WO2006058889A1 - Recepteur couple a la proteine g - Google Patents

Recepteur couple a la proteine g Download PDF

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Publication number
WO2006058889A1
WO2006058889A1 PCT/EP2005/056350 EP2005056350W WO2006058889A1 WO 2006058889 A1 WO2006058889 A1 WO 2006058889A1 EP 2005056350 W EP2005056350 W EP 2005056350W WO 2006058889 A1 WO2006058889 A1 WO 2006058889A1
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Prior art keywords
gpr39
seq
receptor
compound
protein
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PCT/EP2005/056350
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English (en)
Inventor
Diederik Willem Elisabeth Moechars
Benoit Christian Jean-Claude Moreaux
Theophiel Louis Henri Peeters
Inge Irma Thérèse DEPOORTERE
Bernard Coulie
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Janssen Pharmaceutica N.V.
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Priority to EP05817427A priority Critical patent/EP1820026A1/fr
Priority to CA002587534A priority patent/CA2587534A1/fr
Priority to AU2005311321A priority patent/AU2005311321A1/en
Priority to JP2007543845A priority patent/JP2008521419A/ja
Publication of WO2006058889A1 publication Critical patent/WO2006058889A1/fr
Priority to NO20073294A priority patent/NO20073294L/no

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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the present invention relates to the functional characterization of the G protein coupled receptor GPR39 and to compounds, which modify or regulate GPR39 protein activity.
  • the present invention relates to methods of screening for agonists or antagonists of GPR39 in order to identify Gastrointestinal Kinetics regulating agonists or antagonists and to the therapeutic uses of these compounds.
  • the present invention relates to the involvement of GPR39 in metabolism homeostasis, in particular on the cholesterol levels observed in the GPR39 knockout mice.
  • the invention also relates to transgenic animals bearing mutations in the GPR39 gene.
  • GTP-binding proteins act as intermediaries between binding of ligands such as hormones and other chemical mediators to G protein coupled receptors (GPCRs) and activation of intracellular effectors.
  • GPCRs G protein coupled receptors
  • ligands such as hormones and other chemical mediators to G protein coupled receptors (GPCRs)
  • GPCRs G protein coupled receptors
  • intracellular intermediaries such as adenylate cyclase, phospholipase C or ion channels.
  • G protein coupled receptors form a superfamily of integral plasma membrane proteins, each receptor sharing the common feature of seven hydrophobic transmembrane domains, each of which is 20 -30 amino acids long and which are linked by hydrophilic amino acid sequences of varied length.
  • the amino terminus of the receptor is extracellular with the carboxy terminus found in the cytoplasm of the cell .
  • GPCRs are found in a wide range of tissues and cell types and are involved in many different physiological processes. They are activated by a broad range of ligands, for example, hormones such as luteinizing hormone, follicle stimulating hormone, chorionic gonadotrophin, thyrotropin, adrenocorticotrophin, glucagon and vasopressin; neurotransmitters such as 5-HT, acetylcholine (muscarinic AchR) , histamine, prostaglandins, calcitonin, leukotrienes and Ca 2+ .
  • the broad distribution and wide variety of roles of GPCRs indicate that GPCRs may play important roles in a variety of pathological conditions. Indeed, GPCRs have been found to be involved in diseases related to bronchoconstriction, hypertension, inflammation, hormonal disturbance, diabetes, apoptosis, nociception, facilitation of neurotransmission and tremor disorders.
  • G protein-coupled receptor Within the G-protein-coupled receptor superfamily, the putative GPCRs for which the natural ligands are unknown are called "orphan receptors". G protein-coupled receptor have been proven to be valuable drug targets, since they are the target of about 50% of the marketed drugs.
  • GPR39 was identified based upon is sequence similarity with the growth hormone secretagogue receptor (GHS-R) and the neurotensin receptors 1 and 2 (NT-Rl and NT-R2) (McKee et al . , 1997) .
  • the predicted 453-amino acid GRP39 protein contains the 7 transmembrane domains characteristic of GPCRs. By sequence comparison with other GPCRs, McKee et al. (1997) found that the protein sequence of GPR39 is
  • GPR39 has a wide tissue distribution. A single hybridizing mRNA transcript of 1.8-2 kb, was detected in most brain regions tested. However, in addition to this species, an alternate transcript, 3-kb in length, was observed in several peripheral tissues such as stomach and small intestine, and in tissues such as pancreas, thyroid and colon, this 3-kb species was the only transcript detected (McKee et al., 1997) . By fluorescence in situ hybridization, McKee et al. (1997) mapped the GPR39 gene to 2q21-q22. An acidic residue in TM3, essential for the binding and activation of the GHS-R by structurally dissimilar GHSs, is conserved in GPR39.
  • GPR39 has been hypothesized to be involved in cardiovascular disease states (WO2001/081634 & WO2004/004279) , cancers and in particular brain cancers such as glioblastoma (WO2001/036685 & WO01042288), inflammation and neurological disease states (US 2003/ 232769 & WO2004/004279) and in gastrointestinal and liver diseases (WO2004/004279) . Nonetheless, in none of the cited references a functional characterization based on ligand identification for GPR39 has been provided. For said reasons, the present functional annotation of GPR39 is speculative and requires further studies to identify the ligand-binding and functional properties of GPR39. -A-
  • the present invention concerns identification of novel functions of the GPR39 receptor.
  • GPR39 mutations in mammals affect gastric emptying and designate GPR39 as a key element in the regulation of gastrointestinal kinetics.
  • down regulation of the GPR39 gene leads to an increase in gastric emptying it is to be expected that an increase in GPR39 expression will attenuate gastric emptying.
  • This discovery provides an avenue for new therapeutic approaches in the regulation of gastrointestinal kinetics through modulation of G.PR39 activity.
  • This discovery also provides new model systems for studying gastrointestinal kinetics and diseases involving gastrointestinal kinetics disorders, it also provides for new screening methods for identifying compounds useful for the prevention or the treatment of these diseases.
  • GPR39 knockout mice further revealed an involvement of this receptor on cholesterol metabolism.
  • a down regulation of the GPR39 gene leads to increased cholesterol levels. It is accordingly to be expected that GPR39 will be involved in disease conditions with excess cholesterol levels such as the metabolic syndrome, including obesity, diabetes, obesity related cardiovascular diseases, and glaucoma.
  • a first aspect of the invention provides for the use of all or part of GPR39 protein in a method for identifying compounds that modulate gastrointestinal kinetics or which are effective for preventing and/or treating pathologies related with gastrointestinal kinetics disorders.
  • the present invention provides the use of all or part of the GPR39 protein in a method to identify compounds that modulate cholesterol formation or which are effective for preventing and/or treating pathologies related with excess cholesterol formation.
  • the invention provides for the use of cells expressing all or part of the GPR39 protein in such method.
  • GPR39 is an isolated protein having an amino acid sequence selected from the group consisting of SEQ ID No:2, SEQ ID NO:4, a splice variant of the proteins having the aforementioned SEQ ID'S, and an amino acid sequence having at least 50% and preferably at least 60%, 70%, 80%, 90%, 95% or 98% sequence identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
  • Parts of the GPR39 protein are meant to include fragments of the polypeptide of SEQ ID NO:2 or SEQ ID NO:4, said fragments being of at least 10, for example at least 20, 30 40, 50, 75, 100 or 150 or more amino acids in size.
  • Such fragments may be derived from the N-terminal region of SEQ ID NO:2 or SEQ ID NO:4 respectively. Fragments including the N-terminal region may be used to reconstitute the extracellular portion of the receptor to provide receptor binding sites. Preferably, fragments will retain the ability to bind adenine.
