WO1995004821A1 - Utilisation de l'hormone humaine glucagon comme recepteur de peptides 1 - Google Patents

Utilisation de l'hormone humaine glucagon comme recepteur de peptides 1 Download PDF

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Publication number
WO1995004821A1
WO1995004821A1 PCT/US1994/008913 US9408913W WO9504821A1 WO 1995004821 A1 WO1995004821 A1 WO 1995004821A1 US 9408913 W US9408913 W US 9408913W WO 9504821 A1 WO9504821 A1 WO 9504821A1
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WO
WIPO (PCT)
Prior art keywords
receptor
glp
human
cells
glucagon
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Ceased
Application number
PCT/US1994/008913
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English (en)
Inventor
Michael P. Graziano
Doreen A. Borkowski
Gary G. Chicchi
Patricia J. Hey
Catherine D. Strader
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Merck and Co Inc
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Merck and Co Inc
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Publication date
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Priority to JP7506554A priority Critical patent/JPH09501054A/ja
Priority to CA002168448A priority patent/CA2168448A1/fr
Priority to EP94925207A priority patent/EP0716694A4/fr
Publication of WO1995004821A1 publication Critical patent/WO1995004821A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C07K14/575—Hormones
    • C07K14/605—Glucagons

Definitions

  • FIG. 1 Sequence of the human GLP-1 receptor. The 463 residue long amino acid sequence deduced from the cDNA sequence is shown in single letter code. The 337 base pair (bp) fragment (hGLP-1 fragment 1) isolated by degenerate PCR is underlined. Three consensus sequences for N-linked glycoslylation are marked with diamonds.
  • FIG. 1 Comparison of the human and rat GLP-1 receptor cDNA sequences. Comparison of the human (top) and rat GLP-1 receptors was made using the GCG (Genetics Computer Group) Gap program. The 7 putative transmembrane domains are boxed.
  • FIG. 3 Displacement of [ 125j] GLP-1 (7-36) amide binding to transfected COS-7 cells.
  • COS-7 cells (7x1 ⁇ 6 cells) were transfected with 20 ⁇ g of human GLP-1 receptor cDNA in pcDNAI/neo and membranes prepared and frozen from the cells. 27 ⁇ g of membrane protein was incubated with 50 pM [125r GLP-1 (7-36) amide and the indicated concentrations of ligand. Data shown are means +/- S.E.M. of duplicate determinations and are representative of two experiments. Symbols: squares, GLP-1 (7-36) amide; triangles, glucagon; diamonds, gastric inhibitory peptide; circles, secretin.
  • FIG. 4 cAMP accumulation in transfected COS-7 cells.
  • COS-7 cells (7x1 ⁇ 6 cells) were transfected with 100 ⁇ g of human GLP-1 receptor cDNA. Cells were harvested and cAMP accumulation was determined as outlined in the Examples. Data shown are the mean +/- S.E.M. from triplicate determinations from a single experiment and are representative of three experiments. Symbols, triangles, GLP-1 7-36 amide; squares, glucagon. BACKGROUND OF THE INVENTION
  • Glucagon-like 1 peptide is one of several hormones shown to potentiate glucose-induced insulin secretion. Such hormones, known as incretins, are produced in the gut, released in response to a meal, and their interaction with specific receptors on pancreatic islets causes insulin to be secreted in a glucose-dependent manner (H.-C. Fehmann, J. F. Habener, Trends in Endocrinol. and Met. 3, 158-163 (1992)).
  • GLP-1 is produced by postranslational processing of the proglucagon gene in intestinal L cells, through a biologically inactive 37 amino acid form [GLP-1 (1-37)] to either of two biologically active forms, GLP-1 (7-37) and GLP-1 (7-36) amide.
  • GLP-1 (1-37) biologically inactive 37 amino acid form
  • GLP-1 (7-37) and GLP-1 (7-36) amide biologically active forms of GLP-1 are the most potent incretins known, with effects on glucose-mediated insulin secretion being seen at concentrations as low as 10 pM.
  • Infusion of GLP-1 (7-36) amide into patients with type II diabetes leads to increased secretion of insulin which occurs in an glucose-dependent fashion (D. M. Nathan, E. Schreiber, H. Fogel, S. Mojsov, J. F.
