WO2001014888A1 - Procede d'identification de recepteurs urotensine ii antagonistes - Google Patents

Procede d'identification de recepteurs urotensine ii antagonistes Download PDF

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Publication number
WO2001014888A1
WO2001014888A1 PCT/US2000/021171 US0021171W WO0114888A1 WO 2001014888 A1 WO2001014888 A1 WO 2001014888A1 US 0021171 W US0021171 W US 0021171W WO 0114888 A1 WO0114888 A1 WO 0114888A1
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receptor
human
identifying
compound
isolated
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PCT/US2000/021171
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Olivier Civelli
Hans-Peter Nothacker
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The Regents Of The University Of California
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Priority to AU65144/00A priority Critical patent/AU6514400A/en
Publication of WO2001014888A1 publication Critical patent/WO2001014888A1/fr

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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
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/5751—Corticotropin releasing factor [CRF] (Urotensin)

Definitions

  • the present invention relates generally to the field of medicine and, more specifically, to methods and compositions related to the Urotensin II receptor.
  • U II Urotensin II
  • the invention provides a method of identifying a urotensin II (U II) receptor agonist or antagonist.
  • the method consists of contacting an isolated U II receptor with one or more candidate compounds under conditions wherein the U II receptor produces a predetermined signal in response to U II, and identifying a candidate compound that alters production of the signal.
  • a compound is characterized as a U II receptor agonist or antagonist.
  • a method of identifying a U II receptor ligand The method consists of contacting an isolated U II receptor with one or more candidate compounds in the presence of detectably labeled U II, and identifying a compound that decreases binding of the detectably labeled U II to the U II receptor.
  • Such a compound is characterized as a U II receptor ligand.
  • the invention also provides a composition, having an isolated U II receptor and detectably labeled U II. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1A shows the nucleotide sequence (SEQ ID NO:l) and Figure IB shows the deduced amino acid sequence (SEQ ID NO:2) of rat Urotensin II receptor (GPR14 or SENR) (GenBank accession number U23483) .
  • Figure 2A shows the RP-HPLC profile and corresponding activity of fractions of a bovine hypothalamic tissue extract tested for calcium mobilization in GPR14 expressing CHO cells.
  • Figure 2B shows the comparison of calcium kinetics induced by
  • Figure 3A shows the effect of human U II and related peptides on calcium influx in GPR14 expressing CHO cells.
  • Figure 3B shows saturation binding of 125 l- labeled human U II.
  • Figure 4A shows qualitative RT-PCR analysis of GPR14 expression in cardiovascular tissues.
  • M molecular weight marker; 1, negative control; 2, thoracic aorta; 3, abdominal aorta; 4, heart; 5, positive control plasmid.
  • Figure 4B shows the contraction of thoracic aorta strips induced by increasing concentrations of hU II.
  • FIG. 5A shows the tissue distribution of human preprourotensin II (pphU II) on a masterblot. El contains human kidney RNA; B7 contains human spinal cord RNA.
  • Figure 5B shows the RP-HPLC profile of conditioned medium from HEK 293T kidney cells expressing human (pphU II) . DETAILED DESCRIPTION OF THE INVENTION
  • the present invention relates to the identification of the receptor for urotensin II (U II) and its signal transduction pathway.
  • the invention thus provides methods and compositions for identifying compounds that specifically bind to or modulate signaling through the U II receptor. Such compounds can be used therapeutically to prevent or ameliorate U II receptor- associated conditions, including cardiovascular diseases.
  • the invention provides a method of identifying a U II receptor agonist or antagonist. The method consists of contacting an isolated U II receptor with a candidate compound under conditions wherein the U II receptor produces a predetermined signal in response to U II, and identifying a compound that alters production of the predetermined signal. A compound that alters production of the signal is characterized as a U II receptor agonist or antagonist.
  • U II receptor refers to a heptahelical membrane-spanning G protein-coupled polypeptide, previously designated GPR14 (Marchese et al. Genomics 29:335-344 (1995)) or SENR (Tal et al., Bioc. Biophys. Res. Comm. 209:752-759 (1995)), which, as disclosed herein, is the endogenous receptor for urotensin II.
  • GPR14 Septemberese et al. Genomics 29:335-344 (1995)
  • SENR Tal et al., Bioc. Biophys. Res. Comm. 209:752-759 (1995)
  • U II receptor encompasses native U II receptor polypeptides from all vertebrate species including but not limited to human, non-human primate, rat, mouse, rabbit, bovine, porcine, ovine, canine, feline, avian, reptile, amphibian or fish.
  • U II receptor also encompasses polypeptides containing minor modifications with respect to a native U II receptor sequence, and fragments of full-length U II receptor, so long as the modified polypeptide or fragment retains one or more of the biological activities of a native U II receptor.
  • biological activities of a native U II receptor refers to its ability to selectively bind U II, and the ability to couple to and signal through a G protein in response to U II. The ability to selectively bind U II and the ability to couple to signal through a G protein in response to U II can be determined by the binding and signaling assays disclosed herein.
  • a modified U II receptor polypeptide can have one or more additions, deletions, or substitutions of natural or non-natural amino acids relative to the native polypeptide sequence, so long as a biological activity of a native U II receptor is retained.
  • a modification to a polypeptide sequence can be, for example, a conservative change, wherein a substituted amino acid has similar structural or chemical properties, e.g., substitution of an apolar amino acid with another apolar amino acid (such as replacement of leucine with isoleucine) .
  • a modification can also be a nonconservative change, wherein a substituted amino acid has different but sufficiently similar structural or chemical properties so as to not adversely affect the desired biological activity, e.g., replacement of an amino acid with an uncharged polar R group with an amino acid with an apolar R group (such as replacement of glycine with tryptophan) .
  • a minor modification can be the substitution of an L-configuration amino acid with the corresponding D- configuration amino acid with a non-natural amino acid.