  • the present invention also provides the use of an isolated nucleic acid sequence encoding all or part of the polypeptide of SEQ ID NO:2 or SEQ ID NO:4 in a method for identifying compounds that modulate gastrointestinal kinetics or which are effective for preventing and/or treating pathologies related with gastrointestinal kinetics disorders and disease states related to high levels of cholesterol such as the metabolic syndrome, including diabetes and cardiovascular diseases.
  • nucleic acid sequences as used in the methods of the present invention are meant to include the isolated nucleic acid sequences consisting of SEQ ID N0:l or SEQ ID NO:3, and nucleic acid sequences having at least 50% and preferably at least 60%, 70%, 80%, 90%, 95% or 98% sequence identity to the nucleic acid sequence of SEQ ID N0:l or SEQ ID NO:3.
  • Nucleic acids of the invention further include nucleic acids which comprise a sequence having at least 50% and preferably at least 60%, 70%, 80%, 90%, 95% or 98% sequence identity to the nucleic acid sequences of SEQ ID . NO: 1, or SEQ ID NO: 3 or their complements.
  • these sequences will hybridise to the corresponding nucleic acid under conditions controlled to minimise non- specific binding.
  • stringent to moderately stringent hybridisation conditions are preferred. Suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42 °C in 0.25M Na 2 HPO 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55 0 C in 0.
  • nucleic acids do not necessarily encode "full length" polypeptides, and will thus include nucleic acids which represent, for example, mutant forms of the GPR39 gene in which the coding sequence has been prematurely terminated by either a substitution resulting in a stop codon or a frameshift mutation. These are also nucleic acids of the invention.
  • the invention also provides the use of nucleic acids that are fragments of the nucleic acids encoding a polypeptide of the invention.
  • the invention provides nucleic acids primers which consist essentially of from 15 to 50, for example from 15 to 35, 18 to 35, 15 to 24, 18 to 30, 18 to 21 or 21 to 24 nucleotides of a sequence encoding a polypeptide of the invention or its complement.
  • Nucleic acids and polypeptides of the invention may be used therapeutically to treat disease.
  • they may be used to treat diseases, the pathology of which is associated with action at GPR39 receptors, particularly those associated with for preventing and/or treating pathologies related with gastrointestinal kinetics disorders and disease states related to increased cholesterol levels, such as the metabolic syndrome.
  • vectors comprising the sequences of said nucleic acids, particularly expression vectors comprising a promoter operably linked to the nucleic acid sequences of the invention.
  • the vectors may be carried by a host cell, and expressed within said cell. Following said expression, said cells can be used in the methods according to the invention.
  • compounds hereinafter also referred to as agents
  • such compounds are an organic or inorganic assembly of atoms of any size, and includes small molecules (less than about 2500 Daltons) or larger molecules such as peptides, polypeptides, whole proteins and polynucleotides, wherein said compounds may be used in methods of treatment as described above.
  • the present invention opens up the possibility of using GPR39 itself and/or compounds agonising or antagonising this receptor in therapeutic applications. Therefore the invention further extends to a method of treatment of a human or animal body, said method comprising the use of a GPR39 agonist or antagonist.
  • a method of treating disease conditions related with delayed gastric emptying such as for example, gastroparesis post-operative ileus, gastroparesis in diabetic, functional dyspepsia, post-vagotomy gastroparesis, slow transition constipation, constipation- IBS, mixed-IBS and idiopathic intestinal pseudo- obstruction.
  • Said method comprising administering to the human or animal a therapeutically active dosage of a GPR39 receptor antagonist, in particular comprising the use of a GPR39 antagonist identifiable using a method of the present invention. It is also an object to provide a method of treating disease conditions related with increased gastric emptying such as for example, dumping syndrome or increased intestinal motility such as diarrhoea, diarrhoea-IBS and mixed IBS, said method comprising administering to the human or animal a therapeutically active dosage of a GPR39 receptor agonist, in particular comprising the use of a GPR39 agonist identifiable using a method of the present invention.
  • the present invention opens up the possibility of using GPR39 itself and/or compounds agonising or antagonising this receptor in therapeutic applications with a defective cholesterol homeostasis. Therefore the invention also extends to a method of treatment of a human or animal body, said method comprising the use of a GPR39 agonists or antagonist in treating disease conditions related with a defective cholesterol homeostasis.
  • a GPR39 agonist in the treatment of disease conditions related with an excess Cortisol formation such as the metabolic syndrome, including obesity, diabetes, and cardiovascular diseases such as atherosclerosis related to high levels of cholesterol.
  • SEQ ID N0:l is the nucleotide sequence for mice GPR39.
  • SEQ ID NO:2 is the amino acid sequence for mice GPR39.
  • SEQ ID NO:3 is the nucleotide sequence for human GPR39.
  • SEQ ID NO:4 is the amino acid sequence for human GPR39.
  • SEQ ID NO: 5 is the GPR39 forward primer
  • SEQ ID NO: 6 is the GPR39 reverse primer
  • SEQ ID NO:7 is a GPR39 probe sequence
  • SEQ ID No: 8 is human obestatin
  • SEQ ID No: 9 is monkey obestatin
  • SEQ ID No: 10 is mouse obestatin
  • SEQ ID No: 11 is rat obestatin
  • SEQ ID No: 12 is gerbil obestatin
  • SEQ ID No: 13 is pig obestatin
  • SEQ ID No: 14 is cat obestatin
  • SEQ ID No: 15 is dog obestatin
  • SEQ ID No:16 is goat obestatin
  • SEQ ID No:17 is sheep obestatin
  • SEQ ID No:18 is Cattle obestatin
  • SEQ ID No: 19 is the consensus sequence for obestatin
  • Figure IA Real-Time Quantitative Reverse Transcription PCR of GPR39 in tissues derived from wild type mice.
  • Figure IB Real-Time Quantitative Reverse Transcription PCR of GPR39 in four different tissues derived from wild type mice and heterozygote and homozygote GPR39 knock-out mice.
  • FIG. 3 Comparison of the faecal pellets propulsion in wild type mice (A) vis-a-vis GPCR39 knock-out mice (B) .
  • the distribution of pellets is counted in bins of 15% of total colon length and the number of pellets expelled from each bin is counted during a 20 min time interval.
  • Figure 5 Amino acid sequence of preproghrelin from 11 mammalian species are shown with the signal peptide (italicized) , mature ghrelin (shaded) , and the flanking obestatin (underlined) . Consensus basic residues representing putative convertase cleavage sites are shown as white letters on a black background. In the consensus sequence, individual residues with complete conservation are shown in upper case.
  • GenBank (gi) numbers for individual ghrelin genes are 37183224 (human) , 34541890 (monkey), 19224664 (mouse), 11067387 (rat), 27357900 (gerbil), 47523230 (pig), 52782813 (cat), 50978704 (dog), 52782814 (goat), 57526202 (sheep), and 27806613 (cattle) .
  • Nucleic acid as used in the methods of the present invention includes DNA (including both genomic and cDNA) and RNA. Where nucleic acid according to the invention includes RNA, reference to the sequences shown in the accompanying listings should be construed as reference to the RNA equivalent, with U substituted for T.
  • Nucleic acid of the invention may be single or double stranded.
  • Single stranded nucleic acids of the invention include anti-sense nucleic acids.
  • reference to SEQ ID N0:l or sequences comprising SEQ ID N0:l or fragments thereof include complementary sequences unless the context is clearly to the contrary. The same applies to SEQ ID NO:3.
