WO2000064943A1 - Acrp30r1m, homologue d'acrp30 - Google Patents

Acrp30r1m, homologue d'acrp30

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Publication number
WO2000064943A1
WO2000064943A1 PCT/US2000/011606 US0011606W WO0064943A1 WO 2000064943 A1 WO2000064943 A1 WO 2000064943A1 US 0011606 W US0011606 W US 0011606W WO 0064943 A1 WO0064943 A1 WO 0064943A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
sequence
polynucleotide
acrp30r1m
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Application number
PCT/US2000/011606
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English (en)
Inventor
Erding Hu
Randall Forrest Smith
Xiaotong Li
Yuan Zhu
Original Assignee
Smithkline Beecham Corporation
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Publication date
Application filed by Smithkline Beecham Corporation filed Critical Smithkline Beecham Corporation
Priority to EP00928590A priority Critical patent/EP1173483A4/fr
Publication of WO2000064943A1 publication Critical patent/WO2000064943A1/fr

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Classifications

    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • ACRP30R1M A HOMOLOG OF ACRP30
  • 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.
  • the present invention relates to Mus musculus ACRP30R1M, m particular, Mus musculus
  • the invention relates to methods for identifying agonists and antagonists/inhibitors of the Mus musculus ACRP30R1M gene.
  • This invention further relates to the generation of in vitro and in vivo compa ⁇ son data relating to the polynucleotides and polypeptides in order to predict oral absorption and pharmacokmetics m man of compounds that either agonize or antagonize the biological activity of such polynucleotides or polypeptides.
  • the present invention further relates to methods for creating transgemc animals, which overexpress or underexpress or have regulatable expression of a ACRP30R1M gene and "knock-out" animals, preferably mice, in which an animal no longer expresses a ACRP30R1M 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 Mus musculus ACRP30R1M gene functions in these animal models is expected to provide an insight into treating and preventing human diseases including, but not limited to: cancers, inflammation, cell death, obesity, diabetes, heart disease, cell proliferation, immunity, and energy metabolism and homeostasis, hereinafter referred to as "the Diseases", amongst others.
  • the present invention relates to Mus musculus ACRP30R1M polypeptides.
  • 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.
  • polypeptides include those comprising the amino acid of SEQ ID NO:2.
  • polypeptides of the present invention are believed to be members of the Complement C 1 q /
  • TNF Tumor Necrosis Factor family of polypeptides. They are, therefore, of interest, because members of this family are known to play a role in role in inflammation, cell proliferation, cell death, immunity, and energy homeostasis processes.
  • ACRP30R1M shows similarity to one member of this family, ACRP30 (Adipocyte Complement-Related Protein of 30 kDa). ACRP30 is expressed exclusively in adipocytes, and its expression is altered in various forms of obesity (Hu, E, Liang, P and
  • ACRP30 secretion is acutely stimulated by insulin (Scherer, PE et al., J Biol. Chem. 270, 26746-26749, 1995) and is repressed by chronically elevated levels of insulin.
  • Hib27 protein from Siberian chipmunks is also thought to be involved in energy homeostasis, as its expression is specifically extinguished du ⁇ ng hibernation (Takamatsu, N et al , Mol Cell Biol 13 1516-1521, 1993) Recently, it has been shown that the three dimensional structure of ACRP30 can be supe ⁇ mposed with that of TNF's, suggesting that these proteins may have a similar function and mode of action (Shapiro, L and Scherer PE, Current Biology 8, 335-338, 1997).
  • ACRP30R1M is p ⁇ ma ⁇ ly expressed in heart
  • the encoded protein of ACRP30R1M may play a role in cancers, inflammation, cell death, obesity, diabetes, heart disease, cell proliferation, immunity, and energy metabolism and homeostasis
  • 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
  • biological activity of ACRP30R1M a polypeptide of the present invention exhibits at least one biological activity of Mus musculus ACRP30R1M.
  • Polypeptides of the present invention also includes va ⁇ ants of the aforementioned polypeptides, including alleles and splice va ⁇ ants Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative.
  • va ⁇ ants are those m 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.
  • Particularly preferred p ⁇ mers will have between 20 and 25 nucleotides.
  • Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an ammo 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 comp ⁇ smg 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
  • biologically active fragments which are those fragments that mediate activities of ACRP30R1M, including those with a similar activity or an improved activity, or with a decreased undesirable activity.
  • fragments that are antigemc or lmmunogenic in an animal especially in a human
  • fragments comp ⁇ smg receptors or domains of enzymes that confer a function essential for viability of Mus musculus or the ability to initiate, or maintain cause the Diseases in 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 va ⁇ ants 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 pu ⁇ fication, for instance, multiple histidme residues, or an additional sequence for stability during recombinant production
  • the present invention also includes va ⁇ ants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative am o acid substitutions, whereby a residue is substituted by another with like characte ⁇ stics 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 va ⁇ ants in which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added m any combination.
