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  • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
  • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
  • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
• • A—HUMAN NECESSITIES
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  • A61P11/00—Drugs for disorders of the respiratory system
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  • A61P25/16—Anti-Parkinson drugs
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  • A61P25/00—Drugs for disorders of the nervous system
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/22—Anxiolytics
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  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/24—Antidepressants
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  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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  • 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]
  • A61P29/02—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] without antiinflammatory effect
• • A—HUMAN NECESSITIES
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  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
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  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
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  • A61P37/00—Drugs for immunological or allergic disorders
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  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
  • C07B2200/11—Compounds covalently bound to a solid support

Definitions

• This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in diagnosis and in identifying compounds that may be agonists, antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides.
• the drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics,” that is, high throughput genome- or gene-based biology. This approach as a means to identify genes and gene products as therapeutic targets is rapidly superseding earlier approaches based on “positional cloning.” A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position.
• proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 1991, 351 :353-354).
• these proteins are referred to as proteins participating in pathways with G-proteins or PPG proteins.
• Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B.K., et al., Proc. Natl Acad.
• G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M.I., et al., Science, 1991, 252:802- 8).
• effector proteins e.g., phospholipase C, adenyl cyclase, and phosphodiesterase
• actuator proteins e.g., protein kinase A and protein kinase C (Simon, M.I., et al., Science, 1991, 252:802- 8).
• the effect of hormone binding is activation of the enzyme, adenylate cyclase, inside the cell.
• Enzyme activation by hormones is dependent on the presence of the nucleotide, GTP.
• GTP also influences hormone binding.
• a G- protein connects the hormone receptor to adenylate cyclase.
• G-protein was shown to exchange GTP for bound GDP when activated by a hormone receptor.
• 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.
• the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
• the membrane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane ⁇ -helices connected by extracellular or cytoplasmic loops.
• G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.
• G-protein coupled receptors (otherwise known as 7TM receptors) have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops.
• the G-protein family of coupled receptors includes dopamine receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders.
• Other examples of members of this family include, but are not limited to, calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1 , rhodopsins, odorant, and cytomegalovirus receptors.
• TM 1 Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure.
• the 7 transmembrane regions are designated as TM 1 , TM2, TM3, TM4, TM5, TM6, and TM7.
• TM3 has been implicated in signal transduction.
• G-protein coupled receptors Phosphorylation and lipidation (palmitylation or farnesy lation) of cysteine residues can influence signal transduction of some G-protein coupled receptors.
• Most G-protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus.
• G-protein coupled receptors such as the ⁇ -adrenoreceptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.
• the ligand binding sites of G-protein coupled receptors are believed to comprise hydrophilic sockets formed by several G-protein coupled receptor transmembrane domains, said sockets being surrounded by hydrophobic residues of the G-protein coupled
• each G-protein coupled receptor transmembrane helix is postulated to face inward and form a polar ligand bmding site TM3 has been implicated in several G-protem coupled receptors as having a ligand binding site, such as the TM3 aspartate residue TM5 se ⁇ nes, a TM6 asparagme and TM6 or TM7 phenylalamnes or tyrosines are also implicated in ligand binding
• G-protein coupled receptors can be mtracellularly coupled by heterotnme ⁇ c G-proteins to various lntracellular enzymes, ion channels and transporters (see, Johnson et al , Endoc Rev , 1989, 10 317-331 )
• Different G-protein ⁇ -subumts preferentially stimulate particular effectors to modulate va ⁇ ous biological functions in a 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 found in numerous sites within a mammalian host
• the present invention relates to AXOR35, in particular AXOR35 polypeptides and AXOR35 polynucleotides, recombinant matenals and methods for their production
• AXOR35 in particular AXOR35 polypeptides and AXOR35 polynucleotides, recombinant matenals and methods for their production
• Such polypeptides and polynucleotides are of interest in relation to methods of treatment of certain diseases, including, but not limited to bacte ⁇ al, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2, immunodeficiency, transplant rejection, gastrointestinal disorders such as gastric or duodenal ulcer, diarrhea, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, irritable bowel syndrome, vomiting, inflammation such as pru ⁇ tis and atopic dermatitis, allergies and allergic disorders including asthma, allergic rhinitis, autoimmune disorders including, but not limited to delayed type hypersensitivity, rheumato
• agonists bind to and activate the AXOR35 polypeptides (receptors) of the present invention
• antagonists inhibit the interaction of the AXOR35 polypeptides with receptor ligands
• a preferred method for identifying agonist or antagonist of a receptor of the present invention comp ⁇ ses
• preferred method for identifying agonist or antagonist of a receptor of the present invention comp ⁇ ses (a) contacting a cell expressing on the surface thereof the receptor, said receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor, with a compound to be screened under conditions to permit binding to the receptor, and
• the two methods described above further comp ⁇ ses conducting the identification of agonist or antagonist in the presence of labeled or unlabeled histamine or a histamine- ke compound
• the method for identifying agonist or antagonist of a receptor of the present invention compnses determining the inhibition of binding of a ligand to cells which have the receptor on the surface thereof, or to cell membranes containing the receptor, in the presence of a candidate compound under conditions to permit binding to the receptor, and determining the amount of ligand bound to the receptor, such that a compound capable of causing reduction of binding of a ligand is an agonist or antagonist.