  • nucleic acid according to the present invention is provided as an isolate, in isolated and/or purified form, or free or substantially free of material with which it is naturally associated, such as free or substantially free of nucleic acid flanking the gene in the human genome, except possibly one or more regulatory sequence (s) for expression.
  • Nucleic acid may be wholly or partially synthetic and may include genomic DNA, cDNA or RNA.
  • the invention also provides nucleic acids that are fragments of the nucleic acids encoding a polypeptide of the invention.
  • the invention provides nucleic acids primers which consist essentially of from 15 to 50, for example from 15 to 35, 18 to 35, 15 to 24, 18 to 30, 18 to 21 or 21 to 24 nucleotides of a sequence encoding a polypeptide of the invention or its complement.
  • Aconsist essentially of ⁇ refers to nucleic acids which do not include any additional 5 ' or 3 ' nucleic acid sequences.
  • nucleic acids of the invention which consist essentially of from 15 to 30 nucleotides as defined above may however be linked at the 3' but preferably 5' end to short (e.g from 4 to 15, such as from 4 to 10 nucleotides) additional sequences to which they are not naturally linked.
  • additional sequences are preferably linkers which comprise a restriction enzyme recognition site to facilitate cloning when the nucleic acid of the invention is used for example as a PCR primer.
  • Primers of the invention are desirably capable of selectively hybridising to nucleic acids encoding the polypeptides of the invention.
  • Aselective ⁇ it is meant selective with respect to sequences encoding other purine receptors and in particular with respect to receptors other than adenine receptors. The ability of the sequence to hybridise selectively may be determined by experiment or calculated.
  • Tm of a primer is by reference to the formula for calculating the Tm of primers to a homologous target sequence.
  • This formula is generally suitable for primers of up to about 50 nucleotides in length.
  • this formula may be used as an algorithm to calculate a nominal Tm of a primer for a specified sequence derived from a sequence encoding a polypeptide of the invention. The Tm may be compared to a calculated Tm for GPCR sequences of humans and rats, based upon the maximum number of matches to any part of these other sequences.
  • Suitable conditions for a primer to hybridise to a target sequence may also be measured experimentally.
  • Suitable experimental conditions comprise hybridising a candidate primer to both nucleic acid encoding a polypeptide of the invention and nucleic acid encoding other adenine receptors on a solid support under low stringency hybridising conditions (e.g. 6xSSC at 55 0 C), washing at reduced SSC and/or higher temperature, for example at 0.2xSSC at 45°C, and increasing the hybridisation temperature incrementally to determine hybridisation conditions which allow the primer to hybridise to nucleic acid encoding a polypeptide of the invention but not other purine receptor encoding nucleic acids.
  • low stringency hybridising conditions e.g. 6xSSC at 55 0 C
  • washing at reduced SSC and/or higher temperature for example at 0.2xSSC at 45°C
  • increasing the hybridisation temperature incrementally to determine hybridisation conditions which allow the primer to hybridise to nucleic acid encoding a polypeptide of the invention
  • Nucleic acids of the invention may carry a revealing label.
  • Suitable labels include radioisotopes such as 32 P or 35 S, fluorescent labels, enzyme labels, or other protein labels such as biotin. Such labels may be added to polynucleotides or primers of the invention and may be detected using by techniques known per se.
  • Primers of the present invention may be comprised of synthetic nucleic acids, such as those with modified backbone structures intended to improve stability of the nucleic acid in a cell.
  • a number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5 1 ends of the molecule.
  • the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of polynucleotides of the invention.
  • antisense sequences based on the nucleic acid sequences described herein, preferably in the form of oligonucleotides, particularly stabilized oligonucleotides, or ribozymes.
  • Antisense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature iriRNA, interfering with the production of polypeptide encoded by a given target DNA sequence, so that its expression is reduced or prevented altogether.
  • Ribozymes will be designed to cleave mRNA encoded by an GPR39 GPCR encoding nucleic acid sequence of the invention, desirably at a target sequence specific to the GPR39 GPCR, i.e one which is not common to other GPCR sequences .
  • the construction of antisense sequences and their use is described in Peyman and Ulman, Chemical Reviews, 90:543-584, (1990), Crooke, Ann. Rev. Pharmacol.
  • RNA of the invention can be used for induction of RNA interference (RNAi) , using double stranded (dsRNA) (Fire et al. , Nature 391: 806-811. 1998) or short-interfering RNA (siRNA) sequences (Yu et al. , Proc Natl Acad Sci USA. 99:6047-52. 2002) .
  • dsRNA double stranded
  • siRNA short-interfering RNA sequences
  • RNAi is the process by which dsRNA induces homology-dependent degradation of complimentary mRNA.
  • a nucleic acid molecule of the invention is hybridized by complementary base pairing with a "sense" ribonucleic acid of the invention to form the double stranded RNA.
  • the dsRNA antisense and sense nucleic acid molecules are provided that correspond to at least about 20, 25, 50, 100, 250 or 500 nucleotides or an GPR39 coding strand, or to only a portion thereof.
  • the siRNAs are 30 nucleotides or less in length, and more preferably 21- to 23-nucleotides, with characteristic 2- to 3-nucleotide 3 ' -overhanging ends, which are generated by ribonuclease III cleavage from longer dsRNAs. See e.g. Tuschl T. (Nat Biotechnol. 20:446-48. 2002).
  • Intracellular transcription of small RNA molecules can be achieved by cloning the siRNA templates into RNA polymerase III (Pol III) transcription units, which normally encode the small nuclear RNA (snRNA) U6 or the human RNAse P RNA Hl.
  • RNA polymerase III Poly III transcription units
  • snRNA small nuclear RNA
  • Two approaches can be used to express siRNAs: in one embodiment, sense and antisense strands constituting the siRNA duplex are transcribed by- individual promoters (Lee, et al . Nat. Biotechnol . 20, 500-505. 2002); in an alternative embodiment, siRNAs are expressed as stem-loop hairpin RNA structures that give rise to siRNAs after intracellular processing (Brummelkamp et al. Science 296:550-553. 2002) (herein incorporated by reference) .
  • RNAi may be carried out by administering sense and antisense nucleic acids of the invention in the same solution without annealing prior to administration, and may even be performed by administering the nucleic acids in separate vehicles within a very close timeframe.
  • Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a GPR39 or antisense nucleic acids complementary to a GPR39 nucleic acid sequence are additionally provided.
  • Antisense, siRNAs and ribozyme sequences of the invention may be introduced into mammalian cells lines in culture to study the function of GPR39 GPCR, for example by causing down-regulation of this gene and observing phenotypic effects, or the expression or location of proteins described herein which associate with GPR39 GPCR. In cells where aberrant expression of GPR39 GPCR occurs, such antisense, siRNA and ribozyme sequences may be used to down-regulate the expression of the gene.
  • the cDNA sequence of the GPCR of the invention may be cloned using standard PCR (polymerase chain reaction) cloning techniques. This involves making a pair of primers to 5 ' and 3 ' ends on opposite strands of SEQ ID NO: 1, bringing the primers into contact with mRNA or cDNA obtained from a mammalian cortical cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector. The same applies to SEQ ID NO:3.
  • Polynucleotides which are not 100% homologous to the sequence of SEQ ID N0:l or SEQ ID NO:3 but which encode either SEQ ID NO:2 or SEQ ID NO:4 or other polypeptides of the invention can be obtained in a number of ways.
  • site directed mutagenesis of the sequence of SEQ ID NO: 1 or SEQ ID NO:3 may be performed. This is useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides. Further changes may be desirable to represent particular coding changes which are required to provide, for example, conservative substitutions.
  • Nucleic acids of the invention may comprise additional sequences at the 5' or 3' end.
