WO2002007754A2

WO2002007754A2 - New use

Info

Publication number: WO2002007754A2
Application number: PCT/GB2001/003341
Authority: WO
Inventors: John Clapham
Original assignee: SmithKline Beecham Ltd
Current assignee: SmithKline Beecham Ltd
Priority date: 2000-07-25
Filing date: 2001-07-25
Publication date: 2002-01-31
Anticipated expiration: 2003-01-25
Also published as: WO2002007754A3; AU2002224528A1; GB0018248D0

Abstract

The use of UCP3 polypeptides and polynucleotides in the design of protocols for the treatment of obesity, diabetes or body weight disorder, among others.

Description

New Use

Field of the Invention

This invention relates to new uses for mitochondrial uncoupling protein 3 (UCP3) polynucleotides and polypeptides encoded by them, to their use in therapy and in identifying compounds which may be agonists which are potentially useful in therapy.

Background of the Invention

Mitochondrial uncoupling proteins (UCPs) are inner mitochondrial membrane proteins whose function is to uncouple mitochondrial respiration from ADP phosphorylation (see Ricquier et al (1999) J Intern Med 245(6):637-42 for review).

The first member of the family, mitochondrial uncoupling protein 1 (UCP1; Bouillaud et al (1985) Proc Natl Acad Sci 82(2) p445-448; Jacobsson et al (1985) J. Biol. Chem. 260(30) pl6250-16254), is expressed exclusively in the brown adipocyte. It functions to uncouple mitochondrial respiration by dissipating the mitochondrial proton gradient, normally used to drive ATP synthesis, to produce heat as a consequence of fatty acid oxidation. In rodents brown adipose tissue contributes to cold adaptation and body weight regulation via non-shivering thermogenesis and diet-induced thermogenesis respectively. However, since little brown adipose tissue (BAT) is present in adult humans, UCP1 is unlikely to play a major role in either of these important homeostatic functions and although many rodentian tissues display a mitochondrial proton leaks that may subserve these functions the precise molecular mechanism by which these leaks occur are not known. The recent discovery of uncoupling protein homologues with wider tissue distribution in both animals and humans may provide some insight into non-shivering and diet-indiced thermogenesis in humans.

The second member of the uncoupling protein family, uncoupling protein-2, (UCP2)_was reported independently by Fleury et al. (Nature Genetics 15, 269, 1997) and Gimeno, et al. (Diabetes 46, 900-906, 1997). UCP2 shares 59% identity to UCP1 at the amino acid level. However, unlike UCP1, UCP2 is more widely expressed in human tissues predominantly in 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 gain is consistent with it playing some role in weight gain potential (Fleury et al. 1997 supra). In mice, UCP2 maps close to a quantitative trait locus (QTL) on chromosome 7 associated with obesity. Human UCP2 has been mapped to the homologous region of the long arm of chromosome 1 1 (Bouchard et al., Human Molecular Genetics 6, 1887-1889, 1997; Solanes et al., J.Biol.Chem 272 25433-25436, 1997). Shortly after the publication of the sequence for UCP2 a third member of the uncoupling protein family was identified and termed UCP3 (WO98/39432 (SmithKline Beecham); Boss et al, FEBS left 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 UCP1 at the amino acid level. In contrast to the wide tissue distribution 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 in Pima Indians (Schrauwen et al., Diabetes 48 146-149, 1999). 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 evolutionarily very close. UCP3 has also been implicated in wound healing (WO00/02577 SmithKline Beecham pic).

Summary of the Invention

In one aspect, the invention relates to new uses of UCP3 polynucleotides and polypeptides disclosed in WO98/39432 (SmithKline Beecham). Such uses include the treatment of obesity, diabetes and body weight disorders, hereinafter referred to as "the Diseases", amongst others. In a further aspect, the invention relates to methods for identifying agonists using the materials provided by the invention, and treating conditions associated with UCP3 imbalance with the identified compounds.