  • Polypeptides of the present invention can be prepared m 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 prepa ⁇ ng such polypeptides are well understood in the art.
  • the present invention relates to Mus musculus ACRP30R1M polynucleotides.
  • Such polynucleotides mclude isolated polynucleotides comp ⁇ smg a nucleotide sequence encoding a polypeptide having at least a 95% identity, to the ammo acid sequence of SEQ ID 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.
  • Such polynucleotides m clude a polynucleotide comp ⁇ smg the nucleotide sequence contained m SEQ ID NO:l encodmg the polypeptide of SEQ ID NO:2.
  • polynucleotides of the present invention include isolated polynucleotides comp ⁇ smg a nucleotide sequence having at least a 95% identity, to a nucleotide sequence encodmg a polypeptide of
  • 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.
  • Further polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence having at least a 95% identity, to SEQ ID NO: 1 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: 1 , as well as the polynucleotide of SEQ ID NO: 1.
  • the invention also provides polynucleotides which are complementary to all the above described polynucleotides.
  • the nucleotide sequence of SEQ ID NO: 1 shows homology with a Mus musculus EST sequence, GenBank W41045, from 19.5 dpc total fetus (Marra, M., et al., The WashU-HHMI Mouse
  • the nucleotide sequence of SEQ ID NO: 1 is a cDNA sequence and comprises a polypeptide encoding sequence (nucleotide 1 to 855) encoding a polypeptide of 285 amino acids, the polypeptide of SEQ ID NO:2.
  • the nucleotide sequence encoding the 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:l, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.
  • the polypeptide of the SEQ ID NO:2 is structurally related to other proteins of the Complement Clq / Tumor Necrosis Factor (TNF) family, having homology and/or structural similarity with Rattus norvegicus Collagen Alpha 1 type X (EMBL CAA10518.1; Marks, S.C., et al., 1998) and Human 30 Kd Adipocyte Complement-Related Protein (ACRP30; SwissProt ACR_HUMAN; Maeda,K., et al., Biochem. Biophys. Res. Comm. 221, 286-289, 1996).
  • TNF Tumor Necrosis Factor
  • 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 ACRP30R1M 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 Mus musculus 19.5 dpc total fetus, using the expressed sequence tag (EST) analysis (Adams, M.D., et al. Science (1991) 252:1651- 1656; Adams, M.D.
  • EST expressed sequence tag
  • 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, s ⁇ ch 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 NatlAcad Sci 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, ribosome binding sites and sequences that stabilize mRNA.
  • polynucleotides encoding polypeptide variants that comprise 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 1 to 1 or 1 amino acid residues are substituted, deleted or added, in any combination.
  • Particularly preferred probes will have between 30 and 50 nucleotides, but may have between 100 and 200 contiguous nucleotides of the polynucleotide of SEQ ID 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 855 set forth in SEQ ID NO:l, which encodes a ACRP30R1M polypeptide.
  • the invention also includes a polynucleotide consisting of or comprising a polynucleotide of the formula: X-(R 1 ) rn -(R 2 )-(R 3 )n-Y wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of K ⁇ 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, and R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from 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 Ri 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 500
  • Polynucleotides that are identical, or are substantially identical to a nucleotide sequence of SEQ ID NO- 1 may be used as hyb ⁇ dization probes for cDNA and genomic DNA or as p ⁇ mers 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 (mcludmg genes encoding homologs and orthologs from species other than Mus musculus) that have a high sequence identity to SEQ ID NO.1. Typically these nucleotide sequences are 95% identical to that of the referent.
  • Preferred probes or p ⁇ mers will generally comp ⁇ se 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 p ⁇ mers will have between 20 and 25 nucleotides
  • a polynucleotide encodmg a polypeptide of the present invention may be obtained by a process comp ⁇ smg the steps of screening an appropnate library under stringent hyb ⁇ dization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides in length; and isolating full-length cDNA and genomic clones comp ⁇ smg said polynucleotide sequence.
  • hyb ⁇ dization techniques are well known to the skilled artisan.
  • Preferred stringent hyb ⁇ dization conditions include overnight incubation at 42°C m a solution comp ⁇ sing: 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 m length, obtained by screening an appropnate library under stringent hybndization conditions with a labeled probe having the sequence of SEQ ID NO:l or a fragment thereof, preferably of at least 15 nucleotides.
  • isolated polynucleotides preferably of at least 100 nucleotides m length, obtained by screening an appropnate library under stringent hybndization conditions with a labeled probe having the sequence of SEQ ID NO:l or a fragment thereof, preferably of at least 15 nucleotides.
  • Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comp ⁇ smg expression systems. Accordingly, in a further aspect, the present invention relates to expression systems comp ⁇ smg a polynucleotide or polynucleotides of the present mvenhon, to host cells which are genetically engineered with such expression systems and to the production of polypeptides of the invention by recombinant techniques.
