WO2002022801A2 - Nouveaux genes de recepteurs mammaliens et leurs utilisations - Google Patents
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- WO2002022801A2 WO2002022801A2 PCT/US2001/028455 US0128455W WO0222801A2 WO 2002022801 A2 WO2002022801 A2 WO 2002022801A2 US 0128455 W US0128455 W US 0128455W WO 0222801 A2 WO0222801 A2 WO 0222801A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
- C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to biogenic amine receptors from mammalian species and the genes corresponding to such receptors. Specifically, the invention relates to the isolation, cloning and sequencing of complementary DNA (cDNA) copies of messenger RNA (mRNA) encoding a novel mammalian biogenic amine receptor gene.
- the invention also relates to the construction of recombinant expression constructs comprising cDNA of this novel receptor gene, said recombinant expression constructs being capable of expressing receptor protein in cultures of transformed prokaryotic and eukaryotic cells. Production of the receptor protein in such cultures is also provided.
- the invention relates to the use of such cultures of such transformed cells to produce homogeneous compositions of the novel biogenic amine receptor protein.
- the invention also provides cultures of such cells producing this receptor protein for the characterization of novel and useful drugs. Antibodies against and epitopes of this novel biogenic amine receptor protem are also provided by the invention.
- Biogenic amines are a class of naturally-occurring amino acid derivatives having a variety of physiological effects in the peripheral and central nervous systems.
- the parent compound is ⁇ -phenylethylarnine, and derivatives of this compound include the biogenic amines.
- the biogenic amines are a large and diverse class of compounds that include dopamine, noradrenaline, epinephrine, norepinephrine, and serotonin.
- the biogenic amines are implicated in a variety of psychiatric and neurologic disorders.
- biogenic amines are released by the sympathetic nervous system and adrenal medulla and are involved in integrating physiological responses to stress, while in the central nervous system the biogenic amines constitute some of the most important neurotransmitter systems.
- the effects of biogenic amines are mediated through their receptors and their associated cell signaling systems (reviewed in Hoffman & Lefkowitz, 19 2, Ann. Rev. Physiol. 44: 475-484; Civelli et al, 1993, Ann. Rev. Pharm. & Tox. 33: 281-307). These receptors are located in the plasma membrane of biogenic amine-sensitive cells. Structurally, they are characterized by having a pattern of seven transmembrane domains (see, for example, U.S.
- Patent Nos. 5,422,265, 5,569,601, 5,594,108, 5,883,226, 5,880,260, 5,427,942 and 5,686,573) Functionally, certain of these receptors interact with adenylate cyclase, either stimulating or inhibiting the production of cyclic AMP thereby.
- These receptors include the adrenergic receptors (the a-l, a-2, b-l, b-2, and b- 3 adrenergic receptors) and the dopamine receptors (the Di-, D 2 -, D 3 -, D_ 4 -, and D 5 - dopamine receptors).
- epinephrine adrenaline
- norepinephrine synthetic agonists of these biogenic amines which mimic their biological functions, and antagonists which block these biological functions, exert their effects by binding to specific recognition sites (membrane receptors) situated on the cell membranes in the nervous system.
- adrenergic receptors Two principal classes of adrenergic receptors have been defined, the alpha-adrenergic receptors and the beta-adrenergic receptors.
- Five subtypes of adrenergic receptors (a-l, a-2, b-l, b-2, and b-3 adrenergic receptors) have now been distinguished.
- Beta-adrenergic receptors catalyze the activation of a class of G proteins, which in turn stimulates the activity of adenylate cyclase when they bind with biogenic amine agonists, whereas alpha-adrenergic receptor antagonists act in competition with the agonists for the binding to the receptor and prevent the activation of adenylate cyclase.
- adenylate cyclase When adenylate cyclase is activated, it catalyses the production of an intracellular mediator or second messenger, especially cyclic AMP.
- dopamine is a biogenic amine neurotransmitter that modulates neuronal cells involved in movement initiation, appetitive behavior, hormone release, and visual dark adaptation.
- dopamine plays a role in modulating blood pressure and renal function (see generally Cooper et al, 1978, THE BIOCHEMICAL BASIS OF NEU OPHARMACOLOGY, 3d ed., Oxford University Press, New York, pp, 161-195).
- dopamine receptors subtypes that are either "Dl-like” or “D2-like,” which respectively stimulate and inhibitthe enzyme adenylate cyclase (Kebabian & Calne, 1979, Nature 277: 93-96). Alterations in the number or activity of these receptors may be a contributory factor in disease states such as Parkinson's disease (a movement disorder) and schizophrenia (a behavioral disorder) and attention deficit hyperactivity disorder (ADHD).
- Parkinson's disease a movement disorder
- schizophrenia a behavioral disorder
- ADHD attention deficit hyperactivity disorder
- Dl and D2 dopamine receptors have been developed to solubilize and purify these receptor proteins (see Senogles et al, 1986, Biochemistry 25: 749-753; Sengoles et al, 1988, J. Biol. Chem. 263: 18996-19002; Gingrich et al, 1988, Biochemistry 27: 3907-3912).
- the Dl dopamine receptor in several tissues appears to be a glycosylated membrane protein of about 72 kD (Amlaiky et al, 1987, Mol. Pharmacol 31: 129-134; Ninzik et al, 1988, Biochemistry 27: 7594-7599).
- the D2 receptor can also be glycosylated and has been suggested to have a higher molecular weight of about 90-150 kD (Amlaiky & Caron, 1985, J Biol. Chem. 260: 1983-1986; Amlaiky & Caron, 1986, J. Neurochem. 47: 196-204; Jarvie et al, 1988, Mol. Pharmacol. 34: 91-97).
- Dopamine receptors are primary targets in the clinical treatment of psycho- motor disorders such as Parkinson's disease and affective disorders such as schizophrenia (Seeman et al, 1987, Neuropsychopharm. I : 5-15; Seeman, 1987, Synapse : 152-333).
- Five different dopamine receptor genes (Dl, D2, D3, D4 and D5) and various splice variants of their transcripts have been cloned as a result of nucleotide sequence homology which exists between these receptor genes (Bunzow et al, 1988, Nature 336: 783-787; Grandy et al, 1989, Proc. Natl Acad. Sci. USA 86: 9762-9766; Dal Toso et al, 1989, EMBO J.
- biogenic amines are present in much lower quantities (less than 1% of the biogenic amines) and are therefore known as trace amines.
- the trace amines include such compounds as ?_>_-r ⁇ -tyramme (p- tyramine), met ⁇ -tyramine ( /.-tyramme), phenylethylamine, octopamine, and tryptamine.
- ⁇ -phenethylamine ⁇ -phenethylamine
- jo-tyra ine tryptamine
- octopamine ⁇ -phenethylamine
- peripheral tissues ⁇ -phenethylamine
- tryptamine tryptamine
- octopamine ⁇ -phenethylamine
- ⁇ -PEA and / tyramine can be synthesized from phenylalanine or tyrosine by the enzyme amino acid decarboxylase. (Boulton and Dyck, 1974, Life Sci 14: 2497-2506; Tallman et al., 1976, ibid.).
- Sensitive techniques have been developed to detect low concentrations of trace amines in the central nervous system. Such studies have revealed that trace amines in the central nervous system have a high turnover rate (Meek et al, 1970, J. Neurochem. Yf. 1627- 1635 ; Lemberger et al. , 1971 , J. Pharmac. Exp. Ther. YT 169- 176; Wu & Boulton, 1974, Can. J. Biochem. 52: 374-381; Durden & Philips, 1980, J. Neurochem. 34: 1725-1732).
- Trace amines are expressed throughout the brain in a heterogenous pattern and at least two of them can pass easily across the blood-brain- barrier (Boulton, 1974, Lancet ii: 7871; Oldendorf, 1971, Am. J. Physiol 22 ⁇ f. 1629- 1639). Trace amines are also known to potentiate caudate neuronal firing in response to dopamine application and act as sympathomimetics by stimulating release of biogenic amines from brain preparations and synaptosomes when applied at high concentrations. Para-tyramine may act as a "false transmitter" in a manner similar to that of amphetamine by triggering release of neurotransmitters such as dopamine.
- jj-tyramine and ⁇ -PEA The abilities of jj-tyramine and ⁇ -PEA to deplete neurotransmitter from storage vesicles, compete with neurotransmitters for uptake, and stimulate outward neurotransmitter flux through the plasma membrane carriers are similar to the actions of the ⁇ -PEA analog, ⁇ -methyl- ⁇ -phenethylamine, better known as amphetamine (Amara and Sonders, 1998, Drug Alcohol Depend 51:87-96; Seiden et al., 1993). Amphetamines were originally marketed as stimulants and appetite suppressants, but their clinical use is now mostly limited to treating attention deficit hyperactivity disorder (Seiden et al., 1993, Annu Rev Pharmacol Toxicol 33:639-677).
