WO2001040259A2 - Recepteurs de l'orexine-1 de singe - Google Patents

Recepteurs de l'orexine-1 de singe Download PDF

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WO2001040259A2
WO2001040259A2 PCT/US2000/032849 US0032849W WO0140259A2 WO 2001040259 A2 WO2001040259 A2 WO 2001040259A2 US 0032849 W US0032849 W US 0032849W WO 0140259 A2 WO0140259 A2 WO 0140259A2
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Prior art keywords
polypeptide
seq
receptor
sequence
polynucleotide
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PCT/US2000/032849
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WO2001040259A3 (fr
WO2001040259A8 (fr
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Catherine E. Ellis
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Smithkline Beecham Corporation
Smithkline Beecham Plc.
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Publication of WO2001040259A2 publication Critical patent/WO2001040259A2/fr
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Publication of WO2001040259A8 publication Critical patent/WO2001040259A8/fr

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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
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in identifying compounds that may be agonists and/or antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides.
  • G-protems proteins participating in signal transduction pathways that involve G-protems 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-protems or PPG proteins.
  • Some examples of these proteins include the G-protein coupled (GPC) receptors, such as those for adrenergic agents and dopamme (Kobilka, B.K., et al., Proc. Natl Acad.
  • GPC G-protein coupled
  • G-protems themselves, effector proteins, e.g., phospho pase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kmase A and protein kmase C (Simon, M.I., et al., Science, 1991 , 252.802-8).
  • effector proteins e.g., phospho pase C, adenyl cyclase, and phosphodiesterase
  • actuator proteins e.g., protein kmase A and protein kmase 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-protem connects the hormone receptor to adenylate cyclase.
  • G-protem was shown to exchange GTP for bound GDP when activated by a hormone receptor.
  • the GTP-carrymg form then binds to activated adenylate cyclase.
  • Hydrolysis of GTP to GDP catalyzed by the G-protem itself, returns the G-protein to its basal, inactive form.
  • the G-protem 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.
  • 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 ammo acids, connecting at least eight divergent hydrophihc loops.
  • the G-protem 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 calcitonin, adrenergic, endothehn, cAMP, adenosme, musca ⁇ nic, acetylchohne, serotonin, histamme, thrombin, kinm, follicle stimulating hormone, opsms, endothehal differentiation gene-1, rhodopsms, odorant, and cytomegalovirus receptors.
  • G-protem coupled receptors have single conserved cysteme 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 TM1, TM2, TM3, TM4, TM5, TM6, and TM7 TM3 has been implicated m signal transduction.
  • G-protem coupled receptors Most G-protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus
  • G-protem coupled receptors such as the ⁇ -adrenoreceptor, phosphorylation by protein kmase A and/or specific receptor kinases mediates receptor desensitization
  • the hgand binding sites of G-protem coupled receptors are believed to comprise hydrophihc sockets formed by several G-protem coupled receptor transmembrane domains, said sockets being surrounded by hydrophobic residues of the G-protem coupled receptors
  • the hydrophihc side of each G-protein coupled receptor transmembrane helix is postulated to face mward and form a polar hgand binding site TM3 has been implicated in several G-protem coupled receptors as having a hgand binding site, such as the TM3 aspartate residue.
  • TM5 sermes, a TM6 asparagme and TM6 or TM7 phenylalanmes or tyrosmes are also implicated in hgand binding.
  • G-protem coupled receptors can be lntracellularly coupled by heterotrime ⁇ c G-protems to various intracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331). Different G-protem ⁇ -subumts preferentially stimulate particular effectors to modulate various 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- protem coupling of some G-protem coupled receptors. G-protem coupled receptors are found numerous sites within a mammalian host.
  • the present invention relates to Cynomolgous Monkey Orexin 1 Receptor, in particular Cynomolgous Monkey Orexin 1 Receptor polypeptides and Cynomolgous Monkey Orexin 1 Receptor polynucleotides, recombinant materials and methods for their production.
  • the invention relates to methods for identifying agonists and antagonists/inhibitors of the Cynomolgous Monkey Orexin 1 Receptor gene.
  • This invention further relates to the generation of in vitro and in vivo comparison data relating to the polynucleotides and polypeptides m order to predict oral absorption and pharmacokmetics in man of compounds that either agonize or antagonize the biological activity of such polynucleotides or polypeptides.
