EP2030024A1 - Récepteur couplé à la protéine g 39 (gpr39) - Google Patents

Récepteur couplé à la protéine g 39 (gpr39)

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Publication number
EP2030024A1
EP2030024A1 EP07729995A EP07729995A EP2030024A1 EP 2030024 A1 EP2030024 A1 EP 2030024A1 EP 07729995 A EP07729995 A EP 07729995A EP 07729995 A EP07729995 A EP 07729995A EP 2030024 A1 EP2030024 A1 EP 2030024A1
Authority
EP
European Patent Office
Prior art keywords
gpr39
seq
nucleic acid
acid sequence
receptor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07729995A
Other languages
German (de)
English (en)
Inventor
Benoit Christian Jean-Claude Moreaux
Diederik Willem Elisabeth Moechars
Luc August Laurentius Ver Donck
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Janssen Pharmaceutica NV
Original Assignee
Janssen Pharmaceutica NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Janssen Pharmaceutica NV filed Critical Janssen Pharmaceutica NV
Priority to EP07729995A priority Critical patent/EP2030024A1/fr
Publication of EP2030024A1 publication Critical patent/EP2030024A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity

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 for enhancing glucose control in a subject in need of such control which comprises administering to the subject a composition comprising a therapeutically effective amount of a compound capable of modulating or regulating GPR39 protein activity.
  • the present invention relates to the involvement of GPR39 signalling in glucose regulation and thus in glycaemia observed in diabetes and in metabolic syndrome including obesity, diabetes, cardiovascular diseases, atherosclerosis, atherogenic dyslipidemia, hypertension, hypertriglyceridemia and adipose tissue disorders.
  • 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.
  • 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 or 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.
  • GPR39 has a wide tissue distribution.
  • an alternate transcript, 3-kb in length was observed in several peripheral tissues such as stomach and small intestine. In tissues such as pancreas, thyroid and colon, this 3-kb species was the only transcript detected (McKee et al . ,
  • 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) .
  • 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
  • WO2004/004279 gastrointestinal and liver diseases
  • GPR39 has been postulated as a novel anti-obesity target. Nonetheless, in none of the cited references a functional role for GPR39 in energy homeostasis and in particular in carbohydrate metabolism has been provided.
  • Type 2 diabetes the more prevalent type of diabetes, is usually characterized by gradual onset and occurs mainly in people over 40.
  • Type 2 diabetes is a metabolic disorder resulting from the body's inability to make enough insulin or to properly use insulin to meet the body's needs, especially when the person is overweight. It is the most common form of the disease and accounts for 90 to 95 percent cases of diabetes. Initially the combination of dietary measures, weight reduction and oral medication can keep the condition under control for a period of time, but most people with Type 2 diabetes ultimately require insulin injections.
  • Impaired carbohydrate metabolism, and in particular diabetes or metabolic syndrome is also associated with several complications, including (poly) neuropathy (peripheral, autonomic, proximal, focal), nephropathy, renal disease, kidney failure, bladder dysfunction, retinopathy, large- and small vessel vascular complications, stroke,, myocardial infarction, large vessel occlusive disease, coronary (ischemic) artery disease, cerebral vascular disease, heart failure, peripheral arterial disease, hypertension, sexual dysfunction, gastroparesis. Said complications will also benefit from a treatment of the impaired carbohydrate metabolism according to the present invention.
  • the present invention concerns identification of novel functions of the GPR39 receptor.
  • GPR39 mutations in mammals affect blood glucose concentrations and designate GPR39 as a key element in the regulation of carbohydrate metabolism.
  • This discovery provides an avenue for new therapeutic approaches in the treatment of diabetes including associated complications thereof, or of the metabolic syndrome including associated complications thereof through modulation of GPR39 activity.
  • This discovery provides an avenue for new therapeutic approaches in the treatment of diabetes including associated complications thereof, or of the metabolic syndrome including associated complications thereof through modulation of GPR39 activity.
  • This discovery also provides for new screening methods for identifying compounds useful for the prevention or the treatment of diabetes including associated complications thereof, or of the metabolic syndrome including associated complications thereof .