  • GLP-1 receptors have also been described in lung (G. Richter, R. Goke, B. Goke, A. R., FEBS Lett. 267, 78-80 (1990)), adipose (C. Ruiz-Grande, C. Alarcon, E. Merida, I. Vaverde, Peptides 13, 13-16 (1992)); brain (N. S. Hoosein, G. R.S, FEBS. Lett. 178, 83- 86 (1984)); and a gastric tumor cell line, HGT-1 (A. B. Hansen, C. P. Gespach, G. E. Rosselin, J. J. Hoist, FEBS Lett. 236, 119-122 (1988)).
  • a cDNA encoding a GLP-1 receptor has recently been cloned from rat pancreatic islets (B. Thorens, Proceedings of the National Academy of Sciences U.S.A 89, 8641-8645 (1992)). This receptor has seven putative transmembrane domains and belongs to the superfamily of G protein coupled receptors. The GLP-1 receptor is most homologous to other members of a recently defined subclass of G protein coupled receptors that includes the receptors for glucagon and secretin. The GLP-1 receptor acts via stimulation of adenylyl cyclase to raise intracellular levels of cAMP (Fehmann et al., supra).
  • the present invention pertains to the cloning, expression, and pharmacological characterization of a human GLP-1 receptor from the gastric tumor cell line HGT-1.
  • the human glucagon-like 1 peptide (GLP-1) is cloned, expressed and used in an in vitro assay to screen for compounds that bind to the receptor, including compounds which specifically stimulate or inhibit the activity of the receptor.
  • the invention includes the assay, the cloned receptor used in the assay, an isolated human GLP-1 receptor, cells expressing the cloned receptor, and compounds identified through the use of the cloned GLP-1 receptor which selectively bind to the human GLP-1 receptor, including specific agonists or antagonists of the receptor.
  • the human glucagon-like 1 peptide receptor of the present application was cloned from the gastric tumor cell line HGT-1.
  • the cDNA clone encodes a protein of 463 amino acids.
  • the predicted secondary structure places this receptor within the superfamily of seven transmembrane domain G protein coupled receptors.
  • Transfection of the human GLP-1 receptor into COS -7 cells confers upon them high affinity binding for [ 125 j] GLP-1 (7-36) amide.
  • the human glucagon-like 1 peptide receptor (GLP-1) was identified, cloned and expressed in cell cultures by the instant inventors.
  • a partial coding region for this receptor was generated by polymerase chain reaction technology (PCR).
  • PCR polymerase chain reaction technology
  • Degenerate oligonucleotides encoding amino acids present in the rat GLP-1 receptor were used to prime PCR reactions using human HGT-1 cDNA as a template.
  • the predicted sized products were cloned and sequenced. Translation of the amplified cDNA yielded an open reading frame encoding a protein approximately 91 % homologous to the rat GLP-1 receptor. This partial sequence was used to obtain a larger cDNA clone from a human HGT-1 library.
  • the remaining receptor cDNA was obtained by a modification of the PCR- RACE (Rapid Amplification of cDNA ends) protocol (M. A. Frohman, M. K. Dush, G. R. Martin, Proc. Natl. Acad. Sci. U.S. A. 85, 8998-9002 (1988)).
  • a cDNA library was made from HGT-1 cDNA and the plasmid pcDNA I as outlined in Examples. PCR was performed using the HGT-1 library and primers to both the partial fragment of the human GLP-1 receptor and pcDNA I. A series of overlapping cDNA fragments were obtained and sequenced.
  • the HGT-1 cDNA library and primers 5' TGGTGGATTCCTGAACTCC 3' (SEQ ID NO: 3) and 5' CCTGTGGTTTCACAAGAAGC 3' (SEQ ID NO: 4) were used in a PCR reaction to generate the complete receptor sequence ( Figure 1).
  • the open reading frame included in this sequence encodes a 463 amino acid protein that is approximately 91% identical to the rat GLP-1 receptor sequence ( Figure 2).
  • the cloned human GLP-1 receptor when expressed in mammalian cell lines including but not limited to, COS -7, CHO or L cells, is used to discover ligands that bind to the receptor and alter or stimulate its function.
  • the cloned GLP-1 receptor enables quantitation of mRNA levels in human tissues, including the pancreas and gastrointestinal system, by RNase protection assays. For these purposes, a complete coding sequence of the receptor is provided.
  • the specificity of binding of compounds showing affinity for the GLP-1 receptor is shown by measuring the affinity of the compounds to membranes obtained from cells tranfected with the cloned GLP-1 receptor and membranes from tissues known to express GLP-1 receptors.