  • RNAs denved from the DNA constructs of the present invention 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
  • Introduction of polynucleotides into host cells can be effected by methods descnbed m many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook, et al., MOLECULAR CLONING. A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
  • Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, catiomc hpid-mediated transfection, electroporation, transduction, scrape loadmg, ballistic introduction or infection.
  • appropnate hosts include bactenal cells, such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subt ⁇ is cells; fungal cells, such as yeast cells and Aspergillus cells; msect 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.
  • bactenal cells such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subt ⁇ is cells
  • fungal cells such as yeast cells and Aspergillus cells
  • msect 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 va ⁇ ety of expression systems can be used, for instance, chromosomal, episomal and virus-denved systems, e g , vectors de ⁇ ved from bactenal plasrmds, from bactenophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculovrruses, papova viruses, such as SV40, vaccmia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retrovrruses, and vectors denved from combinations thereof, such as those de ⁇ ved from plasmid and bactenophage genetic elements, such as cosmids and phagermds.
  • the expression systems may comp ⁇ se control regions that regulate as well as engender expression.
  • 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 nucleotide sequence may be mserted mto an expression system by any of a va ⁇ ety 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 m screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell.
  • the cells may be harvested pnor to use in the screening assay. If the polypeptide is secreted mto the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced mtracellularly, the cells must first be lysed before the polypeptide is recovered.
  • Polypeptides of the present invention can be recovered and pu ⁇ fied from recombinant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, amon or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lect chromatography. Most preferably, high performance liquid chromatography is employed for pu ⁇ fication Well known techniques for refoldmg proteins may be employed to regenerate active conformation when the polypeptide is denatured du ⁇ ng isolation and/or punfication. Mus musculus ACRP30R1M gene products can be expressed m transgenic animals.
  • mice Animals of any species, including, but not limited to: mice, rats, rabbits, guinea pigs, dogs, cats, pigs, micro- pigs, goats, and non-human pnmates, e g , baboons, monkeys, chimpanzees, may be used to generate ACRP30R1M transgenic animals.
  • This invention further relates to a method of producing transgenic animals, preferably Mus musculus, over-expressing ACRP30R1M, which method may comp ⁇ se the introduction of several copies of a segment comp ⁇ smg at least the polynucleotide sequence encoding SEQ ID NO:2 with a suitable promotor mto the cells of a Mus musculus embryo, or the cells of another species, at an early stage.
  • This invention further relates to a method of producing transgenic animals, preferably Mus musculus, under-expressing or regulatably expressing ACRP30R1M, which method may comp ⁇ se the introduction of a weak promoter or a regulatable promoter (eg , art inducible or repressible promoter) respectively, expressibly linked to the polynucleotide sequence of SEQ ID NO: 1 mto the cells of a Mus musculus embryo at an early stage.
  • This mvention also relates to transgenic animals, characte ⁇ zed m that they are obtained by a method, as defined above.
  • Any technique known in the art may be used to introduce a Mus musculus ACRP30R1M transgene mto animals to produce a founder line of animals.
  • Such techniques include, but are not limited to: pronuclear micromjection (U.S. Patent No. 4,873,191); retrovirus mediated gene transfer mto germ lmes (Van der Putten, et al., Proc Natl Acad Set , USA 82: 6148-6152 (1985); gene targeting m embryonic stem cells (Thompson, et al., Cell 56: 313-321 (1989); electropolation of embryos (Lo, Mol Cell Biol.
  • a further aspect of the present mvention involves gene targeting by homologous recombination m embryonic stem cells to produce a transgenic animal with a mutation in a ACRP30R1M gene ("knock-out" mutation).
  • knock-out animals there is lnactivation of the ACRP30R1M gene or altered gene expression, such that the animals are useful to study the function of the ACRP30R1M gene, thus providing animals models of human disease, which are otherwise not readily available through spontaneous, chemical or irradiation mutagenesis.
  • Another aspect of the present invention mvolves the generation of so-called "knock-m” animals m which a portion of a wild-type gene is fused to the cDNA of a heterologous gene.
  • This mvention further relates to a method of producing "knock-out" animals, preferably mice, no longer expressing ACRP30R1M.
  • "knock-out" animals preferably mice, no longer expressing ACRP30R1M.
  • ACRP30R1M cDNA (SEQ ID NO: 1) can be used as a probe to screen suitable hbranes to obtain the munne ACRP30R1M genomic DNA clone.
  • the method used to create a knockout mouse is characte ⁇ zed m that: a suitable mutation is produced m the polynucleotide sequence of the mu ⁇ ne ACRP30R1M genomic clone, which inhibits the expression of a gene encodmg mu ⁇ ne ACRP30R1M, or inhibits the activity of the gene product; said modified mu ⁇ ne ACRP30R1M polynucleotide is introduced mto a homologous segment of munne genomic DNA, combined with an appropnate marker, so as to obtain a labeled sequence compnsmg said modified mu ⁇ ne genomic DNA; said modified mu ⁇ ne genomic DNA compnsmg the modified polynucleotide is transfected m