  • 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 80% and preferably at least 90%, 95%, 96%, 97%, or 98% sequence identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID N0:4.
  • 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 1 N-terminal region may be used to reconstitute the extracellular portion of the receptor to provide receptor binding sites.
  • fragments will retain the ability to bind an agent known to bind to GPR39, including the natural ligand as well as agonists and/or antagonists of the receptor as defined hereinafter, in particular retaining the capability to bind the putative ligand of GPR39, i.e. to bind obestatin.
  • 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 enhance glucose control in a subject and which are effective for preventing and/or treating pathologies related with an impaired carbohydrate metabolism, in particular in the prevention and/or treatment of diabetes including associated complications thereof, or of the metabolic syndrome including associated complications thereof.
  • 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 80% and preferably at least 90%, 95%, 96%, 97% 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 80% and preferably at least 90%, 95%, 96%, 97% 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 0 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.
  • IX SSC 0.1% SDS.
  • suitable conditions include hybridization overnight at 65 0 C in 0.25M Na 2 HPO 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60 0 C in 0. IX SSC, 0.1% SDS.
  • 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 preventing and/or treating pathologies related with an impaired carbohydrate metabolism, in particular in the prevention and/or treatment of diabetes including associated complications thereof , or of the metabolic syndrome including associated complications thereof.
  • 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.
  • an "antagonist” as used herein refers to agents that attenuate the effects of an agonist for GPR39.
  • Antagonism can be competitive and reversible (i.e. it binds to a region of the receptor in common with the agonist and can be replaced with a sufficient large amount of agonist) or it can be non-competitive and/or irreversible (i.e. the antagonist binds covalently to the binding site and no amount of agonist can overcome the inhibition) .
  • Other types of antagonism are non-competitive antagonism wherein the agent binds to an allosteric site of the receptor or inverse agonism wherein for a constitutive receptor, as reported for GPR39 (Hoist et al . , 2004), the agent binds to the agonist binding site and attenuates the constitutive activity of the receptor.
  • the present invention provides a method of treating disease conditions related with an impaired carbohydrate metabolism, in particular in the prevention and/or treatment of diabetes including associated complications thereof, or of the metabolic syndrome including associated complications thereof. Said method comprising in those conditions wherein a decrease in basal glucose levels is required, administering to the human or animal a therapeutically active dosage of a GPR39 receptor agonist.
  • the use of a GPR39 agonist is of particular use in the treatment of the metabolic syndrome including associated complications thereof , in particular comprising the use of a GPR39 agonist identifiable using a method of the present invention.
  • said method comprises 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.
  • 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 human obestatin
  • SEQ ID No : 6 is monkey obestatin
  • SEQ ID No: 7 is mouse obestatin
  • SEQ ID No: 8 is rat obestatin
  • SEQ ID No: 11 is cat obestatin
  • SEQ ID No: 12 is dog obestatin
  • SEQ ID No: 13 is goat obestatin
  • SEQ ID No: 15 is Cattle obestatin
  • FIG. 1 GPR39-LacZ expression in pancreas.
  • expression was detected in the islets of
  • FIG. 2 Comparison of the body weight of GPRSg ' '' " and WT mice followed in both male and female animals from the age of 8 - 12 up to 16 - 20 weeks. Data are expressed in mean ⁇ SD .
  • the dog and cattle sequences in the alignment differ from the original public domain sequence: the C-terminal amino acids lacking similarity to other GPR39 sequences were removed. The argument is that deleted amino acids were from lower quality sequencing results.
  • the human sequence is used as reference and amino acids are coloured grey. Amino acids of other sequences are coloured if similar to the human sequence.
  • Figure 6B provides a matrix that shows identity, similarity and gaps between every sequence. There are more gaps between dog, cattle sequence and all other sequences, because former sequences are partial. For the overlapping fragments the degree of identity is given by the sum of the %identity with the %gaps . For example, for dog with human the degree of identity for the overlapping fragment is 86%.
  • 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 NO : 1 or sequences comprising SEQ ID NO : 1 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.
