CN101467047A - G protein-coupled receptor 39 (GPR39) - Google Patents

G protein-coupled receptor 39 (GPR39) Download PDF

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CN101467047A
CN101467047A CNA2007800212075A CN200780021207A CN101467047A CN 101467047 A CN101467047 A CN 101467047A CN A2007800212075 A CNA2007800212075 A CN A2007800212075A CN 200780021207 A CN200780021207 A CN 200780021207A CN 101467047 A CN101467047 A CN 101467047A
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gpr39
seq
compound
sequence
diabetes
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B·C·J·-C·莫罗克斯
D·W·E·莫查斯
L·A·L·弗唐克
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Janssen Pharmaceutica NV
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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
    • G01N33/53—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

Abstract

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. In particular the present invention relates to methods of screening for agonists or antagonists of GPR39 in order to identify compounds capable of modulating carbohydrate metabolism and to the therapeutic uses of these compounds. In particular to the use of GPR39 in methods to identify compounds that are capable to 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. Including Type 1 (insulin-dependent or IDDM), Type 2 (non- insulin- dependent diabetes mellitus), maturity-onset diabetes of the young (MODY) and gestational diabetes.

Description

G protein coupled receptor 39
Invention field
The menu that the present invention relates to g protein coupled receptor GPR39 is sought peace and can be modified or regulate the compound of GRP39 protein active.Specifically, the present invention relates to comprise the composition of using the dose therapeutically effective that to regulate the GRP39 protein active to the experimenter to there being the experimenter who needs to strengthen the method for blood sugar (glucose) control.Another embodiment of the present invention relates to the GRP39 signal conduction relevant with hyperglycemia in blood glucose-control and diabetes and the metabolic syndrome, and described metabolic syndrome comprises obesity, diabetes, angiocardiopathy, arteriosclerosis, atherogenic dyslipidemia, hypertension, hypertriglyceridemia and adipose tissue disease.
Background of invention
GTP-is the middle element that parts such as hormone and other chemical mediator combine with g protein coupled receptor (GPCRs) in conjunction with albumen (G albumen), can the interior effector of active cell.Part is with after GPCR combines, and acceptor bag slurry district occurred conformation changes, and makes acceptor and G protein-interacting, thus the interior middle element of active cell, for example adenyl cyclase, phospholipase C or ion channel.This system can amplify initialize signal, and reason is that single part combines the many second messengers of back generation with GPCR.By this mechanism, cell is to the change that can experience its external environment and react.
G protein coupled receptor forms plasmalemma protein matter (integral plasma membrane proteins) superfamily, all acceptors all have common trait: 7 hydrophobicity membrane spaning domains, the long 20-30 of each a domain amino acid residue is by the hydrophile amino acid sequence connection of different length.The amino terminal of acceptor is positioned at the extracellular, and carboxyl terminal is positioned at endochylema.
GPCRs has tissue and cell distribution widely, participates in multiple different physiology course.GPCRs can be activated by multiple part, for example, hormone such as lutropin, follicular stimulating hormone, human chorionic gonadtropin, thyroid-stimulating hormone, corticotropin, hyperglycemic factor, antidiuretic hormone or neurotransmission hormone such as serotonin, acetylcholine (mAChR), histamine, prostaglandin, calcitonin, leukotriene and calcium ion etc.The extensive distribution of GPCRs and multiple effect show that it plays a significant role in multiple pathological state.Really, the multiple disease that GPCRs participates in relating to bronchiostenosis, hypertension, inflammation, hormone disturbance, diabetes, apoptosis, nociception, neurotransmission facilitation and trembles etc..
In the g protein coupled receptor superfamily, the supposition GPCRs of native ligand the unknown is called " orphan receptor ".Confirmed that g protein coupled receptor is the valuable drug target, because the action target of about 50% medicine is GPCRs on the market.Thereby multiple orphan GPCR is just evaluated to find new potential target.
The sequence of GPR39 is similar to secretagogue receptor (GHS-R) and neurotensin (neurotensin) acceptor 1 and 2 (NT-R1 and NT-R2) (McKee et al., 1997).The GRP39 albumen that prediction contains 453 amino acid residues comprises 7 typical GPCRs membrane spaning domains.With other GPCRs sequence contrast, McKee et al. (1997) finds that the homogeneity of GPR39 protein sequence and GSHR, MTLR1 (motilin acceptor), neurotensin receptor-1 is respectively 27%, 29% and 32%.Northern blot analyzes demonstration, and GPR39 has Tissue distribution widely.In great majority experiment brain tissue, find single mRNA spliceosome of 1.8-2kb.Yet, in multiple peripheral tissues such as stomach and small intestine, except that the type, also find the spliceosome of another kind of 3-kb.In tissues such as pancreas, thyroid gland and colon, only find the spliceosome (McKee et al., 1997) of 3-kb.McKee et al. (1997) with fluorescence in situ hybridization with the GPR39 assignment of genes gene mapping in 2q21-q22.The acidic residues of TM3 is guarded among the GPR39.Known this residue for the GHSs of different structure in conjunction with and activate GHS-R and play an important role.
Based on research to the GPR39 Tissue distribution, suppose that GPR39 may participate in angiocardiopathy (WO2001/081634 and WO2004/004279), tumour is brain tumor such as glioblastoma (WO2001/036685 and WO01042288) especially, inflammation, the nervous system disease (US 2003/232769 and WO2004/004279), intestines and stomach and liver diseases (WO2004/004279).Find that recently fat inhibin is the supposition part (Zhang J.V.et al., 2005) of GPR39.Discover that fat inhibin suppresses food intake, reduce gastrointestinal function and comprise gastric emptying and jejunum wriggling.Because GPR39 can make anorexia, supposes that it is a kind of novel anti-fat target.But the list of references that this paper quotes provides GPR39 in the energy equilibrium especially effect in carbohydrate metabolism without any one piece.
Diabetes are diseases that the insulin function deficiency causes blood sugar, protein and abnormalities of sugar/lipid metabolism to cause.The characteristic feature of diabetes comprises that blood sugar level increases unusually, insufficient insulin and glucose in urine occurs.
Diabetes have several clinical subtypes, comprising: type 1 diabetes (insulin-dependent diabetes or IDDM), diabetes B (insulin dependent/non-dependent diabetes), young maturity-onset diabetes (MODY) and gestational diabetes.The cause of disease of dissimilar diabetes, pathology, science of heredity, age of onset and methods of treatment are different.
1 type diabetes, more serious one type, account for the 5%-10% of diabetes, mostly occur the teenager.This type diabetes body does not produce any insulin, if there is not regular injection of insulin, the patient will fall into a coma and be dead.The type 1 diabetes patient is that insulin relies on fully.
2 type diabetes, more common one type, it is characterized in that falling ill gradually, mainly occur in the crowd more than 40 years old.Diabetes B is metabolic disease, the especially obese patient that body can not produce enough insulin or can not suitably utilize insulin to cause to satisfy the body needs.Diabetes B is modal type, accounts for the 90%-95% of diabetes.Beginning unites and use dietary therapy, lose weight and oral drugs can be in a period of time inner control state of an illness, but most of diabetes B patients needs injection of insulin at last.
Diabetes can be controlled with insulin, can pass through diet control in certain case, but need a kind of spinoff minimum and do not have a safe and effective diabetes remedy of the invasive operation of injection of insulin.
Carbohydrate metabolism is impaired, and especially diabetes or metabolic syndrome can cause several complication, comprises DPN (peripheral nerve pathology, autonomic neuropathy, nearly section DPN, local nerve pathology), ephrosis, kidney failure, bladder function disorder, retinopathy, trunk and microvascular complication, apoplexy, miocardial infarction, the inaccessible disease of trunk, coronary artery disease (ischemic), cranial vascular disease, heart failure, peripheral arterial disease, hypertension, sex dysfunction, gastroparesis.The present invention also can improve above-mentioned complication symptom to the impaired treatment of carbohydrate metabolism.
Summary of the invention
As mentioned above, the present invention relates to identify the new function of GPR39 acceptor.Shown in hereinafter embodiment, mammal GPR39 sudden change influences blood sugar concentration, shows that GPR39 is a key factor of regulating carbohydrate metabolism.This discovery provides a kind of and has comprised that by regulating GPR39 active treatment diabetes its related complication or metabolic syndrome comprise the new method of its related complication.This finds also to provide and identifies that can prevent or treat diabetes comprises that its related complication or metabolic syndrome comprise the novel screening methods of the compound of its related complication.
Therefore, a first aspect of the present invention provides GRP39 albumen all or part ofly can strengthen experimenter's glycemic control and can effectively prevent and/or treat purposes in the method with the compound of the impaired relevant pathological state of carbohydrate metabolism (especially prevent and/or treat diabetes and comprise its related complication, or metabolic syndrome comprises its related complication) identifying.In addition, the present invention also provides the purposes of all or part of cell in this method of expressing GPR39 albumen.In one specific embodiment, GPR39 is a kind of protein isolate, amino acid sequence with the protein splice variant that is selected from SEQ IDNo:2, SEQ ID NO:4, above-mentioned SEQ ID, have 80% at least with amino acid sequence, the amino acid sequence of preferred at least 90%, 95%, 96%, 97% or 98% sequence homogeneity with SEQ ID NO:2 or SEQ ID NO:4.
The part of GPR39 albumen mentioned above refers to the fragment of the polypeptide that comprises SEQ ID NO:2 or SEQID NO:4, and this fragment has 10 at least, and for example at least 20,30,40,50,75,100 or 150 or more a plurality of amino acid.This fragment can be derived from the N-end region of SEQ ID NO:2 or SEQ ID NO:4 respectively.The fragment that comprises the N-end region can be used for reformulating the outer part of born of the same parents of acceptor, so that receptor binding site to be provided.Preferably, polypeptide fragment keeps the ability in conjunction with certain medicament, known described medicament can combine with GPR39, comprises native ligand and receptor stimulating agent hereinafter described and/or antagonist, especially keeps in conjunction with GPR39 supposition part promptly in conjunction with the ability of fat inhibin.
The all or part of separated nucleic acid sequence that the present invention also provides polypeptide among coding SEQ ID NO:2 or the SEQ ID NO:4 is being identified the glycemic control that can strengthen the experimenter and being is effectively is being prevented and/or treated purposes in the method with the compound of the impaired relevant pathological state of carbohydrate metabolism (especially prevent and/or treat diabetes and comprise its related complication, or metabolic syndrome comprises its related complication).Used nucleotide sequence comprises a kind of isolated nucleic acid sequences in the inventive method, it is selected from SEQ ID NO:1 or SEQ ID NO:3, have 80% at least with nucleotide sequence, the nucleotide sequence of preferred at least 90%, 95%, 96%, 97% or 98% sequence homogeneity with SEQ ID NO:1 or SEQ IDNO:3.