  • a mutation is generated in a munne ACRP30R1M allele by the introduction of a DNA construct compnsmg DNA of a gene encoding mu ⁇ ne ACRP30R1M, which contains the mutation
  • the mutation is targeted to the allele by way of the DNA construct
  • the DNA of the gene encodmg mu ⁇ ne ACRP30R1M comp ⁇ sed 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 comp ⁇ sed 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 mto cells by, for example, calcium-phosphate DNA co-precipitation It is preferred that a mutation be introduced mto cells using electroporation, microinjection, virus infection, hgand-DNA conjugation, virus-
  • Another aspect of this invention provides for in vitro ACRP30R1M "knock-outs", i e , tissue cultures.
  • Animals of any species, mcludmg, but not limited to: mice, rats, rabbits, guinea pigs, dogs, cats, pigs, micro-pigs, goats, and non-human p ⁇ mates, e g , baboons, monkeys, chimpanzees, may be used to generate in vitro ACRP30R1M "knock-outs”
  • Methods for "knocking out" genes in vitro are desc ⁇ bed m Galh-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 Complement Clq / Tumor Necrosis Factor (TNF). Such animals will be valuable tools to study the functions of a ACRP30R1M gene.
  • Such animal models are expected to provide information about potential toxicological effects m humans of any compounds discovered by an aforementioned screenmg method, among others
  • An understandmg of how a Mus musculus ACRP30R1M gene functions m these animal models is expected to provide an insight mto treating and preventing human diseases mcludmg, but not limited to: cancers, inflammation, cell death, obesity, diabetes, heart disease, cell proliferation, immunity, and energy metabolism and homeostasis
  • Polypeptides of the present mvention are responsible for many biological functions, mcludmg many disease states, m particular the Diseases mentioned herein. It is, therefore, an aspect of the mvention 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, m a further aspect, the present mvention 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 va ⁇ ety of sources, for example, cells, cell-free preparations, chemical hbranes, 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 Cohgan, et al , CURRENT PROTOCOLS IN 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 bea ⁇ ng 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 measunng 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).
  • screening methods may test whether the candidate compound results in a signal generated by an agonist or antagonist of the polypeptide, using detection systems appropnate to cells bea ⁇ ng the polypeptide Antagonists are generally assayed m 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 compnsmg a polypeptide of the present invention, to form a mixture, measunng Mus musculus ACRP30R1M activity m the mixture, and comparing a Mus musculus ACRP30R1M activity of the mixture to a control mixture which contains no candidate compound
  • Polypeptides of the present invention may be employed m 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 desc ⁇ bed by Schullek, et al , Anal Biochem., 246, 20-29, (1997).
  • Fusion proteins such as those made from Fc portion and Mus musculus ACRP30R1M polypeptide, as herein descnbed, can also be used for high-throughput screening assays to identify antagonists of antagonists of the polypeptide of the present mvention (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))
  • 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 ACRP30R1M polypeptide, e.g., a fragment of a ligand, substrate, receptor, enzyme, etc; or small molecules which bind to a ACRP30R1M polypeptide but do not elicit a response, so that an activity of a ACRP30R1M polypeptide is prevented.
  • a ACRP30R1M polypeptide e.g., a fragment of a ligand, substrate, receptor, enzyme, etc
  • small molecules which bind to a ACRP30R1M polypeptide but do not elicit a response, so that an activity of a ACRP30R1M polypeptide is prevented.
  • the present invention relates to a screening kit for identifying agonists, antagonists, inhibitors, ligands, 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 (b) a recombinant cell expressing a polypeptide of the present invention; or (c) a cell membrane expressing a polypeptide of the present invention; which polypeptide is preferably that of SEQ ID NO:2.
  • kits 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 Mus musculus ACRP30R1M polypeptides, polynucleotides, and recombinant materials thereof in selection screens to identify compounds which are neither agonists nor antagonist/inhibitors of Mus musculus ACRP30R1M.
  • 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.
  • the ability to make such a comparison of data will enhance formulation design through the identification of compounds with optimal development characteristics, i.e., high oral bioavailability, UID (once a day) dosing, reduced drug interactions, reduced variability, and reduced food effects, among others.
  • optimal development characteristics i.e., high oral bioavailability, UID (once a day) dosing, reduced drug interactions, reduced variability, and reduced food effects, among others.
  • UID once a day
  • 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 polynucloetide 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 in, for example, improved pharmacokinetic 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 within 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 within 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 amino 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. In general, “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.
  • BESTFIT and GAP may be used to determine the % identity between two polynucleotides and the % identity and the % similarity between two polypeptide sequences.
