WO2004009631A2 - Human gnrh (type 2) receptor - Google Patents

Human gnrh (type 2) receptor Download PDF

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Publication number
WO2004009631A2
WO2004009631A2 PCT/EP2003/007869 EP0307869W WO2004009631A2 WO 2004009631 A2 WO2004009631 A2 WO 2004009631A2 EP 0307869 W EP0307869 W EP 0307869W WO 2004009631 A2 WO2004009631 A2 WO 2004009631A2
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gonadotropin
releasing hormone
type
receptor
polynucleotide
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PCT/EP2003/007869
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French (fr)
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WO2004009631A3 (en
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Alex Smolyar
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Bayer Healthcare Ag
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Publication of WO2004009631A3 publication Critical patent/WO2004009631A3/en

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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
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor

Definitions

  • the invention relates to the regulation of human gonadotropin-releasing hormone (type 2) receptor (GnRH-R).
  • GPCR G protein-coupled receptors
  • GPCRs include receptors for such diverse agents as calcitonin, adrenergic hormones, endothelin, cAMP, adenosine, acetylcholine, serotonin, dopamine, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1, rhodopsins, odorants, cytomegalovirus, G proteins themselves, effector proteins such as phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins such as protein kinase A and protein kinase C.
  • the GPCR protein superfamily now contains over 250 types of paralogues, receptors that represent variants generated by gene duplications (or other processes), as opposed to orthologues, the same receptor from different species.
  • the superfamily can be broken down into five families: Family I, receptors typified by rhodopsin and the ⁇ 2-adrenergic receptor and currently represented by over 200 unique members (reviewed by Dohlman et al, Ann. Rev. Biochem. 60, 653-88, 1991, and references therein); Family H, the recently characterized parathyroid hormone/calcito- nin/secretin receptor family (Juppner et al, Science 254, 1024-26, 1991; Lin et al,
  • GPCRs possess seven conserved membrane-spanning domains connecting at least eight divergent hydrophilic loops. GPCRs (also known as 7TM receptors) have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops. Most
  • GPCRs have single conserved cysteine residues in each of the first two extracellular loops, which form disulfide bonds that are believed to stabilize functional protein structure.
  • the seven transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7.
  • TM3 has been implicated in signal transduction.
  • Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some GPCRs.
  • Most GPCRs contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus.
  • phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.
  • the ligand binding sites of GPCRs are believed to comprise hydrophilic sockets formed by several GPCR transmembrane domains.
  • the hydrophilic sockets are surrounded by hydrophobic residues of the GPCRs.
  • the hydro- philic side of each GPCR transmembrane helix is postulated to face inward and form a polar ligand binding site.
  • TM3 has been implicated in several GPCRs as having a ligand binding site, such as the TM3 aspartate residue.
  • TM5 serines, a TM6 aspara- gine, and TM6 or TM7 phenylalanines or tyrosines also are implicated in ligand binding.
  • GPCRs are coupled inside the cell by heterotrimeric G proteins to various intracellular enzymes, ion channels, and transporters (see Johnson et al., Endoc. Rev. 10, 317-31, 1989).
  • Different G protein alpha subunits preferentially stimulate particular effectors to modulate various biological functions in a cell.
  • Phosphorylation of cytoplasmic residues of GPCRs is an important mechanism for the regulation of some GPCRs.
  • the effect of hormone binding is the activation inside the cell of the enzyme, adenylate cyclase.
  • Enzyme activation by hormones is dependent on the presence of the nucleotide GTP. GTP also influences hormone binding.
  • a G protein connects the hormone receptor to adenylate cyclase. G protein exchanges GTP for bound GDP when activated by a hormone receptor. The GTP-carrying form then binds to activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by the G protein itself, returns the G protein to its basal, inactive form.
  • the G protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
  • the gonadotropin-releasing hormone receptor (GnRH) is a key mediator in the integration of the neural and endocrine systems. See U.S. Patent 5,985,583. Normal reproduction depends on the pulsatile release of physiological concentrations of
  • GnRH which binds to specific high affinity pituitary receptors and triggers the secretion of the gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH).
  • LH gonadotropins luteinizing hormone
  • FSH follicle stimulating hormone
  • the responsiveness and capacity of the gonadotrope GnRH-R is influenced by agonist, concentration and pattern of exposure (Clayton, J. Endocrino 120, 11-19,
  • FIG. 1 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ TD NO:l).
  • Fig. 2 shows the amino acid sequence deduced from the DNA- sequence of Fig.l (SEQ LO NO:2).
  • Fig. 3 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ LD NO:3).
  • Fig. 4 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ ID NO: 1
  • Fig. 5 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ DD NO:5).
  • Fig. 6 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ DD NO:6).
  • Fig. 7 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ LD NO:7).
  • Fig. 8 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ DD NO:8).
  • Fig. 9 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ ID NO: 1
  • Fig. 10 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ ID NO: 10).
  • Fig. 11 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ ID NO: 11).
  • Fig. 12 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ LD NO:12).
  • Fig. 13 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ DD NO:13).
  • Fig. 14 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ
  • Fig. 15 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ LD NO:15).
  • Fig. 16 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ LD NO:16).
  • Fig. 17 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ LO NO:17).
  • Fig. 18 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ LD NO:18).
  • Fig. 19 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ LD NO:18).
  • the invention relates to an isolated polynucleotide from the group consisting of:
  • a novel human GnRH-R is a discovery of the present invention.
  • Human GnRH-R comprises the amino acid sequence shown in SEQ DD NO:2.
  • a coding sequence for human GnRH-R is shown in SEQ DD NO:l.
  • SEQ DD NO:2 is a splice variant of the trembl:AF403014, type ⁇ gonadotropin-releasing hormone (GnRH) receptor gene.
  • SEQ DD NO:2 has a 13 amino acid difference compared to the original gene.
  • Target is localized to chromosome 1, map lq21.1.
  • a seven transmembrane receptor (7tm_l) region is identified.
  • the 13 amino acid difference is located in the second extracellular loop between helices four and five and most likely is involved in ligand binding (see FIG. 1).
  • Related ESTs (SEQ DD NOS:3-19) are expressed in embryonal carcinoma, melanotic melanoma, choriocarcinoma, glioblastoma, placenta, mammary adenocarcinoma, head_neck, aorta, and colon.
  • Human GnRH-R of the invention is expected to be useful for the same purposes as previously identified GnRH-Rs. Human GnRH-R is believed to be useful in therapeutic methods to treat disorders such as obesity. Human GnRH-R also can be used to screen for human GnRH-R activators and inhibitors.
  • One embodiment of the present invention is an expression vector containing any polynucleotide of the present invention.
  • Yet another embodiment of the present invention is a host cell containing any expression vector of the present invention.
  • Still another embodiment of the present invention is a substantially purified Gonadotropin-releasing hormone (type 2) receptor polypeptide encoded by any polynucleotide of the present invention.
  • Yet another embodiment of the present invention is a method of producing a Gonadotropin-releasing hormone (type 2) receptor polypeptide of the present invention, wherein the method comprises the following steps:
  • a culturing the host cells of the present invention under conditions suitable for the expression of the Gonadotropin-releasing hormone (type 2) receptor polypeptide; and b. recovering the Gonadotropin-releasing hormone (type 2) receptor polypeptide from the host cell culture.
  • Yet another embodiment of the present invention is a method for detecting a polynucleotide encoding a Gonadotropin-releasing hormone (type 2) receptor polypeptide in a biological sample comprising the following steps:
  • Still another embodiment of the present invention is a method for detecting a polynucleotide of the present invention or a Gonadotropin-releasing hormone (type
  • receptor polypeptide of the present invention comprising the steps of: a. contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the Gonadotropin-releasing hormone (type 2) receptor polypeptide and b. detecting the interaction
  • Yet another embodiment of the present invention is a diagnostic kit for conducting any method of the present invention.
  • Yet another embodiment of the present invention is a method of screening for agents which decrease the activity of a Gonadotropin-releasing hormone (type 2) receptor, comprising the steps of:
  • a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a Gonadotropin-releasing hormone (type 2) receptor.
  • Still another embodiment of the present invention is a method of screening for agents which regulate the activity of a Gonadotropin-releasing hormone (type 2) receptor, comprising the steps of:
  • a test compound contacting a test compound with a Gonadotropin-releasing hormone (type 2) receptor polypeptide encoded by any polynucleotide of the present invention; and b. detecting a Gonadotropin-releasing hormone (type 2) receptor activity of the polypeptide, wherein a test compound which increases the Gonadotropin- releasing hormone (type 2) receptor activity is identified as a potential therapeutic agent for increasing the activity of the Gonadotropin-releasing hormone (type 2) receptor, and wherein a test compound which decreases the Gonadotropin-releasing hormone (type 2) receptor activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the Gonadotropin-releasing hormone (type 2) receptor.
  • Even another embodiment of the present invention is a method of screening for agents which decrease the activity of a Gonadotropin-releasing hormone (type 2) receptor, comprising the step of: contacting a test compound with any polynucleotide of the present invention and detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of Gonadotropin-releasing hormone (type 2) receptor.
  • Yet another embodiment of the present invention is a method of reducing the activity of a Gonadotropin-releasing hormone (type 2) receptor, comprising the step of: contacting a cell with a reagent which specifically binds to any polynucleotide of the present invention or any Gonadotropin-releasing hormone (type 2) receptor polypeptide of the present invention, whereby the activity of Gonadotropin-releasing hormone (type 2) receptor is reduced.
  • Still another embodiment of the present invention is a reagent that modulates the activity of a Gonadotropin-releasing hormone (type 2) receptor polypeptide or a polynucleotide wherein said reagent is identified by any methods of the present invention.
  • a Gonadotropin-releasing hormone type 2 receptor polypeptide
  • Even another embodiment of the present invention is a pharmaceutical composition, comprising: an expression vector of the present invention or a reagent of the present invention and a pharmaceutically acceptable carrier.
  • Yet another embodiment of the present invention is the use of an expression vector of the present invention or a reagent of the present invention for modulating the activity of a Gonadotropin-releasing hormone (type 2) receptor in a disease, preferably obesity.
  • the invention thus provides a human GnRH-R that can be used to identify test compounds that may act, for example, as activators or inhibitors.
  • Human GnRH-R and fragments thereof also are useful in raising specific antibodies that can block the protein and effectively reduce its activity.
  • Human GnRH-R polypeptides according to the invention comprise at least 6, 10, 15,
  • a GnRH-R polypeptide of the invention therefore can be a portion of a GnRH-R protein, a full- length GnRH-R protein, or a fusion protein comprising all or a portion of a GnRH-R protein.
  • Human GnRH-R polypeptide variants which are biologically active, e.g., retain a functional activity also are human GnRH-R polypeptides.
  • naturally or non-naturally occurring human GnRH-R polypeptide variants have amino acid sequences which are at least about 97, 98, or 99% identical to the amino acid sequence shown in SEQ DD NO: 2 or a fragment thereof.
  • Percent identity between a putative human GnRH-R polypeptide variant and an amino acid sequence of SEQ DD NO:2 is determined by conventional methods. See, for example, Altschul et al, Bull. Math. Bio. 48:603 (1986), and Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA #9:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff & Henikoff, 1992.
  • the "FASTA" similarity search algorithm of Pearson & Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant.
  • the FASTA algorithm is described by Pearson & Lipman, Proc. Nat'l Acad. Sci. USA 55:2444(1988), and by Pearson, Meth. Enzymol. 183:63 (1990).
  • the ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch- Sellers algorithm (Needleman & Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SUM J. Appl. Math.26:7S7 (1974)), which allows for amino acid insertions and deletions.
  • FASTA FASTA program by modifying the scoring matrix file ("SMATPJX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.
  • Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions.
  • Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a human GnRH-R polypeptide can be found using computer programs well known in the art, such as DNASTAR software.
  • the invention additionally, encompasses GnRH-R polypeptides that are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications can be carried out by known techniques including, but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 , acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.
  • Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N- terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression.
  • the GnRH-R polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
  • the invention also provides chemically modified derivatives of GnRH-R polypeptides that may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179,337).
  • the chemical moieties for derivitization can be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, and the like.
  • the polypeptides can be modified at random or predetermined positions within the molecule and can include one, two, three, or more attached chemical moieties.
  • Fusion proteins are useful for generating antibodies against GnRH-R polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins that interact with portions of a human GnRH-R polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • a human GnRH-R polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
  • the first polypeptide segment comprises a GnRH-R polypeptide, such as those described above.
  • the first polypeptide segment also can comprise full-length GnRH-R protein.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include ⁇ -galactosidase, ⁇ - glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VS V- G tags, and thioredoxin (Trx) tags.
  • Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4
  • a fusion protein also can be engineered to contain a cleavage site located between the GnRH-R polypeptide-encoding sequence and the heterologous protein sequence, so that the GnRH-R polypeptide can be cleaved and purified away from the heterologous moiety.
  • a fusion protein can be synthesized chemically, as is known in the art.
  • a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology.
  • Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ DD NO:l in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art.
  • Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA),
  • Species homologs of human GnRH-R polypeptide can be obtained using GnRH-R polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of GnRH-R polypeptide, and expressing the cDNAs as is known in the art.
  • a human GnRH-R polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a GnRH-R polypeptide.
  • a coding sequence for human GnRH-R is shown in SEQ DD NO: 1.
  • nucleotide sequences encoding human GnRH-R polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to the nucleotide sequence shown in
  • SEQ ID NO:l or its complement also are GnRH-R polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2.
  • cDNA Complementary DNA
  • R polynucleotides that encode biologically active GnRH-R polypeptides also are GnRH-R polynucleotides.
  • Polynucleotide fragments comprising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ DD NO:l or its complement also are GnRH-R polynucleotides. These fragments can be used, for example, as hybridi- zation probes or as antisense oligonucleotides.
  • GnRH-R polynucleotides typically, homologous GnRH-R polynucleotide se- quences can be identified by hybridization of candidate polynucleotides to known
  • GnRH-R polynucleotides under stringent conditions as is known in the art.
  • wash conditions 2X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS, 50 °C once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each—homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.
  • GnRH-R polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast.
  • Human variants of the GnRH-R polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast.
  • GnRH-R polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T m of a double-stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al, J. Mol. Biol. 81, 123 (1973). Variants of human GnRH-R polynucleotides or GnRH-R polynucleotides of other species can therefore be identified by hybridizing a putative homologous GnRH-R polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO:l or the complement thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.
  • GnRH-R polynucleotides Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.
  • T m of a hybrid between a GnRH-R polynucleotide having a nucleotide sequence shown in SEQ DD NO:l or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc.
  • Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% formamide, 4X SSC at 42 °C, or 0.5X SSC, 0.1% SDS at 65 °C.
  • Highly stringent wash conditions include, for example, 0.2X SSC at 65 °C.