  • a minor modification can be a chemical or enzymatic modification to the encoded polypeptide, including but not limited to the following: replacement of hydrogen by an alkyl, acyl, or amino group; esterification of a carboxyl group with a suitable alkyl or aryl moiety; alkylation of a hydroxyl group to form an ether derivative; phosphorylation or dephosphorylation of a serine, threonine or tyrosine residue; or N- or O-linked glycosylation .
  • a modified U II receptor polypeptide can be prepared, for example, by recombinant methods, by synthetic methods, by post- synthesis chemical or enzymatic methods, or by a combination of these methods, and tested for its U II receptor biological activity using the signaling and binding assays disclosed herein.
  • the method of identifying a U II receptor agonist, antagonist or ligand is practiced by contacting an "isolated U II receptor" with a candidate compound.
  • isolated U II receptor refers to a U II receptor that is present in a form or composition different from how it naturally exists in tissue.
  • an isolated U II receptor can be present endogenously in an "isolated cell.”
  • isolated cell refers to a cell that is substantially purified from non-cellular tissue components, such as connective tissue fibers. Such a cell can be, for example, a primary cell, either freshly purified from non-cellular tissue components, or cultured for one or many generations.
  • An "isolated cell” can also be, for example, an established cell line.
  • An example of an isolated cell that endogenously expresses U II receptor is a primary cell or established cell line assayed for its expression of U II receptor mRNA or protein, and determined by such criteria to express U II receptor .
  • tissue preparation with uncharacterized U II binding activity is not considered herein to be an "isolated U II receptor" or to be present in an “isolated cell.”
  • a U II receptor present in crude fractions, such as crude membrane fractions, of such tissue preparations is also not considered herein to be an "isolated U II receptor.”
  • a U II receptor can also be an "isolated U II receptor" in that it is present at higher abundance or higher density than in cells as found in nature, or present in a cell that does not naturally express U II receptor.
  • an "isolated U II receptor” can be a U II receptor that is recombinantly expressed, either transiently or stably, in a cell or cell extract.
  • An example of a cell that recombinantly expresses an isolated U II receptor is a Chinese Hamster Ovary (CHO) cell transfected with an expression vector containing a nucleic acid sequence encoding the U II receptor sequence designated SEQ ID NO: 2.
  • a recombinant nucleic acid expression construct generally will contain a constitutive or inducible promoter of RNA transcription appropriate for the host cell or transcription-translation system, operatively linked to a nucleotide sequence that encodes the polypeptide of interest.
  • the expression construct can be DNA or RNA, and optionally can be contained in a vector, such as a plasmid or viral vector.
  • U II receptor polypeptide Based on knowledge of the nucleic acid sequence encoding U II receptor, one skilled in the art can recombinantly express desired levels of a biologically active U II receptor polypeptide using routine laboratory methods, described, for example, in standard molecular biology technical manuals, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
  • Exemplary host cells that can be used to express recombinant U II receptor include isolated mammalian primary cells; established mammalian cell lines, such as COS, CHO, HeLa, NIH3T3, HEK 293-T and PC12; amphibian cells, such as Xenopus embryos and oocytes; and other vertebrate cells.
  • Exemplary host cells also include insect cells (e.g. Drosophila), yeast cells (e.g. S . cerevisiae, S . pombe, or Pi chia pastoris) and prokaryotic cells (e.g. E. coli ) , engineered to recombinantly express U II receptor.
  • recombinant U II receptor can be expressed in extracts that support transcription and translation, such as reticulocyte lysates and wheat germ extracts.
  • An "isolated U II receptor” can also be a U II receptor present in a cell obtained from a transgenic animal, such as a transgenic mouse, that has been engineered by known methods to express recombinant U II receptor in some or all tissues.
  • an appropriate assay for establishing that an isolated cell expresses U II receptor can be determined by those skilled in the art. Such an assay can involve, for example, analysis of expression of U II receptor nucleic acid or expression of U II receptor polypeptide by methods known in the art. Assays for determining expression of U II receptor mRNA include, for example, Northern blots, RT-PCR or in si tu hybrization analysis. Such methods are described, for example, in standard molecular biology manuals such as Sambrook et al . , supra, (1992) and Ansubel et al., supra, (1998). Assays for determining expression of U II receptor protein include, for example, immunoblot analysis, immunoprecipitation, immunofluorescence or immunohistochemistry, using antibodies specific for U II receptor.
  • Antibodies specific for U II receptor can be produced, for example, using whole U II receptor or its peptide fragments as an immunogen.
  • the receptor or peptide can be formulated in an immunogenic composition, such as conjugated to a carrier protein or formulated with an adjuvant, and administered to a laboratory animal.
  • Methods of producing polyclonal or monoclonal antibodies, and their use in detecting protein expression are well known in the art and are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989) .
  • Methods of preparing fragments of such antibodies with specific binding activity, such as Fab fragments, and methods of preparing recombinant, chimeric or humanized antibodies directed against a desired peptide sequence are also well known in the art.
  • a suitable "isolated U II receptor” can also be a U II receptor present in a crude or partially purified fraction of an "isolated cell” expressing a U II receptor, as described above, such as a membrane fraction.
  • a membrane fraction of an isolated cell expressing recombinant U II receptor can be prepared, for example, by the method described in Example III, below.
  • An "isolated U II receptor" useful in the methods of the invention can also be a substantially purified U II receptor, or active fragment thereof having U II binding activity, or G protein coupled signaling activity, or both, depending on the assay.
  • a substantially purified U II receptor can be prepared, for example, by purification of U II receptor from a tissue, cell or cell extract that endogenously or recombinantly expresses U II receptor. Such purification can be accomplished using fractionation methods known in the art including, for example, column chromatography and immunoaffinity procedures. Additionally, a substantially purified U II receptor or active fragment can be prepared by direct chemical synthesis.
