WO2002035231A1 - Procede de tri a l'aide d'un recepteur couple a la g associe a une proteine g specifique - Google Patents

Procede de tri a l'aide d'un recepteur couple a la g associe a une proteine g specifique Download PDF

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WO2002035231A1
WO2002035231A1 PCT/SE2001/002323 SE0102323W WO0235231A1 WO 2002035231 A1 WO2002035231 A1 WO 2002035231A1 SE 0102323 W SE0102323 W SE 0102323W WO 0235231 A1 WO0235231 A1 WO 0235231A1
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adenosine
goif
receptor
receptors
protein
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PCT/SE2001/002323
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Björn KULL
Bertil Fredholm
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Actar Ab
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to a screening method using a G protein coupled receptor. More closely, the invention concerns striatal adenosine A2 A receptors that are specifically associated with the G protein G 0 if.
  • the object of the screening is to find drug candidates that selectively interact with the combination A A-G 0 if to provide highly selective drugs for treatment of, for example, Parkinson's disease and Schizophrenia.
  • the nucleoside adenosine can influence most mammalian cell types via stimulation of four G protein coupled receptors subtypes Ai, A2A, A2B and A3 (Fredholm et al, 1994).
  • the nature of the responses to adenosine and other agonists depends on the selective coupling of the activated receptor to distinct G proteins.
  • the Ai (Freissmuth et al, 1991; Jockers et al, 1994; Munshi et al, 1991) and A3 (Palmer et al, 1995; Zhou et al, 1992) receptors are coupled with Gi proteins inhibiting adenylyl cyclase, whereas the A 2A and A2B receptors (Londos et al, 1980; Pierce et al, 1992; van Calker et al, 1979) couple to Gs-like proteins and activate adenylyl cyclase.
  • Three stimulatory G proteins have been biochemically characterized; G s short, G s long and Goif (Jones et al, 1990).
  • the G s subunits have a widespread distribution, whereas the G 0 if subunit is distributed in a more restricted manner (Herve et al, 1993), and are particularly high in striatum (Drinnan et al, 1991; Herve et al, 1993).
  • the striatal expression may be functionally important since recently developed transgenic mice deficient in Goif are not only anosmic but are also hyperactive (Belluscio et al, 1 98).
  • Goif present in striatum probably plays a key role in the signal transduction mediated by G protein coupled receptors in this area.
  • Di receptors are abundantly expressed in approximately 50% of the striatal neurons, mostly in those projecting to the substantia nigra and containing GABA, substance P and dynorphin (Gerfen et al, 1990; Le Moine et al, 1991).
  • Adenosine A2A receptors are also highly expressed in the striatum (Jarvis and Williams, 1989; Parkinson and Fredholm, 1990).
  • adenosine A2A receptors are segregated from dopamine Di receptors and are selectively expressed in the striatopallidal neurons, that also contain enkephalin and dopamine D2 receptors (Fink et al, 1992; Schiffmann et al, 1991; Svenningsson et al, 1997). This is the other major subpopulation of projection neuron within the striatum. It is also known that A2A receptors stimulate adenylyl cyclase and cAMP-dependent signal transduction in striatum (Fredholm, 1977; Svenningsson et al, 1998).
  • the A2A receptor is particularly abundant in the striatopallidal GABAergic neurons that are also characterized by having a high density of dopamine D2 receptors.
  • Adenosine A2A and dopamine D2 receptors are functionally antagonistic. Under normal physiological conditions, endogenous adenosine exerts a tonic influence on the striatopallidal neurons, causing an increase in neuronal activity. This tonic influence can be counteracted either by dopamine released from the dopaminergic neurons acting on dopamine D 2 receptors or by drugs that inhibit adenosine A2A receptors. In Parkinson's disease the dopaminergic neurotransmission is impaired which leads less dopamine in striatum and consequently to overactive A2A receptors.
  • adenosine receptor When the adenosine receptor is activated it counteracts D2 receptor activation and endogenous dopamine will be less effective (Ferre et al. 1997; Svenningsson et al. 1999). Conversely, if the adenosine A2A receptor is blocked, endogenous dopamine will be more effective, a desired biological mechanism in the treatment of Parkinson's disease. Indeed, there is excellent evidence that blockade of A2A receptors prevents Parkinson-like symptoms in animals and even the progression of the underlying neurodegeneration (Chen et al.). Furthermore, several human superbres with the non-selective antagonist caffeine show that this works also in man (See Fredholm et al. 1999).
