EP1297139A2 - An ein g-protein gekoppelter rezeptor, org10 - Google Patents

An ein g-protein gekoppelter rezeptor, org10

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Publication number
EP1297139A2
EP1297139A2 EP01960347A EP01960347A EP1297139A2 EP 1297139 A2 EP1297139 A2 EP 1297139A2 EP 01960347 A EP01960347 A EP 01960347A EP 01960347 A EP01960347 A EP 01960347A EP 1297139 A2 EP1297139 A2 EP 1297139A2
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EP
European Patent Office
Prior art keywords
polynucleotide
org10
seq
fragments
sequence
Prior art date
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EP01960347A
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English (en)
French (fr)
Inventor
Morag A. Grassie
Emma Jane Grant
Johannes Petrus Gerardus Klomp
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Akzo Nobel NV
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Akzo Nobel NV
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Priority to EP01960347A priority Critical patent/EP1297139A2/de
Publication of EP1297139A2 publication Critical patent/EP1297139A2/de
Withdrawn legal-status Critical Current

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/08Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/14Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/14Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16Anti-Parkinson drugs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/18Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/20Hypnotics; Sedatives
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/22Anxiolytics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/24Antidepressants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/28Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/04Anorexiants; Antiobesity agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/08Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • A61P9/02Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • A61P9/04Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • A61P9/10Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • A61P9/12Antihypertensives
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention provides a full length cDNA sequence that codes for a G- protein coupled receptor, as well as the complete gene and the encoded protein.
  • the present invention provides a recombinant cell line expressing these receptors at appropriate levels such that novel compounds active at these receptors may be identified for therapeutic use.
  • the receptor sequence described in this invention is a member of a novel GPCR receptor sub-family which has no known endogenous ligand. This cDNA can be used to identify novel compounds active at the receptor for therapeutic use especially in the field of CNS disorders, neurodegeneration or analgesia.
  • GPCR G-protein coupled receptor
  • the G-protein coupled receptor (GPCR) superfamily is one of the largest protein families identified to date. This family comprises over 800 cloned members from a wide range of species, and includes at least 300 human members. GPCRs have a proven history as excellent therapeutic targets with between 40-50% of drug targets to date being GPCRs (Murphy et al., 1998). GPCRs are responsive to a wide variety of stimuli and chemical transmitters, including light, biogenic amines, amino acids, peptides, lipids, nucleosides, and large polypeptides. This results in the regulation of multiple processes including neurotransmission, cellular metabolism, secretion, cellular differentiation and growth as well as inflammatory and immune responses.
  • GPCRs are expressed in the brain and may be exploited as therapeutic targets for the treatment of CNS disorders. More significantly, many GPCRs with no known endogenous ligands are still being identified in the public and proprietary databases. These orphan GPCRs represent potential novel therapeutic targets for a range of therapeutic intervention and the treatment of a variety of disorders
  • Orphan GPCRs are predicted to bind ligands, as it is postulated that inactive receptors should have been evolutionary discarded. Orphan receptors may therefore be used as baits to isolate their natural ligands or surrogate ligands.
  • the use of this strategy in identifying novel ligands is exemplified in the identification of orphanin/nociceptin, orexins/hypocretins and prolactin-releasing peptide (Reinscheid et al., 1995, Sakurai et al., 1998 and Hinuma et al., 1998).
  • GPCRs G protein coupled receptors
  • Many known G protein coupled receptors (GPCRs) are well established drug targets with a significant number of currently available drugs targeting such GPCRs (Wilson et al., 1998).
  • the signal is amplified through a range of signal transduction cascades and consequently, regulation of this signal transduction pathway via a ligand binding to a GPCR offers the facility to modulate a tightly controlled biological pathway.
  • GPCRs mediate a wide range of biologically relevant processes and are responsive to a wide variety of stimuli and chemical/neurotransmitters, including light, biogenic amines, amino acids, peptides, lipids, nucleosides, and large polypeptides. How the cloning of a particular receptor has lead to the development of a therapeutic compound is particularly exemplified in the case of the serotonin/adrenergic receptors. Additionally a number of diseases are reported to be associated with mutations in known GPCRs (Wilson et al., 1998). The signalling pathways that mediate the actions of GPCRs have also been implicated in many biological processes significant to the pharmaceutical industry.
  • G proteins G proteins
  • second messengers such as cAMP or calcium (Lefkowitz, 1991)
  • effector proteins such as phospholipase C, adenylyl cyclase, RGS proteins, protein kinase A and protein kinase C (Simon et al., 1991 ).