• the ligand is histamine or a histamine-like compound.
• a method of screening for AXOR35 receptor antagonist or agonist comprises the steps of: (a) incubating a labeled histamine or a histamine-like compound with a whole cell expressing AXOR35 receptor on the cell surface, or cell membrane containing AXOR35 receptor;
• step (c) adding a candidate compound to a mixture of labeled histamine or a histamine-like compound and the whole cell or the cell membrane of step (a) and allowing to attain equilibrium;
• step (d) measuring the amount of labeled histamine or a histamine-like compound bound to the whole cell or the cell membrane after step (c);
• step (e) comparing the difference in the labeled histamine or a histamine-like compound bound in step (b) and (d), such that the compound which causes the reduction in binding in step (d) is an agonist or antagonist.
• the invention relates to agonists and antagonists discovered by any of the present screening methods. Yet in still another aspect, the invention relates to treating conditions associated with AXOR35 imbalance with agonists or antagonists of AXOR35, in particular agonists and antagonists identified by any of the present screening methods. Further, the present invention relates to a method of treating a disease of the invention, in particular asthma, by administering to a patient in need thereof an agonist or an antagoinst of AXOR35. The present invention also relates to a method of agonizing or antagonizing AXOR35 for the treatment of a disease of the invention, in particular asthma, by administering to a patient in need thereof an agonist or an antagonist of AXOR35.
• the present invention relates to a method of treating a disease of the invention, in particular asthma, by administering to a patient in need thereof an agonist or an antagoinst of AXOR35 identified by any of the herein described screening methods. Further the present invention also relates to a method of agonizing or antagonizing AXOR35 for the treatment of a disease of the invention, in particular asthma, by administering to a patient in need thereof an agonist or antagonist of AXOR35 identified by any of the herein described screening methods.
• the present invention relates to AXOR35 polypeptides.
• Such polypeptides include: (a) an isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO:l ;
• Polypeptides of the present invention are believed to be members of the 7TM receptor family of polypeptides They are therefore of interest because G-protein coupled (7TM) receptors, more than other gene family, are targets of pharmaceutical intervention
• AXOR35 biological activity of AXOR35
• AXOR35 activity biological activity of AXOR35
• a polypeptide of the present invention exhibits at least one biological activity of AXOR35
• Polypeptides of the present invention also include vanants of the aforementioned polypeptides, including all allehc forms and splice vanants Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non- conservative, or any combination thereof Particularly preferred variants are those in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acids are inserted, substituted, or deleted, in any combination
• Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO 2, or an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous am o acids truncated or deleted from the amino acid sequence of SEQ ID NO 2
• Preferred fragments are biologically active fragments that mediate the biological activity of AXOR35, including those with
• polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producing the full-length polypeptides of the invention
• the polypeptides of the present invention may be in the form of the "mature" protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amino acid sequence that contains secretory or leader sequences, pro- sequences, sequences that aid in purification, for instance multiple histidine residues, or an additional sequence for stability during recombinant production.
• Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occurring sources, from genetically engineered host cells comprising expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesizers, or a combination of such methods. Means for preparing such polypeptides are well understood in the art.
• the present invention relates to AXOR35 polynucleotides.
• Such polynucleotides include:
• Preferred fragments of polynucleotides of the present invention include an isolated polynucleotide compns g an nucleotide sequence having at least 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQ ID NO 1 , or an isolated polynucleotide compnsing an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ID NO 1
• Preferred vanants of polynucleotides of the present invention include splice vanants, allehc variants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs)
• Polynucleotides of the present invention also include polynucleotides encodmg polypeptide vanants that compnse the amino acid sequence of SEQ ID NO 2 and in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 amino acid residues are substituted, deleted or added, in any combination
• the present invention provides polynucleotides that are RNA transcripts of the DNA sequences of the present invention Accordingly, there is provided an RNA polynucleotide that
• (b) is the RNA transcnpt of the DNA sequence encoding the polypeptide of SEQ ID NO 2
• (c) comprises an RNA transcnpt of the DNA sequence of SEQ ID NO 1 , or
• (d) is the RNA transcnpt of the DNA sequence of SEQ ID NO 1 , and RNA polynucleotides that are complementary thereto
• the polynucleotide sequence of SEQ ID NO 1 shows homology with human histamine H3 receptor (Lovenberg, T W et al Mol Pharm 55 1 101 -1 107, 1999)
• the polynucleotide sequence of SEQ ID NO 1 is a cDNA sequence that encodes the polypeptide of SEQ ID NO 2
• polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence of SEQ ID NO 1 or it may be a sequence other than SEQ ID NO 1 , which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO 2
• the polypeptide of SEQ ID NO 2 is related to other proteins of the 7TM receptor family, having homology and/or structural similarity with human histamine H3 receptor (Lovenberg, T W et al Mol Pharm 55 1 101-1 107, 1999)
• Preferred polypeptides and polynucleotides of the present invention are expected to have inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one AXOR35 activity
• Polynucleotides of the present invention may be obtained using standard cloning and screening techniques from a cDNA library derived from mRNA in cells of human bone marrow, (see for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques.
• the polynucleotide may include the coding sequence for the mature polypeptide, by itself, or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
• a marker sequence that facilitates purification of the fused polypeptide can be encoded.
• the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.
• the polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
• Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SEQ ID NO: 1 may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification reaction (for instance, PCR). Such probes and primers may be used to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence similarity to SEQ ID NO: 1 , typically at least 95% identity.
• Preferred probes and primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50, if not at least 100 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred primers will have between 20 and 25 nucleotides.
• a polynucleotide encoding a polypeptide of the present invention may be obtained by a process comprising the steps of screening a library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
• a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides
• Preferred stringent hybridization conditions include overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM t ⁇ sodmm citrate), 50 mM sodium phosphate (pH 7 6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0 lx SSC at about 65°C
• the present invention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screening a library under stnngent hybndization conditions with a labeled probe having the sequence of SEQ ID NO 1 or a fragment thereof, preferably of at least 15 nucleotides
• an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide does not extend all the way through to the 5' terminus This is a consequence of reverse transc ⁇ ptase, an enzyme with inherently low "processivity" (a measure of the ability of the enzyme to remain attached to the template during the polymerization reaction), failing to complete a DNA copy of the mRNA template during first strand cDNA synthesis
• Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells compnsing expression systems
• the present invention relates to expression systems comprising a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems and to the production of polypeptides of the invention by recombinant techniques
• Cell-free translation systems can also be employed to produce such proteins using RNAs denved from the DNA constructs of the present invention
• host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention
• Polynucleotides may be introduced into host cells by methods descnbed m many standard laboratory manuals, such as Davis et al , Basic Methods in Molecular Biology (1986) and Sambrook et al (ibid)
• Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micro-injection, cationic pid-mediated transfection, electropor
• appropnate hosts include bacte ⁇ al cells, such as Streptococci, Staphylococci, E coh, Streptomyces and Bacillus subtihs cells, fungal cells, such as yeast cells and Asperg ⁇ lus cells, insect cells such as Drosoph ⁇ a S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells, and plant cells
