JP4926952B2 - Use of GPR86 receptor - Google Patents

Description

FIELD The present invention relates to newly identified functions of nucleic acids, polypeptides encoded thereby, and their production and use. More particularly, the nucleic acids and polypeptides relate to certain G protein-coupled receptors (GPCRs), hereinafter referred to as “GPR86”, and members of the purinoceptor family of GPCRs. The invention also relates to the inhibition or activation of the action of such nucleic acids and polypeptides.

Background It is well known that many medically important biological processes are mediated by proteins involved in signaling pathways involving G proteins and / or second messengers such as cAMP (Lefkowitz, Nature , 1991, 351: 353-354). Such a protein is referred to as a protein involved in a pathway having a G protein, or “PPG protein”. Some examples of these proteins include GPC receptors, such as adrenergic and dopamine receptors (Kobilka, BK, et al., Proc. Natl Acad. Sci., USA, 1987, 84: 46-50; Kobilka BK , Et al., Science, 1987, 238: 650-656; Bunzow, JR, et al., Nature, 1988, 336: 783-787), G protein itself, effector proteins such as phospholipase C, adenyl cyclase, and phosphodiesterase, and actuators And proteins such as protein kinase A and protein kinase C (Simon, MI, et al., Science, 1991, 252: 802-8).

  For example, in certain modes of signal transduction, the effect of hormone binding is activation of the enzyme adenylate cyclase in the cell. Enzyme activation by hormones depends on the presence of nucleotide GTP. GTP also affects hormone binding. The G protein links the hormone receptor to adenylate cyclase. The G protein has been shown to exchange GTP for bound GDP when activated by hormone receptors. The GTP-carrying form then binds to activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by the G protein itself, returns the G protein to its basal, inactive form. Thus, the G protein serves a dual role, as an intermediate that relays the signal from the receptor to the effector, and as a clock that controls the duration of the signal.

  The membrane protein gene superfamily of G protein-coupled receptors (GPCRs) has been characterized as having seven putative transmembrane domains. Domains are thought to correspond to transmembrane α-helices connected by extracellular or cytoplasmic loops. G protein-coupled receptors include a wide range of biologically active receptors such as hormones, viruses, growth factors and neuronal receptors.

  G protein-coupled receptors (also known as 7TM receptors) contain these seven conserved hydrophobic stretches of about 20-30 amino acids that connect to at least eight different hydrophilic loops It is characterized as. The G protein family of coupled receptors includes dopamine receptors that bind to neuroleptics used to treat psychiatric and neurological disorders. Examples of other members of this family include, but are not limited to, calcitonin, adrenergic agonist, endothelin, adenosine, muscarinic agonist, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsin, endothelial differentiation gene -1, rhodopsin, odorants, and cytomegalovirus receptors.

  Most G protein-coupled receptors each have a single conserved cysteine residue in the first two extracellular loops, which are thought to form disulfide bonds and stabilize functional protein structures. It has been. The seven transmembrane regions are designated TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction.

  Phosphorylation and lipidation of cysteine residues (palmitine oxidation or farnesylation) can affect the signaling of several G protein coupled receptors. Most G protein-coupled receptors have potential phosphorylation sites in the third cytoplasmic loop and / or the carboxy terminus. For some G protein coupled receptors, such as β-adrenergic receptors, phosphorylation by protein kinase A and / or specific receptor kinases mediates receptor desensitization. For some receptors, the ligand binding site of a G protein coupled receptor includes a hydrophilic socket formed by the transmembrane domains of multiple G protein coupled receptors, the socket being a G protein coupled receptor. It is thought to be surrounded by hydrophobic residues in the body. The hydrophilic side of each G protein-coupled receptor transmembrane helix is thought to face inward to form a polar ligand binding site. TM3 is involved in having multiple ligand binding sites (eg TM3 aspartate residues) in multiple G protein coupled receptors. TM5 serine, TM6 asparagine and TM6 or TM7 phenylalanine or tyrosine are also involved in ligand binding.

G protein-coupled receptors can be conjugated to various intracellular enzymes, ion channels and transporters in cells by heterotrimeric G proteins (Johnson et al., Endoc. Rev., 1989, 10: 317-331). See) Various G protein α subunits selectively stimulate specific effectors to modulate various biological functions within the cell. Phosphorylation of cytoplasmic residues of G protein-coupled receptors has been identified as an important mechanism for the regulation of G protein coupling of some G protein-coupled receptors. G protein-coupled receptors are present at multiple sites within a mammalian host. Over the last 15 years, as many as 350 therapeutics targeting the 7-transmembrane (7 TM) receptor have been successfully marketed.
Lefkowitz, Nature, 1991, 351: 353-354 Kobilka, BK, et al., Proc. Natl Acad. Sci., USA, 1987, 84: 46-50 Kobilka BK, et al., Science, 1987, 238: 650-656 Bunzow, JR, et al., Nature, 1988, 336: 783-787 Simon, MI, et al., Science, 1991, 252: 802-8 Johnson et al., Endoc. Rev., 1989, 10: 317-331

  For this reason, G protein-coupled receptors have a proven and proven history as therapeutic drug targets. Clearly, there is a need for the identification and characterization of additional receptors that can play a role in the prevention, amelioration or correction of dysfunction or disease.

Overview In a first aspect of the invention, the inventors have identified a molecule suitable for the treatment, prevention or alleviation of a GPR86 related disease, particularly an inflammatory disease or pain, wherein the candidate molecule is a GPR86 polypeptide. Determining whether it is an agonist or antagonist, provided that said GPR86 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7, a fragment thereof, or a sequence at least 90% identical thereto Provide the method.

  Preferably, said GPR86 polypeptide is encoded by the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or a sequence at least 90% identical thereto.

  Preferably, the method comprises exposing the candidate molecule to a GPR86 polypeptide and determining whether the candidate molecule binds to a GPR86 polypeptide.

Preferably, the agonist, contacting a cell containing the GPR86 receptor are conjugated with GPR86 preferentially G protein G _i with a candidate compound, and, GPCR sensitivity markers, such as cyclic AMP (cAMP) in the cell By measuring whether or not the level of the blood pressure decreases as a result of the contact.

Preferably, the antagonist contacts a cell comprising a GPR86 receptor conjugated to GPR86 preferential G protein G _i with a candidate compound, and a GPCR sensitive marker such as cyclic AMP (cAMP) in said cell By measuring whether or not the level of increases as a result of the contact.

Preferably, the agonist contacts a cell comprising a GPR86 receptor conjugated to a promiscuous G protein such as _Gα16 with a candidate compound, and as in cyclic AMP (cAMP) in the cell. By measuring whether the level of a sensitive GPCR sensitive marker increases as a result of the contact.

Preferably, the antagonist contacts a cell containing a GPR86 receptor conjugated to a promiscuous G protein such as _Gα16 with a candidate compound, and as in cyclic AMP (cAMP) in the cell. By measuring whether the level of a sensitive GPCR sensitive marker decreases as a result of the contact.

Preferably, the method comprises
(a) providing a wild-type animal or a transgenic non-human animal having a functionally disrupted endogenous GPR86 gene;
(b) exposing said wild-type animal or transgenic non-human animal to a candidate molecule; and
(c) measuring whether the biological parameters of the animal have changed as a result of the contact.

  Preferably, said biological parameter is selected from the group consisting of a response to a stimulus, a response to heat, a response to light, an immune response, an inflammatory response, a response to pain, preferably a response to pain.

Preferably, the method comprises
(a) providing a wild-type cell or a cell having a functionally disrupted endogenous GPR86 gene, preferably a cell isolated from a transgenic non-human animal having a functionally disrupted endogenous GPR86 gene;
(b) exposing the cell to a candidate molecule; and
(c) determining whether the biological activity of the GPR86 polypeptide has changed as a result of said contacting.

  In a second aspect of the invention, a wild-type animal or a disrupted endogenous in a method of identifying an agonist or antagonist of a GPR86 polypeptide for use in the treatment, prevention or alleviation of a GPR86 related disease, particularly an inflammatory disease or pain Use of a transgenic non-human animal having a sex GPR86 gene is provided.

  Use of a transgenic non-human animal having a functionally disrupted endogenous GPR86 gene or an isolated cell or tissue thereof as a model for GPR86-related diseases, particularly inflammatory diseases or pain.

  Preferably, said transgenic non-human animal comprises a disrupted GPR86 gene, preferably a disrupted GPR86 gene having a deletion in the GPR86 gene or a portion thereof.

  Preferably, said transgenic non-human animal exhibits a change in any one or more of the following phenotypes when compared to wild-type animals: response to stimulation, response to heat, response to light, immunity Response, inflammatory response, response to pain, preferably response to pain.

  Preferably, said transgenic non-human animal exhibits at least one of the following: (a) an altered pain sensitivity that is an increased or decreased pain sensitivity when compared to a wild type animal, and (b) an increase Or altered inflammatory pain sensitivity that is reduced inflammatory pain sensitivity.

  Preferably, said transgenic non-human animal is a rodent, preferably a mouse.

  In the third aspect of the present invention, the inventors provide a method for identifying an agonist or antagonist of a GPR86 polypeptide, comprising administering a candidate compound to a wild-type or transgenic non-human animal as described above. And measuring a change in biological parameter.

  As a fourth aspect of the present invention, it is described in SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 for identifying an agonist or antagonist of GPR86 for the treatment or prevention of GPR86-related diseases, particularly inflammatory diseases or pain. The use of a GPR86 polypeptide comprising the amino acid sequence of:

  In a fifth embodiment of the invention, the inventors have identified SEQ ID NO: 1, SEQ ID NO: 2 for identifying GPR86 agonists or antagonists for the treatment, prevention of GPR86 related diseases, in particular inflammatory diseases or pain. Alternatively, the use of a GPR86 polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 4, a fragment thereof, or a sequence at least 90% identical thereto is provided.

  Preferably, the pain is acute pain, chronic pain, skin pain, somatic pain, visceral pain, distant pain, including myocardial ischemia, phantom pain, and neuropathic pain (neuralgia) Pain caused by disease, headache, migraine, cancer pain, pain caused by neurological disorders such as Parkinson's disease, spinal and peripheral nerve surgery, brain tumor, traumatic brain injury (TBI), spinal cord trauma, chronic Pain syndrome, pain due to chronic fatigue syndrome, trigeminal neuralgia, glossopharyngeal neuralgia, neuralgia such as postherpetic neuralgia, and causalgia, one of the following: lupus, sarcoidosis, arachitis, arthritis, rheumatic disease, menstrual pain, back Selected from the group consisting of pain, low back pain, joint pain, abdominal pain, chest pain, labor pain, skeletal muscle disease and skin disease, head trauma, and pain caused by fibromyalgia.

  Preferably, the inflammatory disease is selected from inflammatory disorders, preferably the group consisting of: inflammatory diseases (eg rheumatoid arthritis, multiple sclerosis, Guillain-Barre syndrome, Crohn's disease, ulcerative colitis, Psoriasis, graft-versus-host rejection, systemic lupus erythematosus or insulin-dependent diabetes), autoimmune diseases (e.g. toxic shock syndrome, osteoarthritis, diabetes or inflammatory bowel disease), acute pain, chronic pain, neuropathy Pain, contact dermatitis, atherosclerosis, glomerulonephritis, reperfusion injury, bone resorption injury, asthma, stroke, myocardial infarction, burns, adult respiratory distress syndrome (ARDS), multiple organ disorders secondary to trauma, acute Caused by skin disease with inflammatory components, acute purulent meningitis, necrotizing enterocolitis, syndrome associated with hemodialysis, septic shock, leukopheresis, granulocyte transfusion, smoke inhalation Acute or chronic inflammation of the lung, endometriosis, Behcet's disease, uveitis, ankylosing spondylitis, pancreatitis, cancer, Lyme disease, restenosis following percutaneous transluminal coronary angioplasty, Alzheimer's disease, traumatic arthritis , Sepsis, chronic obstructive pulmonary disease, congestive heart failure, osteoporosis, cachexia, Parkinson's disease, periodontal disease, gout, allergic disease, age-related macular degeneration, infection and pustular fibrosis.

  The present invention, in a sixth aspect, provides an agonist or antagonist of GPR86 identified by the method or use described herein.

  In a seventh aspect of the invention, the use of such molecules for the treatment, prevention or alleviation of inflammatory diseases or pain is provided.

  In an eighth aspect of the invention, the inventors provide an inflammatory disease or a disease comprising any one or more of the following: a GPR86 polypeptide or a portion thereof; an antibody against the GPR86 polypeptide; or a nucleic acid that can encode such. A diagnostic kit for pain or susceptibility thereto is provided.

  In a ninth aspect of the invention, the inventors provide a method for treating an individual suffering from an inflammatory disease or pain, comprising increasing or decreasing the activity or amount of a GPR86 polypeptide in said individual. The method is provided.

  Preferably, the method comprises administering to said individual a GPR86 polypeptide, an agonist of a GPR86 polypeptide or an antagonist of GPR86.

In a tenth aspect of the present invention, there is provided a method for diagnosing an inflammatory disease or pain, comprising the following steps:
(a) detecting the expression level or pattern of GPR86 polypeptide in an animal suffering from or suspected of suffering from such a disease; and
(b) comparing the expression level or expression pattern with that of normal animals;
A method comprising the steps of:

Sequence listing SEQ ID NO: 1 shows the cDNA sequence of human GPR86. SEQ ID NO: 2 shows an open reading frame derived from SEQ ID NO: 1. SEQ ID NO: 3 shows the amino acid sequence of human GPR86. SEQ ID NO: 4 shows the open reading frame of cDNA derived from mouse GPR86. SEQ ID NO: 5 shows the amino acid sequence of mouse GPR86. SEQ ID NO: 6 shows another cDNA sequence of human GPR86. SEQ ID NO: 7 shows another amino acid sequence of human GPR86. SEQ ID NOs: 8 to 20 show the primer sequences of knockout plasmids. SEQ ID NO: 21 shows the sequence of a knockout plasmid.

Methods of Use Unless otherwise indicated, the practice of the present invention uses conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA technology, and immunology, which are within the ability of one skilled in the art. These techniques are explained in publications. See, for example, J. Sambrook, EF Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, 1-3, Cold Spring Harbor Laboratory Press; Ausubel, FM et al. (1995 and periodic complements; Current Protocols in Molecular Biology, Chapters 9, 13, and 16, John Wiley & Sons, New York, NY); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley &Sons; JM Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; MJ Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; DMJ Lilley and JE Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies: A Laboratory Manual: Portable Protocol NO.I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow (editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-314-2), 1855, Lars-Inge Larsson “Immunocytochemistry: Theory and Practice”, CRC Press inc., Baca Raton, Florida, 1988, ISBN 0-8493-6078-1, John D. Pound (ed); “Immunochemical Protocols, vol 80”, in the series: “Methods in Molecular Biology”, Humana Press, Totowa, New Jersey, 1998, ISBN 0-89603- 493-3, Handbook of Drug Screening, Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, NY, Marcel Dekker, ISBN 0-8247-0562-9); Lab Ref: A Handbook of Recipes, Reagents, and Other Edited by Reference Tools for Use at the Bench, Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3; and The Merck Manual of Diagnosis and Therapy (17th edition, Beers, MH, and Berkow , R, ed., ISBN: 0911910107, John Wiley & Sons). Each of these general materials is incorporated herein by reference.

Detailed description
GPR86
This specification generally describes G protein-coupled receptors (GPCRs), particularly the purinoceptor-type G protein-coupled receptors we call GPR86, as well as homologs, variants or derivatives thereof.

  The inventors have identified that transgenic animals lacking functional GPR86 exhibit altered pain sensitivity compared to wild type animals. Specifically, GPR86 knockouts have reduced pain sensitivity. In addition, these animals exhibit altered inflammatory pain sensitivity, particularly reduced inflammatory pain sensitivity.

  Accordingly, we disclose the use of GPR86, homologs, variants or derivatives thereof, and modulators thereof in the treatment, reduction or diagnosis of pain. This and other embodiments of the present invention are described in further detail below.

Expression profile of GPR86
By polymerase chain reaction (PCR) amplification of GPR86 cDNA, expression of GPR86 is detected in varying amounts in bone marrow, thymus, lymph nodes, leukocytes, osteoblasts and chondrocytes of human tissue. The expression was also seen in the human derived cell lines Jurkat CD4 + and Myla CD8 + (both derived from T cells). Low level expression was found in Colo720 (derived from lymphocytes) and THP1 cells (derived from monocytes) (Example 4; FIG. 7).

  In addition, GPR86 expression is found in mouse tissue spleen, salivary gland, spinal cord, tongue, adipose, testis, heart, eye, lung, kidney, thymus, stomach and small intestine, brain, liver and gallbladder, blood, bladder and It was also detected in the adrenal gland (Example 4; FIG. 8).

  The expression profile of GPR86 is shown in FIG.

  Searching human EST data sources with BLASTN using the GPR86 cDNA of SEQ ID NOs: 1 and 6 reveals identical cDNAs from human heart, placenta, and colon, and libraries derived from mouse skin, breast, and hypothalamus. It was issued. This indicates that GPR86 is expressed in these normal or abnormal tissues. Thus, GPR86 polypeptides, nucleic acids, probes, antibodies, expression vectors and ligands can detect, diagnose, treat and other assays for diseases associated with overexpression, underexpression and abnormal expression of GPR86 in these and other tissues. Useful for.

GPR86 related diseases For the methods and compositions described herein, GPR86 GPCRs are useful for the treatment and diagnosis of a range of diseases. These diseases are referred to as “GPR86-related diseases” for convenience.

  Therefore, GPR86-deficient animals can be used as models for GPR86-related diseases. GPR86, fragments, homologues, variants and derivatives thereof, and modulators (including in particular agonists and antagonists) can be used for diagnosis or treatment of GPR86-related diseases. In particular, GPR86 can be used to screen for molecules that can affect its function, and the molecules can be used to treat GPR86-related diseases.

  We have demonstrated herein that human GPR86 maps to Homo sapiens chromosome 3q24. Thus, in certain embodiments, GPR86 can be used to treat or diagnose diseases that map to this locus, chromosomal band, region, arm or the same chromosome.

  Known diseases that are known to be related to the same locus, chromosomal band, region, arm, or chromosome as the chromosomal location of GPR86 (i.e., homo-sapiens chromosome 3q24) include (Inside): Acute myeloid leukemia (3q24), Hermansky-Pudrack syndrome (3q24), platelet ADP receptor abnormality (3q24-q25) and Dundee-Walker syndrome (3q24).

  Thus, in a preferred embodiment, GPR86 and its modulators (such as agonists and antagonists) can be used to treat dopamine related diseases such as Parkinson's disease, heart diseases such as supraventricular arrhythmia or ventricle using any means described herein. Can be used to diagnose or treat sexual arrhythmias, hypotension, nausea, Tourette syndrome, stress and pain.

  Knockout mice lacking GPR86 exhibit a range of phenotypes as demonstrated in the Examples.

  In particular, Example 5 and FIG. 4 show that when tested in the Tail Flick test, knockout animals lacking functional GPR86 are less sensitive to external stimuli and pain compared to wild-type animals. Indicates that Similarly, Example 7 and FIG. 6 show that when tested in the Von Frey Hair test, knockout animals lacking functional GPR86 are less sensitive to external stimuli and pain compared to wild-type animals. It shows that.

