MXPA98010380A - Methods of sizing for useful compounds in length of corpo weight - Google Patents

Abstract

The present invention relates to drug screening assays and to diagnostic and therapeutic methods for the treatment of body weight disorders such as obesity, anorexia and cachexia, using the melanocortin receptor 4 (MC4-R) as an intervention target. The invention also relates to compounds that modulate the activity or expression of MC4-R, and the use of such compounds in the treatment of body weight disorders.

Description

METHODS OF SIZING FOR USEFUL COMPOUNDS IN THE REGULATION OF BODY WEIGHT 1. INTRODUCTION The present invention relates to drug screening assays, as well as methods of diagnosis and therapy for the treatment of body weight disorders such as, for example, obesity, anare ia and cachexia, which involve melanocortin receptor 4 (MC4-R). The invention also relates to compounds that modulate the activity or expression of MC4-R, and to the use of said compounds in the treatment of body weight disorders. 2. BACKGROUND OF THE INVENTION It is known that elanocartins (a variety of different peptide products resulting from post-translational processing of pra-opiomelanocartin) have a wide range of physiological actions. Apart from its well-known effects on cortical adrenal function (for example, by ACTH, adrenocorticotropic hormone), and on elanocytes (for example, by -MSH, melanocyte stimulation hormone), elanocortins affect behavior, learning, and memory, control of the cardiovascular system, analgesia, thermoregulation, and the release of other neuramoral agents including prolactin, luteinizing hormone, and biogenic amines. Peripherally, melanacortins have been identified as having immunomodulatory and neurotrophic properties and have been involved in events related to parturition. Elanocartins measured their effects through elanocortin (MC-R) receptors - a subfamily of G-protein coupled receptors. Other than the MC1-R that was identified as specific for a-MSH, and MC2-R that was identified as specific for ACTH, the mela? acartine receptors cloned and identified to date (MC3-R, MC4-R, MC5-R) are considered co or "orphan" receptors - that is, the identity of the native ligand for each of the receptors remains unidentified, and the physiological function of each of the receptor types remains unknown. Agouti pratein is a gene product expressed in mice which is considered involved in the determination of hair color, but it is also believed to play a role in obesity when its normal expression pattern is deregulated and the protein expressed. in a ubiquitous way. The receptor for agouti has not been identified or cloned; however, it has been observed that agouti antagonizes the MSH-induced activation of two melanocortipa receptors. 2.1. RECEPTORS OF MELAN0C0RTI A The first two cloned melanocortin receptors were the melanocyte MSH receptor, MCL-R, and the adrenscartical ACTH receptor, MC2-R (Mountjay et al., 1992, Science 257: 1248-1251; Chhajlani &; Wikberg, 1992, FEBS Lett 309: 417-420). Subsequently, three additional melanocortin receptor genes were cloned that recognize the core heptapeptide sequence (MEHFRWS) of melanacortin. It has been shown that two of these receptors are expressed primarily in the brain, MC3-R (Roselli-Rehfuss et al., 1993, Proc. Nati, Acad. Sci. USA 90: 8856-8860; Gantz et al., 1993, J. Biol. Chem. 268: 8246-8250) and MC4-R (Gantz et al., 1993, J. Biol. Chem. 268: 15174-15179; Mountjay et al., 1994, Mol. Endo. 8: 1298 -1308). A fifth melanocortin receptor (originally called MC2-R) is expressed in numerous peripheral organs as well as in the brain (Chhajlani et al., 1993, Biochem. Biophys., Res. Commun. 195: 866-873; Bantz et al., 1994, Biochem. Biophs, Res. Commun. 200: 1214-1220). The native ligands and functions of the last three receptors remain unknown. Because of their "orphan" status as receptors without an identified ligand, and due to the absence of any known physiological function for these new receptors, researchers have attempted to characterize receptors in vitro, for their ability to bind and respond (eg , transduce signals) to several known melanocortipas (for example, see Rosel li-Rehfuss, 1993; supra; and Gantz, 1993 supra) or agonists and antagonists derived from amino acid sequences of MSH and ACTH (for example, see Hruby et al., 1995, J. Med. Chem. 38: 3454-3461; and Adán et al., 1994, Eur. J. Pharmacol. 269: 331-337). In another approach, members of the melanocortin receptor family were differed based on their tissue distribution pattern or hypothesis about a function (for example, see Gantz, 1993, supra; and Mountjoy 1994, supra). ). For example, the expression of MCl-R is located in melanocytes, MC2-R is located in adrenal-cortical cells, whereas MC3-R and MC4-R are found primarily in the brain but not in the adrenal cortex or in melanocytes; MC4-R is not expressed in the placenta, a tissue that expresses large amounts of MC3-R. Based on its expression pattern in the hippocampal region of the brain, a function for MC4-R was proposed in terms of learning and memory (Gantz, 1993, supra) but it was observed that there was a "pharmacological paradox" to the extent that that the MC4-R did not respond well to compounds known to have an effect on the retention of learned behaviors. (Mauntjoy, 1994, supra). Mountjoy 1994 further suggests that MC4-R can participate in the modulation of the flow of visual and sensory information, or coordinate aspects of samatomatar control, and / or can participate in the modulation of automatic outflow to the heart. Thus, despite such efforts, the native ligands and the function of MC3-R, MC4-R and MC5-R remain unclear. 2.2. AGUTÍ PROTEIN It is predicted that the agouti gene encodes a secreted protein expressed in hair follicles and in the epidermis, whose expression correlates with the synthesis of the yellow pigment associated with the agouti phenotype (Miller et al., 1993, Gene &; Development 7: 454-467). Some dominant mutations of the agouti gene result in a deregulated, or ubiquitous expression of the agouti protein in mice, demonstrating pleiatropic effects that include obesity and increased susceptibility to tumors. (Miller et al., 1993, supra; Michaud et al., 1993, Genes YES Development 7: 1203-1213). The ectopic expression of agouti protein, wild-type, normal, in transgenic mice results in obesity, diabetes, and the yellow skin color commonly observed in spontaneous obese mutants (Lebig, et al., 1995, Proc. Nati. Acad. Sci. USA 92: 4728-4732). For reviews, see Jackson, 1993, Nature 362: 587-588; Conklin & Bourne, 1993, Nature 364: 110; Siracusa 1994, TIG 10: 423-428; Yen et al., 1994, FASEB J. 8: 479-488; Ezzell, 1994, J. NIH Res. 6: 31-33; and Manne et al., 1995, Proc. Sci. USA 92: 4721-4724. No receptor for agouti has been identified. It has been reported that agouti is a competitive antagonist of the "MSH linkage with MC1-R and MC4-R in vitra (Lu et al., 1994, Nature 371: 799-802), and the authors speculated that the ectopic expression of agouti can lead to obesity by antagonism of melanocortin receptors expressed outside the hair follicle. Regarding this aspect, several theories have been proposed to explain the induction of obesity by ectopic expression of agouti. For example, the expression of agouti in skeletal muscle can result in insulin resistance, hyperinsulinemia and obesity due to elevated Ca ++ levels; alternatively, ectopic expression of agouti in adipocytes may depress lipalysis; conversely, direct effects of agouti on pancreatic islet cells can result in hyperinsulinemia and obesity, another possibility is that the expression of agouti in the brain can result in obesity due to a primary effect on areas of the brain that control regulation of weight and insulin production (see Klebig 1995, supra) In summary, the mechanism of obesity induced by agouti in mice is unknown, and the relevance, eventual entity, of this type of phenomenon for human obese phenotypes has not been 3. COMPENDIUM OF THE INVENTION The present invention relates to drug screening assays for identifying compounds for the treatment of body weight disorders., such as obesity, anorexia and cachexia by using MC4-R as a target. The invention also relates to compounds that modulate body weight through MC4-R. The present invention also relates to treatment of body weight disorders focusing on MC4-R. The invention is based, in part, on the discovery of a specific function for MC4-R in the regulation of body weight. As shown here, mice that do not have MC4-R develop an obesity syndrome at the onset of maturity associated with hyperphagia, hyperinsulinemia, and hyperglycemia. Particularly knockout mice in which the gene encoding MC4-R is defective show a significant increase in weight compared to offspring of the same bait of wild-type females or heterazigssas with MC4-R. The invention is also based, in part, on the discovery that agouti protein, which is known to be involved in an obese phenotype when expressed ectopically in mice, binds to MC4-R. The invention is also based, in part, on the discovery that mutations in MC4-R have been found in extremely obese human patients. A comparison of the signaling response of mutant and wild type receptors indicates an affected signaling of the P receptor, mutant as measured by induction of cAMP in the presence of several agonists. The invention relates to assays designed to screen compounds or compositions that modulate the activity of MC4-R, ie, compounds or compositions that act as agonists or antagonists of MC4-R, and therefore modulate weight control. For this purpose, cell-based assays or non-cell-based assays can be used to detect compounds that interact, for example, that bind to an extracellular domain of MC4-R ("ECD"). Cell-based assays have the advantage of being able to be used to identify compounds that affect the biological activity of MC4-R (ie, signal transduction), including the identification of compounds that do not interact with an ECD of MC4-R, but rather that act on an intracellular component of the signal transduction pathway mediated by MC4-R. The invention also relates to assays designed to screen compounds or compositions that modulate the expression of the MC4-r gene. For example, cell-based assays, or assays of cell lysates (e.g., ip-vitro transcription or translational assays) can be used to screen compounds or compositions that modulate MC4-r transcription (e.g., compounds that modulate expression. , production or activity of transcription factors involved in the expression of the MC4-r gene, palynucleotides that form triple helical structures with a regulatory region of MC4-re inhibit the transcription of the MC4-r gene, etc.). Alternatively, cell-based assays or cell lysate assays can be used to screen compounds composed of compositions that modulate the translation of MC4-R transcripts (e.g., antiseptide and robixima molecules). In another embodiment, cell-based assays or cell lysate assays can be used to test in-cleft pal constructs designed to modify the expression of the MC4-r gene in vivo. Such constructs include polynucleotide constructs designed for gene therapy; for example, expression constructs or gene replacement constructs that place the MC4-r gene under the control of a strong promoter system, an inducible promoter system, or a constitutive promoter system. The invention also encompasses MC4-R agonists and antagonists, including small molecules, large molecules, and antibodies, as well as nucleotide sequences that can be employed to inhibit the expression of the MC4-r gene (e.g., antisense and ribazi molecules). a), and gene or regulatory sequence replacement constructs designed to enhance MC4-r gene expression (eg, expression constructs that place the MC4-r gene under the control of a strong promoter system). Such compounds can be used to treat body weight disorders. In addition, this invention presents methods for evaluating diagnosis and prognosis of body weight disorders, including obesity, cachexia and anorexia, and for the identification of subjects who have a predisposition for such conditions. For example, nucleic acid molecules encoding MC4-R can be used as diagnostic or hybridization probes or for diagnostic polymerase chain reaction analysis for the identification of MC4-R gene mutations, allelic variations and defects Regimens in the MC4-R gene are based, in part, on the identification of MC4-R mutants in obese human patients. The invention also addresses the use of such compounds and compositions, including gene therapy approaches, that modulate MC4-R activity or MC4-r gene expression to treat body weight disorders. 3.1. DEFINITIONS The following terms are used here with the following meanings. Coding sequences or MC4-r nucleotides: this expression refers to DNA sequences that encode MC4-R mRNA transcripts, MC4-R pratein, fragments of MC4-R protein polypeptides or peptides, or MC4-R fusion proteins. The nucleotide sequences of MC4-r encompass DNA, including genomic DNA (eg, the MC4-r gene) or cDNA, MC4-R refers to the MC4-r gene products, eg, transcripts and proteins of MC4 receiver. Polypeptides or peptide fragments of the MC4-R protein are known co or MC4-R polypeptides or MC4-R peptides. Fusions of MC4-R, either MC4-R polypeptides, or fragments of peptides with an unrelated pratein are referred to herein as MC4-R fusion proteins. A functional MC4-R refers to a pratein that binds with melanacortin peptides in vivo to well in vitro. ECD: means "extracellular domain". TM: means "transmembrane domain". CD: means "cytoplasmic domain". 4. DESCRIPTION OF THE FIGURES Figure 1. Amino acid sequences deduced from the melanocortin receptors. The serpentine structure of the melanocortin receptors predicts that the hydrophilic domains located between the TM domains are alternately arranged outside and inside the cell to form the ECD (amino acid residues 1-74, 137-155, 219-231 and 305-316 in Figure 1) and the CD (amino acid residues 102-112, 178-197, 251-280 and 339-final in Figure 1) of the receptor. The predicted transmembrane domains are indicated by bars above and Roman numerals, and the four extracellular domains (ECD1, ECD2, ECD3 and ECD4) and four citaplasmic domains (CD1, CD2, CD3 and CD4) are indicated. Figure 2. Schematic diagram of the construction of the focus vector of MC4-R. Figure 2A. Map of partial restriction of the lac of MC4-R. Figure 2B. The 5 'KO construct of MC4, which contains 3' gene sequences from the MC4-R gene: in the vector pJN2. Figure 2C "The 5 '3' KO construct of MC4-R where 5 'genomic sequences of the MC4-R gene have been inserted into the 5' KO construct of MC4. Figure 2D. The 5'3 'KO neocanstructa of MC4-R where a neo expression cassette has been inserted between the 5' and 3 'flanking sequences of the MC4-R gene. The dotted line represents the vector pJN2. The open box represents the expression cassette of PGK-neo, the shaded box represents the gene of MC4-R and the arrows indicate the direction of transcription. Figure 3. Schematic diagram of the gene approach strategy for the deactivation of MC4-R. Figure 3A. Diagram of the MC4-R locus. The shaded box represents the coding sequences of MC4-R, the solid box indicates the location of the Sanda Sacl-Sphl used to identify homologous recombinants. The arrow indicates the transcription direction of the MC4-R gene. Figure 3B. Diagram of the focus construct of MC4-R. The dashed line represents plasmid sequences of pJN2 and the arrow indicates the direction of the. Neo transcription. Figure 3C. Diagram of the MC4-R locus after homologous reca bination with the focus vector. 3D figure. Restriction fragment lengths predicted for MC4-R and wild-type loci digested with the indicated enzymes and probed with the Sacl-Sphl probe. Figure 3E. Autoradiogram of a Southern Blat analysis of progeny tail DNA F2. The genomic DNA was digested with Apal or Ncol, according to what was indicated and hybridized with a radiolabeled probe illustrated in (A), then removed and rehybridized with a probe, arcade radiation consisting of the human MC4-R coding sequence. . + / +, +/-, and - / - indicates DNA from F2 bait pups of wild type, heterozygosity and hamazigasas, respectively. Figure 4. Weight increase of mice deficient in MC4-R and control chicks of the same bait. Each line represents the weight increase of an individual mouse. Figure 4A. Weight increase of female homszigaso mutant mice (- / -) (filled squares) and wild type (+ / +) F2 controls (empty circles). The heights of 9 mice ho ozigosos and 12 control mice were taken at the indicated times. Figure 4B. Weight increase of mutant (x) female heterozygosity (+/-) and wild type (+ / +) F2 controls (empty circles). The weights of 18 heterazigosas mice and 12 control mice were taken in the indicated times. Figure 4C. Weight increase of mutant mice (full squares) male hamozigosos (- / -) and controls F2 (empty circles) of wild type (+ / +). The weights of 9 hamaziganese mice and 17 control mice were taken at the indicated times. Figure 4D. Weight increase of mutant mice (x) male heterozygotes (+/-) and F2 controls (empty circles) of wild type (+ / +). The weights of 18 heterozygous mice and 17 control mice were taken at the indicated times. Figure 5. Sequence of human MC4-R. The transmembrane domains are underlined. Amino acid differences in the MC4-R rat ds are indicated below the human sequence. Figure 6. Increased linear growth of mice with MC4-R deficiency. The body length of female (empty bars) and males (shaded bars) was measured at the age of approximately 19 weeks (between days 132 and 138). The bars indicate the average length of 12 female F2 mice of wild type (+ / +), and 9 homozygous mutants (- / -), and 15 wild-type F2 mice, 20 male heterozygous F2 mice, and 9 male F2 mutant hamozygous mice. Error bars represent the standard error of the mean, and the asterisks indicate significant difference (p <0.02 by means of the two-tailed Student t test) compared to the wild-type value within a similar sex. Figure 7. Mice that do not have the MC4-R are hyper-logical. The food intake of female mice housed in pairs was measured every day of the week for a period of two weeks. The open bars represent the average of 8 measurements in a cage each of two control C57BL / 6 and das A mice. The shaded bars represent the average of 8 measurements in zad? one of two cages of two hamozygos mutant mice (- / -) and two control mice (+ / +) of wild type F2. The error bars represent the standard error of the mean, and the asterisks indicate a significant difference (p <0.01 in the two-tailed Student's t-test) either of Ay compared to C57BL / 6 or of hamazy mutants MC4-R (- / -) compared with wild type F2 mice MC4-R (+ / +). Figure 8. Levels of serum glucose, insulin and leptin in mice that do not have MC4-R. Glucose, insulin and leptipa were each measured in the same serum samples. The empty bars represent heterozygotes, and the shaded bars represent homozygous mutant mice. The error bars indicate the standard error of the mean. Asterisks indicate significant difference (p <0.05 in the two-tailed Student t test) compared to control within the same sex and age group. For female mice, the "n" for wild-type mice at 4-8 weeks, 10-14 weeks, and 17-23 weeks was 11, 14, and 7, respectively; and in the case of homazigosas mutants, 7, 11 and 3, respectively. In the case of male mice, the "n" for wild type mice at 4-8 weeks, 10-14 weeks and 17-23 weeks was 14, 14, and 6, respectively; and in the case of homazigosos mutants, 8, 8, and 9, respectively. Figures 8A and 8B. Serum glucose levels of female and male mice, respectively. Five μl of serum was analyzed using an axidase glucose assay. Figures 8C and 8D. Serum insulin levels of male and female mice, respectively, were assayed by immunoassay using rat insulin, as a standard. Figures 8E and 8F. The serum leptin levels of male and female mice, respectively, were measured by radioimmunoassay. Figure 9. Removal of the MC4-R gene does not affect basal corticosterone. Serum corticosterone levels were measured in three sets of bait pups with correspondence in sexes that contained a representative animal of each genotype: + / + wild type control, +/- heterozygous, - / - homozygous mutant. The sets are, from left to right, male, female, and male. The males had an age of 15 weeks, the females of 18 weeks. The data indicate the average of measurements made using three serum samples obtained on different days. The measurement in each day was carried out in duplicate. The bars indicate the standard deviation. Analysis of data by two-way ANOVA did not indicate any significant difference in corticosterone levels depending on the genotype. Figure 10. Removal of the MC4-R gene did not affect the mRNA levels of POMC in the brain. Figures 10A, 10B and 10C. Brain sections stained with hematoxylin and eosin from mice deficient in MC4-R wild-type, heterozygous, and homozygous mutant, respectively. Figures 10D, 10E and 10F. Autoradiographs of brain sections of wild-type, heterazigous, and homozygous mutant MC4-R deficient mice, respectively, hybridized with a 35S-P0MC antisense cRNA probe. Figures 11A-11B. Sequence of MC4-R mutant. The mutation is Ilel37Thr mutation (T to C). Figures 12A-12B. Sequence of MC4-R mutant. The mutation is Vall02Ile mutation (G to A). Figures 13A-13B. Sequence of MC4-R mutant. The mutation is Thrll2Met mutation (C to T). Figure 14. Affected mutant receptor signaling I137T. The signaling response of wild type (wt) and mutant (t) receptors was compared to five endogenous elanocortins, α-MSH (alpha), β-MSH (beta), gam al-MSH 9 (ga mal), gamma2- MSH (gamma2) and ACTH. 5. DETAILED DESCRIPTION OF THE INVENTION The invention described in the following subsections encompasses screening methods (e.g., assays) for the identification of compounds that affect weight modulation. The invention also encompasses MC4-R agonists and antagonists, including small molecules, large molecules, and antibodies, as well as nucleotide sequences that can be used to inhibit the expression of MC4-r gene (e.g., antisense molecules and ribazymes) , and genetic or regulatory sequence replacement constructs designed to enhance the expression of the MC4-r gene (for example, expression constructs that place the MC4-R gene under the control of a strong promoter system). Such compounds can be used to treat body weight disorders. Particularly, cellular and non-cellular assays are described which can be used to identify compounds that interact with MC4-F, for example, modulate the activity of MC4-R and / or bind to MC4-R. Cell-based assays can be used to identify compounds or compositions that affect the signal transduction activity of MC4-R, already. whether they bind to MC4-R or act on intracellular factors involved in the transduction pathway. of MC4-R signal.