  • a substantially purified U II receptor or fragment can be incorporated into a membrane composition to ensure correct folding of the U II receptor.
  • a composition can include, for example, a lipid bilayer, such as a natural membrane or liposome, for maintaining the native conformation of the U II receptor.
  • the membrane composition can include cellular fractions or isolated components necessary for transducing and detecting the desired G protein-coupled signal.
  • additional components can include, for example, G proteins, GTP, effector molecules and signal indicator molecules.
  • the method of identifying a U II receptor agonist, antagonist or ligand involves contacting the isolated U II receptor with one or more "candidate compounds.”
  • the term “candidate compound” refers to any molecule that potentially acts as a U II receptor agonist, antagonist or ligand in the screening methods disclosed herein.
  • a candidate compound can be a naturally occurring macromolecule, such as a peptide, nucleic acid, carbohydrate, lipid, or any combination thereof.
  • a candidate compound also can be a partially or completely synthetic derivative, analog or mimetic of such a macromolecule, or a small organic or inorganic molecule prepared partly or completely by combinatorial chemistry methods. If desired a particular assay format, a candidate compound can be detectably labeled or attached to a solid support.
  • candidate compounds to test in the methods of the invention will depend on the application of the method. For example, one or a small number of candidate compounds can be advantageous in manual screening procedures, or when it is desired to compare efficacy among several identified ligands, agonists or antagonists. However, it is generally understood that the larger the number of candidate compounds, the greater the likelihood of identifying a compound having the desired activity in a screening assay. Additionally, large numbers of compounds can be processed in high-throughput automated screening assays. Therefore, "one or more candidate compounds" can contain, for example, greater than about 10 3 different compounds, preferably greater than about 10 5 different compounds, more preferably, greater than about 10 7 different compounds .
  • U II refers to a natural or synthetic cyclic peptide containing the core structure CFWDYC (SEQ ID N0:3), with a disulfide bridge between the Cys residues at positions 1 and 6 of SEQ ID NO: 3, and which activates signaling through the U II receptor at subnanomolar concentrations.
  • U II includes peptides having the core structure CFWDYC, and the flanking amino acid residues of a U II from any species, including, for example, teleost fishes and frog (see Conlon et al . , J. Reg. Peptides 69:95-103 (1997)), as well as human (see Coulauarn et al., Proc . Natl. Acad. Sci. USA 95:15803-15808 (1998)).
  • U II includes cyclic peptides having the goby U II sequence AGTADCFWDYCV (SEQ ID NO: 4), and the human U II sequence ETPDCFWKTCV (SEQ ID NO: 5), which activate signaling in an isolated cell expressing a U II receptor with half-maximal effective concentrations of 0.14 nM and 0.10 nM, respectively.
  • U II also includes peptides with U II activity having the U II core structure and non-naturally occurring N-terminal or C-terminal sequences .
  • Biologically active U II can be prepared by purification from cells or tissues that express U II, such as kidney or spinal cord, or from cells that recombinantly express preprourotensin II and correctly process it into active U II (see Example V, below) . More conveniently, U II can be synthesized by conventional methods of peptide synthesis.
  • U II receptor agonist refers to any compound other than U II that promotes or enhances normal signal transduction through U II receptor with high selectivity and high potency.
  • a U II receptor agonist can promote signaling through U II receptor at high potency, such as half-maximal signaling at concentrations of less than 100 nM, preferably less than 10 nM, most preferably less than 1 nM.
  • U II receptor agonist can promote signaling through U II receptor with high selectivity, such as at concentrations substantially lower, such as at least 100-fold lower or at least 1000- fold lower, than required to promote signaling through other G protein coupled receptors, such as, for example, somatostatin 2A receptor.
  • U II agonists disclosed herein are [I-Tyr q ] human U II and [ 125 I-Tyr 9 ] human U II. As indicated in Example III and Table 1, monoiodination of the core Tyr residue of human U II does not preclude its ability to activate signaling through the U II receptor at subnanomolar concentrations, whereas di-iodinated human U II is a less potent agonist.
  • a further example of a U II agonist, of lesser potency, is [D-Trp 7 ] Urotensin II- (5-10) (SEQ ID NO: 6) .
  • a U II receptor agonist can act by any agonistic mechanism, such as by binding a U II receptor at the normal U II binding site, thereby promoting U II receptor signaling.
  • a U II receptor agonist can also act, for example, by potentiating the binding activity of U II or signaling activity of U II receptor.
  • the methods of the invention can advantageously be used to identify a U II receptor agonist that acts through any agonistic mechanism.
  • the invention also provides a method of identifying U II receptor antagonists.
  • U II receptor antagonist refers to a compound that selectively inhibits or decreases normal signal transduction through the U II receptor, with high potency and high selectivity.
  • a U II receptor antagonist can act by any antagonistic mechanism, such as by binding U II or U II receptor, thereby inhibiting binding between U II and U II receptor.
  • a U II receptor antagonist can also act indirectly, for example, by modifying U II or U II receptor.
  • the methods of the invention can advantageously be used to identify a U II receptor antagonist that acts through any antagonistic mechanism.
  • the method to identify a U II receptor agonist or antagonist is performed under conditions where U II produces a "predetermined signal" in response to U II.
  • predetermined signal refers to a readout, detectable by any analytical means, that is a qualitative or quantitative indication of activation of G protein-dependent signal transduction through U II receptor.
  • G protein refers to a class of heterotrimeric GTP binding proteins, with subunits designated G ⁇ , G ⁇ and Gy, that couple to seven- transmembrane cell surface receptors to transduce a variety of extracellular stimuli, including light, neurotransmitters, hormones and odorants to various intracellular effector proteins. G proteins are present in both eukaryotic and prokaryotic organisms, including mammals, other vertebrates, Drosophila and yeast.