  • a GPCR can be activated by a ligand, binding to the receptor resulting in the activation of a G protein which conveys the message onto the next component of the signal transduction pathway.
  • a component could be adenylyl cyclase.
  • the relevant G protein of which there is a family, must exchange GTP for GDP, which is bound when the G protein is in an inactive state.
  • the exchange of GDP for GTP can occur following the binding of ligand to the GPCR, however, some basal exchange of GDP for GTP can also occur depending on the receptor under investigation.
  • GTP bound at the G protein occurs by hydrolysis and is catalysed by the G protein itself. Following this hydrolysis the G protein is returned to its inactive state. Consequently, the G protein mediates the transfer of the signal from the activated receptor to the intracellular signalling pathway, but also introduces an additional level of control, by controlling the length of time which the receptor can activate the intracellular signalling pathway through the GTP bound G protein.
  • these receptors In general the topology of these receptors is such that they contain 7 transmembrane domains consisting of approximately 20-30 amino acids. Consequently, these receptors are frequently known as 7TM receptors. These 7TM domains can be defined by consensus amino acid sequences and by structural prediction algorithms. Within the putative transmembrane domains, hydrophobic helixes are formed which are connected via extracellular and intracellular loops. The N-terminal end of the polypeptide is on the exterior face of the membrane with the C-terminal on the interior face of the membrane.
  • GPCRs A number of additional features are frequently observed in GPCRs. These include glycosylation of the N-terminal tail. A conserved cysteine in each of the first two extracellular loops, which are modified such that disulphide bonds are formed, which is believed to result in a stabilised functional tertiary structure. Other modifications which occur on GPCRs include lipidation (eg palmityolation and farnesylation) and phosphorylation often in the C terminal tail. Most GPCRs also have sites for phosphorylation in the third intracellular loop, a region, which is believed to contribute to G protein interactions and signal transduction.
  • lipidation eg palmityolation and farnesylation
  • phosphorylation often in the C terminal tail.
  • Most GPCRs also have sites for phosphorylation in the third intracellular loop, a region, which is believed to contribute to G protein interactions and signal transduction.
  • cAPK cAMP dependent protein kinase
  • GRKs GPCR kinases
  • ⁇ - adrenoreceptor Phosphorylation of the third intracellular loop by specific receptor kinases such as cAMP dependent protein kinase (cAPK) or a class of GPCR kinases (GRKs) in several GPCRs such as ⁇ - adrenoreceptor also mediates in the desensitization of such a receptor. Consequently, specific mutations in particular regions of the GPCR can have functional significance.
  • GRKs are known to phosphorylate GPCRs on multiple sites with threonine and serine residues as targets. The phosphorylation not only inactives the receptor but also allows the receptor with an additional inhibitory protein known as ⁇ -arrestin. This interaction can also be used as an indication that the GPCR in question has been activated.
  • the ligand binding sites are believed to comprise hydrophilic pockets formed by some of the transmembrane domains.
  • the amino acid within the ⁇ -helical structure align themselves such that the hydrophilic surface of the amino acid is facing inwards towards the centre of the ligand binding pocket. This results in a postulate polar ligand binding site.
  • the third transmembrane domain of has been reported to be involved in ligand binding in several GPCRs. In particular the aspartate of TM3, serines of TM5, asparagine of TM6 and phenylalanine or tyrosines of TM6 and/or TM7 have been implicated in ligand binding.
  • GPCRs can also couple via G proteins to additional gene families such as ion channels, transporter and enzymes.
  • additional gene families such as ion channels, transporter and enzymes.
  • Many GPCRs are present in mammalian systems exhibiting a range of distribution patterns from very specific to very widespread. For this reason following the identification of a putative novel GPCR by bioinformatics, assigning a therapeutic application to the novel GPCR is not obvious due to this diverse function and distribution of previously reported GPCRs.
  • GPCRs that can function to alter disease status either correction, prevention or amelioration.
  • diseases are diverse and include but are not exclusive to depression, schizophrenia, anxiety, neurological disorders, obesity, insomnia, addiction, neurodegeneration, hypotension, hypertension, acute heart failure, atherothrombosis, atherosclerosis and osteoporosis.
  • the present invention provides a brain expressed gene/protein which we termed ORG10 and which was found to be preferentially expressed in the brain. Analysis of the protein provides evidence that it is a GPCR.