- amphetamines are widely consumed because of their ability to produce wakefulness and intense euphoria.
- amphetamines In addition to the actions of amphetamines at biogenic amine transporters, it is also clear that a subset of amphetamine analogs, especially those with hallucinogenic properties, can act directly on 5 -HT receptors as they have much higher affinities for these sites than for the transporters (Marek and Aghajanian, 1998, Drug Alcohol Depend 51 :189-198).
- biogenic amines and their receptors have created the need for the isolation of additional biogenic amine receptors, particularly trace amine receptors, for the development of therapeutic agents for the treatment of disorders, including disorders of the CNS and most preferably treatment of disorders on mental health such as psychosis, in which biogenic amines and their receptors have been implicated.
- biogenic amines and their receptors have been implicated.
- new tools that will permit identification of new drug lead compounds for developing novel drugs. This is of particular importance for psychoactive and psychotropic drugs, due to their physiological importance and their potential to greatly benefit human patients treated with such drugs. At present, few such economical systems exist.
- Conventional screening methods require the use of animal brain slices in binding assays as a first step.
- the present invention relates to the cloning, expression and functional characterization of a mammalian biogenic amine receptor gene.
- the invention comprises nucleic acids having amicleo tide sequence of a novel mammalian biogenic amine receptor gene that specifically binds to trace amines.
- the nucleic acids provided by the invention comprise a complementary DNA (cDNA) copy of the corresponding mRNA transcribed in vivo from the biogenic amine receptor genes of the invention.
- the mammalian biogenic amine receptor is a human biogenic amine receptor.
- the mammalian biogenic amine receptor is a rat (Rattus norvegicus) biogenic amine receptor.
- This invention in a first aspect provides nucleic acids, nucleic acid hybridization probes, recombinant eukaryotic expression constructs capable of expressing the biogenic amine receptors of the invention in cultures of transformed cells, and such cultures of transformed eukaryotic cells that synthesize the biogenic amine receptors of the invention.
- the invention provides homogeneous compositions of the biogenic amine receptor proteins of the invention, and membrane_and cytosolic preparations from cells expressing the biogenic amine receptor proteins of the invention, as well as antibodies against and epitopes of the biogenic amine receptor proteins of the invention.
- the invention in another aspect provides methods for making said homogenous preparations and membrane and cytosolic preparations using cells transformed with the recombinant expression constructs of the invention and expressing said biogenic amine receptor proteins thereby.
- Methods for characterizing the receptor and biochemical properties of these receptor proteins and methods for using these proteins in the development of agents having pharmacological uses related to these receptors are also provided by the invention.
- the invention provides a nucleic acid having a nucleotide sequence encoding a mammalian biogenic amine receptor.
- the nucleic acid encodes a human biogenic amine receptor.
- the nucleotide sequence comprises 1125 nucleotides of human biogenic amine receptor cDNA comprising 1040 nucleotides of coding sequence, 20 nucleotides of 5' untranslated sequence and 85 nucleotides of 3' untranslated sequence.
- the nucleotide sequence of the biogenic amine receptor is the nucleotide sequence depicted in Figure 1 (SEQ ID No: l).
- FIG. 1 The sequence shown in Figure 1 will be understood to represent one specific embodiment of a multiplicity of nucleotide sequences that encode the human biogenic amine receptor amino acid sequence (SEQ ID No.: 2) of the invention and that these different nucleotide sequences are functionally equivalent and are intended to be encompassed by the claimed invention. Further, it will be understood that the coding sequence comprising 1040 nucleotides can be used to express the cognate protein without inclusion of either the 5' or 3' untranslated sequences.
- allelic variations of the human biogenic amine receptor including naturally occurring and in vitro modifications thereof are within the scope of this invention. Each such variant will be understood to have essentially the same amino acid sequence as the sequence of the human biogenic amine receptor disclosed herein.
- the nucleic acid encodes the rat biogenic amine receptor.
- the nucleotide sequence includes 999 nucleotides of the rat biogenic amine receptor cDNA comprising the coding sequence.
- the nucleotide sequence of the biogenic amine receptor is the nucleotide sequence depicted in Figure 2 (SEQ ID No: 3).
- allelic variations of the rat biogenic amine receptor including naturally occurring and in vitro modifications thereof are within the scope of this invention. Each such variant will be understood to have essentially the same amino acid sequence as the sequence of the human biogenic amine receptor disclosed herein.
- Mammalian biogenic amine receptor proteins corresponding to the human and rat cDNAs of the mvention are a second aspect of the claimed invention.
- the mammalian biogenic amine receptor protein is a human biogenic amine receptor having a deduced amino acid sequence shown in Figure 1 (SEQ ID No.: 2).
- said human biogenic amine receptor protein comprising a membrane or cytosolic preparation from a cell, most preferably a recombinant cell, expressing a nucleic acid encoding a human biogenic amine of the invention.
- the mammalian biogenic amine receptor protein is a rat biogenic amine receptor having a deduced amino acid sequence shown in Figure 2 (SEQ ID No.:4).
- said rat biogenic amine receptor protein comprising a membrane or cytosolic preparation from a cell, most preferably a recombinant cell, expressing a nucleic acid encoding a rat biogenic amine of the invention.
- This invention provides both nucleotide and amino acid probes derived from the sequences herein provided.
- the invention includes probes isolated from either cDNA or genomic DNA, as well as probes made synthetically with the sequence information derived therefrom.
- the invention specifically includes but is not limited to oligonucleotide, nick-translated, random primed, or in vitro amplified probes made using cDNA or genomic clone of the invention encoding a mammalian biogenic amine receptor or fragment thereof, and oligonucleotide and other synthetic probes synthesized chemically using the nucleotide sequence information of cDNA or genomic clone embodiments of the invention.
- nucleic acid hybridization probes to determine the pattern, amount and extent of expression of the biogenic amine receptor gene in various tissues of mammals, including humans. It is also an object of the present invention to provide nucleic acid hybridization probes derived from the sequences of mammalian biogenic amine receptor genes of the invention to be used for the detection and diagnosis of genetic diseases. It is an object of this invention to provide nucleic acid hybridization probes derived from the nucleic acid sequences of the mammalian biogenic amine receptor genes herein disclosed to be used for the detection of novel related receptor genes.
- the present invention also includes synthetic peptides made using the nucleotide sequence information comprising the cDNA embodiments of the invention.
- the invention includes either naturally occurring or synthetic peptides which may be used as antigens for the production of biogenic amine receptor-specific antibodies, or useful as competitors of biogenic amine receptor molecules for agonist, antagonist or drug binding, or to be used for the production of inhibitors of the binding of agonists or antagonists or analogues thereof to such biogenic amine receptor molecules.
- the present invention also provides antibodies against and epitopes of the mammalian biogenic amine receptor molecules of the invention. It is an object of the present mvention to provide antibodies that are immunologically reactive to the biogenic amine receptors of the invention. It is a particular object to provide monoclonal antibodies against these biogenic amine receptors. Hybridoma cell lines producing such antibodies are also objects of the invention. It is envisioned that such hybridoma cell lines may be produced as the result of fusion between a non- immunoglobulin producing mouse myeloma cell line and spleen cells derived from a mouse immunized with a cell line which expresses antigens or epitopes of a mammalian biogenic amine receptor of the invention.
- the present invention also provides hybridoma cell lines that produce such antibodies, and can be injected into a living mouse to provide an ascites fluid from the mouse that is comprised of such antibodies. It is a further object of the invention to provide immunologically-active epitopes of the mammalian biogenic amine receptor proteins of the invention. Chimeric antibodies immunologically reactive against the biogenic amine receptor proteins of the invention are also within the scope of this invention.
- the present invention provides recombinant expression constructs comprising a nucleic acid encoding a mammalian biogenic amine receptor of the invention wherein the construct is capable of expressing the encoded biogenic amine receptor in cultures of cells transformed with the construct.
- a preferred embodiment of such constructs comprises a human biogenic amine receptor cDNA depicted in Figure 1 (SEQ ID No.: 1), such constructs being capable of expressing the human biogenic amine receptor encoded therein in cells transformed with the construct.
- Another preferred embodiment of such constructs comprises a rat biogenic amine receptor cDNA depicted in Figure 2 (SEQ ID No.: 3), such constructs being capable of expressing the rat biogenic amine receptor encoded therein in cells transformed with the construct.
- the invention also provides prokaryotic and more preferably eukaryotic cells transformed with the recombinant expression constructs of the invention, each such cells being capable of and indeed expressing the mammalian biogenic amine receptor encoded in the transfo ⁇ ning construct, as well as methods for preparing mammalian biogenic amine receptor proteins using said transformed cells.
