WO2001043760A2 - Use of ucp3 polynucleotides and polypeptides - Google Patents
Use of ucp3 polynucleotides and polypeptides Download PDFInfo
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- WO2001043760A2 WO2001043760A2 PCT/EP2000/012931 EP0012931W WO0143760A2 WO 2001043760 A2 WO2001043760 A2 WO 2001043760A2 EP 0012931 W EP0012931 W EP 0012931W WO 0143760 A2 WO0143760 A2 WO 0143760A2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
Definitions
- This invention relates to new uses for polynucleotides and polypeptides encoded by them, to their use m therapy and in identifying agonist compounds which are potentially useful m therapy.
- Atherosclerotic plaque rupture and resulting mtracoronary thrombosis are thought to account for most acute coronary syndromes, such as unstable angina, myocardial infarctions as well as many cases of sudden cardiac death. These acute coronary syndromes represent a major cause of overall morbidity and mortality in the developed world.
- the current p ⁇ mary therapy for atherosclerotic disease is aggressive plasma cholesterol lowe ⁇ ng and is dominated by use of the HMG-CoA reductase inhibitors, the statins. Overall, 50% of patients with cardiovascular disease are hypercholesterolaemic.
- the invention relates to new uses of UCP3 polynucleotides and polypeptides disclosed in W098/39432 (SmithKline Beecham Such uses include the treatment of atherosclerosis and hypercholesterolaemia
- the invention relates to methods for identifying UCP3 agonists, and treating conditions associated with UCP3 imbalance with the identified compounds.
- the invention relates to diagnostic assays for detecting, or determining susceptibility to, atherosclerosis or hypercholesterolaemia associated with inapprop ⁇ ate UCP3 activity or levels.
- the present invention relates to the use of a compound selected from:
- UCP3 polypeptides for use m the present invention include isolated polypeptides composing an ammo acid sequence which has at least 95% identity, preferably at least 97-99% identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2.
- Such polypeptides include those composing the ammo acid of SEQ ID NO:2.
- polypeptides for use in the present invention include isolated polypeptides in which the ammo acid sequence has at least 95% identity, preferably at least 97-99% identity, to the ammo acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2.
- Such polypeptides include the polypeptide of SEQ ID NO:2.
- polypeptides for use in the present invention include isolated polypeptides encoded by a polynucleotide composing the sequence contained in SEQ ID NO: 1.
- UCP3 polypeptides for use in the present invention are believed to be members of the mitochondoal uncoupling proteins family of polypeptides.
- Mitochondoal uncoupling proteins are inner mitochondoal membrane proteins whose function is to uncouple mitochondoal respiration from ADP phosphorylation (see Ricquier et al ( 1999) J Intern Med 245(6):637-42 for review).
- the first member of the family mitochondoal uncoupling protein 1 (UCPl; Bouillaud et al (1985) Proc Natl Acad Sci 82(2) P 445-448; Jacobsson et al (1985) J. Biol. Chem. 260(30) pi 6250- 16254), is expressed exclusively in the brown adipocyte. It functions to uncouple mitochondoal respiration by dissipating the mitochondoal proton gradient, normally used to dove ATP synthesis, to produce heat as a consequence of fatty acid oxidation. In rodents brown adipose tissue contobutes to cold adaptation and body weight regulation via non-shiveong thermogenesis and diet-mduced thermogenesis respectively.
- UCP2 uncoupling protem-2
- Fleury et al. Natural Genetics 15, 269, 1997) and Gimeno, et al (Diabetes 46, 900-906, 1997).
- UCP2 shares 59% identity to UCPl at the ammo acid level
- UCP2 is more widely expressed m human tissues predominantly m white adipose tissue, skeletal muscle (a major site of fuel utilisation and thermogenesis) and components of the immune system
- the varying level of expression of UCP2 in mouse strains with differential susceptibility to weight gam is consistent with it playing some role m weight gam potential (Fleury et al. 1997 supra).
- UCP2 maps close to a quantitative trait locus (QTL) on chromosome 7 associated with obesity.
- QTL quantitative trait locus
- Human UCP2 has been mapped to the homologous region of the long arm of chromosome 11 (Bouchard et al., Human Molecular Genetics 6, 1887-1889, 1997; Solanes et al., J.Biol.Chem 272 25433-25436, 1997).