  • the gene may therefore be used in conventional expression systems in order to select compounds that specifically react with ORG10. These compounds may then be used to treat CNS disorders. .
  • the present invention relates to ORG10, in particular ORG10 polypeptides, ORG10 polynucleotides, recombinant materials and methods of their production. Additionally the invention relates to methods which for such polypeptides and polynucleotides can be used to identify compounds such as agonists, partial agonists, inverse agonists or antagonists active at the invention for treatment of disease, such as psychiatric diseases and neurodegeneration, in particular bipolar and unipolar disorders, schizophrenia and anxiety. Use of ligands active at the said invention may be used to correct diseases associated with an imbalance of ORG10 and associated pathways. In particular, this invention relates to a diagnostic assay for identifying modifications in ORG10 gene or expression associated with CNS diseases and especially preferred for bipolar depression, schizophrenia and affective disorders.
  • ORG10 The complete cDNA sequence of ORG10 shown in SEQ ID NO: 4 was translated which resulted in the amino acid sequence of SEQ ID NO: 3 which was then compared with known protein sequences. ORG10 was found to be related to a previously reported GPCR accession number g992582. The genomic sequence of ORG10 is provided in SEQ ID NO: 1.
  • ORG10 cDNA encompassing approximately 800bp of the open reading frame was used to probe a human Multiple Tissue Expression (MTE) array (Clontech) containing human mRNA samples from 76 different tissues.
  • MTE Multiple Tissue Expression
  • the experimental procedure employed is described below. Analysis of the MTE array revealed that ORG10 is highly expressed in the corpus callosum, caudate nucleus, putamen and spinal cord, with lower levels of expression exhibited in other brain regions such as the amygdala and hippocampus. As ORG10 is highly expressed in the basal ganglia and corpus callosum this pattern of distribution makes it likely that this orphan receptor is linked to disorders of the basal ganglia, e.g. movement disorders, neurodegeneration or other impairment of motor function.
  • MTE Multiple Tissue Expression
  • ORG10 in the corpus callosum, which has a large population of Schwann cells, may also implicate a role for ORG10 in neurodegenerative disorders. Therefore expressing this receptor in a recombinant cell line allows screening to identify chemical entities which bind to this receptor. These compounds may then be used to treat Parkinson's disease, Huntington's Chorea, Gilles de la Tourette's syndrome, dystonia, obsessive compulsive disorder and schizophrenia as well as other CNS and neurodegenerative diseases. Peripherally, ORG10 appears to be expressed at much lower levels in a variety of tissues including the heart and skeletal muscle.
  • the 5HT6 receptor has been proposed to be involved in antipsychotic drug action, with various psychotics and antidepressants having a high affinity for this receptor
  • genomic polynucleotides of the present invention may be obtained using standard cloning and screening techniques from a human genomic DNA library, however a full length cDNA product lacking the non-coding region of the invention as described in SEQ ID NO: 1 can only be obtained from a cDNA library such as a brain cDNA library.
  • Polynucleotides detailed in this invention could also be generated from genomic DNA or synthesized using well known and commercially available techniques.
  • sequences of the present invention can be used to derive primers and probes for use in DNA amplification reactions in order to perform diagnostic procedures or to identify further, neighbouring genes that also may contribute to the development of CNS disorders.
  • the sequences of the present invention may also be used to design oligonucleotide probes for more detailed tissue distribution information by in sit ⁇ hybridisation analysis.
  • sequences of the present invention may also be used to design oligonucleotide probes for use in antisense technology
  • the invention also comprises functional equivalents, which are characterised in that they are capable of hybridising to at least part of the ORG10 sequence shown in SEQ ID NO: 1 , preferably under high stringency conditions.
  • Two nucleic acid fragments are considered to have hybridisable sequences if they are capable to hybridising to one another under typical hybridisation and wash conditions, as described, for example in Maniatis, et al., pages 320-328, and 382- 389, or using reduced stringency wash conditions that allow at most about 25-30% basepair mismatches, for example: 2x SSC, 0.1% SDS, room temperature twice, 30 minutes each, then 2x SSC, 0.1% SDS 37 °C once, 30 minutes; then 2X SSC, room temperature twice ten minutes each.
  • homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches. These degrees of homology can be selected by using wash conditions of appropriate stringency for identification of clones from gene libraries or other sources of genetic material, as is well known in the art.