  Thus, in a preferred embodiment of the invention, GPR86 and its modulators (such as agonists and antagonists) can be used to diagnose or treat pain and cancer using any means described herein. . In particular, pain includes neuropathic, postherpetic neuralgia, diabetic neuralgia, trigeminal neuralgia, alcohol (ethanol) related neuralgia, leprosy neuralgia, vascular neuralgia, uremic neuralgia, Guillain-Barre syndrome, multiple sclerosis , Acute immune neuropathy, thoracic outlet syndrome, carpal tunnel syndrome, tarsal tunnel syndrome, sensory abnormal femoral neuralgia, complex local pain syndrome, temporomandibular joint syndrome, atypical facial pain, low back pain, lumbar stenosis, intervertebral disc disease, neck Includes pain, cervical spondylosis myelopathy, cervical spondylosis, cervical disc disease, cervical myelofacial pain, inflammatory pain, osteoarthritis, rheumatoid arthritis, inflammatory bowel disease It is. Cancer pain, particularly cancer, includes breast cancer, prostate cancer, colon cancer, lung cancer, ovarian cancer and osteosarcoma. Includes headache, migraine, tension migraine, cluster migraine, chronic paroxysmal migraine, as well as visceral pain, dysmenorrhea, non-digestive dyspepsia, non-psychogenic chest pain, irritable bowel syndrome Also included are phantom rectum pain, thermal hyperalgesia and postoperative pain.

  Furthermore, the present inventors have demonstrated in the Examples that GPR86 is also expressed in cells derived from immune response cells (Examples 3 and 4). Transgenic animals lacking functional GPR86 exhibit a reduced inflammation tendency as shown in Example 6 and FIG. This indicates that GPR86 is involved in inflammatory responses, including those related to the inflammatory and neuropathic aspects of pain.

  In another embodiment, GPR86 and its modulators (such as agonists and antagonists) are used to treat inflammatory diseases (e.g., rheumatoid arthritis, multiple sclerosis, Guillain-Barre syndrome, clones) using any means described herein. Disease, ulcerative colitis, psoriasis, graft-versus-host rejection, systemic lupus erythematosus or insulin-dependent diabetes), autoimmune diseases (e.g. toxic shock syndrome, osteoarthritis, diabetes or inflammatory bowel disease), acute Pain, chronic pain, neuropathic pain, contact dermatitis, atherosclerosis, glomerulonephritis, reperfusion injury, bone resorption failure, asthma, stroke, myocardial infarction, burn, adult respiratory distress syndrome (ARDS), trauma Secondary organopathy, dermatosis with acute inflammatory component, acute purulent meningitis, necrotizing enterocolitis, syndrome associated with hemodialysis, septic shock, leukocyte transport Act, granulocyte transfusion, acute or chronic inflammation of the lung caused by smoke inhalation, endometriosis, Behcet's disease, uveitis, ankylosing spondylitis, pancreatitis, cancer, Lyme disease, percutaneous transluminal coronary artery formation Subsequent restenosis, Alzheimer's disease, traumatic arthritis, sepsis, chronic obstructive pulmonary disease, congestive heart failure, osteoporosis, cachexia, Parkinson's disease, periodontal disease, gout, allergic disease, age-related macular degeneration, infection And pustular fibrosis can be used to diagnose or treat.

  For convenience, diseases that can be treated and / or diagnosed by the use of GPR86 are referred to as “GPR86-related diseases”. In particularly preferred embodiments, GPR86-related diseases include those with pain as a symptom (see paragraph above).

  As noted above, GPR86 and its modulators (such as agonists and antagonists) may be used to diagnose and / or treat any of these specific diseases using any of the methods and compositions described herein. can do.

  In particular, the inventors have identified nucleic acids, vectors comprising GPR86 nucleic acids, polypeptides (including homologs, variants or derivatives thereof), GPR86 nucleic acids and / or polypeptides for the treatment or diagnosis of the specific diseases described above. Envisage the use of pharmaceutical compositions, host cells as well as transgenic animals. Furthermore, the inventors have used the compounds that interact with or bind to GPR86 and alter the level of endogenous cAMP, antibodies against GPR86, as well as these in the treatment or diagnosis of the specific diseases described above. A manufacturing method or identification method is assumed. In particular, we include the use of any of these compounds, compositions, molecules, etc. in the manufacture of a vaccine for the treatment or prevention of said specific disease. We also disclose a diagnostic kit for the detection of the specific disease in an individual.

  Linkage mapping methods for identifying such specific diseases or additional specific diseases that can be treated or diagnosed by use of GPR86 are known in the art and are described elsewhere herein.

pain
GPR86 and variants thereof and sequences encoding them, antibodies against them, etc. described herein can be used for the diagnosis and / or treatment of many diseases associated with pain.

Acute pain Acute pain is defined as short-term pain or pain that can be easily identified. Acute pain is what the body warns of the current damage to tissue or disease. Acute pain is often rapid and sharp, followed by tingling pain. Acute pain concentrates in an area and then spreads somewhat.

Chronic pain Chronic pain is medically defined as pain that has lasted for more than 6 months. This persistent or intermittent pain often has lost its purpose over time because it does not help the body prevent damage. Chronic pain is often more difficult to treat than acute pain. In general, skilled nursing is required to treat any chronic pain. When opioids are used for a long time, drug resistance, drug addiction and even psychological addiction can occur. Drug resistance and drug addiction are common in opioid users, but psychological addiction is rare.

  Physiological pain experiences can be divided into four categories depending on the source and the nociceptors involved (pain-sensitive nerves).

Skin Pain Skin pain is caused by damage to the skin or surface tissue. Skin nociceptors terminate just below the skin and the density of nerve endings is high, resulting in clear, localized short-term pain. Examples of injuries that cause skin pain include paper cuts, minor burns (first degree), and lacerations.

Somatic pain Somatic pain originates from ligaments, tendons, bones, blood vessels and even the nerve itself and is sensed by somatic pain nociceptors. The rarity of pain receptors in these areas results in pain that is longer, duller and less localized than skin pain; examples include ankle sprains and fractures.

Visceral pain Visceral pain originates from visceral nociceptors of body organs located within the body organs and internal cavities. In these areas, nociceptors are even rarer, resulting in pain that aches and lasts longer than somatic pain. Visceral pain is extremely difficult to locate, and multiple damage to visceral tissue refers to “distant pain”, a pain whose sensation is limited to an area completely unrelated to the site of the injury. Myocardial ischemia (loss of blood flow to a portion of myocardial tissue) is probably the best known example of distant pain; the sensation is as a sensation constrained to the upper thorax, or left shoulder, arm or sometimes hand In addition, it can occur as aching.

Another type of pain phantom limb pain is a pain sensation from the limb that is no longer or no longer from any physical signal from which it is an almost universally appealing experience for limb amputees and limb paralysis. Neuropathic pain (“neuralgia”) can result from damage or disease of the nerve tissue itself. This can interfere with the ability of sensory nerves to transmit the correct information to the thalamus, so the brain reads painful stimuli despite no apparent or confirmed physiological pain source.

  Trigeminal neuralgia (“painful tic”) refers to pain caused by injury or damage to the trigeminal nerve. The trigeminal nerve has three branches: V1 gives sensation to the forehead and eye area, V2 gives sensation to the nose and face, and V3 gives sensation to the jaw and tip area. There is a trigeminal nerve that provides sensation on either side of the face. The pain of only one of the trigeminal neuralgia can extend to the cheeks, mouth, nose and / or jaw muscles. Trigeminal neuralgia generally affects older people, but younger people or those with multiple sclerosis can also experience trigeminal neuralgia.

  The main symptom of trigeminal neuralgia is pain in either the forehead, cheek, chin or lower jaw contour. In severe cases, all three regions or both sides can be affected. Pain attacks are intense, convulsive, short-term, and reminiscent of what is felt with electric shock. This pain can be triggered by common daily activities such as brushing, speaking, chewing, drinking, shaving or even kissing. The frequency of pain attacks increases with time, making it more disturbing and disabling.

  Glossopharyngeal neuralgia is a disease type characterized by sudden onset of pain in the sensory distribution area of the glossopharyngeal nerve. The seizure is identical to trigeminal neuralgia except for the location of pain and the stimulating factor of pain. Typical pain is a repetitive series of electric shock-like punctures on one side of the area of the tonsils or back of the tongue. In addition, pain can be dissipated to the ears or originate from the ears.

  The sensory stimulus that induces this pain is swallowing, and in severe seizures the patient does not move while sitting, bending the head forward and letting the saliva hang down from the mouth. Cardiac arrest, stun (syncope), and seizure have been associated with attacks of glossopharyngeal neuralgia. The cause of glossopharyngeal neuralgia is almost unknown. However, a certain number of cases are attributed to tumors, glossopharyngeal nerve compression by vertebral arteries and vascular malformations.

  Postherpetic neuralgia refers to chronic pain that follows infection with herpes zoster virus. Herpes zoster, also called herpes zoster, is a recurrent infection of varicella-zoster (varicella) virus infection. The virus remains latent in the nerve until the patient's immune system is weakened. The acute disorder of shingles causes pain, which usually heals. However, in some patients, pain continues chronically—this is postherpetic neuralgia.

  Symptoms of herpes zoster include electric shock, deep, continuous pain: pain is 65% on the chest and 20% on the face. When involved in the face, the most common site of virus is the trigeminal eye branch (the top of the face above the eyebrows). Pain usually resolves naturally in 2-4 weeks. However, a few patients have persistent pain. Pain is in the area of the previous rash and is exacerbated by gently stroking the affected skin and relieved by applying pressure to the area. Clothes friction is often very painful. This persistent pain is called postherpetic neuralgia. In cases of herpes zoster on the face, the prevalence of postherpetic neuralgia is higher.

  Causalgia is a rare symptom after partial damage to the peripheral nerve. The pain is characterized by burning pain, autonomic dysfunction, and nutritional changes. Serious cases are called major causalgia. Minor causalgia represents a less serious form, similar to reflex sympathetic dystrophy (RSD). RSD is associated with dominant muscle and joint symptoms, and osteoporosis is common on X-rays.

  Causalgia is caused by peripheral nerve damage, usually brachial plexus damage. Denervation causes hypersensitivity resulting in increased pain, and increased norepinephrine release causes sympathetic findings. Symptoms include pain: pain that is usually scorching and conspicuous on the hands or feet. Most onset is within 24 hours of injury. The median nerve, ulnar nerve and sciatic nerve are most frequently associated. Almost any sensory stimulus makes pain worse. Vascular changes: Increased blood due to vasodilation (warm pink) or decreased blood due to vasoconstriction (cold, mottled blue). Nutritional changes: dry / scaled skin, stiff joints, tapered fingers, stiff uncut nails, long / hard hair or hair loss, sweating changes.

Identity and similarity with GPR86
GPR86 is structurally related to other proteins in the G protein-coupled receptor family, as shown in the results of sequencing the amplified cDNA product encoding human GPR86. The cDNA sequence of SEQ ID NO: 1 comprises an open reading frame (SEQ ID NO: 2, nucleotide numbers 19-1084), which encodes a 354 amino acid polypeptide set forth in SEQ ID NO: 3. We found that human GPR86 maps to homo sapiens chromosome 3q24. Another cDNA of SEQ ID NO: 6 encodes the polypeptide set forth in SEQ ID NO: 7.

P2Y12 platelet ADP receptor [homo sapiens] identity = 154/316 (48%), positive = 211/316 (66%)
KIAA0001 putative G protein-coupled receptor; G protein-coupled receptor for UDP-glucose; identity = 140/295 (47%), positive = 193/295 (64%)
Platelet-activated receptor homolog [Homo sapiens]; identity = 42/144 (29%), positive = 78/144 (54%).

  From the analysis of GPR86 polypeptide (SEQ ID NO: 3) using pfam's HMM structure prediction software (http://www.sanger.ac.uk/Software/Pfam/search.shtml), the GPR86 peptide is a 7TM-1 structural class. (See Fig. 1).

  The mouse homologue of human GPR86 has been cloned and its nucleic acid and amino acid sequences are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively. The mouse GPR86 cDNA of SEQ ID NO: 4 shows a high degree of identity with the human GPR86 (SEQ ID NO: 2) sequence, while the amino acid sequence of mouse GPR86 (SEQ ID NO: 5) is similar to human GPR86 (SEQ ID NO: 3 and SEQ ID NO: 7). Shows a high degree of identity and similarity.

  Human and mouse GPR86 are therefore members of a large family of G protein-coupled receptors (GPCRs).

GPR86 polypeptide As used herein, the term “GPR86 polypeptide” refers to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7, or a homologue, variant or derivative thereof. Is intended. Preferably the polypeptide comprises the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 7 or is a homologue, variant or derivative thereof. Most preferably, the polypeptide comprises the sequence shown in SEQ ID NO: 7 or is a homologue, variant or derivative thereof.

  “Polypeptide” refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, ie, peptide isosteres. “Polypeptide” refers to both short chains, often referred to as peptides, oligopeptides or oligomers, and to longer chains, commonly referred to as proteins. Polypeptides may also contain amino acids other than the 20 amino acids encoded by so-called genes.

  “Polypeptide” includes amino acid sequences modified by natural processes, eg, post-translational processing, or by chemical modification methods (well known in the art). These modifications are described in detail in basic teaching materials and in more detailed monographs as well as in high-volume research literature. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and the amino or carboxy terminus. It will be appreciated that the same type of modification may be made to the same or different degrees at multiple sites in a given polypeptide. Also, a given polypeptide can have many types of modifications.

  Polypeptides may be branched as a result of ubiquitination and may be cyclic with or without branching. Cyclic, branched, and branched cyclic polypeptides can result from natural post-translational processing or can be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent addition of flavins, covalent addition of heme moieties, covalent addition of nucleotides or nucleotide derivatives, covalent addition of lipids or lipid derivatives, phosphatidyl Inositol covalent addition, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-linking formation, cystine formation, pyroglutamic acid formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation , Hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated amino acid addition to proteins (eg arginylation), and ubiquitin Included. For example, Proteins-Structure and Molecular Properties, 2nd edition, TE Creighton, WH Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Posttranslational Covalent Modification of Proteins , BC Johnson, ed., Academic Press, New York, 1983; Seifter et al., “Analysis for protein modifications and nonprotein cofactors”, Meth Enzymol (1990) 182: 626-646 and Rattan et al., “Protein Synthesis: Posttranslational Modifications and Aging” , Ann NY Acad Sci (1992) 663: 48-62.

  As used herein, “variant”, “homologue”, “derivative”, or “fragment” is any substitution, mutation, modification, replacement, deletion of one (or more) amino acids from or to a sequence. Or including additions. Unless stated otherwise, references to “GPR86” and “GPR86 GPCR” include references to such variants, homologues, derivatives and fragments of GPR86.

  Preferably, as applied to GPR86, the resulting amino acid sequence has GPCR activity, more preferably has at least the same activity as GPR86 described in SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7. . In particular, the term “homolog” encompasses identity with respect to structure and / or function, provided that the resulting amino acid sequence has GPCR activity. With respect to sequence identity (ie similarity), there is preferably at least 70%, more preferably at least 75%, more preferably at least 85%, even more preferably at least 90% sequence identity. More preferably there is at least 95% sequence identity, more preferably at least 98% sequence identity. These terms also encompass polypeptides derived from amino acids of allelic variants of the GPR86 nucleic acid sequence.

  When referring to “receptor activity” or “biological activity” of a receptor such as GPR86, these terms refer to the metabolic or physiological function (similar or improved activity, or undesirable) of the GPR86 receptor. Including such activities with reduced side effects). Also included are antigenic and immunogenic activities of GPR86 receptor. Examples of GPCR activity and methods for assaying and quantifying such activity are known in the art and are described elsewhere herein.

  As used herein, a “deletion” is defined as a change in a nucleotide or amino acid sequence that is absent from one or more nucleotides or amino acid residues, respectively. As used herein, an “insertion” or “addition” is a change in a nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are added compared to the natural substance. As used herein, “substitution” occurs by replacing one or more nucleotides or amino acids with different nucleotides or amino acids, respectively.

  The GPR86 polypeptides described herein can also have deletions, insertions or substitutions that produce silent changes and result in functionally equivalent amino acid sequences. Designed amino acid substitutions can be made based on the similarity of residue polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphiphilic nature. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids having uncharged polar head groups with similar hydrophilicity values These include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine.

Conservative substitutions can be made, for example, according to the table below. Amino acids in the same section in the second column, and preferably amino acids in the same row in the third column, can be substituted for each other:

  The GPR86 polypeptide may further comprise a heterologous amino acid sequence, typically at the N-terminus or C-terminus, preferably at the N-terminus. Heterologous sequences can include sequences that affect intracellular or extracellular protein targeting, such as leader sequences. A heterologous sequence can also include sequences that increase the immunogenicity of the polypeptide and / or sequences that facilitate identification, extraction and / or purification of the polypeptide. Further particularly preferred heterologous sequences are polyamino acid sequences such as polyhistidine, which are preferably at the N-terminus. Polyhistidine sequences of at least 10 amino acids, preferably at least 17 amino acids but less than 50 amino acids are particularly preferred.

  The GPR86 polypeptide may be in the form of a “mature” protein or part of a larger protein such as a fusion protein. Often it is convenient to add additional amino acid sequences, including secretory or leader sequences, pro sequences, sequences to aid purification (such as multiple histidine residues) or additional sequences for stability during recombinant production. .

  GPR86 polypeptides are conveniently produced by recombinant methods utilizing known techniques. However, they can also be produced synthetically using techniques well known to those skilled in the art, such as solid phase synthesis. Such polypeptides can also be produced as fusion proteins, eg, to aid extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and / or transcriptional activation domain) and β-galactosidase. It may also be convenient to allow removal of the fusion protein sequence by including a protein cleavage site (such as a thrombin cleavage site) between the fusion protein partner and the protein sequence of interest. Preferably the fusion protein does not interfere with the protein function of the subject sequence.

  The GPR86 polypeptide can be in a substantially isolated form. The term is intended to refer to a change from the natural state by the human hand. If an “isolated” composition or substance is one that occurs in nature, it has been changed or transferred from its original environment, or both. For example, a polynucleotide, nucleic acid or polypeptide naturally occurring in a living animal is not “isolated”, but a polynucleotide, nucleic acid or polypeptide isolated from its natural coexisting material is not As used in the specification, it is “isolated”.

  By the way, it is understood that the GPR86 protein can be mixed with a carrier or diluent that does not interfere with the intended purpose of the protein and still be considered substantially isolated. Such a polypeptide can also be in a substantially purified form, in which case it is generally a preparation in which more than 90% of the proteins in the preparation are, for example, 95%, 98% or 99% GPR86 polypeptide. The protein is contained in the product.

  The present specification also relates to peptides comprising a portion of a GPR86 polypeptide. Accordingly, fragments of GPR86 and homologs, variants or derivatives thereof are encompassed. The peptide is 2 to 200 amino acids in length, preferably 4 to 40 amino acids. The peptide can be derived from the GPR86 polypeptide disclosed herein, for example, by digestion with a suitable enzyme (such as trypsin). Alternatively, the peptides, fragments, etc. can be produced by recombinant methods or synthesized by synthetic methods.

  The term “peptide” includes various synthetic peptide variants known in the art, such as retroinverso D peptides. The peptide can be an antigenic determinant and / or a T cell epitope. The peptide can be immunogenic in vivo. Preferably the peptide is capable of inducing neutralizing antibodies in vivo.

  By aligning GPR86 sequences from different species, which regions of the amino acid sequence are conserved between different species (`` homology regions '') and which regions are different between different species (`` non-homologous regions '') Can be determined.

  A GPR86 polypeptide can thus include a sequence corresponding to at least a portion of a homologous region. A homologous region shows a high degree of homology between at least two organisms. For example, a homologous region may exhibit at least 70%, preferably at least 80%, more preferably at least 90%, more preferably at least 95% identity at the amino acid level using the above-described assay. Peptides containing sequences corresponding to homologous regions can be used in the therapeutic strategies detailed below. Alternatively, the GPR86 peptide can include a sequence corresponding to at least a portion of the heterologous region. Non-homologous regions show a low degree of homology between at least two organisms.

GPR86 Polynucleotides and Nucleic Acids We further elaborate on GPR86 polynucleotides, GPR86 nucleotides and GPR86 nucleic acids, methods for their production, methods of use and the like in the present specification.