  • synthetic or natural 5 ' leader sequences may be attached to the nucleic acid encoding polypeptides of the invention.
  • the additional sequences may also include 5 ' or 3 ' untranslated regions required for the transcription of nucleic acid of the invention in particular host cells.
  • sequences may be obtained by making or obtaining cDNA libraries made from dividing cells or tissues or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of SEQ ID NO:1 or of SEQ ID NO:3 under conditions of medium to high stringency (for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50 N C to about 60 N C) .
  • medium to high stringency for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50 N C to about 60 N C
  • the present invention further extends to an isolated DNA sequence comprising sequences encoding a polypeptide of the invention but in which the encoding sequences are divided up into two or more (preferably no more than five, e.g. four or three) exons.
  • exon sequences may be natural and obtained from genomic clones, or synthetic.
  • Exon sequences may be used in the construction of mini- gene sequences which comprise nucleic acid encoding polypeptides of the invention which sequences are interrupted by one or more exon sequences.
  • Mini-genes may also be constructed using heterologous exons, derived from any eukaryotic source.
  • Isolated polypeptides used in the methods of the present invention will be those as defined above in isolated form, free or substantially free of material with which it is naturally associated such as other polypeptides with which it is found in the cell:
  • the polypeptides may of course be formulated with diluents or adjuvants and still for practical purposes be isolated - for example the polypeptides will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays.
  • the polypeptides may be glycosylated, either naturally or by systems of heterologous eukaryotic cells, or they may be (for example if produced by expression in a prokaryotic cell) unglycosylated.
  • Polypeptides may be phosphorylated and/or acetylated.
  • a polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the polypeptide in the preparation is a polypeptide of the invention.
  • Polypeptides of the invention may be modified for example by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote their secretion from a cell .
  • Polypeptides having at least 50%, for example 60%, 70%, 80%, 90%, 95% or 98% sequence identity to SEQ ID NO:2 or SEQ ID NO:4 may be polypeptides which are amino acid sequence variants, alleles, derivatives or mutants of SEQ ID NO:2 or SEQ ID NO:4 respectively, and are also provided by the present invention.
  • a polypeptide may have an amino acid sequence which differs from that given in SEQ ID NO:2 or SEQ ID NO:4 by one or more of addition, substitution, deletion and insertion of one or more (such as from 1 to 20, for example 2, 3, 4, or 5 to 10) amino acids.
  • the percentage identity of polypeptide sequences can be calculated using commercially available algorithms which compare a reference sequence (e.g. SEQ ID NO:2 or SEQ ID NO:4 of the present invention) with a query sequence. Further details of assessing identity are described below.
  • a query sequence is determined to have an identity to that of SEQ ID NO:2 or SEQ ID NO:4 of at least 50% and preferably at least 60%, 70%, 80%, 90%, 95% or 98% said sequence being that of a polypeptide retaining GPR39 receptor activity, such a sequence forms part of the present invention.
  • a polypeptide according to the present invention may be isolated and/or purified (e.g. using an antibody) for instance after production by expression from encoding nucleic acid.
  • the isolated and/or purified polypeptide may be used in formulation of a composition, which may include at least one additional component, for example a pharmaceutical composition including a pharmaceutically acceptable excipient, vehicle or carrier.
  • a polypeptide according to the present invention may be used as an immunogen or otherwise in obtaining specific antibodies.
  • Antibodies are useful in purification and other manipulation of polypeptides, diagnostic screening and therapeutic contexts.
  • a polypeptide according to the present invention may be used in screening for molecules which bind to it or modulate its activity or function. Such molecules may be useful in a therapeutic (possibly including prophylactic) context.
  • a polypeptide or labelled polypeptide of the invention or fragment thereof may also be fixed to a solid phase, for example the surface of an immunoassay well or dipstick.
  • Such labelled and/or immobilized polypeptides may be packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.
  • Such polypeptides and kits may be used in methods of detection of antibodies to such polypeptides present in a sample or active portions or fragments thereof by immunoassay.
  • Immunoassay methods are well known in the art and will generally comprise: (a) providing a polypeptide comprising an epitope bindable by an antibody against said protein; (b) incubating a biological sample with said polypeptide under conditions which allow for the formation of an antibody-antigen complex; and (c) determining whether antibody-antigen complex comprising said polypeptide is formed.
  • the percentage identity of nucleic acid and polypeptide sequences can be calculated using commercially available algorithms which compare a reference sequence with a query sequence.
  • the following programs may be used to determine homologies/identities: BLAST, gapped BLAST, BLASTN and PSI-BLAST, which may be used with default parameters.
  • GAP Genetics Computer Group, Madison, WI
  • GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps.
  • Another method for determining the best overall match between a nucleic acid sequence or a portion thereof, and a query sequence is the use of the FASTDB computer program based on the algorithm of Brutlag et al (Comp. App. Biosci., 6; 237-245 (1990)) .
  • the program provides a global sequence alignment.
  • the result of said global sequence alignment is in percent identity.
  • Nucleic acid sequences of the present invention may be incorporated into vectors, particularly expression vectors.
  • the vector may be used to replicate the nucleic acid in a compatible host cell.
  • the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the host cell. Suitable host cells are described below in connection with expression vectors.
  • a polynucleotide of the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e.g. phage, phagemid or baculoviral, cosmids, YACs, BACs, or PACs as appropriate.
  • Vectors include gene therapy vectors, for example vectors based on adenovirus, adeno- associated virus, retrovirus (such as HIV or MLV) or alpha virus vectors.
  • the vectors may be provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector.
  • Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
  • the vector may also be adapted to be used in vivo, for example in methods of gene therapy. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.
  • Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others.
  • Promoters and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed.
  • yeast promoters include S. cerevisiae GAL4 and ADH promoters, S. pombe nmtl and adh promoter.
  • Mammalian promoters include the metallothionein promoter which can be induced in response to heavy metals such as cadmium.
  • Viral promoters such as the SV40 large T antigen promoter or adenovirus promoters may also be used. All these promoters are readily available in the art.
  • the vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the polypeptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell.
  • Vectors for production of polypeptides of the invention of for use in gene therapy include vectors which carry a mini-gene sequence of the invention. It is accordingly an object of the present invention to provide a method for treating abnormal conditions related to an under- expression of GPR39 GPCR and its activity, said method comprising the use of a polynucleotide encoding a GPR39 GPCR.
  • a polynucleotide of the invention is used to effect the endogenous production of GPR39 GPCR by the relevant cells in the subject.
  • a polynucleotide encoding a GPR39 GPCR may be engineered for expression in a replication defective retroviral vector as provided above.
  • the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo.
  • gene therapy see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, in Human Molecular Genetics, T. Strachan and A.P. Read, BIOS Scientific Publishers Ltd. (1996) .
  • Vectors may be transformed into a suitable host cell as described above to provide for expression of a polypeptide of the invention.
  • the invention provides a process for preparing polypeptides according to the invention which comprises cultivating a host cell transformed or transfected with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptides, and recovering the expressed polypeptides.
  • Polypeptides may also be expressed in vitro systems, such as reticulocyte lysate.
  • a further embodiment of the invention provides host cells transformed or transfected with the vectors for the replication and expression of polynucleotides of the invention.
  • the cells will be chosen to be compatible with the said vector and may for example be bacterial, yeast, insect or mammalian.
  • the host cells may be cultured under conditions for expression of the gene, so that the encoded polypeptide is produced. If the polypeptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium.