• bacte ⁇ al cells such as Streptococci, Staphylococci, E coh, Streptomyces and Bacillus subtihs cells
• fungal cells such as yeast cells and Asperg ⁇ lus cells
• insect cells such as Drosoph ⁇ a S2 and Spodoptera Sf9 cells
• animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
• a great vanety of expression systems can be used, for instance, chromosomal, episomal and virus-denved systems, e g , vectors denved from bacte ⁇ al plasmids, from bactenophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids
• the expression systems may contain control regions that regulate as well as engender expression Generally, any system or vector that is able to maintain, propagate, or express a polynucleotide to produce a polypeptide in a host may be used The appropnate polynucleo
• a polypeptide of the present invention is to be expressed for use in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested prior to use in the screening assay If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide If produced intracellularly, the cells must first be lysed before the polypeptide is recovered
• Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectm chromatography Most preferably, high performance liquid chromatography is employed for punfication Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured dunng intracellular synthesis, isolation, and/or punfication
• Polynucleotides of the present invention may be used as diagnostic reagents, through detecting mutations in the associated gene Detection of a mutated form of the gene characte ⁇ zed by the polynucleotide of SEQ ID NO 1 in the cDNA or genomic sequence and which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease of the invention, or susceptibility to a disease of the invention, which results from under- expression, over-expression or altered spatial or temporal expression of the gene Individuals carrying mutations in the gene may be detected at the DNA level by a va ⁇ ety of techniques well known in the art
• Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, unne, saliva, tissue biopsy or autopsy material
• the genomic DNA may be used directly for detection or it may be amplified enzymatically by using PCR, preferably RT-PCR, or other amplification techniques pnor to analysis RNA or cDNA may also be used in similar fashion
• Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype
• Point mutations can be identified by hybridizing amplified DNA to labeled AXOR35 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures
• DNA sequence difference may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (see for instance, Myers et al , Science ( 1985) 230 1242) Sequence changes at specific locations may also be revealed by nu
• An array of oligonucleotides probes comprising AXOR35 polvnucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e g , genetic mutations
• Such arrays are preferably high density arrays or gnds
• Array technology methods are well known and have general applicability and can be used to address a va ⁇ ety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability, see, for example, M Chee et al , Science, 274, 610-613 (1996) and other references cited therein
• Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radio-immunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
• the present invention relates to a diagnostic kit comprising:
• a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO: 1 , or a fragment or an RNA transcript thereof;
• polypeptide of the present invention preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or
• kits may comprise a substantial component.
• Such a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly diseases of the invention, amongst others.
• the polynucleotide sequences of the present invention are valuable for chromosome localization studies.
• the sequence is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
• the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library).
• the polynucleotide sequences of the present invention are also valuable tools for tissue expression studies Such studies allow the determination of expression patterns of polynucleotides of the present invention which may give an indication as to the expression patterns of the encoded polypeptides in tissues, by detecting the mRNAs that encode them
• the techniques used are well known in the art and include in situ hybndization techniques to clones arrayed on a grid, such as cDNA microarray hybridization (Schena et al, Science, 270, 467-470, 1995 and Shalon et al, Genome Res, 6, 639-645, 1996) and nucleotide amplification techniques such as PCR
• TAQMAN Trade mark
• Compare studies of the normal expression pattern of mRNAs with that of mRNAs encoded by an alternative form of the same gene for example, one having an alteration in poly
• the polypeptides of the present invention are expressed in peripheral blood monocytes and bone marrow
• the expression of AXOR35 was detected in lymphocytes, macrophages, eosinophils, and neutrophils in asthamtic lung, but not in normal lung
• the present invention relates a method of inhibiting or promoting the function of lymphocytes, macrophages, eosinophils, or neutrophils in diseased tissue, such as, but not limited to, asthmatic lung, by administering to a patient in need thereof AXOR35 agonists or antagonists
• the present invention relates a method of inhibiting or promoting the function of lymphocytes, macrophages, eosinophils, or neutrophils in diseased tissue, such as, but not limited to, asthmatic lung, by administering to a patient in need thereof AXOR35 agonists or antagonists identified by any of the screening methods described herein
• the present invention further relates to a method of agon
• a further aspect of the present invention relates to antibodies.