  The terms “GPR86 polynucleotide”, “GPR86 nucleotide” and “GPR86 nucleic acid” are used interchangeably and include a nucleic acid sequence comprising the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. It is intended to mean a nucleic acid or a homologue, variant or derivative thereof. Preferably the polynucleotide / nucleic acid comprises or is itself a homologue, variant or derivative of the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, SEQ ID NO: 6, most preferably SEQ ID NO: 2.

  These terms are also intended to include nucleic acid sequences that can encode the polypeptides and / or peptides described herein, ie, GPR86 polypeptides. Thus, GPR86 polynucleotides and nucleic acids include a nucleotide sequence that can encode a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or a homologue, variant or derivative thereof. Preferably GPR86 polynucleotides and nucleic acids comprise a nucleotide sequence capable of encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7, or a homologue, variant or derivative thereof.

  “Polynucleotide” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or unmodified DNA or modified RNA or modified DNA. “Polynucleotide” includes, but is not limited to, single stranded and double stranded DNA, DNA that is a mixture of single stranded and double stranded regions, single stranded and double stranded RNA, single stranded. And a hybrid molecule comprising DNA and RNA, which may be a mixture of double stranded regions, RNA, single stranded or more typically double stranded or a mixture of single stranded and double stranded regions included. Furthermore, “polynucleotide” also refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and specialized bases such as inosine. Various modifications are made to DNA and RNA; thus, “polynucleotide” refers to chemically, enzymatically or metabolically modified forms of polynucleotides, such as are typically naturally occurring, and viruses and Includes the chemical forms of DNA and RNA characteristic of cells. “Polynucleotide” also embraces relatively short polynucleotides, often referred to as oligonucleotides.

  One skilled in the art will appreciate that as a result of the degeneracy of the genetic code, a variety of nucleotide sequences can encode the same polypeptide.

  As used herein, the term “nucleotide sequence” refers to nucleotide sequences, oligonucleotide sequences, polynucleotide sequences and variants, homologs, fragments and derivatives thereof (eg, portions thereof). The nucleotide sequence can be genomic or synthetic or recombinantly derived DNA or RNA, but it can be double-stranded or single-stranded, as long as it represents the sense or antisense strand or a combination thereof. A defined nucleotide sequence can be prepared by use of recombinant DNA technology (eg, recombinant DNA).

  Preferably the term “nucleotide sequence” means DNA.

  As used herein, “variant”, “homologue”, “derivative” or “fragment” is any substitution, mutation, modification, replacement of one (or more) nucleic acid from or to the sequence of a GPR86 nucleotide sequence. , Deletions or additions. Unless stated otherwise, references to “GPR86” and “GPR86 GPCR” include references to such variants, homologues, derivatives and fragments of GPR86.

  Preferably, the resulting nucleotide sequence encodes a polypeptide having GPCR activity, preferably having at least as much activity as the GPCR described in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7. Preferably, the term “homolog” is intended to encompass the same structural and / or functional identity such that the resulting nucleotide sequence encodes a polypeptide having GPCR activity. With respect to sequence identity (ie similarity), there is preferably at least 70%, more preferably at least 75%, more preferably at least 85%, even more preferably at least 90% sequence identity. More preferably there is at least 95% sequence identity, more preferably at least 98% sequence identity. These terms also encompass allelic variants of the sequence.

Calculation of Sequence Homology For any sequence described herein, sequence identity can be determined by a simple “eyeball” comparison (ie, a strict comparison) of one or more sequences with another sequence; This makes it possible to check whether the other sequence has, for example, at least 70% sequence identity with the present sequence.

  Relative sequence identity is also determined by any commercially available computer program that can calculate the percent identity between two or more sequences using any algorithm for determining identity, eg, using default parameters. can do. A typical example of such a computer program is CLUSTAL. Other computer programming methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux et al. 1984 Nucleic Acids Research 12: 387) and FASTA (Atschul et al. 1990 J Molec Biol 403-410).

  % Homology can be calculated for contiguous sequences. That is, one sequence is aligned with another sequence and each amino acid in one sequence is directly compared to the corresponding amino acid in another sequence, one residue at a time. This is referred to as “ungapped” alignment. Typically, such ungapped alignments are performed only on a relatively short number of residues.

  This method is a very simple and robust method, for example, in a pair of sequences that are otherwise identical, one insertion or deletion causes the next amino acid residue to be out of alignment, Therefore, it cannot be considered that the homology% may be greatly reduced when the entire alignment is performed. Thus, most sequence comparison methods are designed to generate optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology score. This is accomplished by inserting “gaps” in the sequence alignment to try to maximize local homology.

  On the other hand, these more complex methods assign a “gap penalty” to each gap that occurs in the alignment, so that if the number of identical amino acids is the same, the sequence alignment (as less as possible between two comparison sequences) should be as small as possible. Try to get a higher score than one with many gaps (which reflects high correlation). The commonly used “affin gap cost” imposes a relatively high cost on the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap score system. High gap penalties will of course produce optimized alignments with few gaps. Most alignment programs can change the gap penalty. However, when using such software for sequence comparison, it is preferred to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for one gap and -4 for each extension.

  Thus, the calculation of% maximum homology first requires the generation of an optimal alignment taking into account the gap penalty. A suitable computer program for performing such alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A .; Devereux et al., 1984, Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparison include, but are not limited to, the BLAST package (Ausubel et al., 1999 ibid.-Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and GENEWORKS package software comparison tools are included. Both BLAST and FASTA are available for offline and online searching (Ausubel et al., 1999 ibid, pages 7-58 to 7-60).

  Although it is possible to measure the final% homology in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a generally weighted similarity score matrix is used, which assigns a score based on chemical similarity or evolutionary distance for each pairwise comparison. An example of such a commonly used matrix is an initial setting matrix of a BLAST package software program called a BLOSUM62 matrix. GCG Wisconsin programs generally use either common default values or custom symbol comparison tables if provided. It is preferable to use a common default value for the GCG package or an initial value matrix such as BLOSUM62 for other software.

  Conveniently, the BLAST algorithm is used with parameters set to default values. The BLAST algorithm is described in detail at http://www.ncbi.nih.gov/BLAST/blast_help.html, the contents of which are incorporated herein by reference. Search parameters (defined below) can be conveniently set to predetermined default parameters.

  Conveniently, “substantial identity” is the same as a sequence that matches an EXPECT value of at least about 7, preferably at least about 9, and most preferably 10 or more as assessed by BLAST. The default threshold for EXPECT in a BLAST search is usually 10.

  BLAST (Basic Local Alignment Search Tool) is a heuristic search algorithm performed by the blastp, blastn, blastx, tblastn, and tblastx programs; these programs are based on the statistical methods of Karlin and Altschul (Karlin and Altschul 1990, Proc. Natl. Acad. Sci. USA 87: 2264-68; Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-7; http://www.ncbi.nih.gov/BLAST/ (See blast_help.html) with a slight modification to assign importance to what it finds. The BLAST program is tailored for sequence similarity searches (eg, to identify homologues for query sequences). For a discussion of the basics of sequence database similarity searches, see Altschul et al. (1994) Nature Genetics 6: 119-129.

  The five BLAST programs available from http://www.ncbi.nlm.nih.gov handle the following tasks: blastp-compares amino acid query sequences with protein sequence databases; blastn-nucleotide query sequences Compare with nucleotide sequence database; blastx-Compare conceptual translation product of 6 frames (both strands) of nucleotide query sequence with protein sequence database; tblastn-Compare protein query sequence with all 6 reading frames Compare to nucleotide sequence database dynamically translated (both strands); tblastx-compare 6 frame translations of nucleotide query sequence to 6 frame translations of nucleotide sequence database.

  BLAST uses the following search parameters:

  HISTOGRAM-display a histogram of scores for each search; default is yes. (See parameter H in the BLAST manual).

  DESCRIPTIONS-Limits the number of short descriptions in the matching sequence to the specified number; the default limit is 100 descriptions. (See parameter V in the manual page).

  EXPECT-Statistical validity threshold for reporting matches against database sequences; default is 10, which means that 10 matches are only found probabilistically according to the stochastic model by Karlin and Altschul (1990) Is expected. If the statistical validity attributed to a match is greater than the EXPECT threshold, no match is reported. Lower EXPECT thresholds are more stringent and fewer chance matches are reported. A fractional value can also be used (see parameter E in the BLAST manual).

  CUTOFF-Cut-off score for reporting high score segment pairs (HSP). The default value is calculated from the EXPECT value (see above). An HSP is reported for a database sequence only if the statistical validity attributed to it is at least as high as attributed to a single HSP with a score equal to the CUTOFF value. Higher CUTOFF values increase stringency, resulting in fewer probabilistic match reports. (See parameter S in the BLAST manual). Typically, the effective threshold can be handled more intuitively using EXPECT.

ALIGNMENTS-Limit database sequences to a specified number (high score segment pairs (HSP) are reported for them); the default limit is 50. If more database sequences meet the statistical validity threshold to be reported (see EXPECT and CUTOFF below), only the match that is attributed to the largest statistical validity is reported. (See parameter B in the BLAST manual).

MATRIX-Specify different scoring matrices for BLASTP, BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992). Other valid options include: PAM40, PAM120, PAM250 and IDENTITY. An alternative scoring matrix is not provided for BLASTN; specifying a MATRIX indication in a BLASTN request results in an error response.

STRAND-Limits the TBLASTN search to only the top or bottom strand of the database sequence; or limits the BLASTN, BLASTX, or TBLASTX search to only the reading frame of the top or bottom strand of the query sequence.

FILTER-This masks out segments of the query sequence that have low compositional complexity (determined by the SEG program of Wootton & Federhen (1993) Computers and Chemistry 17: 149-163), or short periodic internal repeats By the XNU program of Claverie & States (1993) Computers and Chemistry 17: 191-201, or by DUST program for Talastov and Lipman (see http://www.ncbi.nlm.nih.gov) for BLASTN Masked). Filtering can eliminate reports that are statistically effective but not biologically interesting from the blast output (e.g. hits to common acidic, basic, or proline-rich regions) and more biological of the query sequence. Interesting regions are available for detailed matching against database sequences.

  Low complexity sequences discovered by the filter program replace the letter “N” in the nucleotide sequence (eg “NNNNNNNNNNNNN”) and the letter “X” in the protein sequence (eg “XXXXXXXXX”).

  Filtering only applies to query sequences (or their translation products), not database sequences. The default filtering is DUST for BLASTN and SEG for other programs.

  When applied to a sequence in SWISS-PROT, it is not uncommon for it to be masked at all by SEG, XNU, or both. Therefore, filtering should not be expected to be effective. Furthermore, in some cases, the sequence is masked in its entirety, suggesting that the statistical validity of every match reported against the unfiltered query sequence is questionable.

NCBI-gi-This causes the NCBI gi identifier to appear in the output in addition to the registration number and / or locus name.

  Most preferably, the sequence comparison is performed using a simple BLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST. In some embodiments, no gap penalty is used when determining sequence identity.

Hybridization We further describe nucleotide sequences that can hybridize to the sequences described herein, or any fragment or derivative thereof, or the complementary sequence of any of the foregoing.

  Hybridization refers to the process by which a nucleic acid strand is brought together with a complementary strand by base pairing (Coombs J (1994) Dictionary of Biotechnology, Stockton Press, New York NY) and the amplification performed by polymerase chain reaction technology. Process (described in Dieffenbach CW and GS Dveksler 1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview NY).

  Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA). And gives the defined "stringency" described below.

  Nucleotide sequences that can selectively hybridize to the nucleotide sequences described herein or their complements are generally at least 20, preferably at least 25 or 30, for example at least 40, 60 or 100 or more consecutive Over a region of nucleotides is at least 70%, preferably at least 75%, more preferably at least 85 or 90% and even more preferably at least 95% or 98% homologous to the corresponding nucleotide sequence described herein . Preferred nucleotide sequences comprise a region homologous to SEQ ID NO: 1, 2 or 4, preferably a region homologous to at least 70%, 80% or 90% and more preferably at least 95% to one of the sequences.

The term “selectively hybridizable” means that the nucleotide sequence used as a probe is used under conditions where the target nucleotide sequence hybridizes to the probe at a level significantly higher than background. . Background hybridization can occur due to other nucleotide sequences present (eg, those present in the cDNA or genomic DNA library being screened). In this event, background is the level of signal produced by the interaction of the probe with a non-specific DNA member of the library, 10 times the intensity of the specific interaction observed with the target DNA. This means that the level is less than, preferably less than 100 times. The strength of the interaction can be measured, for example, by radiolabeling the probe (eg at ³² P).

  Also included within the scope of this specification are nucleotide sequences that can hybridize to the nucleotide sequences described herein under moderate to maximal stringency conditions. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA). And gives the defined "stringency" described below.

  Maximum stringency is typically about Tm-5 ° C (5 ° C below the Tm of the probe); high stringency is about 5 ° C to 10 ° C below the Tm; moderate stringency is about 10 Low stringency is seen at about 20 ° C. to 25 ° C. below Tm. As will be appreciated by those skilled in the art, maximum stringency hybridization can be used to identify or detect identical nucleotide sequences, while moderate (or low) stringency hybridization is similar or related nucleotide sequences. Can be used to identify or detect.

In a preferred embodiment, we hybridize to one or more GPR86 nucleotide sequences under stringent conditions (eg, 65 ° C. and 0.1 × SSC (1 × SSC = 0.15 M NaCl, 0.015 M Na ₃ citrate pH 7.0)). Nucleotide sequences that can be disclosed are disclosed. Where the nucleotide sequence is double stranded, both strands of the duplex are included individually or in combination. When a nucleotide sequence is single stranded, it is to be understood that the complementary sequence of that nucleotide sequence is also included within the scope of this specification.

We further describe nucleotide sequences that can hybridize to the sequences described herein, or sequences complementary to any fragment or derivative thereof. Similarly, nucleotide sequences that are complementary to sequences that are capable of hybridizing to the sequences described herein are also encompassed. These types of nucleotide sequences are examples of variant nucleotide sequences. In this regard, the term “variant” encompasses sequences that are complementary to sequences that are capable of hybridizing to the nucleotide sequences described herein. Preferably, however, the term “variant” is used under conditions that are stringent to the nucleotide sequences described herein (eg, 65 ° C. and 0.1 × SSC {1 × SSC = 0.15 M NaCl, 0.015 Na ₃ citrate pH 7.0}). Includes a sequence complementary to a sequence capable of hybridizing.

Cloning GPR86 and homologues We describe nucleotide sequences that are complementary to the sequences described herein, or any fragment or derivative thereof. If the sequence is complementary to the fragment, the sequence can be used as a probe to identify and clone similar GPCR sequences in other organisms and the like.

  Our disclosure thus makes it possible to achieve the cloning of GPR86, its homologues and other structurally or functionally related genes from humans and other species such as mice, pigs, sheep and the like. A polynucleotide identical or sufficiently identical to the nucleotide sequence contained in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or a fragment thereof can be used as a hybridization probe for cDNA and genomic DNA, and can be used from an appropriate library. Partial or full length cDNA and genomic clones encoding GPR86 can be isolated. These probes also identify cDNA and genomic clones of other genes that have sequence similarity to the GPR86 gene, preferably a high degree of sequence similarity, including genes encoding homologs and orthologs from non-human species. Can be used to release. Hybridization screening, cloning and sequencing techniques are known to those skilled in the art and are described, for example, in Sambrook et al. (Supra).

  Typically, a nucleotide sequence suitable for use as a probe is 70% identical, preferably 80% identical, more preferably 90% identical and even more preferably 95% identical to the subject sequence. A probe generally comprises at least 15 nucleotides. Preferably, such probes have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes range from 150 to 500 nucleotides, more specifically about 300 nucleotides.

  In certain embodiments, a method for obtaining a polynucleotide encoding a GPR86 polypeptide (including homologs and orthologs from non-human species) comprises the steps of: 2. screening with a labeled probe of SEQ ID NO: 4 or a fragment thereof, and isolating partial or full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybridization techniques are well known to those skilled in the art. Stringent hybridization conditions are as defined above or otherwise: 50% formamide, 5XSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate ( Incubate overnight at 42 ° C in a solution containing pH 7.6), 5X Denhart solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA, then wash the filter paper with 0.1XSSC at about 65 ° C To do.

GPR86 Functional Assay Cloned putative GPR86 polynucleotides can be confirmed by sequence analysis or functional assays. For example, a putative GPR86 or homolog can be assayed for receptor activity as follows. An RNA transcript with a cap structure derived from a linearized plasmid template encoding the GPR86 receptor cDNA is synthesized using RNA polymerase in vitro according to standard methods. In vitro transcripts are suspended in water at a final concentration of 0.2 mg / ml. Ovarian leaves are transferred from adult female toads, stage V follicle-removed oocytes are obtained, and RNA transcripts (10 ng / oocytes) are injected as a 50 nl bolus using a microinjection device. Two electrode voltage clamps were used to measure the current from individual Xenopus oocytes in response to agonist exposure. Recording was performed at room temperature in a standard medium consisting of NaCl 115, KCl 2.5, CaCl ₂ 1.8, NaOH-HEPES 10, pH 7.2 (in mM). The Xenopus system can also be used to screen known ligands and tissue / cell extracts for activated ligands, as detailed below.

GPR86 expression assay
In order to design useful therapeutic agents for treating GPR86-related disorders, it is helpful to determine the expression profile of GPR86 (whether wild-type or specific mutant). From this, methods known in the art can be used to determine the organ, tissue and cell type (and developmental stage) in which GPR86 is expressed. For example, traditional or “electronic” Northern analysis can be performed. Reverse transcriptase PCR (RT-PCR) can also be used to assay expression of the GPR86 gene or variant. More sensitive methods for determining the expression profile of GPR86 include RNAse protection assays known in the art.

  Northern analysis is a research technique used to detect the presence of gene transcripts and involves the hybridization of a labeled nucleotide sequence to a membrane to which RNA from a particular cell type or tissue is bound. . (Sambrook, supra, Chapter 7 and Ausubel, F. M. et al., Supra, Chapters 4 and 16). Similar computer techniques ("electronic Northern") that apply BLAST can be used to search for identical or related molecules in nucleotide databases (eg, GenBank or LIFESEQ database (Incyte Pharmaceuticals)). This type of analysis has the advantage of being faster than multiple membrane based hybridization. Furthermore, the sensitivity of computer searches can be varied to determine whether any particular match is classified as accurate or homologous.

  The polynucleotides and polypeptides described herein, including the probes described above, can be used as research reagents and materials for discovering therapeutic and diagnostic agents for animal and human diseases, as described elsewhere herein, Can be used.

Expression of GPR86 Polypeptide The present invention further includes a method for producing a GPR86 polypeptide. This method generally involves culturing a host cell containing a nucleic acid encoding a GPR86 polypeptide or a homolog, variant or derivative thereof under suitable conditions (ie, conditions under which the GPR86 polypeptide is expressed).

  In order to express biologically active GPR86, a nucleotide sequence encoding GPR86 or a homologue, variant or derivative thereof is inserted into an appropriate expression vector (i.e., elements necessary for transcription and translation of the inserted coding sequence). Into the vector).

  Methods which are well known to those skilled in the art are used to construct expression vectors containing sequences encoding GPR86 and appropriate transcriptional and translational control elements. Such methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. These techniques are described in Sambrook, J. et al. (1989; Molecular Cloning, A Laboratory Manual, Chapters 4, 8, and 16-17, Cold Spring Harbor Press, Plainview, NY) and Ausubel, FM et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, Chapters 9, 13, and 16, John Wiley & Sons, New York, NY).

  A variety of expression vector / host systems may be utilized to contain and express the GPR86 coding sequence. These include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; infected with viral expression vectors (eg, baculovirus) Insect cell line; plant cell line transformed with viral expression vector (eg cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or bacterial expression vector (eg Ti or pBR322 plasmid); or animal cell line Is included. Any host cell may be used.