  • Such a comparison of data will enable the selection of drugs with optimal pharmacokmetics in man, i e., good oral bioavailabihty, blood-bram barrier penetration, plasma half life, and minimum drug interaction.
  • the present invention further relates to methods for creating transgenic animals, which overexpress or underexpress or have regulatable expression of a Orexin 1 Receptor gene and
  • mice in which an animal no longer expresses a Orexin 1 Receptor gene. Furthermore, this invention relates to transgenic and knock-out animals obtained by using these methods. Such animal models are expected to provide valuable insight into the potential pharmacological and toxicological effects in humans of compounds that are discovered by the aforementioned screening methods as well as other methods. An understanding of how a
  • Cynomolgous Monkey Orexin 1 Receptor gene functions in these animal models is expected to provide an insight into treating and preventing human diseases including, but not limited to: infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pam; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; nypertension; urinary retention; osteoporosis; angina pecto ⁇ s; myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, delirium, dementia, and severe mental retardation; and dyskmesias, such as
  • the Diseases Huntmgton's disease or Gilles dela Tourett's syndrome, hereinafter referred to as "the Diseases", amongst others.
  • the present invention relates to Cynomolgous Monkey Orexin 1 Receptor polypeptides.
  • Such polypeptides include isolated polypeptides comprising an amino acid sequence having at least a 95% identity, most preferably at least a 97-99% identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2.
  • Such polypeptides include those comprising the amino acid of SEQ ID NO:2.
  • Polypeptides of the present invention are believed to be members of the 7 Transmembrane Receptor family of polypeptides. They are, therefore, of interest, because one can use this polypeptide to validate the animal model and relate the biological activity to man, and 7TM Receptors, more than any other gene family, are the targets of pharmaceutical intervention Furthermore, the polypeptides of the present invention can be used to establish assays to predict oral absorbtion and pharmacokmetics in man and thus enhance compound and formulation design, among others. These properties, either alone or m the aggregate, are hereinafter referred to as
  • a polypeptide of the present invention exhibits at least one biological activity of Cynomolgous Monkey Orexin 1 Receptor.
  • Polypeptides of the present invention also include variants of the aforementioned polypeptides, including alleles and splice variants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative. 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 amino acids are inserted, substituted, or deleted, in any combination. Particularly preferred primers will have between 20 and 25 nucleotides.
  • Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO.2, or an isolated polypeptide comprising an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids truncated or deleted from the ammo acid sequence of SEQ ID NO:2. Also preferred are biologically active fragments that mediate activities of Orexin 1
  • Receptor including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antige c or lmmunogemc in an animal, especially in a human Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viability of Cynomolgous Monkey or the ability to initiate, or maintain cause the Diseases m an individual, particularly a human
  • Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention.
  • polypeptides of the present invention may be in the form of a "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional ammo acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid in purification, for instance, multiple histid e residues, or an additional sequence for stability during recombmant production.
  • the present invention also includes variants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like characteristics Typical substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added in any combination.
  • Polypeptides of the present invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally occurring polypeptides, recombmantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
  • the present invention relates to Cynomolgous Monkey Orexin 1 Receptor polynucleotides
  • polynucleotides include isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide having at least a 95% identity, to the ammo acid sequence of SEQ ED NO:2, over the entire length of SEQ ID NO.2.
  • polypeptides which have at least a 97% identity are highly preferred, while those with at least a 98-99% identity are more highly preferred, and those with at least a 99% identity are most highly preferred.
  • polynucleotides include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO.l encoding the polypeptide of SEQ ID NO:2.
  • polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence having at least a 95% identity, to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, over the entire coding region.
  • polynucleotides which have at least a 97% identity are highly preferred, while those with at least a 98-99% identity are more highly preferred, and those with at least a 99% identity are most highly preferred.
  • polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence having at least a 95% identity, to SEQ ID NO: l over the entire length of SEQ ID NO: 1.
  • polynucleotides which have at least a 97% identity are highly preferred, while those with at least a 98-99% identify are more highly preferred, and those with at least a 99% identity are most highly preferred
  • Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO:l, as well as the polynucleotide of SEQ ID NO: 1.
  • the invention also provides polynucleotides that are complementary to all the above described polynucleotides
  • the nucleotide sequence of SEQ ID NO: 1 shows homology with human Orexin Receptor- 1
  • the nucleotide sequence of SEQ ID NO: 1 is a cDNA sequence and comprises a polypeptide encoding sequence (nucleotide 1 to 1275) encoding the polypeptide set forth in SEQ ED NO:2.