Nucleic acid of the present invention also comprises containing with SEQ ID NO:1 or the nucleotide sequence of SEQ ID NO:3 or their complementary series to have 80% at least, the nucleic acid of the sequence of preferred at least 90%, 95%, 96%, 97% or 98% sequence homogeneity.Preferably, these sequences can be hybridized under controlled condition with corresponding nucleic acids, so that non-specific binding is reduced to is minimum.The hybridization conditions of preferred strictness or moderate strictness.For example, for the sequence that detects about 80%-90% homogeneity, suitable condition comprises: at 0.25M Na 2HPO 4, pH7.2,6.5% SDS, in the 10% dextran sulfate solution, 42 ℃ of hybridization are spent the night, and use 0.1X SSC at last, 0.1% SDS, 55 ℃ of washings down.Detect homogeneity and surpass about 90% sequence, suitable condition comprises: 0.25M Na 2HPO 4, pH7.2,6.5% SDS, in the 10% dextran sulfate solution, 65 ℃ of hybridization are spent the night, and use 0.1X SSC at last, 0.1% SDS, 60 ℃ of washings down.
Should be appreciated that such nucleic acid " total length " polypeptide of may not encoding, thereby they comprise for example nucleic acid of GPR39 gene mutation form, wherein coded sequence is owing to form the replacement of terminator codon or frameshift mutation and crossed early stopping.These nucleic acid also are the nucleic acid among the present invention.
The present invention also provides the purposes of fragment of the nucleic acid of code book invention polypeptide.One aspect of the present invention provides nucleic acid primer, and it is formed as the nucleotide of 15-35,18-35,15-24,18-30,18-21 or 21-24 position basically by 15-50 position in the sequence of code book invention polypeptide or its complementary series.
Nucleic acid among the present invention and polypeptide can be used for treating disease.Specifically, they can be used for treating pathological change and act on the receptor related disease of GPR39, especially prevent and/or treat the impaired diseases associated of pathological change and carbohydrate metabolism, especially prevent and/or treat diabetes and comprise that its related complication or metabolic syndrome comprise its related complication.
On the other hand, the invention provides the carrier that comprises described nucleotide sequence, especially comprise the expression vector of the promoter that is operably connected with nucleotide sequence of the present invention.Carrier can be carried and expressed in this cell by host cell.After the expression, this cell can be used for method of the present invention.
As mentioned above, one of purpose of the present invention provides the experimental technique of authenticating compound (hereinafter also being medicament), and described compound is in conjunction with the activity of polypeptide of the present invention or adjusting polypeptide of the present invention.Especially, this compounds can be the organic or inorganic thing of any size, comprises micromolecule (molecular weight is no more than about 2500 dalton) or big molecule for example peptide, polypeptide, intact proteins and polynucleotide, and wherein this compound can be used for above-mentioned methods of treatment.
Because it is the key factor of regulating carbohydrate metabolism as a kind of acceptor that the present inventor at first finds GPR39, so the present invention has disclosed with the activator of GPR39 acceptor itself and/or this receptor or the possibility that antagonist is treated.Therefore, the present invention further provides the method for the treatment mankind or Animal diseases, this method comprises uses GPR39 activator or antagonist." activator " used herein refers to combination and activates GPR39, promptly produces the medicament of pharmacological reaction, comprises positive allosteric modulators, and its binding site on acceptor is different with receptor stimulating agent, can strengthen the reaction of acceptor to its natural activator." antagonist " used herein refers to the medicament that weakens the effect of GPR39 activator.Antagonist can be competitive and reversible (be that antagonist combines with the receptor binding domain of activator, can be substituted by a large amount of activators) or noncompetitive and/or irreversible (be antagonist and binding site covalent bond, the activator of any dosage all can not overcome its inhibiting effect).The antagonism of other type is a non-competitive antagonism, and wherein medicament combines with the allosteric site of acceptor; Perhaps reverse agonism, wherein for the composition acceptor, as the GPR39 of Holst et al. (2004) report, medicament combines with the binding site of activator, weakens the composition activity of acceptor.
Especially, the invention provides the method for a kind of treatment and the impaired diseases related of carbohydrate metabolism, especially prevent and/or treat diabetes and comprise that its related complication or metabolic syndrome comprise the method for its related complication.This method is included in to be needed to reduce under the situation of basic blood sugar level, gives the GPR39 receptor stimulating agent of human or animal's administering therapeutic active dose.The GPR39 activator comprises that to the treatment metabolic syndrome its related complication is particularly useful, especially comprises the GPR39 activator that uses method of the present invention to confirm.In addition, comprise in its related complication that in the treatment diabetes described method comprises the GPR39 receptor antagonist to human or animal's administering therapeutic active dose, especially comprise the GPR39 antagonist that uses method of the present invention to confirm.
Above aspect of the present invention and others are described in detail in this.
Sequence description
SEQ ID NO:1 is the nucleotide sequence of mouse GPR39.
SEQ ID NO:2 is the amino acid sequence of mouse GPR39.
SEQ ID NO:3 is the nucleotide sequence of people GPR39.
SEQ ID NO:4 is the amino acid sequence of people GPR39.
SEQ ID No:5 is people's a fat inhibin sequence.
EQ ID No:6 is the fat inhibin sequence of monkey.
SEQ ID No:7 is the fat inhibin sequence of mouse.
SEQ ID No:8 is the fat inhibin sequence of rat.
SEQ ID No:9 is the fat inhibin sequence of gerbil jird (gerbil).
SEQ ID No:10 is the fat inhibin sequence of pig.
SEQ ID No:11 is the fat inhibin sequence of cat.
SEQ ID No:12 is the fat inhibin sequence of dog.
SEQ ID No:13 is the fat inhibin sequence of goat.
SEQ ID No:14 is the fat inhibin sequence of sheep.
SEQ ID No:15 is the fat inhibin sequence of ox.
SEQ ID No:16 is the common sequences of fat inhibin.
The accompanying drawing summary
The expression of GPR39-LacZ in Fig. 1 pancreas.At GPR39 -/-In the pancreas, pancreas islet (isletsof Langerhans) (annular inner region) can see expression that blue dyeing is arranged (Figure 1A., 40x).In wild type pancreas, occur dyeing (Figure 1B., 40x).
Fig. 2 contrasts thoughtful 16-20 week of 8-12, GPR39 -/-The body weight of the male and female mice of type and wild type.Data are represented with mean value ± SD.
Fig. 3 contrasts GPR39 -/-Type and wild type male and female mice feed back (A) and empty stomach (B) basic blood sugar concentration.
Fig. 4 contrasts GPR39 -/-Male and the female mice sugar tolerance experiment on an empty stomach of type and wild type.Measure behind the glucose load 15,30,45,60,90 and 120 minutes blood sugar concentration respectively.The result changes number percent with basic blood glucose value and represents.Data are represented with mean value ± SD.
Fig. 5 contrasts GPR39 -/-The physique composition of the male and female mice of type and wild type.Fat body weight and fat-free mass (lean mass) are represented with the number percent of body weight.Data are represented with mean value ± SD.
Fig. 6 A is that the arrangement of people (NP 001499), mouse (AAH85285), rat (XP 222578), dog (part) (XP 853332) and ox (part) (XP 589836) GPR39 protein sequence is compared.People GPR39 protein sequence is in the top in the tabulation, and the similar people's of protein sequence of other species protein sequence is arranged downwards successively.The name of each sequence is reacted its numbering, gene symbol and species name in NCBI nr database.
The protein sequence of dog and ox is different from original common sequence in the tabulation: lack homophylic C-end amino acid with other GPR39 sequence and be removed.Reason is that deleted amino acid is that the inferior quality sequencing result causes.
People's protein sequence is as reference, and its amino acid shows grey.If the amino acid of other sequence is similar to Show Color with people's sequence.
Fig. 6 B shows homogeneity, similarity and the room (gap) between the various sequences.Room between the sequence of dog, ox and other sequence is more, because the former is a partial sequence.For overlapping fragments, its consistent degree is represented by homogeneity number percent and room number percent.For example, the homogeneity of dog and people's overlapping fragments is 86%.
Detailed Description Of The Invention
Nucleic acid
The nucleic acid that is used for the inventive method comprise DNA (comprising genome and cDNA) and RNA. Nucleic acid of the present invention comprises RNA, quote the sequence that provides in the subordinate list should be interpreted as with U replaces the RNA coordinate of T.
Nucleic acid of the present invention is strand or two strands. Single-chain nucleic acid of the present invention comprises antisensenucleic acids. Thereby be to be understood that the sequence of quoting SEQ ID NO:1 or comprising SEQ ID NO:1 or its fragment, The complementary series that comprises them is unless there is in addition opposite explicit stipulation in the context. This is equally suitable Be used for SEQ ID NO:3.
Usually, nucleic acid of the present invention is separator, exists with the isolated or purified form, perhaps trip From or substantially be free on the material of its natural combination, for example free or substantially be free on human genome In the nucleic acid flanking gene, except one or more adjusting sequences of participate in expressing. Nucleic acid can Be all or part of synthetic, also can comprise genomic DNA, cDNA or RNA. In other words Say that " separation " shows that the sequence of natural appearance in the cell breaks away from normal cellular environment. Cause And sequence may be present in the acellular solution or be present in different cellular environments or nucleic acid In the environment. Therefore, the nucleic acid of this claim may be the allos thing, be present in intact cell or In the cell lysate or with the part, basically or the Economical Purification form exist.
The present invention also provides the nucleic acid fragment of code book invention polypeptide. One of them side of the present invention Face provides nucleic acid primer, and it is basically in the sequence or its complementary series by code book invention polypeptide The 15-50 position is such as the nucleotides of 15-35,18-35,15-24,18-30,18-21 or 21-24 position Form.
Term " basically by ... form " refer to and do not comprise 5 ' or the nucleic acid of 3 ' additional nucleotide sequence. Yet, in another aspect of this invention in, nucleic acid of the present invention is basically by 15-30 nucleotides Form, its 3 ' end but preferred 5 ' end are attached with short and small (for example containing 4-15, such as 4-10 nucleotides) Add sequence and connect, but be not to be connected with this appended sequence is natural. This class appended sequence preferably comprises The connector of restriction enzyme site (linker) is when nucleic acid of the present invention drawing as the PCR reaction During thing, this connector is conducive to the clone.
Primer of the present invention can with the nucleic acid selective cross of code book invention polypeptide. " selectively " It is relevant to refer to selective sequence with other purinoceptor of coding, especially with except adenine is subjected to Receptor related outside the body. The ability of sequence selective hybridization can be by experiment or calculative determination.
For example, a kind of method of calculating the primer melting temperature is incorporated in the formula by reference, uses In the Tm that calculates homology target sequence primer. Formula be Tm (℃)=2 (A+T)+4 (G+C)-5. (wherein SSC contains 0.15M NaCl, 0.015M to this formula at 3xSSC and 0.1%SDS Natrium citricum, pH7) Tm under the condition. Usually, this formula is applicable at the most about 50 nucleosides The primer of acid length. In the present invention, this formula can be used as algorithm calculation code polypeptide of the present invention The nominal Tm of primer of appointment nucleotide sequence. Based on any part of these other sequences Maximum coupling amount, this Tm can contrast with the calculating Tm of people and rat GPCR sequence.