Such cell-based assays of the present invention employ cells, cell lines, or manipulated cells or manipulated cell lines that express MC4-R. The cells can be further manipulated to incorporate a reporter molecule bound to the signal transduced by the activated MC4-R to help identify compounds that modulate the signaling activity of MC4-R. The invention also encompasses the use of cell-based assays and assays of cell lysates (e.g., in vitro transcription or translation assays) for. sift compounds or compositions that modulate MC4-r gene expression. For this purpose, constructs containing a reporter sequence linked to a regulatory element of the MC4-r gene can be used in manipulated cells or in extracts of cell lysates to screen compounds that modulate the expression of the reporter gene product at the transcription level. For example, such assays can be used to identify compounds that modulate the expression or activity of transcription factors involved in the expression of the MC4-r gene, or to test the activity of triple helix palynucleotides. Alternatively, manipulated cells or translational extracts can be used to screen compounds (including antisense and ribazyme constructs) that modulate the transcription of mRNA transcripts of MC4-R, and consequently, affect the expression of MC4-R. The invention also encompasses MC4-R proteins, polypeptides (including soluble MC4-R polypeptides or peptides) and MC4-R fusion proteins for use in non-cell-based screening assays, for use in the generation of antibodies , for diagnostic and therapy purposes. It is predicted that MC4-R is a serpentine receptor that traverses the membrane seven times, resulting in four extracellular domains (ECDs) and four cell domains (CDs) (see Figure 1). Peptides corresponding to each ECD, or a polypeptide composed of two or more of the four ECDs joined together can be manipulated in accordance with what is described in section 5.3.1, infra. Alternatively, such peptides or polypeptides can be fused with a heterologous protein, for example, a reporter, an Ig Fc region, etc., to provide a fusion pratein. Such peptides, peptides and fusion proteins can be used in cell-based assays to screen compounds that "interact, for example, modulate, the activity of MC4-R and / a bind with MC4-R. MC4-R can be used to treat weight disorders such as obesity, anorexia or cachexia, such MC4-R protein products include, but are not limited to, soluble derivatives with, for example, peptides or palpeptides corresponding to one or more ECDs. of MC4-R; truncated MC4-R polypeptides that do not have one or more ECD or TM; and MC4-R fusion protein products (especially MC4-R-Ig fusion proteins, ie, MC4-R fusions or an MC4-R domain with an IgFc domain). Alternatively, MC4-R antibodies or anti-idiotypic antibodies that mimic antagonists or agonists (including compounds that modulate signal trapsduction that can act on targets downstream in the MC4 signal transduction pathway) may be used. R) of MC4-R (including Fab fragments), to treat body weight disorders such as obesity, anorexia or cachexia. For example, the administration of an effective amount of soluble MC4-R polypeptide, or an MC4-R fusion protein (eg, ECD-IgFc of MC4-R) or an anti-idiotypic antibody (or its Fab) that mimics the ECD of MC4-R interacts with an endogenous MC4-R ligand and therefore "removes" or "neutralizes" it, and prevents or reduces binding and activation with receptor, causing an increase in weight. In another approach, nucleotide constructs encoding such MC4-R products can be used to genetically manipulate host cells to express such MC4-R products in vivo; these genetically engineered cells can function as "bioreactors" in supplying the body with a continuous supply of MC4-R, MC4-R peptide, soluble MC4-R polypeptide, or MC4-R fusion protein that will "eliminate" or it will neutralize the MC4-R ligand. Approaches to "gene therapy" for the modulation of MC4-R expression and / or its activity for the treatment of body weight disorders are within the scope of the invention. For example, nucleotide constructs encoding functional MC4-Rs, mutant MC4-Rs, as well as antisense and pbazyme molecules can be used to modulate the expression where MC4-r. The invention also encompasses pharmaceutical formulations / methods for the treatment of body weight disorders. 5.1. THE FUNCTION OF MC4-R IN THE REGULATION OF BODY WEIGHT The specific function of the MC4-R protein in vivo was investigated by manipulation of MC4-R from "nacked" mice where the majority of the coding sequence of the Endogenous MC4-R was removed, thus creating mice that could not produce functional MC4-R protein. Unlike studies of MC-R agonist / antagonist, which are complicated studies because each of the MC receptors, instead of only MC4-R, can be affected, this specific elimination of only one MC4-F function allowed an evaluation of the biological function of MC4-R.

In order to produce the knockout mice of MC4-R, human MC4-r gene sequences were used to isolate and clone the murine MC4-r gene. A construct focused towards murine MC4-r was generated after which it was designed to remove the majority of the murine MC4-r coding sequence in homologous recombination with the endogenous murine MC4-r gene. Embryonic (ES) precursor cells containing the disrupted MC4-r gene were produced, said cells were isolated and micransjected into murine blastocysts to provide chimeric mice for cells containing a disrupted MC4-r gene. Descendants of the chimeric mice that resulted from the transmission of the germ line of the ES genome were obtained and heterozygous animals were identified for the disrupted MC4-R. In order to evaluate the function of MC4-R in vivo, the heterazyzate animals for the disrupted MC4-r gene were reared together, producing baits containing wild-type mice, heterozygous mice for the mutation of MC4-r and homazigous mice for the mutation of MC4-r and homozygous mice for the MC4-R mutation. The deactivation of the MC4-R by gene approach results in mice that develop a syndrome of obesity of onset of maturity associated with hyperphagia, hyperinsulinemia, and hyperglycemia.

The increase in weight of the. animals was monitored on a regular basis. Mutant MC4-R mutants showed an increase in weight compared to heterozygous mice for MC4-R removal and wild type mice already. at 25 days of age. At approximately 5 weeks of age, most of the mutant hsmazigasos, both males and females, were heavier than their wild-type siblings of the same sex, and at 7 weeks of age all piutants with null MC4-R were heavier than the control animals (figure 4A and 4C). At 15 weeks of age, the heterozygous mutant females had an average weight twice the weight of their wild-type sisters, whereas the male mutant homazigosas had an overweight of approximately SOX compared to the controls of wild type. Heterazigasos mice for the removal of MC4-R showed an increase of intermediate weight in comparison with the observed in the brothers of wild type and mutants hsmo? Igasos (see figure 4B and 4D), which demonstrates a effect of genetic dosage of the removal of the MC4-R on the regulation of body weight. In addition, as demonstrated in Figure 6, mice deficient in MC4-R are significantly longer than wild-type control animals. The average length of heterozygous mutant females is increased by approximately 117. compared to wild-type F2 mice, and heterozygous females are approximately 7'Á longer than the control animals. Male homozygotes and male heterozygotes are approximately 8% and 2.5 *. longer than the control animals, respectively. The absence of MC4-R also resulted in a significant (46%) increase in food consumption compared to F2 controls of type e. Blood was collected from mice with MC4-R deficiency and tested for serum glucose, insulin and leptin levels. Serum glucose levels remained essentially unchanged in heterazigasas or hozigosas females for the removal of MC4-R, but both heterozygous and hamozygos males were hyperglycemic (Figures 8A and 8B). Both the male and female mice were also hyperinsulinemic (Figures 8C and 8D). Heterozygous mutants were hyperinsulinemic, even at least homozygous mutants. In addition to glucose and insulin levels, serum leptin levels were also altered in mice with MC4-R deficiency (Figures 8R and 8F). Heterozygous mice, essentially, will present intermediate levels of leptin between the levels observed for wild type mice and homozygous mutants. The knockout experiments described here represent definitive evidence of the role of MC4-R in the regulation of body weight. The experimental design is not based on the relationship, if any, of the agouti ligand for the characterization of the MC4-R function. In addition, the role of MC4-R in regulating body weight is demonstrated by the discovery of mutant forms of MC4-R variants in obese human patients.

A comparison of the signaling response of the wild type and mutant receptors indicates one. Accepted signaling of the mutant receptor in accordance with that measured by induction of cAMP in the presence of several agonists. In comparison with the wild-type receptor, the mutant has a much lower minimum activation, i.e., the lowest maximum cAMP level achieved; and generally has a higher EC50, that is, the highest agonist concentration required to achieve half maximal activation. The mutant receptor is only arginally active in the presence of a very high concentration of agonist, which may not be reached under physiological conditions in vivo. 5.2. TESTING TESTS FOR USEFUL DRUGS IN THE REGULATION OF BODY WEIGHT At least three different test systems, described in the following subsections, can be designed and used to identify compounds to compositions that modulate the .7 MC4-R activity to the gene expression of MC4-r, and therefore, modulate weight control. The systems described below can be formulated into sets of elements. For this purpose, MC4-R or cells expressing MC4-R can be packaged in various containers, for example, flasks, tubes, microtiter well plates, bottles, and the like. Other reagents may be included in separate containers and provided with the set of elements; for example, samples of positive controls, samples of negative controls, melanacartin peptides (including, but not limited to, "MSH and ACTH derivatives), regulators, cell culture media, etc. 5.2.1. CELL-BASED TESTS In accordance with the present invention, a cell-based assay system can be used to screen compounds that modulate MC4-R activity and that therefore modulate body weight. For this purpose, cells that endogenously express MC4-R can be used to screen the compounds. Alternatively, cell lines, such as eg 293 cells, COS cells, CHO cells, fibroblasts, and the like, genetically engineered to express MC4-R can be used for screening purposes. Preferably, genetically engineered host cells can be used as the endpoint in the assay to express a functional receptor that responds to. activation by melanocortin peptides; for example, in accordance with that measured by a chemical, physiological or biological or phenotypic change, induction of the host cell gene or a reporter gene, change in cAMP levels, adenylyl cyclase activity, G-cell activity of the host cell, speed of extracellular acidification, kinase activity of host cell, proliferation, differentiation, etc. In addition, cell-based assay systems can be employed for. sift compounds that modulate the activity of MC4-R mutant and that therefore modulate body weight. For example, compounds that increase the activity of mutant MC4-R can be identified thereby alleviating the symptoms of body weight disorders arising from MC4-R mutapte. Cell lines can be engineered such as 293 cells, COS cells, CHO cells, fibroblasts and the like to express mutapte receptor. Alternatively, cells expressing endogenous mutant MC4 receptor can be used to screen compounds. To be useful in screening assays, host cells expressing functional MC4-R must provide a significant response to the MC4-R ligand, preferably a mayar induction response that is 5-fold compared to the background. Host cells should preferably have several characteristics, according to the reading, to optimize the inductive response by melanacortin peptides, as for example, to detect a strong induction of a reporter gene of CRE: (a) a low natural level of cAMP, ( b) proteins 6 capable of interacting with the MC4-R, (c) a high level of adenylyl cyclase, (d) a high level of protein kinase A, (e) a low level of phosphodiesterase, and (f) a high level of cAMP response element binding protein would be helpful. To increase the response to melapacortin peptide, the host cells can be manipulated to express a greater number of favorable factors or a lower amount of unfavorable factors. In addition, alternative ways of inducing the CRE reporter can be eliminated in order to reduce b salt levels. When using such cellular systems, cells expressing the melanocortin receptor are exposed to a test compound or to vehicle controls (eg, placebo). After exposure, cells can be assayed to measure the expression and / or activity of components of the signal transduction pathway of the melanacartin receptor, or the activity of the signal transduction pathway itself can be assayed. For example, after exposure, cell lysates can be assayed for the induction of cAMP. The ability of a test compound to increase cAMP levels, above the levels observed with cells treated with a vehicle control, indicates that the test compound induces signal transduction mediated by the melanocortin receptor expressed by the host cell . In order to determine the intracellular concentrations of cAMP, a scintillation proximity assay (SPA) may be employed (the set of SPA elements is provided by Amersham Life Sciences, Illinois). The assay employs cAMP labeled with 1251, an anti-cAMP antibody, and an incorporated icosphere, with scintillation agent coated with a secondary antibody. When approaching the microsatellite through the labeled cAMP anti-body complex, 1251 excites the scintillation agent to emit light. Unlabeled cAMP extracted from cells competes with cAMP labeled with 1251 to bind to the antibody and thereby decreases scintillation. The assay can be carried out on 96-well plates to allow high-throughput screening and scintillation scourers based on 96 wells can be used, such as those manufactured by Wallac or Packard for reading. In screening for compounds that can act as MC4-R antagonists, it is necessary to include ligands that activate MC4-F :, as for example, a- SH, Í3-MSH to well ACTH, to test the inhibition of signal transduction by the test compound compared to vehicle controls. In a specific embodiment of the invention, constructs containing the responsive element of cAMP linked to any of several different reporter genes can be introduced into cells expressing the melanocortin receptor. Such reporter genes may include, but are not limited to, chloramphenicol acetyltransferase (CAT), luciferaea, GUS, growth hormone, or placental alkaline phosphatase (SEAP). After exposure of the cells to the test compound, the level of reporter gene expression can be quantified to determine the ability of the test compound to regulate the activity of the receptor. Alkaline phosphatase assays are especially useful in the practice of the invention since the enzyme is secreted from the cell. Accordingly, tissue culture supernatant can be assayed for the secreted alkaline phosphatase. In addition, the activity of alkaline fasfatase can be measured by calorimetric, bioluminescent or qui-luminescent assays such as those described in Bronstein, I. et al. (1994, Biotechniques 17: 172-177). Such assays provide a simple, sensitive, easily automated detection system for pharmaceutical screening. When it is desired to discriminate between melanocartin receptacles and to identify compounds that selectively agonize or antagonize MC4-R, the assays described above must be performed using a panel of host cells, each genetically engineered to express one of the melanocartin receptors (MCl-R to MC5-R). The expression of human melanocortin receptors is preferred for drug discovery purposes. For this purpose, host cells can be engineered to express any of the amino acid sequences illustrated for the melanocartin 1 to 5 receptors in Figure 1. The cloning and characterization of each receptor has been described: MCl-R and MC2-R ( Mountjoy., 1992, Science 257: 1248-1251; Chhajlani? Wikberg, 1992 FEBS Lett. 309: 417-420); MC3-R (Roselli-Rehfuss et al., 1993, Proc. Nati, Acad. Sci., USA 90: 8856-8860, Gantz et al., 1993, J. Biol. Chem. 268: 8246-8250); MC4-R (Gantz et al., 1993, J. Bial, Chem. 268: 15174-15179, Mountjoy et al-, 1994, Mol. Endo. 8: 1298-1308); and MC5-R (Chhajlani et al., 1993, Biachem.Biaphys, Res. Ca mun., 195: 866-873, Gantz et al., 1994, Biachem.Biaphys., Commun. 200; 1214-1220), each one of which is incorporated herein by reference in its entirety. Thus, each of the above sequences can be used to manipulate a cell or cell line that expresses one of the melanocortin receptors for use in screening assays described herein. To identify compounds that specifically or selectively regulate the activity of MC4-R, the activation to inhibition of MC4-R activation is compared to the effect of the test compound on the other melanocortin receptors. In a specific embodiment, the cADMs of MCl-R to MC5-R are expressed in 293 cells under the control of transcription of the CMV promoter. Stable cell lines are established. Because transfected human MC2-R (ACTH-R) did not express very well in 293 cells, the human adrenocortical carcinoma cell line H295 (Accession No. ATCC CRL-2128), which expresses endogenous ACTH-R can be used in screening assays in addition to a stable cell line expressing transfected ACTH-R. In the first round of screening, a cell line expressing MC4-R is used to identify candidate compounds that activate MC4-R. Once identified, these candidate compounds can be tested to determine if they selectively activate MC4-R. Activation of melanocortin receptors can be assayed using, for example, the SPA assay described above. In the alternative, if the host cells express more than a melanocortin peptide receptor, the background signal produced by these receptors in rssDue = -aa the melanocortin peptides must be "subtracted" from the signal (see Gantz et al., supra). The response of-when produced by these non-MC4-R melanocortin receptors can be determined by several methods, including the elimination of MC4-R activity by antisense, antibody or antagonist. Regarding this aspect, it should indicate that you wanted wild-type CHO cells to demonstrate a small endogenous response to melapoccrtin peptides that must be subtracted from the bottom. Alternatively, the activity contributed by the other elanocortin receptors must be eliminated by activating host cells with a specific ligand for MC4-R, or by including specific inhibitors of the other melapocortin receptors. 5.2.2. NON-CELL-BASED TESTS In addition to cell-based assays, non-cell-based assay systems can be employed to identify compounds that interact with MC4-R, for example, that bind to MC4-R. Such compounds can act as antagonists or agonists of MC4-R activity and can be used in the treatment of body weight disorders. Isolated membranes can be used to identify compounds that interact with MC4-R. For example, in a ^ 5 In a typical experiment in which isolated membranes are used, 293 cells can be genetically manipulated to express MC4-R. Membranes can be harvested by standard techniques and said membranes can be used in an in vitro binding assay. Ligand labeled with 1251 (eg, α-MSH, β-MSH, or ACTH labeled with 1251) binds to the membranes and is assayed for specific activity; the specific binding is determined by comparison with binding assays performed in the presence of excess unlabeled ligand. To identify MC4-R ligands, labeled ligand membranes are incubated in the presence or absence of test compound. Compounds that bind to the receptor and compete with the labeled ligand for binding to the membranes reduced the signal compared to the vehicle control samples. Alternatively, soluble MC4-R can be recombinantly expressed and used in non-cell-based assays to identify compounds that bind with MC4-R. Recombinantly expressed MC4-R polypeptides or fusion proteins containing one or more of the MC4-R ECDs prepared in accordance with that described in section 5.3.1, infra, can be used in screening assays. based on cells. Alternatively, peptides corresponding to one or more of the MC4-R CDs, or fusion proteins containing one or more of the MC4-R CDs can be used in non-cell-based assay systems to identify binding compounds. can the cytoplasmic portion of MC4-R; Such compounds can be useful for modulating the signal transduction path of the MC4-R. In non-cell-based assays, the replenishingly expressed MC4-R is fixed on a solid substrate such as a test tube, a microtiter well or a column, by means well known to those skilled in the art (see Ausubel et al., Supra). The test compounds are then tested for their ability to bind with MC4-R. In one aspect of the invention, screens can be designed to identify compounds that antagonize the interaction between MC4-R and MC4-R ligands such as a-MSH, (3-MSH and ACTH.) In such screening, the MC4 ligands -R are labeled and the test compounds can be assayed for their ability to antagonize the binding of labeled ligand to MC4-R 5.2.3 TESTING FOR COMPOUNDS OR COMPOSITIONS THAT MODULATE THE EXPRESSION OF MC4-R Cell-based assays vi can be designed to screen compounds that regulate the expression of MC4-R either at the level of transcription or translation.In one embodiment, a DNA encoding a reporter molecule can be linked to a regulatory element of the MC4-gene. ry used in appropriate intact cells, cell extracts or lysates to identify compounds that modulate the expression of the MC4-r gene.Appropriate cells or cell extracts are prepared from any type of e cells expressing narmal epte the MC4-r gene, thus ensuring that the cell extracts contain the transcription factors required for transcription in vitro or in vivo. Screening can be used to identify compounds that modulate the expression of the reporter construct. In such sieves, the level of expression of the distribution gene is determined in the presence of the test compound and compared to the level of expression in the absence of the test compound. To identify compounds that regulate the translation of MC4-R, cells or cell lysates in vitro that contain MC4-R transcripts can be tested to determine the modulation of mRNA translation of MC4-R. For assays for MC4-R translational inhibitors, the test compounds were tested for their ability to modulate the translation of MC4-R mRNA in in vitro translation extracts. Compounds that decrease the expression level of MC4-R, either transcriptionally or translationally they can be useful for the treatment of body weight disorders such as anarexia and cachexia. In contrast, compounds that increase the expression of MC4-R may be useful for the treatment of disorders such as obesity. 5.2.4. COMPOUNDS THAT CAN BE SIZED IN ACCORDANCE WITH THE INVENTION The assays described above can identify compounds that affect the activity of MC4-R. For example, compounds that affect the activity of MC4-R include, without being bound to them, compounds that bind with MC4-R, compounds that inhibit the binding of the natural ligand, and that either activate signal transduction (agonists). ), or block activation (antagonists), as well as compounds that bind to the natural ligand of MC4-R and neutralize the activity of the ligand. Compounds that affect MC4-r gene activity (affecting the expression of MC4-r gene, including molecules, eg, proteins or small organic molecules, that affect transcription to well interfere with splicing events in such a way that the total length or the truncated form of the MC4-R can be modulated) can also be identified in the sieves of the invention. However, it should be noted that the described assays can also identify compounds that modulate the signal transduction of MC4-R (eg, compounds that affect signaling events downstream, such as inhibitors or well-enhancers of protein activities). G involved in the transduction of the signal activated by ligand binding to MC4-R). The identification and use of such compounds that affect the signaling events downstream of MC4-R and therefore modulate the effects of MC4-R on the development of body weight disorders are within the scope of the invention. In some cases, a decrease in the response of G-protein coupled receptors, or a desensitization with a prolonged exposure to the ligand, has been observed. In one embodiment of the invention, assays can be used to identify compounds that block the sensitization of the MC4 receptor, such compounds can be used to sustain the activity of the MC4 receptor. Such compounds can be used as part of a therapeutic method for the treatment of body weight disorders. Compounds that can be screened in accordance with the present invention include, without being bound to them, peptides, antibodies and fragments thereof, and other organic compounds (e.g., peptidamimetics) that bind to the ECD of MC4-R and which mimic the activity triggered by the natural ligand (i.e., agonists), or which inhibit the activity triggered by the natural ligand (i.e., antagonists); as well as peptides, antibodies or fragments thereof, and other organic compounds that include the ECD of MC4-R (or a portion thereof) and bind to a natural ligand and "neutralize" it. Compounds may include, but are not limited to, peptides such as, for example, soluble peptides, including, but not limited to, members of random peptide libraries; (See, for example, Lam, KS et al., 1991, Nature 354: 82-84, Houghten, R. et al., 1991, Nature 354: 84-86), as well as molecular library derived from elaborate combination chemistry. of amino acids in D configuration and / or L configuration, phosphopeptides (including, but not limited to, members of directed, random or partially degenerated fasphapeptide libraries, see, for example, Songyang, Z. et al., 1993, Cell 72 : 767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F (ab ') 2 and FAb expression library fragments, as well as epithelial binding fragments thereof) and small organic or inorganic molecules. Other compounds that can be screened in accordance with the invention include, but are not limited to, small organic molecules capable of crossing the blood-brain barrier, passing into an appropriate cell and affecting the expression of the MC4-R gene or some other gene involved in the signal transduction pathway of MC4-R (for example, by interaction with the regulatory region to transcription factors involved in gene expression); or such compounds that affect the activity of MC4-R or the activity of some other intracellular factor involved in the signal transduction pathway of MC4-R, co or for example the G protein associated with MC4-R. The formation of computerized models and research technologies allow for the research of compounds, or the improvement of already identified compounds, which can modulate the expression of MC4-R or its activity. After having identified such a compound composition, active regions or sites are identified. Such active sites can typically be ligand binding sites. The active site can be identified using methods known in the art including, for example, from amino acid sequences of peptides from nucleic acid nucleic acid sequences, or from the study of complexes of the relevant compound or relevant composition with its natural ligand. . In the latter case, X-ray crystallographic or chemical methods can be used to find the active site by discovering the location of the ligand that forms complexes in the factor.