  `` Control elements '' or `` regulatory elements '' are those of the untranslated regions of the vector that interact with host intracellular proteins to transcribe and translate (i.e. enhancers, promoters and 5 'and 3' untranslated regions). is there. Such elements can vary in their strength and specificity. Depending on the vector system and host used, any number of suitable transcription and translation elements (including constitutive and inducible promoters) can be used. For example, when cloning bacterial systems, inducible promoters such as the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) Or the PSPORT1 plasmid (GIBCO / BRL) hybrid lacZ promoter can be used. In insect cells, the baculovirus polyhedrin promoter can be used. Promoters or enhancers from plant cell genomes (eg, heat shock, RUBISCO and storage protein genes) or plant viruses (eg, viral promoters or leader sequences) can be cloned into vectors. In mammalian cell systems, promoters derived from mammalian genes or from mammalian viruses are preferred. If it is necessary to generate a cell line containing multiple copies of the GPR86 coding sequence, vectors based on SV40 or EBV may be used with an appropriate selectable marker.

  In bacterial systems, many types of expression vectors can be selected depending on the intended use of GPR86. For example, when large amounts of GPR86 are required for antibody induction, vectors that express high levels of easily purified fusion proteins can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), where the GPR86 coding sequence is amino terminal Met followed by 7-galactosidase 7 May be linked to the vector in-frame with the residue, resulting in a hybrid protein), pIN vector (Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509), etc. Is included. pGEX vectors (Promega, Madison, Wis.) can also be used to express foreign polypeptides as fusion proteins with glutathione-S-transferase (GST). In general, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption onto glutathione-agarose beads and subsequent elution in the presence of free glutathione. The protein produced in such a system may be designed to include heparin, thrombin or factor XA protease cleavage sites, thereby optionally freeing the cloned polypeptide of interest from the GST moiety. be able to.

  In the yeast Saccharomyces cerevisiae, a wide variety of vectors containing constitutive or inducible promoters (such as alpha factor, alcohol oxidase and PGH) can be used. For reviews, see Ausubel (supra) and Grant et al. (1987; Methods Enzymol. 153: 516-544).

  When using plant expression vectors, the expression of the GPR86 coding sequence may be driven by any of a variety of promoters. For example, viral promoters (such as CaMV 35S and 19S promoters) can be used alone or in combination with a TMV-derived ω leader sequence. (Takamatsu, N. (1987) EMBO J. 6: 307-311.). Alternatively, a plant promoter such as a small subunit of RUBISCO or a heat shock promoter can be used. (Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broglie, R. et al. (1984) Science 224: 838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17: 85-105.). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in several publicly available reviews. (See, eg, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y .; pp. 191-196.).

  Insect systems can also be used to express GPR86. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as the vector, Spodoptera frugiperda cells or Trichoplusia cells. Express foreign genes in larvae. The GPR86 coding sequence can be cloned into a nonessential region of the virus (eg, the polyhedrin gene) and placed under the control of the polyhedrin promoter. If GPR86 insertion is successful, the polyhedrin gene becomes inactive and a recombinant virus lacking the coat protein is produced. The recombinant virus can then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae, in which GPR86 can be expressed. (Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci. 91: 3224-3227.).

  In mammalian host cells, various virus-based expression systems are available. In cases where an adenovirus is used as an expression vector, the GPR86 coding sequence can be ligated to an adenovirus transcription / translation complex consisting of the late promoter and tripartite leader sequence. Insertion into the nonessential E1 or E3 region of the viral genome can also be used to obtain live viruses capable of expressing GPR86 in infected host cells. (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. 81: 3655-3659.). In addition, transcription enhancers (such as the Rous sarcoma virus (RSV) enhancer) can be used to increase expression in mammalian host cells.

  Thus, for example, the GPR86 receptor can be expressed in human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells. In order to maximize receptor expression, typically all 5 'and 3' untranslated regions (UTR) are removed from the receptor cDNA prior to insertion into the pCDN or pCDNA3 vector. Cells are transfected with individual receptor cDNAs by lipofectin and selected in the presence of 400 mg / ml G418. After 3 weeks of selection, single clones are selected and expanded for further analysis. HEK293 or CHO cells transformed with the vector alone are used as negative controls. To isolate cell lines that stably express individual receptors, typically about 24 clones are selected and analyzed by Northern blot analysis. Receptor mRNA is generally detected from about 50% of the G418 resistant clones analyzed.

  Human artificial chromosomes (HACs) are also available for delivering large DNA fragments that can be contained and expressed in plasmids. Approximately 6 kb to 10 Mb of HAC is constructed and delivered for therapeutic purposes via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles).

  Certain initiation signals can also be used to achieve more efficient translation of the GPR86 coding sequence. Such signals include the ATG start codon and adjacent sequences. If the GPR86 coding sequence and its initiation codon and upstream sequence are inserted into a suitable expression vector, additional transcriptional or translational control signals may not be required. However, if only the coding sequence or fragment thereof is inserted, an exogenous translational control signal containing the ATG start codon should be provided. Moreover, the start codon should be in the correct reading frame to ensure translation of the entire insert. Foreign translation elements and initiation codons can be of various origins, natural and synthetic. Expression efficiency can be enhanced by adding appropriate enhancers (such as those described in the literature) to the specific cell line used. (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162.).

  In addition, a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such polypeptide modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing that cleaves the “prepro” form from the protein can also be used to aid in correct insertion, folding, and / or function. Various host cells (eg CHO, HeLa, MDCK, HEK293, and WI38) with specific intracellular and characteristic mechanisms for post-translational activity are obtained from the American Type Culture Collection (ATCC, Bethesda, Md.) It can be selected to ensure that correct modification and processing of the foreign protein is possible.

  Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, a cell line capable of stably expressing GPR86 can be transformed with an expression vector having a viral origin of replication and / or a foreign expression element and a selectable marker gene on the same or different vectors. . Following the introduction of the vector, the cells may be grown in rich medium for about 1-2 days and then switched to selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows the growth and recovery of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed cells. These include, but are not limited to, the herpes simplex virus thymidine kinase gene (Wigler, M. et al. (1977) Cell 11: 223-32) and the adenine phosphoribosyltransferase gene (Lowy, I. et al. (1980) Cell 22: 817-23), which can be used in tk ⁻ or apr ⁻ cells, respectively. In addition, antimetabolite, antibiotic or herbicide resistance can be used as a basis for selection. For example, dhfr confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77: 3567-70); npt confers resistance to aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14); and als or pat confer resistance to chlorosulfone and phosphinothricin acetyltransferase, respectively (Murry, supra) . Additional selection genes have been described, for example trpB (which allows the cell to use indole instead of tryptophan), or hisD (which allows the cell to use histinol instead of histidine). is there. (Hartman, SC and RC Mulligan (1988) Proc. Natl. Acad. Sci. 85: 8047-51.). Recently, the use of visualization markers has become widespread, and such markers include anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression that can be attributed to a particular vector system. be able to. (Rhodes, CA et al. (1995) Methods Mol. Biol. 55: 121-131.).

  The presence / absence of marker gene expression suggests that the gene of interest is also present, but the presence and expression of the gene may need to be confirmed. For example, if a sequence encoding GPR86 is inserted within the marker gene sequence, transformed cells containing the GPR86 coding sequence can be identified by the absence of marker gene function. Alternatively, the marker gene can be placed in tandem with the GPR86 coding sequence under the control of a single promoter. Expression of a marker gene in response to induction or selection usually indicates that a tandem gene is also expressed.

  Alternatively, host cells comprising a GPR86 encoding nucleic acid sequence and expressing GPR86 can be identified by various methods known to those skilled in the art. Such techniques include, but are not limited to, DNA--DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques (membranes, solutions, or for the detection and / or quantification of nucleic acid or protein sequences. Including chip-based technology).

  The presence of a GPR86-encoding polynucleotide sequence can be detected by DNA-DNA or DNA-RNA hybridization or amplification using a probe or fragment or fragment of the GPR86-encoding polynucleotide. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on GPR86 coding sequences to detect transformants containing DNA or RNA encoding GPR86.

  Various protocols for detecting and measuring the expression of GPR86, whether using polyclonal antibodies specific for the protein or monoclonal antibodies, are known in the art. Examples of such techniques include enzyme immunoassay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site monoclonal antibody-based immunoassay utilizing monoclonal antibodies that react with two non-interfering epitopes on GPR86 is preferred, although competitive binding assays are also available. These and other assay methods are well described in the art, for example, Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, Section IV, APS Press, St Paul, Minn.) And Maddox, DE et al. (1983; J. Exp. Med. 158: 1211-1216).

  A variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes to detect sequences related to GPR86-encoded polynucleotides include oligo-labeling using labeled nucleotides, nick translation, end-labeling, or PCR amplification Is mentioned. Alternatively, the GPR86 coding sequence or any fragment thereof can be cloned into a vector for production of mRNA probes. Such vectors are known in the art and are commercially available and should be used to synthesize RNA probes in vitro by adding appropriate RNA polymerase (such as T7, T3, or SP6) and labeled nucleotides. Can do. These techniques are performed using various commercial kits such as those provided by Pharmacia & Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.), And US Biochemical Corp. (Cleveland, Ohio). Can do. Suitable reporter molecules or labels to facilitate detection include radionuclides, enzymes, fluorescent materials, chemiluminescent materials, or chromogenic materials, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

  Host cells transformed with a GPR86-encoding nucleotide sequence can be cultured under conditions suitable for the expression and recovery of the protein from the cell culture. Depending on the sequence and / or vector used, the protein produced by the transformed cell can be present in the cell membrane, secreted, or contained within the cell. As will be appreciated by those skilled in the art, expression vectors containing GPR86-encoding polynucleotides can be designed to contain signal sequences that direct GPR86 to be secreted through prokaryotic or eukaryotic cell membranes. Other constructs can be used to combine the GPR86 coding sequence with a nucleotide sequence encoding a polypeptide domain that facilitates purification of the soluble protein. These purification facilitating domains include, but are not limited to, metal chelate peptides (such as histidine-tryptophan modules that allow purification on immobilized metal), protein A domains that allow purification on immobilized immunoglobulins And domains utilized in the FLAGS extension / affinity purification system (Immunex Corp., Seattle, Wash.). Adding a cleavable linker sequence (e.g., specific for Factor XA or enterokinase (Invitrogen, San Diego, Calif.), Etc.) between the purification domain and the GPR86 coding sequence is used to facilitate purification. sell. One such expression vector provides for the expression of a fusion protein containing GPR86 and a nucleotide encoding 6 histidine residues preceding the thioredoxin or enterokinase cleavage site. The histidine residue facilitates purification by immobilized metal ion affinity chromatography (described in IMIAC; Porath, J. et al. (1992) Prot. Exp. Purif. 3: 263-281), while enterokinase cleavage. The site provides a means to purify GPR86 from the fusion protein. A discussion of vectors containing fusion proteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol. 12: 441-453).

  GPR86 fragments can be produced not only by recombinant production, but also by direct peptide synthesis using solid phase techniques. (Merrifield J. (1963) J. Am. Chem. Soc. 85: 2149-2154.). Protein synthesis can be performed manually or by automated methods. Automated synthesis can be performed, for example, using an Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Various fragments of GPR86 can be synthesized separately and then combined to produce the full-length molecule.

Biosensor
GPR86 polypeptides, nucleic acids, probes, antibodies, expression vectors and ligands are useful as biosensors (and for their production).

  According to Aizawa (1988), Anal. Chem. Symp. 17: 683, a biosensor is a receptor for molecular recognition (eg, an immobilized antibody or receptor, eg, a GPR86 G protein coupled receptor). , Defined as a unique combination with an amplification means for conveying the measured value. One group of such biosensors detects changes that occur in the optical properties of the surface layer due to the interaction between the receptor and the surrounding medium. Of these techniques, circular dichroism and surface plasmon resonance are particularly mentioned. Biosensors incorporating GPR86 can be used to detect the presence or level of GPR86 ligands (such as nucleotides such as purines or purine analogs) or analogs of these ligands. The construction of such biosensors is well known in the art.

  Thus, cell lines expressing GPR86 receptors are reporter systems for the detection of ligands (such as ATP) via receptor-stimulated [3H] inositol phosphate or other second messenger formation. (Watt et al., 1998, J Biol Chem May 29; 273 (22): 14053-8). Receptor-ligand biosensors are also described in Hoffman et al., 2000, Proc Natl Acad Sci USA Oct 10; 97 (21): 11215-20. Optical biosensors, including GPR86, and other biosensors can also be used to detect the level or presence of G-proteins and other proteins, such as Figler et al., 1997, Biochemistry Dec 23; 36 (51): 16288-99 and Sarrio et al., 2000, Mol Cell Biol 2000 Jul; 20 (14): 5164-74). Sensor units for biosensors are described for example in US 5,492,840.

Screening assay
GPR86 polypeptides (including homologs, variants, and derivatives), whether natural or recombinant, bind to the receptor, or activate GPR86 (agonist) or inhibit activation (antagonist) It can be used in a method for screening a compound.

  Thus, the polypeptides can also be used to assess the binding of small molecule substrates and ligands, such as in cells, cell-free preparations, compound libraries, and natural product mixtures. These substrates and ligands may be natural substrates and natural ligands, or may be structural or functional mimetics. See Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991).

  GPR86 polypeptides are responsible for many biological functions, including many pathologies as described above in “GPR86-related diseases”. Therefore, it is desirable to find compounds and agents that can stimulate GPR86 on the one hand, and on the other hand, can inhibit the function of GPR86. In general, agonists and antagonists are used for therapeutic and prophylactic purposes for conditions such as dopamine related diseases such as Parkinson's disease, heart diseases such as supraventricular arrhythmia or ventricular arrhythmia, hypotension, nausea, Tourette syndrome, stress and pain. used.

  An agonist can activate the GPR86 receptor to any degree. Similarly, antagonists can inactivate GPR86 to any degree or inhibit its activity. Thus, the GPR86 receptor can be partially inactivated by an antagonist to any degree compared to its original, basal, or background level of activity (partial antagonist) or completely to such a level. It can be inactivated (antagonist or full antagonist). Antagonists can further inactivate the receptor, for example to zero activity (adverse agonists). The term “antagonist” thus specifically includes both full antagonists, partial antagonists and adverse agonists.

  Also included within the terms “agonist” and “antagonist” are those that modulate the expression of GPR86 at the transcriptional and / or translational level and those that modulate its activity.

  The rational design of candidate compounds that can potentially interact with the GPR86 protein may be based on structural studies of the molecular shape of the polypeptide. One means of determining which sites interact with a particular other protein is physical structure determination, such as X-ray crystallography or two-dimensional NMR techniques. These provide guidance on which amino acid residues form the molecular contact region. For a detailed description of protein structure determination, see, for example, Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York.

  A rational design alternative uses screening methods that generally involve contacting GPR86 with a candidate modulator and detecting its effects. In general, such methods include generating appropriate cells that express a GPR86 receptor polypeptide (optionally with a partner protein) on their surface, and contacting GPR86 or cells or both with a candidate modulator, and Detecting changes in intracellular levels of related molecules.

  Molecules whose concentrations are affected by the activity of GPCRs (especially GPR86) and in particular molecules that can be used as markers for GPR86 activity are known in the art. These are referred to as “GPCR sensitive markers” for convenience. Examples of such GPCR sensitive markers include intracellular calcium levels, calcium efflux, adenylate cyclase levels, and cyclic AMP levels. In a preferred embodiment, the GPCR sensitive marker comprises intracellular cyclic AMP (cAMP) and the screening comprises detecting changes in intracellular levels of cyclic AMP.

In a particularly preferred embodiment, GPR86 can also be screened in the presence of a partner protein; such partner protein can be co-expressed with a GPR86 receptor polypeptide, preferably intracellularly. The partner protein may preferably comprise a promiscuous G protein, such as a G protein, eg GPR86 preferential G-protein G _i or G _α16 .

The screening using cells with GPR86 and GPR86 preferential G- protein G _i, agonist reduces the level of intracellular cAMP concentration, whereas antagonists increase intracellular cAMP concentration.

When the screening uses promiscuous G proteins such as GPR86 and _Gα16 , agonists increase the level of intracellular cAMP concentration, whereas antagonists decrease intracellular cAMP concentration.

Therefore, the present inventors, discloses a method of identifying agonists of GPR86 when conjugated to GPR86 preferentially G- protein G _i, that this method comprises contacting a candidate compound a cell expressing the GPR86 receptor, And determining whether the intracellular cyclic AMP (cAMP) level decreases as a result of the contact. Alternatively, GPR86 can be co-expressed with a promiscuous G protein such as G _α16 , the method comprising contacting a cell expressing GPR86 and G _α16 with a candidate compound, and Measuring whether the level of click cAMP increases as a result of the contact.

We further disclosed are methods for identifying antagonists of GPR86 when conjugated to GPR86 preferentially G- protein G _i, that this method comprises contacting a candidate compound a cell expressing the GPR86 receptor, And determining whether the intracellular cyclic AMP (cAMP) level increases as a result of the contact. Alternatively, if the GPR86 way to promiscuous stimulatory G protein co-expressed, such as G _Arufa16, be contacted with a candidate compound a cell expressing GPR86 and G _Arufa16, and levels of cyclic cAMP in said cell Measuring whether or not the result of the contact decreases.

  Cells that can be used for screening can be of various cell types. Such cells include animal-derived cells, yeast, Drosophila or E. coli. Cells expressing the receptor (or cell membrane containing the expressed receptor) are then contacted with a test compound to observe binding or inhibition of stimulation or functional response. For example, Xenopus oocytes may be injected with GPR86 mRNA or GPR86 polypeptide, and the current induced by exposure to the test compound is measured using voltage clamp measurements (as used herein). Separately listed).

  In addition, GPR86 receptor activity can be assayed using a microphysiological assay. Activation of various second messenger systems results in a small amount of acid being pushed out of the cell. Most of the acid formed is the result of the increased metabolic activity necessary to stimulate intracellular signaling processes. Although the pH change of the medium surrounding the cells is very small, it can be detected by, for example, a CYTOSENSOR microphysiology measuring instrument (Molecular Devices Ltd., Menlo Park, Calif.). CYTOSENSOR can therefore detect the activation of receptors coupled to energy-utilized intracellular signaling pathways (eg, the G protein coupled receptors described herein).

  Instead of testing each candidate compound individually with the GPR86 receptor, a library or bank of candidate ligands can be advantageously created and screened. From this, for example, a bank of over 200 putative receptor ligands was assembled for screening. Banks include: Transmitters, hormones and chemokines known to act through the human 7 transmembrane (7TM) receptor; putative agonists of the human 7TM receptor Possible natural compounds, non-mammalian biologically active peptides for which no mammalian counterpart has yet been identified; and not found in nature to activate the unknown 7TM receptor of natural ligands Compound. This bank is used to screen receptors for known ligands, including functional assays (i.e. calcium, cAMP, microphysiology, oocyte electrophysiology, etc.) and binding assays Both are used (detailed elsewhere). However, there are still many mammalian receptors for which no cognate activating ligand (agonist) or inactivating ligand (antagonist) has been found. Thus, active ligands for these receptors may not be included in the ligand banks identified to date. Thus, the GPR86 receptor is also functionally screened against tissue extracts (using functional screens such as calcium, cAMP, microphysiology, oocyte electrophysiology) to identify natural ligands . Extracts that produce a positive functional response can then be subfractionated until the activating ligand is isolated and identified by assaying the fractions as described herein.

  The 7TM receptor expressed in HEK 293 has been shown to be functionally coupled to PLC activation and calcium mobilization and / or cAMP stimulation or inhibition. Thus, certain screening methods include extracellular or intracellular pH changes caused by receptor activation of cells expressing GPR86 receptor (e.g., transfected Xenopus oocytes, CHO or HEK293 cells) or Including use in a system to measure intracellular calcium changes. In this technique, the compound can be contacted with a cell that expresses the receptor polypeptide. Second messenger responses, such as signal transduction, pH changes, or changes in calcium levels, are then measured to determine whether potential compounds activate or inhibit the receptor.