The appropraite condition of primer and target sequence hybridization is available determination of experimental method also. Suitable experiment Condition comprises: make primer both with the nucleic acid hybridization of code book invention polypeptide, also with other G-of coding The nucleic acid hybridization of G-protein linked receptor, the hybridization conditions minuent is carried out in reaction at solid support Strictly (for example 6xSSC, 55 ℃); With the SSC of reduced form SSC or higher temperature for example 0.2xSSC, 45 ℃ of washings; The rising hybridization temperature is to determine hybridization conditions, under this hybridization conditions Primer only with the nucleic acid hybridization of code book invention polypeptide, assorted with the nucleic acid of other GPCR of coding Hand over.
Nucleic acid of the present invention, especially primer carry indicative mark. Suitable mark bag For example draw together radio isotope32P or35S, fluorescence labeling, enzyme labeling or other oroteins mark Such as biotin. These marks are connected on polynucleotides of the present invention or the primer, can be by the technology people The known technology for detection of member arrives.
Primer of the present invention can comprise synthetic nucleic acid, for example for strengthening nucleic acid intracellular steady The qualitative nucleic acid that skeleton structure is modified. The multiple different modifying type of oligonucleotides is this area Known. Comprise methyl acid phosphate, the skeleton thiophosphorylation, 3 ' and/or 5 ' end add a word used for translation Pyridine or polylysine chain. To achieve the object of the present invention, should be appreciated that multinuclear described herein Thuja acid can adopt the existing any method in this area to modify. The purpose of modifying is to improve this Activity or life-span in the body of bright polynucleotides.
Antisense sequences based on nucleotide sequence described herein is also included within the scope of the present invention, Oligonucleotides preferably, especially stable oligonucleotides or nuclease.
ASON can with nucleic acid, mRNA precursor or ripe mRNA in complementary series Hybridization disturbs the polypeptide of set target DNA sequence coding to generate, and its expression decreased or quilt are pressed down System. Nuclease can be sheared among the present invention the nucleic acid sequence encoding of coding GPR39 GPCR MRNA, thus the special target sequence of GPR39 GPCR obtained, and namely other GPCR sequence does not have The sequence that has. The structure of antisensenucleic acids and use are at Peyman and Ulman, Chemical Reviews, 90:543-584, (1990), Crooke, Ann.Rev.Pharmacol. Toxicol., 32:329-376, (1992), and Zamecnik and Stephenson, P.N.A.S, 75:280-284 tells about in (1974). The structure of nuclease and use are at Gibson and Shillitoe, Molecular Biotechnology 7 (2): 125-137, tell about in (1997).
In an embodiment, RNA of the present invention can be used for using double-stranded RNA (dsRNA) (Fire et al., Nature 391:806-811.1998) or short interfering rna (siRNA) sequence (Yu Et al., Proc Natl Acad Sci USA.99:6047-52.2002) induce RNA to disturb (RNAi). The process of " RNAi " is that dsRNA induces the homology dependence of complementary mRNA Degraded. In an embodiment, nucleic acid molecules of the present invention is by complementary base pairing and this " justice " northern hybridization of invention forms double-stranded RNA. The antisense of dsRNA and justice Nucleic acid molecules is corresponding at least about 20,25,50,100,250 or 500 nucleotides or GPR39 Coding strand or wherein a part of. In another embodiment, siRNA have 30 or 30 with Lower nucleotides, preferred 21-23 nucleotides, have 3 of 2-3 characteristic nucleotide '-overhanging (overhanging) end is that dsRNA produces after rnase iii is sheared. Referring to Tuschl T. (Nat Biotechnol.20:446-48.2002).
The method that small RNA molecular is transcribed in cell can be that the siRNA template is cloned into The transcript unit of rna plymerase iii (Pol III), the small nut of encoding under this transcript unit's normal condition RNA (snRNA) U6 or people RNAse P RNA H1. Have two kinds of methods can express siRNAs: In an embodiment, form the positive-sense strand of siRNA two strands and antisense strand by separately startup Son is transcribed (Lee, et al.Nat.Biotechnol.20,500-505.2002); In another enforcement side In the case, after processing, siRNA forms stem pili annulati card structure (Brummelkamp et al. in cell Science 296:550-553.2002) (incorporated herein by using).
The method for preparing DsRNA/siRNA commonly used is, before being delivered to organism external Synthetic by justice and the annealing of antisense RNA chain. In another embodiment, the reality of RNAi Applying method is, earlier justice of the present invention and antisensenucleic acids placed same solution but unannealed, Or in the close time, nucleic acid is placed different solutions. Coding GPR39 or and GPR39 The nucleic acid branch of the fragment of the antisensenucleic acids of nucleic acid array complementation, homologue, derivative and analog Son provides in addition.
Antisensenucleic acids of the present invention, siRNA and nuclease sequence can be introduced into the lactation of cultivation and move Thing clone, the function of research GPR39 GPCR for example causes this gene downward modulation and observes it Phenotypic effect, or observe herein expression or the location of the GPR39 GPCR GAP-associated protein GAP of description. In the cell of GPR39 GPCR abnormal expression, these antisensenucleic acidses, siRNA and nuclease Sequence can be used for the expression of down-regulated gene.
The cDNA sequence of GPCR of the present invention can adopt Standard PC R (polymerase chain reaction,PCR) gram Grand technology is synthetic. This technology relates to a pair of 5 ' end and the 3 ' end of synthetic SEQ ID NO:1 opposite strand Primer makes primer contact with mRNA or the cDNA in mammal cortical cell source, one Carry out polymerase chain reaction,PCR under the fixed condition, amplification purpose fragment is separated the fragment of amplification (as using The gel-purified reactant mixture) also reclaims. The primer that relates to contains suitable restriction enzyme digestion mirror Anchor point can make the dna clone of amplification to suitable cloning vector. The method is equally suitable Be used for SEQ ID NO:3.
With SEQ ID NO:1 or non-100% homology of SEQ ID NO:3, but the SEQ ID that can encode The polynucleotides of NO:2 or SEQ ID NO:4 sequence or other polypeptide of the present invention can adopt multiple side Method obtains.
For example, can carry out the rite-directed mutagenesis of SEQ ID NO:1 or SEQ ID NO:3 sequence. This For for example need to the particular host cell of expressing polynucleotide sequence change reticent codon with It is useful optimizing codon preference. Other sequence may also need to change, and introduces restriction with this The property restriction enzyme site, or change character or the function of polynucleotide encoding polypeptide. Coded sequence also needs To further change, replace such as conservative.
Nucleic acid of the present invention can comprise 5 ' or 3 ' terminal appended sequence. For example, that synthesize or natural 5 ' targeting sequencing is attachable on the nucleotide sequence of code book invention polypeptide. Appended sequence also can comprise 5 ' or 3 ' non-translational region, be that particular host cell transcription nucleic acid of the present invention is needed.
In addition, other animal is mammal (such as the mankind or rabbit) especially, and especially spirit is long The class animal comprises the people, and the homologue of its GPR39 can and be used for these with method acquisition of the present invention In the method. The preparation method of these sequences is the cDNA of preparation or acquisition somatoblast or tissue Library, or the genome dna library of other animal species are then with comprising SEQ ID NO:1 Or SEQ ID NO:3 is whole or the probe of any part and library DNA hybridization, the hybridization conditions height Degree strict (such as 0.03M sodium chloride and 0.03M natrium citricum, temperature is about 50 ℃-60 ℃).
The invention still further relates to the DNA isolation sequence of the sequence that comprises code book invention polypeptide, but its Coded sequence is divided into 2 or a plurality of extron (preferably being no more than 5, such as 4 or 3). This class exon sequence can be natural and derive from genomic clone, or synthetic. Extron Can be used for making up the mini gene sequence of the nucleic acid that comprises code book invention polypeptide, its sequence is by one Or a plurality of exon sequences interrupt.
The mini gene also heterologous extron in available any eucaryon source makes up.
Polypeptide
The isolated polypeptide that uses in the inventive method can be above-mentioned unpack format, does not contain or substantially On do not contain the material of its natural combination in the cell, intracellular other polypeptide for example. Polypeptide can with Diluent or adjuvant are prepared together, and still separate for reaching practical purpose-for example, if egg Be used in vain the microtiter plate that coated immunoassays are used, usually with polypeptide and gelatin (gelatin) or Other carrier mixes. Polypeptide can be glycosylated, and glycosylation is natural or through the allos eucaryon Cell system processing also may be nonglycosylated (for example, procaryotic cell expression system table The polypeptide that reaches). Polypeptide can be phosphorylation or acetylizad.
Polypeptide of the present invention also can be the form of basic purifying, in this case, in the preparation Polypeptide is common more than 90%, is desired polypeptides of the present invention such as 95%, 98% or 99%.
Polypeptide of the present invention can be modified, as add histidine residues with auxiliary its purifying or Adding burst promotes it from the cell endocrine.
Shown in the arrangement among Fig. 6, the sequence of mammal GPR39 albumen and people or mouse has At least 80% complete sequence homogeneity. Owing to this reason, the present invention also provides the ID with SEQ NO:2 or SEQ ID NO:4 have 80% at least, and for example at least 90%, 95%, 96%, 97 The polypeptide of % or 98% sequence homogeneity. This polypeptide comprises SEQ ID NO:2 or SEQ ID NO:4 Variant, allele product, derivative or the mutant of amino acid sequence. For example, this The amino acid sequence of the polypeptide of sample can with SEQ ID NO:2 or SEQ ID NO:4 in provide The sequence difference, add therein, replacement, deletion and insertion one or more (1-20 for example, as 2,3,4 or 5-10) amino acid.
The percentage of peptide sequence homogeneity can calculate with commercially available algorithm, with to be measured Sequence and reference sequences (such as SEQ ID NO:2 of the present invention or SEQ ID NO:4) relatively draw. The detailed content of homogeneity assessment is described hereinafter.
If the homogeneity of the sequence of sequence to be measured and SEQ ID NO:2 or SEQ ID NO:4 is at least 80%, preferably at least 90%, 95%, 96%, 97% or 98%, and this polypeptide of sequence keeps the GPR39 receptor active, and this sequence constitutes a part of the present invention.
Behind the encoded expression of nucleic acid of polypeptide of the present invention, can carry out isolated or purified (as using antibody purification).The polypeptide of separation and/or purifying can be used in the composite preparation, and said preparation can comprise a supplementary element at least, for example contains the pharmaceutical composition of pharmaceutically acceptable excipient, medium or carrier.
Polypeptide of the present invention can be used as immunogene or preparation specific antibody.Antibody can be used for purifying and other processing of polypeptide, diagnostic examination and treatment disease.