• the polypeptides of the invention or their fragments, or cells expressing them can be used as immunogens to produce antibodies that are immunospecific for polypeptides of the present invention.
• immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
• Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bearing fragments, or cells to an animal, preferably a non-human animal, using routine protocols.
• any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G.
• antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
• Antibodies against polypeptides of the present invention may also be employed to treat diseases of the invention, amongst others.
• polypeptides and polynucleotides of the present invention may also be used as vaccines. Accordingly, in a further aspect, the present invention relates to a method for inducing an immunological response in a mammal that comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established within the individual or not.
• An immunological response in a mammal may also be induced by a method comprises delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases of the invention.
• nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hyb ⁇ d
• a polypeptide or a nucleic acid vector will be normally provided as a vaccine formulation (composition)
• the formulation may further comprise a suitable carrier Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intra-muscular, intravenous, or mtra- dermal injection)
• parenterally for instance, subcutaneous, intra-muscular, intravenous, or mtra- dermal injection
• Formulations suitable for parenteral administration include aqueous and non-aqueous stenle injection solutions that may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes that render the formulation instonic with the blood of the recipient, and aqueous and non-aqueous stenle suspensions that may include suspending agents or thickening agents
• the formulations may be presented in unit-dose or multi-dose containers,
• Polypeptides of the present invention have one or more biological functions that are of relevance in one or more disease states, in particular the diseases of the invention hereinbefore mentioned It is therefore useful to identify compounds that stimulate or inhibit the function or level of the polypeptide Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those that stimulate or inhibit the function or level of the polypeptide Such methods identify agonists or antagonists that may be employed for therapeutic and prophylactic purposes for such diseases of the invention as hereinbefore mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbranes, collections of chemical compounds, and natural product mixtures Such agonists or antagonists so-identified may be natural or modified substrates, ligands.
• Such small molecules preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons It is preferred that these small molecules are organic molecules
• the screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof, by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve measuring or detecting (qualitatively or quantitatively) the competitive binding of a candidate compound to the polypeptide against a labeled competitor (e.g. agonist or antagonist). Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
• a labeled competitor e.g. agonist or antagonist
• the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring a AXOR35 activity in the mixture, and comparing the AXOR35 activity of the mixture to a control mixture which contains no candidate compound.
• Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screening (HTS) formats.
• HTS formats include not only the well-established use of 96- and, more recently, 384-well micotiter plates but also emerging methods such as the nanowell method described by Schullek et al, Anal Biochem., 246, 20-29, (1997).
• Fusion proteins such as those made from Fc portion and AXOR35 polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al., J Mol Recognition, 8:52-58 ( 1995); and K. Johanson et al., J Biol Chem, 270( 16):9459-9471 ( 1995)).
• Histamine or a histamine-like compound is a ligand for
• the AXOR35 polypeptide of the present invention may be employed in a process for screening for compounds which bind to and activate the AXOR35 polypeptides of the present invention (called agonists), or inhibit the interaction of the AXOR35 polypeptides with receptor ligands (called antagonists).
• screening procedures involve providing appropriate cells which express the receptor polypeptide of the present invention on the surface thereof.
• Such cells include cells from mammals, yeast, Drosophila or E. coli.
• a polynucleotide encoding the receptor of the present invention is employed to transfect cells to thereby express the AXOR35 polypeptide. The expressed receptor is then contacted with a test compound to observe binding, stimulation or inhibition of a functional response.
• One such screening procedure involves the use of melanophores which are transfected to express the AXOR35 polypeptide of the present invention.
• a screening technique is described in PCT WO 92/01810, published February 6, 1992.
• Such an assay may be employed to screen for a compound which inhibits activation of the receptor polypeptide of the present invention by contacting the melanophore cells which encode the receptor with both the receptor ligand, such as histamine or a histamine-like compound, and a compound to be screened
• Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, 1 e , inhibits activation of the receptor
• the technique may also be employed for screening of compounds which activate the receptor by contacting such cells with compounds to be screened and determining whether such compound generates a signal, 1 e., activates the receptor
• Other screening techniques include the use of cells which express the AXOR35 polypeptide (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation.