  • the nucleotide sequence encoding the polypeptide of SEQ ED NO:2 may be identical to the polypeptide encoding sequence of SEQ ED NO:l or it may be a sequence other than SEQ ED NO: l, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ED NO:2.
  • the polypeptide of SEQ ED NO:2 is structurally related to other proteins of the 7 Transmembrane Receptor family, having homology and/or structural similarity with human Orexin Receptor-1 (AF041243).
  • Preferred polypeptides and polynucleotides of the present invention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one Orexin 1 Receptor 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 Cynomolgous Monkey brain, using the expressed sequence tag (EST) analysis (Adams, M.D., et al Science (1991) 252: 1651-1656; Adams, M.D. et al, Nature (1992) 355:632-634; Adams, M.D., et al , Nature (1995) 377 Supp.: 3-174). 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.
  • EST expressed sequence tag
  • the polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide m 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 Set USA (1989) 86:821- 824, or is an HA tag.
  • the polynucleotide may also comprise non-coding 5" and 3 ' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ⁇ bosome binding sites and sequences that stabilize mRNA.
  • polynucleotides encoding polypeptide variants that comprise the ammo 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 1 to 1 or 1 ammo acid residues are substituted, deleted or added, in any combination.
  • Particularly preferred probes will have betw en 30 and 50 nucleotides, but may have between 100 and 200 contiguous nucleotides of the polynucleotide of SEQ ED NO: 1.
  • a preferred embodiment of the invention is a polynucleotide of consisting of or comprising nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 1278 set forth m SEQ ED NO:l, both of which encode a Orexin 1 Receptor polypeptide.
  • the invention also includes a polynucleotide consisting of or comprising a polynucleotide of the formula:
  • R 1 X-(R 1 ) m -(R 2 )-(R 3 ) n -Y
  • X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond
  • Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond
  • each occurrence of R j and R3 is independently any nucleic acid residue or modified nucleic acid residue
  • m is an integer between 1 and 3000 or zero
  • n is an integer between 1 and 3000 or zero
  • R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly the nucleic acid sequence set forth in SEQ ID NO: 1 or a modified nucleic acid sequence thereof.
  • R 2 is oriented so that its 5' end nucleic acid residue is at the left, bound to R and its 3' end nucleic acid residue is at the right, bound to R3.
  • Any stretch of nucleic acid residues denoted by either R j and/or R 2 , where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer
  • the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary
  • m and/or n is an integer between 1 and 1000.
  • Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or
  • Polynucleotides that are identical, or are substantially identical to a nucleotide sequence of SEQ ED NO.1, may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification (PCR) reaction, 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 homologs and orthologs from species other than Cynomolgous
  • probes or primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides, and may even have at least 100 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 an appropriate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ED NO: 1 or a fragment thereof, preferably of at least 15 nucleotides in length; and isolating full-length cDNA and genomic clones comprising said polynucleotide sequence.
  • a labeled probe having the sequence of SEQ ED NO: 1 or a fragment thereof, preferably of at least 15 nucleotides in length; and isolating full-length cDNA and genomic clones comprising said polynucleotide sequence.
  • Preferred stringent hybridization conditions include overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in O.lx SSC at about 65°C
  • the present invention also includes isolated polynucleotides, preferably of at least 100 nucleotides in length, obtained by screening an appropriate 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.
  • an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is cut short at the 5' end of the cDNA. 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 1st strand cDNA synthesis.
  • PCR reaction is then repeated using 'nested' primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence).
  • primers designed to anneal within the amplified product typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence.
  • the products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.
  • Recombmant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising 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 recombmant techniques.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs derived 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.
  • Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY ( 1986) and
  • Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • bacterial cells such as streptococci, staphylococci, E colt, 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
  • plant cells A great variety of expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e g , vectors derived from bacterial plasmids, from bacte ⁇ ophage, 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
  • 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 appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook, et al, MOLECULAR CLONING, A LABORATORY MANUAL (supra).
  • 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 recombmant 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 lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification.
  • Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification
  • the polynucleotide sequences of the present invention are also valuable for chromosome localization studies
  • the polynucleotide sequence, or fragment(s) thereof, is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome
  • the mapping of these sequences to human chromosomes according to the present invention is an important first step in correlating homologous human polynucleotide sequences with gene associated disease in humans
  • RH Radiation Hybrid
  • PCR reactions are performed using primers, designed to the polynucleotide sequence of interest, on the RH DNAs of the panel.