  In such experiments, it has been observed that basal calcium levels in HEK 293 cells of receptor-transfected cells or vector control cells are in the normal range of 100 nM to 200 nM. Fura 2 is added to HEK 293 cells expressing GPR86 or recombinant GPR86 and over 150 selected ligands or tissue / cell extracts are evaluated for agonist-induced calcium mobilization during the same day. Similarly, HEK 293 cells expressing GPR86 or recombinant GPR86 are evaluated using standard cAMP quantitative assays for promoting or inhibiting cAMP production. Agonists exhibiting calcium transients or cAMP fluctuations are tested in vector control cells to determine if the response is specific to transfected cells expressing the receptor.

  Another method involves screening for receptor inhibitors by measuring inhibition or activation of GPR86 receptor-mediated cAMP and / or adenylate cyclase accumulation. Such a method involves transfecting a eukaryotic cell with a receptor to express the receptor on the cell surface. The cells are then exposed to potential antagonists in the presence of the receptor. Then, the amount of cAMP accumulated is measured. If the potential antagonist binds to the receptor and thereby inhibits receptor binding, receptor-mediated cAMP levels or adenylate cyclase activity will be reduced or increased.

  Another method for detecting agonists or antagonists of the GPR86 receptor is the yeast-based technique described in US Pat. No. 5,482,835, which is incorporated herein by reference.

  If the candidate compound is a protein, particularly an antibody or peptide, a library of candidate compounds can be screened using phage display technology. Phage display is a molecular screening protocol that utilizes recombinant bacteriophages. This technique involves transforming a bacteriophage with a gene encoding one compound from a library of candidate compounds to express a candidate compound specific to each phage or phagemid. The transformed bacteriophage, which is preferably linked to a solid support, expresses the appropriate candidate compound and displays it on its own phage coat. Certain candidate compounds that can bind to the GPR86 polypeptide or GPR86 peptide are enriched by selection strategies based on affinity interactions. Successful candidate substances are then characterized. Phage display has advantages over standard affinity ligand screening techniques. The phage surface presents the candidate substance in a three-dimensional arrangement that closely resembles its natural arrangement. This allows for more specific and high affinity binding for screening purposes.

Another method of screening a library of compounds utilizes eukaryotic or prokaryotic host cells that are stably transformed with recombinant DNA molecules that express the library of compounds. Such cells, whether in proliferative or fixed form, can be used in standard binding partner assays. See also Parce et al. (1989) Science 246: 243-247; and Owicki et al. (1990) Proc. Nat'l Acad. Sci. USA 87; 4007-4011 which described a sensitive method for detecting cellular responses. Competitive assays are particularly useful, in which cells expressing a library of compounds are labeled with known antibodies (such as ¹²⁵ I-antibodies) and test samples (such as binding compositions) that are known to bind to GPR86 polypeptides. A candidate compound whose binding affinity to the product has been measured). The bound form of the polypeptide and the free labeled binding partner are then separated to assess the extent of binding. The amount of test sample bound is inversely proportional to the amount of labeled antibody bound to the polypeptide.

  Any of a number of techniques can be used to separate the free binding partner from the bound one to assess the extent of binding. This separation step may typically involve techniques such as attachment to a filter and subsequent washing, attachment to plastic and subsequent washing, or centrifugation of the cell membrane.

  Yet another approach is to use solubilized, unpurified or solubilized purified polypeptides or peptides, such as those extracted from transformed eukaryotic or prokaryotic host cells. This allows for a “molecular” binding assay that has the advantage of increased specificity, can be automated, and has high drug test throughput.

  Another technique for candidate compound screening involves an approach that provides high-throughput screening of novel compounds with appropriate binding affinity (e.g., binding affinity for GPR86 polypeptide), which was released on September 13, 1984. It is described in detail in the published International Publication No. WO 84/03564 (Commonwealth Serum Labs.). First, a number of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or other suitable surface; see Fodor et al. (1991). Next, all pins are reacted with the solubilized polypeptide and washed. The next step relates to detecting the bound polypeptide. Compounds that specifically interact with the polypeptide are thus identified.

  Ligand binding assays provide a direct method for elucidating receptor pharmacology and are applicable to high throughput formats. The purified ligand for the receptor can be radiolabeled to high specific activity (50-2000 Ci / mmol) for binding studies. It is then confirmed that the radiolabeling step does not abolish the activity of the ligand on the receptor. Buffer, ion, pH and other modulator (such as nucleotide) assay conditions are optimized to establish practical signal-to-noise ratios for both membrane and whole cell receptor sources. In these assays, specific receptor binding is defined as the total associated radioactivity minus the radioactivity measured in the presence of excess unlabeled competing ligand. Where possible, two or more competing ligands are used to reveal residual non-specific binding.

  The assay can simply test the binding of the candidate compound, wherein attachment to the receptor-bearing cell is detected by a label that binds directly or indirectly to the candidate compound, or with a label competitor. Detected in an assay for competition. Moreover, these assays can test whether a candidate compound provides a signal resulting from receptor activation, using a detection system appropriate for cells having the receptor on the surface. Activation inhibitors are generally assayed in the presence of a known agonist and the effect of activation of the agonist by the presence of the candidate compound is observed.

  Further, the assay simply comprises mixing a candidate compound with a solution containing a GPR86 polypeptide to form a mixture, measuring GPR86 activity in the mixture, and comparing the GPR86 activity of the mixture to a standard. Steps may be included.

  GPR86 cDNA, protein and antibodies to the protein can also be used to construct assays to detect the effect of added compounds on GPR86 mRNA and protein production in cells. For example, an ELISA can be constructed using monoclonal and polyclonal antibodies for measurement of GPR86 protein secretion or cell association levels (using standard methods known in the art) and using the ELISA. Thus, substances (also called antagonists or agonists, respectively) that can inhibit or promote the production of GPR86 from appropriately engineered cells or tissues can be discovered. Standard methods for performing screening assays are well understood in the art.

  Examples of potential GPR86 antagonists are antibodies or sometimes nucleotides and analogs thereof (including purines and purine analogs), oligonucleotides or proteins closely related to ligands of GPR86 (e.g., fragments of the ligands), or receptors Includes small molecules that bind to the body but do not elicit a response, thereby blocking the activity of the receptor.

  Accordingly, we provide compounds that can specifically bind to GPR86 polypeptides and / or peptides.

  The term `` compound '' refers to a chemical compound (natural or synthetic), e.g. a biological macromolecule (e.g. nucleic acid, protein, non-peptide molecule or organic molecule) or biological material (e.g. bacteria, plants, fungi, Or an extract prepared from an animal (particularly a mammal) or tissue, or an inorganic element or molecule. Preferably the compound is an antibody.

  The materials necessary to perform such screening can be packaged in a screening kit. Such screening kits are useful for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for GPR86 polypeptides, or compounds that reduce or enhance production of GPR86 polypeptides. The screening kit comprises: (a) a GPR86 polypeptide; (b) a recombinant cell that expresses the GPR86 polypeptide; (c) a cell membrane that expresses the GPR86 polypeptide; or (d) an antibody against the GPR86 polypeptide. The screening kit may optionally include instructions for use.

Transgenic animals We further express natural or recombinant GPR86, or homologs, variants or derivatives thereof at normal, increased or reduced levels compared to normal expression levels. A transgenic animal is disclosed. Preferably such transgenic animals are non-human mammals such as pigs, sheep or rodents. Most preferably the transgenic animal is a mouse or a rat.

  We disclose transgenic animals in which all or part of the native GPR86 gene has been replaced with GPR86 sequences from another organism. Preferably the organism is another species, most preferably a human. In a particularly preferred embodiment, we disclose mice in which substantially the entire GPR86 gene has been replaced with the human GPR86 gene. Such transgenic animals and animals in which GPR86 is wild type can be used for screening for agonists and / or antagonists of GPR86.

  For example, such assays expose the wild-type or transgenic animal, or a portion thereof (preferably cells, tissues or organs of the transgenic animal) to a candidate substance, and a GPR86-related phenotype (such as pain or inflammation). ). Cell-based screening using cells from the relevant animal and assaying for effects on intracellular cyclic AMP concentrations can also be performed.

  The inventors further disclose a transgenic animal comprising a functionally disrupted GPR86 gene, wherein one or more functions of GPR86 disclosed herein are partially or completely extinguished. This includes transgenic animals that do not express functional GPR86 ("GPR86 knockout") as a result of one or more loss-of-function mutations (including deletions) of the GPR86 gene.

  Transgenic animals lacking functional GPR86 (GPR86 knockout) show altered pain sensitivity when compared to wild type animals. Specifically, GPR86 knockouts are not very pain sensitive. Moreover, GPR86 knockout animals exhibit altered inflammatory pain sensitivity, particularly reduced inflammatory pain sensitivity.

  Also included are partially loss-of-function mutants, such as incomplete knockouts, which may have deletions in selected portions of the GPR86 gene, for example. Such animals can be produced by selectively substituting or deleting relevant portions of the GPR86 sequence, eg, functionally important protein domains.

  Such fully or partially lost mutants are useful as models for GPR86 related diseases, particularly pain or inflammatory diseases. Animals with partial loss of function can be exposed to candidate substances to identify substances that enhance the phenotype (in other words, increase the reduced pain sensitivity phenotype observed for GPR86) . Other parameters (eg, changes in intracellular cyclic AMP levels) can also be detected using the methods described elsewhere herein.

  Partial and complete knockouts can also be used to identify selective agonists and / or antagonists of GPR86. For example, agonists and / or antagonists can be administered to wild type animals and GPR86 deficient animals (knockouts). Although selective agonists and / or antagonists of GPR86 are observed to affect wild-type animals, they will not be observed in GPR86-deficient animals. Specifically, a specific assay is designed to evaluate a potential agent (candidate ligand or compound) to determine if the potential agent produces a physiological response in the absence of GPR86. This can be accomplished by administering the agent to a transgenic animal as described above and then assaying the animal for a particular response. Similar cell-based methods can also be performed using cells from the relevant animal and assaying for effects on intracellular cyclic AMP concentrations. Such animals can also be used to test the efficiency of agents identified by the screening described herein.

  In another embodiment, transgenic animals with a partial loss of function phenotype are utilized for screening. In such embodiments, screening may involve assaying partial or complete recovery or reversion to the wild type phenotype. Cell-based screens using cells from the animal and assaying for effects on intracellular cyclic AMP concentrations can also be performed. Candidate compounds that are found to have the capability can be considered GPR86 agonists or GPR86 analogs. Such agonists can be used, for example, to restore or increase sensitivity to a stimulus (eg, pain).

  In a preferred embodiment, transgenic GPR86 animals, particularly GPR86 knockout bodies (complete loss of function) exhibit the phenotype described in the examples, which are preferably measured by the test methods described in the examples. From this, GPR86 animals, especially GPR86 knockout bodies, preferably exhibit one or more of the following: lower pain sensitivity (dullness), lower inflammatory sensitivity.

  In a particularly preferred embodiment, the transgenic GPR86 animal, in particular the GPR86 knockout body, is at least 10%, preferably at least 20%, more preferably at least 30%, 40% of the measured parameters compared to the corresponding wild type mouse. 50%, 60%, 70%, 80%, 90%, or 100% (depending on the case) showing high or low values. Thus, for example, the GPR86 knockout body has an increased pain threshold in response to the Tail Flick test described in the Examples, which is 1 second, 2 seconds, 5 seconds when compared to wild type mice. 10 seconds, 30 seconds or more, or 5%, 10%, 20%, 50% or more.

  It will be clear that the phenotype now disclosed for GPR86-deficient transgenic animals can be used effectively in screening with wild-type animals to detect compounds that cause effects similar to loss of function of GPR86. . In other words, modulators of GPR86 function, particularly antagonists, can be identified by exposing wild-type animals to candidate compounds and observing related GPR86 phenotypes (eg, reduced pain sensitivity). Intracellular phenotypes (eg, changes in intracellular cyclic AMP levels) can also be detected using the methods specified elsewhere herein.

  Compounds identified by such screening can be used as antagonists of GPR86 (eg as analgesics), particularly for the treatment or alleviation of GPR86-related diseases.

  The screening can involve observation of any suitable parameter (such as behavioral, physiological or biochemical response). Preferred responses include one or more of the following: altered disease resistance; altered inflammatory response; altered tumor susceptibility; altered blood pressure; altered neovascularization; altered feeding behavior; altered body weight; Changes; changes in body temperature; insulin secretion; gonadotropin secretion; nasal and bronchial secretion; vasoconstriction; memory loss; mental anxiety; hyporeflexia or hyperreflexia;

  Tissue from GPR86 knockout animals can be used in receptor binding assays to determine whether potential agents (candidate ligands or compounds) bind to the GPR86 receptor. Such assays are known to obtain a first receptor preparation from a transgenic animal that has been genetically engineered to lack GPR86 receptor production and bind to any identified GPR86 ligand or compound. Can be carried out by obtaining a second receptor preparation from a known source. In general, the first and second receptor preparations are the same in all respects except the source from which they were obtained. For example, when brain tissue from a transgenic animal (such as those described above and below) is used in the assay, the corresponding brain tissue from a normal (wild type) animal is used as the source of the second receptor preparation. use. Each receptor preparation is incubated with a ligand known to bind to the GPR86 receptor alone or in the presence of a candidate ligand or candidate compound. Preferably, the candidate ligand or candidate compound is examined at a plurality of different concentrations.

  The extent to which binding by a known ligand is displaced by the test compound is determined for both the first and second receptor preparations. Tissue from transgenic animals can be used directly in the assay, or the tissue can be processed to isolate membranes or membrane proteins and used themselves in the assay. A preferred transgenic animal is a mouse. The ligand may be labeled using any means compatible with the binding assay. This includes, but is not limited to, radioactivity, enzyme, fluorescent or chemiluminescent labels (and other labeling techniques detailed hereinabove).

  In addition, GPR86 receptor antagonists can be administered to a wild-type animal that expresses functional GPR86, and any phenotypic characteristics associated with decreased or eliminated expression of GPR86 receptor function. Can be identified.

  Detailed methods for generating non-human transgenic animals are detailed below. Transgenic gene constructs can be introduced into the germ line of animals to produce transgenic animals. For example, one or more copies of the construct can be integrated into the genome of a mammalian embryo by standard transgenic techniques.

  In an exemplary embodiment, the transgenic non-human animals described herein are created by introducing a transgene into the germ line of a non-human animal. Embryonic target cells at various stages of development can be used for transgene introduction. Different techniques are used depending on the stage of embryonic target cell development. The specific line of animals used for this purpose selects for generally good health, good embryo production, good pronuclear visibility in the embryo, and good reproductive adaptability. Furthermore, the haplotype is an important factor.

  Introduction of the transgene into the embryo can be accomplished by any method known in the art, for example, microinjection, electroporation, or lipofection. For example, the GPR86 receptor transgene can be introduced into a mammal by microinjecting the construct into the pronucleus of a fertilized mammalian egg so that one or more copies of the construct are retained in a developing mammalian cell. After introducing the transgene construct into the fertilized egg, the egg can be incubated for various times, or reimplanted into a surrogate host, or both. Maturation by in vitro incubation is within the scope of this specification. One common method is to incubate the embryos in vitro for about 1-7 days, depending on the species, and then reimplant them into a surrogate host.

  Transgenic embryo progeny can be examined for the presence of the construct using Southern blot analysis of tissue pieces. If one or more copies of the exogenous cloning construct remain stably integrated into the genome of such a transgenic embryo, it is possible to establish a permanent transgenic mammalian line that retains the construct added transgeneically. is there.

  Transgeneically modified mammalian littermates can be assayed postnatally for introduction of the construct into the genome of the offspring. Preferably, this assay is performed by hybridizing a probe corresponding to a DNA sequence encoding the desired recombinant protein product or a segment thereof with chromosomal material from the offspring. Mammalian progeny found to have at least one copy of the construct in their genome are grown to maturity.

  For the purposes of the present invention, a zygote is the formation of a diploid cell that can develop into an essentially complete organism. In general, a zygote comprises an egg containing a nucleus formed naturally or artificially by the fusion of two haploid nuclei from gametes. Thus, the gamete nucleus must be a nucleus that is adaptable as it is (ie, produces a viable zygote that can differentiate into a functional organism). In general, an euploid conjugate is preferred. When aneuploid zygotes are obtained, the number of chromosomes should not change by more than 2 relative to the number of euploids of the organism from which any gamete is derived.

  In addition to similar biological considerations, physical considerations also determine the amount (eg, volume) of exogenous genetic material that can be added to the zygote nucleus or to the genetic material that forms part of the zygote nucleus. It depends on you. If genetic material is not removed, the amount of exogenous genetic material that can be added is limited by the amount absorbed without becoming physically destructive. Generally, the volume of exogenous genetic material inserted does not exceed about 10 picoliters. The physical effect of the addition should not be so great as to physically destroy the survival of the conjugate. Biological limits on the number and type of DNA sequences will vary depending on the function of the individual zygote and the exogenous genetic material, which will be readily apparent to those skilled in the art. This is because the genetic material of the zygote obtained (including exogenous genetic material) needs biological ability to initiate and maintain the differentiation and development of the zygote into a functional organism.

  The number of copies of the transgene construct added to the zygote depends on the total amount of exogenous genetic material added and is an amount that allows for genetic transformation. Theoretically only one copy is needed; however, in general, a large number of copies (eg, 1,000-20,000 copies of the transgene construct) are used to ensure that one copy is functional. Having two or more functional copies of each inserted foreign DNA sequence will often be advantageous to enhance phenotypic expression of the foreign DNA sequence.

  Any technique capable of adding exogenous genetic material into the nuclear genetic material may be utilized as long as the technique does not destroy cells, nuclear membranes, or other existing cell or genetic structures. The exogenous genetic material is preferentially inserted into the nuclear genetic material, preferably by microinjection. Microinjection of cells and cell structures is known and used in the art.

  Retransplantation is performed using standard methods. Usually, the surrogate host is anesthetized and the embryo is inserted into the fallopian tube. The number of embryos transferred into a particular host will vary depending on the species, but will usually be comparable to the number of offspring that the species naturally produces.

  Transgenic progeny of the surrogate host can be screened for the presence and / or expression of the transgene by any suitable method. Screening is often performed by Southern blot analysis or Northern blot analysis using a probe complementary to at least a portion of the transgene. Western blot analysis using antibodies against the protein encoded by the transgene may be utilized as an alternative or additional method for screening for the presence of the transgene product. Typically, DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene. Alternatively, tissues or cells that are considered to express the transgene at the highest level are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissue or cell type can be used for this analysis.

  Alternative or additional methods for assessing the presence of a transgene include, but are not limited to, suitable biochemical assays such as enzymes and / or immunological assays, histology for specific markers or enzyme activity. And dyeing, flow cytometry analysis and the like. Analysis of blood can also be useful for detecting the presence of transgene products in the blood and for assessing the effects of the transgene on the levels of various types of blood cells and other blood components.

  The progeny of the transgenic animal can be obtained by crossing the transgenic animal with an appropriate partner or by fertilizing eggs and / or sperm obtained from the transgenic animal in vitro. When mating with a partner, the partner may or may not be transgenic and / or knockout; if it is transgenic, the partner contains the same or different transgene, or both Also good. Alternatively, the partner can be a parent. When performing in vitro fertilization, it is possible to transfer the fertilized embryo to a surrogate host, incubate in vitro, or both. Whichever method is used, the progeny can be assessed for the presence of the transgene using the methods described above or other suitable methods.