Then, the three-dimensional geometric structure of the active site is determined. This can be carried out by known methods, including X-ray crystallography, which can determine a complete molecular structure. On the other hand, NMR of solid or liquid fass can be used to determine certain intramolecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures. These geometric structures can be measured with a ligand that forms complexes, natural to artificial, which can increase the accuracy of the determined active site structure. If an incomplete or insufficiently accurate structure is determined, the numerical model based on computation methods can be used to complete the structure or improve its accuracy. Any recognized modeling method can be used, including specific parameterized models for particular biopolymers, such as proteins or nucleic acids, molecular dynamics made based on the calculation of molecular movements, statistical mechanical models based on thermal assemblies, or combined models . For most types of models, standard molecular force fields, which represent the forces between atoms and constituent groups, are necessary, and can be selected from fields of forces known * in physical chemistry. Incomplete or less precise experimental structures can serve as co-limits of the complete and more precise structures calculated by these models of model formation. Finally, after determining the structure of the active site, either experimentally, by modeling, or by a combination, candidate modulation compounds can be identified by searching databases containing compounds and information on their molecular structure. This search is focused. towards compounds that have structures that correspond to the determined active site structure and that interact with the groups that define the active site. This search can be manual, but it is preferable that it be carried out with the support of computer systems. These compounds found in this search are potential MC4-R modulation compounds. Alternatively, these methods can be employed to identify improved modulation compounds from a known modulation compound or ligand. The composition of the known compound can be modified and the structural effects of the modification can be determined by employing the experimental and encapsulated modeling methods described above applied to the new composition. The altered structure is then compared to the active site structure of the compound to determine whether the result is an improved interaction or adequacy. In this way, systematic variations in composition can be rapidly evaluated, such as, for example, by variation of side groups, in order to obtain ligands or modified modulation compounds of specificity or improved activity. Additional experimental and computer model methods useful for identifying modulation compounds based on the identification of active MC4-R sites, and related transcription and transcription factors will be apparent to those skilled in the art. Examples of molecular model formation systems are the CHARMm and GUANTA programs (Polygen Corporation, Waltham, MA). CHARMm carries out the functions of energy imimization and molecular dynamics. GUANTA carries out the construction, graphic model formation and analysis of the molecular structure. GUANTA allows an interactive construction, modification, visualization, and analysis of the behavior of the molecules between them. Several articles review the formation of computer models of interactive drugs with specific proteins, such as Rativinen, et al.) 1988, Acta Pharmaceutical Fennica 97: 159-166); Ripka (1988 New Scientist 54-57); Mcr-mal and Fassman (l ^? 3, Annu. Re / .Pharmacol Toxicol 29: 111-122); Ferry and Davies, OSAR: fl Ouantitat ve Structure-Activi ty Relationships m Drug Design pages 189-193 Alan R. Liss, Inc. 1989; Lewis and De n (1989, 5 Proc. R. Soc. Land 236: 125-140 and 141-162); and, in relation to a model receptor for nucleic acid components, Asl'ew, et al., (1989, J. Am. Chem. Sac. 111: 1082-1090). Other computer programs that screen and graphically describe chemical substances are available from companies such as BioDesign, Inc. Pasadena, CA.), Allelix, Inc. (Missiissauga, Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario. ). Even though these programs are designed primarily for application with specific drugs for particular proteins, they can be adapted to the design of specific drugs for regions of DNA or RNA, once that region has been identified. Even when described above with reference to the design and generation of compounds that can alter the bond, libraries of known compounds can also be screened, including natural products to synthetic chemicals, and biologically active materials, including proteins, to find compounds that are inhibitors or activators. The compounds identified through tests such as Those described herein may be useful, for example, to elaborate the biological function of the MC4-R gene product, and to improve body weight disorders. Tests to test the efficacy of compounds identified in cell screening can be tested in 5-animal model systems for body weight disorders. Such animal models can be used as test substrates for the identification of drugs, pharmaceutical substances, therapies, and interventions that can be effective for the treatment of these disorders. For example, you can expose animal models to a compound of which you think • that has the ability to improve symptoms of body weight disorders, in a sufficient concentration and for a sufficient time to cause such improvement of the symptoms of body weight disorder in animals exposed. The response of the animals to the exposure can be monitored by evaluating the reversal of the disorders associated with body weight disorders, • Eat, for example, obesity. As for an intervention, any treatment that reverses any aspect of symptoms of bodyweight disorder should be considered a candidate for therapeutic intervention in disorders of body weight in humans. The dosages of the test agent can be determined by derivation of dose-response curves, in accordance can be considered in section 5.5, below.