  • Each of these DNAs contains random genomic fragments from the species of interest.
  • These PCRs result in a number of scores, one for each RH DNA m the panel, indicating the presence or absence of the PCR product of the polynucleotide sequence of interest.
  • These scores are compared with scores created using PCR products from genomic sequences of known location, usually using an on-line resource such as that available at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, USA website (http://www.genome.wi.mit.edu/).
  • a polynucleotide sequence Once a polynucleotide 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 for that species. Also, as a consequence of synteny, where knowledge of the position of a gene on a chromosome of one species can be used to determine the likely position of the orthologous gene on the chromosome of another species, this knowledge can then be used to identify candidate genes for human disease. Thus the localization of a polynucleotide sequence of interest to a specific mouse chromosomal location can be used to predict the localization of the oithologous human gene on the corresponding human chromosome.
  • potential disease association may be inferred from genetic map sources such as, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library).
  • genetic map sources such as, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library).
  • the relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (co- mhe ⁇ tance of physically adjacent genes).
  • Cynomolgous Monkey Orexin 1 Receptor gene products can be expressed in transgenic animals.
  • Animals of any species including, but not limited to: mice, rats, rabbits, guinea pigs, dogs, cats, pigs, micro-pigs, goats, and non-human primates, e g , baboons, monkeys, chimpanzees, may be used to generate Orexin 1 Receptor transgenic animals.
  • This invention further relates to a method of producing transgenic animals, preferably Cynomolgous Monkey, over-expressing Orexin 1 Receptor, which method may comprise the introduction of several copies of a segment comprising at least the polynucleotide sequence encoding SEQ ID NO:2 with a suitable promoter into the cells of a Cynomolgous Monkey embryo, or the cells of another species, at an early stage
  • This invention further relates to a method of producing transgenic animals, preferably Cynomolgous Monkey, under-expressing or regulatably expressing Orexin 1 Receptor, which method may comprise the introduction of a weak promoter or a regulatable promoter (e g., an mducible or repressible promoter) respectively, expressibly linked to the polynucleotide sequence of SEQ ED NO:l into the cells of a Cynomolgous Monkey embryo at an early stage.
  • a weak promoter or a regulatable promoter e g., an mducible or repressible promoter
  • This invention also relates to transgenic animals, characterized in that they are obtained by a method, as defined above. Any technique known m the art may be used to introduce a Cynomolgous Monkey Orexin 1
  • Receptor transgene into animals to produce a founder line of animals include, but are not limited to- pronuclear micromjection (U.S Patent No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten, et al , Proc Natl Acad Set , USA 82. 6148-6152 (1985); gene targeting in embryonic stem cells (Thompson, et al, Cell 56: 313-321 (1989); electropolation of embryos (Lo, Mol. Cell Biol. 3: 1803-1814 (1983); and sperm-mediated gene transfer (Lavitrano, et al, Cell 57: 717-723 (1989); etc.
  • pronuclear micromjection U.S Patent No. 4,873,191
  • retrovirus mediated gene transfer into germ lines Van der Putten, et al , Proc Natl Acad Set , USA 82. 6148-6152 (1985)
  • gene targeting in embryonic stem cells Thompson, e
  • a further aspect of the present invention involves gene targeting by homologous recombination in embryonic stem cells to produce a transgenic animal with a mutation in a Orexin 1 Receptor gene ("knock-out" mutation).
  • knock-out mutation in a Orexin 1 Receptor gene
  • Another aspect of the present invention involves the generation of so-called "knock-in" animals in which a portion of a wild-type gene is fused to the cDNA of a heterologous gene.
  • This invention further relates to a method of producing "knock-out" animals, preferably mice, no longer expressing Orexin 1 Receptor.
  • a Cynomolgous Monkey Orexin 1 Receptor cDNA SEQ ID NO: 1 can be used as a probe to screen suitable libraries to obtain the mu ⁇ ne Orexin 1 Receptor genomic DNA clone.