• compounds may be contacted with cells expressing the receptor polypeptide of the present invention
• a second messenger response e g , signal transduction or pH changes
• Another screening technique involves expressing the AXOR35 polypeptide in which the receptor is linked to phosphohpase C or D
• Representative examples of such cells include, but are not limited to, endothelial cells, smooth muscle cells, and embryonic kidney cells
• the screening may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phosphohpase second signal
• Another method involves screening for compounds which are antagonists or agonists by determining inhibition of binding of labeled ligand, such as histamine or a histamine-like compound, to cells which have the receptor on the surface thereof, or cell membranes containing the receptor
• a method involves transfecting a cell (such as eukaryotic cell) with DNA encoding the AXOR35 polypeptide such that the cell expresses the receptor on its surface
• the cell is then contacted with a potential antagonist or agonist in the presence of a labeled form of a ligand, such as histamine or a histamine-like compound
• the ligand can be labeled, e g , by radioactivity
• the amount of labeled ligand bound to the receptors is measured, e g , by measuring radioactivity associated with transfected cells or membrane from these cells If the compound binds to the receptor, the binding of labeled ligand to the receptor is inhibited as determined by a reduction of labeled
• Another screening procedure involves the use of mammalian or amphibian cells (CHO, HEK 293, Xenopus Oocytes, RBL-2H3, etc) which are transfected to express the receptor of interest
• the cells are loaded with an indicator dye that produces a fluorescent signal when bound to calcium, and the cells are contacted with a test substance and a receptor agonist, such as histamine or a histamine-like compound
• a receptor agonist such as histamine or a histamine-like compound
• Any change in fluorescent signal is measured over a defined period of time using, for example, a fluorescence spectrophoto eter or a fluorescence imaging plate reader.
• a change in the fluorescence signal pattern generated by the ligand indicates that a compound is a potential antagonist or agonist for the receptor.
• Another screening procedure involves use of mammalian or amphibian cells (CHO, HEK293, Xenopus Oocytes, RBL-2H3, etc.) which are transfected to express the receptor of interest, and which are also transfected with a reporter gene construct that is coupled to activation of the receptor (for example, luciferase, beta-galactosidase, or beta-lactamase behind an appropriate promoter).
• the cells are contacted with a test substance and the receptor agonist (ligand), such as histamine or a histamine-like compound, and the signal produced by the reporter gene is measured after a defined period of time.
• a suitable substrate for the reporter gene product is added prior to measurement of the signal.
• luciferin could be added to measure luciferase activity, or CCF2/AM could be added to measure beta-lactamase activity (Zlokarnik et al, Science 1998, 279, 84-88).
• the signal can be measured using a luminometer, spectrophotometer, fluorimeter, or other such instrument appropriate for the specific reporter construct used. Change of the signal generated by the ligand indicates that a compound is a potential antagonist or agonist for the receptor.
• Another screening technique for antagonists or agonits involves introducing polynucleotide (RNA or DNA) encoding the AXOR35 polypeptide into Xenopus oocytes (or CHO, HEK 293, RBL-2H3, etc.) to express the receptor.
• the receptor oocytes are then contacted with the receptor ligand, such as histamine or a histamine-like compound, and a compound to be screened.
• Inhibition or activation of the receptor is then determined by detecting a change in a signal, such as, cAMP, calcium, proton, or other ions.
• one method involves screening for AXOR35 polypeptide inhibitors by determining inhibition or stimulation of AXOR35 polypeptide-mediated cAMP and/or adenylate cyclase accumulation or dimunition.
• Such a method involves transiently or stably transfecting a eukaryotic cell with AXOR35 polypeptide receptor to express the receptor on the cell surface. The cell is then exposed to potential antagonists or agonists in the presence of AXOR35 polypeptide ligand, such as histamine or a histamine-like compound.
• the changes in levels of cAMP is then measured over a defined period of time, for example, by radio-immuno or protein binding assays (for example using Flashplates or a scintillation proximity assay). Changes in cAMP levels can also be determined by directly measuring the activity of the enzyme, adenylyl cyclase. in broken cell preparations. If the potential antagonist or agonist binds the receptor, and thus inhibits AXOR35 polypeptide-ligand binding, the levels of AXOR35 polypeptide-mediated cAMP, or adenylate cyclase activity, will be reduced or increased.