  • an exemplary predetermined signal that is a qualitative or quantitative indication of activation of G protein- dependent signal transduction through U II receptor is
  • Ca 2+ influx which can be measured, for example, using the calcium indicator fluo-3 and fluorescence monitoring system described in Example I, below.
  • a predetermined signal other than Ca 2+ influx can be used as the readout in the methods of the invention.
  • the specificity of a G protein for cell- surface receptors is determined by the C-terminal five ammo acids of the G subunit.
  • the nucleotide sequences and signal transduction pathways of different classes and subclasses of G ⁇ subunits m a variety of eukaryotic and prokaryotic organisms are well known in the art.
  • any convenient G-protem mediated signal transduction pathway can be assayed by preparing a chimeric G ⁇ containing the C-termmal residues of a G ⁇ that couples to U II receptor, such as G ⁇ q, with the remainder of the protein corresponding to a G ⁇ that couples to the signal transduction pathway it is desired to assay.
  • chimeric G ⁇ proteins can be prepared by synthetic methods .
  • G proteins can lead to increased or decreased production or liberation of second messengers, including, for example, arachidonic acid, acetylcholme, diacylglycerol, cGMP, cAMP, mositol phosphate and ions; altered cell membrane potential; GTP hydrolysis; influx or efflux of ammo acids; increased or decreased phosphorylation of mtracellular proteins; or activation of transcription.
  • second messengers including, for example, arachidonic acid, acetylcholme, diacylglycerol, cGMP, cAMP, mositol phosphate and ions
  • altered cell membrane potential including, for example, arachidonic acid, acetylcholme, diacylglycerol, cGMP, cAMP, mositol phosphate and ions
  • altered cell membrane potential including, for example, arachidonic acid, acetylcholme, diacylglycerol, cGMP, cAMP,
  • a variety of cell-based expression systems including bacterial, yeast, baculovirus/insect systems and mammalian cells, useful for detecting G protein coupled receptor agonists and antagonists are described, for example, in Tate et al . , Trends in Biotech. 14:426-430 (1996) .
  • Assays to detect and measure G protein-coupled signal transduction can involve first contacting the isolated cell or membrane with a detectable indicator.
  • a detectable indicator can be any molecule that exhibits a detectable difference in a physical or chemical property in the presence of the substance being measured, such as a color change.
  • Calcium indicators, pH indicators, and metal ion indicators, and assays for using these indicators to detect and measure selected signal transduction pathways are described, for example, in Haugland, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Sets 20-23 and 25 (1992-94).
  • calcium indicators and their use are well known in the art, and include compounds like Fluo-3 AM, Fura-2, Indo-1, FURA RED, CALCIUM GREEN, CALCIUM ORANGE, CALCIUM CRIMSON, BTC, OREGON GREEN BAPTA, which are available from Molecular Probes, Inc., Eugene Oreg., and described, for example, in U.S. Patent Nos. 5,453,517, 5,501,980 and 4, 849, 362.
  • Assays to determine changes in gene expression in response to a U II receptor agonist or antagonist can involve first transducing cells with a promoter-reporter nucleic acid construct such that a protein such as ⁇ - lactamase, luciferase, green fluorescent protein or ⁇ - galactosidase will be expressed in response to contacting U II receptor with U II.
  • a promoter-reporter nucleic acid construct such that a protein such as ⁇ - lactamase, luciferase, green fluorescent protein or ⁇ - galactosidase will be expressed in response to contacting U II receptor with U II.
  • Such assays and reporter systems are well known in the art and are described, for example, at http://www.aurorabio.com/tech_platform- assay_technologies.html (visited August 5, 1999).
  • an assay to determine whether a candidate compound is a U II receptor agonist or antagonist is performed under conditions in which contacting the receptor with U II produces a predetermined signal.
  • the assay can be performed in the presence of a given concentration of U II.
  • the U II concentration will be within 10-fold of the EC 50 .
  • an agonist that competes with U II for signaling through the U II receptor, or indirectly potentiates the signaling activity of U II can be readily identified.
  • an antagonist that prevents U II from binding the U II receptor, or indirectly decreases the signaling activity of U II can also be identified.
  • the invention also provides a method of identifying a U II receptor ligand.
  • the method consists of contacting an isolated U II receptor with one or more candidate compounds in the presence of detectably labeled U II.
  • a compound that reduces binding of the detectably labeled U II to the U II receptor is characterized as a U II receptor ligand.
  • the invention also provides a composition that contains detectably labeled U II and an isolated U II receptor, which is useful, for example, in the method of identifying a U II receptor ligand.
  • detectably labeled U II refers to U II derivatized with, or conjugated to, a moiety that is detectable by any analytical means.
  • Detectably labeled U II useful in the methods disclosed herein generally retains its ability to bind U II receptor at subnanomolar concentrations.
  • a detectable moiety can be a radioisotope, fluorochrome, ferromagnetic substance, or luminescent substance.
  • the detectably labeled U II is radiolabeled.
  • Exemplary radiolabels useful for labeling peptides include 125 I, 1 C and 3 H .
  • an exemplary detectably labeled U II is human U II, radioiodinated at the core Tyr with 125 I, which binds membranes of cells transfected with U II receptor with an apparent K ⁇ of 70 pM.
  • the amount of binding of a given amount of the detectably labeled U II is determined in the absence of the candidate compound.
  • the amount of detectably labeled U II will be less than its K d , for example, 1/10 of its K ⁇ .
  • the amount of binding of the detectably labeled U II in the presence of the candidate compound is determined.
  • a decrease m binding due to a candidate compound characterized as a U II receptor ligand is evidenced by at least 2-fold less, preferably at least 10-fold to at least 100-fold less, such as at least 1000-fold less, binding of detectably labeled U II to U II receptor in the presence of the candidate compound than in the absence of the candidate compound.
  • binding assays can be performed in any suitable assay format including, for example, whole cells or membranes that contain U II receptor, or substantially purified U II receptor polypeptide, either m solution or bound to a solid support.