  • a polypeptide may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e.g. in the formulation of a composition which may include one or more additional components, such as a pharmaceutical composition which includes one or more pharmaceutically acceptable excipients, vehicles or carriers
  • Polynucleotides according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA or ribozymes. Assays
  • the receptor or subunits of the receptor may be employed in a binding assay.
  • Binding assays may be competitive or non-competitive. Such an assay can accommodate the rapid screening of a large number of compounds to determine which compounds, if any, are capable of binding to the polypeptides. Subsequently, more detailed assays can be carried out with those compounds found to bind, to further determine whether such compounds act as agonists or antagonists of the polypeptides of the invention.
  • this invention provides a method for identifying a compound that modulate gastrointestinal kinetics, which method comprises:
  • the present invention provides a method for identifying a compound that modulate gastrointestinal kinetics, which method involves a competitive binding assay wherein:
  • host cells expressing all or part of the GPR39 receptor protein according to the invention are contacted with a compound known to bind to the GPR39 receptor protein both in the presence and absence of the compound to be tested, and (ii) the effect of said compound on the binding of the compound known to bind to the GPR39 receptor protein is being assessed.
  • a decrease in the binding of the compound known to bind to the GPR39 receptor protein in the presence of the compound to be tested is an indication that said compound binds to the GPR39 receptor protein.
  • Obestatin the natural ligand for the GPR39 receptor protein has recently been identified (Zhang, J.V. et al. , 2004 Science VoI.310,-996- 999) as another peptide derived from the same prohormone as ghrelin.
  • the compound known to bind to the receptor consists of obestatin, more in particular selected from one of the obestatin sequences disclosed in Fig.5, i.e. selected from SEQ ID No's 8 to 19, more in particular SEQ ID No.8 or SEQ ID No.10.
  • the obestatin as used herein refers to a peptide having at least 60%, 70%, 80%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10.
  • the aforementioned competitive binding assay is performed on membrane preparations of host cells expressing all or part of the GPR39 receptor protein according to the invention. Methods for preparing said host cells and for preparing membrane preparations from such cells are described hereinafter.
  • the GPR39 receptor protein in the aforementioned binding assays is an isolated protein having an amino acid sequence selected from the group consisting of SEQ ID No:2, SEQ ID NO:4, a splice variant of the proteins having the aforementioned SEQ ID's, and an amino acid sequence having at least 50% and preferably at least 60%, 70%, 80%, 90%, 95% or 98% sequence identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
  • Parts of the GPR39 protein are meant to include fragments of the polypeptide of SEQ ID NO:2 or SEQ ID NO:4, said fragments being of at least 10, for example at least 20, 30 40, 50, 75, 100 or 150 or more amino acids in size.
  • Such fragments may be derived from the N-terminal region of SEQ ID NO:2 or SEQ ID NO:4 respectively. Fragments including the N-terminal region may be used to reconstitute the extracellular portion of the receptor to provide receptor binding sites. Preferably, fragments will retain the ability to bind the compound known to bind to the GPR39 receptor protein.
  • Cell membranes expressing the receptor protein of this invention are useful for certain types of assays including but not restricted to ligand binding assays, GTP- ⁇ -S binding assays and others.
  • the specifics of preparing such cell membranes may in some cases be determined by the nature of the ensuing assay but typically involve harvesting whole cells and disrupting the cell, for example by sonication in ice cold buffer (e.g. 20 mM Tris HCl, 1 mM EDTA, pH 7.4 at 4 0 C) .
  • the resulting crude cell lysate is subsequently cleared of cell debris by low speed centrifugation, for example at 200xg for 5 min at 4 0 C.
  • Cells expressing the receptor of this invention may be used to screen for ligands for said receptor.
  • the same assays may be used to identify agonists or antagonists of the receptor that may be employed for a variety of therapeutic purposes.
  • the final protein concentration in the assay is typically within 12-40 ⁇ g/ml.
  • Membranes are then incubated with radiolabeled ligand either in the presence or absence of competing ligands on ice for 60 min in a total volume of 250 ⁇ l 96 well microtiter plates.
  • the bound ligand is then separated from free ligands by filtration through GF/B filters presoaked in 0.5% polyethyleneimine (PEI), using a Tomtec (Wallac) vacuum filtration device. After addition of Ready Safe (Beckman) scintillation fluid, bound radioactivity is quantitated using a Trilux (Wallac) scintillation counter (approximately 40% counting efficiency of bound counts) . Alternatively, it may be preferable to collect bound ligand and then separate ligand from receptor using procedures well known in the art. Data is fit to non- linear curves using GraphPad prism.
  • agonist or antagonist compounds that bind to the receptor may be identified as they inhibit the binding of the radiolabeled ligand to the membrane protein of cells expressing the said receptor.
  • Non-specific binding is defined as the amount of radioactivity remaining after incubation of membrane protein in the presence of 100 nM of the unlabeled peptide corresponding to the radioligand used.
  • membrane preparations or intact cells transfected with the receptor are incubated in the presence of increasing concentrations of the labeled compound to determine the binding affinity of the labeled ligand.
  • the binding affinities of unlabeled compounds may be determined in equilibrium competition binding assays, using a fixed concentration of labeled compound in the presence of varying concentrations of the displacing ligands .
  • the aforementioned radioligand binding assay is performed using radiolabeled obestatin as defined hereinbefore. Labeling of obestatin and receptor binding has been described in Zhang, J.V. et al . (2004 Science Vol.310;996-999) . Briefly;
  • Iodination of obestatin was performed using the Iodogen (Pierce, Upland, IN) procedure. Mixtures of the peptide (20 ⁇ g) and 1 mCi [ 125 I] NaI were transferred to precoated Iodogen vials and incubated for 4 min. The 1251-labeled peptide was applied to a Sep-Pak C18 cartridge (Waters) before elution with 60% acetonitrile/0.1%TFA.
  • rat jejunum or other tissues were washed with buffer A (20 mM Hepes, 5 mM EDTA, 1 mM dithiothreitol (DTT), 10 ⁇ M amidinophenylmethanesulfonyl fluoride, 5 mg/L leupeptin, 100 mM KCl, pH 7.5), cut into small pieces, and homogenized using a motorized homogenizer. The homogenates were centrifuged at 1,000 g for 5 min. and the supernatant was centrifuged at 300,000 g for 1 hour at 2 0 C.
  • buffer A (20 mM Hepes, 5 mM EDTA, 1 mM dithiothreitol (DTT), 10 ⁇ M amidinophenylmethanesulfonyl fluoride, 5 mg/L leupeptin, 100 mM KCl, pH 7.5
  • the homogenates were centrifuged at 1,000 g for 5 min. and the supern
  • the pellets were resuspended with buffer A without KCl, quickly frozen under liquid nitrogen, and stored at -8O 0 C until use.
  • Tissue homogenates were incubated in 100 ⁇ l of phosphate buffered saline containing 0.1% bovine serum albumin for 18 hours at room temperature with varying concentrations of 125 I-obestatin in the presence or absence of unlabeled obestatin at 1,000-fold excess. After incubation, the tubes were centrifuged for 10 min. at 10,000 g, and pellets were washed twice in ice-cold PBS before quantitation of radioactivity with a ⁇ -spectrophotometer. Specific binding was calculated by subtracting nonspecific binding from total binding. For displacement curves, a fixed concentration of 125 I- obestatin was incubated with or without increasing concentrations of obestatin or other peptides.
  • an assay for identifying compounds that modulate gastrointestinal kinetics, characterized in that said compounds modulate the activity of the GPR39 receptor proteins according to the invention.
  • Such an assay comprises the steps of;
  • said assays can be used to identify compounds that may affect cholesterol homeostasis in a subject, including humans and warm-blooded animals.