For this purpose, transgenic animals expressing the gene products of human MC4-R can be employed. Animals of any species, including, but not limited to, mice, rats, canejos, india guinea pigs, micracerdas, goats, and non-human primates, for example, mandrills, monkeys, and chimpanzees can be used to generate transgenic animals for MC4-R . Any known technique can be used to introduce the transgep of human MC4-R in animals to produce the fundamental lines of transgenic animals, such techniques include, but are not limited to, pronuclear microinjection (Happe, PC and Wagner, 1989, US Patent No. 4,873,191 ); gene transference mediated by retriviruses in germ lines (Van der Putten et al., 1985, Proc. Nati, Acad. Sci., USA 82: 6148-6152); focus on genes in embryonic precursor cells (Thompson et al., 1989, Cell 56: 313-321); embryo electroporation (Lo, 1983, Mol Cell, Biol. 3: 1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57: 717-723); etc. For a review of these techniques, see Grodon, 1989, Trangepic Animáis (Transgenic Animals), Intl. Rev. Cytol, 115: 171-229, which is incorporated herein by reference in its entirety. The present invention provides transgenic animals that carry the transgene of MC4-R in all its cells, as well as animals that carry the transgene in some cells but not in all their cells, that is, mosaic animals. The transgene can be integrated as a single transgene to well in catches, for example, head-to-head tandems to good head-to-tail models. The transgene can also be selectively introduced into a particular cell type and activated following, for example , the teachings of Laska et al. (Laska, M. et al., 1992, Proc. Nati, Acad. Sci. USA 89: 6232-6236). The regulatory sequences required for said specific activation for cell type will depend on the particular cell type of interest, and it will be apparent to experts in the field. When it is desired that the MC4-r transgene be integrated into the chromosomal site of the endogenous MC4-r gene, the gene approach is preferred. In summary, when such a technique should be used, vectors containing nucleotide sequences homologous to the endogenous MC4-R gene sequences flanking the gene for the purpose of integration are designed through recombination with homologous chromosomal sequences and disorder. of the function of the endogenous MC4-r gene. The transgene can also be selectively expressed in a particular cell type with concomitant deactivation of the endogenous MC4-gene in only this cell type, following, for example, the teachings of Gu et al. (Gu, et al., 1994, Science 265: 103-4 ° 106). The regulatory sequences remembered for a specific recombination by cell type will depend on the particular cell type of interest, and will be apparent to the experts in the material. Once base animals are generated, standard techniques such as Southern blot analysis or palimerase chain reaction techniques are used to determine if the integration of the transgene was carried out. The level of mRNA expression of the transgene in the wounds of the foundation animals can also be assessed using techniques including, but not limited to, Northern blat analysis of tissue samples obtained from the animal, analysis by in situ hybridization, and RT- PCR Tissue samples expressing MC4-R gene can also be evaluated immunacitschemically using antibodies specific for the MC4-R transgep product. 5.3. MC4-R PROTEINS, POLYPEPTIDES AND ANTIBODIES MC4-R proteins, polypeptides and fragments of peptides, mutated, truncated or removed forms of MC4-R and / or MC4-R fusion proteins can be prepared for various uses, including, without limiting them, the generation of antibodies such as reagents in diagnostic tests, the identification of other cellular gene products involved in the regulation of body weight, as reagents in assays for screening 5? "compounds. which can be used in the treatment of body weight disorders, and as pharmaceutical reagents useful in the treatment of body weight disorders related to MC4-R. 5.3.1. MC4-R POLYPEPTIDE PRODUCTION The amino acid sequences deduced from the melanocortin receptors, including MC4-R, appear in Figure 1, where predicted transmembrane domains indicated by upper bars and Roman numerals, and the four extracellular domains are indicated (ECD1, ECD2, ECD3, and ECD4) and the four oplysmic ci domains (CD1, CD2, CD3, and CD4). The cerpentine-like structure of the melanacartine receptors predicts that hydraphilic domains located between the TM domains are alternatively arranged outside and inside the cell to form the ECD (amino acid residues 1-74, 137-155, 219-231 and 305-316 in Figure 1) and the CD (amino acid residues 102-112, 178-197m 251-280 and 339-final in Figure 1) of the receptor, Peptides corresponding to one or more domains of MC4-R ( for example, ECDs, TMs or CDs), MC4-R truncated or removed (for example, MC4-R where one or more of the following were removed: ECDs, TMs and CDs) as well as fusion proteins where MC4 -R of full length, a peptide of MC4-R to either truncated MC4-R is fused to an unrelated protein are also within the scope of the invention. Such soluble peptides, proteins, fusion proteins, or antibodies (including ani-idiatipic antibodies) which bind and "neutralize" natural ligand in circulation for MC4-R, can be used in accordance with that described in section 5.5. , infra to achieve an increase in weight. For this purpose, peptides corresponding to individual MC4-R ECDs, soluble MC4-R removal mutants (eg, deltaTM mutants), or the entire MC4-R ECD (manipulated by linking the four ECDs together ds according to what is described below) can be fused with another polypeptide, for example, an IgFc palpeptide). The fusion of the MC4-R or the ECD of MC4-R onto an IgFc polypeptide should not only increase the stability of the preparation but also increase the half-life and the activity of the fusion pratein of MC4-R- Ig in vivo. The Fc region of the Ig portion of the fusion protein can be further modified to reduce the function of the immunoglobulin effector. Such peptides, polypeptides, and fusion proteins can be prepared by recombinant DNA techniques. For example, nucleotide sequences that encode one or more of the four ECD domains of the MC4-R in the serpentine form can be sis-synthesized or cloned and ligated together to encode a soluble ECD of the MC4-R. The. DNA sequence encoding one to several of the four ECDs (ECD1-4 in Figure 1) can be ligated together directly or through an oligonucleotide linker that encodes a peptide spacer. Such linkers can encode 5 amino acid sequences rich in glycine, flexible which thus allows the domains to be linked together to assume a conformation that can bind ligands of MC4-R. Alternatively, nucleotide sequences encoding individual domains within the ECD can be used to express peptides of MC4-R . In addition, mutant MC4-R proteins, such as those illustrated in Figures 11-13, can be expressed by recombinant DNA techniques. Various host-vector expression systems can be used to express nucleotide sequences encoding the appropriate regions of MC4-R to produce such palpeptides. In cases where the resulting polypeptide peptide is a soluble derivative (eg, peptides that correspond to the ECDs, truncated to removed in dande the TMs and / or the CDs are removed) the peptide or The polypeptide can be recovered from the culture medium. In cases where the polypeptide or protein is not secreted, the MC4-R product can be recovered from the host cell itself. The expression vector-host systems also encompass manipulated host cells expressing MC4-R or functional equivalents in situ, i.e., anchored in the cell membrane. Purification or enrichment of MC4-R from such expression systems can be achieved by employing appropriate detergents and lipid micelles and methods well known to those skilled in the art. However, such genetically engineered host cells can themselves be used in situations in which it is important not only to preserve the structural and functional characteristics of MC4-R, but also to evaluate biological activity, for example, in drug screening assays. Alternatively, host-vector expression systems may be employed to manipulate host cells expressing mutant MC4-R protein (see, e.g., Figure 11-13). Such host cells can be used to assess biological activity, for example, in drug screening assays. Host-vector systems of expression that may be employed for the purposes of the present invention include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, expression vectors. of plasmid DNA or cDNAs containing nucleotide sequences of MC4-R; yeast (eg, Saccharomyces, Pichia) transformed with recombinant yeast expression vetars containing the nucleotide sequences of MC4-R; insect cell systems infected with recombinant virus expression vectors (eg, baculovirus) that hold the MC4-R sequences; plant cell systems infected with recombinant virus expression vectors (eg, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg, Ti plasmid) ) that contain the nucleotide sequences of MC4-R; or mammalian cell systems (eg, COS, CHO, BHK, 293, 3T3) containing recombinant expression constructs containing promoters derived from the genama of mammalian cells (eg, metallothianein promoter) or from mammalian virus (eg, the late adenovirus promoter, the 7.5K vaccinia virus promoter). In bacterial systems, several expression vectors can be advantageously selected according to the intended use for the MC4-R gene product being expressed. For example, when a large amount of said protein must be produced, for the generation of MC4-R protein pharmaceutical compositions or to create antibodies to the MC4-R protein, for example, it may be desired to have expression-targeting vectors. of high levels of easily purified pratein fusion products. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2: 1791), wherein the coding sequence of MC4-R can be linked individually to the vector in frame can the lacZ coding region in such a way that a fusion protein is produced; pIN vectors (Inouye? Í Inauye, 1985, Nucleic Acids Res. 13: 3101-3109; Van Heeke &Schuster, 1989, J. Biol. Chem. 264: 5503-5509); and similar. PGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such soluble San fusion proteins can be easily purified from Used cells by adsorption on glutathione-agarose beads followed by elution in the presence of free glutathione. PGEX vectors are designed to include dissociation sites of pratease Xa factor or thrombin of tq so that the cloned target gene product can be released from the GST portion. Alternatively, any fusion pratein can be easily purified using an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht et al. Perpetuate the easy purification of denatured na fusion prateins expressed in human cell lines (Janknecht, et al., 1991, Proc Nati Acad Sci USA 8S: 8972-8976). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid of such an anera that the open reading frame of the gene is fused in a translacian manner with an amine-end marker consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni ++ columns. Nitrilaacetic acid-agarase and histidine-labeled proteins are selectively eluted with imidazole-containing regulators. In an insect system, the nuclear polyhedrosis virus of Autagrapha californica (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence of MC4-R can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (for example, the polyhedrin promoter). Successful insertion of the MC4-R gene coding sequence will result in the inactivation of the polyhedrin gene and the production of na occluded recombinant virus (i.e., the virus that does not have the pratein coating encoded by the polyhedrin gene) . The replenishing viruses are then used to infect cells in which the inserted gene is expressed (for example, see Smith et al., 1983, J. Viral. 46: 584; Smith, US Patent No. 4,215,051 i. In mammalian host cells, various virus-based expression systems can be employed. In cases where an adenovirus is eXpressed as an expression vector, the nucleotide sequence of MC4-R of interest can be ligated to an adenovirus transcription / translation control complex, eg, the late promoter and the leader sequence. tripartite This chimeric gene can then be inserted into the adenovirus genome by recombination in vitro or in vivo. Insertion into a non-essential region of the viral genome (eg, E1 or E3 region) will result in a viable recombinant virus capable of expressing the MC4-R gene product in infected hosts. (For example, see Logan &Shenk, 1984, Proc. Nati, Acad. Sci. USA 81: 3655-3659). Specific initiation signals may also be required for efficient translation of the inserted nucleotide sequences of MC4-R. These signals include the ATG start codon and adjacent sequences. In cases where an entire MC4-R or cDNA gene is inserted, including its own initiation codon and adjacent sequences in the appropriate expression vector, no additional translational control signal may be required. However, in cases where only part of the MC4-R coding sequence is inserted, exogenous translation control signals must be praparcian, including, perhaps, the ATG initiation codon. In addition, the initiation cadon must be in frame with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bittner et al., 1987, Methods in Enzymol, 153: 516-544). In addition, a host cell strain can be selected which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific manner desired. Such modifications (eg, glycosylation) and processing (eg, dissociation) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for past-translational processing and modification of proteins and gene products. Appropriate cell lines or appropriate host systems may be chosen in order to ensure the correct modification and proper processing of the foreign protein expressed. Accordingly, eukaryotic host cells possessing the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be employed. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 and WI38 cell lines. For the high-performance and long-term production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the MC4-R sequences described above can be manipulated. Instead of employing expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (eg, promoter sequences, enhancer, transcription termites, paliadenylazine sites, etc.), and a selectable marker. After the introduction of the foreign DNA, manipulated cells can be allowed to grow for 1-2 days in an enriched medium, and then changed to a selective medium. The selectable marker in the replenishing plasmid confers resistance to selection and allows the cells stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines. This method can be used profitably to manipulate cell lines expressing the MC4-R gene product. Such manipulated cell lines can be especially useful in the screening and evaluation of compounds that affect the endogenous activity of the MC4-R gene product. Several selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase genes (Wigler, et al., 1977, Cell 11: 223), hypoxanthine-guanine phosphoribasyltransferase (Szybalska & amp; amp;; Szybalski, 1962, Prac. Nati Acad. Sci. USA 48: 2026), and of adeninfasforibosyl transferase (Lowy, et al., 1980, Cell 22: 817) which can be employed in the tk-, hgprt- or aprt- cells, respectively. Likewise, antimetabolite resistance can be used as a selection basis for the following genes: dhfr, which provides resistance to metatrexate (Wigler, et al., 1980, Nati Acad Sci USA 77: 3567; O'Hare, et al. al., 1981, Proc. Nati, Acad. Sci. USA 78: 1527); gpt, which provides resistance to mycophenolic acid (Muiigan &Berg, 1981, Proc Nati Acad Sci USA 78: 2072); neo, which provides resistance to aminoglycoside G-418 (Calberre-Garapin, et al., 1981, J. Mal. Biol. 150: 1); and hygro, which provides resistance to hygromycin (Santerre, et al., 1984, Gene 30: 147). 5.3.2. ANTIBODIES FOR MC4-R P0LIPIPTIDES Antibodies that specifically recognize one or more MC4-F epitopes; , or epitopes of conserved variants of MC4-R, or fragments of peptides of MC4-R are also encompassed by the invention. In addition, antibodies that specifically recognize mutant forms of MC4-R, such as those encoded by the DNA sequence illustrated in Figures 11-13, are encompassed by the invention. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') 2 fragments, fragments produced by a Fab expression library, antibodies anti-idiatípicos (anti-Id), and fragments of epitope binding of any of the above. The antibodies of the present invention can be used, for example, to detect MC4-R in a biological sample and can therefore be used as part of a diagnostic or prognostic technique where patients can be tested for abnormal amounts of MC4- R. Antibodies that specifically recognize mutant forms of MC4-R, such as those described below, in Section 8, may be especially useful as part of a diagnostic or prognostic technique. Such antibodies can also be used in combination, for example, with screening schemes of compounds in accordance with what has been described above, for the evaluation of the effect of the test compound on the expression and / or activity of the MC4-R gene product. In addition, such antibodies can be used in combination with the gene therapy techniques described, below, for example, to evaluate MC4-R expression cells that are narcotic and / or manipulated prior to their introduction into the patient. Such antibodies can also be used as a method for the inhibition of the abnormal activity of MC4-R. Thus, such antibodies can, therefore, be used as part of methods of treating weight disorders. For the production of antibodies, several host animals can be immunized by injection with the MC4-R, a peptide of MC4-R (for example, one corresponding to the functional domain of the receptor, such as ECD, TM or CD) , truncated MC4-R polypeptides (MC4-R where one of several domains, eg, TM to CD, has been removed), functional equivalents of MC4-R or mutants of MC4-R. Such host animals may include, but are not limited to, rabbits, mice, guinea pigs, and rats, by way of example only. Various adjuvants may be used to increase the immune response, depending on the host species, including, but not limited to, Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lyisalecithin, pluripic palliae, polyanions. , peptides, emulsions in oil, limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacillus) • from Calmette-Guerin) and Corynebacterium parvum. Polyclonal aodies are heterogeneous populations of 5 aody molecules derived from the sera of the immunized animals. Monachal aodies, which are homogeneous populations of aodies to a particular aen, can be obtained by any technique that provides the production of aody molecules by couous cell lines in cultivars. These include, but are not limited to, the Kohler and Milstein hybridization technique, (1975, Nature 256: 495-497; and North American Patepte No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983). , Immunology Today 4:72; Cole et al., 1983, Proc. Nati Acad. Sci. USA 80: 2026-2030), and the EBV-hybrid technique (Cole et al., 1985, Manoclonal Aodies And Cancer Therapy, Alan • R. Liss. Inc., pp. 77-96). Such aodies can be of any kind of immunoglobulin including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma that produces the mAb of this inven can be cultured in vitro or in vivo. The production of high titers of mAbs in vivo makes this the currently preferred method of production. In addition, techniques developed for the production of "chimeric aodies" (Morrison et al., 1984, Proc. Nati Acad. Sci., 81: 6851-6855; Neuberger et al., 1984, Nature, 312: 604-608; Takeda et al., 1985, Nature, 314: 452-454) by splicing the genes of a mouse aody molecule of appropriate specificity to aen together with genes from a human aody molecule of appropriate biological activity can be used . A chimeric aody is a molecule in which different portions are derived from different animal species such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Alternatively, techniques described for the production of single chain aodies can be adapted (US Patent No. 4,946,778; Bird, 1988, Science 242: 423-426; Huston et al., 1988, Prac. Nati. Acad. Sci. USA 85: 5879-5883; and Ward et al., 1989, Nature 334: 544-546) to produce single chain aodies against the MC4-R gene products. Single chain aens are formed by linking the light and heavy chain fragments of the Fv region through an amino acid bridge, resulting in a single chain palpeptide. Aody fragments that recognize specific epitopes can be generated by known techniques. For example, such fragments include, but are not limited to: F (ab ') 2 fragments that can be produced by pepsin digestion of the aody molecule and Fab fragments that can be regenerated by reducing disulphide bridges the fragments of F (ab ') 2. Alternatively, Fab expression libraries can be constructed (Huse et al., 1989, Science, 246: 1275-1281) to allow rapid and easy ideication of the Fab monoclonal fragments with the desired specificity. Aodies to the MC4-R can be used in turn to generate aidiotype aodies that "mimic" the MC4-F, using techniques well known to those skilled in the art. (See, for example, Greepspan &Bana, 1993, FASEB J 7 (5): 437-444, and Nissinaff, 1991, J. Immunol. 147 (8): 2429-243S). For example, aodies that bind to the ECD of MC4-R and competitively inhibit the binding of melanocortins with MC4-R can be used to generate apti-idiotypes that "mimic" ECD and therefore bind to melanocortins and neutralize them. . Such neutralizing aidiatypes or Fab fragments of such aidiotypes can be employed in therapeutic regimens to neutralize the native ligand and promote weight gain. Alternatively, aodies can be generated for MC4-R, which can act as agonists of MC4-R activity.

Such antibodies will bind to MC4-R and activate the signal transduction activity of the receptor. Such antibodies would be especially useful for the treatment of weight disorders such as obesity. In addition, antibodies that act as an antagonist of MC4-R activity, ie, and that inhibit the activation of MC4-R receptors, can be used to treat weight disorders such as anorexia to cachexia. 5.4. APPROACHES OF GENETIC THERAPY TO CONTROL ACTION OF MC4-RY REGULAR BODY WEIGHT The expression of MC4-R can be controlled in vivo (for example at the level of transcription to translation) using genetic therapy approaches to regulate the activity of MC4-R and treat body weight disorders. Some of the approaches are described below. 5.4.1. SEN REPLACEMENT THERAPY In regard to an increase in the level of normal MC4-R gene expression and / or MC4-R gene product activity, MC4-R nucleic acid sequences can be used for the treatment of disorders of body weight, including obesity. When the cause of obesity is a defective MC4-R gene, the treatment may be administered, for example, in the form of a gene replacement therapy. Specifically, one or more copies of an MC4-F gene; Normal to a part of the MC4-R gene which directs the production of an MC4-R gene product having a normal function can be inserted into the appropriate cells within a patient to animal subject, using vectors including, without limitation to them, vectors of adenoviruses, of virus associated with adeno, of retroviruses and of herpes virus, in addition to other particles that introduce the DNA into cells, such as for example liposomes. Since the MC4-R gene is expressed in the brain, including the cortex, the thalamus, the brain and spinal cord, and the hypatilam, such gene-repletion therapy techniques can deliver MC4-R gene sequences to These types of cells in patients. Thus, techniques for the delivery of the MC4-R gene sequences must be designed to easily cross the hemata-encephalic barrier, which is well known to those skilled in the art (see, for example, the PCT application, publication No. W089 / 10134, which is incorporated herein by reference in its entirety), or alternatively, should involve direct administration of such MC4-R gene sequences to the site of the cells in which the gene sequences of the gene should be expressed. MC4-R. Alternatively, focused homologous recombination can be employed to correct the defective endogenous MC4-R gene in the appropriate tissue; for example, brain tissue. In animals, focused homologous recombination can be used to correct the defect in ES cells in order to generate offspring with a corrected characteristic.

Additional methods that can be employed to increase the overall activity of MC4-R gene expression and / or MC4-R activity include the introduction of appropriate MC4-R expressing cells, preferably autologous cells, into a patient in positions and in a sufficient number to improve the symptoms of body weight disorders, including obesity. Such cells may be either recombinant or recombinant. Among the cells that can be administered to increase the overall level of MC4-R gene expression in a patient are normal cells, either hypothala cells or those expressing the MC4-R gene. The cells can be administered at the anatomical site of the brain, or they can be part of a tissue graft located at a different site in the body. Such cell-based gene therapy techniques are well known to those skilled in the art, véasae, for example, Anderson et al., U.S. Patent No. 5,399,349; Mulligan & Wilson, American Patent Ha. 5,460,959. Finally, composite, identified in the tests described above, which stimulate or enhance the signal transduced by activated MC4-R, for example, by activating signaling proteins downstream in the MC4-R cascade and thereby avoiding the defective MC4-R, can be used in order to achieve weightloss. The formulation and the mode of administration will depend on the physico-chemical properties of the compound. The administration must include known techniques that allow crossing the hemata-encephalic barrier. 5.4.2. INHIBITION OF EXPRESSION MC4-R In an alternative embodiment, weight-increase therapy can be designed to reduce the expression level of endogenous MC4-R gene, for example by the use of antisense or ribasyme approaches in order to inhibit or prevent the transversion of MC4-R mRNA transcripts; triple helix approaches to inhibit transcription of the MC4-R gene; or homologous recombination focused to deactivate or "knock out" the MC4-R gene or its endogenous promoter. Such gene therapy can be used for the treatment of coroparal weight disorders such as for example cachexia and anarexia where the inhibition of the expression of MC4-R is intended to increase body weight. Since the MC4-R gene is expressed in the brain, delivery techniques should preferably be designed to cross the hemata-encephalic barrier (see PCT W098 / 10134, which is incorporated herein by reference in its entirety). Alternatively, the antiseptide, ribosome or DNA constructs described herein should be administered directly to the site containing the target cells. and to form a stable doulex (or trialex.) Antisense approaches include the design of oligonucleotides (either DNA or RNA) that are complementary to mRNA, antisense aligonucleotides will bind to complementary mRNA transcripts and will prevent translation. Although an absolute complementarity is preferred, it is not required A "complementary" sequence with a part of an RNA, in accordance with the one used here, refers to a sequence that has a sufficient complementarity to be able to hybridize with the RNA, forming an established duplex: in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA can therefore be tested, or a triplex formation can be tested.The ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid In general, the longer the hybridizing nucleic acid, the greater the number of mismatches of bases with a DNA that can contain and continue to form a stable duplex (or triplex, depending on the case). A person skilled in the art can determine a tolerable tier of mismatches by using standard procedures to determine the melting point of the hybridized complex. While antisense nucleotides complementary to the coding reaction sequence may be employed, those complementary to the translated transcribed na region are especially preferred. Oligonucleotides that are complementary to the 5 'end of the message, for example, the na sequence translated 5' up to and including the AUG initiation cadon, should function more efficiently to inhibit translation (see Figure 5). However, sequences complementary to the 3 'untranslated sequences of mRNAs have been effective in inhibiting the translation of mRNAs as shown recently. See, generally Wagner, R., 1994, Nature 372: 333-335. Thus, complementary oligonucleotides from either the coding, non-translated 5 'or 3' regions of MC4-R can be employed in an antisense approach to inhibit the translation of the endogenous mRNA. Oligonucleotides complementary to the na translatated region 5 'of the mRNA must include the initiation codon AUG. Antisense oligonucleotides complementary to the mRNA coding regimes are less efficient translational inhibitors but can also be used according to the invention. Whether designed to hybridize with the 5 'or 3' coding region of mRNA of MC4-R, the antisense nucleic acids must be at least 6 nucleotides in length, and are preferably alliganucleotides ranging from 6 to 50 pL lengths. In specific aspects, the oligonucleotide is at least 10 nucleotides, at least 17 pucleotides, at least 25 nucleotides or at least 50 nucleotides, Regardless of the choice of target sequence, it is preferred that the in vitro studies are carried out first for quantify the ability of the antisense ignucleotide to inhibit gene expression. It is preferred that these studies employ controls that distinguish between the antisense gene inhibition and the specific biological effect of the oligonucleotides. It is also preferred that these studies compare the levels of the white RNA or protein with the levels of an internal control RNA or protein. In addition, it is proposed that the results obtained by using the antisense aliganucleotide be compared with the results obtained by showing a control oligonucleotide. It is preferred that the control alliganucleotide be approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differ from the antisense sequence d by no more than necessary to avoid specific hybridization to the target sequence. The oligonucleotides can be DNA or RNA, to chimeric mixtures or derivatives or modified versions thereof, single strand or double strand. The oligonucleotide can be modified in the base portion, the sugar portion, or the phosphate structure, eg, in order to modify the stability of the molecule, hybridization, etc. The oligonucleotide may include other adjoining groups such as for example peptides (for example, to target receptors of host cells in vivo), or agents that facilitate transport across the cell membrane (see, for example, Letsinger et al., 1989, Prac. Nati, Acad. Sci. USA 86: 6553-6556; Le aitre et al., 1987, Proc, Nati. Acad. Sci. 84: 648-652; PCT Publication No. W088 / 09810, published December 15, 1988), or the blood-brain barrier (see, for example, PCT publication No. W089 / 10134, published April 25, 1988), dissociation agents triggered by hybridization (see, for example, Krol et al., 1988, BioTechniques 6: 958-976) or intercalation agents, (see, for example, Zan, 1988, Pharm. Res. 5 539-549). For this purpose, the oligonucleotide can be conjugated to another molecule, for example, a peptide, a cross-linking agent triggered by hybridization, transport agent, dissociation agent triggered by hybridization, etc. The antisense oligonucleotide may comprise at least a portion of modified base selected from the group including, but not limited to, 5-f-lurararacyla, 5-broraracil, 5-claruuracil, 5-iodouration, hypoxanthine, xaphan , 4-acetyl cytosine, 5- (carboxyhydro-imethyl) uracil, 5-carboxymethylaminomethyl-2-t-aiazidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylamine-luracila, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isapenteni ladenine, 1-methylguanine, 1-ethyl-ininasine, 2,2-dimethylguanine, 2-methyladenine, 2-methyl-Iguanine, 3-methylcitasine, 5-methylcytosine, N6-adsynine, 7-methylguanine, 5-methylaminomethyluracil, 5-methyl iaminometi 1-2-thiouracil, ß-D-manasilqueasine, 5'-methoxycarbaximeti luracyl, 'methoxyuracil, 2-methyl thio-N6-isapentenyladenine, uracil-5-oxyacetic acid (v), wibutoxosine, pseudouracila, queasin, 2-thiacytosine, 5-methyl 1-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracyl, uracil-5-oxyacetic acid methyl ester, 1-5-axiacetic uraci acid (v), 5-methyl-2-thiauracyl, 3- (3-amine-3-N-2-carboxypropyl) ) uracil, (acp3), and 2,6-diaminopurine. The antisense oligonucleotide may also comprise at least a selected selected modified sugar portion of the group including, but not limited to, arabinose, 2-f luoroarabinose, xylulose, and hexase. In another embodiment, the antisense oligonucleotide camps at least one modified phosphate structure selected from the group consisting of a phosphatharomate, a phosphoradithiatase, a phospramidothioate, a phosphoramidate, a phosphardiamidate, a methyl phosphanate, an alkyl phosphatriéster, and a fsmartketal or analogue the same.