Description of the Invention

In a first aspect, the present invention relates to the use of a compound selected from:

(a) a UCP3 polypeptide;

(b) a compound which activates a UCP3 polypeptide; or (c) a polynucleotide encoding a UCP3 polypeptide, for the manufacture of a medicament for treating: (i) obesity; (ii) diabetes; or (ii) body weight disorder. Such UCP3 polypeptides include isolated polypeptides comprising an amino 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 comprising the amino acid of SEQ ID NO:2.

Further polypeptides include isolated polypeptides in which the amino acid sequence has at least 95% identity, preferably at least 97-99% identity, to the amino 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. In addition polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 1 are also included.

The polypeptides for use in the present invention may be in the form of the "mature" protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.

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.

In a further aspect, the present invention relates to the use of UCP3 polynucleotides. Such polynucleotides include isolated polynucleotides comprising 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. In this regard, 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. Such polynucleotides include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO:l encoding the polypeptide of SEQ ID NO:2.

Further polynucleotides for use in the present invention include isolated polynucleotides comprising 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. In this regard, 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.

Further polynucleotides for use in the present invention include isolated polynucleotides comprising a nucleotide sequence which has at least 95% identity to SEQ ID NO:l over the entire length of SEQ ID NO: 1. In this regard, 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 comprising the polynucleotide of SEQ ID NO:l as well as the polynucleotide of SEQ ID NO:l.

The nucleotide sequence of SEQ ID NO:l is a cDNA sequence encoding human UCP3 (W098/39432 (SmithKline Beecham); Boss et al., FEBS left 408 39-42, 1997; Vidal-Puig et al., Biochem.Biophys.Res.Commun. 235 79-82, 1997). The nucleotide sequence of SEQ ID NO:l comprises a polypeptide encoding sequence (nucleotide 119 to 1137) encoding a polypeptide of 312 amino acids, the UCP3 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 contained in 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 (WO98/39432 (SmithKline Beecham); Boss et al., FEBS left 408 39-42, 1997; Vidal-Puig et al., Biochem.Biophys.Res.Commun. 235 79-82, 1997).

Preferred polypeptides and polynucleotides for use in 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 UCP3 activity.

Polynucleotides for use in the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library derived from rnRNA in cells of human skeletal muscle and the cell lines rhabdosarcoma, caski and SHSY 5Y (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

(1989)). Polynucleotides for use in 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 polynucleotides described hereinabove may be used for the recombinant production of UCP3 polypeptides for use in the present invention. The polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions. For example, a marker sequence which facilitates purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histjdine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA. Recombinant polypeptides for use in the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression vectors. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.

For recombinant production, host cells can be genetically engineered to incorporate expression vectors or portions thereof for UCP3 polynucleotides. Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al, Basic Methods in Molecular Biology (1986) and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Preferred such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, electroporation, or infection.

Representative examples of appropriate hosts include bacterial cells, yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; or preferably animal cells such as CHO, COS, HeLa or HEK 293.

A great variety of expression vectors can be used, for instance, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenovirases, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, Molecular Cloning, A Laboratory Manual (supra). Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.

UCP3 polypeptides are believed to be involved directly or indirectly with disease states such as obesity, diabetes or body weight disorder. For example, in such disease states the UCP3 polypeptides may be underexpressed or inadequately stimulated. Thus it is desirous to devise screening methods to identify compounds which can stimulate the 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 UCP3 polypeptide. In general, agonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned. Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. Such agonists so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the polypeptide; or may be structural or functional mimetics thereof (see Coligan 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 bearing the polypeptide, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve competition with a labeled competitor. Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring UCP3 activity in the mixture, and comparing the UCP3 activity of the mixture to a standard. Fusion proteins, such as those made from Fc portion and UCP3 polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al, J Mol Recognition, 8:52-58 (1995); and K. Johanson et al, J Biol Chem, 270(16):9459-9471 (1995)).

The UCP3 polynucleotides, polypeptides and antibodies to the UCP3 polypeptide may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells. For example, 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 UCP3 polypeptide from suitably manipulated cells or tissues.

