100 1/min 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 min 20 sec sample exposure.
- drugs in a primary screen are presented to the cells at 10 M final concentration.
- MAP kinase mitogen activated kinase
- mitogen activated kinase may be monitored to evaluate receptor activation.
- MAP kinase is activated by multiple pathways in the cell. A primary mode of 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 also known to activate ras and subsequently produce an activation of MAP kinase.
- Receptors that activate phospholipase C produce diacylglycerol (DAG) as a consequence of phosphatidyl inositol hydrolysis.
- DAG activates protein kinase C which in turn phosphorylates 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) .
- the phosphorylated protein has a slower mobility in SDS- PAGE and can therefore be compared with the unstimulated protein using Western blotting.
- antibodies specific for the phosphorylated protein are available (New England Biolabs) which can be used to detect an increase in the phosphorylated kinase.
- cells 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 Immobilon.
- Immunoreactive bands are detected by standard Western blotting technique. Visible or chemiluminescent signals are recorded on film and may be quantified by densitometry .
- Another approach is based on evaluation of the MAP kinase activity via a phosphorylation assay. Cells are stimulated with the test compound and a soluble extract is prepared. The extract is incubated at 30°C for 10 min with gamma- 32 P-ATP, an ATP regenerating system, and 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 H 3 P0 4 and samples are transferred to ice.
- the chromatography paper is washed and counted for 32 P in a liquid scintillation counter.
- the cell extract is incubated with gamma- 32 P-ATP, an ATP regenerating system, and biotinylated myelin basic protein bound by streptavidin to a filter support.
- the myelin basic protein is a substrate for activated MAP kinase.
- the phosphorylation reaction is carried out for 10 min at 30°C.
- the extract can then by aspirated through the filter, which retains the phosphorylated myelin basic protein.
- the filter is washed and counted for 32 P by liquid scintillation counting.
- Receptor activation of the orphan receptor may lead to a mitogenic or proliferative response which can be monitored via 3 H-thymidine uptake.
- 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.
- cells are grown in culture for 1-3 days. Cells 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 3 H-thymidine at specific activities ranging from 1 to 10 Ci/ml for 2-6 hrs.
- Harvesting procedures may involve trypsinization and trapping of cells by filtration over
- GF/C filters with or without a prior incubation in TCA to extract soluble thymidine.
- the filters are processed with scintillant and counted for 3 H by liquid scintillation counting.
- adherent cells are fixed in MeOH or TCA, washed in water, and solubilized in 0.05% deoxycholate/0.1 N NaOH .
- the soluble extract is transferred to scintillation vials and counted for 3 H by liquid scintillation counting.
- 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, immunoreactivity, DNA hybridization, polymerase chain reaction, etc.
- a GPCR which might normally prefer to couple through a specific signaling pathway (e.g., G s , G i r G q , G 0 , etc.), can be made to couple through the pathway defined by the promiscuous G subunit and upon agonist activation produce the second messenger associated with that subunit's pathway.
- G 15 , G 16 and/or G qz this would involve activation of the G g pathway and production of the second messenger IP 3 .
- it is possible to bias receptor signaling through pathways producing other second messengers such as Ca ++ , cAMP, and K + currents, for example (Milligan and Rees, 1999) .
- oocytes are harvested from Xenopus laevis as previously described (Quick and Lester, 1994; Smith et al . , 1997) .
- Oocytes were injected with a cocktail of T3-driven hSN138 cDNA, T3 - RNA polymerase and dNTP with or without T3-driven CFTR cDNA 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 M ⁇ . Unless otherwise specified, oocytes were voltage-clamped at a holding potential of
- oocytes were bathed in continuously flowing (1-3 ml/min) medium containing 96 mM NaCl, 2 M KC1 , 1.8 mM CaCl 2 , and 5 mM HEPES, pH 7.5 (ND96 Buffer) . Drugs were applied by local perfusion from a 10 ml glass capillary tube at a distance of 0.5 mm from the oocyte.