- UCP3 Shortly after the publication of the sequence for UCP2 a third member of the uncoupling protein family was identified and termed UCP3 (W098/39432 (SmithKline Beecham); Boss et al., FEBS lett 408 39-42, 1997; Vidal-Puig et al., Biochem.Biophys.Res.Commun. 235 79-82, 1997) UCP3 is 73% identical to UCP2 and 59% identical to UCPl at the ammo acid level. In contrast to the wide tissue distobution of UCP2, UCP3 mRNA is predominantly expressed in skeletal muscle. Skeletal muscle is an important site for resting metabolic rate and UCP3 levels in skeletal muscle may be a determinant of energy expenditure and metabolic efficiency m Pima
- UCP3 also maps to 1 lql3 and is adjacent to UCP2 to within 100 kb (Gong et al., Biochem.Biophys.Res.Commun. 256 27-32, 1997, Solanes et al., 1997 supra) suggesting that they are evolutionaoly very close
- mice overexpress g human UCP3 m skeletal muscle are hyperphagic, and yet despite their overeating they remain lean (Clapham, JC et al Nature 2000 Jul 27;406(6794):415-8).
- the present invention is based on further studies which has surposmgly shown that UCP3 overexpressmg mice have significantly reduced total cholesterol (whilst plasma toglyceodes and non-esteofied free fatty acid levels remain normal) and significantly reduced fat content m the liver, despite the overeating exhibited by the mice.
- UCP3 activity or "UCP3 polypeptide activity” or "biological activity of UCP3".
- antigemc and immunogenic activities of said UCP3 polypeptides in particular the antigemc and immunogenic activities of the polypeptide ofSEQ ID NO.2.
- anti-atherosclerotic and cholesterol loweong properties of the UCP-3 polypeptides are included m these terms.
- a polypeptide of the present invention exhibits at least one biological activity of UCP3.
- the UCP3 polypeptides for use m the present invention may be in the form of the "mature" protein or may be a part of a larger protein such as a precursor or a fusion protein It is often advantageous to include an additional ammo acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid m puofication such as multiple histidme residues, or an additional sequence for stability duong recombmant production.
- the invention also includes the use of vaoants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another wi h like characteristics.
- Typical such substitutions are among Ala, Val, Leu and He; among Ser and Tl r; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys a id Arg; or aromatic residues Phe and Tyr.
- Particularly preferred are vaoants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination.
- UCP3 polypeptides for use in the present invention can be prepared in any suitable manner.
- Such polypeptides include isolated naturally occurring polypeptides, recombinantly 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 the use of UCP3 polynucleotides.
- Such polynucleotides include isolated polynucleotides composing a nucleotide sequence encoding a polypeptide which has at least 95% identity to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.
- polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred.
- polynucleotides include a polynucleotide composing the nucleotide sequence contained in SEQ ID NO: 1 encoding the polypeptide of SEQ ID NO:2.
- polynucleotides for use in the present invention include isolated polynucleotides composing a nucleotide sequence that has at least 95% identity to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, over the entire coding region.
- polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred.
- polynucleotides for use in the present invention include isolated polynucleotides composing a nucleotide sequence which has at least 95% identity to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.
- polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identiy are more highly preferred, and those with at least 99% identity are most highly preferred.
- Such polynucleotides include a polynucleotide composing the polynucleotide of SEQ ID NO: 1 as well as the polynucleotide of SEQ ED NO: 1.
- the invention also provides for the use of polynucleotides which are complementary to all the above described polynucleotides.
- the nucleotide sequence of SEQ ID NO:l is a cDNA sequence encoding human UCP3 (W098/39432 (SmithKline Beecham); Boss et al., FEBS lett 408 39-42, 1997; Vidal-Puig et al., Biochem.Biophys.Res.Commun. 235 79-82, 1997).
- the nucleotide sequence of SEQ ID NO:l composes a polypeptide encoding sequence (nucleotide 119 to 1137) encoding a polypeptide of 312 ammo acids, the UCP3 polypeptide of SEQ ED NO:2.
- the nucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained m SEQ ID NO: 1 or it may be a sequence other than the one contained in SEQ ID NO: 1 , which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO.2.
- the polypeptide of the SEQ ID NO:2 is the human UCP3 protein (W098/39432 (SmithKline Beecham); Boss et al., FEBS lett 408 39-42, 1997, Vidal-Puig et al., Biochem.Biophys.Res Commun. 235 79-82, 1997).
- the gene encoding the UCP3 polypeptide of SEQ ID NO:2 has been localised to human chromosome l lql3.