  • isolated indicates that a naturally occurring sequence has been removed from its normal cellular context.
  • the sequence may be in a cell- free solution or placed in a different cellular environment or nucleic acid context. Therefore, the nucleic acids claimed herein can be present as heterologous material in whole cells or in cell lysates or in a partially, substantially or wholy purified form.
  • 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 G-protein coupled receptors on a solid support under low stringency hybridising conditions (e.g. 6xSSC at 55°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 GPCR encoding nucleic acids.
  • Nucleic acids of the invention, particularly primers 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 mRNA, 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.
  • 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 . ,
  • 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) .
  • 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 N0:l, 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.
  • 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 N0:l 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.
  • 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.
  • 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 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:
  • 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 .
  • the algorithm 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.
  • 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.
  • 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 .
  • 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. In particular in a method to treat impaired carbohydrate metabolism, such as diabetes including associated complications thereof. In gene therapy an isolated 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) .
  • 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
  • the assays as provided herein may comprise in addition to all or part of the GPR39 receptor protein, G proteins or other accessory proteins.
  • GPCR's are known to interact with G proteins, as well as with other accessory proteins including arrestins, receptor activity modifying proteins (RAMPs) , PDZ domain containing proteins and others. Interaction of a GPCR with such accessory protein may result in the determination of various important biological properties of a receptor such as transport to or from the membrane, definition of its pharmacology, determination of its glycosylation state or transmission of signal through mechanisms that function independent of G protein coupling (Laburthe et al 2002) .
  • association of calcitonin receptor- like receptor with RAMPl leads to expression of the calcitonin receptor- like receptor able to bind calcitonin gene-reated peptide
  • association with RAMP2 or RAMP3 leads to expression as an adrenomedullin binding receptor (Latchie et al 1998) .
  • a similar interaction of RAMPs has been documented for the VPACl- * , glucagon-, PTHl- and PHT2- receptors (Christopoulos et al 2001, 2003), suggesting a more generalised role for accessory proteins in modulating G protein-coupled receptor signaling.
  • 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 enhances glucose regulation in a subject and which are effective for preventing and/or treating pathologies related with an impaired carbohydrate metabolism, in particular in the prevention and/or treatment of diabetes including associated complications thereof, or of the metabolic syndrome including associated complications thereof, which method comprises: (i) contacting membrane preparations from host cells expressing all or part of the GPR39 receptor protein according to the invention, with said compound under conditions suitable for binding, and (ii) detecting binding of the compound to said receptor protein.
  • ⁇ for preventing and/or treating pathologies related with an impaired carbohydrate metabolism, in particular in the prevention and/or treatment of diabetes including associated complications thereof, or of the metabolic syndrome including associated complications thereof, 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 (i-i) 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 .
  • the compound known to bind to the receptor consists of obestatin, more in particular selected from one of the obestatin sequences selected from SEQ ID No's 5 to 16, more in particular SEQ ID No.5 or SEQ ID No .7.
  • 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 : 5 or SEQ ID NO : 7.
  • 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.
  • 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 80% and preferably at least 90%, 95%, 96%, 97%, 98% or 99% 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.
  • fragments will retain the ability to bind the compound known to bind to the GPR39 receptor protein. More in particular, fragments will retain the ability to bind obestatin as defined hereinbefore.
  • 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 ⁇ t ⁇ M Tris HCl, .1 mM EDTA, pH 7.4 at 4°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) .
  • 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 VoI .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] jjal 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.
  • the pellets were resuspended with buffer A without KCl, quickly frozen under liquid nitrogen, and stored at -80 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.
  • said assays can be used to identify compounds that may affect glucose homeostasis in a subject, including humans and warm-blooded animals.
  • a compound that modulates the activity of a polypeptide of the invention refers to a compound 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.
  • the present invention provides a method for identifying compounds capable to decrease GPR39 activity, said method comprising;
  • test compound identifying the test compound as a compound capable to normalise glucose tolerance in a human or animal, i.e identifying the test compound as useful in the treatment of diabetes including associated complications thereof .
