EP0776364A1 - Recepteur de glutamate - Google Patents

Recepteur de glutamate

Info

Publication number
EP0776364A1
EP0776364A1 EP95926863A EP95926863A EP0776364A1 EP 0776364 A1 EP0776364 A1 EP 0776364A1 EP 95926863 A EP95926863 A EP 95926863A EP 95926863 A EP95926863 A EP 95926863A EP 0776364 A1 EP0776364 A1 EP 0776364A1
Authority
EP
European Patent Office
Prior art keywords
receptor
dna
ala
hmglur
leu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95926863A
Other languages
German (de)
English (en)
Inventor
Peter Josef Flor
Rainer Kuhn
Kristin Lindauer
Irene Püttner
Thomas Knöpfel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis Pharma GmbH Austria
Novartis AG
Original Assignee
Novartis Erfindungen Verwaltungs GmbH
Ciba Geigy AG
Novartis AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novartis Erfindungen Verwaltungs GmbH, Ciba Geigy AG, Novartis AG filed Critical Novartis Erfindungen Verwaltungs GmbH
Publication of EP0776364A1 publication Critical patent/EP0776364A1/fr
Withdrawn legal-status Critical Current

Links

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
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Metabotropic glutamate receptors belong to the class of G-protein (guanine nucleotide binding protein) coupled receptors which upon binding of a glutamatergic ligand may transduce an extracellular signal via an intracellular second messenger system such as calcium ions, a cyclic nucleotide, diacylglycerol, inositol 1,4,5-triphosphate into a physiological response.
  • G-protein guanine nucleotide binding protein
  • second messenger system such as calcium ions, a cyclic nucleotide, diacylglycerol, inositol 1,4,5-triphosphate into a physiological response.
  • Possessing seven putative transmembrane spanning segments, preceded by a large extracellular ammo-terminal domain and followed by a large carboxy-terminal domain metabotropic glutamate receptors are characterized by a common structure.
  • the present invention relates to hmGluR2 having the amino acid sequence depicted in SEQ ID NO:2.
  • HmGluR muteins may be produced from a DNA encoding a hmGluR protein of the invention which DNA has been subjected to in vitro mutagenesis resulting e.g. in an addition, exchange and or deletion of one or more amino acids.
  • substitutional, deletional and insertional variants of a hmGluR subtype of the invention are prepared by recombinant methods and screened for immuno-crossreactivity with the native forms of the hmGluR.
  • a DNA coding for hmGluR2 or a portion thereof is particularly a DNA encoding the hmGluR2 having the amino acid sequence set forth in SEQ ID NO:2, e.g. the DNA with the nucleotide sequence set forth in SEQ ID NO: 1.
  • Preferred regions from which to construct probes include 5' and/or 3' coding sequences, sequences predicted to encode ligand binding sites, and the like.
  • nucleic acid probes of the invention are labeled with suitable label means for ready detection upon hybridization.
  • a suitable label means is a radiolabel.
  • the preferred method of labelling a DNA fragment is by incorporating 3 P-labelled ⁇ -dATP with the Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art.
  • Suitable host cells for expression of an active recombinant hmGluR2 advantageously express endogenous or recombinant G-proteins. Preferred are cells producing little, if any, endogenous metabotropic glutamate receptor. DNA may be stably incorporated into the cells or may be transiently expressed according to conventional methods.
  • the invention relates to an assay for identifying compounds which modulate the activity of hmGluR2, said assay comprising:
  • antagonizing molecules are useful.
  • an antagonist is understood to refer to a molecule that is capable of interacting with hmGluR2, but which does not stimulate a response pathway within a ceU.
  • glutamate antagonists are generally identified by their ability to interact with hmGluR2 of the invention, and thereby reduce the ability of the natural ligand to stimulate a response pathway within a ceU, e.g. by interfering with the binding of L-glutamate to the hmGluR of the invention or by inhibiting other ceUular functions required for the activity of the hmGluR.