Polypeptide of the present invention can be used for screening combination with it or regulates the molecule of its activity or function.This quasi-molecule can be used for treating disease (also may be used for prevent disease).
Polypeptide of the present invention or labeling polypeptide or its fragment also can be fixed on the solid phase carrier, for example the surface of immunoassays micropore or test strips (dipstick).
This class mark and/or fixing polypeptide can be packed in suitable containers with suitable reagent, contrast, instructions etc. and form kit.
This class polypeptide and kit can be used for detecting polypeptide antibody or its active part or the active fragment that exists in the sample by method of immunity.
Method of immunity is known in the art, generally includes:
(a) provide the polypeptide that comprises the epitope (epitope) that can combine with polypeptide antibody;
(b) with biological specimen and this polypeptide incubation under certain condition, form antibody-antigenic compound;
(c) detect whether form the antibody-antigenic compound that comprises this polypeptide.
Sequence homogeneity
The percent of nucleic acid and peptide sequence homogeneity can be used commercially available algorithm computation, and sequence to be measured and reference sequences contrast are drawn.Following procedure (information center provides by the American National biotechnology) can be used for determining homology/homogeneity: BLAST, gapped BLAST, and BLASTN and PSI-BLAST can use default parameters.
(Genetics Computer Group, Madison WI) use Needleman and Wunsch algorithm that two complete sequence are arranged contrast to the GAP algorithm, make the quantity maximum of coupling, the quantity minimum of room (gap).Usually use default parameters, the room generates point penalty=12, and point penalty=4 are expanded in the room.
The method of best overall match is between another kind of definite kernel acid sequence or its partial sequence and the sequence to be measured, uses based on Brutlag et al (Comp.App.Biosci., 6; 237-245 (1990)) the FASTDB computer program of algorithm.This program provides the contrast of the sequence overall situation.The result of this sequence overall situation contrast represents with the homogeneity percent.The dna sequence dna of searching that uses among the FASTDB to calculate the percentile suitable parameters of homogeneity is: matrix=unit matrix, k word=4, mispairing point penalty=1, connect point penalty=30, random packet length=0, critical value=1, gap penalty=5, room size point penalty=0.05, window size=500 or with the sequence length to be measured that nucleoside base is represented are short.Homogeneity and the percentile suitable parameters of similarity of calculating the amino acid arrangement comprise: matrix=PAM150, k word=2, mispairing point penalty=1, connect point penalty=20, random packet length=0, critical value=1, gap penalty=5, room size point penalty=0.05, window size=500 or with the sequence length to be measured that nucleoside base is represented are short.
Carrier
Separated nucleic acid sequence of the present invention can be incorporated on the carrier, especially on the expression vector.Carrier is used in replicating nucleic acid in the compatible host cell.Thereby, in an embodiment, the invention provides a kind of method for preparing polynucleotide of the present invention, comprise separation polynucleotide of the present invention are cloned on the reproducible carrier, carrier is imported compatible host cell, under the condition that carrier is duplicated, cultivate host cell.Carrier can reclaim from host cell.The suitable host cell relevant with expression vector is as described below.
Preferably, separation polynucleotide of the present invention are operably connected to control sequence in the carrier, and this control sequence can make coded sequence express in host cell, and promptly carrier is an expression vector.
Term " is operably connected " to refer to and adjoins, and the position relation of wherein said composition makes them can bring into play the function of expection.The control sequence of coded sequence of " being operably connected " connects in such a way, under the compatible condition of control sequence, can finish the expression of coded sequence.
Suitable carriers can be selected or make up, and comprises suitable adjusting sequence, as promoter sequence, terminator fragment, poly adenine sequence, enhancer sequence, marker gene and other suitable sequence.Carrier can be suitable plasmid, virus, and as bacteriophage, phasmid or baculoviral, clay, YACs, BACs or PACs.Carrier comprises gene therapy vector, adenovirus vector for example, gland relevant viral vector, retroviral vector (for example HIV or MLV) or α viral vectors.
Carrier can have replication origin, adjusting of the promoter of optional these polynucleotide of expression and the promoter of choosing wantonly.
Carrier can comprise one or more selected markers, for example the neomycin drug resistant gene of ampicillin drug resistant gene in the bacterial plasmid or mammal carrier.Carrier can be in external use, as is used for synthetic RNA, or is used for transfection or transformed host cell.Carrier also can use in vivo, for example gene therapy.The system of clone and express polypeptide is known in multiple different host cells.Proper host cell comprises bacterium, eukaryotic such as mammalian cell and yeast, rhabdovirus system.The mammal cell line that can be used for the expressing heterologous polypeptide in this area comprises Chinese hamster ovary cell, human cervical carcinoma go down to posterity (HeLa) cell, baby hamster kidney cell, COS cell and a lot of other clone.
Can select promoter and other to express conditioning signal so that the host cell of itself and expression vector is compatible.For example, Yeast promoter comprises saccharomyces cerevisiae GAL4 and ADH promoter, grain brewer yeast nmt1 and adh promoter.Mammalian promoter comprises metallothionein promoter, and it can be made a response to heavy metal such as cadmium through inducing.Also can use viral promotors for example SV40 large T antigen promoter or adenovirus promoter.All these promoters can both obtain easily in this area.
Carrier can comprise other sequence for example promoter or enhancer, makes the nucleotide sequence of insertion express produce fused polypeptide, and/or comprises the nucleic acid of the secretion signal of encoding, and makes the polypeptide that produces in the host cell secrete from cell.
But express polypeptide and be used for Vectors in Gene Therapy and comprise the carrier that carries mini gene sequence of the present invention among the present invention.Therefore, an object of the present invention is to provide a kind of method, its treatment is expressed and its active relevant pathological state with GPR39 GPCR is low, and this method comprises the polynucleotide that use coding GPR39 GPCR.Especially treat the impaired method of carbohydrate metabolism, for example diabetes comprise its related complication.In gene therapy, it is synthetic that separation polynucleotide of the present invention are used to influence the endogenous of GPR39 GPCR in experimenter's relevant cell.For example, the polynucleotide of coding GPR39 GPCR can be expressed in the replication defect type retroviral vector by genetic engineering, as mentioned above.Separable then retrovirus expression construct, import the incasing cells of retroviral plasmid vector transduction, described retroviral plasmid vector comprises the RNA of code book invention polypeptide, and incasing cells just can produce the infectious viral particle that contains genes of interest like this.These can be produced cell and be administered to the experimenter, be used for through engineering approaches cell in the body, and express polypeptide in vivo.Gene therapy summary is referring to " gene therapy and other methods of treatment based on molecular genetics ", Human Molecular Genetics, T.Strachan andA.P.Read, the 20th chapter of BIOS Scientific Publishers Ltd. (1996).
More details is referring to " molecular cloning: laboratory manual: second edition ", Sambrook et al., 1989, Cold Spring Harbor Laboratory Press.Many known technology and experimental program are at Current Protocols in Molecular Biology, Ausubel et al.eds., John Wiley﹠amp; Sons has a detailed description in 1992, comprises that nucleic acid handles, for example nucleic acids for preparation, sudden change, order-checking, with DNA transfered cell and gene expression etc., and protein analysis.
As mentioned above, carrier can change proper host cell over to and express polypeptide of the present invention.Thereby another aspect of the present invention provides the method for preparing polypeptide of the present invention, comprises cultivating the host cell that conversion or transfection have above-mentioned expression vector under certain condition, makes the vector expression that contains polypeptid coding sequence and reclaims polypeptide expressed.Polypeptide also can be expressed in vitro system (for example reticulocyte lysate).
Another embodiment of the present invention provides conversion or transfection that the host cell of carrier is arranged, and is used for duplicating and expressing of polynucleotide of the present invention.Select can be compatible with this carrier cell, for example bacterium, yeast, insect or mammalian cell.Can cultivate host cell under certain condition,, produce encoded polypeptides with expressing gene.If polypeptide expressed contains the appropriate signals leader peptide, just can be secreted in the nutrient culture media.After the expression, polypeptide can separate and/or purifying from host cell and/or nutrient culture media, be determined on a case-by-case basis, use at the composite preparation that for example can contain one or more supplementary elements (for example containing in the pharmaceutical composition of one or more pharmaceutically acceptable excipient, medium or carrier) as required subsequently.
Polynucleotide of the present invention also can insert above-mentioned carrier by antisense orientation, produce antisense RNA or nuclease.
Test
One of purpose of the present invention provides a kind of test, be used to identify and strengthen experimenter's glycemic control and effectively prevent and/or treat compound with the impaired relevant pathological state of carbohydrate metabolism, especially prevent and/or treat diabetes and comprise that its related complication or metabolic syndrome comprise its related complication, test comprises: all or part of of GPR39 receptor protein of the present invention is provided, this albumen is contacted with the binding compounds of supposition; Determine this compound whether can with this protein-interacting.Known to people, GPCR can with the G protein-interacting, also can with other auxilin effect, test provided herein can comprise G albumen or other auxilin except that GPR39 receptor protein all or part of.
Known GPCR and G protein-interacting also comprise arrestin (arrestin), receptor active modified protein (RAMPs) with other auxilin, contain the interactions such as albumen of PDZ domain.The interaction decision acceptor of GPCR and these auxilins has various biological property, for example be transported on the film or on film and transport, determine its pharmacological property, glycosylation state or the signal transduction mechanism (Laburthe et al2002) that has nothing to do with the G albumen coupling.For example, CTR sample acceptor combines with RAMP1 (a species specific single span memebrane protein), cause the CTR sample receptor expression that can combine with CGRP, and CTR sample acceptor combines with PAMP2 or PAMP3, then causes the expression (Latchie et al 1998) of adrenal medullary hormone bind receptor.Have document proof RAMP can with VPAC1 receptor, glucagon receptor, parathyroid hormone 1 receptor and parathyroid hormone 2 receptors (Christopoulos et al 2001,2003) interact similarly, show that auxilin has generally effect in the signal conduction of regulating g protein coupled receptor.
In an embodiment of test, the subunit of acceptor or acceptor can be used in conjunction with test.In conjunction with test is competitive or noncompetitive.This experiment can a large amount of compounds of rapid screening, to determine which kind of compound (if any) can combine with polypeptide.Subsequently, the compound of available these combinations is tested in more detail, to determine further whether these compounds are the activator or the antagonist of polypeptide of the present invention.
Therefore, an object of the present invention is to provide a kind of method, be used to identify and strengthen effectively preventing and/or treating and the impaired relevant pathological state of carbohydrate metabolism of experimenter's blood glucose-control, especially prevent and/or treat diabetes and comprise that its related complication or metabolic syndrome comprise the compound of its related complication, described method comprises:
(i) all or part of host cell of expressing GPR39 receptor protein of the present invention and described compound are contacted being suitable under the condition of combination,
(ii) detection compound and described receptor protein combines.