  • a human GnRH-R polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated GnRH-R polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments, which comprise GnRH- R nucleotide sequences. Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.
  • Human GnRH-R cDNA molecules can be made with standard molecular biology techniques, using GnRH-R mRNA as a template. Human GnRH-R cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.
  • GnRH-R 5 polynucleotides can be synthesized using synthetic chemistry techniques.
  • the degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a human GnRH-R polypeptide having, for example, an amino acid sequence shown in SEQ DD NO:2 or a biologically active variant thereof.
  • PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements.
  • Sarkar PCR Methods Applic. 2, 318-322, 1993; Triglia et al, Nucleic Acids Res. 16, 8186, 1988; Lagerstrom et al, PCR Methods Applic. 1, 111-119,
  • PCR nested primers
  • PROMOTERFLNDER libraries can be used to walk genomic DNA (CLONTECH, Palo Alto, Calif.). See WO 01/98340.
  • Human GnRH-R polypeptides can be obtained, for example, by purification from human cells, by expression of GnRH-R polynucleotides, or by direct chemical synthesis.
  • Human GnRH-R polypeptides can be purified from any human cell which expresses the receptor, including host cells which have been transfected with GnRH-R polynucleotides.
  • a purified GnRH-R polypeptide is separated from other compounds that normally associate with the GnRH-R polypeptide in the cell, such as
  • a preparation of purified GnRH-R polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.
  • the polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding GnRH-R polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding a human GnRH-R polypeptide.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or ⁇ BR322 plasmids), or animal cell systems. See WO 01/98340.
  • Host cells A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed GnRH-R polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.
  • CHO, HeLa, MDCK, HEK293, and WI38 Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein. See WO 01/98340.
  • marker gene expression suggests that the GnRH-R polynucleotide is also present, its presence and expression may need to be confirmed.
  • a sequence encoding a human GnRH-R polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode an GnRH-R polypeptide can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding an GnRH-R polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the GnRH-R polynucleotide.
  • host cells which contain a human GnRH-R polynucleotide and which express a human GnRH-R polypeptide can be identified by a variety of procedures known to those of skill in the art. Examples include enzyme-linked imrnunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding GnRH-R polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding a human GnRH-R polypeptide can be cloned into a vector for the production of an rnRNA probe.
  • RNA probes are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding a human GnRH-R polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode GnRH-R polypeptides can be designed to contain signal sequences which direct secretion of soluble GnRH-R polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound GnRH-R polypeptide. See WO 01/98340.
  • Sequences encoding a human GnRH-R polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al, Nucl.
  • a human GnRH-R polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • fragments of GnRH-R polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • GnRH-R polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter GnRH-R polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • Antibody as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding an epitope of a human GnRH-R polypeptide.
  • epitopes typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope.
  • epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.
  • An antibody which specifically binds to an epitope of a human GnRH-R polypeptide can be used therapeutically, as well as in irnmunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immuno- precipitations, or other irnmunochemical assays known in the art.
  • irnmunochemical assays such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immuno- precipitations, or other irnmunochemical assays known in the art.
  • Various irnmuno- assays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art.
  • Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to the immunogen.
  • an antibody that specifically binds to a human GnRH-R polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an irnmunochemical assay.
  • antibodies that specifically bind to GnRH-R polypeptides do not detect other proteins in irnmunochemical assays and can immunoprecipitate a human GnRH-R polypeptide from solution. See WO 01/98340.
  • Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of GnRH-R gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al, Chem. Rev. 90, 543-583, 1990.
  • Modifications of GnRH-R gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5', or regulatory regions of the GnRH-R gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons.
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. See WO 01/98340.
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236,
  • Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Patent 5,641,673).
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a human GnRH-R polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the GnRH-R polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988).
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al., EP 321,201). See WO 01/98340.
  • genes whose products interact with human GnRH-R may represent genes that are differentially expressed in disorders including, but not limited to, obesity. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human GnRH-R gene or gene product may itself be tested for differential expression.
  • RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples.
  • Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al, Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subrractive hybridization (Hedrick et al,
  • the differential expression information may itself suggest relevant methods for the treatment of disorders involving the human GnRH-R.
  • treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human GnRH-R.
  • the differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human GnRH-R gene or gene product are up-regulated or down- regulated.
  • the invention provides assays for screening test compounds that bind to or modulate the activity of a human GnRH-R polypeptide or a human GnRH-R polynucleotide.
  • a test compound preferably binds to a human GnRH-R polypeptide or polynucleotide. More preferably, a test compound decreases or increases functional activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.
  • Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity.
  • the compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.
  • Test compounds can be screened for the ability to bind to GnRH-R polypeptides or polynucleotides or to affect GnRH-R activity or GnRH-R gene expression using high throughput screening.
  • high throughput screening many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened.
  • the most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 ⁇ l.
  • many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.
  • free format assays or assays that have no physical barrier between samples, can be used.
  • an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994).
  • the cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose.
  • the combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.
  • Chelsky "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995).
  • Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel.
  • beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.
  • Salmon et al. Molecular Diversity 2, 57-63 (1996).
  • combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.
  • test samples are placed in a porous matrix.
  • One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • the test compound is preferably a small molecule that binds to the GnRH-R polypeptide, such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • either the test compound or the GnRH-R polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • Detection of a test compound that is bound to the GnRH-R polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • binding of a test compound to a human GnRH-R polypeptide can be determined without labeling either of the interactants.
  • a microphysio- meter can be used to detect binding of a test compound with a human GnRH-R polypeptide.
  • a microphysiometer e.g., CytosensorTM
  • LAPS light-addressable potentiometric sensor
  • Determimng the ability of a test compound to bind to a human GnRH-R polypeptide also can be accomplished using a technology such as real-time Bimolecular
  • BIA Interaction Analysis
  • a human GnRH-R polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem. 268,
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • polynucleotide encoding a human GnRH-R polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g. , GAL-4).
  • a DNA sequence that encodes an unidentified protein (“prey" or "sample” can be fused to a polynucleotide that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), wliich is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein that interacts with the GnRH-R polypeptide.
  • a reporter gene e.g., LacZ
  • either the GnRH-R polypeptide (or polynucleotide) or the test compound can be bound to a solid support.
  • Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • any method known in the art can be used to attach the polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
  • Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a human GnRH-R polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • the GnRH-R polypeptide is a fusion protein comprising a domain that allows the GnRH-R polypeptide to be bound to a solid support.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed GnRH-R polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • GnRH-R polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated GnRH-R polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N- hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated
  • 96 well plates (Pierce Chemical).
  • antibodies which specifically bind to a GnRH-R polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include i munodetection of complexes using antibodies which specifically bind to the GnRH-R polypeptide or test compound and SDS gel electrophoresis under non-reducing conditions.
  • Any cell which comprises a GnRH-R polypeptide or polynucleotide can be used in a cell-based assay system.
  • a GnRH-R polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a
  • GnRH-R polypeptide or polynucleotide is determined as described above.
  • Test compounds can be tested for the ability to increase or decrease the functional activity of a human GnRH-R polypeptide. Functional activity can be measured, for example, as described in the specific examples, below. Functional assays can be carried out after contacting either a purified GnRH-R polypeptide, a cell membrane preparation, or an intact cell with a test compound.
  • a test compound that decreases functional activity of a human GnRH-R polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for decreasing GnRH-R activity.
  • a test compound which increases functional activity of a human GnRH-R polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing human GnRH-R activity.
  • test compounds that increase or decrease GnRH-R gene expression are identified.
  • a GnRH-R polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the GnRH-R polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of GnRH-R mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
  • the presence of polypeptide products of a human GnRH-R polynucleotide can be determined, for example, using a variety of techniques known in the art, including irnmunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a human GnRH-R polypeptide.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell that expresses a human GnRH-R polynucleotide can be used in a cell- based assay system.
  • the GnRH-R polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above.
  • Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.
  • compositions of the in-vention can comprise, for example, a human GnRH-R polypeptide, GnRH-R polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a GnRH-R polypeptide, or mimetics, activators, or inhibitors of a human GnRH-R polypeptide activity.
  • the compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
  • Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrcolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpynolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpynolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds can be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers also can be used for delivery.
  • the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation can be a lyopbilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.
  • GnRH-R Human GnRH-R can be regulated to treat obesity.
  • Obesity and overweight are defined as an excess of body fat relative to lean body mass.
  • An increase in caloric intake or a decrease in energy expenditure or both can bring about this imbalance leading to surplus energy being stored as fat.
  • Obesity is associated with important medical morbidities and an increase in mortality.
  • the causes of obesity are poorly understood and may be due to genetic factors, environmental factors or a combination of the two to cause a positive energy balance.
  • anorexia and cachexia are characterized by an imbalance in energy intake versus energy expenditure leading to a negative energy balance and weight loss. Agents that either increase energy expenditure and/or decrease energy intake, absorption or storage would be useful for treating obesity, overweight, and associated comorbidities.
  • Agents that either increase energy intake and/or decrease energy expenditure or increase the amount of lean tissue would be useful for treating cachexia, anorexia and wasting disorders.
  • This gene, translated proteins and agents which modulate this gene or portions of the gene or its products are useful for treating obesity, overweight, anorexia, cachexia, wasting disorders, appetite suppression, appetite enhancement, increases or decreases in satiety, modulation of body weight, and/or other eating disorders such as bulimia.
  • this gene translated proteins and agents which modulate this gene or portions of the gene or its products are useful for treating obesity/overweight-associated comorbidities including hypertension, type 2 diabetes, coronary artery disease, hyperlipidemia, stroke, gallbladder disease, gout, osteoarthritis, sleep apnea and respiratory problems, some types of cancer including endometrial, breast, prostate, and colon cancer, thrombolic disease, polycystic ovarian syndrome, reduced fertility, complications of pregnancy, menstrual irregularities, hirsutism, stress incontinence, and depression.
  • G protein-coupled receptors and obesity treatment including hypertension, type 2 diabetes, coronary artery disease, hyperlipidemia, stroke, gallbladder disease, gout, osteoarthritis, sleep apnea and respiratory problems, some types of cancer including endometrial, breast, prostate, and colon cancer, thrombolic disease, polycystic ovarian syndrome, reduced fertility, complications of pregnancy, menstrual irregularities, hirsut
  • G protein-coupled receptors are integral membrane proteins characterized by seven transmembrane spanning helical domains that mediate the actions of many extracellular signals. GPCRs interact with heterotrimeric guanine nucleotide binding regulatory proteins (G proteins) that modulate a variety of second messenger systems or ionic conductances to effect physiological responses. In fact, almost 50% of currently marketed drugs elicit their therapeutic effects by interacting with GPCRs (Kirkpatrick, Nat. Rev. Drug Disc. 1, 7, 2002).
  • a number of peripherally and centrally acting signaling molecules produce a sense of hunger/satiety or produce elevation in lipid mobilization/oxidation through their interactions with GPCRs.
  • Endocannabinoids, melanin concen- trating hormone, serotonin, dopamine, NPY, ⁇ -MSH, GLP-1, ghrelin and orexin serve as few examples of neurotransmitters/hormones that modulate satiety and/or energy expenditure through GPCRs (Di Marzo et al, Nature 410:822-25, 2001; Marsh et al, Proc. Natl. Acad. Sci.
  • GPCRs also play a critical role in regulating energy expenditure in the periphery.
  • selective agonist ligands of ⁇ 3-adrenergic receptors (AR) induce increase in lipolysis and lipid oxidation in rodents resulting in a decrease in body weight (Arch, Eur. J. Pharmacol. 440: 99-107, 2002).
  • a number of ⁇ 3-AR agonists are cunently being evaluated in clinical trials for their anti-obesity and anti-diabetic effects.
  • GPCRs constitute an attractive drag target for the development of effective anti-obesity agents.
  • This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a human GnRH-R polypeptide binding molecule
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • a reagent which affects GnRH-R activity can be administered to a human cell, either in vitro or in vivo, to reduce GnRH-R activity.
  • the reagent preferably binds to an expression product of a human GnRH-R gene. If the expression product is a protein, the reagent is preferably an antibody.
  • an antibody can be added to a preparation of stem cells that have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.
  • the reagent is delivered using a liposome.
  • the liposome is stable in the animal into which it has been administered for at least about
  • a liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human.
  • the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.
  • a liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell.
  • the transfection efficiency of a liposome is about 0.5 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 6 cells, more preferably about 1.0 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 6 cells, and even more preferably about 2.0 ⁇ g of DNA per 16 nmol of liposome delivered to about 10 6 cells.
  • a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.
  • Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol.
  • a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.
  • a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, for example, U.S. Patent 5,705,151).
  • a reagent such as an antisense oligonucleotide or ribozyme
  • from about 0.1 ⁇ g to about 10 ⁇ g of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 ⁇ g to about 5 ⁇ g of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 ⁇ g of polynucleotides is combined with about 8 nmol liposomes.
  • antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery.
  • Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al Trends in Biotechnol 11, 202-05 (1993);
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases functional activity relative to the functional activity which occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED 50 .
  • compositions that exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun,” and DEAE- or calcium phosphate-mediated transfection.
  • Effective in vivo dosages of an antibody are in the range of about 5 ⁇ g to about
  • effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about
  • the reagent is preferably an antisense oligonucleotide or a ribozyme.
  • Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of a human GnRH-R gene or the activity of a GnRH-R polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent.
  • the effectiveness of the mechanism chosen to decrease the level of expression of a human GnRH-R gene or the activity of a human GnRH-R polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to GnRH-R-specific mRNA, quantitative RT-PCR, immunologic detection of a human GnRH-R polypeptide, or measurement of functional activity.
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents.
  • Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act syner- gistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • Human GnRH-R also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the protein. For example, differences can be determined between the cDNA or genomic sequence encoding GnRH-R in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.
  • Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method.
  • cloned DNA segments can be employed as probes to detect specific DNA segments.
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.
  • DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl. Acad. Sci. USA 85,
  • the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA.
  • mutations can also be detected by in situ analysis. Altered levels of GnRH-R also can be detected in various tissues.
  • Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.
  • the polynucleotide of SEQ DD NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4 gonadotropin-releasing hormone (type 2) receptor polypeptide obtained is transfected into human embryonic kidney 293 cells.
  • the cells are scraped from a culture flask into 5 ml of Tris HCl, 5 mM EDTA, pH 7.5, and lysed by sonication. Cell lysates are centrifuged at 1000 rpm for 5 minutes at 4 °C. The supernatant is centrifuged at 30,000 x g for 20 minutes at 4 °C. The pellet is suspended in binding buffer containing 50 mM Tris HCl, 5 mM MgSO 4 , 1 mM
  • EDTA 100 mM NaCl, pH 7.5, supplemented with 0.1 % BSA, 2 mg/ml aprotinin, 0.5 mg/ml leupeptin, and 10 mg/ml phosphoramidon.