  • the aforementioned activity assays can also be used to identify compounds that would modulate carbohydrate metabolism. Based on the observed phenotype in the glucose tolerance test, it is to be expected that compounds that increase GPR39 activity would result in a lowering of blood glucose tolerance and compounds that decrease GPR39 activity would result in an increase in blood glucose tolerance.
  • the present invention provides a method for identifying compounds capable to decrease blood glucose tolerance said method comprising; (i) contacting host cells expressing all or part of the GPR39 receptor protein with the compound to be tested under conditions permitting the activation of said receptor protein, and (ii) detecting any increase in GPR39 receptor activity, thereby identifying the test compound as a compound capable to decrease blood glucose tolerance.
  • 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.
  • bioassay methods for identifying compounds that modulate the activity of receptors such as polypeptides of the invention generally require comparison to a control.
  • One type of control 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 control cell or culture is not exposed to ' the compound.
  • Another type of control cell or culture that can be employed is a cell or culture that is identical to transfected cells, the exception that the control cell or culture does not express functional GPR39 GPCR. Accordingly, the response of the transfected cell can be compared with that of the control 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)
  • GIRK inward rectifying potassium channels
  • 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-/3S4-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 mM 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. General guidelines for transient receptor expression, cell preparation and microphysiometric recording are described elsewhere (Salon, J. A. and Cwicki, J. A., 1996). Typically cells expressing receptors are harvested and seeded at 3 x 10 5 cells per microphysiometer capsule in complete media 24 hours prior to an experiment. The media is replaced with serum free media 16 hours prior to recording to minimize non-specific metabolic stimulation by assorted and ill-defined serum factors.
  • the cell capsules are transferred to the microphysiometer and allowed to equilibrate in recording media (low buffer RPMI 1640, no bicarbonate, no serum (Molecular Devices Corporation, Sunnyvale, CA) containing 0.1-1% fatty acid free BSA), during which a baseline measurement of basal metabolic activity is established.
  • recording media low buffer RPMI 1640, no bicarbonate, no serum (Molecular Devices Corporation, Sunnyvale, CA) containing 0.1-1% fatty acid free BSA
  • 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.
  • 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.
  • the choice of carrier or diluent will of course depend on the proposed route of administration, which, may depend on the agent and its therapeutic purpose.
  • the 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.
  • 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.
  • 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.
  • 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.
  • diabetes including associated complications thereof
  • Type 1 insulin-dependent or IDDM
  • Type 2 non-insulin-dependent diabetes mellitus
  • MODY maturity-onset diabetes of the young
  • gestational diabetes said composition comprising a GPR39 receptor antagonist.
  • the present inventions also provides for a diagnostic product comprising an isolated nucleic acid sequence selected from the group consisting of;
  • N0:l or SEQ ID NO : 3 or (iii) a nucleic acid sequence having at least 80% sequence identity, and preferably at least 90%, 95%, 96%,
  • DNA, antisense DNA, siRNA of the present invention as probes, a gene abnormality of the DNA or mRNA encoding the GPR39 receptor or parts thereof in mammals including humans, can be detected and therefore these nucleic acid sequences are useful gene diagnostic agents for damage of the DNA or mRNA encoding the GPR39 receptor or parts thereof, mutations thereof, underexpression thereof or overexpression thereof.
  • the above described gene diagnosis can be performed by, for example, publicly known Northern hybridization assay or PCR-SSCP assay.
  • the GPR39 receptor When an underexpression of the GPR39 receptor is detected, it can be diagnosed that the subject suffers from a disease associated with dysfunction of the GPR39 receptor, such as for example metabolic syndrome including associated complications thereof.
  • an overexpression of the GPR39 receptor When an overexpression of the GPR39 receptor is detected, it can be diagnosed that the subject suffers from a disease associated with overexpression of the GPR39 receptor, such as for example diabetes including associated complications thereof.
  • the present invention also provide for a diagnostic product comprising all or part of the GPR39 receptor protein.
  • Said protein can for example be detected in a suitable sample, for example a blood sample, in order to diagnose under- or overexpression of the GPR39 receptor.
  • 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.