- the present invention also includes within its scope protein preparations of prokaryotic and eukaryotic cell membranes containing the biogenic amme receptor protein of the invention, derived from cultures of prokaryotic or eukaryotic cells, respectively, transformed with the recombinant expression constructs of the invention.
- the present invention also includes within its scope protein preparations of prokaryotic and eukaryotic cytoplasmic fractions containing the biogenic amine receptor protein of the invention, derived from cultures of prokaryotic or eukaryotic cells, respectively, transformed with the recombinant expression constructs of the invention.
- the invention also provides methods for screening compounds for their ability to inhibit, facilitate or modulate the biochemical activity of the mammalian biogenic amine receptor molecules of the invention, for use in the in vitro screening of novel agonist and antagonist compounds.
- cells transformed with a recombinant expression construct of the invention are contacted with such a compound, and the binding capacity of the compounds, as well as the effect of the compound on binding of other, known biogenic amine receptor agonists and antagonists, is assayed. Additional preferred embodiments comprise quantitative analyses of such effects.
- the present invention is also useful for the detection of analogues, agonists or antagonists, known or unknown, of the mammalian biogenic amine receptors of the invention, either naturally occurring or embodied as a drug.
- analogues, agonists or antagonists may be detected in blood, saliva, semen, cerebrospinal fluid, plasma, lymph, or any other bodily fluid.
- biogenic amine receptors of the present invention are directly activated by a wide variety of clinically and socially important drugs, including amphetamines, ergot derivatives, and adrenergic agents.
- the receptors of the invention are useful for developing alternative pharmaceutical agents having the beneficial properties of these drugs without at least some of the deleterious effects, for example a propensity for addiction, as well as compounds that can inhibit or overcome said propensity for addition.
- Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.
- Figure 1 illustrates the nucleotide (SEQ ID No.: 1) and amino acid (SEQ ID No.:2) sequences of a human trace amine receptor.
- Figure 2 illustrates the nucleotide (SEQ ID No.: 3) and amino acid (SEQ ID No.:4) sequences of a rat trace amine receptor.
- Figure 3 presents . deduced amino acid sequences for the rat and human trace amine receptors aligned with other homologous G protein-coupled receptors. Identities are outlined in black. Abbreviations are: rTAR, rat trace amine receptor; hTAR, human trace amine receptor; NTR, orphan NeuroTransmitter receptor; orphan GPCR57 and 58; D1R, dopamine Dl receptor; B2aR, B2 adrenergic receptor; 5HT4c, serotonin 5HT4C receptor. Arrowheads indicate positions designated as 5.42 and 5.43.
- Figure 4 is a photograph of an autoradiogram of Northern analysis of total cellular RNA (20 ⁇ g/lane) from human HEK293 cells expressing the human biogenic amine receptor of the invention after transformation with a recombinant expression construct.
- Figure 5 A is a photograph of an ethidium bromide-stained and ultraviolet light irradiated agarose gel containing DNA fragments produced by RT-PCR of RNA from rat brain tissues.
- the PCR products resolved on this gel are from the following rat brain regions, from which cDNA was synthesized from oligo(dT)-primed total RNA: lane 1, pituitary gland; lane 2, hindbrain; lane 3, midbrain; lane 4, locus coeruleus; lane 5, hypothalamus; lane 6, striatum; lane 7, olfactory bulb; lane 8, olfactory tubercle; lane 9, hippocampus; lane 10, cortex; lane 11, cerebellum; lane 12, thalamus; lane 13, 1:100 dilution of human trace amine plasmid DNA.
- Figure 5B is an autoradiogram of a nylon membrane containing DNA fragments transferred from the agarose gel shown in Figure 5 A and probed with 32 P- labeled nucleic acid prepared from the coding sequence of the rat genomic clone encoding the rat trace amine receptor of the invention.
- Figure 6 is a photograph of an autoradiogram of Northern analysis of RNA from various rat cell lines expressing the rat trace amine receptor of the invention after transfection with a recombinant expression construct encoding the rat receptor.
- RNA shown in this gel was obtained from the following cell lines: lane 1, LBP; lane 2, baby hamster kidney (BHK) cells; lane 3, rat insulinoma (RIN5) cells; lane 4, AR42J rat pancreatic tumor cell line; lane 5, CHW cells; lane 6, GH4 rat pituitary cells; lane 7, GH3 rat pituitary cells; lane 8, AtT20 rat pituitary cells; lane 9, PC 12 rat adrenal gland cells; lane 10, SK-N-MC human neuroblastoma cells; lane 11, N4TG1 rat neuroblastoma cells; lane 12, NB4 cells; lane 13, LCS cells; lane 14, R2C rat Ledig cells.
- Figure 7 is a photograph of an autoradiogram of Northern analysis of mRNA expressed in various cell lines expressing a mammalian biogenic amine receptor of the invention after transfection with a recombinant expression construct encoding the rat biogenic amine receptor.
- Figure 8 A is a photograph of an ethidium bromide-stained and ultraviolet light irradiated agarose gel containing DNA fragments produced by RT-PCR of RNA from rat tissues.
- the PCR products resolved on this gel are from the following rat tissues: lane 1, liver (oligo(dT) primed); lane 2, brain (dT); lane 3, spleen (dT); lane 4, lung (dT); lane 5, heart (dT); lane 6, testis (dT); lane 7, kidney (dT); lane 8, intestine (dT); lane 9, COS-7 cell oligo(dT)-selected mRNA from cells transformed with the RC- RSV/rat biogenic amine receptor construct of the invention; lane 10, striatum (dT); lane 11, midbrain (random primed; rp); lane 12, olfactory tubercle (rp); lane 13, cortex (rp + dT); lane 14, midbrain (dT); lane 15,
- Figure 8B is an autoradiogram of a nylon membrane containing DNA fragments transferred from the agarose gel shown in Figure 8A and probed with 32 P- labeled nucleic acid prepared from the full-length rat genomic clone encoding the rat trace amine receptor of the invention.
- Figures 9A through 9D are photographs of fluorescence in situ hybridization analysis of human chromosomes probed with a fluorescently-labeled human artificial chromosome (BAC) containing the human biogenic amine receptor DNA (BAC obtained from Research Genetics, Release IV of DNA pools, Catalog #96001; clone address: plate 278, Row D, Column 22).
- Figure 9E is a schematic diagram of human chromosome 6 denoting the location of the human biogenic amine locus at 6q23.2.
- Figures 10A is a graph showing the ability of various endogenous compounds to stimulate the rat trace amine receptor heterologously expressed in HEK 293 cells in a dose-dependent manner.
- Figure 10B is a graph showing the ability of various synthetic compounds to stimulate the rat trace amine receptor heterologously expressed in HEK 293 cells in a dose-dependent manner.
- FIGS 11 A through 11 C are photographs of immunohistochemical staining of HEK 293 cells expressing epitope-tagged rat trace amine receptor. Ml tagged receptors were bound to anti-FLAG antibodies followed by Cy5 goat anti-mouse IgG in the absence ( Figure 11A) or presence ( Figure 11B) of 0.1% Triton X-100. Control cells shown in Figure 11C express dopamine Dl receptors and were stained with antibodies in the presence of Triton X-100.
- Figures 12A through 12G are graphs showing assays of cAMP production in HEK 293 cells stably transfected with the rat receptor of the invention.
- mammalian biogenic amine receptor and "trace amine receptor” as used herein refer to proteins consisting essentially of, and having substantially the same biological activity as, the protein encoded by the amino acid depicted in Figure 1 (SEQ ID No.: 2) and Figure 2 (SEQ ID No.: 4). This definition is intended to encompass natural allelic variations in the disclosed biogenic, trace amine receptor.
- Cloned nucleic acid provided by the present invention may encode trace amine receptor protein of any species of origin, including, for example, mouse, rat, rabbit, cat, and human, but preferably the nucleic acid provided by the invention encodes trace amine receptors of mammalian, most preferably rat and human, origin.
- the nucleic acids provided by the invention comprise DNA or RNA having a nucleotide sequence encoding a mammalian trace amine receptor. Specific embodiments of said nucleic acids are depicted in Figure 1 (SEQ ID No. : 1) or Figure 2 (SEQ ID NO.: 3), and include any nucleotide sequence encoding a mammalian biogenic amine receptor having an amino acid sequence as depicted in Figure 1 (SEQ ID No.: 2) or Figure 2 (SEQ ID No.: 4).
- Nucleic hybridization probes as provided by the invention comprise any portion of a nucleic acid of the invention effective in nucleic acid hybridization under stringency conditions sufficient for specific hybridization.
- nucleic acid hybridization probes are also within the scope of this embodiment of the invention.