- Prefeoed polypeptides and polynucleotides for use in the present invention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides.
- preferred polypeptides and polynucleotides for use in the present invention have at least one UCP3 activity.
- Polynucleotides for use in the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library deoved from mRNA m cells ofhuman skeletal muscle and the cell lines rhabdosarcoma, caski and SHSY 5 Y (Sambrook et al., Molecular Cloning A Laboratory Manual, 2nd Ed., Cold Spong Harbor Laboratory Press, Cold Spong Harbor, N.Y. (1989)). Polynucleotides for use m the invention can also be obtained from natural sources such as genomic DNA hbraoes or can be synthesized using well known and commercially available techniques.
- polypeptide vaoants which compose the ammo acid sequence of SEQ ID NO:2 and in which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid residues are substituted, deleted or added, m any combination.
- Recombmant polypeptides for use the present invention may be prepared by processes well known m the art from genetically engineered host cells composing expression vectors (see for example Davis et al, Basic Methods in Molecular Biology (1986) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spong Harbor Laboratory Press, Cold Spong Harbor, N.Y. (1989)). Cell-free translation systems can also be employed to produce such proteins using RNAs deoved from the DNA constructs of the present invention.
- polynucleotides into host cells can be effected by methods descobed in 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 Spong Harbor Laboratory Press, Cold Spong Harbor, N.Y. (1989).
- Preferred such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, micromjection, catiomc hpid- mediated transfection, electroporation, transduction, scrape loadmg, ballistic mtroduction or infection.
- bacteoal cells such as E coh 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 and HEK 293 cells
- plant cells such as CHO, COS, HeLa, C127, 3T3, BHK and HEK 293 cells.
- a vaoety of expression vectors can be used, for instance, chromosomal, episomal and virus-deoved systems, e.g., vectors deoved from bacteoal plasmids, from bacteoophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors deoved from combinations thereof, such as those deoved from plasmid and bacteoophage genetic elements, such as cosmids and phagemids.
- the expression vectors may contain control regions that regulate as well as engender expression. Generally, any vector which is able to mamtain, propagate or express a polynucleotide to produce a polypeptide m a host may be used.
- the appropoate nucleotide sequence may be inserted into an expression system by any of a vaoety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., Molecular Cloning, A Laboratory Manual (supra).
- Appropoate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the peoplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals
- the UCP3 polypeptides may be advantageously expressed on the surface of the cell for use in screening for agonist compounds.
- the cells may be harvested poor to use m the screening assay.
- the polypeptides may be secreted into the medium, recovered therefrom and then puofied.
- the UCP3 polypeptides may also be expressed mtracellularly, m which case the cells must first be lysed before the polypeptide is recovered and puofied
- UCP3 polypeptides for use in the present invention can be recovered and puofied 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 lectm chromatography. Most preferably, high performance liquid chromatography is employed for puofication. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured duong isolation and or purification.
- This invention also relates to the use of UCP3 polynucleotides as diagnostic reagents for diagnosis of, or determining the susceptibility to, atherosclerosis or hypercholesterolaemia Detection of a mutated form of the gene characteosed by the polynucleotide of SEQ ID NO 1 which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of, or determine the susceptibility to, atherosclerosis or hypercholesterolaemia which results from under-expression or altered expression of the UCP3 gene Individuals carrying mutations m the gene may be detected at the DNA level by a vaoety of techniques.
- Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, uone, saliva, tissue biopsy or autopsy mateoal.
- the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques poor to analysis.
- RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change m size of the amplified product m compaoson to the normal genotype Point mutations can be identified by hybodizing amplified DNA to labeledUCP3 nucleotide sequences
- DNA sequence differences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments m gels, with or without denatuong agents, or by direct DNA sequencing (ee, e.g., Myers et al, Science (1985) 230: 1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (see Cotton et al , Proc Natl Acad Sci USA (1985) 85 4397-4401).
- an array of ohgonucleotides probes composing UCP3 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e g , genetic mutations.
- Array technology methods are well known and have general applicability and can be used to address a vaoety of questions in molecular genetics including gene expression, genetic linkage, and genetic vaoabihty (see for example: M.Chee et al., Science, Vol 274, pp 610- 613 (1996))
- the diagnostic assays offer a process for diagnosing or determining a susceptibility to atherosclerosis or hypercholesterolaemia through detection of mutation in theUCP3 gene by the methods descobed.