  • A2 A receptors is not only expressed in striatopallidal neurons, but also abundant in endothelial cells, leukocytes, blood plates and blood vessels. For these target tissues and cells, it is not desirable to use drugs affecting the adenosine A2A receptors function since that could lead to unwanted side effects. Abundant reports in the literature convincingly show that endogenous adenosine can activate A2A receptors of importance in blood platelet aggregation and in acute inflammatory reactions. It has been for a long time suggested and demonstrated that adenosine A 2A receptors are coupled to the stimulatory G protein subunit G s in several tissue types.
  • a drug that selectively interacts with the combination of adenosine A2A receptors and Goif would have several potential applications: disorders linked to an altered dopaminergic neurotransmission such as Parkinson's disease, Huntington's chorea, and other CNS diseases involving the dopaminergic or GABA transmittor systems which are modulated by adenosine A2A receptors.
  • Present therapy shows some degree of efficacy, however it is well-known that present therapy is not uniformly successful over the longer term and that additional treatment alternatives are necessary (see e.g. Jankovivic 2000).
  • One additional and interesting possibility is that such a drug could be used to decrease the number of side effects of current therapy in Schizophrenia.
  • the vast majority of these drugs are more or less selective for dopamine D 2 receptors. It is known that the most potent of these drugs when given over the course of several weeks can lead to not only transient movement disorders, but also to so called tardive dyskinesia.
  • the invention relates to a screening method comprising use of an A2A receptor coupled to G 0 if to screen for agents selectively interacting with A2A receptor coupled to G 0 if.
  • the interaction may be directly or indirectly with A2A-G 0 if.
  • the invention relates to use of
  • A2A-G 0 if as a drug target.
  • the drug target may be the A2A receptor either at its ligand binding or G protein-binding site.
  • the drug target may be the Goi f protein.
  • the screening method can, for example, be a high throughput screening method.
  • the inventors will use a cell system to study adenosine A2A receptors coupled to either G s or Goif.
  • the eye- variant of S49 mouse lymphoma cells which is deficient in endogenous stimulatory G protein, provides an ideal experimental system in which to measure the efficacy of adenylyl cyclase activation by exogenous introduced G proteins.
  • a reporter gene might be used to measure receptor activation or inactivation.
  • RevTet retroviral expression
  • Cells are selected with G418 and hygromycin after virus infections and inducible clones isolated.
  • a j ⁇ east two-hybrid system could be used for screening of A2A- Goif selective antagonists or agonists.
  • a further alternative is GTP binding to purified Goif and G s proteins as indicator for screening of Goif selective agonists.
  • the invention in a second aspect, relates to antagonists or agonists for adenosine A2A receptor coupled to Goif obtained by any of the above screening-methods of the invention.
  • Antagonists are intended for the primary indication (Parkinson) and agonists for secondary indication (Schizophrenia) .
  • the invention in a third aspect, relates to a pharmaceutical preparation comprising an antagonist or agonist for adenosine A2A receptor coupled to Goi f .
  • the pharmaceutical preparation is formulated according to conventional practice as concerns additives etc.
  • the pharmaceutical preparation may be used for treatment of disorders linked to dopaminergic neurotransmission, such as Parkinson's disease and
  • a further alternative is to use the pharmaceutical preparation according to invention for treatment of CNS diseases involving dopaminergic or GABA transmittor systems.
  • Digoxgenin- 1 1 -UTP and FuGENETM 6 transfection reagent was purchased from Boehringer Mannheim, Germany.
  • [ 35 S]UTP, [ ⁇ 32 P]GTP and 4-morpholine- propanesulfonic acid (Mops) were purchased from Amersham, (Little Chalfont, England).
  • the supernatant was centrifuged for 50 min (30000 g) and washed once with 50 mM Tris-HCl (pH 7.4). After the second centrifugation the pellet, containing the membranes, was resuspended in an incubation buffer (20 mM MgCh, 400 mM NaCl, 120 mM HEPES and 0.4 mM EDTA) and stored at -80°C in aliquots until used.