In another embodiment, the antisense oligonucleotide is an oligonucleotide -anamic. An α-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA where, unlike the usual β-units, the strands are parallel to each other (Gautier et al., 1987, Nucí Acids Res. 15: 6625-6641) . The oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al., 1987, Nuci Acids Res. 15: 6131-6148), or a chimeric RNA-DNA analogue (Inaue et al., 1987, FEBS Lett. 215: 327-330). Oligonucleotides of the invention can be synthesized by standard methods known in the art, e.g. by the use of an automated DNA synthesizer (such as those commercially available from Biosearc, Applied Biosystems, etc.). As examples, phosphorothialate oligonucleotides can be synthesized by the method of Stein et al. (1988, Nucí Acids Res. 16: 3209), ethylphosphonate oligonucleotides can be prepared by the use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Nati. Acad. Sci. USA 85: 7448-7451), etc. The antisense molecules must be administered to cells expressing MC4-R in vivo, eg, neural tissue. Several methods have been developed for administering antisense DNA or RNA to cells; for example, antisense molecules can be injected directly into the tissue site, either modified antisense molecules, designed to target desired cells (eg, antisense bound to peptides or antibodies that bind specifically to receptors or expressed antigens). on the surface of the target cell) can be administered systemically. However, it is often difficult to achieve sufficient dsl antisense intracellular concentrations to suppress the translation of endogenous mRNAs. Necessarily, a preferred approach employs a recombinant DNA construct wherein the antisense oligonucleotide is placed under the control of a strong PIIII or PIIII promoter. The use of said drug to transfect target cells in a patient will result in the transcription of sufficient quantities of single-stranded RNAs that formed complementary base pairs with the endogenous MC4-R transcripts and will therefore avoid the translation of MC4- R. For example, an in vivo vector can be introduced in such a way that it is picked up by a cell and directs the transcription of an antisense RNA. Said vector can remain episomal to either chromosomally integrated, insofar as it can be trapscribed to produce the desired antisense RNA. Such vectors can be constructed by refill DNA technology methods standard in the art. The vectors can be plasmids, of viral origin, or of another type known in the art, used for replication and expression in mammalian cells. The expression of the sequence encoding the antisense RNA can be any promoter known in the art to act on mammalian cells, preferably human cells. Talss promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 initial promoter region (Bernoist and Chambon, 1981, Nature 290: 304-310), the promoter contained in the 3 'long-end repeat of Raus sarcoma virus (Yamamoto et al. ., 1980, Cell 22: 787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Nati. Acad, Sci. USA 78: 1441-1445), the regulatory sequence of the metalatianein gene (Bripster et al., 1982, Nature 296: 39-42), etc. Any type of plasmid, ida, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site; for example, the choroid plexus to the hypatalama. Alternatively, viral vectors can be used that selectively infect the desired tissue; (for example, in the case of the brain, the herpes virus vectors can be used), in which case the administration can be achieved in another way (for example, systemically).

Ribosome molecule designed to dissociate transcripts from MC4-R mRNAs can also be used to prevent the translation of MC4-R mRNA and the expression of MC4-R. (See, for example, PCT international publication W090 / 11364, published October 4, 1990; Sarver et al., 1990, Science 247: 1222-1225). While ribosomes that dissociate mRNA in sequences of site-specific recognitions can be used to destroy MC4-R mRNAs, the use of hammerhead ribas is preferred. Hammerhead ribosomes dissociate mRNAs in hubs indicated by flank regions that form complementary base pairs with the white mRNA. The only requirement is that the white mRNA has the following sequence of two bases: 5'-UG-3 '. The production of hammerhead ribosomes is well known in the art and is described more fully in Haselsff and Gerlach, 1988, Nature, 334: 585-591. There are hundreds of potential hammerhead ribasima dissociation sites within the nucleotide sequences of human MC4-R cDNA (see Figure 5). Preferably, the ribose is genetically engineered such that the dissociation recognition site is near the 5 'end of the MC4-R mRNA; that is, to increase the efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

The rabzymes of the present invention also include RNA endaribonucleases (hereinafter, "Cech-type ribosims") such as those occurring naturally in Tetrahymena Thermaphila (known as IVS, or IVS L-19 RNA), and which have been described extensively by Tomas Cech and Calaboradores (Zaug, et al., 1984, Science, 224: 574-578, Zaug and Cech, 1986, Science, 231: 470-475, Zaug, et al., 1986, Nature, 324: 429-433, published International Patent Application No. WO 88/04300 by University Patsnts Inc., Been and Cech, 1986, Cell, 47: 207-216). The Cech type ribazymes have an active site of eight base pairs that hybridizes to a target RNA sequence after which the dissociation of the target RNA is carried out. The invention encompasses Cech-type ribozymes that focus on active site sequences of eight base pairs present in MC4-R. As in the antisense approach, ribozymes can be composed of modified oligonucleotides (eg, for improved stability, focus, etc.) and should be administered to cells expressing MC4-R in vivo, eg, hypatlama. A preferred method of delivery includes the use of a DNA strain that "encodes" the ribozy under the control of a strong poly constitutive pol III or poly II promoter such that the transfected cells produce sufficient quantities of the RG, ribazi to destroy the endogenous MC4-R messages and inhibit the translation. Since ribozymes, unlike antisense molecules, are catalytic, a lower intracellular concentration is required to obtain efficiency. The endogenous gene expression of MC4-r can also be reduced by deactivating or "knock-out" of the MC4-r gene by its promoter using focused homologous recombination (eg, see S ithies et al., 1985, Nature 317: 230 -234; Thomas? <Capecchi, 1987, Cell 51: 503-512, Thompson et al., 1989 Cell 5: 313-321, each of which is incorporated herein by reference in its entirety). For example, a non-functional, mutant MC4-R (or a totally unrelated DNA sequence) flanked by a DNA homologous pair to the endogenous MC4-r gene can be used, with or without a selectable marker and / or a selectable marker. negative, to transfect cells expressing MC4-R in vivo. The insertion of the DNA construct, through focused homologous recombination, results in the deactivation of the MC4-r gene. Such approaches are especially suitable in the field of agriculture where modifications to ES cells (embryonic stem) can be employed to generate animal downyers with an inactive MC4-R (see, for example, Thomas & amp;; Capecchi 1987 and Thompson 1989, supra). However, this approach can be adapted for use in humans provided that the recombinant DNA constructs are administered directly or focused to the required site in vivo using appropriate viral vectors, eg, herpes virus vectors 5 for administration to the brain tumor; for example, the hypothalamus and / or choroid plexus. Alternatively, the endogenous MC4-R gene expression can be reduced by the approach of deoxyribonucleotide sequences complementary to the region ^ Regulator of the MC4-R gene (ie, MC4-R promoter and / or enhancers) to form triple helical structures that prevent transcription of the MC4-R gene in target cells in the body. (See, generally, Helene, C. 1991, Anticancer Drug Des., 6 (6): 569-84; Helene, C., et al., 1992, 15 Ann, NY Acad. Sci., 660: 27-36 and Maher, LJ, 1992, Biaassays 14 (12): S07-15). A 5.4.3. ADMINISTRATION OF SOLUBLE MC4-R P0LIPEPTIDES Genetically engineered cells expressing soluble MC4-R ECDs or fusion proteins for example fusion molecules can be administered in vivo where they can function as "bioreactors" that supply soluble molecules. Such soluble MC4-R palpeptides and fusion proteins, when expressed at appropriate concentrations, neutralize or "sweep" the native ligand for MC4-R, and therefore act as inhibitors of MC4-R activity and induce Increased weight. 5.5 PHARMACEUTICAL FORMULATIONS AND METHODS FOR THE TREATMENT OF BODY WEIGHT DISORDERS The invention encompasses methods and compositions for modifying body weight and for treating body weight disorders, including, but not limited to, obesity, cachexia and anorexia. Since a loss of the normal MC4-R gene product function results in the development of an obese phenotype, an increase in MC4-R gene product activity, or activation of the MC4-R pathway (plo axis pair, downstream activation) I should facilitate the progress toward a normal body weight status in obese individuals who have a deficient level of MC4-R gene expression and / or MC4-R activity. In terpative terms, symptoms of some body weight disorders, such as cachexia, involving a lower body weight phenotype than normal, can be improved by decreasing the expression level of MC4-R gene, gene activity of MC4-R, and / or through a down-regulation activity of the MC4-R pathway (for example, by focusing downstream signaling events). Here are several approaches. MC4-R agonists can be used to induce weight loss to treat obesity. MC4-R activity antagonists can be used to induce weight gain to treat conditions such as anorexia to cachexia. < a it is necessary that the composite demonstrates absolute specificity for the MC4-R. For example, compounds that agonize both MC4-R and MCl-R can be employed; such compounds should be administered in such a way that administration to the brain is optimal to achieve weight reduction, and side effects, as for example peripheral production of melamine resulting in a "tan" can be well tolerated. Compounds that do not demonstrate a specificity for MC4-R may be administered in combination with another therapy or drug to control the side effects that may result from the modulation of another melanacortin receptor; however, compounds demonstrating a preference or selectivity for MC4-R compared to MC3-R are preferred since both receptors are expressed in the brain where a localized administration can not be employed to compensate for the lack of receptor-specificity. 5.5.1. DOSAGE DETERMINATIONS The toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD50 (the lethal dose for 50% of the population) and ED50 (the dose therapeutically effective in 507. of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio in LD50 / ED50. Compounds that show large therapeutic indices for the preferred compounds. While compounds that have toxic side effects can be used, care must be taken to design a delivery system that focuses such compounds towards the affected tissue site in order to minimize potential damage to uninfected cells and, consequently, reduce side effects. The data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for use in human beings. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. For any compound employed in the method of the invention, the therapeutically effective dose can be initially drawn from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration within a range that includes the IC50 (ie, the concentration of the test compound that achieves maximum medi-symptom inhibition) in accordance with that determined in cell culture. This information can be used to determine more accurately useful doses in human beings. Plasma levels can be measured, for example, by high performance liquid chromatography. 5.5.2. FORMULATIONS AND USE Pharmaceutical compositions for use in accordance with the present invention can be formulated in conventional manner employing one to several physiologically acceptable carriers or excipients, Thus, the compounds and their physiologically acceptable compounds or solvates can be formulated for administration by inhalation or insufflation (either through the mouth to the nose) to either oral, oral, parenteral or rectal administration. In the case of oral administration, the pharmaceutical compositions may take the form, for example, of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as for example binding agents (eg, pregelatinized maize starch, pal ivinyl pyrralidone or well hydroxypropylmethyl cellulose); fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (for example, potato starch or sodium starch glycolate); or humedecimiepto agents (for example, lauryl sulphite sodium). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or another suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (eg, sarbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (for example, lecithin or acacia); non-aqueous vehicles (for example, almond oil, fatty esters, ethyl alcohol, or else vegetable oils that are broken); and canservatives (for example, methyl or propyl-p-hydraxibepzoates or sorbic acid). The preparations may also contain reagents, salts, flavors, colorants, sweetening agents as appropriate. Preparations for oral administration can be suitably formulated to provide a controlled release of the active compound. For buccal administration, compositions may have ß: the form of tablets or pills formulated in a conventional manner. For administration by inhalation, the compounds for use in accordance with the present invention are conveniently administered in the form of an aerosol spray presentation from low pressure or spray packings, with the use of a suitable impeller, for example, diclarodif luoromethane, tri cloraf luoro tana, dichlorotetraf luoroethane, carbon dioxide or another suitable gas. In the case of a low pressure aerosol, the dosage unit can be determined by supplying a valve to administer a smaller amount. Capsules and cartridges, for example gelatin for use in an inhaler or insufflator can be formulated, which contain a powder mixture of the compound and a suitable powder base such as for example lactose or starch. The compounds can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection can be pressed in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The fields may have suspensions, solutions or emulsions in oil or aqueous vehicles, and may contain formulating agents such as suspension, stabilization and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile, pyrogen-free water, before use. The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, eg exemplary, which contain conventional suppository bases such as cocoa butter, or other glycerides. In addition to the formulations described previously, the compounds can also be formulated as depot preparation. Such long-acting action formulations can be administered by implant (e.g., subcutaneous or intramuscular) to either intramuscular injection. Thus, for example, the compounds can be formulated with suitable palmeric or hydrophobic materials (for example, as an emulsion in an acceptable ac ite) or ion exchange resins, or as sparingly soluble derivatives, for example, as a scant salt soluble entity. The compositions may, if desired, be presented in a package or delivery device which may contain one or more unit dosage forms which contain the active ingredient. The package can, for example, comprise a sheet of metal or plastic, for example a blistsr type packaging. The packaging to supply device can be accompanied by instructions for administration. 5.6. DIAGNOSIS OF ABNORMALITIES OF BODY WEIGHT DISORDER Mutations at several different genetic loci have been identified which lead to phenotypes related to body weight disorders. Ideally, the treatment of patients suffering from such body weight disorders will be designed to focus on the particular genetic loci that hold the mutation. Accordingly, diagnostic methods that identify mutations in specific genes related to disorders of body weight, for example the MC4-r gene, will allow the treatment of body weight disorders through the focus of the mutated gene. Several methods can be used for evaluating diagnosis and prognosis of body weight disorders, including bowel disease, cachexia and anorexia, and for the identification of subjects who have a predisposition for such disorders. Such methods may employ, for example, reagents such as the MC4-r gene nucleotide sequences and antibodies focused against MC4-r gene products, including peptide fragments thereof.

Specifically, such reagents can be used, for example, for: (1) detecting the presence of MC4-r gene mutations, or detecting either the taste-expression or the mRNA sub expression of MC4-r gene in relation to the na status of body weight disorder; and (2) the detection of either the over-abundance or the sub-abundance of the MC4-r gene product in relation to the unsettled body weight disorder. The methods described here can be carried out, for example, by the use of sets of pre-packaged diagnostic elements comprising at least one nucleic acid reagent of MC4-r gene specific to either an anti-MC4-antibody reagent. r, which can be conveniently employed, for example, in clinical settings, to diagnose patients showing abnormalities of body weight disorder. For the detection of MC4-r mutations, any nucleated cell can be used as the initial source for genomic nucleic acid. For the detection of MC4-R gene expression or MC4-R gene products, any type of cell or tissue in which the MC4-r gene is expressed as an axis of cerebral cells can be used. Next, in section 5.6.1. detection techniques based on nucleic acid are described. Peptide detection techniques are described, below, in section 5.6.2. 5.6.1. DETECTION OF NUCLEIC ACID MOLECULES OF MC4-r GENE Predisposition to body weight disorders can be determined by testing any tissue to determine mutations of the MC4-r gene. For example, a person who has inherited a germline MC4-4 mutation will likely develop an obese phenotype. In addition, a prenatal diagnosis can be carried out by testing fetal cells, placental cells, or fluid to nyotics for mutations of the MC4-R gene. Alterations in the MC4 ~ r allele can be detected using any of the methods presented here. Mutacians with the MC4-r gene can be detected by using several techniques. Nucleic acid can be used from any nucleated cell as the starting point for such assay techniques, and can be isolated in accordance with standard nucleic acid preparation procedures well known from those in the art. DNA can be used in hybridization assay or amplification of biological samples to detect anamalities that involve the MC4-r gene structure, including point mutations, insertions, deletions and chromosomal rearrangements. Such assays may include, but are not limited to, Southern analysis, single-strand conformalnal polymorphism analysis fSSCP), and polymerase chain reaction analysis. Techniques well known in the art can be used to explore regions of a selected gepam apart from it, to determine the presence of variants to mutations, either known or unknown. Methods that can be used to detect such variants to mutations in the MC4-r gene include the direct sequence (Maxam «• Gilbert, 1980, Methods in Enzymalag / 65: 499-560; Sanger et al., 1977, Proc. Acad. Sci. 74: 5463); the sequence is observed by sets of a3 igonucleotides (Fador et al., 1993, Nature 364 s 555-556); Southern blot analysis or in field-pulsed gel (PFGE) (Schwartz et al., 1984, Cell 37:67); single strand conformation analysis ("SSCA") (Opta eet al., 1989, Prac. Nati. Acad. Sci. USA 86: 2776-2770); heteroduplex analysis (HA) (een et al., 1991, Trends Genet, 7: 5); denaturing gradient gel electrophoresis ("DGGE") (Wartell et al., 1990, Nucí Acids Res. 18: 2699-2705; Myers st al., 1985, Nucleic Acid Research 13: 3131-3145); Denaturing HPLC (Underhill, P.A., 1997, Proc.Nat.Acid Sci USA 93: 196-200); RNase protection assays (Fin elstein et al., 1990), Genotics 7: 167-172; msler te al., 1991, Science 251: 1366-1370; Myers, R.M. et al., 1985, Science 230: 1242-1246); hybridization of allele-specific aliqanucleotides ("ASO") (Conner et al., 1983, Proc Nati Acad Sci 80: 278-282; Wallace, RB, et al., 1979, Nuci Acids Res. 6: 3543-3557; Sait-i, Rr et al., 1989, Proc. Nati. Acad. Sci. USA 86: 6230-6234); Oligonucleotide binding assay ("OLA") (Landegren, U., et al., 1988, Science 241: 1077-1080; Tobe, VO, et al., 1996, Nucí. Acids Res. 24: 3728- 3732); sequence-specific amplification (Newton, C.P. et al., 1989, Nucí Acids Res. 17: 2503-2516); chemical dissociation of mismatches (CMC) (Catttan, R.G.H. et al., 1988, Nucí Acids Res. 17: 4223-4233); Enzymatic Dissociation of Mismatches (EMC) (Boabon J.J., R. et al., 1995, Nucí Acids Res. 23: 5082-5084, Marshall, R.D. et al., 1995, Nat. Genet. 9: 177-183); and the use of proteins that recover nucleotide mismatches, such as, for example, the E. coli mutS protein (Modpch, 1991, Ann. Rev. Genet, 25: 229-253). Diagnostic methods that can be used to detect mutations at the MC4-r locus can be classified into two general categories of use. The first category of diagnostic methods involves diagnostic methods designed to explore a region for an unknown vapant. These diagnostic methods can also be applied to the detection of known variants. The second category of diagnostic methods includes methods designed specifically for variants of the previously identified type, but not usually employed for the detection of unknown variants. Diagnostic methods that can be employed for variants of previously identified type can include, but are not limited to, allele-specific oligonucleotide (ASO) hybridization, and alliganucleotide (OLA) ligation assay, as well as sequence-specific amplification. In one embodiment, allele-specific oligonucleotide hybridization is employed in order to detect a previously identified variant (s) or allele (s) of MC4-r. The hybridization of oligonucleotides specific for allele comprises the hybridization separated from a pair of oligonucleotides, specific for the previously identified allele, with DNA or gepomic RNA amplified by polymerase chain reaction, under conditions that discriminate between complete correspondences and mismatches of single base. The pair of oligonucleotides should cover the base variant. Under the appropriate reaction conditions, the target DNA is not amplified if there is a base mismatch (eg, a nucleotide substitution caused by a mutation) or a small removal or insertion, at the 3 'end of the primer (Okayama et al. al., 1989, J. Lab. Clin. Med. 114: 105-113; Sommer et al., 1992, BioTechniques 12: 82-87). In a specific embodiment of the invention, the following oligonucleotide = or its complements can be used to identify variants of MC4-r. To identify the Ilel37Thr mutation, the following nucleotides can be used: 5'-CACTT-3 '5'-ATCCACTTGC-3' 5'-TGCATCCACTTGCAG-3 '5'-GCTTGCATCCACTTGCAGCC-3' 5'-CT GCTTGCATCCACTTGCAGCCTG-3 '" 5'-CTCCTTGCTTGCATCCACTTGCAGCCTGCT-3 '.