- Nucleic acid probes as provided herein are useful for isolating mammalian species analogues of the specific embodiments of the nucleic acids provided by the invention.
- Nucleic acid probes as provided herein are also useful for detecting mammalian trace amine receptor gene expression in cells and tissues using techniques well-known in the art, including but not limited to Northern blot hybridization, in situ hybridization and Southern hybridization to reverse transcriptase - polymerase chain reaction product DNAs.
- the probes provided by the present invention are also useful for Southern hybridization of mammalian, preferably human, genomic DNA for screening for restriction fragment length polymorphism (RFLP) associated with certain genetic disorders.
- RFLP restriction fragment length polymorphism
- Nucleic acid encoding a trace amine receptor may be obtained, in view of the instant disclosure, by chemical synthesis, by screening reverse transcripts of mRNA from appropriate cells or cell line cultures, by screening genomic libraries from appropriate cells, or by combinations of these procedures, in accordance with known procedures as illustrated below. Additionally, sequences of such receptors can be obtained from any species in which the content of the species genomic DNA has been determined and assembled in a database or other searchable compilation, using search programs l ⁇ iown in the art and the sequences of the trace amine receptors disclosed herein. Screening of mRNA or genomic DNA may be carried out with oligonucleotide probes generated from the nucleic acid sequence information from mammalian trace amine receptor nucleic acid as disclosed herein.
- Probes may be labeled with a detectable group such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with l ⁇ iown procedures and used in conventional hybridization assays, as described in greater detail in the Examples below.
- mammalian biogenic amine receptor nucleic acid sequences may be obtained by use of the polymerase chain reaction (PCR) procedure, using PCR oligonucleotide primers corresponding to nucleic acid sequence information derived from a biogenic amine receptor as provided herein. See U.S. Patent Nos.4,683 , 195 to Mullis et al. and 4,683,202 to Mullis.
- Mammalian trace amine receptor protein may be synthesized in host cells transformed with a recombinant expression construct comprising a nucleic acid encoding said receptor and comprising genomic DNA or cDNA.
- a recombinant expression construct can also be comprised of a vector that is a replicable DNA construct.
- Vectors are used herein either to amplify DNA encoding a trace amine receptor and/or to express DNA encoding a trace amine receptor gene.
- a recombinant expression construct is a replicable DNA construct in which a nucleic acid encoding a trace amine receptor is operably linked to suitable control sequences capable of effecting the expression of the receptor in a suitable host.
- control sequences include a transcriptional promoter, an optional operator or enhancer sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation.
- Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. See, Sambrook et al, 1990, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press: New York).
- Vectors useful for practicing the present invention include plasmids, viruses (including phage and mammalian DNA and RNA viruses), retroviruses, and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination).
- the vector can replicate the gene of interest and function independently of the host genome, or can, in some instances, integrate into the genome itself. Suitable vectors will contain replicon and control sequences which are derived from species compatible with the intended expression host.
- a preferred vector is RcRSV (obtained from Invitrogen, San Diego, CA).
- Another preferred vector is pcDNA3.1/V5 His-TOPO (Invitrogen, San Diego, CA).
- the pcDNA3.1/V5/His-TOPO vector expresses a receptor preceded at its amino terminus by a cleavable 16 amino acid signal sequence of the influenza hemaglutinin virus immediately followed by the 8 amino acid M 1 -Flag epitope and then a two amino acid linker (MetGly) just before the initiation methionine (Guan et al. , 1992, JBiol Chem, 267:21995-21998).
- Transformed host cells are cells that have been transformed or transfected with recombinant expression constructs made using recombinant DNA techniques and comprising nucleic acid encoding a trace amine receptor protein.
- Cultures of cells derived from multicellular organisms are a desirable host for recombinant biogenic amine receptor protein synthesis. In principal, any higher eukaryotic cell culture is useful, whether from vertebrate or invertebrate culture. However, mammalian cells are preferred, as illustrated in the Examples. Propagation of such cells in cell culture has become a routine procedure. See Tissue Culture. Academic Press, Kruse & Patterson, editors (1973).
- Examples of useful host cell lines are human embryonic kidney (HEK) 293 cells, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, mouse Ltk " cell lines and WI138, BHK, COS-7, CV, and MDCK cell lines.
- Preferred host cells are HEK293 cells, COS-7 cells (Gluzman, 1981, Cell 23: 175- 182) and Ltk " cells.
- Transformed host cells may express the trace amine receptor protein, but host cells transformed for purposes of cloning or amplifying nucleic acid hybridization probe DNA need not express the receptor.
- the trace amine receptor of the invention can be located in the host cell cytosol.
- the invention provides preparations of cell cytosolic fractions comprising the trace amine receptor protein of the invention, as well as purified, homogeneous preparations of the receptor protein itself. See, Sambrook et al, ibid.
- the receptor of the invention may also be located in membranes from the host cell. Therefore, the invention provides preparations of said cell membranes comprising the trace amine receptor protein of the invention. See, Sambrook et al, ibid.
- the invention provides homogeneous compositions of mammalian trace amine receptor protem produced by transformed eukaryotic cells as provided herein.
- Each such homogeneous composition is intended to be comprised of a trace amine receptor protein that comprises at least 75%, more preferably at least 80%, and most preferably at least 90% of the protein in such a homogenous composition; in said homogeneous preparations, individual contaminating protein species are expected to comprise less than 5%, more preferably less than 2% and most preferably less than 1% of the preparation.
- the invention also provides membrane and cytosolic preparations from cells expressing mammalian trace amine receptor protein as the result of transformation with a recombinant expression construct, as described herein.
- Mammalian trace amine receptor proteins made from cloned genes in accordance with the present invention may be used for screening trace amine analogues, or trace amine receptor agonists or antagonists of trace amine binding, or for determining the amount of such agonists or antagonists are present in a solution of interest (e.g. , blood plasma, cerebrospinal fluid or serum).
- host cells may be transformed with a recombinant expression construct of the present invention, a mammalian trace amine receptor expressed in those host cells, and the cells, membranes or cytosolic fractions thereof used to screen compounds for their effect on trace amine receptor agonist binding activity.
- agonists (also referred to herein as stimulators) of the receptor of the present invention can be endogenous neurotransmitters or drugs.
- Neurotransmitters and drugs that activate the receptor are further described in the Examples section herein, and include -tyramine, phenylethylamine, tryptamine, octopamine, synephrine, dopamine, serotonin, m- tyramine, amphetamines, methamphetamines, MDMA, j-chloroamphetamine, betahistine, 1-phenylpiporazine, phenylephrine, apomorphine, metergoline, and ergot alkaloids.
- Agonists for the trace amine receptor include, but are not limited to ⁇ - phenethylamine (PEA), hordenine , L-tyrosinol, S,R-amphetamine (+ and -), 4-OH- R(-)-amphetamine, methamphetamine (+ and -), ( ⁇ )DOI, phenelzine, tranylcypromine, 3,4-DiMeO-PEA>Mescaline, ( ⁇ )MDMA, 3,4-dihydroxybenzylguanidine, 3- phenylpropylamine, l-methyl-3-phenylpropylarnine, N,N-dimethylpropiophenone, N- phenylethylenediamine, kynuramine, 4-phenylbutylamine, tryptamine, 2- thiopheneethylamine, betahistine, 2>4>3-pyridylethylamine, 1-phenylpiperazine, 1- (
- Antagonists of the trace amine receptor include, but are not limited to phenylalanine, ( ⁇ )N-ethylamphetamine, propylhexedrine, fenfluramine, deprenyl, norepinephrine, epinephrine, N,N,N-trimethyldopamine, dopamine-guanidine, dimethylsulfonium-DA, benzylamine, pargyline, tryptophan, 5- carbooxamidotyptamine, histamine, 2-(2a ⁇ _inoethyl)l,3-dioxolane, iproniazid, isoniazid, l,l-dimethyl-4-phenylpiperazinium, trans- 1-cinnamylpiperazine, 1- (4Acetophenone)piperazine, quipazine, SH-I-101, PAPP (LY 165, 163), 4-OH,4- phenylpiperidine, HA-1, HA-2
- a compound identified in a screen may be useful for treating various conditions associated with effects of unregulated trace amine activity as a result of endogenous or exogenous stimulation.
- the present invention provides a pharmaceutical composition comprising the compound in admixture with a pharmaceutically acceptable carrier.
- a therapeutically effective amount of the pharmaceutical composition is administered to a patient with a condition associated with unregulated trace amine activity.
- the pharmaceutical composition of the present invention can be used to reduce sympathomimetic effects of enhanced trace amine transmission induced by elevated levels of trace amines or certain drugs. Common sympathomimetic effects include rapid heart rate, high blood pressure, agitation, cardiac arrythmia, seizures, and coma.