- diseases may be diagnosed by methods composing dete ⁇ rnnmg from a sample deoved from a subject an abnormally decreased level of polypeptide or mRNA.
- Decreased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
- the present invention relates to a diagonostic kit which comprises
- a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO 1 , or a fragment thereof ;
- polypeptide of the present invention preferably the polypeptide of SEQ ID NO.2 or a fragment thereof;
- any such kit may compose a substantial component
- Such a kit will be of use in diagnosing or determining suspectabihty to atherosclerosis or hypercholesterolaemia
- polypeptides of the invention or their fragments or analogs thereof, or cells expressing them, can also be used as lmmunogens to produce antibodies lmmunospecific for polypeptides of the present invention.
- lmmunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides
- Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-beaong fragments, analogs or cells to an animal, preferably a non-human animal, using routine protocols.
- any technique which provides antibodies produced by continuous cell line cultures can be used Examples include the hybodoma technique (Kohler, G and Milstem, C , Nature (1975) 256 495- 497), the t ⁇ oma technique, the human B-cell hybodoma technique (Kozboret al , Immunology Today (1983) 4.72) and the EBV-hyb ⁇ doma technique (Cole et al , Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc , 1985).
- antibodies may be employed to isolate or to identify clones expressing the polypeptide or to puofy the polypeptides by affinity chromatography.
- Antibodies agamst polypeptides of the present invention may also be employed to treat atherosclerosis or hypercholesterolaemia
- the present invention relates to genetically engineered soluble fusion proteins composing a polypeptide of the present invention, or a fragment thereof, and vaoous portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
- IgG, IgM, IgA, IgE immunoglobulins of various subclasses
- an lmmunoglobuhn is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region.
- the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa.
- this invention relates to processes for the preparation of these fusion proteins by genetic engmeeong, and to the use thereof for drug screening, diagnosis and therapy.
- a further aspect of the invention also relates to polynucleotides encoding such fusion proteins Examples of fusion protein technology can be found in International Patent Application Nos. W094/29458 and W094/22914
- Another aspect of the invention relates to a method for inducing an immunological response in a mammal which composes inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response to protect said animal from the Diseases hereinbefore mentioned amongst others.
- Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which composes, dehveong a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
- a further aspect of the invention relates to an lmmunological/vaccme formulation (composition) which, when introduced into a mammalian host, induces an immunological response m that mammal to a polypeptide of the present invention wherein the composition composes a polypeptide or polynucleotide of the present invention
- the vaccine formulation may further compose a suitable earner Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or mtradermal injection).
- Formulations suitable for parenteral administration include aqueous and non-aqueous steole injection solutions which may contain anti-oxidants, buffers, bacteoostats and solutes which render the formulation mstonic with the blood of the recipient; and aqueous and non-aqueous steole suspensions which may include suspending agents or thickening agents
- the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-doed condition requiong only the addition of the steole liquid earner immediately poor to use.
- the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-m water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine expeomentation.
- UCP3 polypeptides of use m the present invention are involved m reducing the osk or development of atherosclerosis and minimising hypercholesterolaemia It is therefore desirous to devise screening methods to identify compounds which stimulate the expression or function of the UCP3 polypeptide. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate the function of the polypeptide.
- agonists may be employed for therapeutic and prophylactic purposes for the prevention or treatment of atherosclerosis or hypercholesterolaemia
- Compounds may be identified from a vaoety of sources, for example, cells, cell-free preparations, chemical hbraoes, and natural product mixtures.
- agon ⁇ sts,so- ⁇ dent ⁇ f ⁇ ed 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 l(2):Chapter 5 (1991)).
- the screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes beaong the polypeptide, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve competition with a labeled competitor.
- these screening methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide, using detection systems appropoate to the cells beaong the polypeptide Further, the screening methods may simply compose the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring UCP3 activity in the mixture, and compaong the UCP3 activity of the mixture to a standard.
- the polynucleotides, polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells.
- an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents which may enhance the production of polypeptide (also called agonist) from suitably manipulated cells or tissues.
- the present invention relates to a screening kit for identifying agonists for polypeptides of the present invention; or compounds which enhance the production of such polypeptides, which composes: (a) a polypeptide of the present invention;
- polypeptide of the present invention may also be used in a method for the structure-based design of an agonist of the polypeptide, by:
- the present invention provides methods of treating atherosclerosis or hypercholesterolaemia, related to an under-expression of UCP3 polypeptide activity.