  • Optimal membrane suspension dilutions defined as the protein concentration required to bind less than 10 % of an added radioligand are added to 96-well polypropylene microtiter plates containing ligand, non-labeled peptides, and binding buffer to a final volume of 250 ml.
  • membrane preparations are incubated in the presence of increasing concentrations (0.1 nM to 4 nM) of 125 I ligand.
  • Binding reaction mixtures are incubated for one hour at 30 °C. The reaction is stopped by filtration through GF/B filters treated with 0.5% polyethyleneimine, using a cell harvester. Radioactivity is measured by scintillation counting, and data are analyzed by a computerized non-linear regression program. Non-specific binding is defined as the amount of radioactivity remaining after incubation of membrane protein in the presence of 100 nM of unlabeled peptide. Protein concentration is measured by the Bradford method using Bio-Rad Reagent, with bovine serum albumin as a standard. The gonadotropin-releasing hormone (type 2) receptor activity of the polypeptide comprising the amino acid sequence of SEQ DD NO: 2 is demonstrated. EXAMPLE 2
  • the Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant human GnRH-R polypeptides in yeast.
  • the GnRH-R-encoding DNA sequence is derived from SEQ DD NO.T.
  • the DNA sequence is modified by well known methods in such a way that it contains at its 5 '-end an initiation codon and at its 3 '-end an enterokinase cleavage site, a His6 reporter tag and a termination codon.
  • the yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea.
  • the bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San
  • GnRH-R polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution.
  • Human GnRH-R polypeptides comprise the amino acid sequence shown in SEQ ID NO:2.
  • the test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.
  • the buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a human GnRH-R polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound that increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a human GnRH-R polypeptide.
  • test compound is administered to a culture of human cells transfected with a GnRH-R expression construct and incubated at 37 °C for 10 to 45 minutes.
  • a culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.
  • RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18, 5294-99, 1979).
  • Northern blots are prepared using 20 to 30 ⁇ g total RNA and hybridized with a 32 P-labeled GnRH-R-specific probe at 65 ° C in Express-hyb
  • the probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ DD NO:l.
  • a test compound that decreases the GnRH- R-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of GnRH-R gene expression.
  • the qualitative expression pattern of GnRH-R in various tissues is determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Quantitative expression profiling
  • GnRH-R is involved in the disease process of obesity
  • expression is determined in the following tissues: subcutaneous adipose tissue, mesenteric adipose tissue, adrenal gland, bone manow, brain (cerebellum, spinal cord, cerebral cortex, caudate, medulla, substantia nigra, and putamen), colon, fetal brain, heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle small intestine, spleen, stomach, testes, thymus, thyroid trachea, and uterus.
  • Quantitative expression profiling is performed by the form of quantitative PCR analysis called "kinetic analysis” firstly described in Higuchi et al, BioTechnology 10, 413-17, 1992, and Higuchi et al, BioTechnology 11, 1026-30, 1993.
  • the principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies.
  • the probe is cleaved by the 5 '-3' endonuclease activity of Taq DNA polymerase and a fluorescent dye released in the medium (Holland et al, Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991). Because the fluorescence emission will increase in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al, Genome Res. 6, 986-94, 1996, and Gibson et al, Genome Res.
  • TaqMan probe internally quenched fluorescent oligonucleotide
  • the amplification of an endogenous confrol can be performed to standardize the amount of sample RNA added to a reaction.
  • the control of choice is the 18S ribosomal RNA. Because reporter dyes with differing emission spectra are available, the target and the endogenous, control can be independently quantified in the same tube if probes labeled with different dyes are used. All "real time PCR" measurements of fluorescence are made in the ABI Prism 7700.
  • RNA extraction and cDNA preparation Total RNA from the tissues listed above are used for expression quantification. RNAs labeled "from autopsy” were extracted from autoptic tissues with the TRIzol reagent (Life Technologies, MD) according to the manufacturer's protocol.
  • RNA Fifty ⁇ g of each RNA were treated with DNase I for 1 hour at 37°C in the following reaction mix: 0.2 U/ ⁇ l RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/ ⁇ l RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tris-HCl pH 7.9; lOmM
  • RNA is extracted once with 1 volume of ⁇ henol:chloroforrn:iso- amyl alcohol (24:24:1) and once with chloroform, and precipitated with 1/10 volume of 3 M sodium acetate, pH5.2, and 2 volumes of ethanol.
  • RNA from the autoptic tissues Fifty ⁇ g of each RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, TX). After resuspension and spectrophoto- metric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufacturer's protocol. The final concentration of RNA in the reaction mix is 200ng/ ⁇ L. Reverse transcription is carried out with 2.5 ⁇ M of random hexamer primers.
  • TaqMan quantitative analysis Specific primers and probe are designed according to the recommendations of PE Applied Biosystems; the probe can be labeled at the 5' end FAM (6-carboxy-fluorescein) and at the 3' end with TAMRA (6-carboxy-tetramethyl-rhodamine). Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate.
  • FAM 6-carboxy-fluorescein
  • TAMRA 6-carboxy-tetramethyl-rhodamine
  • the assay reaction mix is as follows: IX final TaqMan Universal PCR Master Mix (from 2X stock) (PE Applied Biosystems, CA); IX PDAR control - 18S RNA (from 2X stock) (PE Applied Biosystems, CA); IX PDAR control - 18S RNA (from 2X stock) (PE Applied Biosystems, CA); IX PDAR control - 18S RNA (from 2X stock) (PE Applied Biosystems, CA); IX PDAR control - 18S RNA (from
  • the experiment is performed on an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, CA).
  • fluorescence data acquired during PCR are processed as described in the ABI Prism 7700 user's manual in order to achieve better background subtraction as well as signal linearity with the starting target quantity.
  • Human embryonic kidney 293 cells transfected with a polynucleotide which expresses human GnRH-R are scraped from a culture flask into 5 ml of Tris HCl, 5 mM EDTA, pH 7.5, and lysed by sonication. Cell lysates are centrifuged at 1000 rpm for 5 minutes at 4 °C. The supernatant is centrifuged at 30,000 x g for 20 minutes at 4 °C. The pellet is suspended in binding buffer containing 50 mM Tris
  • Optimal membrane suspension dilutions defined as the protein concentration required to bind less than 10 % of the added radioligand, are added to 96-well polypropylene microtiter plates containing 125 I-labeled ligand or test compound, non- labeled peptides, and binding buffer to a final volume of 250 ⁇ l.
  • Binding reaction mixtures are incubated for one hour at 30 °C.
  • the reaction is stopped by filtration through GF/B filters treated with 0.5% polyethyleneimine, using a cell harvester. Radioactivity is measured by scintillation counting, and data are analyzed by a computerized non-linear regression program.
  • Non-specific binding is defined as the amount of radioactivity remaining after incubation of membrane protein in the presence of 100 nM of unlabeled peptide. Protein concentration is measured by the Bradford method using Bio-Rad Reagent, with bovine serum albumin as a standard.
  • a test compound which increases the radioactivity of membrane protein by at least 15% relative to radioactivity of membrane protein which was not incubated with a test compound is identified as a compound which binds to a human GnRH-R polypeptide.
  • Receptor-mediated inhibition of cAMP formation can be assayed in host cells which express human GnRH-R.
  • Cells are plated in 96-well plates and incubated in Dulbecco's phosphate buffered saline (PBS) supplemented with 10 mM HEPES,
  • Radioactivity is quantified using a gamma counter equipped with data reduction software.
  • a test compound which decreases radioactivity of the contents of a well relative to radioactivity of the contents of a well in the absence of the test compound is identified as a potential inhibitor of cAMP formation.
  • a test compound which increases radioactivity of the contents of a well relative to radioactivity of the contents of a well in the absence of the test compound is identified as a potential enhancer of cAMP formation.
  • Intracellular free calcium concentration can be measured by microspecfrofluorometry using the fluorescent indicator dye Fura-2/AM (Bush et al, J. Neurochem. 57, 562- 74, 1991).
  • Stably transfected cells are seeded onto a 35 mm culture dish containing a glass coverslip insert. Cells are washed with HBS , incubated with a test compound, and loaded with 100 ⁇ l of Fura-2/AM (10 ⁇ M) for 20-40 minutes. After washing with HBS to remove the Fura-2/AM solution, cells are equilibrated in HBS for 10- 20 minutes. Cells are then visualized under the 40X objective of a Leitz Fluovert FS microscope.
  • Fluorescence emission is determined at 510 nM, with excitation wavelengths alternating between 340 nM and 380 nM.
  • Raw fluorescence data are converted to calcium concentrations using standard calcium concentration curves and software analysis techniques.
  • a test compound which increases the fluorescence by at least 15% relative to fluorescence in the absence of a test compound is identified as a compound which mobilizes intracellular calcium.
  • the 3 H-inositol phosphate accumulation from inositol phospholipid metabolism is started by adding 10 ⁇ ml of a solution containing a test compound. To the first well 10 ⁇ l are added to measure basal accumulation. Eleven different concentrations of test compound are assayed in the following 11 wells of each plate row. All assays are performed in duplicate by repeating the same additions in two consecutive plate rows.
  • the plates are incubated in a CO 2 incubator for one hour.
  • the reaction is terminated by adding 15 ⁇ l of 50% v/v trichloroacetic acid (TCA), followed by a 40 minute incubation at 4 °C.
  • TCA 50% v/v trichloroacetic acid
  • the content of the wells is transferred to a Multiscreen HN filter plate (Millipore) containing Dowex AG1-X8 (200-400 mesh, formate form).
  • the filter plates are prepared by adding 200 ⁇ l of Dowex AG1-X8 suspension (50% v/v, wate ⁇ resin) to each well.
  • the filter plates are placed on a vacuum manifold to wash or elute the resin bed.
  • Each well is washed 2 times with 200 ⁇ l of water, followed by 2 x 200 ⁇ l of 5 mM sodium tetraborate/60 mM ammonium formate.
  • the 3 H-IPs are eluted into empty 96-well plates with 200 ⁇ l of 1.2 M ammonium formate/0.1 formic acid.
  • the content of the wells is added to 3 ml of scintillation cocktail, and radioactivity is determined by liquid scintillation counting.
  • Binding assays are carried out in a binding buffer containing 50 mM HEPES, pH 7.4, 0.5% BSA, and 5 mM MgCl 2 .
  • the standard assay for radioligand binding to membrane fragments comprising GnRH-R polypeptides is carried out as follows in 96 well microtiter plates (e.g., Dynatech
  • Radioligand is diluted in binding buffer+ PMSF/Baci to the desired cpm per 50 ⁇ l, then 50 ⁇ l aliquots are added to the wells. For non-specific binding samples, 5 ⁇ l of 40 ⁇ M cold ligand also is added per well. Binding is initiated by adding 150 ⁇ l per well of membrane diluted to the desired concentration (10-30 ⁇ g membrane protein/well) in binding buffer+ PMSF/Baci.
  • binding assays to obtain membrane pellets for studies on solubilization of receptordigand complex and for receptor purification are also carried out. These are identical to the standard assays except that (a) binding is carried out in polypropylene tubes in volumes from 1-250 ml, (b) concentration of membrane protein is always 0.5 mg/ml, and (c) for receptor purification, BSA concentration in the binding buffer is reduced to 0.25%, and the wash step is done with binding buffer without BSA, which reduces BSA contamination of the purified receptor.
  • membrane pellets are resuspended in 200 ⁇ l per microtiter plate well of ice-cold binding buffer without BSA. Then 5 ⁇ l per well of 4 mM N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS, Pierce) in DMSO is added and mixed. The samples are held on ice and UV-irradiated for 10 minutes with a Mineralight R-52G lamp (UVP Inc., San
  • Membrane solubilization is carried out in buffer containing 25 mM Tris , pH 8, 10% glycerol (w/v) and 0.2 mM CaCl 2 (solubilization buffer).
  • the highly soluble detergents including Triton X-100, deoxycholate, deoxycholate ysolecithin, CHAPS, and zwittergent are made up in solubilization buffer at 10% concentrations and stored as frozen aliquots. Lysolecithm is made up fresh because of insolubility upon freeze-thawing and digitonin is made fresh at lower concentrations due to its more limited solubility.
  • washed pellets after the binding step are resuspended free of visible particles by pipetting and vortexing in solubilization buffer at 100,000 x g for 30 minutes.
  • solubilization buffer at 100,000 x g for 30 minutes.
  • the supematants are removed and held on ice and the pellets are discarded.
  • the intact R:L complex can be assayed by four different methods. All are carried out on ice or in a cold room at 4-10 °C).
  • PEG- precipitated receptor : 125 1-ligand complex is determined by gamma counting of the filters.
  • Whatman GF/B glass fiber filters are soaked in 0.3% polyethyleneimine (PEI, Sigma) for 3 hours. Samples of solubihzed membranes (25-100 ⁇ l) are replaced in 12 x 75 mm polypropylene tubes. Then 4 ml of solubilization buffer without detergent is added per sample and the samples are immediately filtered through the GFB/PEI filters (1-3 seconds) and washed with 4 ml of solubilization buffer. CPM of receptor : 125 I-ligand complex adsorbed to filters are determined by gamma counting.PAR.4. Charcoal/Dextran (Paul and Said, Peptides 7[Suppl. 77,147-149, 1986). Dextran T70 (0.5 g, Pharmacia) is dissolved in 1 liter of water, then 5 g of activated charcoal (Norit A, alkaline; Fisher Scientific) is added. The suspension is stirred for
  • Binding of biotinyl-receptor to GH 4 Cl membranes is carried out as described above. Incubations are for 1 hour at room temperature. In the standard purification protocol, the binding incubations contain 10 nM Bio-S29. I ligand is added as a tracer at levels of 5,000-100,000 cpm per mg of membrane protein. Control incubations contain 10 ⁇ M cold ligand to saturate the receptor with non-biotinylated ligand.
  • Solubilization of recepto ⁇ ligand complex also is carried out as described above, with 0.15% deoxycholate ysolecithin in solubilization buffer containing 0.2 mM MgCl 2 , to obtain 100,000 x g supematants containing solubihzed R:L complex.
  • Immobilized streptavidin (streptavidin cross-linked to 6% beaded agarose, Pierce Chemical Co.; "SA-agarose”) is washed in solubilization buffer and added to the solubihzed membranes as 1/30 of the final volume. This mixture is incubated with constant stirring by end-over-end rotation for 4-5 hours at 4-10 °C. Then the mixture is applied to a column and the non-bound material is washed through. Binding of radioligand to SA-agarose is determined by comparing cpm in the 100,000 x g supernatant with that in the column effluent after adsorption to SA-agarose.
  • GTP-gamma-S (Sigma)+0.15% (wt/vol) deoxycholatedysolecithin +1/1000 (vol/vol) 100.times.4pase.