  • a compound that is determined to be a U II receptor ligand in the methods of the invention can be assayed for its effect on U II receptor signaling, using the biological assays described herein.
  • a "U II receptor ligand" can be determined to be a direct agonist or antagonist of U II receptor, as described above, or can have little or no effect on U II receptor signaling in the absence of U II.
  • a U II receptor ligand that has little or no effect on U II receptor signaling is useful, for example, in many of the same applications as a U II receptor antibody.
  • a U II receptor ligand can be used to specifically target a diagnostic or therapeutic moiety to a tissue that expresses U II receptor.
  • a U II receptor ligand can be labeled with a detectable moiety, such as a radiolabel, fluorochrome, ferromagnetic substance, or luminescent substance, and used to detect expression of U II receptor polypeptide in an isolated sample or in in vivo diagnostic imaging procedures.
  • a U II receptor ligand can be labeled with a therapeutic moiety, such as a cytotoxic or cytostatic agent or radioisotope, and administered in an effective amount to arrest proliferation or kill a cell or tissue that expresses U II receptor.
  • a therapeutic moiety such as a cytotoxic or cytostatic agent or radioisotope
  • U II receptor ligands, agonists and antagonists identified using the methods and compositions of the invention can be isolated and administered to an individual, such as a human or other mammal, in an effective amount to prevent or ameliorate a U II receptor-associated condition.
  • U II receptor-associated condition refers to any abnormal physiological or psychological condition in which a quantitative or qualitative alteration in signaling through the U II receptor contributes to the symptoms of the condition.
  • a "U II receptor-associated condition” can also be any physiological or psychological condition in which altering signaling through the U II receptor has a beneficial effect in the individual.
  • Exemplary conditions for which administration of a U II receptor ligand, agonist or antagonist are contemplated include diseases of the circulatory system.
  • the U II receptor is expressed m cardiovascular tissues, and human U II acts as a vasoconstrictor in aorta.
  • administration of a compound that acts as a U II receptor agonist is beneficial.
  • administration of a compound that acts as a U II receptor antagonist is beneficial.
  • the U II receptor agonists, antagonists and ligands identified by the methods of the invention can be used to prevent or treat circulatory disorders including, but not limited to, ischemia, hypertension, hypotension, angina pectoris, myocardial infarction, stroke, congestive heart failure, shock, stroke and impotence.
  • circulatory disorders including, but not limited to, ischemia, hypertension, hypotension, angina pectoris, myocardial infarction, stroke, congestive heart failure, shock, stroke and impotence.
  • mRNA encoding the U II receptor has been observed in skeletal muscle, as well as in neural and sensory tissue, including retma, circumvallate papillae (taste buds), olfactory epithelium, cerebellum, and choroid plexus (Tal et al . , supra (1995)).
  • uses of the agonists, antagonists and ligands identified by the methods of the invention in treating disorders of the nervous system, muscle and eye are also provided.
  • Disorders of the nervous system and muscle amenable to treatment by the compounds of the invention include disorders that result from trauma (e.g. spinal cord injury, hemorrhage), infectious agents (e.g.
  • disorders of the eye amenable to treatment by the methods of the invention particularly include diseases of the retma, such as diabetic retinal disease and retmoblastoma .
  • the agonists, antagonists and ligands identified by the methods of the invention are useful m treating psychiatric disorders, such as depression; anxiety disorders, such as generalized anxiety disorder, panic attacks, obsessive-compulsive disorder, phobias, acute stress disorder, post-traumatic stress disorder; and psychotic disorders, such as unipolar mania or depression, bipolar disorder and schizophrenia.
  • the agonists, antagonists and ligands identified by the methods of the invention are useful in treating disorders of behavior, memory and learning, and sleep. Disorders of behavior include, but are not limited to, autistic disorder, Asperger's disorder, aggression, pervasive developmental disorders, Tourette's syndrome, attention-deficit hyperactivity disorder and addiction.
  • Disorders of memory and learning include, but are not limited to, dementia, including dementia due to neurodegenerative diseases, infectious disease, proliferative diseases, endocrine disease, tumors, metabolic disorders, and toxins; and developmental learning disabilities.
  • Disorders of sleep and of the sleep-wake cycle include, but are not limited to, insomnia, bedwetting, sleepwalking, sleep apnea and narcolepsy.
  • U II receptor agonists, antagonists and ligands of the present invention can be conveniently formulated for therapeutic administration together with a pharmaceutically acceptable carrier.
  • suitable pharmaceutical carriers for the methods of the invention are well known and include, for example, aqueous solutions such as physiologically buffered saline, and other solvents or vehicles such as glycols, glycerol, oils or injectable organic esters.
  • a pharmaceutical carrier can contain a physiologically acceptable compound that acts, for example, to stabilize or increase the solubility of a pharmaceutical composition.
  • Such a physiologically acceptable compound can be, for example, a carbohydrate, such as glucose, sucrose or dextrans; an antioxidant, such as ascorbic acid or glutathione; a chelating agent; a low molecular weight protein; or another stabilizer or excipient.
  • a carbohydrate such as glucose, sucrose or dextrans
  • an antioxidant such as ascorbic acid or glutathione
  • a chelating agent such ascorbic acid or glutathione
  • a chelating agent such ascorbic acid or glutathione
  • a chelating agent such ascorbic acid or glutathione
  • the blood-brain barrier excludes many highly hydrophilic compounds.
  • the therapeutic compounds of the invention can be formulated, for example, in liposomes, or chemically derivatized.
  • they can be formulated, for example, in liposomes, or chemically derivatized.
  • the choice of the pharmaceutical formulation and the appropriate preparation of the composition will depend on the intended use and mode of administration .
  • Methods of introduction of a therapeutic compound of the invention include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, intranasal, intraspinal and intracerebral routes. Methods of introduction can also be provided by rechargable or biodegradable devices, particularly where gradients of concentrations of drug in a tissue is desired.