- G s -coupled adenylyl cyclase (AC) Therefore, activation of G s -coupled AC pathway is assayed by applying test compound in ND96 solution to oocytes and observing the changes of inward CFTR currents at a holding potential of -80 mV.
- activation of the phospholipase C (PLC) pathway is assayed by applying a test compound in ND96 solution to oocytes previously injected with mRNA or cDNA for the human SNORF138 and observing inward currents at a holding potential of approximately -80 mV.
- the appearance of currents that reverse at -25 mV and display other properties of the Ca ++ -activated Cl " channel is indicative of receptor-acitivation of PLC and release of IP 3 and intracellular Ca ++ .
- Such activation is exhibited by GPCRs that couple to G q or G 1X .
- Measurement of inwardly rectifying K + (potassium) channel (GIRK) activity may be monitored in oocytes that have been co- injected with mRNA or cDNA encoding the mammalian 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 to G A or G 0 (Kubo et al., 1993; Dascal 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 + .
- IP inositol phosphate
- COS- 7 cells expressing the receptor of interest are plated at a density of 70,000 cells per well and allowed to incubate for 24 hours .
- the cells are then labeled with 0.5 Ci [H] myo- inositol overnight at 37°C, 5% C0 2 .
- the medium is removed and replaced with 180 L of Phosphate-Buffered Saline (PBS) containing 10 mM LiCl .
- PBS Phosphate-Buffered Saline
- the plates are then incubated for 20 min at 37°C, 5% C0 2 .
- the cells are challenged with agonist (20 1/well; lOx concentration) for 30 min at 37 G C.
- the challenge is terminated by the addition of 100 L of 5% v/v trichloroacetic acid, followed by incubation at 4°C for greater than 30 minutes.
- Total IPs are isolated from the lysate by ion 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 L of Dowex AG1-X8 suspension (50% v/v, water: resin) to each well. The filter plates are placed on a vacuum manifold to wash or elute the resin bed.
- the coding region of 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.
- 0.5 g of viral DNA (BaculoGold) and 3 g of DNA construct encoding a polypeptide may be co-transfected into 2 x 10 6 Spodoptera frugiperda insect Sf9 cells 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 recombinant virus plaque purified.
- the procedure to infect cells with virus, to prepare stocks of virus and to titer the virus stocks are as described in Pharmingen ' s manual.
- Quantitative PCR using a, fluorogenic probe with real time detection Quantitative PCR using fluorogenic probes used to characterize the distribution of SNORF138 RNA.
- 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.
- FAM (6- carboxyfluorescein) was used as the reporter
- BH1 Black Hole Quencher-1TM
- the labeled oligonucleotide probe hybridizes to the gene sequence between the two oligonucleotides used for amplification.
- the nuclease activity of Tag 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 of amplicon generated.
- This labeled probe confers a high degree of specificity. Non-specific amplification is not detected as the labeled probe does not hybridize and as a consequence is not cleaved. All experiments were conducted in a PE7700 Sequence Detection System (PE Biosystems, Foster City, CA) .
- Quantitative RT-PCR Quantitative RT-PCR was used for the detection of SNORF138 RNA.
- cDNA was synthesized by reverse transcription from total human RNA. Reverse transcription by SuperScriptll RNAse H " and (GibcoBRL/life Technologies) was primed using random hexamers . Parallel reactions included 32 P labeled dCTP to allow quantification of the cDNA. Following reverse transcription, cDNA was phenol/chloroform extracted and precipitated. Incorporation of 3 P dCTP was assessed after precipitation with trichloroacetic acid and the amount of cDNA synthesized was calculated.
- Primers were designed to amplify a region of human SNORF138. Primers with the following sequences were used:
- Fluorogenic oligonucleotide probe SNORF138-370T 5 ' (6-FAM) -CAGGTCGACACTGCTTTGGCTGCTT - ⁇ BHQ-1 ) 3 ' (SEQ ID NO: 9) Using these primer pairs, amplicon length is 72 bp for human SNORF138.