  • the method used to create a knockout mouse is characterized in that: a suitable mutation is produced m the polynucleotide sequence of the mu ⁇ ne Orexin 1 Receptor genomic clone, which inhibits the expression of a gene encoding mu ⁇ ne Orexin 1 Receptor, or inhibits the activity of the gene product; said modified mu ⁇ ne Orexin 1 Receptor polynucleotide is introduced into a homologous segment of murme genomic DNA, combined with an appropriate marker, so as to obtain a labeled sequence comprising said modified murme genomic DNA; said modified murme genomic DNA comprising the modified polynucleotide is transfected into embryonic stem cells and correctly targeted events selected in vitro; then said stem cells are remjected into a mouse embryo; then said embryo is implanted into a female recipient and brought to term as a chimera which transmits said mutation through the germhne; and homozygous recombmant mice are obtained at the
  • a mutation is generated in a murine Orexin 1 Receptor allele by the introduction of a DNA construct comprising DNA of a gene encoding murine Orexin 1 Receptor, which murine gene contains the mutation.
  • the mutation is targeted to the allele by way of the DNA construct.
  • the DNA of the gene encoding murine Orexin 1 Receptor comprised in the construct may be foreign to the species of which the recipient is a member, may be native to the species and foreign only to the individual recipient, may be a construct comprised of synthetic or natural genetic components, or a mixture of these.
  • the mutation may constitute an insertion, deletion, substitution, or combination thereof.
  • the DNA construct can be introduced into cells by, for example, calcium-phosphate DNA co-precipitation. It is preferred that a mutation be introduced into cells using electroporation, microinjection, virus infection, hgand-DNA conjugation, virus- hgand-DNA conjugation, or hposomes.
  • Another embodiment of the instant invention relates to "knock-out" animals, preferably mice, obtained by a method of producing recombmant mice as defined above, among others.
  • Another aspect of this invention provides for in vitro Orexin 1 Receptor "knock-outs", i e., tissue cultures. Animals of any species, including, but not limited to: mice, rats, rabbits, guinea pigs, dogs, cats, pigs, micro-pigs, goats, and non-human primates, e g , baboons, monkeys, chimpanzees, may be used to generate in vitro Orexin 1 Receptor "knock-outs". Methods for "knocking out" genes in vitro are described m Galli-Tahadoros, et al , Journal of Immunological Methods 181 : 1-15 (1995).
  • Transgenic, "knock-in”, and “knock-out” animals are a particularly advantageous model, from a physiological point of view, for studying the 7 Transmembrane Receptor. Such animals will be valuable tools to study the functions of a Orexin 1 Receptor gene. Moreover, such animal models are expected to provide information about potential toxicological effects in humans of any compounds discovered by an aforementioned screening method, among others.
  • Polypeptides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases mentioned herein. It is, therefore, an aspect of the invention to devise screening methods to identify compounds that stimulate (agonists) or that inhibit (antagonists) the function of the polypeptide, such as agonists, antagonists and inhibitors. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those that stimulate or inhibit the function of the polypeptide. In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for the Diseases mentioned herein mentioned.
  • Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures
  • Such agonists and antagonists so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc , as the case may be, of the polypeptide; or may be structural or functional mimetics thereof (see Coligan, et al , CU_RRENT PROTOCOLS T IMMUNOLOGY 1(2). Chapter 5 (1991)).
  • 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
  • a screening method may involve measuring or, qualitatively or quantitatively, detecting the competition of binding of a candidate compound to the polypeptide with a labeled competitor (e g , agonist or antagonist). Further, screening methods may test whether the candidate compound results in a signal generated by an agonist or antagonist of the polypeptide, using detection systems appropriate to cells bearing the polypeptide.
  • Antagonists are generally assayed in the presence of a known agonist and an effect on activation by the agonist by the presence of the candidate compound is observed Further, screening methods may simply comprise the steps of mixing a candidate compound with a solution comprising a polypeptide of the present invention, to form a mixture, measuring Cynomolgous Monkey Orexin 1 Receptor activity m the mixture, and comparing a Cynomolgous Monkey Orexin 1 Receptor 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 m high-throughput screening (HTS) formats.
  • HTS formats include not only the well-established use of 96- and, more recently, 384-well microtiter 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 Cynomolgous Monkey Orexin 1 Receptor polypeptide, as herein described, can also be used for high-throughput screening assays to identify antagonists of antagonists of 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)).
  • One screening technique includes the use of cells which express the receptor of this invention (for example, transfected CHO cells) m a system which measures extracellular pH or intracellular calcium 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, pH changes, or changes m calcium level, is then measured to determine whether the potential compound activates or inhibits the receptor.
  • Another method involves screening for receptor inhibitors by determining inhibition or stimulation of receptor-mediated cAMP and/or adenylate cyclase accumulation.