  • Pancreas was dissected from WT and GPR39 ⁇ / ⁇ mice and whole- mount LacZ staining was performed. Briefly, tissue was dissected into PBS solution on ice, fixed for 2 h at 4°C in 0.5% glutaraldehyde in PBS. Next tissue was rinsed 3 times in PBS with gentle shaking. Staining was performed overnight at 30 0 C in a staining buffer supplemented with lmg/ml X-gal, 5mM potassium ferricyanide and 5mM potassium ferrocyanide . After staining, tissue was rinsed in PBS wash buffer, dehydrated in alcohol, cleared in xylol embedded in paraffin and sectioned at 5 ⁇ m. Histological examination was performed on lO ⁇ m tissue sections with and without Harris H&E counterstaining.
  • Animals were housed individually in cages at 22 ⁇ 1°C with a 12h-12h light-dark cycle. Animals were fed ad libitum with a standard diet (22% proteins, 4.3% fat and 4% cellulose from U.A. R. , France) . Body weight was followed in these animals starting at the age of 8-12 up to 16-20 weeks .
  • Glucose tolerance test in WT and GPR39 -/ ⁇ mice GPR39 ⁇ / ⁇ and WT mice (male and female) were fasted overnight (starting at 5.30 p.m.) and then injected subcutaneousIy with 2 g/kg glucose (saline solution at a final concentration of 200 mg/ral) . Blood samples were taken by tail venesection. Firstly baseline was taken before weighing animals, then glucose was administered. Blood glucose concentration was determined at 15, 30, 45, 60, 90 and 120 min post glucose administration by means of One Touch Ultra (Lifescan Inc., USA) .
  • mice Body weight was followed in these animals starting at the age of 8-12 up to 16-20 weeks. Male GPR39 '/" mice gained more weight than the corresponding male wild type mice
  • Body composition was measured in mice at the age of 13-17 and 16-20 weeks. In 13-16 weeks-old mice no difference was observed in body composition between both genotypes fed with standard diet. Three weeks later, in the same animals, body fat in male GPRSi-T'' " mice appeared increased compared to male wild type mice (15.76+5.30% vs. 11.97+2.60%) while body lean mass appeared decreased in male GPR39 "7" compared to wild type mice (41.22+3.41% vs. 43.45 ⁇ 1.78%) (Fig.5) . Discussion
  • Ghrelin/motilin-related peptide is a potent prokinetic to reverse gastric postoperative ileus in rat. Am. J. Physiol. 282, G948-G952.

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Abstract

La présente invention concerne la caractérisation fonctionnelle du récepteur couplé à la protéine G GPR39 ainsi que des composés qui modifient ou régulent l'activité de la protéine GPR39. En particulier, la présente invention concerne des méthodes de criblage d'agonistes ou antagonistes du GPR39 afin d'identifier des composés capables de moduler le métabolisme des hydrates de carbone, ainsi que les utilisations thérapeutiques de ces composés, en particulier l'utilisation du GPR39 dans des méthodes d'identification de composés qui sont capables d'améliorer la régulation du glucose chez un sujet et qui sont efficaces dans la prévention et/ou le traitement de pathologies liées à une compromission du métabolisme des hydrates de carbone, en particulier dans la prévention et/ou le traitement du diabète, y compris les complications associées à ce dernier, ou du syndrome métabolique, y compris les complications associées à ce dernier, comprenant le diabète de type 1 (insulinodépendant ou DID), le diabète de type 2 (non insulinodépendant ou DNID), le syndrome MODY (maturity-onset diabetes of the youth) et le diabète gestationnel.
EP07729995A 2006-06-08 2007-06-08 Récepteur couplé à la protéine g 39 (gpr39) Withdrawn EP2030024A1 (fr)

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EP4070797A4 (fr) * 2019-11-01 2023-11-29 Peking University Utilisation d'acides biliaires et de dérivés de ceux-ci dans la préparation d'un agoniste gpr39
US20230174484A1 (en) 2020-04-30 2023-06-08 Oregon Health & Science University Antagonists of gpr39 protein
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CA2652398A1 (fr) 2007-12-13

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