A kind of method is provided in another embodiment of the present invention, evaluation can strengthen experimenter's blood glucose-control and effectively prevention or treatment and the impaired relevant pathological state of carbohydrate metabolism, especially prevention or treatment diabetes comprise that its related complication or metabolic syndrome comprise the compound of its related complication, and this method comprises:
(i) all or part of host cell membrane product of expressing GPR39 receptor protein of the present invention and this compound are contacted being suitable under the condition of combination,
(ii) detection compound and this receptor albumen combines.
A kind of method is provided in another embodiment of the present invention, evaluation can strengthen experimenter's blood glucose-control and effective prevention or treatment and weaken relevant pathological state with carbohydrate metabolism, especially prevention or treatment diabetes comprise that its related complication or metabolic syndrome comprise the compound of its related complication, this method relates to competitive in conjunction with test, wherein:
(i) all or part of host cell of expressing GPR39 receptor protein of the present invention is contacted with a kind of compound, known this compound all can combine with the GPR39 receptor protein when having or not testing compound to exist,
(ii) detect the influence of this compound pair and the known compound binding ability that can combine with the GPR39 receptor protein.
When testing compound existed, the combination of this known compound that can combine with the GPR39 receptor protein weakened, and showed that this testing compound is attached on the GPR39 receptor protein.Fat inhibin, the native ligand of GPR39 receptor protein is considered to have identical incretogenous another kind of peptide (Zhang, J.V.et al., 2004 Science Vol.310 with GHRP recently; 996-999).In a preferred embodiment, knownly can comprise fat inhibin, preferably be selected from one of fat inhibin sequence of SEQ ID No ' s5-16, more preferably SEQ ID No.5 or SEQ ID No.7 with the compound of receptors bind.In addition, fat inhibin used herein refers to the peptide that has consensus amino acid sequence among 60%, 70%, 80%, 90%, 95%, 98% or 99% sequence and SEQ ID NO:5 or the SEQ ID NO:7 at least.
In another embodiment, above-mentioned competitiveness is carried out on the membrane product of all or part of host cell of expressing GPR39 receptor protein of the present invention in conjunction with test.Preparing this host cell describes hereinafter with the method for preparing the host cell membrane product.
In an embodiment, GPR39 receptor protein in the above-mentioned combination test comprises, contain and be selected from SEQ ID No:2, SEQ ID NO:4, the protein splice variant of above-mentioned SEQ ID ' s, have 80% at least with amino acid sequence, the amino acid sequence of preferred at least 90%, 95%, 96%, 97%, 98% or 99% sequence homogeneity with SEQ ID NO:2 or SEQ ID NO:4.
The part of GPR39 albumen mentioned above comprises the fragment of polypeptide among SEQ ID NO:2 or the SEQ ID NO:4, and this fragment contains 10 for example at least 20,30,40,50,75,100 or 150 or more a plurality of amino acid residue at least.These fragments are derived from the N-end of SEQ ID NO:2 or SEQ IDNO:4.The fragment that comprises the N-end region can be reformulated the extracellular region of acceptor, and receptor binding site is provided.Preferably, fragment keeps the ability in conjunction with the known compound that can combine with the GPR39 receptor protein.More preferably, fragment keeps the ability in conjunction with fat inhibin, and is as indicated above.
Membrane product
The cell membrane of expressing receptor protein of the present invention can be used for the several types test, comprises part in conjunction with test, and GTP-γ-S is in conjunction with test and other test, but is not limited to these.The details of preparation cell membrane depends on the character of experiment, hatches intact cell but relate generally to, and destroys cell, for example ultrasonic degradation cell (as 20mM Tris HCl, 1mM EDTA, pH7.4,4 ℃) on ice in the cold buffer liquid.Low-speed centrifugal is removed the cell fragment in the cell lysate, 200xg for example, 4 ℃, 5 minutes.Last high speed centrifugation is further removed fragment makes membrance concentration, and for example 40,000xg, 4 ℃, 20 minutes, suspension cell on ice in the cold buffer liquid repeated high speed centrifugation.Last membrane granule is resuspended in the experiment damping fluid.Bradford (1976) method is measured protein concentration, with bovine serum albumin standard reference in vain.Film can use or freezing preservation immediately.
The labelled with radioisotope part is in conjunction with test
The cell of expressing acceptor of the present invention is used for the part of examination this receptor.Can identify the activator or the antagonist of acceptor with multiple therapeutical uses with same experiment.
The labelled with radioisotope part in conjunction with the method for test is, the film that is come by the cell preparation of expressed receptor dilutes with suitable damping fluid, for example contain 2.1% bovine serum albumin(BSA) (Sigma), protease inhibitors bestatin (0.005mg/ml, Boehringer Mannheim) and Aprotinin (01.mM, Sigma) 50mM Tris damping fluid (pH=7.4,0 ℃).The final concentration of albumen is 12-40 μ g/ml in the experiment.
Having or not in the presence of the competitive part, with the part of film and labelled with radioisotope, in 96 hole microtiter plates, every hole cumulative volume is 250 μ l, hatches on ice 60 minutes then.Use Tomtec (Wallac) vacuum apparatus, the GF/B filter that is soaked in 0.5% polyethyleneimine (PEI) in advance filters, and binding partner is separated with free ligand.Add Ready Safe (Beckman) scintillation solution, with the radioactivity (combination rate is about 40%) of Trilux (Wallac) scintillation counter quantitative measurement combination.In addition, preferable methods is that the part of collection combination makes it separate with acceptor with program well known in the art then.Use curve fitting software such as GraphPad prism to analyze data and be nonlinear curve.
According to this method, can identify and the activator or the antagonist of receptors bind, because suppressing the labelled with radioisotope part, they combine with the epicyte protein of expressing this receptor.Non-specific bond refers to when having the 100nM unmark peptide, the radioactivity amount that still has after memebrane protein is hatched.In conjunction with in testing, the intact cell of the film of preparation or transfection acceptor under the situation that tagged compound concentration increases, is hatched the affinity of measuring tagged ligand in equilibrium saturation.The affinity of non-labelled compound can be with the balance competition in conjunction with measurements determination, and tagged compound concentration is certain, changes the concentration of displaced ligands.
In a preferred embodiment, the labelled with radioisotope part is the fat inhibin of labelled with radioisotope in conjunction with what test use.The mark of fat inhibin and receptors bind be at Zhang, J.V.et al. (2004 Science Vol.310; Existing concise and to the point description the 996-999).
(IN) iodide process is carried out iodine labeling to fat inhibin for Pierce, Upland to use Iodogen.Peptide (20 μ g) and 1mCi[ 125I] potpourri of sodium iodide adds in the Iodogen bottle of precoated shet, hatched 4 minutes. 125The peptide of I mark adds in the Sep-Pak C18 post, uses 60% acetonitrile/0.1%TFA eluant solution then.The labelled with radioisotope part in conjunction with method of testing is, the jejunum of rat or other tissue buffer A (20mM Hepes, 5mM EDTA, 1mM dithiothreitol (DTT) (DTT), 10 μ M methanesulfonyl fluorides, 5mg/L leupeptin, 100mM KCl, pH7.5) after the cleaning, tissue is cut into equally distributed fritter with automatic homogenizer.Tissue homogenate 1,000g, centrifugal 5 minutes.Supernatant 300,000g, 2 ℃, centrifugal 1 hour.The resuspended membrane granule of buffer A (rough membrane-bound fragment) that does not contain KCl, the liquid nitrogen snap frozen ,-80 ℃ store for future use.The phosphate buffer 1 00 μ l of 0.1% bovine serum albumin(BSA) has or not excessive 1,000 times unmarked fat inhibin to exist down, and room temperature was hatched tissue homogenate 18 hours, changed 125The concentration of the fat inhibin of I mark.After hatching, 10,000g, centrifugal 10 minutes, cold PBS is washed twice on ice, γ-spectrophotometer measurement radioactivity.Deduct non-specific bond amount in the total binding and obtain specific binding capacity.For obtaining among the displacement curve figure, that concentration is certain 125Fat inhibin of I mark and concentration have or not the fat inhibin of increase or other peptide to hatch jointly.
Except above-mentioned combination test, one of purpose of the present invention provides the compound functions experiment that evaluation can strengthen experimenter's glycemic control, it is characterized in that the activity of this compound adjusting GPR39 receptor protein of the present invention.This experiment comprises the following steps:
(i) all or part of and testing compound of GPR39 receptor protein is contacted,
(ii) detect the activity of GPR39 receptor protein, wherein when testing compound existed, the GPR39 activity change showed that compound has the function of regulating carbohydrate metabolism.
Consider that GPR39 participates in carbohydrate metabolism, can combine and regulate with GPR39 in the open embodiment of its activity that can adopt this experimental identification can influence the compound of experimenter's blood glucose balance, the experimenter comprises people and homeothermal animal in all evaluations.
The compound of regulating polypeptide active of the present invention refers to, and changes polypeptide active, and the effect when the effect when having compound to exist of making it exists with no compound is different.The compound that influence is regulated comprises activator and antagonist.Activator comprises the compound that activates GPR39 GPCR function.Antagonist comprises the compound that disturbs GPR39 GPCR function.The effect of antagonist is the receptor activation of blocking-up agonist induction, yet for composition active acceptor GPR39, disturb the compound of GPR39 GPCR function also to comprise inverse agonist, promptly have identical receptor binding site but bring into play the medicament of opposite pharmacological action with receptor stimulating agent.Antagonist comprises competitiveness and noncompetitive antaganist.Competitive antagonist (competitive blocking agent) and the specific bond site of activator or near site interaction it.Other site beyond noncompetitive antaganist or blocking agent and the activator binding site interacts, and makes the function of receptors inactivation.
Therefore, one of purpose of the present invention provides a kind of method of identifying the compound that can strengthen experimenter's glycemic control, and this method comprises:
(i) all or part of host cell of expressing the GPR39 receptor protein is contacted under the condition that can activate this receptor albumen with testing compound and contacts,
(ii) detecting the GPR39 receptor active increases, thereby identifies that testing compound is a kind of compound that can strengthen experimenter's glycemic control.
In another embodiment of the present invention, a kind of method of identifying the compound that can reduce the GPR39 activity is provided, this method comprises:
(i) all or part of host cell of expressing the GPR39 receptor protein is contacted under the condition that can activate this receptor albumen with testing compound,
(ii) detect the GPR39 receptor active and reduce, thereby identify that testing compound is a kind of compound that can make human or animal's sugar tolerance normalization, promptly this testing compound comprises in its related complication it being useful in the treatment diabetes.
The combination test of above mentioning, activity experiment also can be used for identifying the compound of regulating carbohydrate metabolism.Based on observed phenomenon in the sugar tolerance experiment, can think that the compound that increases the GPR39 activity can make the blood sugar tolerance reduce, the compound that reduces the GPR39 activity can make the blood sugar tolerance raise.In a preferred embodiment of the present invention, a kind of method of identifying the compound of energy blood sugar lowering tolerance is provided, this method comprises:
(i) all or part of host cell of expressing the GPR39 receptor protein is contacted under the condition that can activate this receptor albumen with testing compound,
(ii) detecting the GPR39 receptor active increases, thereby identifies that testing compound is a kind of compound of energy blood sugar lowering tolerance.