  • a compound that Amodulates the activitys of a polypeptide of the invention refers to a compound o that alters the activity of the polypeptide so that it behaves differently in the presence of the compound than in the absence of the compound.
  • Compounds affecting modulation include agonists and antagonists.
  • An agonist encompasses a compound which activates GPR39 GPCR function.
  • an antagonist includes a compound that interferes with GPR39 GPCR function.
  • the effect of an antagonist is observed as a blocking of agonist-induced receptor activation, however in the case of GPR39, which has recently been described as a constitutive active receptor, the compounds that interferes with GPR39 GPCR function also include inverse agonists, i.e.
  • Antagonists include competitive as well as non-competitive antagonists.
  • a competitive antagonist (or competitive blocker) interacts with or near the site specific for agonist binding.
  • a non-competitive antagonist or blocker inactivates the function of the receptor by interacting with a site other than the agonist interaction site. It is thus an object of the present invention to provide a method for identifying compounds capable to increase gastric emptying and/or colonic motility said method comprising;
  • the present invention provides a method for identifying compounds capable to decrease gastric emptying and/or colonic motility said method comprising;
  • the aforementioned activity assays can also be used to identify compounds that would modulate cholesterol metabolism. Based on the observed phenotype it is to be expected that compounds that increase GPR39 activity would result in a lowering of cholesterol levels and compounds that decrease GPR39 activity would result in an increase in cholesterol levels.
  • the present invention provides a method for identifying compounds capable to decrease cholesterol levels said method comprising;
  • GPR39 belongs to the class of proteins known as G-protein coupled receptors (GPCRs) .
  • GPCRs transmit signals across cell membranes upon the binding of the ligand.
  • the ligand- bound GPCR activates intracellular signalling events mediated by heterotrimeric G proteins, such as activation of the adenylate cyclase pathway or activation of the phospholipase C- ⁇ pathway.
  • Assay to assess the activation of the aforementioned intracellular signalling events are generally known in the art and include amongst others cell based assays for signal transduction comprising chimeric ligand-inducible transcription factors, binding assays for G-protein-coupled receptors using fluorescence intensity distribution analysis, cell-signaling assays using cyclic nucleotides coupled to luminophores or measurement of responses from G protein coupled receptors using a multiple response element or cAMP response element- directed reporter assay. Description of several such assays follows.
  • the present invention also provides a bioassay for identifying compounds which modulate the regulatory regions of the GPR39 GPCR gene.
  • Such an assay is conducted utilising rat or human cells capable of expressing a polypeptide of the invention (preferably of SEQ ID NO:2 or SEQ ID NO:4) .
  • the cells are contacted with at least one compound wherein the ability of said compound to modulate the regulatory region is known. Thereafter, the cells are monitored for expression of the nucleic acid of the invention.
  • the promoter may be linked to a reporter gene. Suitable reporter genes that may be employed include, for example, the chloramphenicol acetyltransferase gene, the luciferase gene, and the like.
  • bioassay methods for identifying compounds that modulate the activity of receptors such as polypeptides of the invention generally require comparison to a control.
  • Acontrol ⁇ is a cell or culture that is treated substantially the same as the test cell or test culture exposed to the compound, with the distinction that the Acontrol ⁇ cell or culture is not exposed to the compound.
  • Acontrol ⁇ cell or culture that can be employed is a cell or culture that is identical to transfected cells, the exception that the Acontrols cell or culture does not express functional GPR39 GPCR. Accordingly, the response of the transfected cell can be compared with that of the Acontrols cell or culture to the same compound under the same reaction conditions.
  • assays include binding assays and functional assays which may be performed as follows:
  • Over-expression of nucleic acid encoding polypeptides of the invention in cell lines may be used to produce membrane preparations bearing said polypeptides (referred to in this section as GPR39 GPCR for convenience) for ligand binding studies.
  • These membrane preparations can be used in conventional filter-binding assays (eg. Using Brandel filter assay equipment) or in high throughput Scintillation Proximity type binding assays (SPA and Cytostar-T flashplate technology; Amersham Pharmacia Biotech) to detect binding of radio-labelled ligand and displacement of such radio-ligands by competitors for the binding site.
  • Radioactivity can be measured with Packard Topcount, or similar instrumentation, capable of making rapid measurements from 96-, 384-, 1536- microtitre well formats.
  • SPA/Cytostar-T technology is particularly amenable to high throughput screening and therefore this technology is suitable to use as a screen for compounds able to displace standard ligands.
  • GPR39 GPCR acts via Gi or Go (inhibitory G protein), which usually interacts with GIRK (inward rectifying potassium channels), potassium ion flux should result on activation of these receptors.
  • This flux of ions may be measured in real time using a variety of techniques to determine the agonistic or antagonistic effects of particular compounds. Therefore, recombinant GPR39 GPCR receptors expressed in cell lines or, for example, Xenopus oocytes can be characterised using whole cell and single channel electrophysiology to determine the mechanism of action of compounds of interest.
  • Electrophysiological screening, for compounds active at GPR39 GPCR may be performed using conventional electrophysiological techniques and when they become available, novel high throughput methods currently under development.
  • Fluorescence - Calcium and sodium fluxes are measurable using several ion-sensitive fluorescent dyes, including fluo-3, fluo-4, fluo-5N, fura red, Sodium Green, SBFI and other similar probes from suppliers including Molecular Probes. Calcium and sodium influx as a result of GPR39 GPCR can thus be characterised in real time, using fluorometric and fluorescence imaging techniques, including fluorescence microscopy with or without laser confocal methods combined with image analysis algorithms .
  • FLIPR® FLuorescence Imaging Plate Reader
  • the FLIPR assay is designed to measure fluorescence signals from populations of cells before, during and after addition of compounds, in real time, from all 96-/384-wells simultaneously.
  • the FLIPR assay may be used to screen for and characterise compounds functionally active at recombinant GPR39 GPCR, eg rat or human GPR39 GPCR, expressed in cell lines. As described below, calcium transients in cells transfected with GPR39 GPCR were measured using the FLIPR assay in order to measure activation of the receptors by various substrates in order to determine the natural ligand of the receptor.
  • Cyclic AMP (cAMP) assay The receptor-mediated stimulation or inhibition of cyclic AMP (cAMP) formation may be assayed in cells expressing the receptor.
  • An exemplary protocol for a cAMP assay is provided hereinafter.
  • COS-7 cells are transiently transfected with the receptor gene using the DEAE-dextran method and plated in 96-well places. 48 hours after transfection, cells are washed twice with Dulbecco's phosphate buffered saline (FES) supplemented with 10 iriM HEPES, 10 mM glucose and 5 mM theophylline and are incubated in the same buffer for 20 min at 37 0 C, in 5% CO 2 - Test compounds are added and cells are incubated for an additional 10 min at 37 0 C. The medium is then aspirated and the reaction stopped by the addition of 100 mM HCl.
  • FES Dulbecco's phosphate buffered saline
  • Microphysiometric assay Because cellular metabolism is intricately involved in a broad range of cellular events (including receptor activation of multiple messenger pathways) , the use of microphysiometric measurements of cell metabolism can in provide a generic assay of cellular activity arising from the activation of any orphan receptor regardless of the specifics of the receptor's signaling pathway.
  • a standard recording protocol specifies a 100 ⁇ l/min flow rate, with a 2 min total pump cycle which includes a 30 sec flow interruption during which the acidification rare measurement is taken.
  • Ligand challenges involve a 1 min 20 sec exposure to the sample just prior to the first post challenge rate measurement being taken, followed by two additional pump cycles for a total of 5 min 20 sec sample exposure.