  • the invention also includes sequences coding for the same amino acid sequences as the sequences disclosed herein. Also portions of the coding sequences coding for individual domains of the expressed protein are part of the invention as well as allelic and species variations thereof. Sometimes, a gene expresses different isoforms in a certain tissue which includes splicing variants, that may result in an altered 5' or 3' mRNA or in the inclusion of an additional exon sequence. Alternatively, the messenger might have an exon less as compared to its counterpart. These sequences as well as the proteins encoded by these sequences all are expected to perform the same or similar functions and form also part of the invention.
  • sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases.
  • the specific sequence disclosed herein can be readily used to isolate further genes which in turn can easily be subjected to further sequence analyses thereby identifying sequencing errors.
  • the present invention provides for isolated polynucleotides encoding a novel gene, disrupted in schizophrenia.
  • the DNA according to the invention may be obtained from cDNA.
  • the coding sequence might be genomic DNA, or prepared using DNA synthesis techniques.
  • the polynucleotide may also be in the form of RNA.
  • the polynucleotide may be in single stranded or double stranded form.
  • the single strand might be the coding strand or the non-coding (anti-sense) strand.
  • the present invention further relates to polynucleotides which have at least 80%, preferably 90% and more preferably 95% and even more preferably at least 98% identity with SEQ ID NO:1.
  • polynucleotides encode polypeptides which retain the same biological function or activity as the natural, mature protein.
  • Identity is the term given in the art, to the degree of similarity or relationship between 2 sequences determined by direct comparison along the length of those 2 sequences.
  • algorithms and computer packages available for the automated calculation of percentage identity between pairs of DNA sequences. These algorithms and computer applications may also compare a single sequence with all the members of a sequence database to determine which sequence in that database has the highest identity.
  • DNAMAN Large Scalable Computer Application
  • DNA according to the invention will be very useful for in vivo or in vitro expression of the novel gene according to the invention in sufficient quantities and in substantially pure form.
  • polypeptides comprising the amino acid sequence encoded by the above described DNA molecules.
  • polypeptides according to the invention comprise at least part of the amino acid sequences as shown in SEQ ID NO: 3.
  • polypeptides according to the present invention include the polypeptides comprising SEQ ID NO:3 but also their isoforms, i.e. polypeptides with a similarity of 70%, preferably 90%, more preferably 95%. Also portions of such polypeptides still capable of conferring biological effects are included. Especially portions which still bind to ligands form part of the invention. Such portions may be functional per se, e.g. in solubilized form or they might be linked to other polypeptides, either by known biotechnological ways or by chemical synthesis, to obtain chimeric proteins. Such proteins might be useful as therapeutic agent in that they may substitute the gene product in individuals with aberrant expression of the ORG10 gene.
  • sequence of the gene may also be used in the preparation of vector molecules for the expression of the encoded protein in suitable host cells.
  • host cell and cloning vehicle combinations may be usefully employed in cloning the nucleic acid sequence coding for the ORG10 gene of the invention or parts thereof.
  • useful cloning vehicles may include chromosomal, non-chromosomal and synthetic DNA sequences such as various known bacterial plasmids and wider host range plasmids and vectors derived from combinations of plasmids and phage or virus DNA.
  • Vehicles for use in expression of the genes or a ligand-binding domain thereof of the present invention will further comprise control sequences operably linked to the nucleic acid sequence coding for a ligand-binding domain.
  • control sequences generally comprise a promoter sequence and sequences which regulate and/or enhance expression levels.
  • control and other sequences can vary depending on the host cell selected.
  • Suitable expression vectors are for example bacterial or yeast plasmids, wide host range plasmids and vectors derived from combinations of plasmid and phage or virus DNA. Vectors derived from chromosomal DNA are also included. Furthermore an origin of replication and/or a dominant selection marker can be present in the vector according to the invention.
  • the vectors according to the invention are suitable for transforming a host cell.
  • Recombinant expression vectors comprising the DNA of the invention as well as cells transformed with said DNA or said expression vector also form part of the present invention.
  • Suitable host cells according to the invention are bacterial host cells, yeast and other fungi, plant or animal host such as Chinese Hamster Ovary cells or monkey cells.
  • a host cell which comprises the DNA or expression vector according to the invention is also within the scope of the invention.
  • the engineered host cells can be cultured in conventional nutrient media which can be modified e.g. for appropriate selection, amplification or induction of transcription.
  • the culture conditions such as temperature, pH, nutrients etc. are well known to those ordinary skilled in the art.