  • Expression of the cloned GLP-1 receptor screening for compounds that inhibit the binding of radiolabeled GLP-1 (7-36) amide or compounds that stimulate cAMP production in these cells provides a rational way for selection of compounds and discovery of new compounds with predictable pharmacological activities.
  • the recombinant GLP-1 receptor is free of other human proteins.
  • the membranes from the recombinant cells expressing human GLP-1 receptor are then isolated according to methods well known in the art and may be used in a variety of membrane associated receptor binding assays.
  • One example of such an assay is described by Strader et al., ⁇ Proc. Natl. Acad. Sci. USA 84, 4384-4388, 1987).
  • a compound of interest is used to compete with the binding of a known, quantifiable GLP-1 receptor ligand.
  • radiolabeled [ 125 I] GLP (7-36) amide or [ H]-GLP may be used for this purpose. Because of the ease of 125j detection, [125j] GLP (7-36) amide is preferred for this purpose. By increasing the amount of unlabeled test compound, the labeled compound is competed off the receptor. From these experiments, IC50 values for each test compound and receptor subtype is determined.
  • agonist ligands that activate the receptor may be detected by measuring the ability of added compounds to increase cAMP production mediated by the receptor expressed in COS -7 or CHO cells.
  • cAMP can be measured directly by radioimmunoassay or by stimulation of adenylylcyclase in membranes prepared from the cells (Salomon, Y., Landos, C. and Rodbell, M. 1974. Anal. Biochemistry, Vol. 58, 541-548) by methods that are well-known in the art.
  • a method for identifying compounds specific for the human GLP-1 receptor comprising the following steps: a. Cloning the human glucagon-like 1 peptide (GLP-1) receptor; b.
  • HGT-1 cells (cell line C1.19A, a human gastric carcinoma cell line) were obtained from Dr. C. L. Laboisse and cultured as described (Laboisse et al., Cancer Research 42, 1541-1528 (1982)). COS -7 cells were cultured in a manner identical to HGT-1 cells.
  • RNA was isolated from HGT-1 cells using the Fast-Track system (Invitrogen).
  • cDNA was prepared from 5 ⁇ g of HGT-1 poly A + RNA by simultaneous priming with random hexanucleotides and oligo dT primers using the Riboclone cDNA synthesis system (Promega).
  • HGT-1 cDNA and degenerate primers based on the rat cDNA sequence were mixed in a PCR reaction to amplify a partial fragment of the human GLP-1 receptor cDNA ( Figure 1, underlined). The protocol is described below:
  • SEQ ID NO:5 1 ⁇ l 20 uM primer [5' AT(AG) TCN GT(AC) TT(AG) CAC AT 3';
  • Reaction conditions 40 cycles at 95°C, 1 min.; 45°C, 0.5 min.; 72°C,
  • telomere sequence was determined by the dideoxy chain termination method.
  • RNA was isolated from HGT-1 cells using the Fast-Track system (Invitrogen).
  • cDNA was prepared from 5 ⁇ g of HGT-1 poly A + RNA by simultaneous priming with random hexanucleotides and oligo dT primers using the Riboclone cDNA synthesis system (Promega).
  • the cDNA was ligated with non-palindromic BST XI linkers (Invitrogen). Excess linkers were removed by gel-filtration over a cDNA sizing column (Gibco-BRL).
  • BST XL cDNA sizing column
  • the cDNA was ligated into plasmid pcDNA I (Invitrogen) after restriction with BST XL
  • a series of primers were made to sequences in hGLP-fragment 1 (Example 2) and to sequences in pcDNA I. PCR was performed as follows:
  • Reaction conditions 35 cycles at 95°C, 1 min.; 55°C, 0.5 min.; 72°C, 1 min.
  • 5X SSC (IX SSC is 0.15 M sodium chloride, 0.015 M sodium citrate)
  • 5X Denharts solution 1% Ficoll, 1% polyvinylpyrrolidone
  • a single molecule encoding the entire hGLP-1 receptor sequence was obtained by amplification using the polymerase chain reaction. The following conditions were employed:
  • Reaction mixtures were heated at 96°C for 5 min. and then 35 thermal cycles performed: 1 min. at 95°C, 0.5 min. at 55°C, 2 min. at 72°C.
  • COS -7 cells were transfected by electroporation with the human GLP-1 receptor cDNA subcloned into the eukaryotic expression vector pcDNA I/neo (Invitrogen). Cells were harvested after 60-72 h. Membranes containing the expressed receptor protein were prepared as described (C. D. Strader et al, Proc. Natl. Acad. Sci. U.S.A. 84, 4384- 4388 (1987). Membranes prepared from the COS-7 cells transfected with the vector containing the human GLP-1 receptor cDNA specifically bound the GLP-1 receptor agonist [ 12 5i] GLP-1 (7-36) amide ( Figure 3).