  • elution buffer 0.15% (wt/vol) deoxycholatedysolecithin +1/1000 (vol/vol) 100.times.4pase.
  • one column volume of elution buffer is passed through the column and flow is stopped for 20-30 minutes. Then 3-4 more column volumes of elution buffer are passed through. All the eluates are pooled. Eluates from the streptavidin column are incubated overnight (12-15 hours) with immobilized wheat germ agglutinin (WGA agarose, Vector Labs) to adsorb the receptor via interaction of covalently bound carbohydrate with the WGA lectin.
  • WGA agarose immobilized wheat germ agglutinin
  • the ratio (vol/vol) of WGA-agarose to streptavidin column eluate is generally 1 :400. A range from 1:1000 to 1:200 also can be used.
  • the resin is pelleted by centrifugation, the supernatant is removed and saved, and the resin is washed 3 times (about 2 minutes each) in buffer containing 50 mM HEPES, pH 8, 5 mM MgCl 2> and 0.15% deoxycholatedysolecithin.
  • the resin is extracted three times by repeated mixing (vortex mixer on low speed) over a 15-30 minute period on ice, with 3 resin columns each time, of 10 mM
  • N-N'-N"-triacetylchitotriose in the same HEPES buffer used to wash the resin. After each elution step, the resin is centrifuged down and the supernatant is carefully removed, free of WGA-agarose pellets. The three, pooled eluates contain the final, purified receptor. The material non-bound to WGA contain G protein subunits specifically eluted from the streptavidin column, as well as non-specific contaminants. All these fractions are stored frozen at -90 °C.
  • This protocol is to determine the effect of chronic administration of an unknown compound on body weight and food and water consumption in obese Zucker fa/fa rats.
  • Obese Zucker fa/fa rats are frequently used in the determination of compound efficacy in the reduction of body weight.
  • This animal model has been successfully used in the identification and characterization of the efficacy profile of compounds that are or have been used in the management of body weight in obese humans 1 ' 2 ' 3 ' 4 * 5 .
  • Animals are orally gavaged (2ml/kg) daily before the dark phase of the LD/cycle for a pre-determined number of days (typically 8-14 days) with their assigned dose/compound. At this time, body weight, food and water consumption are measured. On the final day, animals are euthanized using CO 2 inhalation.
  • the purpose of this protocol is to determine the effect of chronic administration of an unknown compound on body weight of mice made obese by exposure to a 45% kcal/g high fat diet during more than 10 weeks.
  • the body weight of mice selected for the studies is higher than three standard deviations from the weight of a control group of mice fed standard low fat (5-6% fat) mouse chow.
  • Diet-induced obese (DIO) animals are frequently used in the determination of compound efficacy in the reduction of body weight 1 ' 2 ' 3 ' 4 .
  • This animal model has been successfully used in the identification and characterization of the efficacy profile of compounds that are or have been used in the management of body weight in obese humans 1 ' 2 ' 3 .
  • Animals are orally gavaged (5ml/kg) daily before the dark phase of the LD/cycle for a pre-determined number of days (typically 8-14 days) with their assigned dose/compound. At this time, body weight, food and water consumption are measured. Data is analyzed using appropriate statistics following the research design. On the final day, animals are euthanized using CO 2 inhalation.
  • Sibutramine reduces feeding, body fat and improve insulin resistance in dietary-obese Wistar rats independently of hypothalamic neuropeptide Y.
  • Diazoxide restores beta-3 adrenergic receptor function in diet-induced obesity and diabetes. Endocrinology, 141 (10), 3630-7.
  • This protocol is to determine the effect of a single dose of an unknown compound on food consumption of lean overnight fasted rats.
  • the fasted-refed rat model is frequently used in the field of obesity to identify compounds with potential for anorectic effects.
  • This animal model has been successfully used in the identification and characterization of the efficacy profile of compounds that are or have been used in the management of body weight in obese humans 1 ' 2 ' 3 & 4 .
  • the efficacy test The rats are fasted overnight during the dark phase (total of approx. 16-18 hrs). The animal is dosed orally with his assigned treatment (2mg/ml). One hour after dosing, pre-weighed food jars are returned to the cage. Food intake is recorded 30, 60, 90, 180, 240 minutes post food return. At each time point, spillage is returned to the food jar and then the food jars are weighed. The amount of food consumed is determined for each time point. Difference between treatment group is determined using appropriate statistical analysis.

Abstract

Reagents that regulate human GnRH-R and reagents which bind to human GnRH-R gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to obesity.

Description

REGULATION OF HUMAN GONADOTROPIN- RELEASING HORMONE (TYPE 2) RECEPTOR
FIELD OF THE INVENTION
The invention relates to the regulation of human gonadotropin-releasing hormone (type 2) receptor (GnRH-R).
BACKGROUND OF THE INVENTION
Many medically significant biological processes are mediated by signal transduction pathways that involve G proteins (Lefkowitz, Nature 351, 353-54, 1991). The family of G protein-coupled receptors (GPCR) includes receptors for hormones, neurotrans- mitters, growth factors, and viruses. Specific examples of GPCRs include receptors for such diverse agents as calcitonin, adrenergic hormones, endothelin, cAMP, adenosine, acetylcholine, serotonin, dopamine, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1, rhodopsins, odorants, cytomegalovirus, G proteins themselves, effector proteins such as phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins such as protein kinase A and protein kinase C.
The GPCR protein superfamily now contains over 250 types of paralogues, receptors that represent variants generated by gene duplications (or other processes), as opposed to orthologues, the same receptor from different species. The superfamily can be broken down into five families: Family I, receptors typified by rhodopsin and the β2-adrenergic receptor and currently represented by over 200 unique members (reviewed by Dohlman et al, Ann. Rev. Biochem. 60, 653-88, 1991, and references therein); Family H, the recently characterized parathyroid hormone/calcito- nin/secretin receptor family (Juppner et al, Science 254, 1024-26, 1991; Lin et al,
Science 254, 1022-24, 1991); Family DI, the metabotropic glutamate receptor family in mammals (Nakanishi, Science 258, 597-603, 1992); Family IV, the cAMP receptor family, important in the chemotaxis and development of D. discoideum (Klein et al, Science 241, 1467-72, 1988; and Family V, the fingal mating pheromone receptors such as STE2 (reviewed by Kurjan, Ann. Rev. Biochem. 61, 1097-129, 1992).
GPCRs possess seven conserved membrane-spanning domains connecting at least eight divergent hydrophilic loops. GPCRs (also known as 7TM receptors) have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops. Most
GPCRs have single conserved cysteine residues in each of the first two extracellular loops, which form disulfide bonds that are believed to stabilize functional protein structure. The seven transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction.
Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some GPCRs. Most GPCRs contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus. For several GPCRs, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.
For some receptors, the ligand binding sites of GPCRs are believed to comprise hydrophilic sockets formed by several GPCR transmembrane domains. The hydrophilic sockets are surrounded by hydrophobic residues of the GPCRs. The hydro- philic side of each GPCR transmembrane helix is postulated to face inward and form a polar ligand binding site. TM3 has been implicated in several GPCRs as having a ligand binding site, such as the TM3 aspartate residue. TM5 serines, a TM6 aspara- gine, and TM6 or TM7 phenylalanines or tyrosines also are implicated in ligand binding. GPCRs are coupled inside the cell by heterotrimeric G proteins to various intracellular enzymes, ion channels, and transporters (see Johnson et al., Endoc. Rev. 10, 317-31, 1989). Different G protein alpha subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of GPCRs is an important mechanism for the regulation of some GPCRs. For example, in one form of signal transduction, the effect of hormone binding is the activation inside the cell of the enzyme, adenylate cyclase. Enzyme activation by hormones is dependent on the presence of the nucleotide GTP. GTP also influences hormone binding. A G protein connects the hormone receptor to adenylate cyclase. G protein exchanges GTP for bound GDP when activated by a hormone receptor. The GTP-carrying form then binds to activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by the G protein itself, returns the G protein to its basal, inactive form. Thus, the G protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
Over the past 15 years, nearly 350 therapeutic agents targeting GPCRs receptors have been successfully introduced onto the market. This indicates that these receptors have an established, proven history as therapeutic targets.
Gonadotropin-releasing hormone receptor
The gonadotropin-releasing hormone receptor (GnRH) is a key mediator in the integration of the neural and endocrine systems. See U.S. Patent 5,985,583. Normal reproduction depends on the pulsatile release of physiological concentrations of
GnRH which binds to specific high affinity pituitary receptors and triggers the secretion of the gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH). Whereas physiological concentrations of GnRH orchestrate normal reproduction, high levels of agonist lead to an opposite response, the suppression of gonadotropin secretion. The capacity of GnRH analogues both to activate and to inhibit the hypothalamic-pituitary-gonadal axis has led to their wide clinical utility in the treatment of a variety of disorders ranging from infertility to prostatic carcinoma.
The responsiveness and capacity of the gonadotrope GnRH-R is influenced by agonist, concentration and pattern of exposure (Clayton, J. Endocrino 120, 11-19,
1989). Both in vivo and in vitro studies have demonstrated that low concentration pulsatile GnRH is trophic to the receptor and that a high concentration of agonist induces receptor down-regulation and desensitization. The binding of GnRH to its receptor stimulates phospholipase C and generates inositol-l,4,5-triphosphate and diacylglycerol (Huckle & Conn, Endocrine Reviews 9, 387-95, 1988). These second messengers, in turn, release calcium from intracellular stores and activate protein kinase C. Receptor up-regulation appears to involve both protein kinase C and calcium (Huckle & Conn, 1988; Huckle et αl., J. Biol. Chem. 263, 3296-302, 1988; Young et αl, J. Endocrinol. 107, 49-56, 1985). It is not certain which effectors underlie down-regulation.
Because of gonadotropin-releasing hormone receptor's diverse biological effects, there is a need in the art to identify additional members of the gonadotropin-releasing hormone receptor GCPR family whose activity can be regulated to provide therapeutic effects.
It is an object of the invention to provide reagents and methods of regulating a human GnRH-R. This and other objects of the invention are provided by one or more of the embodiments described below.
BRIEF DESCRIPTION OF THE FIGURES Fig. 1 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ TD NO:l). Fig. 2 shows the amino acid sequence deduced from the DNA- sequence of Fig.l (SEQ LO NO:2). Fig. 3 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ LD NO:3).
Fig. 4 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ
ID NO:4).
Fig. 5 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ DD NO:5). Fig. 6 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ DD NO:6).
Fig. 7 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ LD NO:7).
Fig. 8 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ DD NO:8).
Fig. 9 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ
LD NO:9).
Fig. 10 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ ID NO: 10). Fig. 11 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ ID NO: 11).
Fig. 12 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ LD NO:12). Fig. 13 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ DD NO:13). Fig. 14 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ
LD NO:14). Fig. 15 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ LD NO:15). Fig. 16 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ LD NO:16). Fig. 17 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ LO NO:17).
Fig. 18 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ LD NO:18). Fig. 19 shows the DNA-sequence encoding a gonadotropin- releasing hormone (type 2) receptor Polypeptide (SEQ
DD NO:19).
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to an isolated polynucleotide from the group consisting of:
a) a polynucleotide encoding a Gonadotropin-releasing hormone (type 2) receptor polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 97% identical to the amino acid sequence shown in SEQ LD NO: 2; and the amino acid sequence shown in SEQ ID NO: 2. b) a polynucleotide comprising the sequence of SEQ ID NO: 1 ; c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b) and encodes a Gonadotropin-releasing hormone (type 2) receptor polypeptide; d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a Gonadotropin-releasing hormone (type 2) receptor polypeptide; and e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a Gonadotropin- releasing hormone (type 2) receptor polypeptide.
A novel human GnRH-R is a discovery of the present invention. Human GnRH-R comprises the amino acid sequence shown in SEQ DD NO:2. A coding sequence for human GnRH-R is shown in SEQ DD NO:l. SEQ DD NO:2 is a splice variant of the trembl:AF403014, type π gonadotropin-releasing hormone (GnRH) receptor gene. SEQ DD NO:2 has a 13 amino acid difference compared to the original gene. Target is localized to chromosome 1, map lq21.1. Furthermore, a seven transmembrane receptor (7tm_l) region is identified. The 13 amino acid difference is located in the second extracellular loop between helices four and five and most likely is involved in ligand binding (see FIG. 1). Related ESTs (SEQ DD NOS:3-19) are expressed in embryonal carcinoma, melanotic melanoma, choriocarcinoma, glioblastoma, placenta, mammary adenocarcinoma, head_neck, aorta, and colon.
Human GnRH-R of the invention is expected to be useful for the same purposes as previously identified GnRH-Rs. Human GnRH-R is believed to be useful in therapeutic methods to treat disorders such as obesity. Human GnRH-R also can be used to screen for human GnRH-R activators and inhibitors. One embodiment of the present invention is an expression vector containing any polynucleotide of the present invention.
Yet another embodiment of the present invention is a host cell containing any expression vector of the present invention.
Still another embodiment of the present invention is a substantially purified Gonadotropin-releasing hormone (type 2) receptor polypeptide encoded by any polynucleotide of the present invention.
Even another embodiment of the present invention is a method of producing a Gonadotropin-releasing hormone (type 2) receptor polypeptide of the present invention, wherein the method comprises the following steps:
a. culturing the host cells of the present invention under conditions suitable for the expression of the Gonadotropin-releasing hormone (type 2) receptor polypeptide; and b. recovering the Gonadotropin-releasing hormone (type 2) receptor polypeptide from the host cell culture.
Yet another embodiment of the present invention is a method for detecting a polynucleotide encoding a Gonadotropin-releasing hormone (type 2) receptor polypeptide in a biological sample comprising the following steps:
a. hybridizing any polynucleotide of the present invention to a nucleic acid material of a biological sample, thereby forming a hybridization complex; and b. detecting said hybridization complex.
Still another embodiment of the present invention is a method for detecting a polynucleotide of the present invention or a Gonadotropin-releasing hormone (type
2) receptor polypeptide of the present invention comprising the steps of: a. contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the Gonadotropin-releasing hormone (type 2) receptor polypeptide and b. detecting the interaction
Even another embodiment of the present invention is a diagnostic kit for conducting any method of the present invention.
Yet another embodiment of the present invention is a method of screening for agents which decrease the activity of a Gonadotropin-releasing hormone (type 2) receptor, comprising the steps of:
a. contacting a test compound with a Gonadotropin-releasing hormone (type 2) receptor polypeptide encoded by any polynucleotide of the present invention; b. detecting binding of the test compound to the Gonadotropin-releasing hormone (type 2) receptor polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a Gonadotropin-releasing hormone (type 2) receptor.