  • Various slow release polymeric devices are known in the art for the controlled delivery of drugs, and include both biodegradable and non- degradable polymers and hydrogels.
  • an effective dose of a therapeutic compound of the invention can be determined, for example, by extrapolation from the concentration required for modulating U II receptor signaling or binding in in vi tro assays described herein, or from the dose required for efficacy in the aorta vasoconstriction assay described herein.
  • an appropriate dose of a therapeutic compound of the invention can be in the range of 0.001- 100 mg/kg of body weight, such as in the range of 0.01- 10 mg/kg of body weight.
  • An appropriate dose can be determined by those skilled in the art based on the bioactivity of the particular compound, the desired route of administration, the gender and health of the individual, the number of doses and duration of treatment, and the particular condition being treated.
  • This example shows the identification of Urotensin II (UII) as the endogenous ligand for the orphan G-protein coupled receptor GPR14.
  • UI Urotensin II
  • This example also shows a signaling assay that can be used in a method of identifying a U II receptor agonist or antagonist.
  • peptide extracts from a variety of different mammalian tissues were prepared.
  • the peptide extracts were prepared by mincing, under liquid nitrogen, tissue obtained from a slaughterhouse and immediately boiling the tissue for 10 min. in deionized water at a 1:2 tissue/water (g/ml) ratio. After cooling to 10°C, acetic acid was added to 1M final concentration. The homogenate was further treated with a Polytron for 2 min. After centrifugation for 30 min at 12,000 x g, the supernatant was removed, and the pellet re-extracted with one volume of 1M acetic acid. The supernatants were combined, and three volumes of acetone were added.
  • the precipitate was removed at 15,000 x g for 30 min. After concentration of the supernatant by rotor evaporation, the remainder was extracted twice with two volumes of ethyl ether, then frozen and lyophilized. The lyophilized material was resuspended in 5% CH 3 CN, 0.1% trifluoroacetic acid (TFA) and applied on a PrepPak-Delta-Pak C18, 15 ⁇ m, 300 A, 25 x 100 mm (Waters) preequilibrated with 5% CH ? CN/0.1% TFA.
  • TFA trifluoroacetic acid
  • the material was eluted with a linear gradient from 5% to 39% CH 3 CN/0.1% TFA, with the active fractions eluting at 34%-38% CH 3 CN .
  • the tissue extracts were fractionated by preparative reverse-phase high performance liquid chromatography (rpHPLC) into 72 individual one-minute fractions. Aliquots of each fraction were tested for induction of mtracellular calcium changes using Chinese hamster ovary cells transiently transfected with GPR14 cDNA and loaded with the calcium-sensitive dye fluo-3. Changes m fluorescence were monitored over 240 seconds using a fluorescence imaging plate reader (FLIPR) system, following procedures described by Coward et al., Proc . Natl. Acad. Sci. USA 95:352-357 (1998). The maximal fluoresence increment generated by each fraction on GPR14 transfected cells was normalized to the maximal control value seen m mock-transfected cells.
  • FLIPR fluorescence imaging plate reader
  • the active component was purified over a seven-step purification strategy combining reverse-phase and cationic-exchange HPLC columns. One single activity peak was detected, indicating that the activity can be attributed to a unique molecular entity.
  • the bioactive compound was found to be extremely scarce, precluding a total structural analysis.
  • the calcium response to the active material depicted a distinctive time course, as shown by the open circles Figure 2B.
  • the active material was determined to be sensitive to trypsin and reducing agents, indicating that the biological activity could be attributed to a peptide containing basic amino acid(s) and cysteine residues.
  • GPR14 is distantly related to the somatostatin receptors (SSTRs) , sharing the highest similarity to SST4R (41% in the transmembrane regions), somatostatin- like, cysteine bridge-containing peptides were screened in the above system for their ability to activate calcium signaling through GPR14.
  • the peptides tested were human melanin-concentrating hormone (MCH) (SEQ ID NO:7), somatostatin-14 (SST-14) (SEQ ID NO:8), cortistatin-14 (SEQ ID NO: 9) and goby urotensin II (U II) (SEQ ID NO : 4 ) (see Table 1) .
  • This example shows the specificity and potency of U II for the Urotensin II receptor, GPR14.
  • This example also shows a binding assay that can be used in a method of identifying a U II receptor ligand.
  • [D-Trp]U II-(5-10) behaved as an agonist with a 190-fold lower potency compared to human U II, but with no antagonistic activity at concentrations up to 1 ⁇ M .
  • Somatostatm-14 and the somatostatin analogs RC-160 (SEQ ID NO: 10) and octreotide (SEQ ID NO: 11) were also tested for their ability to activate GPR14 (see
  • RC-160 activated the receptor with an EC 50 of 338 +/- 53.6 nM.
  • Octreotide structurally similar to RC-160, showed even lower potency.
  • Somatostatm-14 was active on GPR14 expressing cells but at physiologically non-relevant concentrations (see Figure 3A and Table 1) . This activity was not observed in untransfected cells.
  • Cort ⁇ statm-14 which differs from somatostatm-14 in a C-termmal lysine residue, exhibited even lower affinity, pointing out the importance of having a non-charged carboxy terminus for GPR14 reactivity.
  • GPR14 with high affinity and in a saturable manner.
  • human U II was initially labeled with cold iodine. Human U II contains one tyrosine residue at position 9 that can be used for iodination. Tyr 9 was iodinated with Nal according to the method of Markwell et al., Anal. Biochem. 125:427-432 (1982), using IODO-BEADS ⁇ (Pierce, Rockford, IL) .
  • Monoiodo-hU II was shown to induce a concentration-dependent transient increase in [Ca 2+ ] in CHO cells stably transfected with GPR14 with an EC 5C similar to the non-modified peptide ( Figure 3A, Table 1) .