  • the proteins according to the invention can be recovered and purified from recombinant cell cultures by common biochemical purification methods including ammonium sulfate precipitation, extraction, chromatography such as hydrophobic interaction chromatography, cation or anion exchange chromatography or affinity chromatography and high performance liquid chromatography. If necessary, also protein refolding steps can be included.
  • ORG10 gene products according to the present invention can be used for the in vivo or in vitro identification of novel ligands or analogues thereof.
  • binding studies can be performed with cells transformed with DNA according to the invention or an expression vector comprising DNA according to the invention, said cells expressing the ORG10 gene products according to the invention.
  • ORG10 gene products according to the invention as well as ligand-binding domains thereof can be used in an assay for the identification of functional ligands, endogenous ligands or analogues for the ORG10 gene products.
  • the present invention provides for a method for identifying ligands for ORG10 gene products, said method comprising the steps of: a) introducing into a suitable host cell a polynucleotide according to the invention, b) culturing cells under conditions to allow expression of the DNA sequence c) optionally isolating the expression product d) bringing the expression product (or the host cell from step b)) into contact with potential ligands which will possibly bind to the protein encoded by said DNA from step a); e) establishing whether a ligand has bound to the expressed protein. f) Optionally isolating and identifying the ligand
  • signal transduction capacity may be measured.
  • the present invention thus provides for a quick and economic method to screen for therapeutic agents for the prevention and/or treatment of diseases related to CNS disorders.
  • the method is especially suited to be used for the high throughput screening of numerous potential ligands.
  • Compounds which activate or inhibit the function of ORG10 gene products may be employed in therapeutic treatments to activate or inhibit the polypeptides of the present invention.
  • antibodies especially monoclonal antibodies raised against the polypeptide molecule according to the invention.
  • Such antibodies can be used therapeutically to inhibit ORG10 gene product function and diagnostically to detect ORG10 gene products.
  • the invention furthermore relates to the use of the ORG10 gene products as part of a diagnostic assay for detecting psychiatric abnormalities or susceptibility to psychiatric disorders related to mutations in the nucleic acid sequences encoding the ORG10 gene.
  • Such mutations may e.g. be detected by using PCR (Saiki et al., 1986).
  • the relative levels of RNA can be determined using e.g. hybridization or quantitative PCR technology.
  • the presence and the levels of the ORG10 gene products themselves can be assayed by immunological technologies such as radioimmuno assays, Western blots and ELISA using specific antibodies raised against the gene products. Such techniques for measuring RNA and protein levels are well known to the skilled artisan.
  • the determination of expression levels of the ORG10 gene products in individual patients may lead to fine tuning of treatment protocols.
  • transgenic animals may be prepared in which the expression of the ORG10 gene is altered or abolished.
  • Full and partial cDNA encoding ORG10 were amplified by PCR using proof reading Expand polymerase (Roche) and AmplitaqGold Taq DNA polymerase (Perkin Elmer) respectively, and oligonucleotide primers based upon the sequence of ORG10.
  • the template DNA used for the PCR reactions was Marathon-ready human whole brain cDNA (Clontech).
  • the cycling conditions used in all reactions were identical, differing only in annealing temperature, and were as follows: Following an initial denaturation step at 94°C for 5 minutes, the reaction was allowed to cycle 33 times through a sequence of temperatures: 1) Denaturation of template 94°C for 1 minute, 2) primer annealing at 55°C, for 1 minute 30 seconds, 3) DNA polymerisation at 72°C for 2 minutes. A final elongation step at 72°C for 10 minutes was also performed to ensure generation of full-length products.
  • the full length ORG10 cDNA generated in the PCR reaction described above was ligated into the prokaryotic expression vector pCR 2.1 (Invitrogen). Following chemical transformation and mini-prep DNA isolation, restriction digestion was performed using the restriction endonuclease EcoR I to excise the ORG10 cDNA from the vector. This restriction enzyme was used as the vector pCR 2.1 contains two EcoR I sites on either side of the multiple cloning site, which enable the sub- cloned insert to be excised effectively
  • ORG10 clones were selected for sequence analysis.
  • the selected ORG10 clones were completely sequenced in both directions using the Big Dye terminator cycle sequencing kit and ABI310 Genetic Analyser (PE Biosystems) and following the manufacturers instructions.
  • the primers used to sequence the selected clones were M13 forward and reverse and internal forward and reverse ORG10 specific primers.
  • the relative abundance of ORG10 in a broad range of human tissues was analysed using a Multiple Tissue Expression (MTE) array purchased from Clontech.