Still another embodiment of the present invention is a method of screening for agents which regulate the activity of a Gonadotropin-releasing hormone (type 2) receptor, comprising the steps of:
a. contacting a test compound with a Gonadotropin-releasing hormone (type 2) receptor polypeptide encoded by any polynucleotide of the present invention; and b. detecting a Gonadotropin-releasing hormone (type 2) receptor activity of the polypeptide, wherein a test compound which increases the Gonadotropin- releasing hormone (type 2) receptor activity is identified as a potential therapeutic agent for increasing the activity of the Gonadotropin-releasing hormone (type 2) receptor, and wherein a test compound which decreases the Gonadotropin-releasing hormone (type 2) receptor activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the Gonadotropin-releasing hormone (type 2) receptor.
Even another embodiment of the present invention is a method of screening for agents which decrease the activity of a Gonadotropin-releasing hormone (type 2) receptor, comprising the step of: contacting a test compound with any polynucleotide of the present invention and detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of Gonadotropin-releasing hormone (type 2) receptor.
Yet another embodiment of the present invention is a method of reducing the activity of a Gonadotropin-releasing hormone (type 2) receptor, comprising the step of: contacting a cell with a reagent which specifically binds to any polynucleotide of the present invention or any Gonadotropin-releasing hormone (type 2) receptor polypeptide of the present invention, whereby the activity of Gonadotropin-releasing hormone (type 2) receptor is reduced.
Still another embodiment of the present invention is a reagent that modulates the activity of a Gonadotropin-releasing hormone (type 2) receptor polypeptide or a polynucleotide wherein said reagent is identified by any methods of the present invention.
Even another embodiment of the present invention is a pharmaceutical composition, comprising: an expression vector of the present invention or a reagent of the present invention and a pharmaceutically acceptable carrier. Yet another embodiment of the present invention is the use of an expression vector of the present invention or a reagent of the present invention for modulating the activity of a Gonadotropin-releasing hormone (type 2) receptor in a disease, preferably obesity.
The invention thus provides a human GnRH-R that can be used to identify test compounds that may act, for example, as activators or inhibitors. Human GnRH-R and fragments thereof also are useful in raising specific antibodies that can block the protein and effectively reduce its activity.
Polypeptides
Human GnRH-R polypeptides according to the invention comprise at least 6, 10, 15,
20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 381 contiguous amino acids selected from the amino acid sequence shown in SEQ DD
NO:2 or a biologically active variant thereof, as defined below. A GnRH-R polypeptide of the invention therefore can be a portion of a GnRH-R protein, a full- length GnRH-R protein, or a fusion protein comprising all or a portion of a GnRH-R protein.
Biologically active variants
Human GnRH-R polypeptide variants which are biologically active, e.g., retain a functional activity, also are human GnRH-R polypeptides. Preferably, naturally or non-naturally occurring human GnRH-R polypeptide variants have amino acid sequences which are at least about 97, 98, or 99% identical to the amino acid sequence shown in SEQ DD NO: 2 or a fragment thereof. Percent identity between a putative human GnRH-R polypeptide variant and an amino acid sequence of SEQ DD NO:2 is determined by conventional methods. See, for example, Altschul et al, Bull. Math. Bio. 48:603 (1986), and Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA #9:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff & Henikoff, 1992.
Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson & Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant. The FASTA algorithm is described by Pearson & Lipman, Proc. Nat'l Acad. Sci. USA 55:2444(1988), and by Pearson, Meth. Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ DD NO: 2) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if krup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff value (calculated by a predetermined formula based upon the length of the sequence the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch- Sellers algorithm (Needleman & Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SUM J. Appl. Math.26:7S7 (1974)), which allows for amino acid insertions and deletions. Preferred parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATPJX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990). FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.
Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a human GnRH-R polypeptide can be found using computer programs well known in the art, such as DNASTAR software.
The invention additionally, encompasses GnRH-R polypeptides that are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications can be carried out by known techniques including, but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.
Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N- terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The GnRH-R polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
The invention also provides chemically modified derivatives of GnRH-R polypeptides that may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179,337). The chemical moieties for derivitization can be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, and the like. The polypeptides can be modified at random or predetermined positions within the molecule and can include one, two, three, or more attached chemical moieties.
Whether an amino acid change or a polypeptide modification results in a biologically active GnRH-R polypeptide can readily be determined by assaying for functional activity, as described in the specific examples, below.
Fusion proteins Fusion proteins are useful for generating antibodies against GnRH-R polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins that interact with portions of a human GnRH-R polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
A human GnRH-R polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment comprises a GnRH-R polypeptide, such as those described above. The first polypeptide segment also can comprise full-length GnRH-R protein. The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include β-galactosidase, β- glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VS V- G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4
DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the GnRH-R polypeptide-encoding sequence and the heterologous protein sequence, so that the GnRH-R polypeptide can be cleaved and purified away from the heterologous moiety.
A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ DD NO:l in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA),
Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA- KITS). Identification of species homologs
Species homologs of human GnRH-R polypeptide can be obtained using GnRH-R polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of GnRH-R polypeptide, and expressing the cDNAs as is known in the art.
Polynucleotides
A human GnRH-R polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a GnRH-R polypeptide.
A coding sequence for human GnRH-R is shown in SEQ DD NO: 1.
Degenerate nucleotide sequences encoding human GnRH-R polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to the nucleotide sequence shown in
SEQ ID NO:l or its complement also are GnRH-R polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2. Complementary DNA (cDNA) molecules, species homologs, and variants of GnRH-
R polynucleotides that encode biologically active GnRH-R polypeptides also are GnRH-R polynucleotides. Polynucleotide fragments comprising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ DD NO:l or its complement also are GnRH-R polynucleotides. These fragments can be used, for example, as hybridi- zation probes or as antisense oligonucleotides.
Identification of polynucleotide variants and homologs
Variants and homologs of the GnRH-R polynucleotides described above also are
GnRH-R polynucleotides. Typically, homologous GnRH-R polynucleotide se- quences can be identified by hybridization of candidate polynucleotides to known
GnRH-R polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions~2X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS, 50 °C once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each—homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.
Species homologs of the GnRH-R polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of
GnRH-R polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the Tm of a double-stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al, J. Mol. Biol. 81, 123 (1973). Variants of human GnRH-R polynucleotides or GnRH-R polynucleotides of other species can therefore be identified by hybridizing a putative homologous GnRH-R polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO:l or the complement thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.
Nucleotide sequences which hybridize to GnRH-R polynucleotides or their complements following stringent hybridization and/or wash conditions also are
GnRH-R polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.
Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20 °C below the calculated Tm of the hybrid under study. The Tm of a hybrid between a GnRH-R polynucleotide having a nucleotide sequence shown in SEQ DD NO:l or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc.
Natl. Acad. Sci. U.S.A. 48, 1390 (1962):
Tm = 81.5 °C - 16.6(log10[Na+]) + 0.41 (%G + C) - 0.63(%formamide) - 600/0, where / = the length of the hybrid in basepairs.
Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% formamide, 4X SSC at 42 °C, or 0.5X SSC, 0.1% SDS at 65 °C. Highly stringent wash conditions include, for example, 0.2X SSC at 65 °C.
Preparation of polynucleotides
A human GnRH-R polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated GnRH-R polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments, which comprise GnRH- R nucleotide sequences. Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.
Human GnRH-R cDNA molecules can be made with standard molecular biology techniques, using GnRH-R mRNA as a template. Human GnRH-R cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.
Alternatively, synthetic chemistry techniques can be used to synthesize GnRH-R 5 polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a human GnRH-R polypeptide having, for example, an amino acid sequence shown in SEQ DD NO:2 or a biologically active variant thereof.
10. Extending polynucleotides
Various PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements. Sarkar, PCR Methods Applic. 2, 318-322, 1993; Triglia et al, Nucleic Acids Res. 16, 8186, 1988; Lagerstrom et al, PCR Methods Applic. 1, 111-119,
15 1991; Parker et al, Nucleic Acids Res. 19, 3055-3060, 1991). Additionally, PCR, nested primers, and PROMOTERFLNDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA (CLONTECH, Palo Alto, Calif.). See WO 01/98340.
20 Obtaining Polynucleotides
Human GnRH-R polypeptides can be obtained, for example, by purification from human cells, by expression of GnRH-R polynucleotides, or by direct chemical synthesis.
25 Protein purification
Human GnRH-R polypeptides can be purified from any human cell which expresses the receptor, including host cells which have been transfected with GnRH-R polynucleotides. A purified GnRH-R polypeptide is separated from other compounds that normally associate with the GnRH-R polypeptide in the cell, such as
30 certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
A preparation of purified GnRH-R polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.
Expression of polynucleotides
To express a human GnRH-R polynucleotide, the polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding GnRH-R polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.
A variety of expression vector/host systems can be utilized to contain and express sequences encoding a human GnRH-R polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or ρBR322 plasmids), or animal cell systems. See WO 01/98340.
Host cells A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed GnRH-R polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein. See WO 01/98340.
Detecting expression
Although the presence of marker gene expression suggests that the GnRH-R polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a human GnRH-R polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode an GnRH-R polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding an GnRH-R polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the GnRH-R polynucleotide.
Alternatively, host cells which contain a human GnRH-R polynucleotide and which express a human GnRH-R polypeptide can be identified by a variety of procedures known to those of skill in the art. Examples include enzyme-linked imrnunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting
(FACS). Hampton et al., SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) andMaddox et al, J. Exp. Med. 158, 1211-1216, 1983). See also WO 01/98340.
A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding GnRH-R polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding a human GnRH-R polypeptide can be cloned into a vector for the production of an rnRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Expression and purification of polypeptides Host cells transformed with nucleotide sequences encoding a human GnRH-R polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode GnRH-R polypeptides can be designed to contain signal sequences which direct secretion of soluble GnRH-R polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound GnRH-R polypeptide. See WO 01/98340.
Chemical synthesis
Sequences encoding a human GnRH-R polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al, Nucl.
Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nuc Acids Res. Symp. Ser.
225-232, 1980). Alternatively, a human GnRH-R polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of GnRH-R polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.
Production of altered polypeptides
As will be understood by those of skill in the art, it may be advantageous to produce GnRH-R polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
The nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter GnRH-R polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
Antibodies
Any type of antibody known in the art can be generated to bind specifically to an epitope of a human GnRH-R polypeptide. "Antibody" as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab')2, and Fv, which are capable of binding an epitope of a human GnRH-R polypeptide.
Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.
An antibody which specifically binds to an epitope of a human GnRH-R polypeptide can be used therapeutically, as well as in irnmunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immuno- precipitations, or other irnmunochemical assays known in the art. Various irnmuno- assays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to the immunogen.
Typically, an antibody that specifically binds to a human GnRH-R polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an irnmunochemical assay. Preferably, antibodies that specifically bind to GnRH-R polypeptides do not detect other proteins in irnmunochemical assays and can immunoprecipitate a human GnRH-R polypeptide from solution. See WO 01/98340.
Antisense oligonucleotides
Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of GnRH-R gene products in the cell. Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al, Chem. Rev. 90, 543-583, 1990.
Modifications of GnRH-R gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5', or regulatory regions of the GnRH-R gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al, in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES, Fuτura Publishing Co., Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. See WO 01/98340.
Ribozymes Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236,
1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
The coding sequence of a human GnRH-R polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the GnRH-R polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al., EP 321,201). See WO 01/98340.
Differentially expressed genes
Described herein are methods for the identification of genes whose products interact with human GnRH-R. Such genes may represent genes that are differentially expressed in disorders including, but not limited to, obesity. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human GnRH-R gene or gene product may itself be tested for differential expression.
The degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques. Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis. To identify differentially expressed genes total RNA or, preferably, mRNA is isolated from tissues of interest. For example, RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al, ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Patent 4,843,155.
Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al, Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subrractive hybridization (Hedrick et al,
Nature 308, 149-53; Lee et al, Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), and, preferably, differential display (Liang & Pardee, Science 257, 967-71, 1992; U.S. Patent 5,262,311).
The differential expression information may itself suggest relevant methods for the treatment of disorders involving the human GnRH-R. For example, treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human GnRH-R. The differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human GnRH-R gene or gene product are up-regulated or down- regulated.
Screening methods
The invention provides assays for screening test compounds that bind to or modulate the activity of a human GnRH-R polypeptide or a human GnRH-R polynucleotide.
A test compound preferably binds to a human GnRH-R polypeptide or polynucleotide. More preferably, a test compound decreases or increases functional activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.
Test compounds
Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.
Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann etal, J. Med. Chem. 37, 2678, 1994; Cho et al, Science 261, 1303, 1993; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al, J. Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution
(see, e.g., Houghten, BioTechniques 13, 412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S. Patent 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. U.S.A. 89, 1865-1869, 1992), orphage (Scott & Smith, Science 249, 386-390, 1990; Devlin, Science 249, 404-406, 1990); Cwirla et al, Proc. Natl. Acad. Sci. 97, 6378-6382,
1990; Felici, J. Mol. Biol. 222, 301-310, 1991; and Ladner, U.S. Patent 5,223,409). High throughput screening
Test compounds can be screened for the ability to bind to GnRH-R polypeptides or polynucleotides or to affect GnRH-R activity or GnRH-R gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.
Alternatively, "free format assays," or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.
Another example of a free format assay is described by Chelsky, "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change. Yet another example is described by Salmon et al., Molecular Diversity 2, 57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.
Another high throughput screening method is described in Beutel et al, U.S. Patent
5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together.
Binding assays
For binding assays, the test compound is preferably a small molecule that binds to the GnRH-R polypeptide, such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules.
In binding assays, either the test compound or the GnRH-R polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
Detection of a test compound that is bound to the GnRH-R polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
Alternatively, binding of a test compound to a human GnRH-R polypeptide can be determined without labeling either of the interactants. For example, a microphysio- meter can be used to detect binding of a test compound with a human GnRH-R polypeptide. A microphysiometer (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a human GnRH-R polypeptide (McConnell et al, Science 257, 1906-1912, 1992).
Determimng the ability of a test compound to bind to a human GnRH-R polypeptide also can be accomplished using a technology such as real-time Bimolecular
Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal Chem. 63, 2338-2345, 1991, and Szabo et al, Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BlAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
In yet another aspect of the invention, a human GnRH-R polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem. 268,
12046-12054, 1993; Bartel et al, BioTechniques 14, 920-924, 1993; Iwabuchi et al, Oncogene 8, 1693-1696, 1993; and Brent W094/10300), to identify other proteins which bind to or interact with the GnRH-R polypeptide and modulate its activity.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one construct, polynucleotide encoding a human GnRH-R polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g. , GAL-4). In the other construct a DNA sequence that encodes an unidentified protein ("prey" or "sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), wliich is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein that interacts with the GnRH-R polypeptide.