  • the diiodo-form of hU II showed a 25-fold decrease in potency compared to hU II. Consequently, the monoiodinated form of human U II was used as a radioligand for the determination of physical constants.
  • [ 125 I-Tyr 9 ] hU II was prepared as described above, using 0.5 nmol hU II and 500 ⁇ Ci Na 125 I (Amersham) .
  • Ligand binding assays were performed in 96-well glass fiber type B filtration plates using a Multiscreen filtration system (Millipore) pre-coated with 0.5% BSA at room temperature for one hour.
  • CHO cells stably expressing GPR14 were harvested with a cell scraper and resuspended in binding buffer containing 20 M Tris-HCl, pH 7.4, 2 mM MgCl 2 , 0.25% BSA and 0.25 mg/ml bacitricin.
  • Membranes were prepared using a Polytron tissue homogenizer. Total membrane particulate was obtained after centrifugation at 20,000 x g for 60 min at 4°C.
  • Figure 3B shows saturation binding of 125 I-labeled hU II.
  • Membranes of CHO cells stably expressing GPR14 were incubated with various concentrations of ⁇ " I-labeled hU II and the bound ligand separated by filtration, as described above. Concentrations of free ligand were calculated by subtracting the amount of specifically bound ligand from the total amount of radioligand added. The data represent the average of two independent experiments done in triplicate. The insert shows a Scatchard transformation of the specific binding data.
  • [ :25 I-Tyr 9 ] hU II displayed saturable and displaceable binding to membranes of cells transfected with GPR14 with an apparent dissociation constant (K d ) of 70 pM and a maximal binding capacity (B ma ) of 350 fmol per milligram of membrane protein.
  • This example shows the distribution of GPR14 mRNA in cardiovascular tissue and the physiological activation of GPR14 by human U II.
  • GPR14 As demonstrated above, activation of GPR14 by U II stimulates Ca 2+ influx, suggesting a possible physiological role for GPR14 as an excitatory receptor, in contrast to somatostatin receptors, which are coupled to inhibition of adenylate cyclase.
  • Physiological actions of fish U II have been described in the mammalian cardiovascular system (Gibson et al . , Gen. Comp. Endocrinol. 64:435-439 (1986)), and specific fish U II binding sites on rat thoracic aortic membranes have been reported (Itoh et al., Eur. J. Pharm. 149:61-66 (1988)).
  • RT-PCR reverse transcription-polymerase chain reaction
  • Reverse transcription reactions were performed with Superscript II reverse transcriptase (Life Technologies) using 5 ⁇ g of total tissue RNA. 10% of the final reaction products were used in PCR reactions containing 0.2 ⁇ M each of primers having the sequences 5'-CTGAGCCTGGAGTCTACAACAAGC-3' (SEQ ID NO: 12) and 5'- TAGGTGGCTATGATGAAGGGAATG-3' (SEQ ID NO: 13).
  • Reactions were carried out using Goldtaq polymerase (Perk Elmer) and buffer conditions recommended by the manufacturer. The PCR conditions were 94°C for 10 mm, followed by 34 cycles of 94°C for 30 sec, 58°C for 30 sec and 72°C for 60 sec. Reaction products were separated on a 1% agarose gel.
  • GPR14 is expressed rat thoracic aorta (lane 2), abdominal aorta (lane 3), and heart (lane 4) . Because GPR14 was shown to be expressed m thoracic aorta, hU II was tested for its ability to cause vascular smooth muscle contractions in thoracic aorta.
  • thoracic aorta tissue segments To prepare thoracic aorta tissue segments, four month old male Fischer 344 rats were sacrificed by decapitation and their thoracic aorta removed. Arteries were cut into 3 mm segments and mounted on platinum wires m an oxygenated tissue bath containing 40 ml of Krebs ' solution (118 mM NaCl, 5.2 mM KC1, 1.6 mM CaCl , 1.2 mM KHP0 4 , 25.5 mM NaHC0 3 , 1.2 mM MgS0 4 , 0.027 mM disodium EDTA, and 115 mM glucose) , and kept at 37°C and oxygenated with 95% 0,/5% CO,.
  • Krebs ' solution 118 mM NaCl, 5.2 mM KC1, 1.6 mM CaCl , 1.2 mM KHP0 4 , 25.5 mM NaHC0 3 , 1.2 mM MgS0
  • Tissue segments were slowly stretched to 1 gram resting tension, then allowed to equilibrate for 1 hour, with fresh Krebs' solution added every 20 mm. Contractile responses to cumulative addition of urotensin (10 "1C1 to 10 7 M) were recorded using Fort 10 force transducers and a MacLab analog-digital converter. Maximal contraction of each arterial segment was then determined by addition of 10 4 M norepmephrme, and endothelial integrity confirmed by measuring relaxation to acetylcholine (10 ⁇ 6 M) . The contraction induced by human U II is expressed in Figure 4B as the percentage of the maximum change in tension induced by norepinephrine (NE) , with the bars indicating the S.E.M of four independent experiments.
  • NE norepinephrine
  • GPR14 is the receptor responsible for hU II-directed contraction of rat thoracic aorta.
  • This example shows the expression of human preprourotensin II in human kidney, and the ability of kidney cells to process preprourotensin II into biologically active urotensin II.
  • the tissues shown on the human masterblot (Clontech) in Figure 5A are: Al, whole brain; A2, amygdala; A3, caudate nucleus; A4 , cerebellum; A5, cerebral cortex; A6, frontal lobe; A7 , hippocampus; A8 , medulla oblongata; Bl, occipital lobe; B2, putamen; B3, substantia nigra B4, temporal lobe; B5, temporal lobe; B6, subthalamic nucleus; B7, spinal cord; Cl, heart; C2, aorta; C3, skeletal muscle; C4, colon; C5, bladder; C6, uterus; C7, prostate; C8, stomach; Dl, testis; D2 , ovary; D3, pancreas, D4 , pituitary; D5, adrenal; D6, thyroid; D7 , salivary; D8, mammary gland; El kidney; E2, liver; E3, small intestin
  • Hybridization conditions were as recommended by the manufacturer.