- the pharmaceutical composition can also be used to treat peripheral effects of drugs, such as amphetamine.
- the pharmaceutical composition of the present invention can be used to treat hyperthermia caused by amphetamine action.
- Some conditions that can be treated using a pharmaceutical composition of the present invention are pathological, such as schizophrenia, depression, etc.
- the pharmaceutical composition of the present invention can be used to treat drug addiction in a mammal, preferably a human.
- compositions of the present invention can be manufactured in a manner that is itself l ⁇ iown, e.g., by means of a conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
- compositions of the compounds of the present invention can be formulated and administered through a variety of means, including systemic, localized, or topical administration. Techniques for formulation and administration can be found in "Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA. Pharmaceutical compositions for use in accordance with the present invention thus can be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
- Non-toxic pharmaceutical salts include salts of acids such as hydrochloric, phosphoric, hydrobromic, sulfuric, sulfinic, formic, toluenesulfonic, methanesulfonic, nitic, benzoic, citric, tartaric, maleic, hydroiodic, alkanoic such as acetic, HOOC- (CH 2 ) n -CH 3 where n is 0-4, and the like.
- Non-toxic pharmaceutical base addition salts include salts of bases such as sodium, potassium, calcium, ammonium, and the like. Those skilled in the art will recognize a wide variety of non-toxic pharmaceutically acceptable addition salts.
- the compounds of the invention can be formulated in appropriate aqueous solutions, such as physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
- physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
- penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally l ⁇ iown in the art.
- the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well l ⁇ iown in the art.
- Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
- Pharmaceutical preparations for oral use can be obtained with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
- Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
- disintegrating agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
- Dragee cores are provided with suitable coatings.
- concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
- Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
- Pha ⁇ naceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
- the push-fit capsules can contain the active compounds in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
- the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
- stabilizers can be added. All formulations for oral administration should be in dosages suitable for such administration.
- the compositions can take the form of tablets or lozenges formulated in conventional manner.
- the active compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifiuoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifiuoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifiuoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifiuoromethane, trichlorofluoromethane, dichlorotetrafluoroethan
- the active compounds can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
- Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
- the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
- the active compounds can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
- a suitable vehicle e.g., sterile pyrogen-free water
- the active compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
- the active compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
- the active compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
- compositions also can comprise suitable solid or gel phase carriers or excipients.
- suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
- the mode of administration can be selected to maximize delivery to a desired target site in the body.
- Suitable routes of administration can, for example, include oral, rectal, fransmucosal, franscutaneous, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
- parenteral delivery including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
- one can administer the compound in a local rather than systemic manner for example, via injection of the compound directly into a specific tissue, often in a depot or sustained release formulation.
- compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
- the therapeutically effective dose can be estimated initially from cell culture assays, as disclosed herein.
- a dose can be formulated in animal models to achieve a circulating concentration range that includes the EC50 (effective dose for 50% increase) as determined in cell culture, i. e. , the concentration of the test compound which achieves a half-maximal inhibition of tumor cell growth in vitro.' Such information can be used to more accurately determine useful doses in humans.
- the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination, the severity of the particular disease undergoing therapy and the judgment of the prescribing physician.
- the drug or a pharmaceutical composition containing the drug may also be added to the animal feed or drinking water. It will be convenient to formulate animal feed and drinking water products with a predetermined dose of the drug so that the animal takes in an appropriate quantity of the drug along with its diet. It will also be convenient to add a premix containing the drug to the feed or drinking water approximately immediately prior to consumption by the animal.
- Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
- the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50.
- Compounds that exhibit high therapeutic indices are preferred.
- the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
- the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
- the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
- the recombinant expression constructs of the present invention are useful in molecular biology to transform cells that do not ordinarily express a trace amine receptor to thereafter express this receptor. Such cells are useful as intermediates for making cell membrane or cytosolic preparations useful for receptor binding activity assays, which are in turn useful for drag screening.
- the recombinant expression constructs of the present invention thus provide a method for screening potentially useful drugs at advantageously lower cost than conventional animal screening protocols. While not completely eliminating the need for ultimate in vivo activity and toxicology assays, the constructs and cultures of the invention provide an important first screening step for the vast number of potentially useful drugs synthesized, discovered or extracted from natural sources each year.
- the recombinant expression constructs of the present invention are useful in molecular biology to detect, isolate, characterize and identify novel endogenous trace amine receptor agonists and antagonists found in plasma, serum, lymph, cerebrospinal fluid, seminal fluid, or other potential sources of such compounds.
- This utility thereby enables rational drug design of novel therapeutically-active drugs using currently- available techniques (see Walters, "Computer-Assisted Modeling of Drugs", in Klegerman & Groves, eds., 1993, Pharmaceutical Biotechnology. Interpharm Press: Buffalo Grove, IL, pp. 165-174).
- the recombinant expression constructs of the present invention may also be useful in gene therapy.
- Cloned genes of the present invention, or fragments thereof, may also be used in gene therapy carried out homologous recombination or site- directed mutagenesis. See generally Thomas & Capecchi, 1987, Cell 5_1: 503-512; Bertling, 1987, Bioscience Reports 7: 107-112; Smithies et al, 1985, Nature 317: 230-234.
- Nucleic acid and oligonucleotide probes as provided by the present invention are useful as diagnostic tools for probing trace amine receptor gene expression in tissues of humans and other animals.
- tissues are probed in situ with oligonucleotide probes carrying detectable groups by conventional autoradiographic or other detection techniques, to investigate native expression of this receptor or pathological conditions relating thereto.
- chromosomes can be probed to investigate the presence or absence of the corresponding trace amine receptor gene, and potential pathological conditions related thereto.
- the invention also provides antibodies that are immunologically reactive to the trace amine receptor protein or epitopes thereof provided by the invention.
- the antibodies provided by the invention may be raised, using methods well l ⁇ iown in the art, in animals by inoculation with cells that express a trace amine receptor or epitopes thereof, cell membranes from such cells, whether crude membrane preparations or membranes purified using methods well known in the art, cytosolic preparations, or purified preparations of proteins, including fusion proteins, particularly fusion proteins comprising epitopes of the trace amine receptor protein of the invention fused to heterologous proteins and expressed using genetic engineering means in bacterial, yeast or eukaryotic cells, said proteins being isolated from such cells to varying degrees of homogeneity using conventional biochemical methods.
- Synthetic peptides made using established synthetic methods in vitro and optionally conjugated with heterologous sequences of amino acids, are also encompassed in these methods to produce the antibodies of the invention.
- Animals that are useful for such inoculations include individuals from species comprising cows, sheep, pigs, chickens, mice, rats, rabbits, hamsters, goats and primates.
- Preferred animals for inoculation are rodents (including mice, rats, hamsters) and rabbits. The most preferred animal is the mouse.
- Cells that can be used for such inoculations, or for any of the other means used in the invention include any cell line which naturally expresses the trace amine receptor provided by the invention, or more preferably any cell or cell line that expresses the trace amine receptor of the invention, or any epitope thereof, as a result of molecular or genetic engineering, or that has been treated to increase the expression of an endogenous or heterologous trace amine receptor protein by physical, biochemical or genetic means.
- Preferred cells are mammalian cells, most preferably cells syngeneic with a rodent, most preferably a mouse host, that have been transformed with a recombinant expression construct of the invention encoding a trace amine receptor protein, and that express the receptor therefrom.
- the present invention also provides monoclonal antibodies that are immunologically reactive with an epitope derived from a trace amine receptor of the invention, or fragment thereof, present on the surface of such cells. Such antibodies are made using methods and techniques well l ⁇ iown to those of skill in the art. Monoclonal antibodies provided by the present invention are produced by hybridoma cell lines, that are also provided by the invention and that are made by methods well l ⁇ iown in the art.
- Hybridoma cell lines are made by fusing individual cells of a myeloma cell line with spleen cells derived from animals immunized with cells expressing a trace amine receptor of the invention, as described above.
- the myeloma cell lines used in the invention include lines derived from myelomas of mice, rats, hamsters, primates and humans.
- Preferred myeloma cell lines are from mouse, and the most preferred mouse myeloma cell line is P3X63-Ag8.653.
- the animals from which spleens are obtained after immunization are rats, mice and hamsters, preferably mice, most preferably Balb/c mice.
- Spleen cells and myeloma cells are fused using a number of methods well l ⁇ iown in the art, including but not limited to incubation with inactivated Sendai virus and incubation in the presence of polyethylene glycol (PEG).
- PEG polyethylene glycol
- the most preferred method for cell fusion is incubation in the presence of a solution of 45% (w/v) PEG-1450.