It may be desirable to immobilize either the GnRH-R polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the GnRH-R polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a human GnRH-R polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
hi one embodiment, the GnRH-R polypeptide is a fusion protein comprising a domain that allows the GnRH-R polypeptide to be bound to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed GnRH-R polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either a human
GnRH-R polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated GnRH-R polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N- hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated
96 well plates (Pierce Chemical). Alternatively, antibodies which specifically bind to a GnRH-R polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include i munodetection of complexes using antibodies which specifically bind to the GnRH-R polypeptide or test compound and SDS gel electrophoresis under non-reducing conditions.
Screening for test compounds which bind to a human GnRH-R polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a GnRH-R polypeptide or polynucleotide can be used in a cell-based assay system. A GnRH-R polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a
GnRH-R polypeptide or polynucleotide is determined as described above.
Functional activity
Test compounds can be tested for the ability to increase or decrease the functional activity of a human GnRH-R polypeptide. Functional activity can be measured, for example, as described in the specific examples, below. Functional assays can be carried out after contacting either a purified GnRH-R polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound that decreases functional activity of a human GnRH-R polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for decreasing GnRH-R activity. A test compound which increases functional activity of a human GnRH-R polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing human GnRH-R activity.
Gene expression
In another embodiment, test compounds that increase or decrease GnRH-R gene expression are identified. A GnRH-R polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the GnRH-R polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
The level of GnRH-R mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide products of a human GnRH-R polynucleotide can be determined, for example, using a variety of techniques known in the art, including irnmunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a human GnRH-R polypeptide.
Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell that expresses a human GnRH-R polynucleotide can be used in a cell- based assay system. The GnRH-R polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.
Pharmaceutical compositions
The invention also provides pharmaceutical compositions that can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the in- vention can comprise, for example, a human GnRH-R polypeptide, GnRH-R polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a GnRH-R polypeptide, or mimetics, activators, or inhibitors of a human GnRH-R polypeptide activity. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.
In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrcolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpynolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers. Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery.
Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation can be a lyopbilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.
Therapeutic indications and methods Human GnRH-R can be regulated to treat obesity. Obesity and overweight are defined as an excess of body fat relative to lean body mass. An increase in caloric intake or a decrease in energy expenditure or both can bring about this imbalance leading to surplus energy being stored as fat. Obesity is associated with important medical morbidities and an increase in mortality. The causes of obesity are poorly understood and may be due to genetic factors, environmental factors or a combination of the two to cause a positive energy balance. In contrast, anorexia and cachexia are characterized by an imbalance in energy intake versus energy expenditure leading to a negative energy balance and weight loss. Agents that either increase energy expenditure and/or decrease energy intake, absorption or storage would be useful for treating obesity, overweight, and associated comorbidities.
Agents that either increase energy intake and/or decrease energy expenditure or increase the amount of lean tissue would be useful for treating cachexia, anorexia and wasting disorders.
This gene, translated proteins and agents which modulate this gene or portions of the gene or its products are useful for treating obesity, overweight, anorexia, cachexia, wasting disorders, appetite suppression, appetite enhancement, increases or decreases in satiety, modulation of body weight, and/or other eating disorders such as bulimia. Also this gene, translated proteins and agents which modulate this gene or portions of the gene or its products are useful for treating obesity/overweight-associated comorbidities including hypertension, type 2 diabetes, coronary artery disease, hyperlipidemia, stroke, gallbladder disease, gout, osteoarthritis, sleep apnea and respiratory problems, some types of cancer including endometrial, breast, prostate, and colon cancer, thrombolic disease, polycystic ovarian syndrome, reduced fertility, complications of pregnancy, menstrual irregularities, hirsutism, stress incontinence, and depression. G protein-coupled receptors and obesity treatment
G protein-coupled receptors (GPCRs) are integral membrane proteins characterized by seven transmembrane spanning helical domains that mediate the actions of many extracellular signals. GPCRs interact with heterotrimeric guanine nucleotide binding regulatory proteins (G proteins) that modulate a variety of second messenger systems or ionic conductances to effect physiological responses. In fact, almost 50% of currently marketed drugs elicit their therapeutic effects by interacting with GPCRs (Kirkpatrick, Nat. Rev. Drug Disc. 1, 7, 2002).
A number of peripherally and centrally acting signaling molecules produce a sense of hunger/satiety or produce elevation in lipid mobilization/oxidation through their interactions with GPCRs. There are numerous examples of neurotransmitters and hormones acting on central satiety pathways. Endocannabinoids, melanin concen- trating hormone, serotonin, dopamine, NPY, α-MSH, GLP-1, ghrelin and orexin serve as few examples of neurotransmitters/hormones that modulate satiety and/or energy expenditure through GPCRs (Di Marzo et al, Nature 410:822-25, 2001; Marsh et al, Proc. Natl. Acad. Sci. USA °9:3240-45, 2002; Nonogaki et al, Nat. Med. 4:1152-56, 1998; Gadde et al, Obes. Res. 9:544-51, 2001; Danielsa et al, Peptides 22:483-91, 2001; Hinney et al, J. Clin. Endocrin. Metabol. 84: 1483-86,
1999; Meier et al, Eur. J. Pharmacol. 440:269-19, 2002; Nakazato et al, Nature 409: 194-98, 2001; Haynes et al, Regul. Pept. 104: 153-59, 2002). Small molecule agonists or antagonists ligands of these GPCRs would serve as effective anti-obesity therapeutics.
h addition to modulation of central pathways, GPCRs also play a critical role in regulating energy expenditure in the periphery. For example, selective agonist ligands of β3-adrenergic receptors (AR) induce increase in lipolysis and lipid oxidation in rodents resulting in a decrease in body weight (Arch, Eur. J. Pharmacol. 440: 99-107, 2002). A number of β3-AR agonists are cunently being evaluated in clinical trials for their anti-obesity and anti-diabetic effects. In summary, GPCRs constitute an attractive drag target for the development of effective anti-obesity agents.
This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a human GnRH-R polypeptide binding molecule) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
A reagent which affects GnRH-R activity can be administered to a human cell, either in vitro or in vivo, to reduce GnRH-R activity. The reagent preferably binds to an expression product of a human GnRH-R gene. If the expression product is a protein, the reagent is preferably an antibody. For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells that have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.
In one embodiment, the reagent is delivered using a liposome. Preferably, the liposome is stable in the animal into which it has been administered for at least about
30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin. A liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell. Preferably, the transfection efficiency of a liposome is about 0.5 μg of DNA per 16 nmole of liposome delivered to about 106 cells, more preferably about 1.0 μg of DNA per 16 nmole of liposome delivered to about 106 cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome delivered to about 106 cells. Preferably, a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.
Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.
Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, for example, U.S. Patent 5,705,151). Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 μg of polynucleotides is combined with about 8 nmol liposomes.
In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al Trends in Biotechnol 11, 202-05 (1993);
Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICAΉONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al, J. Biol. Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci. US.A. 87, 3655-59 (1990); Wu et al, J. Biol Chem. 266, 338-42 (1991).
Determination of a therapeutically effective dose
The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases functional activity relative to the functional activity which occurs in the absence of the therapeutically effective dose.
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
Therapeutic efficacy and toxicity, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun," and DEAE- or calcium phosphate-mediated transfection.
Effective in vivo dosages of an antibody are in the range of about 5 μg to about
50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding single-chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about
100 μg of DNA. If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
Preferably, a reagent reduces expression of a human GnRH-R gene or the activity of a GnRH-R polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of a human GnRH-R gene or the activity of a human GnRH-R polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to GnRH-R-specific mRNA, quantitative RT-PCR, immunologic detection of a human GnRH-R polypeptide, or measurement of functional activity.
In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act syner- gistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
Diagnostic methods Human GnRH-R also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the protein. For example, differences can be determined between the cDNA or genomic sequence encoding GnRH-R in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.
Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.
Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl. Acad. Sci. USA 85,
4397-4401, 1985). Thus, the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA. In addition to direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis. Altered levels of GnRH-R also can be detected in various tissues. Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.
All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention.
EXAMPLE 1
Detection of gonadotropin-releasing hormone (type 2) receptor activity
The polynucleotide of SEQ DD NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4 gonadotropin-releasing hormone (type 2) receptor polypeptide obtained is transfected into human embryonic kidney 293 cells. The cells are scraped from a culture flask into 5 ml of Tris HCl, 5 mM EDTA, pH 7.5, and lysed by sonication. Cell lysates are centrifuged at 1000 rpm for 5 minutes at 4 °C. The supernatant is centrifuged at 30,000 x g for 20 minutes at 4 °C. The pellet is suspended in binding buffer containing 50 mM Tris HCl, 5 mM MgSO4, 1 mM
EDTA, 100 mM NaCl, pH 7.5, supplemented with 0.1 % BSA, 2 mg/ml aprotinin, 0.5 mg/ml leupeptin, and 10 mg/ml phosphoramidon. Optimal membrane suspension dilutions, defined as the protein concentration required to bind less than 10 % of an added radioligand are added to 96-well polypropylene microtiter plates containing ligand, non-labeled peptides, and binding buffer to a final volume of 250 ml.
In equilibrium saturation binding assays, membrane preparations are incubated in the presence of increasing concentrations (0.1 nM to 4 nM) of 125I ligand.
Binding reaction mixtures are incubated for one hour at 30 °C. The reaction is stopped by filtration through GF/B filters treated with 0.5% polyethyleneimine, using a cell harvester. Radioactivity is measured by scintillation counting, and data are analyzed by a computerized non-linear regression program. Non-specific binding is defined as the amount of radioactivity remaining after incubation of membrane protein in the presence of 100 nM of unlabeled peptide. Protein concentration is measured by the Bradford method using Bio-Rad Reagent, with bovine serum albumin as a standard. The gonadotropin-releasing hormone (type 2) receptor activity of the polypeptide comprising the amino acid sequence of SEQ DD NO: 2 is demonstrated. EXAMPLE 2
Expression of recombinant human GnRH-R
The Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant human GnRH-R polypeptides in yeast. The GnRH-R-encoding DNA sequence is derived from SEQ DD NO.T. Before insertion into vector pPICZB, the DNA sequence is modified by well known methods in such a way that it contains at its 5 '-end an initiation codon and at its 3 '-end an enterokinase cleavage site, a His6 reporter tag and a termination codon. Moreover, at both termini recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pPICZ B with the conesponding restriction enzymes the modified DNA sequence is ligated into pPICZB. This expression vector is designed for inducible expression in Pichia pastoris, driven by a yeast promoter. The resulting pPICZ/md-His6 vector is used to transform the yeast.
The yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea. The bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San
Diego, CA) according to manufacturer's instructions. Purified human GnRH-R polypeptide is obtained.
EXAMPLE 3 Identification of test compounds that bind to GnRH-R polypeptides
Purified GnRH-R polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. Human GnRH-R polypeptides comprise the amino acid sequence shown in SEQ ID NO:2. The test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.
The buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a human GnRH-R polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound that increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a human GnRH-R polypeptide.
EXAMPLE 4
Identification of a test compound which decreases GnRH-R gene expression
A test compound is administered to a culture of human cells transfected with a GnRH-R expression construct and incubated at 37 °C for 10 to 45 minutes. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.
RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30 μg total RNA and hybridized with a 32P-labeled GnRH-R-specific probe at 65 ° C in Express-hyb
(CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ DD NO:l. A test compound that decreases the GnRH- R-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of GnRH-R gene expression.
EXAMPLE 5
Tissue-specific expression of GnRH-R
The qualitative expression pattern of GnRH-R in various tissues is determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Quantitative expression profiling
To demonstrate that GnRH-R is involved in the disease process of obesity, expression is determined in the following tissues: subcutaneous adipose tissue, mesenteric adipose tissue, adrenal gland, bone manow, brain (cerebellum, spinal cord, cerebral cortex, caudate, medulla, substantia nigra, and putamen), colon, fetal brain, heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle small intestine, spleen, stomach, testes, thymus, thyroid trachea, and uterus. Neuroblastoma cell lines SK-Nr-Be (2), Hr, Sk-N-As, HTB-10, IMR-32, SNSY-5Y, T3, SK-N-D2, D283, DAOY, CHP-2, U87MG, BE(2)C, T986, KANTS, MO59K, CHP234, C6 (rat), SK-N-F1, SK-PU-DW, PFSK-
1, BE(2)M17, and MCIXC also are tested for GnRH-R expression. As a final step, the expression of GnRH-R in cells derived from normal individuals with the expression of cells derived from obese individuals is compared.
Quantitative expression profiling is performed by the form of quantitative PCR analysis called "kinetic analysis" firstly described in Higuchi et al, BioTechnology 10, 413-17, 1992, and Higuchi et al, BioTechnology 11, 1026-30, 1993. The principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies.
If the amplification is performed in the presence of an internally quenched fluorescent oligonucleotide (TaqMan probe) complementary to the target sequence, the probe is cleaved by the 5 '-3' endonuclease activity of Taq DNA polymerase and a fluorescent dye released in the medium (Holland et al, Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991). Because the fluorescence emission will increase in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al, Genome Res. 6, 986-94, 1996, and Gibson et al, Genome Res. 6, 995-1001, 1996). The amplification of an endogenous confrol can be performed to standardize the amount of sample RNA added to a reaction. In this kind of experiment, the control of choice is the 18S ribosomal RNA. Because reporter dyes with differing emission spectra are available, the target and the endogenous, control can be independently quantified in the same tube if probes labeled with different dyes are used. All "real time PCR" measurements of fluorescence are made in the ABI Prism 7700.
RNA extraction and cDNA preparation. Total RNA from the tissues listed above are used for expression quantification. RNAs labeled "from autopsy" were extracted from autoptic tissues with the TRIzol reagent (Life Technologies, MD) according to the manufacturer's protocol.
Fifty μg of each RNA were treated with DNase I for 1 hour at 37°C in the following reaction mix: 0.2 U/μl RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/μl RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tris-HCl pH 7.9; lOmM
MgCl2; 50 mM NaCl; and 1 mM DTT.
After incubation, RNA is extracted once with 1 volume of ρhenol:chloroforrn:iso- amyl alcohol (24:24:1) and once with chloroform, and precipitated with 1/10 volume of 3 M sodium acetate, pH5.2, and 2 volumes of ethanol.
Fifty μg of each RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, TX). After resuspension and spectrophoto- metric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufacturer's protocol. The final concentration of RNA in the reaction mix is 200ng/μL. Reverse transcription is carried out with 2.5μM of random hexamer primers.
TaqMan quantitative analysis. Specific primers and probe are designed according to the recommendations of PE Applied Biosystems; the probe can be labeled at the 5' end FAM (6-carboxy-fluorescein) and at the 3' end with TAMRA (6-carboxy-tetramethyl-rhodamine). Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate.
Total cDNA content is normalized with the simultaneous quantification (multiplex
PCR) of the 18S ribosomal RNA using the Pre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE Applied Biosystems, CA).