  • pphU II mRNA was found to be expressed at high levels only in human kidney (see Figure 5A) . Moderate to low expression was found in spinal cord and medulla oblongata respectively, but these are not expected to be a source of circulating U II. All other tissues examined either expressed none or very low amounts of the UII precursor. The expression and processing of the UII precursor potentially varies from individual to individual, which may be a reflection of the overall health and condition of the individual. Based on the high expression of pphU II mRNA in kidney, an investigation was made of whether biologically active hU II could be secreted by cells derived from the kidney.
  • the human U II precursor cDNA was obtained from Research Genetics (IMAGE clone # 926809) and sequenced from both ends. The insert which contained the full open reading frame was subcloned into pcDNA3.1(+) (Invitrogen, San Diego) . The expression construct was transfected into human embryonic kidney cells (HEK 293T cells) grown in DMEM containing 10% fetal bovine serum. For peptide expression studies the medium was replaced by Opti-MEM (Life Technologies) with or without added serum, and the cells grown for 24 hours.
  • the conditioned medium from HEK 293T cells transfected with pphU II cDNA was removed from the cell layer and applied to a SePak C18 cartridge (Waters). Absorbed material was eluted with 60% CH 3 CN/0.1% TFA and fractionated using a Nova-Pak C18 3.9x150 mm analytical column (Waters) equilibrated with 5% CH 3 CN/0.1% TFA. The material was eluted with a linear gradient to 60%
  • kidney may be the peripheral source of human U II which modulates vascular function.
  • human U II can be processed from its precursor in the same molecular form as that the biologically active peptide shown as SEQ ID NO: 5 in Table 1.

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Abstract

L'invention concerne un procédé d'identification de récepteur urotensine II (U II) agoniste ou antagoniste. Ce procédé consiste à mettre en contact un récepteur U II isolé (avec un ou plusieurs composés candidats, dans des conditions où le récepteur U II produit un signal prédéterminé en réponse à U II, et à identifier un composé candidat modifiant la production de ce signal. Ce composé est caractérisé en tant que récepteur U II agoniste ou antagoniste. L'invention concerne également un procédé d'identification d'un ligand de récepteur U II. Ce procédé consiste à mettre en contact un récepteur U II isolé avec un ou plusieurs composés candidats, en présence de U II bien étiqueté, et à identifier un composé qui réduit la liaison du U II bien étiqueté avec le récepteur U II. Ce composé est caractérisé en tant que ligand de récepteur U II. L'invention concerne encore une composition qui possède un récepteur U II isolé et un U II bien étiqueté.
PCT/US2000/021171 1999-08-24 2000-08-03 Procede d'identification de recepteurs urotensine ii antagonistes WO2001014888A1 (fr)

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Cited By (1)

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WO2003003018A2 (fr) * 2001-06-29 2003-01-09 Aventis Pharma Deutschland Gmbh Procede d'identification de composes permettant de traiter le processus de vieillissement du systeme cardiovasculaire

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EP0859052A1 (fr) * 1997-01-27 1998-08-19 Smithkline Beecham Corporation Clonage du récepteur humain GPR14
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WO1999040192A1 (fr) * 1998-02-09 1999-08-12 Smithkline Beecham Corporation Recepteur gpr14 de l'homme et un procede de detection d'agonistes et antagonistes des recepteurs gpr14 de l'homme et du rat

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WO1995034651A2 (fr) * 1994-06-14 1995-12-21 Neurocrine Biosciences, Inc. Recepteurs du facteur2 liberant la corticotropine
EP0859052A1 (fr) * 1997-01-27 1998-08-19 Smithkline Beecham Corporation Clonage du récepteur humain GPR14
WO1999035266A2 (fr) * 1998-01-09 1999-07-15 Smithkline Beecham Corporation Urotensine humaine ii
WO1999040192A1 (fr) * 1998-02-09 1999-08-12 Smithkline Beecham Corporation Recepteur gpr14 de l'homme et un procede de detection d'agonistes et antagonistes des recepteurs gpr14 de l'homme et du rat

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GIBSON A: "COMPLEX EFFECTS OF GILLICHTHYS UROTENSIN II ON RAT AORTIC STRIPS", BRITISH JOURNAL OF PHARMACOLOGY, vol. 91, no. 1, 1987, pages 205 - 212, XP000978590, ISSN: 0007-1188 *
GRIGORIADIS DIMITRI E ET AL: "125I-Tyr-0-sauvagine: A novel high affinity radioligand for the pharmacological and biochemical study of human corticotropin-releasing factor-2alpha receptors.", MOLECULAR PHARMACOLOGY, vol. 50, no. 3, September 1996 (1996-09-01), pages 679 - 686, XP000978435, ISSN: 0026-895X *
LUTHIN DAVID R ET AL: "Synthesis and biological activity of oxo-7H-benzo(e)perimidine-4-carb oxylic acid derivatives as potent, nonpeptide Corticotropin Releasing Factor (CRF) receptor antagonists.", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 9, no. 5, 8 March 1999 (1999-03-08), pages 765 - 770, XP000978428, ISSN: 0960-894X *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003003018A2 (fr) * 2001-06-29 2003-01-09 Aventis Pharma Deutschland Gmbh Procede d'identification de composes permettant de traiter le processus de vieillissement du systeme cardiovasculaire
WO2003003018A3 (fr) * 2001-06-29 2004-01-08 Aventis Pharma Gmbh Procede d'identification de composes permettant de traiter le processus de vieillissement du systeme cardiovasculaire
US7348155B2 (en) 2001-06-29 2008-03-25 Sanofi-Aventis Deutschland Gmbh Method for identifying compounds for treating aging processes in the coronary circulatory system

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