  • GLP-1 (7-36) amide inhibits the binding of [ 12 5i] GLP-1 (7- 36) amide to the receptor with an IC50 of 4 nM.
  • Glucagon, gastric inhibitory peptide, and secretin inhibit [125i] GLP-1 (7-36) amide binding with a potency at least a 100-fold lower, consistent with the identification of the receptor as a GLP-1 receptor.
  • Binding reactions were performed in a final volume of 200 ⁇ l of PBS (10 mM sodium phosphate, 1 mM potassium phosphate, 2.7 mM potassium chloride, 137 mM sodium chloride, pH 7.0 ) under the following conditions:
  • Membranes were incubated at room temperature with shaking for one hour. Membranes were harvested on GF/C filters (Whatman) that had been presoaked in 0.5% polyethylenimine/0.1% BSA. The filters were washed three times with ice-cold PBS and bound radioactivity determined by gamma counting. Data were analyzed using the Inplot program (Graphpad Software).
  • the human GLP-1 receptor is functionally coupled to adenylyl cyclase in transiently transfected COS-7 cells (Figure 4).
  • Incubation of COS-7 cells expressing the human GLP-1 receptor with GLP-1 (7-36) amide leads to a 4-fold increase in cyclic AMP (cAMP) over basal levels.
  • cAMP cyclic AMP
  • mock transfected COS cells show no significant increase in cAMP over basal levels.
  • GLP-1 (7-36) amide stimulates cAMP accumulation with an EC50 of 25 pM.
  • Glucagon also stimulates cAMP accumulation in COS -7 cells transfected with the human GLP-1 receptor but with a 200-fold decrease in potency compared with GLP-1 (7-36) amide ( Figure 4).
  • the decreased potency for glucagon is consistent with its acting via the human GLP-1 receptor.
  • Stimulation of cAMP accumulation in intact COS-1 cells that had been transfected with the hGLP-1 receptor assays were carried out in 120 ⁇ l volume of ACC (75 mM Tris pH 7.4, 250 mM sucrose, 12.5 mM magnesium chloride, 1.5 mM ethylenediamine- tetraacetic acid (EDTA), 0.1 mM of the phosphodiesterase inhibitor Ro- 201724) containing the following additions:
  • Transfected cells expressing recombinant human GLP-1 receptor may be used to identify compounds that are agonists for it. This is done by incubating cells with test compounds in range of concentrations such that an ED50 for the test compound is obtained.
  • the ED50 is defined as the concentration of compound which increases intracellular cyclic AMP (cAMP) to 50% of the maximal quantity of cAMP produced by interaction of the test compound with the human GLP-1 receptor.
  • Tyr lie lie Tyr Thr Val Gly Tyr Ala Leu Ser Phe Ser Ala Leu Val 145 150 155 160 lie Ala Ser Ala lie Leu Leu Gly Phe Arg His Leu His Cys Thr Arg 165 170 175
  • Trp Glu Arg Trp Arg Leu Glu His Leu His lie Gin Arg Asp Ser Ser 420 425 430
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Diabetes (AREA)
  • Endocrinology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Emergency Medicine (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
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  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Obesity (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Saccharide Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Le récepteur humain de GLP-1 est cloné et utilisé dans un essai in vitro pour cribler les composés qui se lient spécifiquement au récepteur humain de GLP-1, y compris les composés efficaces pour réduire les symptômes du diabète. L'invention porte sur l'essai, sur le récepteur humain cloné utilisé pour l'essai, sur un récepteur humain isolé de GLP-1 ne contenant pas d'autres protéines humaines, et sur des composés identifiés grâce à l'utilisation de ce nouveau récepteur cloné qui se lie sélectivement au récepteur humain de GLP-1.
PCT/US1994/008913 1993-08-09 1994-08-03 Utilisation de l'hormone humaine glucagon comme recepteur de peptides 1 Ceased WO1995004821A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP7506554A JPH09501054A (ja) 1993-08-09 1994-08-03 ヒトグルカゴン様１ペプチドレセプター
CA002168448A CA2168448A1 (fr) 1993-08-09 1994-08-03 Recepteur de glp-1 humain
EP94925207A EP0716694A4 (fr) 1993-08-09 1994-08-03 Utilisation de l'hormone humaine glucagon comme recepteur de peptides 1