  • a suitable assay e.g.
  • An in vitro assay for a glutamate agonist or antagonist may require that the hmGluR of the invention is produced in sufficient amounts in a functional form using recombinant DNA methods.
  • An assay is then designed to measure a functional property of the hmGluR2 protein, e.g. interaction with a glutamatergic ligand. Production of the hmGluR of the invention is regarded as occurring in sufficient amounts, if activity of said receptor results in a measurable response.
  • a suitably labeled Ugand is e.g. a radioactively labeled Ugand, such as [ 3 H]glutamate, or a Ugand which can be detected by its optical properties, such as absorbance or fluorescence.
  • a radioactively labeled Ugand such as [ 3 H]glutamate
  • a Ugand which can be detected by its optical properties, such as absorbance or fluorescence After removing unbound ligand and test compound the amount of labeled Ugand bound to hmGluR2 is measured. If the amount of labeled Ugand is reduced in the presence of the test compound this compound is said to be bound to the target molecule.
  • a competitive binding assay may be performed e.g. with transformed or transfected host cells expressing the hmGluR of the invention or a membraneous ceUular fraction comprising teh hmGluR of the invention.
  • •Ca 2+ signals resulting from functional interaction of compounds with the target molecule can be transient if the compound is appUed for a limited time period, e.g. via a perfusion system. Usin transient appUcation several measurements can be made with the same cells aUowing for internal controls and high numbers of compounds tested.
  • hmGluR2 functional coupling of hmGluR2 to Ca 2+ signaUing may be achieved by transfection of the hmGluR of the invention if these ceUs naturally express (i) voltage gated Ca channels, activity of which is functionaUy linked to activity of mGluRs or ( ⁇ ) Ca 2+ -permeable cAMP dependent ion channels.
  • GH3 ceUs which naturaUy express voltage-gated Ca channels, directly aUow appUcation of Ca 2+ assays to test for hmGluR2 functional activity by cotransfection of hmGluRs.
  • the invention provides polyclonal and monoclonal antibodies generated against hmGluR2.
  • Such antibodies may useful e.g. for immunoassays including immunohistochemistry as weU as diagnostic and therapeutic appUcations.
  • antibodies specific for the extraceUular domain, or portions thereof, of hmGluR2 can be appUed for blocking the endogenous hmGluR subtype.
  • the ampUfied DNAs are gel purified, cloned into the Smal site of pBluescript SK, and characterized by DNA sequencing (Sequenase T7 polymerase Kit United States Biochemicals).
  • 2x 10 ⁇ plaques of human fetal brain and human adult hippocampus cDNA libraries, constructed in Lambda-ZAPII (Stratagene) from oUgo-(dT) and randomly primed poly(A) + RNA, are screened sequentiaUy with N- and C-terminal rat mGluR2 probes. Metabotropic GluR2 probes are generated by random priming of gel purified fragments using [ ⁇ -- ⁇ 2 P]dCTP.
  • HmGluR2 is negatively coupled to adenylate cyclase when expressed in CHO cells. Agonist binding leads to an inhibition of forskolin induced cAMP accumulation.
  • CeUs transformed with a hmGluR2 expression plasmid are loaded with a calcium sensitive fluorescent dye such as fura-2 or fluro-3.
  • a calcium sensitive fluorescent dye such as fura-2 or fluro-3.
  • To achieve this ceUs are plated in single wells, single weUs containing a coversUp, or 96- well plates and grown for 1 to 5 days until a 50-100 % confluent layer of cells is obtained.
  • Wells are washed d ree times with a balance salt solution (BBS) and incubated for lh in BBS followed by three additional washings with BBS.
  • BBS balance salt solution

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Neurology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Compositions Of Macromolecular Compounds (AREA)
  • Cosmetics (AREA)