To identify the Val1021 mu mutation, the following oligonucleotides can be used: 5'-CCATT-3 '5'-AAACCATTAT-3' 5'-CAGAAACCATTATCA-3 '5'-GATCAGAAACCATTATCATC-3' 5'-ATGGATCAGAAACCATTATCATCAC-3 '5 '-CAAATGGATCAGAAACCATTATCATCACCC-3'. To identify the Thrll2Met mutation, the following oliganucleotides can be used; '-TATGG-3 '5'-AGATATGGAT-3' 5'-TACAGATATGGATGC-3 '5'-CAGTACAGATATGGATGCAC-3' 5'-GTACAGTACAGATATGGATGCACAG-3 '5'-ACAGTACAGTACAGATATGGATGCACAGAG-3'. In another embodiment, an oligonucleotide ligation assay (OLA) is used to discriminate a single base mismatch, through the use of a pair of oligonucleotides complementary to the preselected alleles. However, ^^ in an alliganucleotide ligation assay, the two dsl oligonucleotides are bound in a manner dependent on the binding at the site of the variant base. The white or tempered sequence is typically a region of the gene that spans the variant base and is preferably amplified by PCR from DNA or genomic RNA. In a manner similar to that described above, a specific amplification by ? The sequence is based on palimerase chain reaction primers designed in such a way that the variant base is located at the 3 'end of one of the polymerase chain reaction primers. Thus, amplification by polymerase chain reaction depends on the complete tempering of this primer and will not be carried out if there is a single base mismatch. In specific embodiments, the direct sequence of polymerase chain reaction products amplified from DNA or genomic RNA can be achieved using either fluorescent or radioactive methods well known to those skilled in the art. In another embodiment, the resequencing of these products of the chain reaction of palimerase is achieved more rapidly by hybridization, with high density assemblies, of oligonucleotides representing the wild-type sequence of the MC4-R gene (Ha tori, M., 1993, Genomics 15: 415-417). In another embodiment, a Southern blot-driven field gel analysis (PF6E) can be employed to identify sequence variants that cancel or create new restriction sites. In another modality, variants are identified by single-strand canformation analysis (SSCA) by virtue of their effect on the canformaci? P of the DNA molecule. Regianes containing known variants or regions searched for variants are amplified by polymerase chain reaction. These products are denatured and subjected to electrophorei = through polyacrylamide gels under denaturing conditions that allow the single-stranded molecule to retain its interactions between strands. Changes in single base affect these interactions and therefore alter the speed of migration of the molecule through the gel. Thus, variants are identified as fragments of altered mobility. In another embodiment a method related to SSCA of heteroduplex analysis detects unique mismatches of base pairs in the sequences of double-stranded molecules assembled by detecting the altered conformations of such molecules. Such conformational changes will affect the migration of the double-stranded molecules assembled through polyacrylamide gels under non-denaturing conditions. In another embodiment, denaturing gradient gel electrophoresis (DGGE) can be used to identify variants based on the difference in melting temperature between two DNA fragments with a single base pair difference. Dable strand molecules are subjected to electrophoresis through a polyacrylamide gel containing a gradient of increasing denaturant, for example, denaturant, such that at a defined point a molecule begins to denature and migrate more slowly. case of SSCA and HA, variants are identified by their altered mobility in the gel. In another embodiment, denaturing HPLC can be used to reveal variants that alter the conformation of the DNA fragment and therefore affect the rate of migration through the chromatography columns. Some methods of denaturing HPLC include denaturation and binding of a DNA or test RNA in a control- to allow the formation of heteroduplex, as in HA analysis. Subsequently, chemical substances or enzymes are used to dissociate one or two strands at or near the mismatch. In one embodiment, chemical mismatch dissociation (CMC) is employed, which employs osmium tetroxide, hydroxyla and piperidine to dissociate one or both strands.

In another embodiment, enzymatic mismatch dissociation (EMC) is employed employing mismatch dissociation enzymes such as T4 endonuclease VII, in order to dissociate one or both strands. In another embodiment, RNas protection assays are used to detect an RNA: RNA mismatch. The RNase protection assays exploit the ability of RNase A to dissociate RNA: RNA mismatches. DNA amplified from genomic DNA or RNA is reverse transcribed in cRNA. The test sample cRNA is linked onto a wild-type cNN of the control. It is then possible to detect variants of co sequences or mismatches between the two cRNA molecules dissociated by RNase. The dissociation is revealed by the electrophoretic determination of the size of the products. Among the preferred MC4-r nucleic acid sequences for such hybridization and / or analysis in PCR, are the sequences that detect the presence of MC4-r gene mutations described below in section 8.2. The gene expression level of MC4-r can also be tested. For example, RNA can be isolated from a well-suspected type of cell or tissue known to express the MC4-r gene, such as the brain, and can be tested using hybridization or polymerase chain reaction techniques. such as those described above. The isolated cells may be derived from cell culture or from a patient. Analysis of the cells taken from the culture may be a necessary step in the evaluation of the cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the MC4 gene. -r. Such analyzes can reveal quantitative and qualitative aspects of the expression pattern of the MC4-r gene, including activation or deactivation of MC4-r gene expression. In one embodiment of said detection scheme, a cDNA molecule is synthesized from an RNA molecule of interest (e.g., by reverse transcription of the RNA molecule in cDNA). A sequence within the cDNA is then used as annealing for a nucleic acid amplification reaction, e.g., amplification reaction by polymerase chain reaction, to like. The nucleic acid reagents employed as synthetic initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from the MC4-r gene nucleic acid reagents described in section 5.1. The preferred lengths of such nucleic acid reagents are at least 9-30 nucleotides. For the detection of the amplified product, nucleic acid amplification can be carried out using radioactively labeled or non-radioactively labeled nucleotides. Alternatively, a sufficient amount of amplified product can be made in such a manner that the product can be visualized by standard ethidium bromide staining or else by the use of any other suitable method of nucleic acid staining. Additionally, it is possible to carry out MC4-r gene expression assays "in situ", that is, directly on tissue sections (fixed and / or frozen) of tissue from a patient obtained from biopsies or resections, in such a way no nucleic acid purification is necessary. Nucleic acid reagents such as those described in section 5.1 can be used as probes and / or primers for such in situ procedures (see, for example, Nuova, 6.J., 1992, "PCR In Situ Hybridization: Pratacals and Applications" , Raven Press, NY). Alternatively, if a sufficient amount of the appropriate cells can be obtained, a standard Northern analysis can be carried out to determine the level of mRNA expression of the MC4-r gene. 5,6.2. DETECTION OF MC4-r GENE PRODUCTS Antibodies directed against wild-type or mutant MC4-r gene products or conserved variants or peptide fragments thereof can also be used as diagnostic and prognostic indicators of body-weight disorder, in accordance with the one described here. Such diagnostic methods can be used to detect anomialities in terms of the level of expression of the MC4-r gene, or abnormalities in structure and / or temporal, tissue, cellular or subcellular localization of the gene product of MC4-r. Antibodies, or fragments of antibodies, such as those described below, can be used to screen patencially therapeutic compounds in vitro to determine their effects on MC4-r gene expression and the production of MC4-r peptide and the production of MC4 peptide. -r. Compounds that have beneficial effects on body weight disorders such as obesity, cachexia and anarexia, can be identified, and a therapeutically effective dose can be determined. Immunoassays in vitro can also be used, for example, to evaluate the efficacy of a gene therapy based on cells for body weight disorders, including obesity, cachexia and aporexia. Antibodies directed against MC4-r peptides can be used in vitro to determine the level of MC4-r gene expression achieved in cells genetically engineered to produce MC4-r peptides. Such analysis will allow the determination of the number of transformed cells necessary to reach the therapeutic efficacy in vivo, as well as the aptimization?; of the gene replacement protocol. The tissue or cell type to be analyzed will generally include those of which it is known or suspected to express the MC4-r gene, such as for example brain cells. The protein isolation methods planted here may, for example, be such as those described in Harlow and Lane (Harla, E. and Lane, D., 1988, "Antibadies: A Laboratory Manual", Cold Spring Harbor Labaratary Press, Cold Spring Harbor, New York), which is hereby incorporated by reference in its entirety. The isolated cells can be derived from cell culture to a patient's well. Cell analysis taken from cult can be a necessary step in the evaluation of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compound on the gene expression. of MC4-r. Preferred diagnostic methods for the detection of wild type mutant MC4-r gene products or conserved variants or peptide fragments thereof may involve, for example, immunoassays where the MC4-ra gene products are well-conserved variants or Peptide fragments are detected due to their interaction with an antibody specific for anti-MC4-r gene product. For example, antibodies, or fragments of antibodies, such as those described above in section 5.3, useful in the present invention can be used to quantitatively or qualitatively detect the presence of wild type or mutant MC4-r gene products at conserved variants or fragments of peptides thereof. This can be achieved, for example, by immunofluorescence techniques employing a fluorescently labeled antibody (see below this section) coupled with microscopic detection of light, flow cytometric, or fluorescence. Such techniques are especially preferred if such MC4-r gene products are expressed on the surface of the cell. Antibodies (or fragments thereof) useful in the present invention can be further employed, histologically as in immunofluorescence to immunaelectronic microscopy, for the in situ detection of mutant wild-type MC4-r gene products or variants conserved or peptide fragments thereof. In situ detection can be achieved by the reoction of a histological sample from a patient, and by the application of a labeled antibody of the present invention. The antibody (or fragment) is preferably applied by placing the labeled antibody (or fragment) in a biological sample. Through the use of such a method, it is possible to determine only the presence of MC4-r gene product, or of conserved variants to peptide fragments, but also its distribution in the one examined. Employing the present invention, persons with certain knowledge in the art will readily determine that any of several histological methods (such as staining procedures) can be modified in order to achieve such detection in situ. Immunoassays for wildtype or mutant MC4-r gene products or knockout variants or peptide fragments thereof will typically comprise incubation of a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or Used from cells that have been incubated in cell culture, in the presence of a detectably labeled antibody capable of identifying wild-type or mutant MC4-r gene products or conserved variants or peptide fragments thereof, and detecting the antibody bound by any of several techniques well known in the art. The biological sample may be contacted with a solid-phase carrier vehicle or it may be immobilized in said carrier or vehicle as an example of nitrocellulose, or else another solid support capable of immobilizing cells, cell particles, or soluble proteins. The support can then be washed with suitable regulators followed by treatment with the antibody specific for MC4-r gene detectably labeled. The solid phase support can then be washed with the regulator a second time to remove the bound na antibody. The amount of label bound on the solid support can then be detected by conventional means. By "solid phase carrier or vehicle", we mean any carrier capable of binding an antigen or an antibody. Supports to well-known vehicles include glass, polystyrene, polypropylene, polyethylene, dextran, nylap, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the vehicle may be either soluble to some extent or insoluble for the purposes of the present invention. The support material can have virtually any possible structural configuration insofar as the coupled molecule can bind on an antigen or antibody. Thus, the configuration of soup can be spherical, eat in a pearl, well cylindrical, co or in the case of the internal surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, a test band, etc. Preferred supports include palmateirane beads. Those skilled in the art will know other vehicles suitable for binding aptigepo or antibody, or may determine them by the use of routine experimentation. The binding activity of a given batch of anti-MC4-r gene product antibody will be determined in accordance with well-known methods. Those skilled in the art will be able to determine the optimal and operative test conditions for each determination by employing routine experiments. One of the ways in which the antibody specific for MC4-r gene peptide can be detectably labeled is by linking said antibody with an enzyme and the use of an enzyme immunoassay (EIA) (Vol ler, A ., "The Enzyme Linked Immunasarbent Assay (ELISA)", 1978, Diagnostic Horizons 2: 1-7, Micrabialogical Associates Quarterly Publicatian, Waikersville, MD); Valler, A. et al., 1978, J. Clin. Pathol. 31: 507-520; Butler, J.E., 1981, Meth. Enzymol. 73: 482-523; Maggia E. (ed), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL; Ishikawa, E. et al., (Eds.), 1981, Enzyme Immunoaesay, Kgaku Shain, Tokia). The enzyme bound on the antibody will react with a. Suitable substrate, preferably, a cramatogenic substrate, in such a way that a chemical portion that can be detected is produced, for example, by means of spectra-photic, optical, or visual means. Enzymes that may be used to detectably label the antibody include, but are not limited to, alata dehydrogenase, staphylococcal nuclease 1, α-5-esterase-yeast, yeast-alcohol dehydrogenase, f-glyraphosphate, dehydrogenase. , triosphosphataisamerase, horseradish peroxidase, alkaline phosphatase, asparagmase, glucose axidase, beta-galactosides, pbanuclease, ureas, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcalmesterase. Detection can be achieved by calorimetric methods that employ a chromosomal substrate for the enzyme. Detection can also be achieved by visual comparison of the magnitude of the enzymatic reaction of a substrate compared to standards prepared in a similar manner. Detection can also be achieved by the use of any of several other immunoassays. For example, by radioactive labeling of antibodies or antibody fragments it is possible to detect MC4-ra gene peptides through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioi munoassays, Seventh Training Caurse on Fíadialigand Assay Techniques, The Endacrine Society, March 1986, which is incorporated by reference here). The radioactive isotope can be detected by means such as the use of a gamma counter to a scintillation recorder or by autoradiography. It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of an appropriate length of walking, its presence can be detected due to fluorescence. Among the fluorescent marker compounds most frequently used are the isothiocyanate of luorescein, rhodamine, phycoerythrin, phycocyanin, alsficacianipa, a-phthaldehyde and luorescamine. The antibody can also be detectably labeled using metals that emit fluorescence such as 152Eu, to others of the lanthanide series. These metals can be fixed on the antibody using metal chelation groups such as diethylenetriaminpentaacetic acid (DTPA) or ethylenediaetraeacetic acid (EDTA). The antibody can also be detectably labeled by its coupling to a chemoluminiscept compound. The presence of the chemo-luminescent-labeled antibody is then determined by detecting the presence of the luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemoxide labeling compounds are luminol, isolu inol, therameric acridinium ester , imidazole, acridinium salt and axalata ester.