  • BESTFLT uses the "local homology" algorithm of Smith and Waterman (J. Mol. Biol., 147:195-197, 1981, Advances in Applied Mathematics, 2, 482- 489, 1981) and finds the best single region of similarity between two sequences.
  • BESTFIT is more suited to comparing two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer.
  • GAP aligns two sequences, finding a "maximum similarity", according to the algorithm of Neddleman and Wunsch (J. Mol. Biol., 48, 443-453, 1970). GAP is more suited to comparing sequences that are approximately the same length and an alignment is expected over the entire length.
  • the parameters "Gap Weight” and “Length Weight” used in each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively.
  • % identities and similarities are determined when the two sequences being compared are optimally aligned.
  • the BLOSUM62 amino acid substitution matrix (Henikoff S. and Henikoff 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 amino acid sequences before compa ⁇ son.
  • 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 descnbed
  • a polynucleotide sequence having, for example, at least 95%o 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, mterspersed 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 descnbed.
  • % 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 mclude up to five point mutations per each 100 ammo acids of the reference sequence.
  • Such point mutations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion. These point mutations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids m the reference sequence or in one or more contiguous groups withm the reference sequence.
  • a sequence polypeptide sequence having at least 95% identity to a reference polypeptide sequence up to 5% of the ammo acids of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore descnbed.
  • % identities such as 96%), 97%, 98%, 99%, and 100%).
  • Polynucleotide embodiments further include an isolated polynucleotide comp ⁇ smg a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO.l, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO.l or may include up to a certam 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 m the reference sequence or in one or more contiguous groups withm the reference sequence, and wherein said number of
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides m SEQ ID NO: 1
  • 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-integer product of x n and y is rounded down to the nearest integer pnor 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 compnsmg 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 certam 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 m the reference sequence or m one or more contiguous groups within the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of ammo acids in SEQ ID NO.2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of
  • 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 recombinant method is "isolated” even if it is still present in said organism, which organism may be living or non-living.
  • “Knock-in” 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.
  • RNA splicing occurs when a primary RNA transcript undergoes splicing, generally for the removal of introns, which results in 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 in its embryonic stage. In most cases, the transgenic approach aims at specific modifications of the genome, e.g., by introducing whole transcriptional units into the genome, or by up- or down-regulating pre-existing cellular genes. The targeted character of certain of these procedures sets transgenic technologies apart from experimental methods in which random mutations are conferred to the germline, such as administration of chemical mutagens or treatment with ionizing solution.
  • Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, 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 compnsmg DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions
  • polynucleotide refers to tnple-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, tntylated bases and unusual bases such as mosine. A vanety of modifications may be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as ohgonucleotides.
  • Polypeptide refers to any peptide or protein compnsmg two or more ammo acids jomed 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 ohgomers, and to longer chains, generally referred to as proteins. Polypeptides may compnse ammo acids other than the 20 gene-encoded ammo acids.
  • Polypeptides include ammo acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art.
  • Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chams 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 comp ⁇ se 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-nbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a hpid or lipid denvative, covalent attachment of phosphotidylmositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-lmks, formation of cysteme, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, mynstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins such as argmylation, and ubiquitmation (
  • 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 amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or mserted amino 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 allelic 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.
  • ACRP30R1L mRNAs The expression pattern of ACRP30R1L mRNAs in 8 human tissues (heart, brain, placenta, lung, liver, muscle, kidney and pancreas) was examined by Northern analysis.
  • a probe to ACRP30R1L hybridizes to two mRNAs: one is ⁇ 1.2 kb (kilobases) in size, the other is -2.4 kb.
  • the 1.2 kb mRNA is most abundant in heart; while the 2.4kb mRNA is most abundant in placenta.