  • drugs in a primary screen are presented to the cells at 10 ⁇ M final concentration.
  • GPR39 is involved in the regulation of gastrointestinal kinetics and cholesterol homeostasis.
  • this includes diseases related to delayed gastric emptying such as for example, gastroparesis post-operative ileus, gastroparesis in diabetic, functional dyspepsia, post-vagotomy gastroparesis and idiopathic intestinal pseudoobstruction.
  • diseases related to an increased gastric emptying such as for example, dumping syndrome or increased motility such as diarrhoea, diarrhoae-IBS and mixed-IBS.
  • GPR39 GPCR and related receptors can be used as a valuable tool for drug development in a variety of therapeutic areas.
  • the invention further provides novel binding agents, including modulatory agents obtained by an assay according to the present invention, and compositions comprising such agents.
  • Agents which bind to the receptor and which may have agonist or antagonist activity may be used in methods of treating diseases as characterized hereinbefore, whose pathology is characterised by action via the GPR39 GPCR receptor, and such use forms a further aspect of the invention.
  • the agents may be administered an effective amount of an agent of the invention. Since many of the above-mentioned conditions are chronic and often incurable, it will be understood that Atreatment ⁇ is intended to include achieving a reduction in the symptoms for a period of time such as a few hours, days or weeks, and to include slowing the progression of the course of the disease.
  • compositions comprising an agent together with a pharmaceutically acceptable carrier or diluent.
  • the agent may in the form of a physiologically functional derivative, such as an ester or a salt, such as an acid addition salt or basic metal salt, or an N or S oxide.
  • Compositions may be formulated for any suitable route and means of administration.
  • Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal, inhalable, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration.
  • formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, starch, magnesium stearate, sodium saccharin, talcum, glucose, sucrose, magnesium carbonate, and the like may be used.
  • the active compound as defined above may be formulated as suppositories using, for example, polyalkylene glycols, acetylated triglycerides and the like, as the carrier.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc, an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
  • a carrier such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like
  • the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.
  • wetting or emulsifying agents such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbit
  • composition or formulation to be administered will, in any event, contain a quantity of the active compound(s) in an amount effective to alleviate the symptoms of the subject being treated.
  • Dosage forms or compositions containing active ingredient in the range of 0.25 to 95% with the balance made up from non-toxic carrier may be prepared.
  • a pharmaceutically acceptable non-toxic composition is formed by the incorporation of any of the normally employed excipients, such as, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, sodium crosscarmellose, starch, magnesium stearate, sodium saccharin, talcum, glucose, sucrose, magnesium, carbonate, and the like.
  • excipients such as, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, sodium crosscarmellose, starch, magnesium stearate, sodium saccharin, talcum, glucose, sucrose, magnesium, carbonate, and the like.
  • Such compositions take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • Such compositions may contain l%-95% active ingredient, more preferably 2-50%, most preferably 5-8%.
  • Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly or intravenously.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like.
  • the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as ' wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, triethanolamine sodium acetate, etc .
  • the percentage of active compound contained in such parental compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject. However, percentages of active ingredient of 0.1% to 10% in solution are employable, and will be higher if the composition is a solid which will be subsequently diluted to the above percentages.
  • the composition will comprise 0.2-2% of the active agent in solution.
  • Another object of the invention is a pharmaceutical composition for the treatment of increased cholesterol levels, increased gastric emptying, diseases related to increased gastric emptying such as for example dumping syndrome or increased intestinal motility such as diarrhoea, diarrhoea-IBS and mixed-IBS, said composition comprising a GPR39 agonist. It is accordingly an object of the present invention to provide the use of a GPR39 antagonist in the manufacture of a medicament for the treatment of a disease condition related to delayed gastric emptying. In particular in the treatment of gastroparesis post-operative ileus, gastroparesis in diabetic, functional dyspepsia, post- vagotomy gastroparesis and idiopathic intestinal pseudo- obstruction.
  • the present invention provides the use of a GPR39 agonist in the manufacture of a medicament for the treatment of a disease condition related to increased gastric emptying.
  • a disease condition related to increased gastric emptying In particular in the treatment of dumping syndrome, diarrhoea, diarrhoea- IBS and mixed-IBS.
  • GPRC39 knock-out mice were obtained from Lexicon Genetics Inc. Disruption of the open reading frame of GPR39 was performed by replacing the coding region of the first coding exon of the GPR39 gene with an IRES LacZ/MCl-Neo reporter gene/selection cassette. Animals were maintained in an SPF facility that meets all Belgian and European requirements for animal care. Mice were group-housed in a climate-controlled animal colony with a 12h dark-light cycle (light on 7:00 EST) with free access to food and water, unless indicated differently. Adequate measures were taken to minimize pain or discomfort. All experiments were carried out in accordance with the European Communities Council Directives (86/609/EEC) and were approved by the local ethical committee.
  • RNA from different tissues dissected from wild type (brain, liver, spinal cord, stomach, kidney, spleen, colon, lung, heart, oesophagus, pancreas, ileum, jejenum) and GPR39 knock-out (liver, stomach, jejenum, colon) mouse were analysed using real-time quantitative reverse transcription PCR to study the tissue distribution of GPR39 and confirm the GPR39 knock-out.
  • First strand cDNA synthesis was performed on 1,0 mg total RNA using random hexamer primers and Superscript II RT (Invitrogen Life Technologies) .
  • Quantitative PCR was performed on an ABIPrism 7000 cycler (Applied Biosystems) using a Taqman PCR kit. Serial dilutions of cDNA were used to generate standard curves of threshold cycles versus the logarithms of concentrations for ⁇ -actin.
  • the RTQ specific primer pairs and probes are enlisted below.
  • Tissues pancreas, stomach, ileum, jejenum and colon
  • homozygous GPR39 knock-out mice that express the bacterial ⁇ -galactosidase gene (LacZ) under the regulatory control of the endogenous mouse GPR39 promoter.
  • Whole-mount staining on these tissues was performed according to described protocols (InvitroGen) , tissues were next paraffin imbedded and sectioned.
  • the solid test meal consisted of 0.1 g baked scrambled egg yolk, doped with 0.01 ⁇ Ci 14 C-octanoic acid sodium salt (American Radiolabeled chemicals Inc, St Louis, MO, USA) and mixed with 0. Ig standard mice chow. Tap water was added and mixed to form a homogenous paste. Before the start of the experiments, fasted mice (19h) were trained twice weekly, for 2 weeks, on a fixed time schedule, to eat spontaneously the test meal, without the radioactive marker. After this training the test meal was ingested within 60s by most of the animals.
  • ti /2 Gastric half excretion time (ti /2 ) , time at which 50% of the total amount of 14 CO 2 was excreted.
  • the ti /2 -value therefore not only contains the gastric half emptying time but also includes the time necessary for postgastric processes such as absorption and metabolism of the 14 C octanoic acid and the excretion of 14 CO 2 in the breath.
  • Formula 1: t 1/2 (-1/k) *ln(l-2 ⁇ 1/ ⁇ )
  • Formula 2: ti /2 60* (b/c)
  • the lag phase (ti ag ) delay in gastric emptying due to the time required for the stomach to grind the meal in fine particles.
  • a panel of 6 wild type (WT) and 6 homozygous GPR39 knockout (GPR 3>9 ⁇ ' ⁇ ) female mice were used to investigate the gastric secretion.