The principle of compounds affect GPR39 activity may be to increase or reduce the interior second messenger of the cell that regulated by GPR39 and form, for example intracellular Ca2+, cAMP or acceptor gene product.GPR39 is the member of g protein coupled receptor (GPCRs) family.GPCRs combines with part, strides film and transmits signal.The signal transduction mechanism that regulated by heterotrimeric G protein, for example activated adenyl cyclase path or phospholipase C-β path.It is normally known in the art to assess the experiment that signal transduction mechanism activates in the above-mentioned cell, and this class experiment comprises, different intercellular signal conduction experiments such as the experiment of chimeric part induced transcription factor; The g protein coupled receptor that uses the fluorescence intensity distributional analysis is in conjunction with test; The signal conduction experiments is used the cyclic adenosine monophosphate in conjunction with luminophor, or the reaction of many response elements or cAMP response element detection g protein coupled receptor.This class experiment is described hereinafter.
The present invention also provides a kind of biologicall test, and evaluation can be regulated the compound in GPR39 gpcr gene scalable district.This class experimental technique uses rat or the people's cell that can express polypeptide of the present invention (preferred SEQ IDNO:2 or SEQ ID NO:4).Cell contacts with at least a compound, and wherein the ability in this compound regulatory gene scalable district is known.Then, expression of nucleic acids of the present invention in the monitoring cell.Also promoter can be connected on the reporter gene.Suitable reporter gene comprises chloramphenicol acetyl transferasegene, luciferase gene etc.
As known to persons skilled in the art, evaluation can be regulated the bioassay method of the compound of acceptor such as polypeptide active of the present invention, needs contrast usually.Wherein a kind of contrast is and the cell to be measured that is exposed to compound or nutrient culture media cell or the nutrient culture media through same treatment that difference is that control cells or nutrient culture media are not exposed to compound.Another kind of contrast is cell or the nutrient culture media identical with transfectional cell, but control cells or nutrient culture media expressive function GPR39 GPCR not.Therefore, can be under same reaction conditions, contrast transfectional cell and control cells or nutrient culture media are to the reaction with a kind of compound.
Preferred experiment is that method is as follows in conjunction with test and functional experiment:
In conjunction with test
The clone that the nucleic acid of code book invention polypeptide is crossed expression (comprises mammal HEK 293 clones, Chinese hamster ovary celI system and sf9 insect cell line) can be used for preparing membrane product, produce this polypeptide (this part is represented with GPR39 GPCR for simplicity) and be used for part in conjunction with research.Membrane product can be used for traditional filter membrane and (for example, uses Brandel filter membrane experiment instrument or high flux to get close to flicker in conjunction with test (SPA and Cytostar-T flashplate technology in conjunction with test; AmershamPharmacia Biotech), the combination of detection of radioactive isotope labeling part with and binding site by the replacement of competitor.Radioactivity can be measured with Packard Topcount or analogous instrument, can carry out fast detecting to 96,384,1536 micropores.The SPA/Cytostar-T technology is particularly useful for the high flux examination, and therefore this technology can be used for the compound that examination replaces the standard part.
Another kind of research part is near in the natural environment, to utilize the plasma resonance effect that shows (Malmqvist M., the Biochem Soc Trans.1999 Feb of Biacore instrument generation in conjunction with the method for GPR39 gpcr protein; 27 (2): 335-40).GPR39 GPCR in membrane product or the intact cell is fixed on the Biacore biologic sensor chip, is having or not in the presence of the compound combination of detector ligand, thereby the competitor of evaluation binding site.
Functional experiment
GPR39 GPCR by with the interactional G of GIPK iOr Go (inhibition type G albumen) plays a role, so the potassium ion flux can activate these acceptors.This ionic flux can be measured with multiple technologies in good time, determines the excitement or the antagonistic effect of special compound.Therefore, the feature of the reorganization GPR39GPCR acceptor of expressing in available intact cell and single channel electrophysiology reacting cells system or the xenopus leavis oocytes is determined the reaction mechanism of purpose compound.Electric physiology examination to the compound that acts on GPR39GPCR can be used traditional electrophysiological technique, and at present, novel high-throughput techniques is is researched and developed.
Fluorescence-calcium and sodion flux can be measured with several ion-sensitive fluorochromes, comprise that the similar probe of fluo-3, fluo-4, fluo-5N, fura red, Sodium Green, SBFI and other manufacturer-supplied comprises molecular probe.Use fluorophotometer and fluorescence imaging technology to detect calcium and sodion flux, the fluorescence imaging technology comprises fluorescent microscope and the imaging analysis algorithm that has or not laser focusing in good time.
Another kind method is high flux examination experiment, and examination can influence the activator or the correctives compound of calcium transient.What this method was used is fluorescence imaging plane reader
Figure A200780021207D00261
Molecular Devices Corporation).FLIPR can excite and measure the fluorescence that fluorochrome sends.FLIPR adopts Argon ion laser fluorescence excitation element, and optical system is scanned up to the bottom fast from the top of 96/384 micropore, and producing wavelength is the high-energy fluorescence signal of 488nm, and the CCD camera of precooling is caught the fluorescence that sends.FLIPR also has the pipettor of 96/384 micropore, and liquid medicine to be measured is added in 96/384 microwell plate.FLIPR experiment can measure simultaneously institute in 96/384 microwell plate porose in the fluorescence signal of cell, before compound adds, in the adition process and measurement in good time after adding.The FLIPR experiment can be used for examination and acts on the compound of reorganization GPR39 GPCR and react its feature, the rat or the people GPR39 GPCR that express in reorganization GPR39 GPCR such as the clone.As described below, the calcium transient of transfection GPR39 GPCR cell can be used the FLIPR experiment measuring, and the activity that detects acceptor with multiple substrate is to determine the native ligand of acceptor.
CAMP experiment (cAMP) experiment-can in the cell of expressed receptor, carry out receptor-mediated CAMP (cAMP) to form activation or suppress experiment.A kind of cAMP experimental program hereinafter is provided.In this scheme, adopt DEAE-glucosan method that the acceptor gene transient transfection is gone in the COS-7 cell, and cell is added in 96 orifice plates.After the transfection 48 hours, with containing 10mMHEPES, Dulbecco ' s phosphate buffer (FES) washed cell twice of 10mM glucose and 5mM theophylline, in this damping fluid 37 ℃ then, 5%CO 2, hatched 20 minutes.Add testing compound, continue 37 ℃ and hatched 10 minutes.The sucking-off nutrient culture media adds 100mM hydrochloric acid cessation reaction.Culture plate-20 ℃ storage 2-5 days.When cAMP measures, culture plate is thawed, cAMP gets close to flicker experiment (Amersham Pharmacia Biotech) and measures cAMP content in every hole.Microbeta Trilux calculating instrument (Wallac) quantitative measurment radioactivity.
Little physiological function (microphysiomeric) determination experiment-because cellular metabolism relates to cell incident (receptor activation that comprises a plurality of signal paths) widely, little physiological function measurement method is measured cellular metabolism can provide a kind of general experimental technique that detects cytoactive, the activity that can be used for any orphan receptor active cell detects, and does not relate to the receptor signal conduction path.
Acceptor moment expression, the guide of cell preparation and little physiological function measurement record is described in (Salon, J.A.and Cwicki, J.A., 1996).Before the experiment, collect the cell of expressed receptor, be inoculated in little physiograph vesica 3 x 10 5Cell/vesica was hatched in the complete medium 24 hours.Before the record, serum free medium continues to cultivate 16 hours, will reduce to minimum by the non-specific metabolism stimulation that multiple unclear blood plasma factor causes.Test the same day, cell vesicle is transferred in little physiograph, (low-buffer RPMI 1640 to be equilibrated at the registering instrument nutrient culture media, no hydrocarbonate, serum-free (Molecular Devices Corporation, Sunnyvale, CA), 0.1-1% free fatty acid, no BSA), determine the baseline of basic metabolism activity.
The standard recording scheme is specified, flow velocity 100 μ l/min, and total cycle time is 2 minutes, interrupts therebetween flowing 30 seconds, adopts rare acidifying to measure.Before for the first time fast measuring the part flow rate, earlier with ligand exposed in sample 1 minute 20 seconds, carry out 2 circulations then, the T.T. that is exposed to sample is 5 minutes 20 seconds.Representational is that when screening first, the medicine final concentration that acts on cell is 10 μ M.
The dose dependent of detection of active compound subsequently, method is that cell is contacted with the medicine of variable concentrations scope, the drug concentration scope surpasses the amount that produces reaction needed, from the threshold value to the maximum.The wash-out part reacts the percentage composition that exceeds the baseline ratio that records before the experiment with peak value and represents acidifying rate then.
The compound of regulating polypeptide active of the present invention has multiple therapeutical uses, according to being the adjusting that GPR39 participates in carbohydrate metabolism.Specifically, these diseases comprise with blood sugar level increases diseases associated, and for example diabetes (comprising its related complication) comprise type 1 diabetes (insulin-dependent or IDDM), diabetes B (insulin independent form), young maturity-onset diabetes (MODY) and gestational diabetes.In a word, GPR39 GPCR and associated receptor are the useful tools of a plurality of treatments field Chinese traditional medicine exploitation.
In conjunction with medicament
The invention provides novelly in conjunction with medicament, comprise the correctives that obtains according to experiment of the present invention, its composition comprises in conjunction with medicament.With the medicament of receptors bind, have the medicament of excitement or antagonistic activity, can be used for treating the disease of above describing, its pathological characteristics is to play a role by the GPR39GPCR acceptor, the purposes of these medicaments is other a part of contents of the present invention.
Give the dose therapeutically effective of patient's medicament of the present invention.Because above-mentioned disease majority is chronic and can not cures that therefore it should be understood that " treatment " comprises, mitigation symptoms in a period of time for example several hrs, several days or a few week, also comprises the progression of slowing down disease.
This class medicament can be formed preparation with pharmacology acceptable carrier or thinning agent compatibility.Medicament can be the derivant that physiological function is arranged, for example ester or salt, acidic salt or alkaline metal salt, or nitrogen or oxysulfide.Preparation can carry out compatibility according to any suitable route of administration and mode.Pharmacology acceptable carrier or thinning agent can be used in the pharmaceutical preparation, be suitable for oral, per rectum, via intranasal application, suck (comprising) through oral cavity and sublingual gland, transvaginal or intestines and stomach outer (comprise subcutaneous, in the muscle, intravenous, intracutaneous, the interior and exterior dura of sheath) administering mode.The administering mode of recommendation is depended in the selection of carrier or thinning agent, and administering mode depends on medicament and its therapeutic purposes.Pharmaceutical preparation can be an one-pack type, also can be by any method preparation known in the art.This method comprises, adds the carrier of forming one or more assistants in active component.Usually, the preparation method of preparation is, with active component and fluid carrier or the solid phase carrier that separates or carrier that both all have evenly, mix nearly, if necessary, makes the shape of converted products.