Thus, in another aspect, the present invention relates to a screening kit for identifying agonists for UCP3 polypeptides of the present invention; or compounds which enhance the production of such polypeptides, which comprises: (a) a UCP3 polypeptide;

(b) a recombinant cell expressing a UCP3 polypeptide;

(c) a cell membrane expressing a UCP3 polypeptide; or

(d) antibody to a UCP3 polypeptide; which UCP3 polypeptide is preferably that of SEQ ID NO:2. It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.

It will be readily appreciated by the skilled artisan that a polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, by: (a) determining in the first instance the three-dimensional structure of the polypeptide;

(b) deducing the three-dimensional structure for the likely reactive or binding site(s) of an agonist;

(c) synthesing candidate compounds that are predicted to bind to or react with the deduced binding or reactive site; and (d) testing whether the candidate compounds are indeed agonists.

It will be further appreciated that this will normally be an iterative process.

In a further aspect, the present invention provides methods of treating abnormal conditions such as, for instance, obesity, diabetes or body weight disorder, related to an under-expression of UCP3 polypeptide and/or UCP3 polypeptide activity. For treating such abnormal conditions several approaches are also available. One approach comprises 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. Alternatively, gene therapy may be employed to effect the endogenous production of UCP3 by the relevant cells in the subject. For example, 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. For an overview of gene therapy, see Chapter 2_0_j_ Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996). Another approach is to administer a therapeutic amount of a polypeptide of the present invention in combination with a suitable pharmaceutical carrier. In a further aspect, the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of an agonist peptide or small molecule compound, in combination with a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The invention further relates to pharmaceutical packs and kits comprising 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.

The 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 intraperitoneal, 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. In addition, if a polypeptide or other compounds of the present invention can be formulated in an enteric or an encapsulated formulation, oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in 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 attending practitioner. Suitable dosages, however, are in the range of 0.1-100 μg kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above. Thus, for example, 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.

The following definitions are provided to facilitate understanding of certain terms used frequently hereinbefore.

"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library. "Isolated" means altered "by the hand of man" from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal 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. "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 comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term "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, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino 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 ubiquitination, 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-ribosylation, amidation, biotinylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance, Proteins - Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Wold, F., Post-translational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Post-translational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al, "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al, "Protein Synthesis: Post-translational Modifications and Aging", Ann NY Acad Sci (1992) 663:48-62). "Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Hei je, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity 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. Biol. 215: 403-410 (1990). 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 algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following: 1) Algorithm: 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 program useful with these parameters is publicly available as the "gap" program from

Genetics Computer Group, Madison WI. The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps). Preferred parameters for polynucleotide comparison include the following:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches = +10, mismatch = 0

Gap Penalty: 50

Gap Length Penalty: 3 Available as: The "gap" program from Genetics Computer Group, Madison WI. These are the default parameters for nucleic acid comparisons.

By way of example, 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 within the reference sequence. The number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the numerical percent of the respective percent identity(divided by 100) and subtracting that product from said total number of nucleotides in SEQ ID NO:l, or: ^{≤ x}n - (^Xn ^β y)> wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO: 1, 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-integer product of x_n and y is rounded down to the nearest integer prior to subtracting it from x_n. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.

Similarly, 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 amino 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 amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the numerical percent of the respective percent identity(divided 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 amino acid alterations, x_a is the total number of amino 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-integer product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a.

"Fusion protein" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. In one example, EP-A-0 464 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, 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]. On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified.

Examples

Example 1 - Transgenic mice overexpressing human UCP3

The human α-skeletal actin promoter was used to drive tissue-directed expression of a human UCP3 transgene in C57Bl/6xCBA mice. The transgenic mice were prepared as described in patent application number GB 9923334.8, SmithKline Beecham. A number of founders were generated and three independent lines were bred to homozygosity. Out of these lines, two independent lines showed a significant reduction in body weight. The third line expressed low levels of human UCP3 protein. The line expressing the highest levels of human UCP3 was used to further examine the phenotype. Mice were housed in groups of 12 (in cages of 3) and maintained on a 12 hour light/12 hour dark light cycle (lights on at 06:00hr GMT). Normal diet was Teklad 2018 (13.7 KJ/g) and the palatable diet (12.6 KJ/g) was prepared according to Widdowson et al. Am.J.Physiol 249, E639-E645 (1985). The animals were implanted with Implantable Programmable Temperature Transponder (IPTT-100; Biomedic Data Systems Inc) chips over the rear thigh muscles for identification and muscle temperature estimation.