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Abstract

L'invention concerne des polypeptides et polynucléotides ACRP30R1M de Mus musculus, ainsi que des procédé de production de ces polypeptides par des techniques de recombinaison. L'invention porte également sur des procédés de criblage de composés agonistes ou antagonistes de ACRP30R1M de Mus musculus. De tels composés sont susceptibles d'être utiles dans le traitement de maladies humaines, y compris, entre autres, le cancer, les inflammations, la mort cellulaire, l'obésité, le diabète, les cardiopathies, la prolifération cellulaire, l'immunité, et le métabolisme et l'homéostasie énergétiques.
PCT/US2000/011606 1999-04-28 2000-04-28 Acrp30r1m, homologue d'acrp30 WO2000064943A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6620909B1 (en) 1999-04-20 2003-09-16 Zymogenetics, Inc. Adipocyte complement related protein homolog zacrp2
WO2003097689A1 (fr) * 2002-05-17 2003-11-27 Serono Genetics Institute S.A. Fragments d'obg3 inhibant la conversion d'obg3 actif en obg3 de plus faible activite et autres compositions pour le traitement de troubles metaboliques

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009386A2 (fr) * 1994-09-21 1996-03-28 The Government Of The United States Of America, Re Variantes alleliques du recepteur 5ht2c de la serotonine

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AU758074B2 (en) * 1998-04-29 2003-03-13 Genesis Research And Development Corporation Limited Polynucleotides isolated from skin cells and methods for their use
JP2003527067A (ja) * 1998-05-21 2003-09-16 スミスクライン ビーチャム コーポレーション Ａｃｒｐ３０（３０ｋｄの脂肪細胞補体関連タンパク質）の相同体ａｃｒｐ３０ｒ１ｌ

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WO1996009386A2 (fr) * 1994-09-21 1996-03-28 The Government Of The United States Of America, Re Variantes alleliques du recepteur 5ht2c de la serotonine

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Title
See also references of EP1173483A4 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6620909B1 (en) 1999-04-20 2003-09-16 Zymogenetics, Inc. Adipocyte complement related protein homolog zacrp2
US6921649B2 (en) 1999-04-20 2005-07-26 Zymogenetics, Inc. Polynucleotide encoding adipocyte complement related protein homolog zacrp2
US7208578B2 (en) 1999-04-20 2007-04-24 Zymogenetics, Inc. Adipocyte complement related protein homolog zacrp2
WO2003097689A1 (fr) * 2002-05-17 2003-11-27 Serono Genetics Institute S.A. Fragments d'obg3 inhibant la conversion d'obg3 actif en obg3 de plus faible activite et autres compositions pour le traitement de troubles metaboliques

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