  Transgenic animals produced according to the description herein contain exogenous genetic material. As noted above, exogenous genetic material is a DNA sequence that, in certain embodiments, results in production of the GPR86 receptor. Furthermore, in such embodiments, the sequence is preferably linked to a transcriptional control element (eg, a promoter) that allows expression of the transgene product in a particular type of cell.

  Retroviral infection can also be used to introduce transgenes into non-human animals. Developing non-human embryos can be cultured in vitro to the blastocyst stage. During this time, blastomeres can be targeted for retroviral infection (Jaenich, R. (1976) PNAS 73: 1260-1264). Enzymatic treatment that removes the zona pellucida results in efficient infection of the blastomeres (Manipulating the Mouse Embryo, edited by Hogan (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986), used to introduce transgenes) Viral vector systems are typically replication-deficient retroviruses that retain the transgene (Jahner et al. (1985) PNAS 82: 6927-6931; Van der Putten et al. (1985) PNAS 82: 6148-6152). Transfection is easily and efficiently performed by culturing blastomeres on a monolayer of virus producing cells (Van der Putten, supra; Stewart et al., (1987) EMBO J. 6: 383- 388) Alternatively, infection can be performed late, and virus or virus-producing cells can be injected into the blastocoele (Jahner et al. (1982) Nature 298: 623-628). A subset of cells that form animals Most founders are mosaic for the transgene since it only occurs in the genome, and the founders are located at various positions in the genome that segregate the various retroviral inserts of the transgene, generally in the offspring. Furthermore, it is also possible to introduce a transgene into the germline by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982), supra).

  A third type of target cell for transgene introduction is embryonic stem cells (ES). ES cells are obtained from preimplantation embryos cultured in vitro and fused with the embryo (Evans et al., 1981, Nature, 292, 154-156; Bradley et al. (1984), Nature, 309, 255-258; Gossler et al. (1986), PNAS, 83, 9065-9069; and Robertson et al. (1986), Nature, 322, 445-448). Transgenes can be effectively introduced into ES cells by DNA transfection or retrovirus-mediated transduction. Such transformed ES cells can then be integrated with placental vesicles derived from non-human animals. The ES cells then colonize the embryo and contribute to the resulting chimeric animal germline. For a review, see Jaenisch, R. (1988) Science 240: 1468-1474.

  We also provide non-human transgenic animals, where the transgenic animals have a modified GPR86 gene (preferably as described above) as a model for GPR86 receptor function. It is characterized by that. Modifications to the gene include the introduction of a foreign gene having nucleotides with deletions or other loss-of-function mutations, target mutations or random mutations, the introduction of foreign genes from another species, or combinations thereof. included. The transgenic animals can be homozygous or heterozygous for modification. These animals and their derived cells are useful for screening biologically active substances that can modulate GPR86 receptor function. The screening method is particularly useful for determining the specificity and action of potential therapeutics for pain and inflammatory diseases such as osteoarthritis, rheumatoid arthritis, asthma, irritable bowel syndrome and allergies. . The animals are useful as models for investigating the role of GPR86 receptors in normal brain, heart, spleen and liver functions.

  Another embodiment relates to a transgenic non-human animal that has a functionally disrupted endogenous GPR86 gene while retaining and expressing a transgene encoding a heterologous GPR86 protein (i.e., GPR86 from another species) in the genome. . Preferably, the animal is a mouse and the heterologous GPR86 is human GPR86. Such animals or cell lines derived therefrom that have been reverted by human GPR86 can be used to identify substances that inhibit human GPR86 in vivo and in vitro. For example, a stimulus that induces signal transduction through human GPR86 can be applied to the animal or cell line in the presence or absence of the substance being tested, and the response of the animal or cell line can be measured. A substance that inhibits human GPR86 in vivo or in vitro can be identified based on a decrease in the response in the presence of the substance compared to the response in the absence of the substance.

  The present inventors also provide a GPR86-deficient transgenic non-human animal (“GPR86 knockout body”). Such animals are those that exhibit reduced or absent GPR86 activity (preferably as a result of disruption or deletion of the endogenous GPR86 genomic sequence). Preferably such animals do not exhibit GPCR activity. More preferably, the animal does not exhibit GPR86 activity as set forth in SEQ ID NO: 3 or SEQ ID NO: 5. GPR86 knockout bodies can be made by various techniques known in the art, as detailed below.

  The present invention also relates to a nucleic acid construct for functionally disrupting the GPR86 gene in a host cell. The nucleic acid construct comprises (a) a non-homologous replacement portion; (b) a first homologous region located upstream of the non-homologous replacement portion and having a nucleotide sequence substantially identical to the first GPR86 gene sequence. A first homologous region; and (c) a second homologous region located downstream of the non-homologous replacement portion and having a nucleotide sequence substantially identical to the second GPR86 gene sequence, provided that the second GPR86 gene sequence is native In the endogenous GPR86 gene at a position downstream from the first GPR86 gene sequence). In addition, the first and second homologous regions are of sufficient length for homologous recombination between the nucleic acid construct and the endogenous GPR86 gene in the host cell when the nucleic acid molecule is introduced into the host cell. In a preferred embodiment, the heterologous replacement portion comprises an expression reporter, preferably comprising lacZ and a positive selection expression cassette, preferably comprising a neomycin phosphotransferase gene operably linked to regulatory elements.

  Preferably said first and second GPR86 gene sequences are derived from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or a homologue, variant or derivative thereof.

  Another aspect relates to recombinant vectors that incorporate the nucleic acid constructs described herein. In yet another embodiment, the nucleic acid construct described herein is introduced into a host cell, thereby homologous recombination between the nucleic acid construct and the endogenous GPR86 gene of the host cell, and functional disruption of the endogenous GPR86 gene. Said host cell. Host cells can be mammalian cells from liver, brain, spleen or heart that normally express GPR86, or pluripotent cells, such as mouse embryonic stem cells. When the embryonic stem cell into which the nucleic acid construct has been introduced and homologously recombined with the endogenous GPR86 gene is further developed, a transgenic non-human having a cell that is a descendant of the embryonic stem cell and thus retains the GPR86 gene disruption in its genome Animals are obtained. Animals that retain the GPR86 gene disruption in their germline can then be selected and bred to obtain animals in which all somatic and germ cells are GPR86 gene disrupted. These mice can then be bred to homozygosity for the GPR86 gene disruption.

Antibody For the purposes of the present invention, the term “antibody” unless otherwise specified includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and Fab expression libraries. Fragment to be included. These fragments include whole antibody fragments, Fv, F (ab ′), and F (ab ′) ₂ fragments that retain binding activity to the target substance, as well as single chain antibodies (scFv), fusion proteins and antibody fragments. Other synthetic proteins that contain an antigen binding site are included. The antibodies and fragments thereof may be humanized antibodies (eg as described in EP-A-239400). further,
Antibodies with fully human variable regions (or fragments thereof) (eg, those described in US Pat. Nos. 5,545,807 and 6,075,181) can also be used. Neutralizing antibodies, ie antibodies that inhibit the biological activity of the substance amino acid sequence, are particularly preferred for diagnosis and therapy.

  Antibodies can be produced by standard methods, such as by immunization or by use of phage display libraries.

  Antibodies can be developed by known techniques using GPR86 polypeptides or peptides. Such antibodies may have the ability to specifically bind to GPR86 protein or homologues, fragments and the like.

  If a polyclonal antibody is desired, a selected mammal (eg, mouse, rabbit, goat, horse, etc.) can be immunized with an immunogenic composition comprising a GPR86 polypeptide or peptide. Depending on the host species, various adjuvants can be used to increase the immune response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (eg, aluminum hydroxide), and surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin As well as dinitrophenol. BCG (Bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants, which are immunocompromised for the purpose of stimulating systemic defenses with purified substance amino acid sequences. It can be used when administered to an individual.

  Serum is collected from the immunized animal and processed by known methods. When serum containing polyclonal antibodies against an epitope that can be obtained from a GPR86 polypeptide contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order to be able to make such antibodies, we further provide GPR86 amino acid sequences for use as immunogens in animals or humans, or fragments thereof haptenized to other amino acid sequences.

  Monoclonal antibodies against epitopes that can be obtained from GPR86 polypeptides or peptides can also be readily produced by those skilled in the art. General methods for producing monoclonal antibodies by hybridomas are well known. Immortal antibody-producing cell lines can be generated by cell fusion and can also be generated by other techniques, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. it can. A panel of monoclonal antibodies raised against the orbit epitope may be screened for various properties (ie, isoforms and epitope affinity).

  Monoclonal antibodies may be prepared using any technique that results in the production of antibody molecules by continuously cultured cell lines. These techniques include, but are not limited to, hybridoma technology, trioma technology, human B cell hybridoma technology (Kosbor et al., (1983) Immunol) first described in Koehler and Milstein (1975 Nature 256: 495-497). Today 4:72; Cote et al. (1983) Proc Natl Acad Sci 80: 2026-2030), and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc. , 1985).

  Furthermore, splicing of a mouse antibody gene to a human antibody gene to obtain a molecule having an appropriate antigen specificity and biological activity, a technique developed for the production of “chimeric antibodies” can be used (Morrison (1984) Proc Natl Acad Sci 81: 6851-6855; Neuberger et al. (1984) Nature 312: 604-608; Takeda et al. (1985) Nature 314: 452-454). Alternatively, a substance-specific single chain antibody can be prepared by applying the technique described for the production of a single chain antibody (US Pat. No. 4,946,779).

  Antibodies (both monoclonal and polyclonal) against epitopes that can be obtained from GPR86 polypeptides or peptides are particularly useful for diagnosis, and neutralizing antibodies are useful in passive immunotherapy. In particular, monoclonal antibodies can be used to elicit anti-idiotype antibodies. An anti-idiotype antibody is an immunoglobulin that retains an “internal image” of a substance and / or drug (for which protection is desired). Techniques for eliciting anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may also be useful for therapy.

  Antibodies can also be generated by inducing production in lymphocyte populations in vivo, or by screening a panel of recombinant immunoglobulin libraries or highly specific binding reagents, which can be found in Orlandoi et al. (1989). Proc Natl Acad Sci 86: 3833-3837) and Winter G and Milstein C (1991, Nature 349: 293-299).

Antibody fragments that contain specific binding sites for the polypeptide or peptide can also be generated. For example, such fragments include, but are not limited to, F (ab ′) ₂ fragments that can be generated by pepsin digestion of antibody molecules, and reduction of disulfide bridges of F (ab ′) ₂ fragments. Fab fragments are included. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al. (1989) Science 256: 1275-1281).

  Single chain antibodies against GPR86 polypeptide can also be produced by applying single chain antibody production techniques (US Pat. No. 4,946,778). In addition, transgenic mice or other organisms (including other mammals) can be used to express humanized antibodies.

  The above-described antibodies can be used to isolate or identify clones that express the polypeptide, or to purify the polypeptide using affinity chromatography.

  Antibodies against GPR86 polypeptides can also be used to treat pain and inflammatory diseases such as osteoarthritis, rheumatoid arthritis, asthma, irritable bowel syndrome and allergies.

Diagnostic Assays We further describe the use of GPR86 polynucleotides and polypeptides (and their homologs, variants and derivatives) for use as diagnostic reagents in diagnosis or in genetic analysis. Nucleic acids that are complementary or capable of hybridizing to GPR86 nucleic acids (including homologs, variants and derivatives), and antibodies to GPR86 polypeptides are also useful in such assays.

  Detection of a mutant form of the GPR86 gene associated with dysfunction can be in addition to or a diagnosis of a disease resulting from or under susceptibility to GPR86 underexpression, overexpression or altered expression Provide a diagnostic tool that can define a diagnosis. Individuals carrying mutations in the GPR86 gene (including control sequences) can be detected at the DNA level by various techniques.

  For example, DNA can be isolated from a patient and the DNA polymorphism pattern of GPR86 can be determined. The identified pattern is compared to a control patient who is known to have a disease associated with GPR86 over-, under-, or aberrant expression. Patients can then be identified that show genetic polymorphism patterns associated with GPR86-related diseases. Genetic analysis of the GPR86 gene may be performed by any technique known in the art. For example, individuals may be screened by determining the DNA sequence of the GPR86 allele and performing RFLP or SNP analysis or the like. Identified that a patient has a genetic predisposition to a disease associated with GPR86 over-, under-, or aberrant expression by detecting the presence of a DNA polymorphism in the GPR86 gene sequence or any sequence that controls its expression can do.

  The patient thus identified can then be treated to prevent the onset of GPR86-related disease or more actively at an early stage of GPR86-related disease to prevent further onset or progression of the disease. . GPR86 related diseases include dopamine related diseases such as Parkinson's disease, heart diseases such as supraventricular or ventricular arrhythmias, hypotension, nausea, Tourette syndrome, stress and pain.

  We further disclose kits for identifying genetic polymorphism patterns in patients associated with GPR86-related diseases. The kit includes a DNA sample collection means and a genetic polymorphism pattern measurement means, and the measured polymorphism pattern is then compared to a control sample to determine a patient's susceptibility to a GPR86 related disease. A GPR86-related disease diagnostic kit comprising a GPR86 polypeptide and / or an antibody (or fragment thereof) against such a polypeptide is also provided.

The diagnostic nucleic acid can be obtained from a subject's cells, for example from blood, urine, saliva, tissue biopsy or autopsy material. In a preferred embodiment, DNA is obtained from blood cells obtained by collecting blood on absorbent paper by a patient's finger puncture. In a further preferred embodiment, blood is collected with an AmpliCard ^™ (University of Sheffield, Department of Medicine and Pharmacology, Royal Hallamshire Hospital, Sheffield, England S10 2JF).

  The DNA can be used directly for detection or can be amplified enzymatically using PCR or other amplification techniques prior to analysis. Oligonucleotide DNA primers that target specific polymorphic DNA regions within the gene of interest are prepared so that amplification of the target sequence is achieved in the PCR reaction. RNA or cDNA may be used as a template in a similar manner. The DNA sequence amplified from the template DNA can then be analyzed by restriction enzymes to determine the genetic polymorphism present in the amplified sequence, thereby obtaining the patient's genetic polymorphism profile. The length of the restriction fragment can be identified by gel analysis. Alternatively or in combination, techniques such as SNP (single nucleotide polymorphism) analysis may be used.

  Deletions and insertions can be detected by a change in the size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled GPR86 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or differences in melting temperatures. DNA sequence differences can also be detected by changes in electrophoretic mobility of DNA fragments in gels with or without denaturing agents, or by direct DNA sequencing. See, for example, Myers et al., Science (1985) 230: 1242. Sequence changes at specific positions can also be revealed by nuclease protection assays such as RNase and S1 protection or by chemical cleavage methods. See Cotton et al., Proc Natl Acad Sci USA (1985) 85: 4397-4401. In other embodiments, an array of oligonucleotide probes containing GPR86 nucleotide sequences or fragments thereof can be constructed to perform efficient screening, eg, for gene mutations. Array techniques are well known and have a variety of applications that can be used to address various problems in molecular genetics, including gene expression, genetic linkage, and genetic variability. (See, for example, M. Chee et al., Science, Vol 274, pp 610-613 (1996)).

  Single strand conformation polymorphism (SSCP) can be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al., (1989) Proc Natl. Acad. Sci USA: 86: 2766, Cotton (1993) Mutat Res 285: 125-144; see also Hayashi (1992) Genet Anal Tech Appl 9: 73-79). Single-stranded DNA fragments of sample and control GPR86 nucleic acids can be denatured and regenerated. The secondary structure of a single-stranded nucleic acid varies depending on the sequence, and the resulting change in electrophoretic mobility makes it possible to detect even a single base change. The DNA fragment may be labeled, or the DNA fragment may be detected using a labeled probe. The sensitivity of the assay can be enhanced by using RNA whose secondary structure is susceptible to sequence changes (rather than DNA). In a preferred embodiment, the method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules based on changes in electrophoretic mobility (Keen et al., (1991) Trends Genet 7: 5).

  Diagnostic assays provide a method of diagnosing or determining susceptibility to infection through detection of mutations in the GPR86 gene by the methods described.

  The presence of GPR86 polypeptide and nucleic acid in the sample can be detected. Accordingly, the above-mentioned infections and diseases can be diagnosed by a method comprising identifying an abnormal decrease or increase in the level of GPR86 polypeptide or GPR86 mRNA from a sample derived from a subject. The sample is a cell or tissue sample from an organism with or suspected of having a disease associated with increased, decreased, or otherwise abnormal GPR86 expression (including spatial or temporal changes in expression levels or patterns) Can be included. The expression level or pattern of GPR86 in an organism with or suspected of having such a disease can be effectively compared to the expression level or pattern in a normal organism as a diagnostic tool for the disease.

  Thus, in general, we are a method of detecting the presence of a nucleic acid containing a GPR86 nucleic acid in a sample, wherein the sample is contacted with at least one nucleic acid probe specific for the nucleic acid; And disclosing the method by monitoring the sample for the presence of the nucleic acid. For example, a nucleic acid probe can be specifically bound to a GPR86 nucleic acid or a portion thereof to detect binding between the two; the presence of the complex itself may be detected. In addition, the inventors provide a method for detecting the presence of a GPR86 polypeptide, contacting a cell sample with an antibody capable of binding to the polypeptide, and monitoring the sample for the presence of the polypeptide. The method is disclosed. This can be conveniently done by monitoring the presence of the complex formed between the antibody and the polypeptide or by monitoring the binding between the polypeptide and the antibody. Methods for detecting binding between the two are known in the art and include FRET (fluorescence resonance energy transfer), surface plasmon resonance, and the like.

  The decrease or increase in expression is measured at the RNA level using any polynucleotide quantification method known in the art (eg, PCR, RT-PCR, RNase protection, Northern blot, and other hybridization methods). Can be measured. Assay techniques that can be used to identify levels of a protein (eg, GPR86) in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.

  The present specification also relates to a diagnostic kit for a disease or susceptibility to a disease (including infection), such as a dopamine related disease such as Parkinson's disease, a heart disease such as supraventricular arrhythmia or ventricular arrhythmia, These include hypotension, nausea, Tourette syndrome, stress and pain. The diagnostic kit comprises a GPR86 polynucleotide or fragment thereof; a complementary nucleotide sequence; a GPR86 polypeptide or fragment thereof, or an antibody to a GPR86 polypeptide.

Chromosome assay
The GPR86 nucleotide sequence is also useful for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on a particular human chromosome. As mentioned above, human GPR86 has been found to map to homo sapiens chromosome 3q24.

  Mapping related sequences to chromosomes is an important first step in correlating those sequences with gene-related diseases. Once a sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic mapping data. Such data is found, for example, in V. McKusick, Mendelian heritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between the genes mapped to the same chromosomal region and the disease is then identified by linkage analysis (co-inheritance of physically adjacent genes).

  Differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals, but not in normal individuals, the mutation is likely a causative factor of the disease.

Prophylaxis and Treatment We provide a method for treating abnormal conditions associated with both excess and deficiency of GPR86 activity.

  If GPR86 activity is excessive, several approaches are available. One method involves activating a subject with an inhibitor compound (antagonist) as described herein above, together with a pharmaceutically acceptable carrier, by blocking the binding of the ligand to GPR86 or suppressing a second signal. Administration in an amount effective to prevent the disease, thereby alleviating said abnormal condition.

  Alternatively, a soluble form of a GPR86 polypeptide that can still bind to a ligand in competition with endogenous GPR86 may be administered. An exemplary embodiment of such a competitor comprises a fragment of GPR86 polypeptide.

  In yet another method, expression of the gene encoding endogenous GPR86 may be suppressed using expression blocking techniques. Such known techniques involve the use of antisense sequences, which are generated internally or administered separately. See, for example, O'Connor, J Neurochem (1991) 56: 560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla, 1988. Alternatively, an oligonucleotide that forms a triple helix with the gene may be supplied. See, for example, Lee et al., Nucleic Acids Res (1979) 6: 3073; Cooney et al., Science (1988) 241: 456; Dervan et al., Science (1991) 251: 1360. These oligomers may be administered per se or the relevant oligomers may be expressed in vivo.