- Each PCR reaction contained 3.0 ng cDNA.
- Oligonucleotide concentrations were: 500 nM of forward and reverse primers, and 200 nM of fluorogenic probe.
- PCR reactions were carried out in 50 ml volumes 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 (Foster City, CA) . Reactions were carried in a PE7700 sequence detection system (PE Applied Biosystems) using the following thermal cycler profile: 50EC 2 min., 95EC 10 min., followed by 40 cycles of: 95EC, 15 sec, 60EC 1 min.
- ROX Perkin Elmer proprietary passive reference I
- Standard curves for quantification of human SNORF138 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 DNA polymerase. Integrity of RNA was assessed by amplification of RNA coding for cyclophilin or glyceraldehyde 3 -phosphate dehydrogenase (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 fitted a standard. curve to obtain relative quantities of
- RNA samples were obtained from diabetic donors that were insulin dependant for a minimum of five years. Control samples were obtained from normal donors (perirenal adipose) , or purchased (skeletal muscle; Clonetech) . Tissue was homogenized in TRIZOL (Invitrogen) , 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 DNA was removed from the samples using DNAse .
- Reverse transcription was carried out using Superscript II (Invitrogen) .
- PCR reactions were carried out in 50 1 volumes using Taq DNA polymerase (Perkin Elmer) .
- the primer, probe and PCR conditions used were the same as previously described.
- Each human SNORF138 PCR reaction contained cDNA from either a diabetic or normal donor pool. Expression levels assayed in cDNA from individual diseased donors was compared to cDNA from a pool of normal donors. For perirenal adipose cDNA, the normal pool contained cDNA synthesized two donors, and skeletal muscle pool contained cDNA from eight donors. To equalize the amount of diabetic and normal cDNA in each reaction the amounts of cDNA normalized to cyclophilin. Each cDNA was assayed for cyclophilin in increasing quantities ranging from an estimated 0.25ng to 3.0ng. The cycle threshold values of the diabetic samples were compared to those of pooled 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 in each reaction was IX TaqMan Universal PCR Master Mix which contains AmpliTaq Gold DNA Polymerase, AmpErase UNG, dNTPS with dUTP, passive reference, and optimized buffer component s ' . Perkin Elmer proprietary enzymes and buf fers ) .
- Betamethasone Valerate The IUPAC chemical name for Betamethasone Valerate is fluoro-llb, 17 , 21 - trihydroxy-16b -methylpregna -l , 4 -diene - 3 , 20-dione 17 -valerate; registry number: 2152-44-5. Betamethasone Valerate can be purchased for research use as a white, crystallized powder from Pharmacia Fine Chemicals or from a number of other commercial vendors. The structure for Betamethasone Valerate is as follows:
- Betamethasone Valerate is classified under the corticost eroid chemical class and has known therapeutic uses as an anti -inflammatory and/or immunosuppressive agent .
- the full-length coding sequence of human SNORF138 receptor was isolated from a human kidney cDNA library. PCR primers used to screen the cDNA library were designed based on the sequence of an unannotated putative novel GPCR fragment found in the human genome database using datamining techniques.
- the resultant putative full-length hSNORF138 sequence consists of a 993 bp nucleotide coding region which can encode a 330 amino acid protein.
- the predicted hSNORF138 amino acid sequence contains seven putative transmembrane domains and sequence motifs characteristic of the rhodopsin GPCR superfamily. Searches of sequence databases using BLAST analysis (GCG Wisconsin Package Version 10.3, Accelrys Inc., San Diego, CA) show that the most closely related human sequences are GPCRs with the following overall amino acid identities: gpr27 orphan receptor, 36%; edg-8 (sphingosine-1-phosphate receptor), 33%; beta3 adrenergi ⁇ receptor, 33%.