  • MTE Multiple Tissue Expression
  • the ORG10 cDNA was amplified by PCR using the ORG10 5' primer and internal reverse primer which produced a 800bp probe corresponding to the 5' most end of the cDNA.
  • the PCR product was purified on QIAquick columns (Qiagen) and the DNA concentration was estimated by agarose gel electrophoresis.
  • the cDNA (100 ng) was radiolabelled using the High Prime DNA labelling method (Boehringer Mannheim), and the probe was subsequently purified away from unincorporated nucleotides using ProbeQuant G-50 micro columns (Amersham Pharmacia Biotech).
  • a prehybridisation mixture was prepared containing 10 ml of prewarmed ExpressHyb solution (Clontech) and 1 mg of sheared salmon sperm DNA (Sigma).
  • the salmon sperm DNA was heat-denatured at 100°C for 10 min and chilled quickly on ice prior to its addition to the ExpressHyb solution.
  • the MTE array was placed in a Hybaid glass tube (25cm) and the ExpressHyb mixture was added immediately.
  • Prehybridisation was performed for 2 hours at 65°C with continuous rotation.
  • the purified labelled ORG10 cDNA probe was heat-denatured at 100°C for 5 min and then combined with 1 ml of pre-hybridisation mixture, before adding this back to the remaining hybridisation solution in the glass tube.
  • Hybridisation was performed overnight at 65°C with continuous rotation.
  • the MTE array was subjected to a series of washing steps as follows; four 20 min washes at 65°C in 2 x SSC plus 1 % SDS, two 20 min washes at 55°C in 0.1 x SSC plus 0.5 % SDS, and a final 30 min wash at 65°C in 0.1 x SSC plus 0.5 % SDS. All washing steps were performed with continuous agitation in a large plastic containers with a lid.
  • the MTE was wrapped in Saran wrap and exposed to X-ray film with an intensifying screen at -70°C overnight.
  • the receptors of the present invention are expressed in e.g human embryonic kidney 293 (HEK293) cells or adherent CHO cells.
  • HEK293 human embryonic kidney 293
  • pCDNA3 an appropriate eukaryotic expression vector
  • the cells are transfected with individual receptor cDNAs by lipofectin and selected in the presence of selected antibiotic (e.g hygromycin). After 3 weeks of selection, individual clones are picked and expanded for further analysis.
  • HEK293 or CHO cells transfected with the vector alone serve as negative controls.
  • To isolate cell lines stably expressing the individual receptors about 24 clones are typically selected and analysed by Northern blot analysis. Receptor mRNAs are generally detectable in about 50% of the antibiotic resistant clones analysed.
  • Microphvsiomietric assays Activation of a wide variety of second messenger systems results in extrusion of small amounts of acid from a cell.
  • the acid formed is largely as a result of the increased metabolic activity respired to fuel the intracellular signalling process.
  • the pH changes in the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd, Menlo Park, CA).
  • the CYTOSENSOR is thus capable of detecting the activation of a receptor which is coupled to an energy utilising intracellular signalling pathway such as the G-protein coupled receptor of the present invention.
  • Extract/Cell Supernatant screening A large number of mammalian receptors exist for which there remains, as yet, no cognate activating ligand (agonist). Thus, active ligands for these receptors may not be included within the ligand banks as identified to date. Accordingly, the 7TM receptor of the invention is also functionally screened (using calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) against tissue extracts to identify natural ligands. Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated and identified.
  • HEK293 or CHO cells Calcium and cAMP functional assays 7TM receptors which are expressed in e.g HEK293 or CHO cells have been shown to be coupled functionally to activation of PLC and calcium mobilisation and /or cAMP stimulation or inhibition. Basal calcium levels in the HEK293 or CHO cells in receptor-transfected or vector control cells were observed to be in the normal, 100nM to 200nM range.
  • HEK293 or CHO cells expressing recombinant receptors are loaded with fura 2 and in a single day >150 selected ligands or tissue/cell extracts are evaluated for agonist induced calcium mobilisation.
  • HEK293 or CHO cells expressing recombinant receptors are evaluated for the stimulation or inhibition of cAMP production using cAMP quantitation assays.
  • Agonists presenting a calcium transient or cAMP fluctuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor.
  • Additional detection methods for functional receptor activation by cAMP stimulation/inhibition and/or calcium mobilisation may include readouts from reporter gene constructs e.g luciferase luminescence and secreted alkaline phosphatase (SEAP).