For solid pharmaceutical preparation, traditional nontoxic solid phase carrier comprises, sweet mellow wine for example, and lactose, cellulose, cellulose derivative, starch, dolomol, saccharin sodium, talcum, glucose, sucrose, magnesium carbonate, etc.Reactive compound can be made suppository, polyolefin diols for example, and acetoglyceride three fat etc., carrier is same.Accessible fluid preparation preparation method is, dissolving, dispersed activity compound and adjuvant in carrier, water for example, glucose saline, glycerine, ethanol etc., thereby form solution or suspension.If needed, pharmaceutical composition also can comprise a small amount of nontoxic adjuvant, as wetting agent or emulsifying agent, and pH buffering agent, sodium acetate for example, sorbierite list lauryl, triethanolamine sodium acetate, triethanolamine oleate etc.The practical methods for preparing this formulation is well known by persons skilled in the art, for example, and referring to Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 15th Edition, 1975.
In any case the dosage of the reactive compound that preparation comprises can effectively be alleviated experimenter's symptom.
Can prepare active component content and be 0.25-95% and with the formulation or the preparation of non-toxic carrier content balance.
During oral administration, in the acceptable toxic formulation of pharmacology, contain excipient commonly used, for example, sweet mellow wine, lactose, cellulose, cellulose derivative, crosslinked sodium cellulose glycolate, starch, dolomol, saccharin sodium, talcum, glucose, sucrose, magnesium carbonate etc.The formulation of this preparation is a solution, suspension, tablet, pill, capsule, powder, sustained release preparation etc.This preparation can contain the active component of 1%-95%, preferred 2-50%, more preferably 5-8%.
The characteristics of intestines and stomach external administration are to adopt method, hypodermic injection, intramuscular injection or the intravenous injection of injection.Injection can prepare by traditional form, as fluid solution or suspension, or is dissolved in the solid forms of making solution or suspension in the fluid before the injection, or emulsion.Suitable excipient comprises water, salt, glucose, glycerine, ethanol etc.In addition, if needed, pharmaceutical composition also can comprise the nontoxic adjuvant of less dosage, for example wetting agent or emulsifying agent, pH buffering agent, sodium acetate for example, sorbic alcohol list lauryl, triethanolamine oleate, triethanolamine sodium acetate etc.
The percentage composition height of reactive compound depends on special nature, activity and the experimenter's of compound needs in this parenteral formulations.Yet the percentage composition of active component is 0.1%-10% in solution, increases in solid dosage forms, is diluted to above-mentioned content subsequently.Preferably, preparation comprises the active agents of 0.2-2% in the solution.
Therefore, one of purpose of the present invention provides pharmaceutical composition, being used for the treatment of experimenter's carbohydrate metabolism weakens, diabetes (comprising its related complication) for example, comprise type 1 diabetes (insulin-dependent or IDDM), diabetes B (insulin independent form), young maturity-onset diabetes (MODY) and gestational diabetes, said preparation comprises the GPR39 receptor antagonist.
Diagnostic products
The present invention also provides a kind of diagnostic products, comprises being selected from following separated nucleic acid sequence:
(i) all or part of nucleotide sequence of coding SEQ ID NO:2 or SEQ ID No:4 polypeptide,
The (ii) nucleotide sequence of forming by SEQ ID NO:1 or SEQ ID NO:3, perhaps
(iii) the nucleotide sequence with SEQ ID NO:1 or SEQ ID NO:3 has 80% at least, the nucleotide sequence of preferred at least 90%, 95%, 96%, 97% or 98% sequence identity.
Use DNA of the present invention, antisense DNA, siRNA is as probe, mammal be can detect and philtrum coding GPR39 acceptor or the DNA of its part fragment or the gene unconventionality of mRNA comprised, therefore, these nucleotide sequences are useful gene therapy medicaments, can be used for destroying coding GPR39 acceptor or its fragment or its mutant, the DNA or the mRNA of low expression or high expressed.
The said gene diagnostic method comprises known Northern hybrid experiment or PCR-SSCP experiment.
The GPR39 acceptor is low expresses if detect, and can diagnose the experimenter to suffer from and GPR39 function of receptors defective diseases associated, and for example metabolic syndrome comprises its related complication.
If detect GPR39 acceptor high expressed, can diagnose the experimenter to suffer from and GPR39 acceptor high expressed diseases associated, for example diabetes comprise its related complication.
The present invention also provides a kind of diagnostic products, comprises all or part of of GPR39 receptor protein.Can detect this albumen in suitable sample, blood for example is with the low expression or the high expressed of diagnosis GPR39 acceptor.
Be attached to by reference hereinafter during experiment describes in detail, the present invention may be better understood, but those skilled in the art is willing to thank the illustration of describing in detail in the claim hereinafter.In addition, by this application, various publications are cited.During the disclosure of these publications is attached to by reference and should uses, the state in the affiliated field of the present invention is described fully.
Embodiment
The following example is for example understood the present invention.According to these examples, those skilled in the art will operate other embodiment.
Material and method
The GPR39 knock-out mice
The GPRC39 knock-out mice is from Lexicon Genetics Inc.With IRESLacZ/MCl-Neo reporter gene/selection site, replace the code area of GPR39 gene first extron, can destroy the open reading frame of GPR39.Letting animals feed in the SPA level facility of Belgium and European animal feeding requirement.Mouse is gregarious the raising in controllable environment, and is unglazed and have light to hocket (lighting time is at 7:00 EST), mouse ad libitum access and drinking-water, except as otherwise noted.Take measures to make misery of mouse or discomfort to minimize.Experiment is carried out according to the council of European Community indication (86/609/EEC), and through the approval of local Ethics Committee.
The histologic analysis that GPR39 expresses in pancreas
From wild type and GPR39 -/-Isolate pancreas in the mouse, carry out whole mount LacZ dyeing.Briefly, on ice, separate tissue in PBS solution, is contained among the PBS of 0.5% glutaraldehyde and fixes 2 hours.Then, PBS, gentleness is shaken, rinsing tissue 3 times.Containing 1mg/mlX-gal, in the dyeing damping fluid of the 5mM potassium ferricyanide and 5mM potassium ferrocyanide, 30 ℃, dyeing is spent the night.After the dyeing, tissue PBS damping fluid rinsing 3 times, dehydration of alcohol, dimethylbenzene is fixed, and paraffin embedding is made 5 μ m section.Histotomy with 10 μ m carries out histology, Harris H﹠amp; E redyes or does not redye.
Contrast wild type and GPR39 -/- Type mouse body weight
Animal is raised separately, and temperature is 22 ± 1 ℃, have light and unglazed each 12 hours.(from U.A.R., France) arbitrarily raise by 22% albumen, 4.3% fat and 4% cellulose with standard feed for animal.From beginning to 16-20 week, follow the trail of to measure the weight of animals 8-12 week.
Wild type and GPR39 -/- The experiment of type mouse sugar tolerance
GPR39 -/-Type and wild-type mice (male and female) overnight fasting (since afternoon 5.30), hypodermic injection 2g/kg glucose (final concentration is the salt solusion of 200mg/ml) then.Get mouse tail vein blood.Baseline is set earlier before weighing, gives mouse glucose then.Use OneTouch Ultra blood glucose meter, 15,30,45,60,90 and 120 minutes difference measuring blood concentration behind glucose load.
Contrast wild type and GPR39 -/- Type mouse physique composition
(Minispec mq10 NMR analyser, Bruker Germany), between 9 and 11 of mornings, measure conscious GPR39 to adopt quantitative nuclear magnetic resonance technique (NMR) -/-Type and wild-type mice fat and muscle content.NMR is a kind of for measuring the noninvasive method that mouse physique composition consciously grows up fast.Non-fat (lean body), fat and body fluid content can be used the NMR quantitative measurement.First weighing the weight of animals before NMR measures.
The result
The histologic analysis that GPR39 expresses in pancreas
GPR39 -/-Mouse, the code area of its first extron is replaced by the LacZ reporter gene, therefore, detects the indicator that the LacZ expression can be used as endogenous GPR39 expression pattern in the pancreas.At GPR39 -/-In the pancreas of type mouse (Figure 1A), whole pancreas islet, promptly the endocrine district of pancreas can detect expression, does not express (Figure 1B) and have in wild-type mice.
Contrast wild type and GPR39 -/- Type mouse body weight
From beginning in 8-12 week to 16-20 week, follow the trail of the body weight of measuring mouse.Male GPR39 -/-The body weight rising (Fig. 2) of the more male wild-type mice of body weight of type mouse.In this age bracket, the body weight indifference of female mice.
Wild type and GPR39 -/- The experiment of type mouse sugar tolerance
After 16 hours, compare GPR39 on an empty stomach with wild-type mice (male and female) -/-The blood sugar concentration of type mouse (male and female) (12-16 week) does not have obvious change.After the feed, the blood sugar concentration of two kinds of mouse genotypes is indifference (Fig. 3) also.
Behind the glucose load (glucose administration), male GPR39 -/-The weight increase of type mouse is starkly lower than male wild-type mice (12-16 week) (P＜0.05).Two kinds of genotypic female mices (12-16 week) blood sugar increases indifference (Fig. 4).
Contrast wild type and GPR39 -/- Type mouse physique composition
Measure the physique composition of 13-17 week and 16-20 week mouse.Feed mouse with standard feed, when 13-16 is all, the physique composition indifference of two kinds of mouse genotypes.After 3 weeks, male GPR39 -/-The fat content of the more male wild-type mice of fat content of type mouse increases (15.76 ± 5.30% to 11.97 ± 2.60%), and male GPR39 -/-The non-fatty body weight of type mouse is lost weight (41.22 ± 3.41% to 43.45 ± 1.78%) (Fig. 5) than the non-fat of wild-type mice.
Discuss
Earlier results shows, GPR39 acceptor high expressed in pancreatic tissue, GPR39 -/-Type mouse phenotype analytical result shows, compares with littermate wild-type mice, and the GPR39 knock-out mice cholesterol levels of two kinds of sexes increases (data is not published), based on these results, should study GPR39 acceptor may act in control of blood sugar is too much.Confirm that GPR39 participates in hyperglycemic control, will make this receptor become the fine target of treatment diabetes and complication and all metabolic syndrome relevant diseases.
Owing to these reasons the objective of the invention is, at first use the lacZ expression technology in pancreatic tissue, to locate the GPR39 acceptor, next contrasts littermate GPR 39 -/-The sugar tolerance experiment of type mouse and wild-type mice.For making these results convincing, in the sugar tolerance experiment, before and after glucose load, measure insulin concentration, to determine GPR39 may act in control of blood sugar is too much.With parallel the carrying out of these experiments, use the NMR instrument to measure the physique composition.For making PRELIMINARY RESULTS have cogency, dispose the littermate GPR 39 of comparative study in (cage set-up) at the Standard Metabolism cage -/-The metabolism situation of type mouse and wild-type mice.