  • Such a method involves transfectmg a eukaryotic cell with the receptor of this invention to express the receptor on the cell surface. The cell is then exposed to potential antagonists in the presence of the receptor of this invention. The amount of cAMP accumulation is then measured. If the potential antagonist binds the receptor, and thus inhibits receptor binding, the levels of receptor-mediated cAMP, or adenylate cyclase, activity will be reduced or increased.
  • Another method for detecting agonists or antagonists for the receptor of the present invention is the yeast based technology as described in U.S. Patent No. 5,482,835.
  • Examples of potential polypeptide antagonists include antibodies or, in some cases, ohgopeptides or proteins that are closely related to hgands, substrates, receptors, enzymes, etc., as the case may be, of a Orexin 1 Receptor polypeptide, e.g., a fragment of a hgand, substrate, receptor, enzyme, etc.; or small molecules which bind to a Orexin 1 Receptor polypeptide but do not elicit a response, so that an activity of a Orexin 1 Receptor polypeptide is prevented.
  • Orexin 1 Receptor polypeptide e.g., a fragment of a hgand, substrate, receptor, enzyme, etc.
  • small molecules which bind to a Orexin 1 Receptor polypeptide but do not elicit a response, so that an activity of a Orexin 1 Receptor polypeptide is prevented.
  • the present invention relates to a screening kit for identifying agonists, antagonists, inhibitors, hgands, receptors, substrates, enzymes, etc. for polypeptides of the present invention; or compounds which decrease or enhance the production of such polypeptides, which compounds comprise a member selected from the group consisting of:
  • polypeptide of the present invention (c) a cell membrane expressing a polypeptide of the present invention; which polypeptide is preferably that of SEQ ID NO:2.
  • any such kit, (a), (b) or (c) may comprise a substantial component.
  • polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by:
  • the present invention relates to the use of Cynomolgous Monkey Orexin 1 Receptor polypeptides, polynucleotides, and recombmant materials thereof m selection screens to identify compounds which are neither agonists nor antagonist/inhibitors of Cynomolgous Monkey Orexin 1 Receptor.
  • the data from such a selection screen is expected to provide in vitro and in vivo comparisons and to predict oral absorption, pharmacokmetics in humans.
  • Allele refers to one or more alternative forms of a gene occurring at a given locus in the genome.
  • “Fragment” of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide. “Fragment” of a polynucleotide sequence refers to a polynucleotide sequence that is shorter than the reference sequence of SEQ ID NO: 1.
  • Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof.
  • EP-A-0 464 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting m, for example, improved pharmacokmetic properties [see, e.g., EP-A 0232 262].
  • “Homolog” is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence. Such relatedness may be quantified by determining the degree of identity and/or similarity between the two sequences as hereinbefore defined. Falling withm this generic term are the terms, "ortholog", and “paralog”. "Ortholog” refers to polynucleotides/genes or polypeptide that are homologs via speciation, that is closely related and assumed to have commend descent based on structural and functional considerations. "Paralog” refers to polynucleotides/genes or polypeptide that are homologs via gene duplication, for instance, duplicated variants withm a genome.
  • Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences.
  • identity refers to an exact nucleotide to nucleotide or ammo acid to amino acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared.
  • a "% identity” may be determined.
  • the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment.
  • a % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length. "Similarity" is a further, more sophisticated measure of the relationship between two polypeptide sequences.
  • similarity means a comparison between the amino acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact correspondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact correspondence, whether, on an evolutionary basis, one residue is a likely substitute for the other This likelihood has an associated 'score' from which the "% similarity" of the two sequences can then be determined
  • the BLOSUM62 ammo acid substitution matrix (Hemkoff S and Hemkoff J G , Proc Nat Acad Sci USA, 89 10915-10919 (1992)) is used in polypeptide sequence comparisons including where nucleotide sequences are first translated into ammo acid sequences before comparison
  • the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a polynucleotide or a polypeptide sequence of the present invention, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore described.
  • a polynucleotide sequence having, for example, at least 95% identity to a reference polynucleotide sequence is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference sequence.
  • Such point mutations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion.
  • point mutations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups withm the reference sequence.
  • a polynucleotide sequence having at least 95% identity to a reference polynucleotide sequence up to 5% of the nucleotides of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described.
  • % identities such as 96%, 97%, 98%, 99% and 100%.
  • a polypeptide sequence having, for example, at least 95% identity to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include up to five point mutations per each 100 amino acids of the reference sequence
  • Such point mutations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion. These point mutations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups withm the reference sequence.