The assay reaction mix is as follows: IX final TaqMan Universal PCR Master Mix (from 2X stock) (PE Applied Biosystems, CA); IX PDAR control - 18S RNA (from
20X stock); 300 nM forward primer; 900 nM reverse primer; 200 nM probe; 10 ng cDNA; and water to 25 μl.
Each of the following steps are carried out once: pre PCR, 2 minutes at 50° C, and 10 minutes at 95°C. The following steps are carried out 40 times: denaturation,
15 seconds at 95°C, annealing/extension, 1 minute at 60°C.
The experiment is performed on an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, CA). At the end of the run, fluorescence data acquired during PCR are processed as described in the ABI Prism 7700 user's manual in order to achieve better background subtraction as well as signal linearity with the starting target quantity.
EXAMPLE 6 Radioligand binding assays
Human embryonic kidney 293 cells transfected with a polynucleotide which expresses human GnRH-R are scraped from a culture flask into 5 ml of Tris HCl, 5 mM EDTA, pH 7.5, and lysed by sonication. Cell lysates are centrifuged at 1000 rpm for 5 minutes at 4 °C. The supernatant is centrifuged at 30,000 x g for 20 minutes at 4 °C. The pellet is suspended in binding buffer containing 50 mM Tris
HCl, 5 mM MgSO4, 1 mM EDTA, 100 mM NaCl, pH 7.5, supplemented with 0.1 % BSA, 2 μg/ml aprotinin, 0.5 mg/ml leupeptin, and 10 μg/ml phosphoramidon. Optimal membrane suspension dilutions, defined as the protein concentration required to bind less than 10 % of the added radioligand, are added to 96-well polypropylene microtiter plates containing 125I-labeled ligand or test compound, non- labeled peptides, and binding buffer to a final volume of 250 μl.
In equilibrium saturation binding assays, membrane preparations are incubated in the presence of increasing concentrations (0.1 nM to 4 nM) of 125I-labeled ligand or test compound (specific activity 2200 Ci/mmol). The binding affinities of different test compounds are determined in equilibrium competition binding assays, using 0.1 nM
125I-peptide in the presence of twelve different concentrations of each test compound.
Binding reaction mixtures are incubated for one hour at 30 °C. The reaction is stopped by filtration through GF/B filters treated with 0.5% polyethyleneimine, using a cell harvester. Radioactivity is measured by scintillation counting, and data are analyzed by a computerized non-linear regression program.
Non-specific binding is defined as the amount of radioactivity remaining after incubation of membrane protein in the presence of 100 nM of unlabeled peptide. Protein concentration is measured by the Bradford method using Bio-Rad Reagent, with bovine serum albumin as a standard. A test compound which increases the radioactivity of membrane protein by at least 15% relative to radioactivity of membrane protein which was not incubated with a test compound is identified as a compound which binds to a human GnRH-R polypeptide.
EXAMPLE 7
Effect of a test compound on human GnRH-R -mediated cyclic AMP formation
Receptor-mediated inhibition of cAMP formation can be assayed in host cells which express human GnRH-R. Cells are plated in 96-well plates and incubated in Dulbecco's phosphate buffered saline (PBS) supplemented with 10 mM HEPES,
5 mM theophylline, 2 μg/ml aprotinin, 0.5 mg/ml leupeptin, and 10 μg/ml phosphoramidon for 20 minutes at 37 °C in 5% CO2. A test compound is added and incubated for an additional 10 minutes at 37 °C. The medium is aspirated, and the reaction is stopped by the addition of 100 mM HCl. The plates are stored at 4 °C for 15 minutes. cAMP content in the stopping solution is measured by radioimmuno- assay.
Radioactivity is quantified using a gamma counter equipped with data reduction software. A test compound which decreases radioactivity of the contents of a well relative to radioactivity of the contents of a well in the absence of the test compound is identified as a potential inhibitor of cAMP formation. A test compound which increases radioactivity of the contents of a well relative to radioactivity of the contents of a well in the absence of the test compound is identified as a potential enhancer of cAMP formation.
EXAMPLE 8
Effect of a test compound on the mobilization of intracellular calcium
Intracellular free calcium concentration can be measured by microspecfrofluorometry using the fluorescent indicator dye Fura-2/AM (Bush et al, J. Neurochem. 57, 562- 74, 1991). Stably transfected cells are seeded onto a 35 mm culture dish containing a glass coverslip insert. Cells are washed with HBS , incubated with a test compound, and loaded with 100 μl of Fura-2/AM (10 μM) for 20-40 minutes. After washing with HBS to remove the Fura-2/AM solution, cells are equilibrated in HBS for 10- 20 minutes. Cells are then visualized under the 40X objective of a Leitz Fluovert FS microscope.
Fluorescence emission is determined at 510 nM, with excitation wavelengths alternating between 340 nM and 380 nM. Raw fluorescence data are converted to calcium concentrations using standard calcium concentration curves and software analysis techniques. A test compound which increases the fluorescence by at least 15% relative to fluorescence in the absence of a test compound is identified as a compound which mobilizes intracellular calcium. EXAMPLE 9
Effect of a test compound on phosphoinositide metabolism
Cells which stably express human GnRH-R cDNA are plated in 96-well plates and grown to confluence. The day before the assay, the growth medium is changed to
100 μl of medium containing 1% serum and 0.5 μCi H-myinositol. The plates are incubated overnight in a CO incubator (5% CO2 at 37 °C). Immediately before the assay, the medium is removed and replaced by 200 μl of PBS containing 10 mM LiCl, and the cells are equilibrated with the new medium for 20 minutes. During this interval, cells also are equilibrated with antagonist, added as a 10 μl aliquot of a 20- fold concentrated solution in PBS.
The 3H-inositol phosphate accumulation from inositol phospholipid metabolism is started by adding 10 μml of a solution containing a test compound. To the first well 10 μl are added to measure basal accumulation. Eleven different concentrations of test compound are assayed in the following 11 wells of each plate row. All assays are performed in duplicate by repeating the same additions in two consecutive plate rows.
The plates are incubated in a CO2 incubator for one hour. The reaction is terminated by adding 15 μl of 50% v/v trichloroacetic acid (TCA), followed by a 40 minute incubation at 4 °C. After neutralizing TCA with 40 μl of 1 M Tris, the content of the wells is transferred to a Multiscreen HN filter plate (Millipore) containing Dowex AG1-X8 (200-400 mesh, formate form). The filter plates are prepared by adding 200 μl of Dowex AG1-X8 suspension (50% v/v, wateπresin) to each well. The filter plates are placed on a vacuum manifold to wash or elute the resin bed. Each well is washed 2 times with 200 μl of water, followed by 2 x 200 μl of 5 mM sodium tetraborate/60 mM ammonium formate. The 3H-IPs are eluted into empty 96-well plates with 200 μl of 1.2 M ammonium formate/0.1 formic acid. The content of the wells is added to 3 ml of scintillation cocktail, and radioactivity is determined by liquid scintillation counting.
EXAMPLE 10
Receptor Binding Methods
Standard Binding Assays. Binding assays are carried out in a binding buffer containing 50 mM HEPES, pH 7.4, 0.5% BSA, and 5 mM MgCl2. The standard assay for radioligand binding to membrane fragments comprising GnRH-R polypeptides is carried out as follows in 96 well microtiter plates (e.g., Dynatech
Immulon II Removawell plates). Radioligand is diluted in binding buffer+ PMSF/Baci to the desired cpm per 50 μl, then 50 μl aliquots are added to the wells. For non-specific binding samples, 5 μl of 40 μM cold ligand also is added per well. Binding is initiated by adding 150 μl per well of membrane diluted to the desired concentration (10-30 μg membrane protein/well) in binding buffer+ PMSF/Baci.
Plates are then covered with Linbro mylar plate sealers (Flow Labs) and placed on a Dynatech Microshaker II. Binding is allowed to proceed at room temperature for 1-2 hours and is stopped by centrifuging the plate for 15 minutes at 2,000 x g. The supematants are decanted, and the membrane pellets are washed once by addition of 200 μl of ice cold binding buffer, brief shaking, and recentrifugation. The individual wells are placed in 12 x 75 mm tubes and counted in an LKB Gammamaster counter (78% efficiency). Specific binding by this method is identical to that measured when free ligand is removed by rapid (3-5 seconds) filtration and washing on poly- ethyleneimine-coated glass fiber filters.
Three variations of the standard binding assay are also used.
1. Competitive radioligand binding assays with a concentration range of cold
1 ligand vs. I-labeled ligand are carried out as described above with one modification. All dilutions of ligands being assayed are made in 40X
PMSF Baci to a concentration 40X the final concentration in the assay. Samples of peptide (5 μl each) are then added per microtiter well. Membranes and radioligand are diluted in binding buffer without protease inhibitors. Radioligand is added and mixed with cold ligand, and then binding is initiated by addition of membranes. 2. Chemical cross-linking of radioligand with receptor is done after a binding step identical to the standard assay. However, the wash step is done with binding buffer minus BSA to reduce the possibility of non-specific cross-linking of radioligand with BSA. The cross-linking step is carried out as described below. 3. Larger scale binding assays to obtain membrane pellets for studies on solubilization of receptordigand complex and for receptor purification are also carried out. These are identical to the standard assays except that (a) binding is carried out in polypropylene tubes in volumes from 1-250 ml, (b) concentration of membrane protein is always 0.5 mg/ml, and (c) for receptor purification, BSA concentration in the binding buffer is reduced to 0.25%, and the wash step is done with binding buffer without BSA, which reduces BSA contamination of the purified receptor.
EXAMPLE 11 Chemical Cross-Linking of Radioligand to Receptor
After a radioligand binding step as described above, membrane pellets are resuspended in 200 μl per microtiter plate well of ice-cold binding buffer without BSA. Then 5 μl per well of 4 mM N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS, Pierce) in DMSO is added and mixed. The samples are held on ice and UV-irradiated for 10 minutes with a Mineralight R-52G lamp (UVP Inc., San
Gabriel, Calif.) at a distance of 5-10 cm. Then the samples are transferred to Eppendorf microfuge tubes, the membranes pelleted by centrifugation, supematants removed, and membranes solubihzed in Laemmli SDS sample buffer for polyacrylamide gel electrophoresis (PAGE). PAGE is carried out as described below. Radiolabeled proteins are visualized by autoradiography of the dried gels with Kodak
XAR film and DuPont image intensifier screens. EXAMPLE 12
Membrane Solubilization
Membrane solubilization is carried out in buffer containing 25 mM Tris , pH 8, 10% glycerol (w/v) and 0.2 mM CaCl2 (solubilization buffer). The highly soluble detergents including Triton X-100, deoxycholate, deoxycholate ysolecithin, CHAPS, and zwittergent are made up in solubilization buffer at 10% concentrations and stored as frozen aliquots. Lysolecithm is made up fresh because of insolubility upon freeze-thawing and digitonin is made fresh at lower concentrations due to its more limited solubility.
To solubilize membranes, washed pellets after the binding step are resuspended free of visible particles by pipetting and vortexing in solubilization buffer at 100,000 x g for 30 minutes. The supematants are removed and held on ice and the pellets are discarded.
EXAMPLE 13
Assay of Solubilized Receptors
After binding of 125I ligands and solubilization of the membranes with detergent, the intact R:L complex can be assayed by four different methods. All are carried out on ice or in a cold room at 4-10 °C).
1. Column chromatography (Knuhtsen et al, Biochem. J. 254, 641-647, 1988). Sephadex G-50 columns (8 x 250 mm) are equilibrated with solubilization buffer containing detergent at the concentration used to solubilize membranes and 1 mg/ml bovine serum albumin. Samples of solubihzed membranes (0.2- 0.5 ml) are applied to the columns and eluted at a flow rate of about 0.7 ml/minute. Samples (0.18 ml) are collected. Radioactivity is determined in a gamma counter. Void volumes of the columns are determined by the elution volume of blue dextran. Radioactivity eluting in the void volume is considered bound to protein. Radioactivity eluting later, at the same volume as free 125I ligands, is considered non-bound.
2. Polyethyleneglycol precipitation (Cuatrecasas, Proc. Natl. Acad. Sci. USA 69, 318-322, 1972). For a 100 μl sample of solubihzed membranes in a 12 x
75 mm polypropylene tube, 0.5 ml of 1% (w/v) bovine gamma globulin (Sigma) in 0.1 M sodium phosphate buffer is added, followed by 0.5 ml of 25% (w/v) polyethyleneglycol (Sigma) and mixing. The mixture is held on ice for 15 minutes. Then 3 ml of 0.1 M sodium phosphate, pH 7.4, is added per sample. The samples are rapidly (1-3 seconds) filtered over Whatman
GF/B glass fiber filters and washed with 4 ml of the phosphate buffer. PEG- precipitated receptor : 125 1-ligand complex is determined by gamma counting of the filters.
3. GFB/PEI filter binding (Brims et al, Analytical Biochem. 132, 74-81, 1983).
Whatman GF/B glass fiber filters are soaked in 0.3% polyethyleneimine (PEI, Sigma) for 3 hours. Samples of solubihzed membranes (25-100 μl) are replaced in 12 x 75 mm polypropylene tubes. Then 4 ml of solubilization buffer without detergent is added per sample and the samples are immediately filtered through the GFB/PEI filters (1-3 seconds) and washed with 4 ml of solubilization buffer. CPM of receptor : 125 I-ligand complex adsorbed to filters are determined by gamma counting.PAR.4. Charcoal/Dextran (Paul and Said, Peptides 7[Suppl. 77,147-149, 1986). Dextran T70 (0.5 g, Pharmacia) is dissolved in 1 liter of water, then 5 g of activated charcoal (Norit A, alkaline; Fisher Scientific) is added. The suspension is stirred for
10 minutes at room temperature and then stored at 4 °C. until use. To measure R:L complex, 4 parts by volume of charcoal dextran suspension are added to 1 part by volume of solubihzed membrane. The samples are mixed and held on ice for 2 minutes and then centrifuged for 2 minutes at 11,000 x g in a Beckman microfuge. Free radioligand is adsorbed charcoal/dextran and is discarded with the pellet. Receptor : I-ligand complexes remain in the supernatant and are determined by gamma counting.
EXAMPLE 14 Receptor Purification
Binding of biotinyl-receptor to GH4 Cl membranes is carried out as described above. Incubations are for 1 hour at room temperature. In the standard purification protocol, the binding incubations contain 10 nM Bio-S29. I ligand is added as a tracer at levels of 5,000-100,000 cpm per mg of membrane protein. Control incubations contain 10 μM cold ligand to saturate the receptor with non-biotinylated ligand.
Solubilization of receptoπligand complex also is carried out as described above, with 0.15% deoxycholate ysolecithin in solubilization buffer containing 0.2 mM MgCl2, to obtain 100,000 x g supematants containing solubihzed R:L complex.