  • an incubation buffer (20 mM MgCh, 400 mM NaCl, 120 mM HEPES and 0.4 mM EDTA
  • 35 S- or digoxigenin-labeled antisense and sense cRNA probes were prepared by in vitro transcription from cDNA clones corresponding to fragments of rat adenosine A2A receptor (Fink et al, 1992), rat G protein alpha subunit G s (Jones and Reed, 1987) and rat G protein alpha subunit Goif (Jones and Reed, 1989). The transcription was performed using MAXI-scriptTM in vitro transcription kit according to the manufacturer's protocol (Ambion Inc., Austin, TX, USA). Single in situ hybridization
  • the adenosine A2A receptor cRNA probe was labeled with [ 35 S]UTP, whereas the probes against G s and Goif mRNAs were labeled with digoxigenin-11-UTP.
  • Cryostat sections were pretreated as described above. The sections were hybridized overnight at 55° C with a combination of 35 S- and digoxigenin-labeled probes (10 6 c.p.m. of 35 S-labeled probe and approximately 20 ng of digoxigenin-labeled probe in 50 ⁇ l of hybridization solution). The slides were washed in RNAse A and various concentrations of SSC, but without DTT. At the end of the washes, the slides were put in O.
  • the sections were then incubated overnight in the dark at room temperature in 0.1 M buffer B (pH 9.5) containing 0.34 mg/ml nitroblue tetrazolium and 0.18 mg/ml bromochloro-indolylphosphate.
  • the sections were thereafter rinsed in 0.1 M buffer B (pH 9.5), then in 1 x SSC, dried and dipped into Ilford K5 emulsion. After being exposed for seven to eleven weeks the sections were developed and mounted without counterstaining.
  • radioactive m-AcAGTP The preparation of radioactive m-AcAGTP was performed as described by Zor et al. (1995). Briefly, [ ⁇ 32 P]GTP (3000 Ci/mmol) was freeze-dried and redissolved in 50 ⁇ l of 0.125 M MES buffer (pH 6.5). EDC (10 ⁇ mol) was then dissolved in this solution and m-aminoacetophenone (20 ⁇ mol) in 20 ⁇ l of 1,4-dioxane was added. After 5 h at room temperature the mixture was freeze dried and redissolved in 10 mM Mops buffer (pH 7.0). Insoluble m- aminoacetophenone was removed by centrifugation. The product was stored at -18°C until used.
  • the solubilized photolabeled membranes were diluted 1 : 1 with immunoprecipitation buffer (10 mM Tris-HCl (pH 7.4), 1% Triton X- 100, 1% sodium deoxycholate, 0.5% SDS, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 10 ⁇ g/ml aprotinin and 0.2 mM PMSF) and centrifuged at 12000 g for 10 min at 4°C.
  • immunoprecipitation buffer 10 mM Tris-HCl (pH 7.4), 1% Triton X- 100, 1% sodium deoxycholate, 0.5% SDS, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 10 ⁇ g/ml aprotinin and 0.2 mM PMSF
  • the pellet from this centrifugation was discarded and the supernatant was mixed with 3 ⁇ l of non-diluted subtype-specific G protein subunit Goif antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA.), which does not cross-react with the G protein ⁇ subunit G s , and was incubated for 1 h at 4° C under constant rotation. Then 60 ⁇ l of protein A-Sepharose (4 mg) in immunoprecipitation buffer was added to each sample and incubated overnight at 4°C under constant rotation.
  • Sepharose beads were pelleted (1 min 12000 g, 4°C) and washed twice with 1 ml of washing buffer A (50 mM Tris/HCl, 600 mM NaCl, 0.5% SDS and 1% Tergitol NP-40, pH 7.4) and twice with washing buffer B (100 mM Tris/HCl, 300 mM NaCl and 10 mM EDTA, pH 7.4).
  • washing buffer A 50 mM Tris/HCl, 600 mM NaCl, 0.5% SDS and 1% Tergitol NP-40, pH 7.4
  • washing buffer B 100 mM Tris/HCl, 300 mM NaCl and 10 mM EDTA, pH 7.4
  • the washed Protein A-Sepharose was then resuspended in 100 ⁇ l Laemmli buffer and heated at 80° C for 5 min and then centrifuged as above. Fifty microliters of the supernatant was then subjected to SDS/PAGE
  • the rat G alpha subunit Goif cDNA was amplified with polymerase chain reaction (PCR) using a plasmid (Bluescript, Stratagene) containing the rat Goif cDNA sequence (a gift from Dr. A.G. Gilman).