  • SEAP alkaline phosphatase
  • Each PCR contained 1X PCR buffer, 1.5mM Magnesium chloride, 200 ⁇ M dNTP mix, 1 ⁇ M each primer, 10% DMSO, 2.5 units Amplitaq Gold Taq DNA polymerase (Perkin Elmer)and 5 ⁇ l human marathon ready cDNA (Clontech) in a total volume of 50 ⁇ l.
  • the human cDNAs investigated for expression of ORG10 were: heart, kidney, skeletal muscle, spleen, ovary, lung, liver, thymus, testis, small intestine and brain (Clontech). A positive control reaction with human genomic DNA (Promega) was also set up.
  • PCR amplification of the housekeeping gene G3PDH was performed as described above using sequence- specific primers purchased from Clontech, and this was used as a positive control for each cDNA template. Reactions were cycled in a MJ Research PTC-200 Thermal Cycler using the following conditions: 94°C, 5min and 33 cycles of 94°C for 1 min, 55°C for 1 min 30sec, 72°C for 2 min, followed by an extension of 72°C for 10min. PCR products were separated on 1% agarose gels containing ethidium bromide (10mg/ml) and visualised under UV light. Following 33 cycles of amplification an intense band of 1104bp corresponding to the full length ORG10 cDNA was observed in brain, with very faint bands present in heart, skeletal muscle, lung, liver and testis. ( Figure 3).
  • the CNS distribution profile of ORG10 was investigated in coronal and transverse sections of rat brain utilising 33 P-labelled oligonucleotide probes targeted against ORG10.
  • 20 m coronal sections were cryostat-cut onto superfrost slides (BDH) throughout the rostrocaudal extent of the rat brains. For coronal sections, a series of slides (with 3 sections on each slide) were collected from 4.7 mm through to -14.08 mm from Bregma.
  • a synthetic oligonucleotide probe was designed from the human ORG10 sequence to also be complementary to the rodent orthologue of ORG10.
  • the specificity of this oligonucleotide probe was assessed using the National Centre for Biotechnology Information (NCBI) database utilising a Basic Local Alignment Search Tool (BLAST, Altschul et al., 1990) in order to verify that the selected sequence was non- complementary to any other known sequences.
  • NCBI National Centre for Biotechnology Information
  • BLAST Altschul et al., 1990
  • the 45-mer oligonucleotide selected (sequence 5'- TGG CCG GAG GGG CGG CAA GAA GGA GAG GCG GCT GTC CAG AGA GTC -3') was 90% homologous to the human ORG10 sequence and demonstrated homology to bases 695-652 of SEQ ID NO: 4.
  • 3'-end labelling of the oligonucleotide probe was carried out using 33 P-dATP.
  • the oligonucleotide probe at a concentration of 4 pmol, was radio-labelled by the isotope at an incubation temperature of 37°C in a mixture containing, 8 ⁇ l terminal transferase reaction buffer, 2 ⁇ l terminal deoxynucleotide transferase pH7.2 (all reagents Promega) with 3.2 ⁇ l of 33 P-dATP (3000 Ci/mmol, NEN) and made up to a final volume of 40 ⁇ l with diethylpyrocarbonate (DEPC)-treated H 2 0. Following 1 hour incubation, the reaction was stopped by heating the reaction mixture to 70°C.
  • DEPC diethylpyrocarbonate
  • the labelled probe was subsequently purified using Micro Bio-Spin chromatography columns (P-30 Tris RNase-free, Bio-Rad) centrifuged at 4000 r.p.m. for 4 minutes (microcentrifuge 5415C, Eppendorf). 1 ⁇ l of the probe was removed and counted by a liquid scintillation counter (Tricarb, 1500 Packard) beta-counter to assess the efficiency of radioactive labelling. Only oligonucleotide probes with counts in the range 100,000-300,000cpm/ ⁇ l were utilised for further hybridisation studies.
  • Sections were removed from the -80°C freezer and thawed at room temperature for 30 minutes. Sections were then fixed in a 4% paraformaldehyde solution for 10 minutes, rinsed in DEPC treated water and incubated in 0.25% solution of acetic anhydride in 0.1 M triethalolamine and 0.9% saline (pH 8.0) for a further 10 minutes. Sections were then dehydrated in a series of ascending concentrations of ethanol (1 min in 70%, 1 min in 80%, 2 min in 90%, and 1 min in 100%), defated for 5 minutes in 100% chloroform, rehydrated for 1 min in 100% and 1 min in 95% ethanol and finally air dried at room temperature.