Example 2 - RNA analysis and immunoblotting.

Quantitation of mRNA transcripts was performed using the PCR-based 5' nuclease assay (Holland, PM et al Proc. Natl Acad. Sci. 88, 7276-7280 (1991) utilising gene-specific fluorogenic TaqMan® hydrolysis probes (Livak, KJ et al PCR Methods Appl. 4, 357-362 (1995). Total RNA was treated with deoxyribonuclease I (Promega) and reverse transcribed using random nonamers (Stratagene) and MMLV reverse transcriptase (Life Technologies). After first-strand cDNA synthesis, transcript cDNAs were measured using TaqMan® assay oligonucleotide primers and fluorogenic probes designed for human and mouse UCP3 (amplicon 915 - 1011 of GenBank accession U84763 and amplicon 51 - 128 of GenBank accession AF032902 respectively) and mouse UCP1 (amplicon 11 - 109 of GenBank accession U63419).

Immunoblots of human UCP3 were carried out on mouse mitochondrial fractions (Ernster, L et al Methods Enzymol 10, 86 -94 (1967) separated on SDS-PAGE using rabbit anti human UCP3 antibodies (Alpha Diagnostic Inc), an anti-rabbit IgG horse radish peroxidase secondary antibody (Amersham) and ECL detection kit (Pierce-Warriner).

Quantitative RT-PCR of mRNA for the human transgene in a single UCP3tg (transgenic) mouse confirmed that expression was largely confined to skeletal muscle with little or no ectopic expression in other tissues (eg, stomach smooth muscle <1% of skeletal muscle) other than brown adipose tissue (BAT). However, measurement of transgenic UCP3 expression in BAT in a larger study (n=10 animals) demonstrated that the expressed transgene was only 1% of that in skeletal muscle. Total UCP3 expression in the transgenic mouse was increased 66-fold in skeletal muscle but only 50% in BAT (representing 3% of the levels of endogenous UCP1). The phenotype described here is therefore due primarily to expression of the transgene in skeletal muscle. As expected, protein expression was confined to the mitochondrial fraction of transgenic muscle.

Example 3 - Blood Analytes.

Leptin and insulin were measured in plasma taken from 5 h fasted mice using commercially available ELISA (Crystal Chem Inc). Plasma glucose, triglycerides, NEFA and total cholesterol were measured in the same samples spectrophotometrically using a Cobas Mira plus Clinical Chemistry Analyser (Roche Diagnostics). Oral glucose tolerance tests was performed at 8 weeks of age according to Young, P et al Diabetes 44, 1087-1092 (1995). The results are shown in Table 1.

The results show that UCP-3tg mice displayed reduced fasting plasma glucose levels, increased glucose clearance following an oral glucose load and reduced plasma insulin levels, suggesting that they were more insulin sensitive than their wildtype littermates.