In the same way, a bialuminescent compound can be used to label the antibody of the present invention. The bial or ipiscence is a type of qui ialuminiscence found in biological systems even though a catalytic protein increases the efficiency of the chemiluminescence reaction. The presence of an in vivo bialu pratein is determined by detecting the presence of the luminescence. Important bialuminescent compounds for labeling purposes are luci ferin, luciferase and aecarin. 6. EXAMPLE: GENERATION OF A DEFICIENT MOUSE IN MC4-R The following example describes the genetic manipulation and the generation of "knocked out" mice in which the endogenous MC4-r is deactivated. The results show that knocked down ratans gain weight, which demonstrates the role of MC4-R in regulating body weight. 6.1. MATERIALS AND METHODS 6.1.1. IDENTIFICATION OF THE MURINE MC4-R GENE The murine melanacartin 4 (MC4-r) receptor gene was isolated from a genomic phage library of mouse strain 129 / Sv, obtained from Stratagene, using a human MC4-r probe. . The human probe was generated by amplification by polymerase chain reaction of the MC4-r coding sequences from human genetic DNA using the following primers: 5 '-ATA GTC GAC ATS GTG AAC TCC ACC CAC CGT-3; and 5 '-TAT AAG CTT TTA ATA TCT GCT AGA CAA TTC-3'. Two positive phage clones containing the MC4-r gene were identified, and the MC4 ~ r locus was subcloned from the phage in pBluescript II as a Hmd III fragment of about 5 μl; b, and a Sac I fragment of approximately 4.7 r. These subclauses were mapped by restriction and partially sequenced to produce the lac map of MC4-r illustrated in Figure 2A. In order to deactivate MC4-r, a focus construct was constructed that was able to remove the mavone from the MC4-r coding sequences after homologous recollection with the endogenous MC4-r locus. 6.1.2. GENERATION OF THE FOCUS CONSTRUCTION The focus construct of MC4-r was constructed in the following manner. The 1.4 Kb Eco RI-Ava I fragment from pBR322 was replaced with the following synthetic oligonucleotides: 5 '-AAT TAG CGG CCG CAG TAT GCA AAA AAA AGC CCG CTC ATT AGG CGG GCT-3'; and 5 '-CCG AAG CCC GCC TAA TGA GCG GGC TTT TTT TTG CAT ACT GCG GCC GCT-3'. The resulting plasmid, called pJNI, was digested with Not I and the following oligonucleotides were ligated into the Not I site. 5 '-GGC CGG CAT GCA TCA AGC TTA TCT CGA GAT CGT CGA CTA CCA TGG TAC ATC GAT CAG GTA CCA TCC CGG GGC-3 '; and 5 'GGC CGC CCC GGG ATG GTA CCT GAT CGA TGT ACC ATG GTA GTC GAC GAT CTC GAG ATA AGC TTG ATG CAT GCC-3'. The resulting plasmid was called pJN2. The 1.2 Kb Sph I-Hipd III 3 'fragment of the MC4-r gene (see Figure 2A) was subclained in pJN2 digested by Sphl-Hind III to generate the plasmid 3' KO of MC4-r (Figure 2B). This fragment represents the 3 'region of genomic homology in the focus vector. A 3.4 Kb Ncal-Hind III fragment, including the first 20 nucleotides of the MC4-r gene (see Figure 2A), was removed as Ncal-Asp718 fragment from the MC4-r subclone locus. The Asp718 site was derived from pBluescript II polyadense sequences that immediately flank the native 5 'Hind III site of approximately 3.4 Kb of the MC4-r gene (Figure 2A). This fragment, which represents the 5 'region of genomic homology in the approach canstructa, was ligated into 5' KO of MC4 ~ r digested by Ncol-Asp 718 to produce 5 '3' KO of MC4-r (FIG. 2C). The PG -neo expression cassette of plasmid p Jl (Tybulewicz et al., Cell 65, 1153-1163, 1991), which contains the nea gene lowers the transcriptional control of the mouse phosphaglyceratinase (F'GK-1) promoter and the site of poly (A) addition of PGK-i was subclained as Eco RI-Hind III fragment in pGEM 7-Zf (+) digested with Eco RI-Hind III to generate pSEM 7 (KJi). The 1.7 Kb fragment that contains the n: PGK-neo expression cassette was removed by: 1) digestion of pGEM 7 (KJ1) with Xho I, which cuts into the 5 'palienlazadar of PGK promoter, and by filling the XA I site's flattened Xha I with polymerase of Klenow, and 5 2) by digestion with Sea I that letter within the 3 'genomic mouse sequence of the polyadenylation signal of PGK. This fragment was ligated into 5 '3' KO of MC4-r digested by Xho I that had also been subjected to extreme flattening with Klenow polymerase, fl 10 to generate the 5 '3' KO focus vector of MC4-r (figure 2D). A schematic map of the gene approach strategy for deactivation of the MC4-r locus with this vector is illustrated in Figures 3A-3D. 6.1.3. GENERATION OF CELLS IS FOCUSED 15 The RF-8 ES cell line (obtained at the Gladstane Institute of Cardiovascular Disease, UCSF) was culti- vated in f Clu ...... «nt-dor-mitotic-nte des cti as SNL76 / 7 in accordance with what is described in McMahan and Bradley (199, Cell 62: 1071; 1085). For electropsing, the cells were trypsinized and resuspended at a concentration of 1.1 x 10,000,000 / ml in PBS (free of Ca ++ and MG ++, Gibca). One was added to the 0.9 ml icuota (1 x 10,000,000 cells) with 20 μg of 5 '3' DNA of K0 of MC4-r, which had been linearized by digestion with Not I, and boosted at 250 V, 25 500 μ .F (Bio-Rad Gene Pulser), after which the cells were diluted in culture medium, placed in dishes at a density of 1 x 1,000,000 per 100-m dish containing food cells, and placed under selection. hours later in sulfate G41S (400 μg / ml of powder., Gibca) for 6 days. 427 clones resistant to G418 were collected which were dissociated with trypsin and divided into one well each of two 96-well dishes. At the confluence, the ES cells were frozen in one of the 96-well plates in accordance with that described by Ramirez-Sal is et al., (Methads in Enzy slaqy, vol.225, Waesar an, PM, DePamphilis, ML ( eds.) Academic Press, pp. 855-878, 1992) and 'expanded into a dish of 24 pazos. Upon reaching the confluence, the DNA was prepared for Southern blot analysis. The genomic DNA was prepared in situ from ES cells in 24-well dishes by the method of Laird et al. (1991, Nucleic Acids Research 19: 4293). To screen homogeneous recombination between the vector and the locus of endogenous MC4-r, ss was subjected to digestion approximately 20 μg of genetic DNA with Apa I, subjected to electrophoresis through an agarase gel at 1"Á, and transferred to a Hyband N + membrane (Amersham), and hybridized with the Sac I-Sph I probe labeled with 32 P (see Figure 3A) 6.1.4 GENERATION OF MISCONDUCT MICE IN MC4-R Clone 155 was injected into C57BL blastocysts / 6J to generate chimeric mice in accordance with that described in (Bradley, A. In Rabertsan, EJ (ed) Teratacarcinamas and Embryonic Stem Cells, IF'L Press, Oxford, England, pp. 113-151, 1987.) Oui eras Male animals were reared with C57BL / 6J females, and agouti offspring (representing the germline transmission of the ES genome) were screened for the presence of MC4-r gene focused by Southern blot hybridization of digested dike calf DNA. Apa I as well as Nca I using the sanda illust In Fig. 3A, heterazigosas for the mutation were identified either due to the presence of a band of 7.6 Kb of Apa I in addition to the band of 2.2 Kb of wild type to good due to the presence of a band of 1.9 Kb of Neo I in addition to the wild type bands of 2.6 Kb and 2.8 Kb (Figure 3D). Heterozygous mice were crossed and the offspring generated by these crosses were screened by Southern Blot hybridization of tail DNA digested by Apa I as well as pair Nca I. Hamazigane mice for the removed MC4-r gene were identified due to the absence of the band of 2.2 Kb of Apa I of wild type and of the band of 2.8 Kb of Hca I, and due to the presence of the 7.6 Kb band of Apa I and the 1.9 Kb band of Neo I focused. To verify the crest of the MC4-r gene, the stains digested by Apa I and by Neo I were removed and retested with the human MC4-r coding sequence. No band was observed. Hybridization in the DNA of homozygous mice = a = for the MC4-r mutation, which verifies the absence of the MC4-r gene in these mice. 6.1.4. WEIGHT AND LENGTH MEASUREMENTS The weight increase was measured regularly, starting at 3-4 weeks of age, using a Sartorius Na models. 14800 P. The length was measured by manual immobilization and extension of the mouse to its full length, always by the same person, and the distance from the nose to the anus was measured in centimeters. 6.1.5. FOOD CONSUMPTION Food intake was measured for two A, two C57BL / 6J, four hamozy mutants for MC4-R, and four wild type F2 controls, all housed in two cages. The mice were housed for at least a week before taking a measurement. In a period of two weeks, we weighed and provided a sufficient amount of food for the week to the mice ad libitum. Each morning of the days of the week the remaining food was measured, for a total of 8 measurements. The cages were carefully monitored to account for spills that were negligible. The Ay and C57BL / 6J mice were 9 weeks old at the time of the beginning of the measurement of food consumption; both the four ratonss deficient for MC4-R 11" co or the F2 controls were 15, 15. 17.5 and 20.5 weeks of age. 6.1.6. ANALYSIS OF SERUM For the measurements of glucose, insulin and leptin, the blood was collected by retroorbital sinus function from animals that received food and water ad libitum. The mice were manipulated regularly (three times a week for several weeks) before bleeding to minimize stress, and the mice were moved one at a time to a separation location at the time of bleeding. For the measurement of glucose levels, 5 μl of serum was analyzed in a YSI Model 27 glucose analyzer (Yellow Sppings Instrument Company, Inc.) using a glucose oxidase assay. The results are expressed in mg / dl. The detection range is 0-500 mg / dl, with a coefficient of variation of less than 1%. The serum insulin concentration was measured in duplicate in a volume of 10 μl by means of a specific competitive pratein binding assay using insulin ds rat as standard. The results are expressed as ng / ml. The detection range is 0.1-25 ng / ml with a coefficient of variation of less than 107 .. The duplicated pair leptipa was measured in 20 μl of serum using a set of radioimmunoassay elements for mouse leptin with mouse leptin. Recombinant as standard (Lineo Research, Inc). ne In the case of serum corticosterone measurements, the mice were housed in cages of three animals with food and water ad libitu. To avoid the elevation caused by the stress of the carthesterone levels, the mice were manipulated 2-3 times a day for 3 days before the blood was taken. The cages were taken one by one in a separate room, the mice were weighed and then held as to draw blood. On the fourth day the mice were handled in a similar manner, and blood was drawn between 8:00 and 9:00 A.M. within 30 seconds of handling. The cages were not returned to the lodging room until all the samples were obtained. Blood was obtained from a cut at the tip of the calla and blood was collected in a Multivette S Gel tube (Sarstedt). The tubes were placed in a freeze for 20-40 minutes and centrifuged for 3-4 minutes at 14,000 revolutions per minute to separate the serum. Two aliquots of 1 μl of serum from each sample were then assayed for corticosterase levels using a set of ImmuChem Double Antibody Corticasterone 1251 RIA (ICN Biomedicals, Inc.) elements. 6.1.7. HISTOLOGY For analysis of in situ hybridization of P0MC gene expression, heterozygous, homozygous and wild-type mutant mice were maintained under a cycle of 12 hours of light and 12 hours of darkness at a constant temperature. Feed (feed for Purina mouse) and water ad libitum were provided. The anesthetized animals (avertin) were sacrificed between 1500 and 1700 hours before the lights were turned off by cardiac puncture and perfusion with a saline solution (20 ml) and then 50 ml of fixation regulator at ice temperature (4"/. of formaldehyde in borate buffer, pH 9.5) Whole brains were quickly removed and then fixed overnight at 107. sucrose / fixative buffer Blocked hypothalamic sections were cangelated in dry ice powder and then stored at a temperature of -80 ° C before cutting into sections An antisense F'OMC probe was prepared by linearization of the plasmid mP0MCE3ribo (kindly provided by Dr. Malcalm Law), which contains exop 3 of the mouse POMCd gene, can I. I. Probes (35S) cRNA were prepared by transcribing 1 μg of each linearized DNA with T7 DNA palimerase for 1 hour at a temperature of 37 ° C in accordance with the description or (Promega) The hypothalamic brain blocks were assembled in a frozen stage and sectioned in series in 4 series of slices of 20 μm with a sliding microtome. Sections were prepared which were hybridized for 20 hours at a temperature of 5S ° C with probes 12? marked with 353 (5 x 1,000,000 cpm / ml) in form at 65"/., NaCl 0.26 M, 1.3 x Denhardt's solution, 1.3 M EDTA, dextr ana sulphate at 137., 13 mM Tris, pH 8. sections were then digested with RNase (20 μg / ml) for 30 minutes at a temperature of 37 ° C, and then the salt was removed in a series of washes from 4 and SSC'l mM DTT until a final condition of 0.1 and SSC / 1 mM DTT at 65 ° C for 30 minutes The sections were dehydrated in ascending ethanol, dried under vacuum at room temperature for 30 minutes, and then e-placed on a Dupant Cranex film for several days. submerged in an NTB-2 emulsion (! &odat), and developed after 6 days 6.2.RESULTS 6.2.1 GENERATION OF DECEIVING MICE IN MC4-R The mupna MC4-R gene consists of approximately 1 i- b of coding sequence contained within a single exon (figure 1A). A focus vector was designed to remove vi All the coding sequence of MC4-R after recombination homologous with the lacus in precursor cells e ionic b (ES). Camo is shown in Figure 1A, the vector consists of a total of about 4.5 I 'b of mouse genomic DNA of strain 129 / Sv flanking a riffle of 1.5 μ-b. This removal extends from the Neo I site located approximately 20 nucleotides downstream of the initiation codon of MC4-P to the Hmd III site located approximately 0.5 b from the 3 'end of the gene. The removed MC4-R sequences have been replaced by the neo gene under control of the fosfagl? Ceratqu? Nase-1 (PGH-1) promoter. A total of 809 G418-resistant colonies were screened for homologous recapitulation by means of Southern blct hybridization of Apa I-digested genomic DNA to the flank probe illustrated in Figure 3A. One clone showed the Apa I DNA fragment focused on 7 ,. r b predicted in addition to the wild type fragment of 2.2 kb expected. Injection of this clone in C57BL / 6J blastacistas yielded several male sex quiras that, when coupled with C57BL / 6J females, transmitted the MC4-P allele focused on their offspring Fl 129 / B6. The heterozygotes Fl were crossed between them and their descendants genotyped by hybridization by Southern blot of tail DNA digested with Apa I or Nca I with the flank probe »As described above, Apa I digestion generates a wild-type fragment 2.2 - b and a 7.6 Ib fragment (note that this 7.6 ib is different from the background band of a slightly smaller molecular weight that is present in all samples, Figure 3E). Digestion with Nca I generates two wild type fragments of 2.7 ', 2.9! < b, since the Neo I site is located within 12 sequences recognized by the flank probe (figure 3). The Mea I fragment of 2.7 kb represents gene sequences that extend at the 3 'end of the probe na affected by the MC4-R approach, while the 2.9 kb band includes the MC4-F gene sequences;, After the focus, this last fragment is reduced to a 2 kb band diagnosis of the mutated MC4-R allele. As shown in Figure 3E, between heterozygous crosses produced homazigase mutant, heterozygous differences and offspring of wild-type F2. To verify the removal of the MC4-F gene; In haemozygous mutants, the filters were washed and rehybridized with a MC4-R probe. No hybridization of MC4- was detected in homozygous mutant mice, whereas the 2.2 kb and 5.1 kb bands of Apa I predicted (d Apa I cuts within the MC4-R gene that generates two fragments containing MC4-R, see Figure 3A) and a Neo I fragment of 2.9 kb were observed in both heterazigosas and wild-type chicks (Figure 3E). 6.2.2. BODY WEIGHT AND MASS OF DEFICIENT MICE IN MC4-R Animals f2 were kept on a diet of mice feed ad libitum and their weight was monitored regularly. The weights of mice deficient in MC4-R and their wild-type bait partners were essentially indistinguishable during the first 4 weeks of life. NeverthelessAt approximately 5 weeks of age, most male and female Hamaziga mutants were heavier than their wild-type siblings of the same sex, and at 3 and 7 weeks of age all mutants were heavier than animals. of control (figure 4A and 4C). At 15 weeks of age, the female mutants hamazigo = as had an average weight of the weight of their sisters of wild type, whereas the mutant males homazigosas had a weight approximately 50"/. Higher than the controls of wild type The weight increase in both the case of male and female mutant mice seemed to reach a high plateau at approximately 24 weeks of age at this time the weight of the female mice averaged approximately 63 grams (n = 3) , and the males reached an average of approximately 65 grams (n = 8). The heterozygous mice for the removal of MC4-P presented a weight increase intermediate to that observed for their wild-type and homozygous mutants (figure 4B and 4D) , which demonstrates the effect of gene dosage of the removal of MC4-R on the regulation of body weight.One of the distinguishing characteristics of the obese yellow phenotype is an increase in growth skeletal Typically, Ay mice had a mean length about 5% greater than their wild-type siblings (Castle, 1941, Genetics 26: 177191; Carpenter and Meyer, 1958, Am. J. Physiol. 193: 499-504). In order to determine if the mice that have the MC4-R have an enhanced linear growth, the corporal lengths of the offspring of F2 were measured at approximately 19 weeks of age (between 132 and 138 days). As shown in Figure 6, mice deficient in MC4-R are significantly longer than controls of wild type. The average length of the hamozygos mutant females is increased by approximately 11. compared to the wild-type F2 mice, and the heterozygous females are approximately 7% longer than the control animals. Male homozygotes and heterozygotes are approximately 87. and 2.5"/ longer than control animals, respectively 6.2.3 FOOD CONSUMPTION To determine if food intake increased in mice that did not have MC4-R , homozygous mutant females and F2 controls of wild type were manipulated in terms of food intake in a period of 2 weeks, A mutants were also administered, on a background of C57BL / 6, and controls C57BL / 6. Bed previously documented ( Fpgep et al., 1988, Endocpnalogy 113: 2097-2105; Shimizu et al., 1989, L fe Sciences 45: 543-552), A and F mice were hyperphagic, eating 36% more than the C57BL / 6J control animals. Similarly, the absence of 'MC4-R also resulted in a significant increase (46%) in food consumption compared to the wild-type F2 controls (Figure 7) 6.2.4 ANALYSIS OF SERUM Collected blood from mice deficient in MC4-R and control s of wild type in three time intervals (4-8 weeks, 10-14 weeks, 17-23 weeks) and serum was assayed to determine glucose and insulin levels. The glucose levels in the serum were essentially unchanged in heterozygous females to well hamozigasas for the formation of MC4-R, but both the heterozygous and haemozygous patches were hyperglycemic (Figures 8A and SB). This was evident first for the ho hoigasos males in the range of 10-24 ssmanas at that time the glucose levels were elevated more than 2 times by the levels observed in the control animals, up to 390 mg / dl, pear Heterazigas mutants present only a slight elevation of serum glucose at this age. At 17-23 weeks of age, both male and female heterozygous mutant mice have a doubling of normal serum glucose levels (334 and 361 mg / dl, respectively) compared to control animals (156 mg / dl). ). Both the male and female mutant mice were 12c hipepnsul mérmeos (Figures 8C and SDK) Increases of nine times and 5 times in the levels of insulin were observed in the sera of female and male homazigasas mutants, respectively, at 4-8 weeks of age. dramatically with the passage of time, such that at 17-23 weeks of age the mean concentration of insulin in the serum of homszygous mutant females was approximately 65 ng / ml, and in the case of males, approximately 130 ng / ml, which represents approximately increases of 60 and 14 times, respectively, in comparison to osservatized insulin levels in wild type F2 control animals.Heterazigosos mutants also presented hypepnsulinemia, although in lower tier than mutants In the house of the heteracigatas machas as females, a significant difference was observed in the interval of IO-14 weeks in terms of insulin levels compared to females. with the levels observed in the control animals; at 17-23 weeks, the mean insulin levels of the heteracygrates were elevated to approximately 10 ng / ml (females) and 85 ng / ml (males). In addition to glucose and insulin, serum levels of leptma and corticosterone were also determined. Leptin levels are high in A mice (Maffei et al., 1995, Nat. Med. 1: 1155-1161; Mizuno et al., 1996, Prac.