- Monoclonal antibodies produced by hybridoma cell lines can be harvested from cell culture supernatant fluids from in vitro cell growth; alternatively, hybridoma cells can be injected subcutaneously and/or into the peritoneal cavity of an animal, most preferably a mouse, and the monoclonal antibodies obtained from blood and/or ascites fluid.
- Monoclonal antibodies provided by the present invention are also produced by recombinant genetic methods well l ⁇ iown to those of skill in the art, and the present invention encompasses antibodies made by such methods that are immunologically reactive with an epitope of a trace amine receptor of the invention.
- the present invention also encompasses fragments, including but not limited to F(ab) and F(ab) 2 fragments, of such antibody. Fragments are produced by any number of methods, including but not limited to proteolytic or chemical cleavage, chemical synthesis or preparation of such fragments by means of genetic engineering technology.
- the present invention also encompasses single-chain antibodies that are immunologically reactive with an epitope of a trace amine receptor, made by methods l ⁇ iown to those of skill in the art.
- the present invention also encompasses an epitope of a trace amine receptor of the invention, comprised of sequences and/or a conformation of sequences present in the receptor molecule.
- This epitope may be naturally occurring, or may be the result of chemical or proteolytic cleavage of a receptor molecule and isolation of an epitope- containing peptide or may be obtained by chemical or in vitro synthesis of an epitope- containing peptide using methods well known to those skilled in the art.
- the present invention also encompasses epitope peptides produced as a result of genetic engineering technology and synthesized by genetically engineered prokaryotic or eukaryotic cells.
- the invention also includes chimeric antibodies, comprised of light chain and heavy chain peptides immunologically reactive to a biogenic amine receptor-derived epitope.
- the chimeric antibodies embodied in the present invention include those that are derived from naturally occurring antibodies as well as chimeric antibodies made by means of genetic engineering technology well l ⁇ iown to those of skill in the art.
- the Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They set forth for explanatory purposes only, and are not to be taken as limiting the invention.
- cDNA prepared from total cellular RNA obtained from a rat pancreatic tumor cell line AR42 J was used as template for a polymerase chain reaction (PCR)-based random cloning experiment.
- PCR polymerase chain reaction
- PCR was performed using a pair of degenerate oligonucleotide primers corresponding to a consensus sequence of the third and sixth transmembrane regions of l ⁇ iown G-coupled receptors.
- PCR products obtained in this experiment were characterized by nucleotide sequencing.
- a full length clone was obtained by screening a rat genomic library using a cloned PCR product encoding a novel G-protein coupled receptor as deduced by nucleotide sequencing and comparison with a sequence database (GenBank).
- rat cDNA preparation was then subjected to 35 cycles of PCR amplification using 500 picomoles of degenerate oligonucleotide primers having the following sequence: Primer III (sense): GAGTCGACCTGTG(C/T)G(C/T)(C/G)AT(C/T)(A/G)CIIT(G/T)GAC(C/A)G(C/G)T AC
- PCR products were identified by screening the GenBank database and identified a cloned fragment having a high degree of homology 5 to l ⁇ iown biogenic amine receptors, as well as containing sequence motifs that are common to the G-protein coupled family of receptors, but that was not identical to any previously-identified biogenic amine receptor sequence.
- Example 1 The cloned PCR product obtained in Example 1 was used to isolate a full- length clone from a rat genomic DNA library (obtained from Clonetech, Palo Alto, CA) as follows.
- the 0.4 kb DNA fragment generated by PCR having high homology to known 5 biogenic amine receptors was 32 P-labeled using the random priming technique (Stratagene, San Diego CA). This probe was used to screen a rat genomic library that had been transferred to nylon membranes (Gene Screen Plus, NEN, Boston MA). Hybridization was performed in 50% formamide, 5X SSC, 1% SDS, 5X Denhardt' solution, and salmon sperm DNA (50 ⁇ g/mL) with the radioactive probe at 2xl0 6 0 cpm/mL at 37°C for overnight.
- nylon filters were then washed as follows: at room temperature in a solution of 2X SSC/ 0.1% SDS for 10 minutes, followed by a wash at 55°C in a solution of 2X SSC/ 0.1% SDS for 15 minutes, and finally a wash at 55°C in a solution of 0.5X SSC/ 0.1% SDS for 5 minutes. Filters were then exposed to XOMAT X-ray film (Kodak) overnight. Filter hybridization was performed in duplicate to confirm positive signals. Secondary and tertiary screens were performed until single homogenous clones were isolated.
- the sequence immediately 5' to the proposed initiation codon was found to contain several translation termination codons in-frame with the open reading frame, supporting the assignment of the translation start site.
- Predicted transmembrane domains (using the algorithm of Eisenberg et al. (1984, J. Molec. Biol. 179: 125-142)) are boxed and identified by Roman numerals (I- VII), and two sites of possible N-linked glycosylation are identified in the amino-te ⁇ ninal portion of the protein with solid triangles.
- a potential protein kinase C site was also found in the C-terminal tail.
- the predicted amino acid sequences of the transmembrane domains were also compared with corresponding sequences in human D 1 dopamine receptor, human D2 dopamine receptor, rat serotonin lc receptor, rat ⁇ l-b adrenergic receptor, rat serotonin 4 receptor, rat serotonin la receptor, human a-2 adrenergic receptor, and human H-2 histamine receptor (Probst et al, 1992, DNA Cell Biology 11 : 1-20).
- Table I shows the percentage extent of homology in pairwise fashion between the different biogenic amine receptors.
- sequence DRY (amino acids 120-123 in the human sequence and amino acids 119-122 in the rat sequence) is conserved in the majority of G-protein coupled receptors. Expression of this receptor in a rat insulinoma suggests that biogenic amines may play a role in pancreatic cell function.
- the Asp in TMIII is thought to be a counterion to the positively charged amino group present in biogenic amines.
- the deduced amino acid sequence predicts a Ser in TMV, which would be able to form a hydrogen bond with the para- hydroxyl group of molecules such as the dopamine, norepinephrine, and epinephrine, as well as the trace amines para-tyramine, octopamine, and synephrine.
- One Ser was found in the receptor compared with the adrenergic and dopamine receptors, which contain an additional one or two Ser residues N-terminal to the "SerPheTyrXaaPro" (where "Xaa” is any residue) motif in TMV.
- Thr is found directly N-terminal to the Ser that might hydrogen bond with ligands.
- Trp that is found in the rhodopsin family of G-protein coupled receptors. Distal and two residues proximal to this Trp, the receptor displays significant homology to members of the biogenic amine receptor family.
- Pro residue 6 amino acids N-terminal of the generally conserved Pro residue found in TMIV of biogenic amine receptors.
- the two Ser residues in TMIV that are conserved among GPCRs activated by biogenic amines are not present in the novel receptor of the invention.
- novel G-protein coupled receptor genes of the invention are biogenic amine receptors.
- rat cDNA was cloned into a mammalian expression construct (pRcRSVneo, obtained from Invitrogen, San Diego, CA), the resulting recombinant expression construct transfected into COS-7 cells (for transient expression assays) and human embryonic kidney cells (HEK293) for stable expression assays, and cell membranes (COS-7) or cell lines (HEK293) were generated that expressed the receptor protein in cellular membranes at the cell surface.
- pRcRSVneo obtained from Invitrogen, San Diego, CA
- the entire coding region of the receptor DNA insert was amplified using PCR as described above with primers specific for flanking sequences; such PCR primers advantageously contained restriction enzyme digestion recognition sites at the 5' termini such that digestion with said restriction enzymes allowed facile cloning of the receptor cDNA into the RcRSVneo mammalian expression constract.
- PCR products generated in this way were subcloned in to the RcRSV vector using conventional techniques (see Sambrook et al, ibid) and the orientation of the inserted cDNA confirmed by restriction enzyme digestion analysis of insert-containing subclones.
- Such recombinant expression constructs were introduced into COS-7 cells using the calcium-phosphate precipitation technique (Chen & Okayama, 1987, Molec. Cell.
- recombinant expression constructs were also introduced into HEK293 cells using the calcium-phosphate precipitation technique, and stably-transfected clones were selected by growth in the mammalian neomycin analog G418 (Grand Island Biological Co., Long Island, NY), as the vector RcRSV contains a functional copy of a bacterial neomycin resistance gene. Stable cell lines were then selected for membrane binding studies based on mRNA expression levels of individual neomycin- resistant transfected clones determined by Northern analysis (see Sambrook et al, ibid). Cell membranes were prepared and used as described above for COS-7 cell transfectants.
- RNA Easy kit obtained from Qiagen, Valencia, CA.
- 10 ⁇ g of total cellular RNA was subjected to electrophoresis in a 1.2% agarose gel using HEPES/ EDTA buffer (pH 7.8) overnight.