  Several methods are available to treat abnormal conditions associated with underexpression of GPR86 and its activity. One method involves administering to a subject a therapeutically effective amount of a compound that activates GPR86 (ie, the agonist) in combination with a pharmaceutically acceptable carrier, thereby alleviating the abnormal condition. . Alternatively, gene therapy may be used to promote endogenous production of GPR86 by the relevant cells of the subject. For example, a GPR86 polynucleotide can be engineered to be expressed in a replication defective retroviral vector, as described above. The retroviral expression construct is then isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding the GPR86 polypeptide, which packaging cell subsequently contains the gene of interest. Produce infectious virus particles. This producer (virus producing) cell can be administered to a subject to manipulate the cell in vivo and to express the polypeptide in vivo. For an overview of gene therapy, see Human Molecular Genetics, T Strachan and AP Read, BIOS Scientific Publishers Ltd (1996), Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches (and references cited therein).

Formulation and administration peptides (eg, soluble forms of GPR86 polypeptides, agonists and antagonist peptides) or small molecules can be formulated in combination with a suitable pharmaceutical carrier. Such formulations comprise a therapeutically effective amount of the polypeptide or compound and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration, which is well known to those skilled in the art. The present description also relates to pharmaceutical packs and kits comprising one or more containers filled with one or more components of the above composition.

  GPR86 polypeptides and other compounds can be used alone or in combination with other compounds, such as therapeutic compounds.

  A preferred mode of systemic administration of the pharmaceutical composition includes injection, typically intravenous injection. Other injection routes such as subcutaneous, intramuscular, or intraperitoneal may be utilized. Alternatives to systemic administration include transmucosal and transdermal administration, which use penetrants such as bile salts or fusidic acids or other surfactants. Furthermore, oral administration is also possible if it is appropriately formulated as an enteric preparation or an encapsulated preparation. These compounds may be administered externally and / or topically in the form of ointments, pastes, gels and the like.

  The required dosage range depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the patient's symptoms and the judgment of the attending physician. However, suitable dosages range from 0.1 to 100 μg / kg (subject's body weight). However, given the variety of available compounds and the differences in efficiency of various administration routes, the required dosage is expected to vary greatly. For example, oral administration is expected to require higher dosages than administration by intravenous injection. These dosage level variations can be adjusted using standard rules of thumb for optimization, as is well understood in the art.

  Polypeptides used for therapy can also be produced endogenously in a subject, as described above, in a treatment modality often referred to as “gene therapy”. Thus, for example, cells from a subject can be engineered to encode a polypeptide ex vivo using a polynucleotide such as DNA or RNA and using, for example, a retroviral plasmid vector. The cells are then introduced into the subject.

Pharmaceutical Compositions We also provide a therapeutically effective amount of a GPR86 polypeptide, polynucleotide, peptide vector or antibody (described herein) for administration, and optionally a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a diluent or excipient (including combinations thereof).

  The pharmaceutical composition can be used in humans or animals as a pharmaceutical for humans and animals, and typically includes one or more pharmaceutically acceptable diluents, carriers, or excipients. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical industry and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro ed., 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition can contain any suitable binder, lubricant, suspending agent, coating agent, solubilizer, as or in addition to a carrier, excipient or diluent.

  Preservatives, stabilizers, dyes and even flavors may be added to the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents can be also used.

  There may be different composition / formulation requirements for different delivery systems. By way of example, the pharmaceutical compositions described herein can be obtained using a minipump or by mucosal route, for example as an aerosol or nasal spray for inhalation, or as a solution for oral ingestion, or parenterally (in this case the composition The product can be formulated in an injectable form), eg, delivered by intravenous, intramuscular, or subcutaneous routes. Alternatively, the formulation can be designed to be delivered from both routes.

  When delivering a drug by the mucosal route through the gastrointestinal mucosa, it must remain stable while it passes through the gastrointestinal tract; for example, the drug resists proteolysis and is stable at acidic pH And should be resistant to bile surfactant effects.

  Where appropriate, the pharmaceutical composition may be by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment, or dusting powder, by the use of a skin patch, such as starch or lactose. An elixir, solution or suspension containing orally in the form of a tablet containing an excipient, or alone or as a mixture with an excipient, in a capsule or ovule, or containing a flavor or colorant Or can be injected parenterally (eg, intravenously, intramuscularly, or subcutaneously). For parenteral administration, it is best to use the composition in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or monosaccharides to make the solution isotonic with blood. I will. For buccal or sublingual administration, the composition may be administered in the form of a tablet or lozenge that can be formulated in conventional manner.

Another embodiment of a vaccine is a method of inducing an immune response in a mammal, wherein the mammal produces antibodies and / or generates a T cell immune response to protect the animal from GPR86 related diseases. It relates to such a method comprising inoculating sufficient GPR86 polypeptide or fragment thereof.

  Yet another embodiment is a method for inducing an immune response in a mammal, wherein the GPR86 polynucleotide is generated in vivo for the purpose of inducing an immune response and producing antibodies to protect the animal from disease. Delivering the GPR86 polypeptide via a vector that expresses.

  A further embodiment is an immunological / vaccine formulation (composition) wherein the formulation elicits an immune response against a GPR86 polypeptide in the mammal when introduced into the mammalian host (the composition is a GPR86 Containing peptide or GPR86 gene). The vaccine formulation may further comprise a suitable carrier.

  Since GPR86 polypeptide can be broken down in the stomach, it is preferably administered parenterally (injection subcutaneously, intramuscularly, intravenously, intradermally, etc.). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile infusion solutions (which may include antioxidants, buffers, bacteriostats and solutes that make the formulation isotonic with the recipient's blood); Aqueous sterile suspensions (which may include suspending or thickening agents) are included. Formulations may be provided as unit dose or multi-dose containers (eg, sealed ampoules and sealed vials) and stored in a freeze-dried state that requires only the addition of a sterile liquid carrier just prior to use. Can do. Vaccine formulations may also include adjuvant systems that enhance the immunogenicity of the formulation, such as oil-in-water systems and other systems known in the art. The dose depends on the specific activity of the vaccine and can be readily determined by routine experimentation.

  A vaccine can be prepared from one or more GPR86 polypeptides or peptides.

  The preparation of vaccines containing immunogenic polypeptides or peptides as active ingredients is known to those skilled in the art. Typically, such vaccines are prepared as injectables, as liquid solutions or suspensions; they may be prepared in solid form suitable for making solutions or suspensions prior to injection. The preparation may also be emulsified or the protein may be encapsulated in liposomes. The active immunogenic ingredients are often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like and combinations thereof.

  In addition, if desired, the vaccine may contain minor amounts of adjuvants such as wetting or emulsifying agents, pH buffering agents, and / or adjuvants that enhance the effectiveness of the vaccine. Potentially effective adjuvants include, but are not limited to, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N- Acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, called nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2 '-Dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP 19835A, called MTP-PE), and RIBI (three components extracted from bacteria, monophosphoryl lipid A, trehalose dimyco Rate (trehalose dimycolate) and cell wall skeleton (MPL + TDM + CWS) in 2% squalene / Tween 80 emulsion).

  Further examples of adjuvants and other substances include aluminum hydroxide, aluminum phosphate, aluminum phosphate, potassium aluminum sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsion, oil-in-water emulsion, muramyl Dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotide, sodium alginate, lanolin, lysolecithin, vitamin A, saponins, liposomes, levamisole, DEAE-dextran, block copolymers or other synthetic adjuvants, which are commercially available from various sources, such as Merck Adjuvant 65 (Merck and C ompany, Inc., Rahway, N.J.) or Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, Michigan).

  Typically, adjuvants such as Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel are used. Only aluminum hydroxide is approved for human use.

The ratio of immunogen to adjuvant can vary widely as long as both are present in effective amounts. For example, aluminum hydroxide may be present in an amount of about 0.5% (based on Al ₂ O ₃ ) of the vaccine mixture. Conveniently, the vaccine is formulated to contain a final concentration of immunogen in the range of 0.2-200 μg / ml, preferably 5-50 μg / ml, most preferably 15 μg / ml.

  After formulation, the vaccine can be incorporated into a sterile container, which can then be sealed and stored at low temperature (eg, 4 ° C.) or lyophilized. Freeze drying allows long-term storage in a stable state.

  The vaccine can be administered in a conventional manner, parenterally, by injection (eg, subcutaneously or intramuscularly). Additional formulations suitable for other modes of administration include suppositories and, in some cases, oral formulations. Conventional binders and carriers for suppositories may include, for example, polyalkylene glycols or triglycerides; such suppositories may comprise from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%. Can be formed. Oral formulations include such commonly used excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. These compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% active ingredient, preferably 25% to 70%. When the vaccine composition is freeze-dried, the freeze-dried product can be prepared before use, for example, as a suspension before administration. Preparation at the time of use is preferably performed in a buffer.

  Capsules, tablets and pills for oral administration to patients can also be prepared with enteric coatings, such as Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate or hydroxy Contains propylmethylcellulose.

  GPR86 polypeptides can be formulated into vaccines in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (salt formation with free amino groups of the peptide) and inorganic acids (e.g. hydrochloric acid or phosphoric acid) or organic acids (e.g. acetic acid, oxalic acid, tartaric acid and maleic acid). Salt. Salts formed with free carboxyl groups also include inorganic bases (e.g. sodium, potassium, ammonium, calcium or ferric hydroxide) and organic bases (e.g. isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine and Procaine).

Administration Typically, the physician will determine the actual dosage that is most appropriate for an individual subject, but this may vary with the age, weight and response of the particular patient. The following dosage is an illustration of an average case. Of course, higher or lower dosage ranges may be advantageous.

  The pharmaceutical and vaccine compositions described herein can be administered by direct injection. The composition may be formulated for parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular, or transdermal administration. Typically, each protein can be administered at a dose of 0.01-30 mg / kg body weight, preferably 0.1-10 mg / kg body weight, more preferably 0.1-1 mg / kg body weight.

  The term “administered” also includes delivery by viral or non-viral techniques. Viral delivery mechanisms include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retrovirus vectors, lentivirus vectors, and baculovirus vectors. Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic surface amphiphiles (CFA), and combinations thereof. Such delivery mechanism routes include, but are not limited to, mucosal, intranasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.

  The term “administered” includes, but is not limited to, delivery by a mucosal route (eg, as an aerosol or nasal spray for inhalation or as a solution for oral ingestion); Delivery by an internal, intramuscular, or subcutaneous route).

  The term “co-administered” means that the administration site and duration of each additional substance, such as, for example, GPR86 polypeptide and adjuvant, is such that it achieves the required modulation of the immune system. means. Thus, while the polypeptide and adjuvant can be administered simultaneously and at the same site, it may be advantageous to administer the polypeptide at a different time and at a different site than the adjuvant. It is even possible to deliver the polypeptide and the adjuvant in the same delivery vehicle, and the polypeptide and antigen may or may not be bound and / or genetically bound. Good.

  The GPR86 polypeptide, polynucleotide, peptide, nucleotide, or antibody thereof, and optionally an adjuvant, may be administered separately or co-administered to the host subject in a single dose or multiple doses.

  The vaccine and pharmaceutical compositions described herein are available in a number of ways, including injection (including parenteral, subcutaneous, and intramuscular injection), intranasal, mucosal, oral, intravaginal, urethral, or intraocular administration. It can be administered by different routes.

  The vaccines and pharmaceutical compositions described herein can be administered by conventional methods, parenterally, by injection (eg, subcutaneously or intramuscularly). Additional formulations suitable for other dosage forms include suppositories, and in some cases oral formulations. Conventional binders and carriers for suppositories may include, for example, polyalkylene glycols or triglycerides; such suppositories are formed from mixtures containing the active ingredient in the range of 0.5% to 10%, or 1% to 2%. can do. Oral formulations include such commonly used excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. These compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% active ingredient, preferably 25% to 70%. When the vaccine composition is freeze-dried, the freeze-dried product can be prepared before use, for example, as a suspension before administration. Preparation at the time of use is preferably performed in a buffer.

Further Aspects Further aspects and embodiments of the invention are described in the numbered paragraphs below; it is to be understood that the invention encompasses these aspects.

  Paragraph 1 GPR86 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, or a homologue, variant or derivative thereof.

  Paragraph 2. A nucleic acid encoding the polypeptide of Paragraph 1.

  Paragraph 3. The nucleic acid of Paragraph 2, comprising the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or a homologue, variant or derivative thereof.

  Paragraph 4. A polypeptide comprising a fragment of the polypeptide of Paragraph 1.

  Paragraph 5. The polypeptide of Paragraph 3, comprising one or more regions homologous to SEQ ID NO: 3 and SEQ ID NO: 5, or comprising one or more regions non-homologous to SEQ ID NO: 3 and SEQ ID NO: 5.

  Paragraph 6. A nucleic acid encoding the polypeptide of Paragraph 4 or 5.

  Paragraph 7. A vector comprising the nucleic acid according to any one of paragraphs 2, 3, or 6.

  Paragraph 8. A host cell containing the nucleic acid according to any one of Paragraphs 2, 3, or 6, or the vector according to Paragraph 7.

  Paragraph 9. A transgenic non-human animal containing the nucleic acid according to any one of Paragraphs 2, 3, or 6, or the vector according to Paragraph 7.

  Paragraph 10. The transgenic non-human animal of Paragraph 9, which is a mouse.

  Paragraph 11. Use of a polypeptide according to any one of Paragraphs 1, 4 or 5 in a method for identifying a compound capable of specifically interacting with a G protein coupled receptor.

  Paragraph 12. Use of a transgenic non-human animal according to Paragraph 9 or 10 in a method for identifying a compound capable of specifically interacting with a G protein-coupled receptor.

  Paragraphs for identifying a compound capable of increasing the endogenous level of cyclic AMP in a cell, comprising contacting a GPR86-expressing cell with a candidate compound, and as a result of said contacting, cyclic AMP (cAMP in the cell) Determining) whether the level of) increases.

  Paragraph 14 A method for identifying a compound capable of reducing an endogenous level of cyclic AMP in a cell, comprising contacting a GPR86-expressing cell with a candidate compound, and, as a result of the contact, cyclic AMP ( determining whether the level of cAMP) decreases.

  Paragraph 15 A method of identifying a compound capable of binding to a GPR86 polypeptide comprising contacting a GPR86 polypeptide with a candidate compound and determining whether the candidate compound binds to a GPR86 polypeptide Said method.

  Paragraph 16. A compound identified by the method according to any one of Paragraphs 11 to 15.

  Paragraph 17. A compound capable of specifically binding to the polypeptide of Paragraph 1, 4 or 5.

  Paragraph 18. Use of the polypeptide according to Paragraph 1, 4 or 5 or a part thereof, or the nucleic acid according to Paragraph 2, 3 or 6 in a method for producing an antibody.

  Paragraph 19. An antibody capable of specifically binding to the polypeptide according to Paragraph 1, 4 or 5, or a part thereof, or the polypeptide encoded by the nucleotide according to Paragraph 2, 3 or 6, or a part thereof.

  Paragraph 20. A pharmaceutical composition comprising one or more of the following together with a pharmaceutically acceptable carrier or diluent: a polypeptide according to Paragraph 1, 4 or 5, or a part thereof; a nucleic acid according to Paragraph 2, 3 or 6, or a nucleic acid thereof The vector according to paragraph 7, the cell according to paragraph 8, the compound according to paragraph 16 or 17, and the antibody according to paragraph 19.

  Paragraph 21: A vaccine composition comprising one or more of the following: the polypeptide of Paragraph 1, 4 or 5 or a part thereof; the nucleic acid of Paragraph 2, 3 or 6 or a part thereof; the vector of Paragraph 7; The cell according to paragraph 8, the compound according to paragraph 16 or 17, and the antibody according to paragraph 19.

  Paragraph 22. A kit for diagnosing a disease or susceptibility to a disease comprising one or more of the following: the polypeptide of Paragraph 1, 4 or 5 or a part thereof; the nucleic acid of Paragraph 2, 3 or 6 or a part thereof The vector according to paragraph 7, the cell according to paragraph 8, the compound according to paragraph 16 or 17, and the antibody according to paragraph 19.

  Paragraph 23. A method for treating a patient suffering from a disease associated with enhanced GPR86 activity comprising administering to the patient an antagonist of GPR86.

  Paragraph 24. A method for treating a patient suffering from a disease associated with reduced GPR86 activity comprising administering to the patient an agonist of GPR86.

  Paragraph 25. The method of Paragraph 23 or 24, wherein GPR86 comprises a polypeptide having the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5.

  Paragraph 26. A method of treating and / or preventing a disease in a patient comprising administering to the patient one or more of the following: the polypeptide of Paragraph 1, 4 or 5 or a portion thereof; Paragraph The nucleic acid according to 2, 3 or 6 or a part thereof; the vector according to paragraph 7; the cell according to paragraph 8; the compound according to paragraph 16 or 17; the antibody according to paragraph 19; The composition; and the vaccine of paragraph 20.

  Paragraph 27 A drug for use in the treatment or prevention of a disease, the polypeptide according to Paragraph 1, 4 or 5, or a part thereof; the nucleic acid according to Paragraph 2, 3 or 6 or a part thereof; The agent of claim 7, comprising the vector of paragraph 7, the cell of paragraph 8, the compound of paragraph 16 or 17, and / or the antibody of paragraph 19.

  Paragraph 28. The polypeptide of Paragraph 1, 4 or 5 or a part thereof in the manufacture of a pharmaceutical composition for the treatment or prevention of disease; the nucleic acid or Paragraph thereof according to Paragraph 2, 3 or 6; Paragraph 7 Use of the vector according to paragraph; the cell according to paragraph 8; the compound according to paragraph 16 or 17; and the antibody according to paragraph 19.

  Paragraph 29. A non-human transgenic animal comprising a modified GPR86 gene.

  Paragraph 30 Said modification is: Deletion of GPR86, GPR86 mutation resulting in loss of function, introduction of foreign gene having nucleotide sequence with target mutation or random mutation into GPR86, derived from another species 30. The non-human transgenic animal according to paragraph 29, which is selected from the group consisting of introduction of a foreign gene into GPR86, and any combination thereof.

  Paragraph 31. A non-human transgenic animal having a functionally disrupted endogenous GPR86 gene, wherein said transgenic animal has and expresses in its genome a transgene encoding a heterologous GPR86 protein.

  Paragraph 32. A nucleic acid construct for functionally disrupting the GPR86 gene in a host cell, comprising: (a) a non-homologous replacement portion; (b) a first homologous region located upstream of the non-homologous replacement portion, comprising: Said first homologous region having a nucleotide sequence substantially identical to the GPR86 gene sequence of; and (c) having a nucleotide sequence substantially identical to the second GPR86 gene sequence located downstream of the non-homologous replacement portion The nucleic acid construct comprising a second homologous region (wherein the second GPR86 gene sequence is located downstream from the first GPR86 gene sequence in the native endogenous GPR86 gene).

  Paragraph 33 A method for producing a GPR86 polypeptide, comprising culturing the host cell according to Paragraph 8 under conditions where a nucleic acid encoding the GPR86 polypeptide is expressed.

  Paragraph 34. A method for detecting the presence of a nucleic acid according to Paragraph 2, 3 or 6 in a sample, wherein the sample is contacted with at least one nucleic acid probe specific for the nucleic acid, and the presence of the nucleic acid. Monitoring the sample for.

  Paragraph 35. A method for detecting the presence of the polypeptide of Paragraph 1, 4 or 5 in a sample, contacting the sample with the antibody of Paragraph 19, and monitoring the sample for the presence of the polypeptide. Said method comprising:

  Paragraph 36 Methods for diagnosing a disease or syndrome caused by, or associated with, increased, decreased or otherwise aberrant expression of GPR86: (a) an animal suffering from or suspected of suffering from such disease Detecting the expression level or expression pattern of GPR86 therein; and (b) comparing the expression level or expression pattern to that of a normal animal.