  • sequence polypeptide sequence having at least 95% identity to a reference polypeptide sequence up to 5% of the ammo acids of the m the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described.
  • % identities such as 96%, 97%, 98%, 99%, and 100%.
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ED NO: 1, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ED NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5 ' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or m one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides m SEQ ED NO: l by the integer defining the percent identity divided
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ED NO: l
  • y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-mteger product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
  • Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO:2 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of ammo acids m SEQ ED NO:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in S
  • n a is the number of ammo acid alterations
  • x a is the total number of ammo acids in SEQ ID NO:2
  • y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-mteger product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • isolated means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombmant method is "isolated” even if it is still present m said organism, which organism may be living or non-living.
  • “Knock-” refers to the fusion of a portion of a wild-type gene to the cDNA of a heterologous gene
  • “Knock-out” refers to partial or complete suppression of the expression of a protein encoded by an endogenous DNA sequence in a cell
  • the “knock-out” can be affected by targeted deletion of the whole or part of a gene encoding a protein, in an embryonic stem cell As a result, the deletion may prevent or reduce the expression of the protein in any cell in the whole animal in which it is normally expressed.
  • “Splice Variant” as used herein refers to cDNA molecules produced from RNA molecules initially transcribed from the same genomic DNA sequence but which have undergone alternative RNA splicing.
  • RNA splicing occurs when a primary RNA transcript undergoes splicing, generally for the removal of mtrons, which results m the production of more than one mRNA molecule each of that may encode different amino acid sequences
  • the term splice variant also refers to the proteins encoded by the above cDNA molecules
  • Transgenic animal refers to an animal to which exogenous DNA has been introduced while the animal is still its embryonic stage
  • the transgenic approach aims at specific modifications of the genome, e g , by introducing whole transc ⁇ ptional units into the genome, or by up- or down-regulatmg pre-existing cellular genes
  • the targeted character of certain of these procedures sets transgenic technologies apart from experimental methods m which random mutations are conferred to the germlme, such as administration of chemical mutagens or treatment with ionizing solution.
  • Polynucleotide generally refers to any polyribonucleotide or polydeox ⁇ bonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA
  • polynucleotide also includes DNAs or RNAs comprising one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosme A variety of modifications may be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as ohgonucleotides.
  • Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i e , peptide isosteres.
  • Polypeptide refers to both short chains, commonly referred to as peptides, ohgopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may comprise amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may comprise many types of modifications.
  • Polypeptides may be branched as a result of ubiquitmation, and they may be cyclic, with or without branching Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP- ⁇ bosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a hpid or hpid derivative, covalent attachment of phosphotidylmositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins such as argmylation, and ubiquitination (see, for instance, PROTEINS
  • Variant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations m the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs m ammo acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted ammo acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally.
  • Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
  • All publications including, but not limited to, patents and patent applications, cited in this specification or to which this patent application claims priority, are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.
  • the receptors of the present invention are expressed in either human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells.
  • HEK293 human embryonic kidney 293
  • adherent dhfr CHO cells typically all 5' and 3' untranslated regions (UTRs) are removed from the receptor cDNA prior to insertion into a pCDN or pCDNA3 vector.
  • the cells are transfected with individual receptor cDNAs by hpofectin and selected in the presence of 400 mg/ml G418. After 3 weeks of selection, individual clones are picked and expanded for further analysis.
  • HEK293 or CHO cells transfected with the vector alone serve as negative controls.
  • To isolate cell lines stably expressing the individual receptors about 24 clones are typically selected and analyzed by Northern blot analysis. Receptor mRNAs are generally detectable in about 50% of the G418-res ⁇ stant clones analyzed.
  • Example 2 Ligand bank for binding and functional assays.
  • a bank of over 600 putative receptor hgands has been assembled for screening.
  • the bank comprises: transmitters, hormones and chemokmes known to act via a human seven transmembrane (7TM) receptor; naturally occurring compounds which may be putative agonists for a human 7TM receptor, non-mammalian, biologically active peptides for which a mammalian counterpart has not yet been identified; and compounds not found in nature, but which activate 7TM receptors with unknown natural hgands.
  • This bank is used to initially screen the receptor for known hgands, using both functional (i.e. calcium, cAMP, microphysiometer, oocyte electrophysiology, etc, see below) as well as binding assays.
  • Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format.