Abstract

L'invention concerne un sous-type de récepteur du glutamate métabotrope (hmGluR), des acides nucléiques isolés codant pour ce dernier, des cellules hôtes produisant une protéine selon l'invention, des procédés de préparation de ladite protéine, desdits acides nucléiques et cellules hôtes, et leurs utilisations. L'invention porte également sur des anticorps dirigés contre la protéine hmGluR.
EP95926863A 1994-08-19 1995-07-12 Recepteur de glutamate Withdrawn EP0776364A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB9416554A GB9416554D0 (en) 1994-08-19 1994-08-19 Glutamate receptor
GB9416554 1994-08-19
PCT/EP1995/002728 WO1996006167A1 (fr) 1994-08-19 1995-07-12 Recepteur de glutamate

Publications (1)

Publication Number Publication Date
EP0776364A1 true EP0776364A1 (fr) 1997-06-04

Family

ID=10759939

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95926863A Withdrawn EP0776364A1 (fr) 1994-08-19 1995-07-12 Recepteur de glutamate

Country Status (9)

Country Link
EP (1) EP0776364A1 (fr)
JP (1) JPH10504198A (fr)
AU (1) AU3110095A (fr)
CA (1) CA2196997A1 (fr)
FI (1) FI970633L (fr)
GB (1) GB9416554D0 (fr)
HU (1) HUT76969A (fr)
NO (1) NO970740L (fr)
WO (1) WO1996006167A1 (fr)