  Paragraph 37 The kit according to Paragraph 22, the method according to any one of Paragraphs 23 to 26, the drug according to Paragraph 27, the use according to Paragraph 28, or the method according to Paragraph 36, wherein the disease Said kit, method, medicament, or use associated with an inflammatory disease, preferably selected from the following group: inflammatory diseases (eg rheumatoid arthritis, multiple sclerosis, Guillain-Barre syndrome, Crohn's disease) Ulcerative colitis, psoriasis, graft-versus-host rejection, systemic lupus erythematosus or insulin-dependent diabetes), autoimmune diseases (e.g. toxic shock syndrome, osteoarthritis, diabetes or inflammatory bowel disease), acute pain Chronic pain, neuropathic pain, contact dermatitis, atherosclerosis, glomerulonephritis, reperfusion injury, bone resorption disorder, asthma, stroke, myocardial infarction, burn, adult respiratory distress syndrome (ARDS), trauma Multiple organ damage, skin disease with acute inflammatory components, acute purulent meningitis, necrotizing enterocolitis, syndrome associated with hemodialysis, septic shock, leukopheresis, granulocyte transfusion, smoke inhalation caused by smoke inhalation Acute or chronic inflammation, endometriosis, Behcet's disease, uveitis, ankylosing spondylitis, pancreatitis, cancer, Lyme disease, restenosis following percutaneous transluminal coronary angioplasty, Alzheimer's disease, traumatic arthritis, sepsis, Chronic obstructive pulmonary disease, congestive heart failure, osteoporosis, cachexia, Parkinson's disease, periodontal disease, gout, allergic disease, age-related macular degeneration, infection and pustular fibrosis.

Example 1 Transgenic GPR86 Knockout Mouse: Construction of GPR86 Gene Targeting Vector
A PAC containing the GPR86 gene was identified from the PAC library using a radiolabeled probe derived from a portion of its coding sequence. The 8.0 kb genomic contig was assembled using an in-house restriction site anchor PCR method similar to GeneWalker (Clontech). From further bio-informatic studies, the contig size was increased to 300 kb. This contig provided sufficient flanking sequence information to allow the design of homology arms to be cloned into the targeting vector.

  The mouse GPR86 gene has one coding exon. The targeting strategy was designed to delete most of the coding sequence (including the entire transmembrane domain). The 3.9 kb 5 'and 1.2 kb 3' homology arms on either side of the region to be deleted are amplified by PCR and the fragment is cloned into a targeting vector. The 5 'end of each oligonucleotide primer used to amplify the arm contains another restriction site for the rare cutting restriction enzyme (a site compatible with the cloning site of the vector polylinker and not present in the arm itself). To synthesize. For GPR86, the primers are designed as shown in the primer table below, where the 5 'arm cloning site is Sal / Not and the 3' arm cloning site is Asc / Mfe (the structure of the targeting vector used). Is shown in FIG. 2 including the relevant restriction sites).

In addition to the arm primer pair (5'armF / 5'armR) and (3'armF / 3'armR), additional primers specific for the GPR86 locus were designed for the following purposes: isolated Two short 150-300 bp fragments of non-repetitive genomic DNA outside each arm and extending beyond each arm to allow Southern analysis of target loci in a putative target clone When used for multiplex PCR with 5 'and 3' probe primer pairs (5'prF / 5'prR and 3'prF / 3'prR) for amplification; vector specific primers (in this case Asc350) A mouse genotyping primer pair (hetF and hetR) that allows discrimination between wild type, heterozygous and homozygous mice; and finally, annealing downstream of the end of the 3 ′ arm region and vector (TK5IBLMNL ) 3 'end specific primer (in this case Asc236) Events specific 1.25kb target screening primers which give rise to amplification products (amplimer) (3'scr). This amplified product can only be derived from template DNA from cells that have undergone the desired genomic modification, and can also identify correctly targeted cells from the background of a clone containing a randomly integrated copy of the vector To. The positions of these primers and the genomic structure of the region of the GPR86 locus used for the targeting strategy are shown in SEQ ID NO: 21.

  The position of the homology arm is chosen so as to disrupt the GPR86 gene. A selection cassette composed of a neomycin phosphotransferase (neo) gene operably linked to a promoter arranged in the same direction as the GPR86 gene, and an endogenous gene expression reporter upstream thereof. A targeting vector substituted with a heterologous sequence composed of (frame independent lacZ gene) is prepared.

  Once the 5 'and 3' homology arms have been cloned into the targeting vector TK5IBLMNL (see Figure 2), large quantities of high purity DNA preparations are prepared using standard molecular biology techniques. Although 20 μg of freshly prepared endotoxin-free DNA is restricted with another rare cutting restriction enzyme SwaI, cleavage occurs at a unique site between the ampicillin resistance gene of the vector backbone and the bacterial origin of replication. The linearized DNA is then sedimented, resuspended in 100 μl phosphate buffered saline and ready for electroporation.

  After 24 hours of electroporation, the transfected cells are cultured for 9 days in a medium containing 200 μg / m neomycin. Clones are selected and placed in a 96-well plate, replicated and expanded, then screened by PCR (using primers 3'scr and Asc236 as described above) to make homology between the endogenous GPR86 gene and the targeting construct A clone in which recombination has occurred is identified. Positive clones can be identified at a rate of 1-5%. Increasing these clones to freeze replicas, and to prepare sufficiently high quality DNA for Southern blot confirmation of targeting events using external 5 'and 3' probes, All using standard methods (Russ et al., Nature 2000 Mar 2; 404 (6773): 95-99). When a Southern blot of DNA digested with diagnostic restriction enzymes is hybridized with an external probe, homologously targeted ES cell clones are confirmed by the presence of mutant and unmodified wild-type bands. For example, with a 5 ′ probe, HindIII digested genomic DNA yields a 14.9 kb wild type band and a 12.0 kb targeting band; for a 3 ′ probe, HinDIII digested DNA yields a 14.9 kb wild type band and a 7.0 kb targeting band.

Example 2 Transgenic GPR86 knockout mice: generation of GPR86-deficient mice
C57BL / 6 female and male mice were mated and blastocysts were isolated on day 3.5 of pregnancy. 10-12 cells from selected clones were injected per blastocyst and 7-8 blastocysts were transplanted into pseudopregnant F1 female uterus. Chimerized offspring were born, including multiple males with high levels (up to 100%) of agouti (the color of the striped hair indicates the contribution of cells that are offspring of the target clone). This male chimera was mated with female MF1 and 129 mice and germline transmission was confirmed by striped hair color and PCR genotyping, respectively.

  PCR genotyping was performed on hair lysates cut from the tail using primers hetF and hetR and a third vector specific primer (Asc350). This multiplex PCR allowed amplification from the wild type locus (if present) with primers hetF and hetR, resulting in a 207 bp band. This amplification does not occur from the target allele because the hetF site is deleted in the knockout mouse. In contrast, the Asc350 primer amplifies a 380 bp band from the target locus in combination with a hetR primer that anneals to the region just inside the 3 ′ arm. Thus, this multiplex PCR reveals litter genotypes as follows: wild-type samples show a single 207 bp band; heterozygous DNA samples generate two bands at 207 bp and 380 bp And the homozygous sample shows only the target specific 380 bp band.

Example 3 FIG. Biological data: Gene expression pattern RT-PCR
RT-PCR was used to show expression of the gene in liver and leukocytes (FIG. 3).

Example 4 Biological data: GPR86 expression by RT-PCR RT-PCR using cDNA libraries from both humans and mice was used to examine GPR86 mRNA expression. For human sequences, primers:
Forward 5'-GGTGTTTGTTCACATCCCCAGC-3 '
Reverse 5'-TGGTGTTGCTTCCTTGTTGCTC-3 '
Can be used to obtain a 364 bp product.

The reaction conditions are as follows:
85 ℃ hot start
94 ℃ 15 seconds
60 cycles at 60 ° C for 30 seconds
72 ° C 60 seconds
4 ℃ hold

  Products were separated on a Tris acetate EDTA agarose gel containing ethidium bromide and viewed using a UV light source.

  Expression in human tissue and cells was found in bone marrow, thymus, lymph nodes, osteoblasts and chondrocytes (FIG. 7). Expression was also seen in the human cell lines Jurkat CD4 + and Myla CD8 + (both derived from T cells). Low levels of expression were seen in lymphocyte-derived Colo720 and monocyte-derived THP1 cells.

  In mouse tissue, expression is found in spleen, salivary gland, spinal cord, tongue, adipose, testis, heart, eye, embryo, kidney, thymus, stomach and small intestine, brain, liver and gallbladder, blood, bladder and adrenal gland. (FIG. 8).

  The expression of GPR86 was also found in cells derived from immune responsive cells, suggesting that GPR86 is involved in immune cell function (including those related to the inflammatory and neurogenic aspects of pain).

Example 5 FIG. GPR86 knockout mice with external stimulus and pain sensitivity test (analgesic test): Tail Flick Test
A tail flick analgesia test was performed using a tail flick analgesia measuring machine. This device provides a simple method for accurately and reproducibly determining rodent pain sensitivity (D'Amour, FE and DL Smith, 1941, Expt. Clin. Pharmacol., 16: 179-184). This appliance has a shutter control lamp as a heat source. The lamp is placed under the animal to provide a less confined environment. Tail-climbing behavior (tail flick) is detected by an automatic sensing circuit, which allows the user to handle the animal freely by hand. The animal is restrained in a ventilated tube and its tail is placed over the sensing groove on the device.

  Activating a powerful light beam that hits the tail through the opening of the shutter causes discomfort at some point, when the animal hits its tail out of the light beam. In automatic mode, the light sensor senses the movement of the tail and stops the clock and closes the shutter. Record the total elapsed time from when the shutter opens until the animal reacts.

  The response of mutant transgenic mice is compared to wild type mice of matching age and sex. Single animals can be subjected to different heat settings to increase tail temperature without exceeding 55 ° C.

  Knockout and wild type control animals were tested on a tail flick device. There was a significant difference in the time to tail until the knockout animals (6.88 ± 0.26 sec) compared with the wild type control (5.95 ± 0.28). Therefore, knockout animals are not as sensitive as wild type animals (Figure 4).

Example 6 GPR86 knockout mice with external stimulus and pain sensitivity test (analgesic test): formalin test The formalin test measures the response to toxic substances injected into the hind paw. A 20 μl volume of 5% formalin solution is injected subcutaneously into the back of one hind paw using a thin standard needle. The behavior of licking, shaking and biting the hind legs is quantified as the cumulative number of seconds spent on that behavior. Use the following rating scale: 1 = lightly lay the formalin-injected foot on the floor and not overweight; 2 = lift the injected foot; 3 = lick, bite or shake the injected foot.

  The formalin test has two response phases. Phase 1 begins immediately after injection and lasts about 10 minutes and represents an acute onset of pain fiber-derived activity. Phase 2 begins about 20 minutes after injection and lasts about 1 hour. This phase appears to represent a response to tissue damage, including inflammatory hyperalgesia.

  The GPR86 variant was tested in the formalin test and was found to be less sensitive as measured by the number of times the injected foot was licked, bitten or shaken. Paw inflammatory edema in response to injected formalin was also 12% less in KO mice compared to wild type edema (FIG. 5).

Example 7 External stimulation and pain sensitivity test in GPR86 knockout mice (analgesic test): Von Frey Hair test The haptic test used to measure pain threshold uses von Frey hair. This hair is a set of very fine gauged wires. The withdrawal threshold for mechanical stimulation is measured. The animal is placed on a raised platform whose surface is a wide wire mesh. Insert Von Frey hair from below through the holes in the mesh and poke the lower surface of the hind legs. At the threshold, the mouse responds to move the paw away from the hair, generally raising the paw next, licking the paw and / or speaking. The mechanical withdrawal threshold is defined as the minimum gauge wire stimulus that causes two withdrawal responses during three consecutive runs.

  Knockout and wild type control animals were tested with Electronic Vonfrey (5 tests for each hind paw). The latency to paw withdrawal was significantly different between knockout animals (5.9 ± 0.2 s) and wild type controls (5.2 ± 0.2, p = 0.03, t test). Therefore, knockout animals are not as sensitive as wild type animals (FIG. 6).

Example 8 FIG. External stimulation and pain sensitivity test in GPR86 knockout mice (analgesic test): Neuropathic pain Neuropathic pain can be induced by tightly ligating the L5 spinal nerve in anesthetized mice (Kim and Chung 1992 ). After recovery, neuropathic pain development and maintenance can be measured for allodynia (pain perception of non-noxious stimuli) and hyperalgesia (increased response to noxious stimuli).

  Allodynia is measured over 4 weeks using von Frey filament (as described in Example 7). Each hind paw is tested and the ipsilateral (injured) and contralateral (naïve) paw responses are compared in knockout and wild type mice. Hyperalgesia can be tested with harmful heat, harmful cold, and harmful mechanical stimuli. In both these tests, knockout mice were found to be less sensitive than wild type controls.

  The patent applications and patents mentioned herein, as well as the respective documents cited or referenced in each of the above patent applications and patents (including those cited or referenced during examination of each patent application and patent) (`` Documents cited in the application '') and any manufacturer's instructions or catalogs of any products cited or described in the respective patent applications and patents and in the documents cited in any application are hereby incorporated by reference. Incorporated into the specification. In addition, all references mentioned in this specification, all references cited or referenced in references mentioned in this specification, and any manufacturer's instructions or catalogs of any products referenced or described in this specification. Is incorporated herein by reference.

  Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, the invention as claimed should not be unduly limited to such specific embodiments, and various modifications to the invention It should be understood that and additions may be made within the scope of the present invention. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims. Furthermore, various combinations of the configurations of the dependent claims can be made using the configurations of the independent claims without departing from the scope of the present invention.

Sequence listing
SEQ ID NO: 1
SEQ ID NO: 1 shows the cDNA sequence of human GPR86

SEQ ID NO: 2
SEQ ID NO: 2 shows the open reading frame derived from SEQ ID NO: 1.

SEQ ID NO: 3
SEQ ID NO: 3 shows the amino acid sequence of human GPR86

SEQ ID NO: 4
SEQ ID NO: 4 shows the open reading frame of mouse GPR86 cDNA

SEQ ID NO: 5
SEQ ID NO: 5 shows the amino acid sequence of mouse GPR86

SEQ ID NO: 6
SEQ ID NO: 6 shows another cDNA sequence of human GPR86

SEQ ID NO: 7
SEQ ID NO: 7 shows another amino acid sequence of human GPR86

SEQ ID NOs: 8-20
SEQ ID NOs: 8 to 20 show the knockout plasmid primer sequences

SEQ ID NO: 21
SEQ ID NO: 21 shows the knockout plasmid sequence

The analysis result of human GPR86 polypeptide (sequence number 3) using the HMM structure prediction software (http://www.sanger.ac.uk/Software/Pfam/search.shtml) of pfam is shown. A description of the knockout plasmid is given. Figure 3 shows the expression profile of human GPR86 generated by reverse transcription polymerase chain reaction (RT-PCR). A graph of the data from the Tail Flick Test showing the results of GPR86 knockouts versus wild type animals compared is shown. Figure 6 shows a graph of data from the formalin test showing the results expressed as a percentage increase in paw width for contrasted GPR86 knockouts (white) and wild type animals (black). Figure 5 shows a graph of electronic Vonfrey test results (-/-knockout animals; + / + wild type controls). The RT-PCR analysis result of GPR86 expression in several human-derived tissues is shown. Lane 1: Bone Marrow; Lane 2: Thymus; Lane 3: Lymph Node; Lane 4: Jurkat CD4 +; Lane 5: Myla CD8 +; Lane 6: Colo 720; Lane 7: THP1; Lane 8: Osteoblast; Lane 9: Cartilage Cells; lane 10: negative control; lane 11: positive control (brain). The RT-PCR analysis result of GPR86 expression in several mouse-derived tissues is shown. Lane 1: Spleen; Lane 2: Salivary gland; Lane 3: Spinal cord; Lane 4: Muscle; Lane 5: Tongue; Lane 6: Ovary; Lane 7: Pancreas; Lane 8: Adipose tissue; Lane 9: Testis; Lane 10: Heart Lane 1: eye; lane 12: lung; lane 13: kidney; lane 14: thymus; lane 15: stomach + SI (small intestine) ;: lane 16: brain: lane 17: liver + Gb (gallbladder); lane 18: Lane 19: bladder; lane 20: adrenal gland; lane 21: C57BL6J genomic DNA; lane 22: 129SvEv genomic DNA.

Claims (7)

A method for in vitro identification of candidate molecules for the treatment, prevention or alleviation of inflammatory diseases or pain comprising:
The candidate molecule is a GPR86 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7 or a sequence having at least 98% identity thereto, and the sensitivity of the mouse knocked out of the GPR86 polypeptide to external stimulation and pain is wild type exposing the GPR86 polypeptide having the property of reduced compared to, and,
By measuring the binding of the candidate molecule to the GPR86 polypeptide to the candidate molecules to determine whether an agonist or antagonist of the GPR86 polypeptide,
Wherein the candidate molecule binds to the GPR86 polypeptide is a candidate molecule for the treatment of inflammatory diseases or pain, prevention or alleviation, the method. A method for in vitro identification of molecules suitable for the treatment, prevention or alleviation of inflammatory diseases or pain comprising:
(a) a GPR86 polypeptide comprising a sequence having at least 98% identity the amino acid sequence or to that of the described G protein G _i is conjugate to that SEQ ID NO: 7, an external knockout mice the GPR86 polypeptide (B) contacting a cell comprising a GPR86 polypeptide having a property of reducing sensitivity to irritation and pain compared to wild type with a candidate compound; and
Measuring cyclic AMP (cAMP) levels in the cells;
Wherein the candidate compound is identified as a molecule suitable for the treatment, prevention or alleviation of inflammatory disease or pain when the level is increased as a result of the contact. A method for in vitro identification of molecules suitable for the treatment, prevention or alleviation of inflammatory diseases or pain comprising:
(a) a GPR86 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7 conjugated to G _α16 or a sequence having at least 98% identity thereto, comprising external stimulation of a mouse knocked out of the GPR86 polypeptide; (B) contacting a cell comprising a GPR86 polypeptide having a property that reduces sensitivity to pain compared to wild-type with a candidate compound; and
Measuring intracellular calcium levels in the cells;
Wherein the candidate compound is identified as a molecule suitable for the treatment, prevention or alleviation of an inflammatory disease or pain when the level is reduced as a result of the contact.   4. The method of any one of claims 1-3, wherein the GPR86 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7. Further comprising the method of claim 1 in vitro tests for determining that the candidate molecule is an antagonist of the GPR86 polypeptide. The test is
(a) a GPR86 polypeptide comprising a sequence having at least 98% identity the amino acid sequence or to that of the described G protein G _i is conjugate to that SEQ ID NO: 7, an external knockout mice the GPR86 polypeptide (B) contacting a cell comprising a GPR86 polypeptide having a property of reducing sensitivity to irritation and pain compared to wild type with a candidate compound; and
Measuring cyclic AMP (cAMP) levels in the cells;
6. The method of claim 5, wherein said candidate compound is identified as a molecule suitable for the treatment, prevention or alleviation of inflammatory disease or pain when said level is increased as a result of said contact. The test is
(a) a GPR86 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7 conjugated to G _α16 or a sequence having at least 98% identity thereto, comprising external stimulation of a mouse knocked out of the GPR86 polypeptide; (B) contacting a cell comprising a GPR86 polypeptide having a property that reduces sensitivity to pain compared to wild-type with a candidate compound; and
Measuring intracellular calcium levels in the cells;
6. The method of claim 5, wherein the candidate compound is identified as a molecule suitable for the treatment, prevention or alleviation of inflammatory disease or pain when the level is reduced as a result of the contact.

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