  • Mice were anesthetized using isoflurane. Under anesthesia the pylorus was closed by means of a suture (vicryl 4-0, Ethicon) then the abdominal cavity was sutured. The animal was replaced individually in its cage for 4h. Four hours after the pylorus closing mice were sacrificed and the esophagus was closed just above the cardia. The stomach was withdraw from the abdominal cavity. The stomach was weighed and opened to take the gastric secretion. The gastric secretion was measured and then diluted in 5 ml of water (Water Molecular Biology Grade, Eppendorf) . The pH was measured using a pH electrode (pH 597, Profi Lab) .
  • mice and stomach weight and gastric secretion were compared between wild type (WT) and GPR39 ⁇ / ⁇ mice using a non parametric Wilcoxon test.
  • the pH of water (5 ml) was firstly measured then added to the gastric secretion and the pH was again measured.
  • the difference between the pH of water and the pH of solution with gastric secretion was calculated and then reported to 1 ml of the solution and then compared between both groups using a non parametric Wilcoxon test.
  • mice were asphyxiated by CO 2 followed by decapitation.
  • the colon was cut out from the caecum till the rectum, and freed of adhering tissues.
  • the tissue was transferred to a glass beaker containing 100 ml of Krebs-Henseleit solution, gassed with a mixture of 95 % O 2 and 5 % C O 2 and maintained at 37° C. Prior to the start of the experiment, the length of the colon was measured. The pellets in the colon were counted and their distance to the rectum was measured. Then the time to expulsion of each pellet was recorded for 20 min.
  • the Open Field Test was performed during the light phase of a normal light/dark cycle. Each mouse was subjected to a 30-min testing session in an automated open field system. Locomotion in the horizontal and vertical pane was recorded. During this session, the following parameters were recorded: duration of moves, number of moves, total distance traveled, distance traveled in the centre, distance traveled in the margin, relative distance traveled in the centre, time spent in the centre, time spent in the margin, relative time spent in the centre, distance traveled in centre vs. distance traveled in margin, time spent in the centre vs. time spent in the margin, number of rearings, and duration of rearing. A Single Factor ANOVA was used as statistical analysis of the results obtained.
  • the Elevated Zero Maze (0-maze) was performed during the light phase of a normal light/dark cycle. Each mouse was subjected to a 5-min testing session in the elevated zero maze. During this session, the following parameters were recorded: move time in closed and open arms, the relative time spent in closed and open arms, total distance traveled, distance traveled in closed and open arms. A Single Factor ANOVA was used as statistical analysis of the results obtained.
  • a panel of six wild type and six homozygous male GPR39 knock-out mice were habituated to AIN-93G Rodent Purified Diet (Dyets, Inc., USA) prior to putting them into the metabolic cages.
  • Mice were individually housed in type-2 cages under the same conditions as for the metabolic cages (see below) for a minimum of 1 week prior to measurement. For two consecutive days, all mice were housed individually in metabolic cages under controlled temperature (28.8°C), humidity (55-60 %) and light (12:12 h light/dark cycle, lights on 06:00 h) . The animals had free access to rodent chow and water. The following parameters were measured and expressed as mean: respiratory quotient (RER), VO2 and heat production.
  • RER respiratory quotient
  • Plasmatic level of cholesterol and triglycerides were determined in 6 month old male and female GPR39 "7" mice versus GPR39+/+ littermates after 12h food deprivation after a ad libitum availability of a standard chow diet. Cholesterol and triglyceride levels were determined by means a Hitachi Modular random access analyzer (Roche Diagnostics, Basel, Switzerland) using reagents and test methods from the same supplier.
  • Cumulative food intake analysis during 6h in ad libitum fed and fasted young (17 weeks) and old (56 weeks) GPR39 "7" mice and GPR39 +/+ littermates (n 6 per group) was started at 10 a.m. Each mouse was put in an individual cage, placed in a dark room (unless stated otherwise) , where it received a pre-weighed amount of food. Cumulative food intake was measured by subtracting the uneaten food at 30 min, 1, 2, 3, 4, 5 and 6 hours.
  • Real-Time Quantitative Reverse Transcription PCR of GPR39 in multiple mouse tissues derived from wild type mice showed a wide tissue distribution (Fig. IA) .
  • Real-Time Quantitative Reverse Transcription PCR of GPR39 in four different tissues (liver, stomach, jejunum, colon) derived from wild type mice and heterozygote and homozygote GPR39 knock-out mice confirmed the absence of the GPR39 transcript in the GPR39 knock-out mouse. Intermediate levels were obtained in the heterozygote mice (Fig. IB) .
  • GPR39 was detected in the exocrine (Islets of Langerhans) and endocrine portion of the pancreas, the parietal cells located in the upper region of the mucosal glandular tissue in the pylorus and in the mucous neck cells in the corpus of the stomach, the enterocytes in the small intestine and the colon as well as in the neurons that innervate the muscle layers of the intestine.
  • Triglyceride levels appeared increased in both male (162.5 ⁇ 14.7) and female (84.6 ⁇ 7.2) GPR39 "7" mice compared to GPR39 +/+ male (123.9 ⁇ 20.8) and female (71.4 ⁇ 12.0) littermates, but the differences did not reach significance.

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Abstract

La présente invention porte sur la caractérisation fonctionnelle du récepteur GPR39 couplé à la protéine G et sur des composés qui modifient ou régulent l'activité de la protéine GPR39. L'invention porte notamment sur des méthodes de recherche systématique des agonistes ou antagonistes de GPR39 afin d'identifier des composés capables de moduler la cinétique gastro-intestinale et/ou le métabolisme du cholestérol, l'invention portant également sur les utilisations thérapeutiques de ces composés. L'invention porte, en outre, sur des animaux transgéniques portant des mutations dans le gène GPR39.
PCT/EP2005/056350 2004-12-01 2005-11-30 Recepteur couple a la proteine g WO2006058889A1 (fr)

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EP05817427A EP1820026A1 (fr) 2004-12-01 2005-11-30 Recepteur couple a la proteine g
CA002587534A CA2587534A1 (fr) 2004-12-01 2005-11-30 Recepteur couple a la proteine g
AU2005311321A AU2005311321A1 (en) 2004-12-01 2005-11-30 G protein coupled receptor
JP2007543845A JP2008521419A (ja) 2004-12-01 2005-11-30 Ｇタンパク質共役型受容体
NO20073294A NO20073294L (no) 2004-12-01 2007-06-28 G-proteinkoplet reseptor

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EP1743944A1 (fr) * 2004-04-23 2007-01-17 Takeda Pharmaceutical Company Limited Nouveau procédé de criblage
WO2007141322A1 (fr) * 2006-06-08 2007-12-13 Janssen Pharmaceutica N.V. Récepteur couplé à la protéine g 39 (gpr39)

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CN111443209B (zh) * 2020-03-26 2022-12-20 中国中医科学院医学实验中心 一种筛选非激动剂型PPARγ配体的方法

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WO2004040000A2 (fr) * 2002-09-09 2004-05-13 Nura, Inc Recepteurs couples a la proteine g et leurs utilisations
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Cited By (3)

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EP1743944A1 (fr) * 2004-04-23 2007-01-17 Takeda Pharmaceutical Company Limited Nouveau procédé de criblage
EP1743944A4 (fr) * 2004-04-23 2011-01-05 Takeda Pharmaceutical Nouveau procédé de criblage
WO2007141322A1 (fr) * 2006-06-08 2007-12-13 Janssen Pharmaceutica N.V. Récepteur couplé à la protéine g 39 (gpr39)

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AU2005311321A1 (en) 2006-06-08
NO20073294L (no) 2007-08-29
EP1820026A1 (fr) 2007-08-22
KR20070086003A (ko) 2007-08-27
ZA200704526B (en) 2008-11-26

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