- the electrophoresed RNA was then transferred to a GeneScreen Plus membrane (New England Nuclear, Boston, MA) by capillary transfer, and fixed to the membrane by baking at 85°C for lh.
- the membrane was then prehybridized overnight at 37°C in the following buffer: 50% formamide, 1% sodium dodecyl sulfate (SDS), 5X SSC (where IX SSC is 0.15M NaCl/ 0.015M sodium citrate, pH 7), 50 ⁇ g/mL denatured salmon spermDNA, and 5X P-buffer (comprising 0.25M Tris, pH 7.5, 0.5% sodium pyrophosphate, 0.5% SDS, 1% bovine serum albumin, 1% polyvinylpyrrolidone and 1% Ficoll (400,000 MW)).
- SDS sodium dodecyl sulfate
- 5X SSC where IX SSC is 0.15M NaCl/ 0.015M sodium citrate, pH 7
- 5X P-buffer comprising 0.25M Tris, pH 7.5, 0.5% sodium pyrophosphate, 0.5% SDS, 1% bovine serum albumin, 1% polyvinylpyrrolidone and 1% Ficol
- 32 P-labeled DNA prepared from the full-length genomic receptor clone described above was added at a concentration of 3 x 10 6 cpm/mL and the membrane hybridized overnight at 37°C.
- the hybridized membrane was then washed using the following high-stringency washing conditions: 10 min at room temperature in a wash solution of 2X SSC/ 1% SDS; 10 min at 60°C in 2X SSC/ 1% SDS; and finally 5 min at 60°C in 0.5X SC/ 1% SDS, where the washing solutions were changed between each washing step.
- the washed membrane was then exposed overnight to X-ray film (X-omat, Kodak, Rochester, NY).
- RNA corresponding to expression of the biogenic amine receptor gene in various regions of the rat brain was determined by reverse transcription/polymerase chain reaction (RT-PCR) performed as follows. Total RNA from various rat brain sections was isolated using the RNA Easy kit (Qiagen) described in Example 3 and converted to single-stranded cDNA using reverse transcriptase (BRL, Gaithersburg, MD) primed by oligo dT or random primers or a combination of both these primers. PCR was then performed using the 5' sense primer (TCT CTG AGT GAT GCA TCT TTG; SEQ ID No.
- an antisense primer (AGC AGT GCT CAACTG TTC TCA CCA TGC; SEQ ID No.: 8) having its 3' end at nucleotide residue 243 of the SEQ ID No. 3 (resulting in a PCR product of about 250bp in length) or an antisense primer (GCA CGA TTA ATT GAC CTC GCT TG; SEQ ID No.: 9) having its 3' end at nucleotide residue 650 of the SEQ ID No. 3 (resulting in a PCR product of about 650bp in length).
- PCR was performed for 35 cycles, wherein one cycle consisted of incubations at 94°C for 90 sec (denaturation), 55°C for 90 sec (annealing), and 72°C for 120 sec (extension).
- the resulting fragments were resolved from 30 ⁇ L reaction mixture using 1% agarose gel electrophoresis and visualized by ethidium bromide staining and UV illumination. The fragments were then transferred onto a nylon membrane (GeneScreen Plus,
- Hybridized fragments were detected using a phosphoimager (Molecular Devices, Mountain View, C A).
- Figures 5 A and 5B show a photograph of an ethidium bromide stained 1% agarose gel viewed under ultraviolet light illumination.
- PCR product (lO ⁇ L of a 30 ⁇ L reaction mixture) was electrophoresed as described above, and bands specific for the predicted fragments of the rat biogenic amine receptor of the invention (250 or 650bp) were detected.
- Figure 5B shows the results of the hybridization assay, which results in greater sensitivity of detection of PCR-amplified fragments. These results indicated that the biogenic amine receptor was expressed strongly in midbrain and olfactory tubercle, less strongly in the olfactory bulb, moderately in the striatum and weakly in the hypothalamus.
- the transcript was widely distributed throughout the brain, with the highest levels of expression detected in the olfactory bulb, nucleus accumbens/olfactory tubercle, prefrontal cortex and other cortical regions, midbrain regions consisting of substantia nigra and ventral tegmentum, cerebellum, and pons/medulla.
- the highest level was observed in the liver, with lesser expression detected in kidney, gastrointestinal tract, spleen, pancreas, and heart.
- olfactory tubercle > intestine D midbrain, cortex, spleen > heart, kidney
- the receptor was also expressed at detectable levels in lung, transfected COS cells, and olfactory bulb. These results are consistent with known patterns of trace amine receptor expression in olfactory tubercle and midbrain.
- the novel mammalian trace amine receptor cDNA obtained in Example 2 was used to isolate a partial genomic clone from a library of human genomic DNA cloned in lambda EMBL3 (obtained from Clontech, Palo Alto, CA) as follows.
- the full- length rat receptor cDNA ( ⁇ 1 kb in length) was 32 P-labeled by the random priming technique a kit obtained from Stratagene (San Diego, CA) according to the manufacturer's instructions. This probe was then used to screen the human genomic library., which had been plated and then transferred to nylon membranes (Gene Screen Plus, NEN, Boston, MA).
- Hybridization was performed in a solution of 50% formamide, 5XSSC, 1% SDS, 5X Denhardt solution, and salmon sperm DNA (50 micro grams/mL) with the radioactive probe at 2 x 10 6 cpm/mL and at a temperature of 37°C overnight.
- the nylon filters were then washed at room temperature in a solution of 2X SSC/ 0.1% SDS for 10 minutes, followed by a wash at 55°C in a solution of 2X SSC/ 0.1% SDS for 15 minutes, and finally a wash at 55°C in a solution of 0.5X SSC/ 0.1 % SDS for 5 minutes . Filters were then exposed to XOMAT X-ray film (Kodak) overnight at -80°C. Filter hybridization was performed in duplicate to confirm positive signals. Secondary and tertiary screens were performed until single homogenous clones were identified.
- BAC bacterial artificial chromosome
- Each PCR amplification cycle consisted of incubations at 94°C for 90 sec (denaturation), 50°C for 90 sec (annealing), and 72°C for 120 sec (extension) for 35 cycles. Amplified products of the PCR reaction were separated on a 1.0% agarose gel
- the novel human trace amine receptor is encoded by a single coding exon.
- the sequence of the human receptor is presented in Figure 1.
- the open reading frame of the human homologue of the trace amine receptor gene is 21 bases longer than the rat (1017 vs 996, respectively) which translates into a human receptor that is 339 amino acids long compared to a receptor of 332 amino acids in the rat (shown in Figure 2).
- a comparison between the primary amino acid sequences of the human and rat receptors is presented in Figure 3.
- the chromosomal locus of the human trace amine receptor gene of the invention was mapped by fluorescence in situ hybridization as follows.
- BAC DNA encoding the human trace amine receptor described in Example 5 was nick-translated using digoxigenin-11-UTP for use as a probe for in situ chromosomal mapping to localize the gene.
- This fluorescently labeled DNA was hybridized in situ to denatured human metaphase chromosomes for 16 hours. Signal was detected in the presence of DAPI (4,6-diamidino-2-phenylindole) counter staining and the chromosome was identified by sequential G-banding. The hybridization signal appeared to be consistent with a chromosomal location on the distal long arm of chromosome 6.
- DAPI 4,6-diamidino-2-phenylindole
- 3-MT is the major metabolite of dopamine produced by the enzyme COMT, a variant of which was recently found to be transmitted with greater frequency to schizophrenic offspring in a family based association study (Egan et al., 2001, Proc Natl Acad Sci USA 98: 6917-6922).
- G-protein coupled receptors are capable of stimulating the MAP (microtubule-associated protein) kinase assay in mammalian cells.
- MAP microtubule-associated protein
- activation of the pathway by ligand binding to receptor results in increased phosphorylation of mammalian transcription factor Elk by the MK kinase.
- the phosphorylated Elk transcription factor then binds to promoters containing cis- sequences responsive to this transcription factor. Transcription factor binding results in increase transcription of sequences operatively linked and under the transcriptional control of such Elk-responsive promoters.
- reporter genes such as ⁇ -galactosidase or firefly luciferase are operatively linked to such Elk-responsive promoters, thereby permitting ligand binding to a receptor to be linked with expression of the reporter gene.
- HEK 293 cells were transfected with the full-length human clone encoding the trace amine receptor of the invention contained in the pcDNA 3.1 expression vector (Invitrogen), wherein the first 22 nucleotides of the 5' untranslated region is followed by an initiation codon (ATG, Met), followed by nucleotides encoding an 8-amino acid FLAG sequence (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys; SEQ ID No. : 12), followed by a nucleotide sequence encoding the 21 amino acids of the human D2 receptor (as disclosed in co-owned U.S. Patent No.