Families Citing this family (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5912122A (en) * 1993-06-04 1999-06-15 Sibia Neurosciences, Inc. Nucleic acids encoding and method for detecting nucleic acid encoding human metabotropic glutamate receptor subtype mGluR6
AU695641B2 (en) 1993-09-20 1998-08-20 Novartis Ag Human metabotropic glutamate receptor subtypes (HMR4, HMR6, HMR7) and related DNA compounds
US6017697A (en) * 1994-11-14 2000-01-25 Eli Lilly And Company Excitatory amino acid receptor protein and related nucleic acid compounds
WO1999021992A2 (fr) * 1997-10-23 1999-05-06 Ganimed Pharmaceuticals Gmbh Molecules d'acides nucleiques codant pour un recepteur de glutamate
US7262280B1 (en) 1998-04-03 2007-08-28 Nps Pharmaceuticals, Inc. G-protein fusion receptors and constructs encoding same
GB9807722D0 (en) * 1998-04-08 1998-06-10 Smithkline Beecham Plc Novel compounds
AU3952699A (en) * 1998-05-19 1999-12-06 Compugen Ltd. Metabotropic glutamate receptor-like protein and encoding cdna
CA2497356A1 (fr) * 2002-09-11 2004-03-25 Merck & Co., Inc. Sequences d'acides nucleiques codant des mutations ponctuelles de mglur2 et mglur3
WO2005076014A1 (fr) * 2004-01-28 2005-08-18 Bayer Healthcare Ag Diagnostic et traitement de maladies associees au recepteur 2 metabotropique du glutamate humain (mglur2)
AR059898A1 (es) 2006-03-15 2008-05-07 Janssen Pharmaceutica Nv Derivados de 3-ciano-piridona 1,4-disustituida y su uso como moduladores alostericos de los receptores mglur2
TW200845978A (en) 2007-03-07 2008-12-01 Janssen Pharmaceutica Nv 3-cyano-4-(4-tetrahydropyran-phenyl)-pyridin-2-one derivatives
TW200900065A (en) 2007-03-07 2009-01-01 Janssen Pharmaceutica Nv 3-cyano-4-(4-pyridinyloxy-phenyl)-pyridin-2-one derivatives
SI2203439T1 (sl) 2007-09-14 2011-05-31 Ortho Mcneil Janssen Pharm 1',3'-disubstituirani 4-fenil-3,4,5,6-tetrahidro-2H-1'H-(1,4')bipiridinil-2'-oni
EP2344470B1 (fr) 2008-09-02 2013-11-06 Janssen Pharmaceuticals, Inc. Dérivés de 3-azabicyclo[3.1.0]hexyle comme modulateurs des récepteurs métabotropiques du glutamate
JP5690277B2 (ja) 2008-11-28 2015-03-25 ジャンセン ファーマシューティカルズ， インコーポレイテッド． 代謝型グルタミン酸受容体の調節因子としてのインドールおよびベンゾオキサジン誘導体
MY153913A (en) 2009-05-12 2015-04-15 Janssen Pharmaceuticals Inc 7-aryl-1,2,4-triazolo[4,3-a]pyridine derivatives and their use as positive allosteric modulators of mglur2 receptors
BRPI1010831A2 (pt) 2009-05-12 2016-04-05 Addex Pharmaceuticals Sa derivados de 1,2,4-triazolo[4,3-a]piridina e seu como moduladores alostéricos positivos de receptores de mglur2
CN102439008B (zh) 2009-05-12 2015-04-29 杨森制药有限公司 1,2,4-三唑并[4,3-a]吡啶衍生物及其用于治疗或预防神经和精神病症的用途
ES2536433T3 (es) 2010-11-08 2015-05-25 Janssen Pharmaceuticals, Inc. Derivados de 1,2,4-triazolo[4,3-a]piridina y su uso como moduladores alostéricos positivos de receptores mGluR2
US9271967B2 (en) 2010-11-08 2016-03-01 Janssen Pharmaceuticals, Inc. 1,2,4-triazolo[4,3-a]pyridine derivatives and their use as positive allosteric modulators of mGluR2 receptors
AU2011328203B2 (en) 2010-11-08 2015-03-19 Janssen Pharmaceuticals, Inc. 1,2,4-triazolo[4,3-a]pyridine derivatives and their use as positive allosteric modulators of mGluR2 receptors
JO3368B1 (ar) 2013-06-04 2019-03-13 Janssen Pharmaceutica Nv مركبات 6، 7- ثاني هيدرو بيرازولو [5،1-a] بيرازين- 4 (5 يد)- اون واستخدامها بصفة منظمات تفارغية سلبية لمستقبلات ميجلور 2
JO3367B1 (ar) 2013-09-06 2019-03-13 Janssen Pharmaceutica Nv مركبات 2،1، 4- ثلاثي زولو [3،4-a] بيريدين واستخدامها بصفة منظمات تفارغية موجبة لمستقبلات ميجلور 2
MX386697B (es) 2014-01-21 2025-03-19 Janssen Pharmaceutica Nv Combinaciones que comprenden agonistas ortostericos o moduladores alostericos positivos del receptor glutamatergico metabotropico de subtipo 2 y su uso
ME03518B (fr) 2014-01-21 2020-04-20 Janssen Pharmaceutica Nv Combinaisons comprenant des modulateurs allostériques positifs de sous-type 2 de récepteurs glutamatergiques métabotropes et leur utilisation

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL105587A0 (en) * 1992-05-08 1993-09-22 Lilly Co Eli Human metabotropic glutamate receptor and related dna compounds
US5521297A (en) * 1993-06-04 1996-05-28 Salk Institute Biotechnology/Industrial Associates Nucleic acids encoding human metabotropic glutamate receptors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9606167A1 *

Also Published As

Publication number Publication date
JPH10504198A (ja) 1998-04-28
NO970740L (no) 1997-04-14
HUT76969A (hu) 1998-01-28
AU3110095A (en) 1996-03-14
FI970633A7 (fi) 1997-02-14
GB9416554D0 (en) 1994-10-12
WO1996006167A1 (fr) 1996-02-29
CA2196997A1 (fr) 1996-02-29
FI970633A0 (fi) 1997-02-14
MX9701267A (es) 1997-11-29
FI970633L (fi) 1997-02-14
NO970740D0 (no) 1997-02-18

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