- One approach composes administering to a subject a therapeutically effective amount of a compound which activates a polypeptide of the present invention, i.e., an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition.
- gene therapy may be employed to effect the endogenous production ofUCP3 by the relevant cells in the subject.
- a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above.
- the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest.
- These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo.
- Another approach is to administer a therapeutic amount of a polypeptide of the present invention in combination with a suitable pharmaceutical carrier.
- the prese nt invention provides for pharmaceutical compositions composing a therapeutically effecti ⁇ e amount of a polypeptide, such as the soluble form of a polypeptide of the present invention agonistpeptide or small molecule compound, in combination with a pharmaceutically acceptable earner or excipient.
- a pharmaceutically acceptable earner or excipient include, but are not limited to, saline, buffered salme, dextrose, water, glycerol, ethanol, and combinations thereof.
- the invention further relates to pharmaceutical packs and kits composing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
- Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
- composition will be adapted to the route of administration, for instance by a systemic or an oral route.
- Preferred forms of systemic administration include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or mtrapeotoneal, can be used.
- Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
- oral administration may also be possible. Administration of these compounds may also be topical and or localized, m the form of salves, pastes, gels, and the like.
- the dosage range required depends on the choice of peptide or other compounds of the present invention, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attendmg practitioner. Suitable dosages, however, are in the range of 0.1-100 ⁇ g/kg of subject. Wide vaoations in the needed dosage, however, are to be expected in view of the vaoety of compounds available and the differing efficiencies of vaoous routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection.
- Vaoations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art
- Polypeptides used m treatment can also be generated endogenously m the subject, in treatment modalities often referred to as " gene therapy" as descobed above.
- cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector The cells are then introduced into the subject.
- Antibodies as used herein includes polyclonal and monoclonal antibodies, chimeoc, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobulm expression library.
- Isolated means altered “ by the hand of man” from the natural state If an " isolated” composition or substance occurs m nature, it has been changed or removed from its oog al environment, or both.
- a polynucleotide or a polypeptide naturally present a living animal is not “ isolated,” but the same polynucleotide or polypeptide separated from the coexisting mateoals of its natural state is “ isolated” , as the term is employed herein
- Polynucleotide generally refers to any polyobonucleotide or polydeoxobonucleotide, 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 smgle- and double-stranded regions, hybrid molecules composing 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 composing RNA or DNA or both RNA and DNA.
- polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
- Modified bases include, for example, totylated bases and unusual bases such as mosme. A vaoety 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 characteostic of viruses and cells.
- Polynucleotide also embraces relatively short polynucleotides, often referred to as ohgonucleotides.
- Polypeptide refers to any peptide or protein composing two or more ammo 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 ohgomers, and to longer chains, generally referred to as proteins. Polypeptides may contain 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 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 m 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 contain 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-obosylation, amidation, biotinylation, covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide deovative, covalent attachment of a hpid or hpid deovative, covalent attachment of phosphotidylmositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystme, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, myostoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as argmylation, and ubiquitmation (see
- Vaoant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties.
- a typical vaoant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the vaoant 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 the polypeptide encoded by the reference sequence, as discussed below.
- a typical vaoant of a polypeptide differs ammo acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the vaoant are closely similar overall and, in many regions, identical.
- a vaoant and reference polypeptide may differ m ammo acid sequence by one or more substitutions, additions, deletions in any combination.
- a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
- a vaoant of a polynucleotide or polypeptide may be a naturally occurong such as an allehc vaoant, or it may be a vaoant that is not known to occur naturally.
- Non-naturally occurong vaoants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
- Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by compaong the sequences.
- identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between stongs of such sequences.
- Preferred methods to determine identity are designed to give the largest match between the sequences tested
- Methods to determine identity and similanty are codified in publicly available computer programs
- Preferred computer program methods to determine identity and similanty between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S F. et al., J. Molec.
- the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al , J Mol Biol. 215: 403-410 (1990).
- the well known Smith Waterman algoothm may also be used to determine identity.
- polypeptide sequence compaoson examples include the following- 1) Algoothm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix. BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad. Sci USA 89:10915-10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4
- a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: l, that is be 100% identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence.
- Such 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 in one or more contiguous groups withm the reference sequence.