• yeast Saccharomyces cerevisiae
• Heterothallic strains of yeast can exist in two mitotically stable haploid mating types, MATa and MATa. Each cell type secretes a small peptide hormone that binds to a G-protein coupled receptor on opposite mating-type cells which triggers a MAP kinase cascade leading to Gl arrest as a prelude to cell fusion.
• Genetic alteration of certain genes in the pheromone response pathway can alter the normal response to pheromone, and heterologous expression and coupling of human G-protein coupled receptors and humanized G-protein subunits in yeast cells devoid of endogenous pheromone receptors can be linked to downstream signaling pathways and reporter genes (e.g., U.S. Patents 5,063,154; 5,482,835; 5,691,188).
• Such genetic alterations include, but are not limited to, (i) deletion of the STE2 or STE3 gene encoding the endogenous G-protein coupled pheromone receptors; (ii) deletion of the FAR1 gene encoding a protein that normally associates with cyclin-dependent kinases leading to cell cycle arrest; and (iii) construction of reporter genes fused to the FUS 1 gene promoter (where FUS 1 encodes a membrane-anchored glycoprotein required for cell fusion).
• Downstream reporter genes can permit either a positive growth selection (e.g., histidine prototrophy using the FUS 1-HIS3 reporter), or a colorimetric, fluorimetric or spectrophotometric readout, depending on the specific reporter construct used (e.g., b-galactosidase induction using a FUS l-LacZ reporter).
• a positive growth selection e.g., histidine prototrophy using the FUS 1-HIS3 reporter
• a colorimetric, fluorimetric or spectrophotometric readout depending on the specific reporter construct used (e.g., b-galactosidase induction using a FUS l-LacZ reporter).
• the yeast cells can be further engineered to express and secrete small peptides from random peptide libraries, some of which can permit autocrine activation of heterologously expressed human (or mammalian) G-protein coupled receptors (Broach, J.R. and Thorner, J. Nature 384: 14-16, 1996; Manfredi et al., Mol. Cell. Biol. 16: 4700-4709, 1996).
• This provides a rapid direct growth selection (e.g, using the FUS 1-HIS3 reporter) for surrogate peptide agonists that activate characterized or orphan receptors.
• yeast cells that functionally express human (or mammalian) G-protein coupled receptors linked to a reporter gene readout can be used as a platform for high-throughput screening of known ligands, fractions of biological extracts and libraries of chemical compounds for either natural or surrogate ligands.
• Functional agonists of sufficient potency can be used as screening tools in yeast cell-based assays for identifying G-protein coupled receptor antagonists. For example, agonists will promote growth of a cell with FUS- HIS3 reporter or give positive readout for a cell with FUSl-LacZ.
• a candidate compound which inhibits growth or negates the positive readout induced by an agonist is an antagonist.
• the yeast system offers advantages over mammalian expression systems due to its ease of utility and null receptor background (lack of endogenous G-protein coupled receptors) which often interferes with the ability to identify agonists or antagonists.
• the present invention also provides a method for identifying new ligands not known to be capable of binding to an AXOR35 polypeptides. The screening assays described above for identifying agonists may be used to identify new ligands.
• the present invention also contemplates agonists and antagonists obtainable from any of the above described screening methods.
• AXOR35 polypeptide receptor antagonists examples include peptidomimetics, synthetic organic molecules, natural products, antibodies, etc. which bind to the receptor but do not elicit a second messenger response such that the activity of the receptor is prevented.
• Potential antagonists also include proteins which are closely related to the ligand of the
• AXOR35 polypeptide receptor i.e. a fragment of the ligand, which have lost biological function, and when they bind to the AXOR35 polypeptide receptor, elicit no response.
• the present invention relates to particular antagonists and further agonists (apart from histamine and histamine-like compounds as defined below) identified by the screening methods described herein as shown in Table I and Table II.
• the compounds above have IC50 range of 0.195 - 0.323 uM.
• the assay for IC50 is provided in Example 9.

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