List of references:
Bradford, M.M., a kind of albumen-dyestuff combination principle that utilizes carries out quantitative quick and responsive method .Anal.Biochem.1976 to albumen microgram content; 72:248-254.
Holst, B., Holliday, N.D., Bach, A., Elling C.E., Coc, H.M.andSchwartz, T.W., the common structure basis .J.Biol.Chem.2004 of Ghrelin receptor family composition activity, 279 (51): 53806-53817.
Date Y, Nakazato M, Murakami N, Kojima M, Kangawa K, MatsukuraS.Ghrelin act on central nervous system gastric acid secretion .Biochem Biophys ResCommun.2001 Jan 26; 280 (3): 904-7.
Ghoos Y, Maes B, Geypens B, Mys G, Hiele M, the sad expiration experimental evaluation stomach solid rate of evacuation .Gastroenterol.1993 of Rutgeerts P andVantrappen G. carbon markings; 104:1640-7.
Inui A, Asakawa A, Bowers CY, Mantovani G, Laviano A, MeguidMM, Fujimiya M.Ghrelin, appetite and stomach power: stomach is as endocrine organ's effect .FASEB J.2004Mar; 18 (3): 439-56.
Maes B, Ghoos Y, Geypens B, Mys G, Hiele M, Rutgeerts P andVantrappen G. associating 13The C-aminoacetic acid/ 14The sad expiration experiment of C-: two carbon markingses of monitoring gastric liquid and solid rate of evacuation are exhaled and are tested .J.Nucl.Med.1994; 35:824-31.
McKeeK K, Tan C P, Palyha O C, Liu J, Feighner S D, Hreniuk D L, Smith R G, the clone and the feature description .Genomics 46:426-434 of two kinds of g protein coupled receptor genes (GPR38 andGPR39) that Howard A D and Van der Ploeg L H T. is relevant with secretagogue receptor and neurotensin receptor, 1997.
Trudel L, Tomasetto C, Rio M C, Bouin M, Plourde V, Eberling P andPoitras P (2002) Ghrelin/motilin-related peptides is the effective power .Am.J.Physiol.282 that reverses the rat stomach postoperative ileus, G948-G952.
Peeters TL.Ghrelin regulates the maincenter and the periphery mechanism .J PhysiolPharmacol.2003 Dec of intestinal motive force; 54 Suppl 4:95-103.
The in good time detection of Salon J.A.and Owicki J.A. receptor active: use little physiograph and detect receptor biological behavior .Meth.Neurosci.1996; 25:201-224.
Zhang J.V., Ren P-G., Avsian-Kretchmer O., Luo, C-W., Rauch R., Klein C., the fat inhibin of Hsueh A.J.W. (2004), the peptide of ghrelin gene code, the effect .Science Vol.310 of antagonism ghrelin in food intake; 996-999.
The acceptor VPAC.Receptors ﹠amp of Laburthe M.Couvineau A.Marie J C.VIP and PACAP; Channels.8:137-153,2002.
McLatchie LM.Fraser NJ.Main MJ.Wise A.Brown J.Thompson N.Solari R.Lee MG.Foord SM.RAMPs regulates the transportation and the ligand specificity .Nature.393:333-339 of CTR sample acceptor, 1998.
Christopoulos A.Christopoulos G.Morfis M.Laburthe M.CouvineauA.Sexton PM. receptor active modified protein (RAMPS) and VPAC1 acceptor interaction: show the difference modulation .Proceedings Society for Neuroscience of many signal transduction pathway, 2001.abstract 5508 (http://sfn.scholarone.com/itin2001/).
The function .Journal of Biological Chemistry.278:3293-3297 of Christopoulos A.Christopoulos G.Morfis M.Udawela M.Laburthe M.Couvineau A.Kuwasako K.Tilakaratne new receptor companion of N.Sexton PM. and receptor active modified protein, 2003.
Sequence table
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Figure A200780021207D00361
Figure A200780021207D00371
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Figure A200780021207D00372
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Figure A200780021207D00373
Figure A200780021207D00381
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＜213〉people
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Figure A200780021207D00382
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Figure A200780021207D00383
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Figure A200780021207D00391
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Figure A200780021207D00392
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Figure A200780021207D00393
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Figure A200780021207D00401
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<213>Sus?scrofa
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Figure A200780021207D00402
<210>11
<211>24
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<213>Felis?catus
<400>11
Figure A200780021207D00403
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<211>24
<212>PRT
＜213〉domesticated dog
<400>12
Figure A200780021207D00411
<210>13
<211>24
<212>PRT
<213>Capra?hircus
<400>13
<210>14
<211>24
<212>PRT
<213>Ovis?aries
<400>14
Figure A200780021207D00413
<210>15
<211>24
<212>PRT
<213>Bos?taurus
<400>15
Figure A200780021207D00421
<210>16
<211>24
<212>PRT
＜213〉artificial sequence
<220>
＜223〉fat inhibin consensus sequence
<400>16
Figure A200780021207D00422

Claims (21)

1. the method for an authenticating compound, described compound strengthens experimenter's glycemic control and effectively prevents and/or treats and the impaired relevant pathologic state of carbohydrate metabolism, especially prevent and/or treat diabetes and comprise its related complication, or metabolic syndrome comprises its related complication, and described method comprises uses all or part of of GPR39 receptor protein.
2. the method for claim 1, wherein the GPR39 receptor protein is selected from SEQ ID No:2, SEQ ID No:4, has the splicing variants of the albumen of above-mentioned SEQ ID, have 80% at least with amino acid sequence, the amino acid sequence of preferred at least 90%, 95%, 96% or 98% sequence homogeneity with SEQ IDNo:2 or SEQ ID No:4.
3. claim 1 or 2 method, wherein the part of GPR39 receptor protein is made up of at least 10 amino acid whose fragments of the polypeptide of SEQ IDNo:2 or SEQ ID No:4.
4. the method for an authenticating compound, described compound strengthens experimenter's blood glucose-control and effectively prevents and/or treats and the impaired relevant pathologic state of carbohydrate metabolism, especially prevent and/or treat diabetes and comprise its related complication, or metabolic syndrome comprises its related complication, and described method comprises:
(i) all or part of host cell of expressing GPR39 receptor protein of the present invention and described compound are contacted being suitable under the condition of combination,
(ii) detection compound and described receptor protein combines.
5. the method for an authenticating compound, described compound strengthens experimenter's blood glucose-control and effectively prevents and/or treats and the impaired relevant pathologic state of carbohydrate metabolism, especially prevent and/or treat diabetes and comprise its related complication, or metabolic syndrome comprises its related complication, and described method comprises:
(i) membrane product that makes all or part of host cell of expressing GPR39 receptor protein of the present invention and described chemical combination contact under the condition of combination being suitable for,
(ii) detection compound and described receptor protein combines.
6. claim 4 or 5 method, wherein compound detects with the method for the available direct or indirect mark testing compound of combining of GPR39 receptor protein or detects in conjunction with test with the competitiveness of mark competition thing.
7. the method for claim 6, wherein mark competition thing is the fat inhibin of mark.
8. each method of claim 4-7, wherein in the step (i), compound with express claim 2 and 3 in all or part of host cell of GPR39 albumen of definition contact.
9. method of identifying the compound of regulating carbohydrate metabolism, the method comprising the steps of:
(i) all or part of and testing compound of GPR39 receptor protein is contacted, and
(ii) measure the activity of GPR39 receptor protein, the change of GPR39 activity showed that described compound has the ability of regulating carbohydrate metabolism when wherein testing compound existed.
10. the method for claim 9, wherein in the step (i), compound with express claim 2 and 3 in all or part of host cell of GPR39 albumen of definition contact.
11. the method for claim 9 or 10, the wherein activity of GPR39 receptor protein courier's in measurement is pair cell regulating action.
12. the method for claim 11, wherein the second messenger is cAMP, calcium or reporter gene product in the cell.
13. be selected from the purposes of following separated nucleic acid sequence in each method of claim 1-12:
(i) all or part of nucleotide sequence of the polypeptide of coding SEQ ID NO:2 or SEQ ID No:4,
The (ii) nucleotide sequence of forming by SEQ ID NO:1 or SEQ ID No:3, or
The nucleotide sequence that (iii) has 80% sequence homogeneity at least with the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.
14. comprise the carrier of the nucleotide sequence of claim 13 definition, the purposes in claim 1,4,5,8,9 or 10 each methods.
15. comprise the purposes of host cell in claim 1,4,5,8,9 or 10 each methods of the carrier of the nucleotide sequence of claim 13 definition or claim 14 definition.
16. be used for the treatment of the impaired pharmaceutical composition of human or animal's glycemic control, it comprises GPR39 receptor stimulating agent or antagonist.
17.GPR39 activator or antagonist are used for the treatment of purposes in the medicine with the impaired diseases related of carbohydrate metabolism in preparation, described disease is diabetes (comprising its related complication) particularly, comprise type 1 diabetes (insulin-dependent or IDDM), diabetes B (insulin non-insulin dependent diabetes), young maturity-onset diabetes (MODY) and gestational diabetes.
18. comprise the diagnostic products that is selected from following separated nucleic acid sequence:
(i) all or part of nucleotide sequence of the polypeptide of coding SEQ ID NO:2 or SEQ ID No:4,
The (ii) nucleotide sequence of forming by SEQ ID NO:1 or SEQ ID No:3, or
The nucleotide sequence that (iii) has 80% sequence homogeneity at least with the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.
19. the diagnostic products of claim 18, wherein the part of polypeptide is claim 3 definition in the step (i).
20. comprise all or part of diagnostic products of GPR39 receptor protein.
21. the diagnostic products of claim 20, wherein the GPR39 receptor protein is claim 2 definition, and the part of GPR39 receptor protein is claim 3 definition.
CNA2007800212075A 2006-06-08 2007-06-08 G protein-coupled receptor 39 (GPR39) Pending CN101467047A (en)

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CN115996908A (en) 2020-04-30 2023-04-21 俄勒冈健康科学大学 Antagonists of GPR39 proteins
WO2023076219A2 (en) 2021-10-26 2023-05-04 Vasocardea, Inc. Antagonists of gpr39 protein

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EP1280937A2 (en) * 2000-04-26 2003-02-05 Millennium Pharmaceuticals, Inc. Methods and compositions for the diagnosis and treatment of cardiovascular and tumorigenic disease using 4941
US20030232769A1 (en) * 2002-06-17 2003-12-18 Isis Pharmaceuticals Inc. Antisense modulation of G protein-coupled receptor 39 expression
US20040071708A1 (en) * 2002-09-26 2004-04-15 Immusol, Inc. GPR 39 modulators that control cancerous cell growth
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