Example 4 - Physiological and activity measurements. a) - Food intake

For food intake measurements, mice were transferred to cages with grid bottoms for 24 hr and one measurement made each week. Body weight was also determined once weekly. Results: When UCP3tg and wild-type animals were moved to an energetically equivalent but palatable diet (composition: 41.5% normal diet, 41.5% condensed milk, 9% sucrose, 8% corn oil) to encourage further consumption (UCP3tg intake 96.5 KJ/24h on palatable diet vs 78.6 KJ/24h on normal diet) the wild-type animals showed accelerated weight gain whereas UCP-3tg animals showed no weight gain; indeed weight gain in UCP3tg tended to plateau at the point of diet change. A surprising finding was that in spite of their markedly lower body weight, UCP3tg mice were hyperphagic, consuming between 15 and 28% more food energy than wildtype mice on normal diet between 4 and 8 weeks of age, and 33 - 54% more on the palatable diet between 8 and 12 weeks of age. Palatable diets invoke increases in sympathetic nerve activity (Bellisle, F. et al. Am .Physiol. 249, E639-E645 (1985); Leblanc, J. & Brondel, L., Am.J.Physiol. 248, E333- E336 (1985); Diamond, P., et al. Am.J.Physiol. 248, E75-E79 (1985)), which stimulate thermogenesis in brown adipose tissue and, together with the effects of UCP3 overexpression, may account for the plateau of body weight gain despite increased energy intake. Moreover, further activation of transgenic UCP3 via products of sympathetically-mediated lipolysis (e.g. fatty acids) cannot be ruled out. The differential in energy intake between transgenic mice and their wildtype littermates was maintained when the animals were returned to normal diet. Despite a 50% increase in food consumption, plasma triglycerides and non-esterified fatty acids (NEFAs) were similar between the UCP3tg mice and wildtype controls, suggesting that increased fat combustion was occurring in UCP3tg (Table 1). b) Respiration rate and membrane potential Respiration rate and membrane potential of isolated skeletal muscle mitochondria were measured in 6 independent paired experiments (Cadenas, S. et al FEBS Lett. 462, 257-260 (1999)). Results: UCP3 overexpression was associated with uncoupling, measured as a decrease in respiratory control ratio from 3.4 ± 0.1 to 2.4 ± 0.1 (PO.001). This was caused by a 26 ± 9% increase in state 4 (plus oligomycin to inhibit ATP synthesis) respiration (P<0.003) from a control value of 109 ± 6 nmol 02/min/mg protein, and a 12 ± 6%, decrease in state 3 respiration P = 0.04). In parallel experiments (nigericin present to allow exchange of H⁺ with K⁺), the mitochondrial membrane potential of 168 ± 5 mV decreased by 12 ± 3% (P = 0.001). Increased state 4 respiration accompanied by decreased membrane potential is diagnostic of mitochondrial uncoupling. However, changes in mitochondrial proton conductance following physiologically- mediated changes in UCP3 levels have not been described and a mitochondrial transport function of UCP3 cannot be ruled out. c) Locomotor activity (LMA)

Locomotor activity was tested in unhabituated mice as described elsewhere (Rogers, DC et al Behav. Brain. Res. 105, 207-217 (1999). Mice (one per cage) were placed in the activity monitor for a period of 24 hours with food and water available ad lib. LMA was recorded automatically every 30 min. Rotorod experiments, were conducted as described elsewhere (Jones, BJ et al J. Pharm. Pharmacol. 20, 302-304 (1968). d) Oxygen consumption

Oxygen consumption was measured using a Servomax oxygen analyser model 580A. Samples were monitored continuously in 20 min blocks with room air as reference. Mice were placed n the chamber during the light phase and the data collected from a 5-hour period after the animals had settled into the new environment, but before increased activity due to anticipation of the dark phase, to reflect basal oygen consumption.

Results: the phenotype of the UCP3tg mouse is consistent with an increase in energy expenditure and this was confirmed directly by these oxygen consumption and activity experiments. Resting oxygen consumption was increased by 25% on normal diet at 8 weeks of age (30.5 ± 2.2 ml/animal/h in wildtype vs 38.3 ± 1.6 ml/animal/h in UCP-3tg; PO.05) and by 40% on palatable diet at 12 weeks of age (39.8 ± 3.0 ml/animal/h in wildtype mice vs 55.9 ± 4.0 ml/animal/h in UCP3tg mice; P=0.03), although locomotor activity was not significantly increased (Table 1). If oxygen consumption is corrected by body weight, the increased consumption of the transgenic mice was 77% (PO.02) on normal diet and 91% (PO.005) on palatable diet. Time spent on an accelerating Rotorod (Table 1), as an index of muscle motor co-ordination, was also unaffected by UCP3 overexpression. Core temperature was not affected by the presence of the transgene (38.32 ± 0.07 °C in wildtype mice vs 38.33 ± 0.13 °C for UCP-3tg mice), though muscle temperature was increased (37.52 ± 0.32 °C in wildtype mice vs 38.72 ± 0.38 °C in UCP-3tg; P<0.05) when measured at 14 weeks of age.