  • Hybrislips were floated off using 2 x SSC and the slides were returned to racks for further processing. Stringent washes were carried out for 30 minutes at room temperature in 1 x SSC, 30 minutes at 55°C in 1 x SSC, and 10 minutes at 55°C in 0.1 x SSC. Sections were then dehydrated for 2 minutes in 70% and 95% ethanol and allowed to air dry. Once dry, the sections were placed in a cassette and exposed to X-ray film (BioMax MR-1 , Kodak) for 21 days at RT. An autoradiographic 14 C micro-scale (Amersham) of known radioactivity (range 31-833 nCi/g) was also placed in each cassette. The film was then developed in using a Kodak automatic developer (M-35M X-OMAT Processor).
  • M-35M X-OMAT Processor Kodak automatic developer
  • RNAse pre- treatment of the sections was further assessed by RNAse pre- treatment of the sections or by the addition of 100-fold excess non-labelled probe.
  • brain sections were incubated in RNAse A solution (ribonuclease A (Sigma) 10 ⁇ g/ml) at 37°C for 30 minutes prior to hybridisation.
  • RNAse A solution ribonuclease A (Sigma) 10 ⁇ g/ml
  • the sections were then processed for in situ hybridisation as previously described.
  • 50 pmol of non-labelled ORG10 oligonucleotide probe was added to the appropriate hybridisation buffer prior to hybridisation.
  • In situ hybridisation was then performed as previously described. No specific in situ hybridisation signal was observed following pre-treatment of the sections with RNAse A or following competition by addition of 100-fold excess non-labelled oligonucleotide probe.
  • Densitometric analysis of autoradiographs was performed using a Microcomputer Imaging Device (MCID) system. Optical density measurements were obtained from coronal levels throughout the rostrocaudal extent of each rat brain. High levels of expression of ORG10 signal were restricted predominantly to white matter structures such as the forceps minor and major corpus callosum, anterior commussure, intrabulbar, lateral olfactory tubercle, genu corpus callosum, anterior commisure, corpus callosum, white matter tracts throughout the striatum, fimbria hippocampus, internal capsule, fornix, stria medullaris thalamus, deep cerebral white matter, mammillothalamic tract, optic tract, superior thalamic radiation and white matter tracts of the cerebeilar cortex ( Figure 4, 5). Legends to the figures
  • Figure 1 Kyte-Doolittle hydrophobicity plot of predicted protein of ORG 10, showing the 7 transmembrane domains, the first 5 of which are separated from the last 2 by the long intracellular loop.
  • the red lines indicate the areas covered by Incyte templates 077146.1 (covering 5'UTR and first few amino acids) and 446715.1 (covering TMs 4 to 7).
  • ORF upstream of predicted methionine start site is notionally translated.
  • the bright green arrow indicates the predicted actual translational start site.
  • Detailed CNS ISH demonstrates expression of ORG10 in corpus callosum and areas of white matter indicating a potential role for ORG10 in neuronal support cells such as glia.
  • ORG10 neuronal support cells
  • Glial cells have been shown to actively modulate neuronal synaptic transmission (Smit et al., 2001) and enhance CNS synaptic formation and function (Temburni et al., 2001), suggesting a role for glia in the regulation of essential brain functions.
  • Expression of ORG10 in macroglia may suggest that this receptor may have a role in the additional functions of these cells.
  • Figure 2 Diagrammatic representation of phylogenetic relationship between ORG10 and its relatives
  • ORG10 is not closely related to any other known orphan GPCRs.
  • branch lengths in this diagram are not to scale, and are not indicative of any quantitative measure of evolutionary distance
  • FIG. 3 The tissue cDNAs investigated using the ORG10 specific primers which yield a 1104bp PCR product are labelled 1-11. The corresponding 900bp G3PDH postive control reaction for each cDNA sample is denoted by G.
  • FIG. 4 Autoradiographic images of in situ hybridisation demonstrating the distribution of ORG10 mRNA in a coronal section of rat brain at 0.2mm from Bregma according to the atlas of Paxinos and Watson (1982).
  • ORG 10 signal is localised predominantly to white matter structures such as the corpus callosum (cc), anterior commissure (aca), lateral olfactory tubercle (lo) and white matter fibre tracts throughout the striatum (str).
  • Kitada, C Masuo, Y., Asano, T., Matsumoto, H., Sekiguchi, A., Kurokawa, T., Nishimura, O., Onda, M., Fujino, M. (1998;. Nature 393:272-276.

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