- the number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the numeocal percent of the respective percent ⁇ dent ⁇ ty(d ⁇ v ⁇ ded by 100) and subtracting that product from said total number of nucleotides in SEQ ID NO.1 , or n n ⁇ x n - (x n • y), wherein n n is the number of nucleotide alterations, x n is the total number of nucleotides in SEQ ID NO: l, and y is, for instance, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etc, and wherein any non-mteger product of x n and y is rounded down to the nearest integer poor to subtracting it from x n .
- Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ED NO:2 may create nonsense, missense or frameshift mutations m this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
- a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2, that is be 100% identical, or it may include up to a certain integer number of ammo acid alterations as compared to the reference sequence such that the % identity is less than 100%.
- Such alterations are selected from the group consisting of at least one amino 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 in one or more contiguous groups withm the reference sequence.
- the number of ammo acid alterations for a given % identity is determined by multiplying the total number of ammo acids m SEQ ID NO:2 by the numeocal percent of the respective percent ⁇ dent ⁇ ty(d ⁇ v ⁇ ded by 100) and then subtracting that product from said total number of amino acids in SEQ ID NO'2, or n a ⁇ x a - (x a * y)» wherein n a is the number of ammo acid alterations, x a is the total number of ammo acids in SEQ ID NO:2, and y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein any non-mteger product of x a and y is rounded down to the nearest integer poor to subtracting it from x a .
- “Homolog” is a geneoc tenrt used m the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a subject sequence. Such relatedness may be quantified by determining the degree of identity and/or similaoty between the sequences being compared as hereinbefore descobed. Falling within this geneoc term are the terms “ortholog”, meaning a polynucleotide or polypeptide that is the functional equivalent of a polynucleotide or polypeptide m another species, and "paralog” meaning a functionally similar sequence when considered within the same species
- Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof
- EP-A-0 464 discloses fusion proteins composing vaoous portions of constant region of immunoglobulm molecules together with another human protein or part thereof.
- employing an immunoglobulm 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].
- mice overexpressmg human UCP-3 in skeletal muscle were used in this study and were housed and maintained in accordance with procedures outlined in the Home Office Animals (Scientific Procedures) Act 1986, UK Mice were housed m groups of 12 (m cages of 3) and maintained on a 12 hour hght/12 hour dark light cycle (lights on at 06:00hr GMT). Animals were allowed free access to food (Teklad 2018 (13.7 KJ/g)) and water.
- Total cholesterol was measured in terminal blood samples spectrophotometocally using a Cobas Mira plus Clinical Chemistry Analyser.
- Table 1 Body weight, 24 hour food consumption and plasma total cholesterol levels in 14 week old male transgenic mice overexpressing UCP3 protein in skeletal muscle.
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Abstract
Description
Claims
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AU26747/01A AU2674701A (en) | 1999-12-17 | 2000-12-18 | New use |
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Publication number | Priority date | Publication date | Assignee | Title |
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WO1998039432A1 (en) * | 1997-03-05 | 1998-09-11 | Smithkline Beecham Plc | Polynucleotides and polypeptides belonging to the uncoupling proteins family |
WO1998045313A1 (en) * | 1997-04-04 | 1998-10-15 | Amylin Pharmaceuticals, Inc. | Novel uncoupling protein and methods of use |
WO2000078941A2 (en) * | 1999-06-23 | 2000-12-28 | University Of Vermont And State Agricultural College | Methods and products for manipulating uncoupling protein expression |
WO2001024625A1 (en) * | 1999-10-01 | 2001-04-12 | Smithkline Beecham P.L.C. | Transgenic rodent comprising a polynucleotide emcoding a human ucp3 polynucleotide |
-
1999
- 1999-12-17 GB GBGB9930100.4A patent/GB9930100D0/en not_active Ceased
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2000
- 2000-12-18 WO PCT/EP2000/012931 patent/WO2001043760A2/en active Application Filing
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WO1998039432A1 (en) * | 1997-03-05 | 1998-09-11 | Smithkline Beecham Plc | Polynucleotides and polypeptides belonging to the uncoupling proteins family |
WO1998045313A1 (en) * | 1997-04-04 | 1998-10-15 | Amylin Pharmaceuticals, Inc. | Novel uncoupling protein and methods of use |
WO2000078941A2 (en) * | 1999-06-23 | 2000-12-28 | University Of Vermont And State Agricultural College | Methods and products for manipulating uncoupling protein expression |
WO2001024625A1 (en) * | 1999-10-01 | 2001-04-12 | Smithkline Beecham P.L.C. | Transgenic rodent comprising a polynucleotide emcoding a human ucp3 polynucleotide |
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