Immobilized streptavidin (streptavidin cross-linked to 6% beaded agarose, Pierce Chemical Co.; "SA-agarose") is washed in solubilization buffer and added to the solubihzed membranes as 1/30 of the final volume. This mixture is incubated with constant stirring by end-over-end rotation for 4-5 hours at 4-10 °C. Then the mixture is applied to a column and the non-bound material is washed through. Binding of radioligand to SA-agarose is determined by comparing cpm in the 100,000 x g supernatant with that in the column effluent after adsorption to SA-agarose. Finally, the column is washed with 12-15 column volumes of solubilization buffer+0.15% deoxycholate ysolecithin +1/500 (vol/vol) 100 x 4pase.PAR.The streptavidin column is eluted with solubilization buffer+0.1 mM EDTA+0.1 mM EGTA+0.1 mM
GTP-gamma-S (Sigma)+0.15% (wt/vol) deoxycholatedysolecithin +1/1000 (vol/vol) 100.times.4pase. First, one column volume of elution buffer is passed through the column and flow is stopped for 20-30 minutes. Then 3-4 more column volumes of elution buffer are passed through. All the eluates are pooled. Eluates from the streptavidin column are incubated overnight (12-15 hours) with immobilized wheat germ agglutinin (WGA agarose, Vector Labs) to adsorb the receptor via interaction of covalently bound carbohydrate with the WGA lectin. The ratio (vol/vol) of WGA-agarose to streptavidin column eluate is generally 1 :400. A range from 1:1000 to 1:200 also can be used. After the binding step, the resin is pelleted by centrifugation, the supernatant is removed and saved, and the resin is washed 3 times (about 2 minutes each) in buffer containing 50 mM HEPES, pH 8, 5 mM MgCl2> and 0.15% deoxycholatedysolecithin. To elute the WGA-bound receptor, the resin is extracted three times by repeated mixing (vortex mixer on low speed) over a 15-30 minute period on ice, with 3 resin columns each time, of 10 mM
N-N'-N"-triacetylchitotriose in the same HEPES buffer used to wash the resin. After each elution step, the resin is centrifuged down and the supernatant is carefully removed, free of WGA-agarose pellets. The three, pooled eluates contain the final, purified receptor. The material non-bound to WGA contain G protein subunits specifically eluted from the streptavidin column, as well as non-specific contaminants. All these fractions are stored frozen at -90 °C.
EXAMPLE 15
In vivo testing of compounds/target validation Evaluation of a Test Compound's Efficacy on the Reduction of Body Weight and
Food and Water Consumption in Obese Zucker fa/fa Rats
The purpose of this protocol is to determine the effect of chronic administration of an unknown compound on body weight and food and water consumption in obese Zucker fa/fa rats. Obese Zucker fa/fa rats are frequently used in the determination of compound efficacy in the reduction of body weight. This animal model has been successfully used in the identification and characterization of the efficacy profile of compounds that are or have been used in the management of body weight in obese humans 1'2'3'4*5.
A typical study includes 60-80 male Zucker fa/fa, (n=10/treatment group) with an average body weight of approximately 550g. Rats are kept in standard animal rooms under controlled temperature and humidity and a 12/12 light dark cycle. Water and food are continuously available. Rats are single housed in large rat shoeboxes containing grid floor. Animals are adapted to the grid floors and sham dosed with study vehicle for at least four days before the recording of two-days baseline measurement of body weight and 24 hr food and water consumption. Rats are assigned to one of 6-8 treatment groups based upon their body weight on baseline. The groups are set up so that the mean and standard enor of the mean of body weight were similar.
Animals are orally gavaged (2ml/kg) daily before the dark phase of the LD/cycle for a pre-determined number of days (typically 8-14 days) with their assigned dose/compound. At this time, body weight, food and water consumption are measured. On the final day, animals are euthanized using CO2 inhalation.
1. Al-Barazanji KA, Arch JR, Buckingham RE and Tadayyon, M. (2000). Central exedin-4 reduces body weight without altering plasma leptin in (fa/fa)
Zucker rats. Obes Res. 8 (4), 317-23.
2. Assimacopoulos-Jeannet F, et al., (1991). Effect of a peroxisome proliferator on b-oxidation and iverall energy balance in obese (fa/fa) rats. Am J Physiol, 260 (2 Pt 2):R278-83. 3. Dryden S, Brown M, King P and Williams G. (1999). Decreased plasma leptin in lean and obese Zucker rats after treatment with the serotonin reuptake inhibitor fluoxetine. Horm Metab Res, 31 (6), 363-6.
4. Edwards S and Stevens R. (1994). Effects of chronic systemic administration of 5-HT on food intake and body weight in rats. Pharmacology Biochem Behav. 47 (4), 865-72.
5. Grinker JA, Drewnowski A, Enns M and Kissuleff H (1980). Effects of d- amphetamine and fenfluramine on feeding patterns and activity of obese and lean Zucker rats. Pharmacol Biochem Behav. 12 (2), 265-75. Evaluation of a Test Compound's Efficacy on the Reduction of Body Weight in Diet-Induced Obese Mice
The purpose of this protocol is to determine the effect of chronic administration of an unknown compound on body weight of mice made obese by exposure to a 45% kcal/g high fat diet during more than 10 weeks. The body weight of mice selected for the studies is higher than three standard deviations from the weight of a control group of mice fed standard low fat (5-6% fat) mouse chow. Diet-induced obese (DIO) animals are frequently used in the determination of compound efficacy in the reduction of body weight1' 2' 3' 4. This animal model has been successfully used in the identification and characterization of the efficacy profile of compounds that are or have been used in the management of body weight in obese humans1' 2' 3.
A typical study include 60-80 male C57bl/J6 mice (n=10/treatment group) with an average body weight of approximately 45 g. Mice are kept in standard animal rooms under controlled temperature and humidity and a 12/12 light dark cycle. Water and food are continuously available. Mice are single housed in shoeboxes. Animals are sham dosed with study vehicle for at least four days before the recording of two-days baseline measurement of body weight and 24 hr food and water consumption. Mice are assigned to one of 6-8 treatment groups based upon their body weight on baseline. The groups are set up so that the mean and standard error of the mean of body weight were similar.
Animals are orally gavaged (5ml/kg) daily before the dark phase of the LD/cycle for a pre-determined number of days (typically 8-14 days) with their assigned dose/compound. At this time, body weight, food and water consumption are measured. Data is analyzed using appropriate statistics following the research design. On the final day, animals are euthanized using CO2 inhalation.
1. Brown M, Bing C, King P, Pickavance L, Heal D and Wilding J. (2001).
Sibutramine reduces feeding, body fat and improve insulin resistance in dietary-obese Wistar rats independently of hypothalamic neuropeptide Y.
British Journal of Pharmacology, 132, 1898-1904. 2. Guene-Millo m, et al., (2000). Peroxisome Proliferator-activated receptor a activators improve insulin sensitivity and reduce adiposity. The Journal of Biological Chemistry, 275 (22), 16638-16642.
3. Han LK, Kirnura Y and Okuda H. (1999). Reduction in fat storage during chitin-chitosan treatment in mice fed a high-fat diet. Int J Obes Relat Metab Disord, 23 (2) 174-9.
4. Surwit RS, Dixon TM, Petro AE, Daniel KE and Collins S. (2000). Diazoxide restores beta-3 adrenergic receptor function in diet-induced obesity and diabetes. Endocrinology, 141 (10), 3630-7.
Evaluation of a Test Compound's Efficacy on the Reduction of Food Intake in Lean Overnight Fasted Rats
The purpose of this protocol is to determine the effect of a single dose of an unknown compound on food consumption of lean overnight fasted rats. The fasted-refed rat model is frequently used in the field of obesity to identify compounds with potential for anorectic effects. This animal model has been successfully used in the identification and characterization of the efficacy profile of compounds that are or have been used in the management of body weight in obese humans1' 2' 3 & 4.
A typical study includes 60-80 male rats (n=10/treatment group) with an average body weight of approximately 280 g. Rats are kept in standard animal rooms under controlled temperature and humidity and a 12/12 light dark cycle. Rats are single housed in suspended cages with a mesh floor. Water and food are continuously available unless the animals are being fasted for the study.
The efficacy test: The rats are fasted overnight during the dark phase (total of approx. 16-18 hrs). The animal is dosed orally with his assigned treatment (2mg/ml). One hour after dosing, pre-weighed food jars are returned to the cage. Food intake is recorded 30, 60, 90, 180, 240 minutes post food return. At each time point, spillage is returned to the food jar and then the food jars are weighed. The amount of food consumed is determined for each time point. Difference between treatment group is determined using appropriate statistical analysis.
1. Blavet N DeFeudis FV and Clostre F (1982). Studies on food intake in fasted rat. Gen Pharmacology, 13(4), 293-7.
2. Grignaschi G, Fanelli E, Scagnol I, and Samanin R (1999). Studies on the role of serotonin receptors in the effect of sibutramine in various feeding paradigms in rats. Br. J. Pharmacol., 127(5), 1190-1194.
3. McTavish D and Heel RC. (1992). Dexfenfluramine: A review of its pharma- cological properties and therapeutic potential in obesity. Drug. 43 (5), 713-
733.
4. Rowland NE, Antelnian SM, Bartness TJ (1985). comparison of the effects of fenfluramine and other anorectic agents in different feeding and drinking paradigms in rats. Life Science, 36, 2295-2300.
REFERENCE
The genes encoding the type II gonadotropin-releasing hormone receptor and the ribonucleoprotein RBM8A in humans overlap in two genomic loci. Genomics 2001 Nov;78(l-2):15-8.

Claims

1. An isolated polynucleotide being selected from the group consisting of: a. a polynucleotide encoding a gonadotropin-releasing hormone (type 2) receptor polypeptide comprising an amino acid sequence selected form the group consisting of: i. amino acid sequences wliich are at least about 97% identical to the amino acid sequence shown in SEQ DD NO: 2; and ii. the amino acid sequence shown in SEQ DD NO: 2. b. a polynucleotide comprising the sequence of SEQ ID NO: 1 ; c. a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b) and encodes a gonadotropin- releasing hormone (type 2) receptor polypeptide; d. a polynucleotide the sequence of which deviates from the poly- nucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a gonadotropin-releasing hormone (type 2) receptor polypeptide; and e. a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a gonadotropin-releasing hormone (type 2) receptor polypeptide.
2. An expression vector containing any polynucleotide of claim 1.
3. A host cell containing the expression vector of claim 2.
4. A substantially purified gonadotropin-releasing hormone (type 2) receptor polypeptide encoded by a polynucleotide of claim 1.
5. A method for producing a gonadotropin-releasing hormone (type 2) receptor polypeptide, wherein the method comprises the following steps: a. culturing the host cell of claim 3 under conditions suitable for the expression of the gonadotropin-releasing hormone (type 2) receptor polypeptide; and b. recovering the gonadotropin-releasing hormone (type 2) receptor polypeptide from the host cell culture.
6. A method for detection of a polynucleotide encoding a gonadotropin- releasing hormone (type 2) receptor polypeptide in a biological sample comprising the following steps :
a. hybridizing any polynucleotide of claim 1 to a nucleic acid material of a biological sample, thereby forming a hybridization complex; and b. detecting said hybridization complex.
7. The method of claim 6, wherein before hybridization, the nucleic acid material of the biological sample is amplified.
8. A method for the detection of a polynucleotide of claim 1 or a gonadotropin- releasing hormone (type 2) receptor polypeptide of claim 4 comprising the steps of:
a. contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the gonadotropin-releasing hormone (type 2) receptor polypeptide and b. detecting the interaction
9. A diagnostic kit for conducting the method of any one of claims 6 to 8.
10. A method of screening for agents which decrease the activity of a gonadotropin-releasing hormone (type 2) receptor, comprising the steps of: a. contacting a test compound with any gonadotropin-releasing hormone (type 2) receptor polypeptide encoded by any polynucleotide of claiml; b. detecting binding of the test compound to the gonadotropin-releasing hormone (type 2) receptor polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a gonadotropin-releasing hormone (type 2) receptor.
11. A method of screening for agents which regulate the activity of a gonadotropin-releasing hormone (type 2) receptor, comprising the steps of:
a. contacting a test compound with a gonadotropin-releasing hormone (type 2) receptor polypeptide encoded by any polynucleotide of claim
1; and b. detecting a gonadotropin-releasing hormone (type 2) receptor activity of the polypeptide, wherein a test compound which increases the gonadotropin-releasing hormone (type 2) receptor activity is identified as a potential therapeutic agent for increasing the activity of the gonadotropin-releasing hormone (type 2) receptor, and wherein a test compound which decreases the gonadotropin-releasing hormone (type 2) receptor activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the gonadotropin- releasing hormone (type 2) receptor.
12. A method of screening for agents which decrease the activity of a gonadotropin-releasing hormone (type 2) receptor, comprising the step of: contacting a test compound with any polynucleotide of claim 1 and detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of gonadotropin-releasing hormone (type 2) receptor.
13. A method of reducing the activity of gonadotropin-releasing hormone (type 2) receptor, comprising the step of: contacting a cell with a reagent which specifically binds to any polynucleotide of claim 1 or any gonadotropin-releasing hormone (type 2) receptor polypeptide of claim 4, whereby the activity of gonadotropin-releasing hormone (type 2) receptor is reduced.
14. A reagent that modulates the activity of a gonadofropin-releasing hormone (type 2) receptor polypeptide or a polynucleotide wherein said reagent is identified by the method of any of the claim 10 to 12.
15. A pharmaceutical composition, comprising: the expression vector of claim 2 or the reagent of claim 14 and a pharmaceutically acceptable carrier.
16. Use of the expression vector of claim 2 or the reagent of claim 14 in the preparation of a medicament for modulating the activity of a gonadotropin- releasing hormone (type 2) receptor in a disease.
17. Use of claim 16 wherein the disease is obesity.
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Publication number Priority date Publication date Assignee Title
WO2002070701A2 (en) * 2001-03-01 2002-09-12 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) Novel human gnrh receptor

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002070701A2 (en) * 2001-03-01 2002-09-12 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) Novel human gnrh receptor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FAURHOLM ET AL: "The genes encoding the type II gonadotropin-releasing hormone receptor and the ribonucleoprotein RBM8A in humans overlap in two genomic loci" GENOMICS, vol. 78, November 2001 (2001-11), pages 15-18, XP004468703 ISSN: 0888-7543 *
MILLAR ET AL: "A novel mammalian receptor for the evolutionarily conserved type II GnRH" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, vol. 98, 14 August 2001 (2001-08-14), pages 9636-9641, XP002178834 ISSN: 0027-8424 *
NEILL: "Minireview: GnRH and GnRH receptor genes in the human genome" ENDOCRINOLOGY, vol. 143, March 2002 (2002-03), pages 737-743, XP002264358 *

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