Nati Acad. Sci. 93: 3434-3438), which indicates that the syndrome does not result in defects in the production of leptma. Consistently with the postulated role of the hormone in signaling fat deposition levels (Ca pfield et al., 1 ^ 5, Science 269: 546-549, Halaas et al., 1995, Science 269: 543-546; F'el Lev Aether et al., 1995, Science 269: 540-543), the leptma is also elevated in mice deficient in MC4-R (Figures 8E and 8F). At 4-8 weeks of age, the leptme was elevated 4.5 times and 1.5 times in the serum of male / female hamozygus mutants, respectively, compared to wild-type control animals. At 17-23 weeks of age, leptin levels in serum in females had reached approximately 97 ng / ml, in males such levels reached approximately 58 ng / ml, representing increases of 6.5 and 2.5 times, respectively. . Heterozygous mice showed essentially intermediate lepton levels between the level observed in the wild-type rat and the level observed in the hamaziganese mutants. Since glucacorticoids can profoundly affect weight homeostasis and somatic growth, basal serum corticosterone was measured in three joint baiting partners with sex correspondence, each set containing a wild-type animal, a heterozygous mutant animal, and a mutant hamazigoso animal (figure 9). No effect of the knockout of the MC4-F gene on the carthesterone-based levels was detected. 6.2.5. EXPRESSION OF POMC GENE To establish if the observed effects of MC4-R removal on hameastasis of the weight can be attributed to the induction of changes in the only known source of ligand for MC4-P, the FOMC gene, the expression of the gene Central POMC was examined in wild-type mice, mice heteraza gasos for the removal of MC4-R and homazigosas mutants by in situ hybridization i FIGS. 10A-10F). No new sites of POMC gene expression were detected or any consistent changes in POMC mRNA levels at its primary expression site, the arcuate nucleus of hypatala a, in this assay. In addition, important neuraanthic defects will not be obese in brain sections stained with tianma for animals deficient in MC4-R homozygous to heterozygous mutants by histological analysis (Figures 10A-10C). 7. EXAMPLE: AGUTÍ PROTEIN BINDS DIRECTLY WITH MCL-R AND MC4-P The following example describes experiments that demonstrate that agouti protein binds directly to melanacortin receptors. 7.1. MATERIAL AND METHODS Transplacement of human melanocortic receptor 4 cDNA (hMC4-r), to control the promoter CM'f, in cell line 293, and stable clones (293 / MC4-P) were selected. The stable clones were tested to determine the reduction of intracellular cAMP levels in the presence of 5nM of agouti pratein. COS-7 cells were transfected into hMCl-r or hMC4-r using the DEAE-De trana method. A plasmid containing the adenovirus VA1 and VA2 RNA genes was used to co-transfect COS cells to enhance the expression of transient protein by increasing the translation start. The plasmids of the cDNA MC4-r and VA1 / 2 will be used in a ratio of 10 to 1. Some control plates received only the plasmid VA1 / 2. 48 hours after transfection, the cells were rinsed and culture supernatant containing 15 nM AP-Ag was added. AP-Ag is a truncated agouti pratein that has the cysteine-rich domain labeled with alkaline phosphatase at its N-terminus. AP-Ag binding on transfected cells was carried out at room temperature for 90 minutes with gentle agitation. The cells were then washed 7 times before fixing and developing color using an NBT / BCIP substrate. 7.2. RESULTS When the 5 nM agouti pratein was added to line 293 / MC4-R, a reduction of 187 was observed in terms of the level of intracellular cAMP, in relation to the 293 parental cell line. The results indicate that MC4-R mediates the decrease caused by agouti of the level of intracellular cAMP and confers agouti response to 293 cells. A truncated agouti protein at the N-terminus, which only has the C-rich C-terminus domain retains the activity of antagonism of full-length agouti. A truncated agouti protein, containing only the cysteine-rich domain and labeled with alkaline phosphatase at its N-terminus, was used to test the direct link between the agouti protein and the MCI and MC4 receptors in C0S7 cells. The efficiency of transfection, in accordance with that monitored by the reporter plasmid (3-galactosidase, was typically 12-16"/ .. 10-137, of C0S7 cells transfected with MC1-r and 3-47 of the transfected C0S7 cells. with MC4-r bound to agouti pratein according to what was determined by AP staining The difference observed in percentage of positive cells between C0S7 cells transfected with MCl-r and MC4-r could be attributed to differences in affinity of linkage and / or level of expression To evaluate the affinity of the agouti protein for the MCl-R receptor, a Scatchard analysis was carried out on transfected C0S7 cells MCL-r using culture sabrenate containing up to 30 nM AP -Ag. Kd was estimated within a range of 20-30 nM 7.3 DISCUSSION The dominant agouti alleles that give rise to obesity result from the deregulated constitutive synthesis of the wild-type agouti protein in the animal, and explained a likely among the other characteristics of pleiotrotic obesity syndrome such as hyperphagia, hyperinsulinemia, and hyperglycemia. A possible mechanism by which syctotic expression of agouti induces obesity is the aberrant antagonism of melanocortin receptors, such as MC4-R, expressed in regions of the cebrera which are known to be involved in the regulation of feeding. The data presented here demonstrate that the agouti protein binds directly to MC4-R. In addition, in accordance with that described in Example 6, supra, transgenic mice that do not have the MC4-R produce an obesity syndrome that looks surprisingly similar to the agouti syndrome. The recapitulation of many of the features of agouti syndrome in mice deficient in MC4-R demonstrates that antagonism of melanacartin signaling through MC4-R is the primary cause of the agouti obesity syndrom. 8. EXAMPLE: IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF MELANOCORTIN 4 HUMAN RECEPTOR Ilel37Thr MUTANT Several mutant genes have been identified which cause obesity in the mouse; In humans, however, only one mutation in the adrenergic receptor gene has been associated in a non-definitive manner (33 with obesity.) Identification of the Ilel37 Thr mutation MC4-R in obese humans and the finding of their affected signaling support the hypothesis that poses that MC4-R is a causative genetic factor that contributes to human obesity, and validates the receptor as a potential drug target. 8.1. MATERIALS AND METHODS 8.1.1. DETECTION OF MC4-R MUTANT RECEPTORS A human genomic DNA isolated from total white blood cells was amplified by polymerase chain reaction using the following primer pairs: C4flb 5'-TGTAAAACGACGGCCAGTCTGACCCAGGAGGTTAAATC-3 'C4rlb 5'-CAGGAAACAGCTATGACCGCTGCAGATGAAAAAGTACATG-3' MC4f2b 5'-TGTAAAACGACGGCCAGTTGCTACGAGCAACTTTTTCTC-3 'MC4r2b 5'-CAGGAAACAGCTATGACCGGTACTGGAGAGCATAGAAG-3' MC4f3 5'-TGTAAAACGACGGCCAGTTGGTGAGCGTTTCAAATGGAT-3 'MC4r3 5'-CAGGAAACAGCTATGACCGAGCCAGCATGGTGAAGAAC-3' MC4f4 5'-TGTAAAACGACGGCCAGTATCTTCTATGCTCTCCAGTAC-3 'MC4r4 5'-CAGGAAACAGCTATGACCTTCTGAGGACAAGAGATGTAG-3' MC4f5b 5'-TGTAAAACGACGGCCAGTTTCTCTCTATGTCCACATGTTC-3 'MC4r5b 5'-CAGGAAACAGCTATGACCGAGTGAAAAAGTCTCTTATGCATG-3' The following conditions were used for the 1: amplification pair chain reaction of palimerase; polymerase chain reactions of 25 μL contained a regulator (10 mM Tps-HCl, pH 8.3, 50 mM I <C1, 1.5 M MgC12), the four dNTPs at 100 μM each, 1 μM of each primer, 0.5 U Taq polymerase, and 50-100 ng of genomic DNA. The fragments were amplified in a polymerase chain reaction (94 ° C for 30 seconds, 65 ° C-55 ° C for 30 seconds (-l'C / cidla), 72 ° C for 40 seconds), followed by 30 cycles of 94 * 0 for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 40 seconds. The amplifications were treated with Exonuclease I to remove the residual single-strand initiators and shrimp alkaline phosphatase was used to remove the unincorporated dNTPs (Joint of tempering purification elements for sequencing, Amersham US70995). The purified fragments were then digested with restriction enzymes MspI and Hinfl to provide fragments smaller than 250bp, more suitable sizes for SSCP pair analysis. The digested polymerase chain reaction products were diluted 1: 9 in charge buffer (95% pharmacid, 20 mM EDTA, 0.057 bromfenal blue, 0.057 ml xylene), thermally denatured at 98 μl. ! C for 2 minutes, and cooled in an ice paste. 2-3 μL were loaded in one gallon of 10% acrylamide (50: 1 acp lamid: bis-acrylamide) with 10% glycerol and the experiment was carried out at 25 watts for 4 hours at 4 ° C. The gel was stained with Green I and II SYBR to detect both single-stranded and double-stranded DNA fragments, and visualized in a fluorine image. 8.1.2. RECOMBINANT EXPRESSION OF MC4-R MUTANT Genomic DNA containing the wild-type MC4-R and Ilel37Thr variants of MC4-R were used as annealing for an amplification reaction by polymerase chain reaction. The primers flanking the coding region of MC4-R were used for the polymerase chain reaction of the entire MC4 including the Ilel37Thr mutation of MC4-R. The polymerase chain reaction primers and conditions were as follows: HMC4 top primer 5 '- CGTAGGATCCATGGTGAACTCCACCCACCTG-3' HMC4 bottom primer 5'-AGCCTCGAGTTAATATCTGCTAGACAAGTC-3 'polymerase chain reaction conditions: 96 ° C for 15 minutes minutes; 94 ° C for 1 minute; 55ßC during 1 minute; 73 ° C for 2 minutes for 35 cycles; 73"C for 10 minutes; 4 ° C. The resultant polymerase chain reaction fragments of i kb were subsequently cloned into pCADN3 (Invitrogen) and sequenced to confirm the correct DNA sequence.Expression vectors that captienep human-type cDNAs wild for MC4-R and I137T mutants, respectively, were transfected into 293T cells (HEK 293 cells expressing the SV40T antigen) using Lif pofecta ina (BRL). 48 hours later, the transfected cells were placed in 96-well dishes. After an additional 24 hours, the transfected cells were incubated at 37 ° C for 15 minutes (or at room temperature for 1 hour) with several melanacortins (BACHEM). The intracellular levels of cAMP were then determined by running a trial based on SPA (Amersham). To manipulate and normalize the transfection efficiency of the wild-type and mutant cDNAs, an expression vector containing the (3-galactosidase gene was always transfected with the MC4-R expression vectors (cDNA of (3-galactosidase and MCR-R in a ratio of 1: 9) The (3-galactosidase assay performed 72 hours after transfection confirmed that the MC4-Rs of wild type and mutant and 137T had the same transfection efficiency 8.2 RESULTS 216 individuals were screened na related to sequence variation within the coding region of the MC4-R gene.The 216 individuals comprised 96 extreme obese according to the one determined by the * body mass index (BMI >).50), 24 obese (BMI: 30-50), 18 intermediate (BMI 25-30), 54 thin (BMI, 25), and 24 patients are polycystic ovarian syndrome (PCOS). There will be 3 amino acid variants in MC4-R in this sample. The 3 variants are Vall02Ile (G to A, see Figures 12A-B), Ileí37Thr (T to C, see Fiquras HA-A), and Thrll2Met (C to T, see Figures 13A-B). 5 The Val 1021 substitution was found among all the BMI categories within the sample. The following individuals were heterazigasas for this variant: 2 extreme obese (BMI = 74.2, BMI = 57.2), 2 obese (BMI = 43, BMI-26), 2 thin (BMI = 22.5, BMI + 21). átk lO The Ilei37Thr variant was found only once in an extremely obese person (BMI = 57.3). This person is heterozygous for this variant. Substitution of a threonine (polar) with an isoleucine (non-polar) may have an effect on the reception. It is possible that this substitution decrease the activity of receiving MC4 which contributes to the obesity of this patient. The Trhll2Met variant was found only once (BMI-17.9). This person is heterozygous for this variant. The. replacement of one. methionine (not polar) by a threonine (polar) can have an effect on the receiver. It is possible that this substitution increases MC4 receptor activity, contributing to the thin character of this person. In order to test if the. variant Ilel37Thr was able to function normally, the activity of the receive mutant Ilel37Thr with a wild type receptor 13 in a signaling test. Figure 14 compares the response to five endogenous melanocortins, a-MSH (alpha), 13-MSH (beta), gammal-MS (gam), gamma2-MSH (gamma2), and ACTH. The results demonstrate the affected signaling of the mutant receptor Ilel37Thr measured pair induction of cAMP in the presence of several agonists. In comparison with the wild type receptor, the mutant has a much lower maximum activation, that is, a lower maximum cAMP level was reached; and generally has a higher EC50, that is, the highest agonist concentration required to achieve maximum mean activation. The receptor mutant na is completely inactive in the in vitro assay; but it is marginally active only in the presence of a very high concentration of agonist that can not be reached under physiological conditions in vivo. The present invention is not limited as to its. The scope of the specific embodiments described which are intended to illustrate individual aspects of the invention, and functionally equivalent methods and catches are within the scope of the invention. Obviously several modifications to the invention, in addition to those illustrated and described here, will be apparent to those skilled in the art from the foregoing description and the appended drawings. Such modifications fall within the scope of the appended claims.

Claims (44)

1. CLAIMS 1. A method for identifying compounds that regulate body weight, comprising: (a) contacting a test compound with a cell that expresses a functional melanocartin receptor 4, and (b) rmining whether the test compound active melanocortin receptor 4, in dande the test compounds that activate melapocartin receptor 4 are identified as compounds for the induction of weight loss.
2. 2. A method for identifying compounds that regulate body weight, which comprises: (a) contacting a peptide of elanocartin in the presence and absence of a test compound with a cell expressing a functional melanacartin receptor 4, and (b) the rmination of whether the test compound inhibits the melanocartipa peptide-induced activation of melanocortin receptor 4, wherein the test compounds that inhibit melanacartin receptor 4 activation are identified as compounds to induce weight increase .
3. 3. The method of claim 1 or claim 2 wherein the activation of the melanacartin receptor 4 is rmined by measuring the induction of cAMP.
4. 4. The method of claim 3 wherein the cell further contains a reporter gene operatively associated with 5 an element that resists cAMP, and the induction of cAMP is indicated by expression of the delivery gene.
5. 5. The method of claim 4 wherein the gene splits is alkaline phosphatase, claranfenical acetyltransferase, luciferase, glucuranidsintetas, growth hormone, or «P 10 well placental alkaline phosphatase !.
6. 6. The method of claim 2 wherein the mel nocortin peptide is -MSH.
7. 7. A method for identifying compounds that regulate body weight, comprising: (a) contacting a test compound with melanocortin receptor 4, and; (b) the rmination of whether the test compound interacts with the melanacartin receptor 4, in which the test compounds that interact with the melanocortin receptor 4 are ideptified as compounds that regulate body weight.
8. 8. A method for identifying compounds that regulate body weight, comprising: (a) contacting a melanocortin peptide in 25 presence and absence of a test compound with a functional melanocortin 4 receptor; and (b) the rmination of whether the test compound inhibits the • interaction of melanocortin peptide with melanocortin receptor 4, where test compounds that inhibit the interaction of melanocartin peptide with melanocartin receptor 4 are identified as compounds that regulate body weight.
9. 9. The method according to claim 7 or claim 10 wherein the melanacartin receptor 4 is contained in an isolated membrane or is expressed recombinantly.
10. 10. The method according to claim 8 wherein the melanacartipa peptide is MSH.
11. 11. A method for identifying compounds that regulate body weight, comprising: (a) contacting a test compound with a • cell or a cell lysate containing a reporter gene operatively associated with a regulatory element of 20 melanocortin receptor 4; and (b) ction of reporter gene product expression.
12. 12. A method to identify compounds that regulate the body weight, comprising: (a) contacting a test compound with a cell or cell lysing containing transcripts of receiving 4 elanacartin; and (b) the ction of the translation of the transcript of me lanocartin.
13. 13. A method for the treatment of body weight disorders, comprising the modulation of melanacortin receptacle 4 activity.
14. 14. A method for the treatment of body weight disorders comprising administration of an effective amount of a compound that agonizes to antaganiz the activity of melanocartin receptor 4.
15. 15. The method of claim 14 wherein the compound activates melanocartin receptor 4 and induces weight loss.
16. 16. The method of claim 15 wherein the compound is an agonist or an antibody that binds melanocortin receptor 4 and active receptor.
17. 17. The method of claim 14 wherein the compound inhibits the activation of melanacortin receptor 4 and induces weight gain.
18. 18. The method of claim 17 wherein the compound is an antibody or a soluble extracellular domain of melanacartin receptor 4.
19. 19. A method for the treatment of body weight disorders, which includes the modulation of the activity of 14: melanocortin receptor 4 gene.
20. 20. A method for the treatment of body weight disorders comprising the administration of an effective amount of a compound that decreases the expression of melanocortin receptor 4 and induces the increase in weight.
21. 21. The method of claim 20 wherein the compound is an oligonucleotide that encodes an antisense or ribazyme molecule that targets transcripts of melanocortin receptor 4 and inhibits translation.
22. 22. The method of claim 20 wherein the compound is an aligonucleotide that forms a triple helix with the promoter of the melanocortin receptor 4 gene and inhibits transcription.
23. 23. A pharmaceutical formulation for the treatment of body weight disorders, comprising a compound that activates melanocartin receptor 4, mixed with a pharmaceutically acceptable carrier.
24. The pharmaceutical formulation of claim 23 wherein the compound is a melanacartin peptide agonist to an antibody specific for melanocortin receptsr 4.
25. 25. A pharmaceutical formulation for the treatment of body weight disorders, comprising a compound that inhibits the selective activation of melanocortin receptor 4, mixed with a pharmaceutically acceptable carrier.
26. 26. The pharmaceutical formulation of claim 25 wherein the compound is an antagonist or an antibody specific for melanocortin receptor 4.
27. 27. The pharmaceutical formulation of claim 25 wherein the compound is an aliganucleotide that encodes an antisense or ribazyme molecule that targets transcripts of melapacartin 4 receptors and inhibits translation.
28. 28. The pharmaceutical formulation of claim 25 wherein the compound is an aliganucleotide that forms a triple helix with the promoter of the melanocortin receptor 4 gene and inhibits transcription.
29. 29. A genetically engineered obese animal in which the melanocortin receptor 4 gene has been deactivated.
30. 30. A transgenic animal that expresses a human melanocartin receptor 4 gene.
31. 31. A set of elements that catches a melanocartin receptor 4 or cells that express the melanocortin receptor 4 packaged in a container.
32. 32. A method for diagnosing disorders of body weight in a mammal, comprising the measurement of MC4-r gene expression in a patient sample.
33. The method of claim 32 wherein the expression is measured by the detection of mRNA transcripts of the MC4-r gene.
34. 34. The method of claim 32 wherein the expression is measured by detection of the gene product of MC4-r.
35. 35. A method for diagnosing disorders of body weight in a mammal, comprising the detection of an MC4-r gene mutation contained in the mammalian genome,
36. 36. The method of claim 35 wherein the mutation is located at the nucleus? 137 of the MC4 ~ r gene.
37. 37. An isolated DNA consisting of an altered sequence of MC4-r having a G at nucleotide 102.
38. 38. An isolated DNA consisting of an altered sequence of MC4-r having a C at nucleotide 137.
39. 39 An isolated DNA consisting of an altered sequence of MC4-r having a T at nucleotide 112,
40. 40. A nucleic acid probe complementary to the human altered MC4 ~ r gene sequences, wherein said nucleic acid probe is hybridizes to a mutant MC4-r gene under conditions that prevent hybridization of said nucleic acid probe with an MC4-r gene having a wild-type sequence.
41. 41. The nucleic acid probe of claim 40 wherein the mutant MC4-r gene has a G ep the nucleotide corresponding to the Na base. 102 in the gene sequence of MC4-r.
42. 42. The nucleic acid probe of claim 40 wherein the mutant MC4-r gene has a C in the nucleotide corresponding to the bass Na, 137 in the gene sequence of MC4-r.
43. 43. The nucleic acid probe of claim 40 wherein the mutant MC4-r gene has a T in the nucleotide corresponding to the Na base. 112 in the gene sequence of MC4-r.
44. 44. A method for identifying compounds that regulate body weight, comprising: (a) contacting a test compound with a cell expressing a mutant melanocortin receptor 4, and (b) determination of the test compound itself, active, melanocortin receptor 4, wherein the test compounds that activate the melanocortipase receptor 4 identify compounds or compounds to induce weight loss.