Table 1 Summary of biological data wild type UCP-3tg P mRNA expression (cDNA units/ng total RNA x lθ3 ) Skeletal Muscle endogenous UCP-3 1.15 ± 0.13 0.93 ± 0.10 ns hUCP-3 transgene - 74.4 ± 8.25 BAT endogenous UCP-1 26.0 ± 1.29 28.4 ± 2.40 ns endogenous UCP-3 1.63.± 0.10 1.68 ± 0.13 ns hUCP-3 transgene - 0.85 ± 0.28

5 hour-fasted plasma lipid and horm moonnee lleevveellss triglycerides (mmol.H) 1.70 ± 0.08 2.02 ± 0.19 ns NEFA (mmol.l-¹) 1.22 ± 0.06 1.19 ± 0.1 ns total cholesterol (mmol.H) 4.63 ± 0.13 2.90 ± 0.17 <0.001 leptin (ng.mH) 4.50 ± 1.33 3.72 ± 0.84 ns insulin (ng.ml'l) 6.16 ± 1.71 1.78 ± 0.34 O.02 Activity measurements Locomotor Activity 26233 ± 1796 33677 ± 2940 ns (24 h total activity)

Rotorod 183 ± 27 196 ± 17 ns

(seconds)

Example 5 - Magnetic Resonance Imaging (MRI) analysis. Images were obtained on a Bruker AMX300 interfaced to a 18.3 cm 7 tesla magnet. Animals were anaesthetised using 5% isoflurane and maintained using 1.5% isoflurane and 100% O2. Heart rate and respiratory monitoring was achieved using ECG and a fibre-optic mechanical probe respectively. Data acquisitions were triggered to the flat part of the respiratory cycle as recorded by the fibre-optic probe. A 3-D multi-echo sequence was used, acquiring a field of view of 6 x 6 x 6 cm and a matrix size of 128 x 96 x 96. Experimental time was 38 minutes using 8 echoes with an echo time of 6.6 ms, a TR of approximately 2 s and a spectral width of 100 kHz. The data set was zero filled to 256 x 128 x 128 for reconstruction.

All 3-D images were quantified using 7 sequential transverse sections, 2 mm apart. The total area quantified and the anatomical boundaries were consistent between each animal and quantification of the fat depots within each mouse was achieved using the standard software tools available on ParaVision 2.0 (Bruker, Karlsruhe, Germany).

MRI analysis following 4 weeks on the palatable diet revealed a striking reduction in adipose tissue mass in UCP3tg mice such that a 44 and 57% decrease in adipose tissue volume to total animal volume observed in the MRI field of view was seen in both males and females respectively. These alterations may be the predominant factor contributing to reduced weight in these animals. It is noted that despite the reduction in adipose tissue content of the UCP3tg mice, plasma leptin levels were not significantly reduced (Table 1).