- transcripts for this receptor are expressed in relatively low amounts in all tissues assayed. None of the 31 tissues assayed showed very high expression levels. The highest levels of SNORF138 transcripts were found in placenta and spleen. The presence of relatively high expression in the placenta, combine with the significant levels detected in the uterus suggest that SNORF138 may play a role in gestational disorders. The relatively high levels detected in the spleen suggest a role in modulating the immune system.
- adipose tissue Expressing at a somewhat lower level are adipose tissue, kidney medulla, large intestine and lung. Moderate expression levels are detected in three subtypes of adipose tissue: subcutaneous, peri-renal, and omental . Although absolute expression levels are not high as compared to other GPCRs (in adipose tissue or elsewhere) , the expression levels in adipose tissues are fairly high when compared to the expression of SN0RF138 in other tissues. This is important because of the potential role that adipose tissue, especially omental and peri-renal has in metabolic disorders, as well as obesity and lipid metabolism.
- SNORF138 The moderate levels of SNORF138 detected in the lung imply a role in modulation respiratory function, or perhaps in the regulation of the smooth muscle surrounding the airways. All regions of the gastrointestinal tract (GI) that were assayed express SNORF138 RNA. This suggests a role in modulation gastric motility of other aspects of GI function. Expression in the kidney is notable. Despite the fact that expression levels in the kidney are relatively low, SNORF138 is one of the few genes examined that has a differential expression pattern in the kidney cortex vs. medulla. Most GPCRs assayed by our group have equivalent expression levels in both kidney cortex and medulla. Expression levels of SNORF138 are 4-5 fold higher in the medulla than in the cortex. This suggests that SNORF138 may play a role in regulation of water reabsorbtion or other aspects of renal homeostasis .
- GI gastrointestinal tract
- the localization data for RNA encoding the human SNORF138 receptor indicates that the DNA encoding the human SNORF138 receptor can be used to predict the likelihood that a tissue sample of unknown tissue origin is of spleen origin with respect to a given individual .
- the DNA encoding the human SNORF138 receptor can be used to predict the likelihood that a tissue sample of unknown tissue origin is of spleen origin with respect to a given individual .
- RNA coding for human SNORF138 receptor using qRT- PCR Distribution of RNA coding for human SNORF138 receptor using qRT- PCR
- RNA encoding SNORF138 is expressed as copies/ng cDNA
- SNORF138 The expression of SNORF138 was assayed in perirenal adipose tissue (five donors) and skeletal muscle (7 donors) obtained from diabetic doors. All donors were insulin dependant for a minimum of five years. The levels of SNORF138 in diabetic donors was compared to the levels detected in a pool of normal controls.
- SNORF138 is up-regulated (approximately 2-3 fold) in four of five donors.
- the fifth donor shows a modest decrease which is not distinguishable from the normal controls.
- Skeletal muscle from diabetic donors shows an inconsistent pattern of up-regulation.
- Four of the seven donors are indistinguishable from the normal pool, and the remaining three show a greater than four fold up-regulation over the normal pool .
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PCT/US2003/031659 WO2005059550A1 (fr) | 2003-10-06 | 2003-10-06 | Adn codant snorf138 et ses utilisations |
AU2003279855A AU2003279855A1 (en) | 2003-10-06 | 2003-10-06 | Dna encoding snorf138 and uses thereof |
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WO2002008258A2 (fr) * | 2000-07-21 | 2002-01-31 | Millennium Pharmaceuticals, Inc. | 65494, un nouveau membre de la famille des recepteurs couples a la proteine g chez l'homme et utilisation de celui-ci |
WO2002010387A2 (fr) * | 2000-07-27 | 2002-02-07 | Incyte Genomics, Inc. | Récepteurs couplés à la protéine g |
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WO2002008258A2 (fr) * | 2000-07-21 | 2002-01-31 | Millennium Pharmaceuticals, Inc. | 65494, un nouveau membre de la famille des recepteurs couples a la proteine g chez l'homme et utilisation de celui-ci |
WO2002010387A2 (fr) * | 2000-07-27 | 2002-02-07 | Incyte Genomics, Inc. | Récepteurs couplés à la protéine g |
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