  • the PCR product was subcloned into the vector pCI-neo (Promega, Scandinavian Diagnostic Services, Falkenberg, Sweden).
  • the subcloned cDNA fragments were sequenced using an automated DNA sequencer, ABI 373A (Applied Biosystems Inc. Foster City, Ca, USA).
  • both Goif cDNA-transfected cells and control cells were sown out in 12-well plates (200,000 cells per well) and were allowed to grow for 36 h. Cells were then washed 2 times with HEPES- buffered (20 mM) ⁇ -MEM (pH 7.4). The cells were incubated at 37° for 10 min in 0.9 ml of HEPES buffered medium. The adenosine A2A agonist CGS 21680 was added in 0.1 ml of medium and the cells were incubated for another ten min. The reactions were terminated by the addition of perchloric acid to a final concentration of 0.4 M.
  • the acidified cell suspensions were transferred to tubes and neutralized with 4 M KOH/ 1 M Tris-HCl.
  • the cAMP content in the samples was determined using a competitive radioligand-binding assay (Nordstedt and Fredholm, 1990). Radioactivity was measured in an LKB/ Pharmacia scintillation counter with 3 ml of ReadySafe (LKB /Pharmacia) scintillation fluid.
  • adenosine A2A receptor mRNA was detected in e.g. the pyramidal cell layer of piriform cortex and in the Islands of Calleja (Fig. lb). These neurons in the Islands of Calleja are known to be interconnected with the olfactory tubercle (Fallon, 1983) and to express dopamine Di and D3 receptors (Le Moine and Bloch, 1996; Svenningsson et al, 1997). Gs mRNA was most abundant in areas not expressing adenosine A2A receptor mRNA, such as pyramidal cells of the piriform cortex, cerebral cortex, claustrum, endopiriform nucleus and the diagonal band of Broca.
  • G s mRNA was found throughout septum, whereas mRNA encoding A2A receptors were found only in occasional cells in lateral septum. Moderate levels of G s mRNA were detected in nucleus accumbens, whereas caudate putamen only exhibited very low expression (Fig. lc).
  • striatal neurons There are three major subcategorize of striatal neurons; medium-sized (app. 20 ⁇ m in diameter) neurons that are further subdivided into spiny and aspiny neurons and large-sized (app. 40 ⁇ m in diameter) neurons. 95 % of the striatal neurons are medium-sized spiny neurons and the remaining 5 % are medium-sized aspiny neurons and large-sized neurons. Since no staining for detecting spines was used in the present study, we could only distinguish between neurons based on their size.
  • adenosine A2A receptor mR In order to determine the co-expression of adenosine A2A receptor mR with that of Goif or G s in caudate putamen and nucleus accumbens we performed double in situ hybridization. Quantifications were made in the lateral and medial parts of striatum and in the core and shell regions of nucleus accumbens. As expected from the above-mentioned data, the large majority of the adenosine A2A receptor mRNA-containing neurons also expressed Goif mRNA in the lateral and medial parts of striatum as well as in the core and shell regions of nucleus accumbens (95, 91 , 91 and 88 %, respectively) (Fig. 2a, 3).
  • the crude photolabeled membranes gave a broad band with an apparent molecular mass of 40-50 kDa (Fig. 4a).
  • the Goif subunits were immunoprecipitated with a specific antibody directed against Goif, and separated on SDS-PAGE.
  • the incorporated [ ⁇ 32 P]m-AcAGTP label was determined by autoradiography (Fig. 4b).
  • the immunoprecipitate gave a single band with an apparent molecular mass of 44-45 kDa, a size identical to the molecular mass of the protein visualized in immunoblot experiments (data not shown).
  • Albright and McCune -Albright syndromes which are associated with mutations of the G s gene, do not exhibit any extrapyramidal neurological symptoms (Schwindinger et al, 1992; Weinstein et al, 1990).
  • Herve and co-workers (1993) have shown that selective lesioning of striatonigral neurons, using a retrograde neurotoxin, markedly decreases the levels of dopamine Di receptors and Goif in striatum.
  • 6-OHDA lesioning of the dopaminergic axons in neonatal rats induced hypersensitivity to dopamine receptor agonists in adulthood without any change in Di receptor binding — but there is an increase in Goif expression.