Applications Claiming Priority (2)

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US10451793A 1993-08-09 1993-08-09
US104,517 1993-08-09

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997044357A1 (fr) * 1996-05-22 1997-11-27 Merck & Co., Inc. Proteines synthetiques de liaison au glucagon
WO1998025955A3 (fr) * 1996-12-13 1998-07-30 Allelix Biopharma Recepteurs clones de peptides-2 semblables au glucagon
US5889167A (en) * 1996-05-22 1999-03-30 Merck & Co., Inc. Synthetic glucagon binding proteins
US8592553B2 (en) 1996-12-13 2013-11-26 Nps Pharmaceuticals, Inc. Cloned glucagon-like peptide-2 receptors

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK39892D0 (da) * 1992-03-25 1992-03-25 Bernard Thorens Peptid

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DIABETES, Volume 42, Number 8, issued 1993, M. STOFFEL et al.: "Human Glucagon-Like Peptide-1 Receptor Gene", pages 1215-1218, especially the abstract. *
M.A. INNIS et al., "PCR Protocols: A Guide to Methods and Applications", published 1990 by ACADEMIC PRESS INC. (NEW YORK), chapter 11, pages 84-91, see entire chapter. *
MOLECULAR ENDOCRINOLOGY, Volume 4, Number 2, issued February 1990, M. BIRNBAUMER et al.: "Development and Characterization of a Mouse Cell Line Expressing the Human V2 Vasopressin Receptor Gene", pages 245-254, especially page 251, col. 2. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, Volume 89, issued September 1992, B. THORENS: "Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1", pages 8641-8645, especially page 8641, col. 1; page 8642; and page 8644. *
See also references of EP0716694A4 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997044357A1 (fr) * 1996-05-22 1997-11-27 Merck & Co., Inc. Proteines synthetiques de liaison au glucagon
US5889167A (en) * 1996-05-22 1999-03-30 Merck & Co., Inc. Synthetic glucagon binding proteins
WO1998025955A3 (fr) * 1996-12-13 1998-07-30 Allelix Biopharma Recepteurs clones de peptides-2 semblables au glucagon
US8592553B2 (en) 1996-12-13 2013-11-26 Nps Pharmaceuticals, Inc. Cloned glucagon-like peptide-2 receptors

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EP0716694A4 (fr) 1997-06-04
CA2168448A1 (fr) 1995-02-16
EP0716694A1 (fr) 1996-06-19
JPH09501054A (ja) 1997-02-04

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