- Enhanced expression of the receptor was achieved by cloning the full-length rat cDNA into the mammalian expression vector pcDNA3.1/V5/His-TOPO (Invitrogen).
- a PCR product was generated that upon expression produced the rat receptor sequence preceded at its amino terminus by a cleavable 16 amino acid signal sequence of the influenza hemaglutinin virus immediately followed by the 8 amino acid Ml -Flag epitope and then a two amino acid linker (MetGly) just before the initiation methionine (Guan et al, 1992, J Biol Chem. 267: 21995-21998).
- HEK293 cells were transfected using the Lipofectamine transfection reagent and cells stably expressing the construct were selected in G418.
- the flag-tagged receptor was analyzed by immunofluorescence to determine cellular localization.
- localization of the Dl receptor in HEK293 cells stably expressing the cloned flag-tagged human D 1 receptor was also examined.
- Cells were maintained in DMEM media containing 10% fetal calf serum and 700 ⁇ g/mL G418 (Life Technologies, Bethesda, MD).
- Confluent cells were detached with PBS solution containing 0.05% trypsin and 0.53 mM EDTA, harvested, diluted 1 :10, and plated on glass microscope coverslips coated with poly-D-lysine and grown at 37°C for 48 hours. Cells were washed twice with PBS and fixed with 2.5% paraformaldehyde in PBS for 20 minutes. Cells were then incubated for 30 minutes with anti-FLAG monoclonal antibody (1:500; Sigma) in blocking buffer solution (3% dry milk, 1 mM CaCl 2 , 50 mM Tris HClPh 7.5) with or without 0.1% Triton X-100.
- HEK293 cells stably transfected with the pcDNA3.1/V5/His-TOPO expression vector containing the full-length rat cDNA clone described above were assayed for cAMP production in response to various ligands.
- HEK293 cells were harvested in Krebs- Ringer buffer (KRH; Sigma) and preincubated in KRH with 200 ⁇ M IBMX.
- KRH Krebs- Ringer buffer
- test compound or 10 ⁇ M forskolin
- the cells were then boiled for 20 min after adding an equal volume of 0.5 mM sodium acetate buffer, centrifuged to remove cell debris, and the resulting extract was analyzed for cAMP content using competitive binding of 3 H-cAMP to a cAMP binding protein (Diagnostic Products Corp., Los Angeles, CA).
- these molecules had EC 50 s in the following rank order (lowest to highest):
- the rank order of potencies observed for the human trace amine receptor indicates that a hydroxyl group at the meta position on ⁇ -PEA analogs or at the 5- position on tryptamine has deleterious effects on agonist potency, a trend that is contrary to that observed for catecholamine receptors.
- Comparison of the amino acid sequence of the trace amine receptor with those of catecholamine and 5-HT receptors suggests a structural basis for this change in selectivity. It has been proposed from mutagenesis studies of the ⁇ 2 -AR and the 5-HT ⁇ A receptor (Ho et al., 1992, FEBSLett
- endogenous agonists of the trace amine receptors of the invention may include some "inactive" catecholamine metabolites such as 3-methoxytyramine, the principal extracellular metabolite of dopamine (Wood and Altar, 1988, Pharmacol Rev 40 : 163 - 187) .
- the trance amine receptor is more potently activated by the presumably "inactive" catecholamine metabolites 3-methoxytyramine (3-MT), normetanephrine and metanephrine than by the neurotransmitters dopamine, norepinephrine, and epinephrine themselves.
- amphetamine analogs including methamphetamine and its congener MDMA ("ecstasy") could activate the trace amine receptors of the invention.
- ecstasy methamphetamine and its congener MDMA
- These and several other amphetamine analogs potently stimulated cAMP production in recombinant cells expressing this receptor.
- Amphetamines act directly on the receptor, since these drugs (at 1 ⁇ M concentrations) produced no cAMP stimulation in control cells transfected either with an empty vector or with the human Dl receptor.
- Amphetamine analogs that activate the receptor include both classic neurotransmitter transporter substrates as well as a prototypical hallucinogenic amphetamine, 2-amino,(l-[2,5-dimethoxy-4-iodophenyl]propane, which has poor affinity for transporters but high affinity for 5-HT 2 receptors (Marek and Aghajanian, 1998, ibid).
- N-ethyl analogs ( ⁇ )fenfluramine and ( ⁇ )N-ethylamphetamine, had substantially lower activities than the N-methyl congeners, methamphetamine and MDMA or than the primary amine congeners.
- tryptamine to activate the rTARl suggested that some ergot alkaloids might act as agonists.
- Antagonists of biogenic amine receptors and transporters were also found to stimulate camp production in recombinant cells expressing the trace amine receptors of the invention.
- Such compounds include the adrenergic antagonists phentolamine and tolazoline, the serotonergic antagonists cyproheptadine, dihydroergotamine, and metergoline, and the nonsubsfrate inhibitors of dopamine transporter protein nomifensine and l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine.
- the antipsychotic drug chlorpromazine typically considered to be a dopamine receptor antagonist, also acted as a weak agonist with the trace amine receptors of this invention.
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CA002422288A CA2422288A1 (fr) | 2000-09-12 | 2001-09-12 | Nouveaux genes de recepteurs mammaliens et leurs utilisations |
EP01972993A EP1319070A2 (fr) | 2000-09-12 | 2001-09-12 | Genes de recepteurs mammaliens et leurs utilisations |
AU2001292617A AU2001292617A1 (en) | 2000-09-12 | 2001-09-12 | Mammalian receptor genes and uses |
US10/380,614 US7381542B2 (en) | 2001-07-09 | 2001-12-19 | Nucleic acids encoding a biogenic amine receptor |
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US09/659,519 US6783973B1 (en) | 2000-09-12 | 2000-09-12 | Mammalian catecholamine receptor genes and uses |
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WO2004083851A2 (fr) * | 2003-03-18 | 2004-09-30 | Bayer Healthcare Ag | Diagnostics et traitements contre des maladies associees au recepteur 1 d'amine sous forme de traces (ta1) |
EP1621552A1 (fr) * | 2004-07-08 | 2006-02-01 | F.Hoffmann-La Roche Ag | Récepteur humain associé aux amines neurogènes (hTAAR) |
EP1626057A1 (fr) * | 2004-07-30 | 2006-02-15 | F.Hoffmann-La Roche Ag | Récepteurs du chimpanzé associés aux "trace amines" |
WO2007085558A1 (fr) * | 2006-01-27 | 2007-08-02 | F. Hoffmann-La Roche Ag | Utilisation de 2-imidazoles pour le traitement de troubles du snc |
WO2007085557A2 (fr) * | 2006-01-27 | 2007-08-02 | F. Hoffmann-La Roche Ag | Utilisation de 2-imidazole substitue de derives d'imidazoline |
WO2007090720A2 (fr) * | 2006-01-27 | 2007-08-16 | F. Hoffmann-La Roche Ag | Utilisation de 2-imidazoles pour le traitement de troubles du système nerveux central |
US7368541B2 (en) | 2004-12-01 | 2008-05-06 | Merck & Co., Inc. | Trace amine receptor 1 of the African green monkey |
US7652055B2 (en) | 2007-07-02 | 2010-01-26 | Hoffman-La Roche Inc. | 2-imidazolines |
US7812047B2 (en) | 2007-07-03 | 2010-10-12 | Hoffman-La Roche Inc. | 4-imidazolines |
US7834044B2 (en) | 2006-11-02 | 2010-11-16 | Hoffman-Laroche Inc. | Substituted-2-imidazoles |
US7858653B2 (en) | 2006-12-13 | 2010-12-28 | Hoffman-La Roche Inc. | 2-imidazoles |
US7858652B2 (en) | 2006-11-16 | 2010-12-28 | Hoffmann-La Roche Inc. | Substituted 4-imidazoles |
US7902238B2 (en) | 2007-02-15 | 2011-03-08 | Hoffmann-La Roche Inc. | 2-aminooxazolines as TAAR1 ligands |
US8008305B2 (en) | 2007-08-03 | 2011-08-30 | Hoffmann-La Roche Inc. | TAAR1 ligands |
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US10508107B2 (en) | 2016-03-17 | 2019-12-17 | Hoffmann-La Roche Inc. | Morpholine derivative |
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US9452980B2 (en) | 2009-12-22 | 2016-09-27 | Hoffmann-La Roche Inc. | Substituted benzamides |
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- 2001-09-12 WO PCT/US2001/028455 patent/WO2002022801A2/fr not_active Application Discontinuation
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EP1319070A2 (fr) | 2003-06-18 |
CA2422288A1 (fr) | 2002-03-21 |
WO2002022801A9 (fr) | 2003-07-03 |
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