  • G s protein There is also an increase in G s protein, but mainly in glial cells (Penit-Soria et al, 1997). Furthermore, mice deficient in G 0 if are hyperactive (Belluscio et al, 1998). These results could indicate that G 0 if may, in striatum, play some of the role(s) otherwise attributed to G s .
  • Photoaffinity labeling is a method used for identification of distinct molecular components in biochemical processes.
  • the simplest procedure for labeling G proteins is by u.v. irradiation of [ ⁇ 32 P]GTP (Basu and Modak, 1987).
  • this method has low sensitivity.
  • the most widely used photoaffinity label for G proteins is azidoanilido-GTP and was developed by Pfeuffer (Pfeuffer, 1977).
  • this compound has some disadvantages: it is instable and its synthesis, purification and application must be performed in complete darkness.
  • the photoaffinity label we have used m-acetylanilido-GTP, developed by Zor and co-workers (1995), has several advantages: (1) No need for purification; (2) Quantitative conversion of GTP into m-AcAGTP; (3) Excellent stability in solution; (4) Resistant to hydrolysis and remains bound to the G protein during centrifugal washing.
  • This photolabeling method we could demonstrate that the adenosine A2 A agonist CGS 21680 induced an increased incorporation of [ ⁇ 32 P]m- AcAGTP in immunoprecipitated Goif subunits. The magnitude of the increase was GDP-concentration-dependent, with the largest increase seen at a high GDP concentration (100 ⁇ M).
  • CGS 21680 increased labeling in a dose-dependent manner with a calculated EC50 value of 16 nM (4.7-54). This increase was receptor mediated since it was blocked by the A2A receptor antagonist SCH 58261 (data not shown). The CGS 21680 potency is in agreement with the potency in the cAMP assay.
  • adenosine A2A receptor mRNA was co- expressed with Goif mRNA in striatal medium-sized neurons to a much higher extent than with G s mRNA.
  • Using a photolabeling technique we showed that activation of adenosine A2A receptors in striatal membranes led to activation of Goif.
  • transfection of Goif cDNA into cells that express human adenosine A2A receptors led to an increase of the maximal agonist stimulated cAMP level.
  • Jarvis MF and Williams M (1989) Direct autoradiographic localization of adenosine A2 receptors in the rat brain using the A2-selective agonist
  • Fisone G (1998) Activation of adenosine A2A and dopamine Di receptors stimulates cyclic AMP-dependent phosphorylation of DARPP-32 in distinct populations of striatal projection neurons. Neuroscience 84:223-228.
  • Histograms showing the numbers of neurons in the lateral and medial caudate-putamen and in the core and shell of nucleus accumbens that are labeled (+) or not labeled (-) with probes for adenosine A2A receptor mRNA, Goif mRNA and G s mRNA. All neurons within the examined areas were counted.
  • Agonist-stimulated photoaffinity labeling of Goif is influenced by GDP.
  • Membranes from striatum were photolabeled with [ ⁇ 32 P]m-AcAGTP at various GDP concentrations in the absence (-) or presence (+) of CGS 21680 (10 ⁇ M).
  • A autoradiogram showing solubilized labeled membranes directly subjected to SDS-PAGE or
  • B one representative autoradiogram out of three independent experiments showing immunoprecipitated membranes with the subtype-specific G protein ⁇ subunit Goif antibody subjected to SDS-PAGE.

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Abstract

La présente invention concerne des procédés de tri comportant l'utilisation d'un récepteur A2A couplé à la Golf pour sélectionner des agents coopérant sélectivement avec un récepteur A2A couplé à la Golf. L'utilisation d'un récepteur A2A couplé à la Golf comme cible de médicament permet de trouver un antagoniste ou un agoniste du récepteur A2A d'adénosine couplé à la Golf. Cet antagoniste ou cet agoniste peuvent être utilisés dans une préparation pharmaceutique pour traiter des troubles liés à la neurotransmission dopaminergique, telle que la maladie de Parkinson et la schizophrénie. Une telle préparation pharmaceutique pourrait également servir au traitement de maladies du système nerveux central, y compris des systèmes dopaminergiques ou transmetteurs de GABA .
PCT/SE2001/002323 2000-10-26 2001-10-24 Procede de tri a l'aide d'un recepteur couple a la g associe a une proteine g specifique WO2002035231A1 (fr)

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