SEQUENCE INFORMATION SEQ ID NO:l

CGCCCGGGCAGGTCAAGGAGGGGCCATCCAATCCCTGCTGCCACCTCCTGGGATGGAGCCCTAGGGAGCCCC TGTGCTGCCCCTGCCGTGGCAGGACTCACAGCCCCACCGCTGCACTGAAGCCCAGGGCTGTGGAGCAGCCTC TCTCCTTGGACCTCCTCTCGGCCCTAAAGGGACTGGGCAGAGCCTTCCAGGACTATGGTTGGACTGAAGCCT TCAGACGTGCCTCCCACCATGGCTGTGAAGTTCCTGGGGGCAGGCACAGCAGCCTGTTTTGCTGACCTCGTT ACCTTTCCACTGGACACAGCCAAGGTCCGCCTGCAGATCCAGGGGGAGAACCAGGCGGTCCAGACGGCCCGG CTCGTGCAGTACCGTGGCGTGCTGGGCACCATCCTGACCATGGTGCGGACTGAGGGTCCCTGCAGCCCCTAC AATGGGCTGGTGGCCGGCCTGCAGCGCCAGATGAGCTTCGCCTCCATC CGCATCGGCCTCTACGACTCCGTCAAGCAGGTGTACACCCCCAAAGGCGCGGACAACTCCAGCCTCACTACC CGGATTTTGGCCGGCTGCACCACAGGAGCCATGGCGGTGACCTGTGCCCAGCCCACAGATGTGGTGAAGGTC CGATTTCAGGCCAGCATACACCTCGGGCCATCCAGGAGCGACAGAAAATACAGCGGGACTATGGACGCCTAC AGAACCATCGCCAGGGAGGAAGGAGTCAGGGGCCTGTGGAAAGGAACTTTGCCCAACATCATGAGGAATGCT ATCGTCAACTGTGCTGAGGTGGTGACCTACGACATCCTCAAGGAGAAGCTGCTGGACTACCACCTGCTCACT GACAACTTCCCCTGCCACTTTGTCTCTGCCTTTGGAGCCGGCTTCTGTGCCACAGTGGTGGCCTCCCCGGTG GACGTGGTGAAGACCCGGTATATGAACTCACCTCCAGGCCAGTACTTCAGCCCCCTCGACTGTATGATAAAG ATGGTGGCCCAGGAGGGCCCCACAGCCTTCTACAAGGGATTTACACCCTCCTTTTTGCGTTTGGGATCCTGG AACGTGGTGATGTTCGTAACCTATGAGCAGCTGAAACGGGCCCTGATGAAAGTCCAGATGTTACGGGAATCA CCGTTTTGAACAAGACAAGAAGGCCACTGGTAGCTAACGTGTCCGAAACCAGTTAAGAATGGAAG

SEQ ID NO:2

MVG KPSDVPPTMAVKFLGAGTAACFADLVTFPLDTAKVRLQIQGENQAVQTARVQYRGVLGTILTMVRTE GPCSPYNGLVAGLQRQMSFASIRIG YDSVKQVYTPKGADNSS TTRILAGCTTGAMAVTCAQPTDWKVRF QASIHLGPSRSDRKYSGTMDAYRTIAREEGVRGLWKGTLPNIMRAI^'VNCAEWTYDILKEKLLDYHLLTDN FPCHFVSAFGAGFCATWASPVDWKTRYMNSPPGQYFSPLDCMIKMVAQEGPTAFYKGFTPSFLRLGS NV VMFVTYEQLKRALMKVQMLRESPF

Claims

1. The use of a compound selected from:

(a) a UCP3 polypeptide; (b) a compound which activates a UCP3 polypeptide; or

(c) a polynucleotide encoding a UCP3 polypeptide, for the manufacture of a medicament for treating: (i) obesity; (ii) diabetes; or (iii) body weight disorder.

2. The use according to claim 1 wherein the medicament is used in the treatment of obesity.

3. The use according to claim 1 wherein the medicament is used in the treatment of diabetes.

4. The use according to claim 1 wherein the medicament comprises an isolated polypeptide which comprises a polypeptide having at least 95% identity to the UCP3 polypeptide of SEQ ID NO:2.

5. The use according to claim 4 wherein the isolated polypeptide is the UCP3 polypeptide of

SEQ ID NO:2.

6. The use according to claim 1 wherein the medicament comprises a compound which activates a UCP3 polypeptide.

7. The use according to claim 1 wherein the medicament comprises a polynucleotide encoding a polypeptide having at least 95% identity with the amino acid sequence of SEQ ID NO:2.

8. The use according to claim 7 wherein the polynucleotide comprises a polynucleotide having at least 95% identity with the polynucleotide of SEQ ID NO:l.

9. The use according to claim 7 or 8 wherein the polynucleotide has the polynucleotide sequence of SEQ ID NO: 1.