  • the purified ligand for a receptor is radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies. A determination is then made that the process of radiolabehng does not dim ish the activity of the ligand towards its receptor.
  • Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell receptor sources.
  • specific receptor binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding.
  • RNA transcripts from linearized plasmid templates encoding the receptor cDNAs of the invention are synthesized in vitro with RNA polymerases in accordance with standard procedures
  • In vitro transcripts are suspended in water at a final concentration of 0.2 mg/ml.
  • Ovarian lobes are removed from adult female toads, Stage V defolhculated oocytes are obtained, and RNA transcripts (10 ng/oocyte) are injected m a 50 nl bolus using a micromjection apparatus.
  • Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes m response to agonist exposure. Recordings are made in Ca2+ free Barth's medium at room temperature.
  • the Xenopus system can be used to screen known hgands and tissue/cell extracts for activating hgands.
  • Example 5 Microphysiomet ⁇ c Assays Activation of a wide variety of secondary messenger systems results in extrusion of small amounts of acid from a cell.
  • the acid formed is largely as a result of the increased metabolic activity required to fuel the intracellular signaling process.
  • the pH changes in the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, CA).
  • the CYTOSENSOR is thus capable of detecting the activation of a receptor which is coupled to an energy utilizing intracellular signaling pathway such as the G-protem coupled receptor of the present invention.
  • the 7TM receptor of the invention is also functionally screened (using calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) against tissue extracts to identify natural hgands. Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated and identified.
  • HEK 293 cells which are expressed in HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and/or cAMP stimulation or inhibition.
  • Basal calcium levels in the HEK 293 cells in receptor-transfected or vector control cells were observed to be m the normal, 100 nM to 200 nM, range.
  • HEK 293 cells expressing recombmant receptors are loaded with fura 2 and m a single day > 150 selected hgands or tissue/cell extracts are evaluated for agonist induced calcium mobilization.
  • HEK 293 cells expressing recombmant receptors are evaluated for the stimulation or inhibition of cAMP production using standard cAMP quantitation assays. Agonists presenting a calcium transient or cAMP fluctuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor.

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Abstract

L'invention concerne des polypeptides et des polynucléotides du récepteur d'orexine-1 de singe Cynomolgus, ainsi que des procédés de production desdits polypeptides par des techniques de recombinaison. L'invention concerne également des méthodes de criblage de composés agonistes ou antagonistes du récepteur d'orexine-1 de singe Cynomolgus. Ces composés sont susceptibles d'être utiles dans le traitement de maladies humaines comprenant notamment, mais pas exclusivement: des infections telles que des infections bactériennes, fongiques, protozoaires et virales, en particulier des infections provoquées par le VIH-1 ou le VIH-2; des douleurs; des cancers; le diabète, l'obésité; l'anorexie; la boulimie; l'asthme; la maladie de Parkinson; l'insuffisance cardiaque aiguë; l'hypotension; l'hypertension; la rétention d'urine; l'ostéoporose; l'angor; l'infarctus du myocarde; l'attaque; les ulcères; l'asthme; les allergies; l'hypertrophie bénigne de la prostate; les migraines; les vomissements; les troubles psychotiques et neurologiques, y compris l'anxiété, la schizophrénie, les troubles maniaco-dépressifs, le délire, la démence et le retard mental grave; et des dyskinésies telles que la maladie de Huntington ou le syndrome de Gilles de la Tourette.
PCT/US2000/032849 1999-12-02 2000-12-04 Recepteurs de l'orexine-1 de singe WO2001040259A2 (fr)

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US7763638B2 (en) 2004-03-01 2010-07-27 Actelion Pharmaceuticals Ltd. Substituted 1,2,3,4-tetrahydroisoquinoline derivatives

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US6020157A (en) * 1997-04-30 2000-02-01 Smithkline Beecham Corporation Polynucleotides encoding HFGAN72X receptor

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US6020157A (en) * 1997-04-30 2000-02-01 Smithkline Beecham Corporation Polynucleotides encoding HFGAN72X receptor

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SAKURAI T. ET AL.: 'Orexins and Orexin Receptors: A Family of Hypothalamic Neuropeptides and G Protein-Coupled Receptors that Regulate Feedings Behavior' CELL vol. 92, 20 February 1998, pages 573 - 585, XP002943277 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7763638B2 (en) 2004-03-01 2010-07-27 Actelion Pharmaceuticals Ltd. Substituted 1,2,3,4-tetrahydroisoquinoline derivatives

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