MXPA99004184A - Method for the detection of compounds that modulate the effects of the obese (ob) protein - Google Patents

Abstract

A method for the detection of a compound that mimics, potentiates or inhibits the physiological effect of the ob-protein, which method comprises:(a) for a compound which mimics the physiological effect of the ob-protein, assessing the effect of the compound upon an ob-protein activated signal transducer and activator of transcription (STAT) DNA response element coupled to a reporter gene;or (b) for a compound which potentiates or inhibits the physiological effect of the ob-protein, assessing the effect of the compound upon the response provided by ob-protein upon an ob-protein activated STAT DNA response element coupled to a reporter gene;wherein, the response element and the reporter being expressed in an ob-protein responsive cell line, which cell line is selected from the list consisting of:a hypothalamic derived cell line;a pheochromocytoma derived cell line;a haematopoietic derived cell line;a pancreatic derived cell line;a liver derived cell line;a preadipocyte derived cell line;a skeletal muscle derived cell line;and an ovarian derived cell line;or the response element and the reporter are expressed in a cell line, which cell line (the engineered cell line) is also transfected with a polypeptide which is capable of mediating the stimulation by ob-protein of an ob-protein activated STAT DNA response element and contains the appropriate STAT proteins.

Description

METHOD FOR THE DETECTION OF COMPOUNDS THAT MODULATE THE EFFECTS OF THE PROTEIN OF OBESITY (OB) DESCRIPTIVE MEMORY The invention relates to a new method and very particularly to a method for the detection of compounds that mimic, potentiate or inhibit the physiological effects of the ob protein. The ob (or leptin) protein is a secreted hormone that acts as a signal from adipose tissue to other organs to regulate weight and energy balance (Zhang et al., Nature, 1995, 372, 425). Additional roles for the ob protein in hematopoietic and reproductive function have been suggested (Cioffi et al. Nature Medicine, 1996, 2 (5), 585). Protein molecules that contain a core composed of four helices a form an unequal topology package comprising a family of cytokines and growth factors. The proteins of this family cause homo- and heterooligomerization of the membrane receptors known to activate the kinase cascades that result in gene transcription. Family receptors that are activated by oligomerization are found in two broad classes; those such as the epidermal growth factor receptor, which possesses integral tyrosine kinase activity in its intracellular domains (A. Ullrich &J. Schlessinger, Cell, 1990, 61, 203-212), and those such as IL4 and irotropoietin receptors, which lack such activity and adjust their response by an associated protein tyrosine kinase (J.N. Ihle et al. TIBS, 1994,19,222-227). The receptor subtypes are activated by cytosines, but the four-helical packaging proteins activate only the non-integral tyrosine kinase subtype. The non-integral tyrosipa kinase protein receptors generally act through a pathway involving the Janus kinase (JAK) and its associated signal transducers and their transcriptional protein activators (STATs). By activating the STAT proteins, the DNA response elements are linked by the gene transcription control. Oligonucleotide sequences comprising DNA regulatory elements of the general sequence TT (N) nAA have been identified (Seidel et al., Proc.A Nat.Acad.Sci.USA., 1995, 92.3041) as STAT response elements. STAT proteins link elements in response to signaling molecules such as cytosines. In the UK co-pending patent application number 95091641.1 the discovery is disclosed that the ob protein is characterized by a four-helical tertiary packaging structure. It is believed that the ob protein interacts with a membrane binding receptor that is activated as a JAK-STAT clase cascade and thus forms the basis of a test system for the detection of compounds that mimic, potentiate or inhibit physiological effects of the ob protein. Said assay has the utility of selecting compounds for weight treatment, energy balance, hematopoietic, fertility and other disorders modulated by the ob protein. The test is especially useful for selecting the compounds for the treatment of such disorders related to obesity, anorexia, cachexia and diabetes. International co-pending patent application number PCT / EP96 / 02291 refers to a new detection method using JAK-STAT technology. A particularly advantageous detection method has been discovered which also uses said technology. Accordingly, the invention provides a method for the detection of a compound that mimics, enhances or inhibits the physiological effect of the ob protein, which method comprises: (a) for a compound that mimics the effect the physiological effect of the protein ob , evaluating the effect of the compound on an ob activated signal transducer protein and a transcription activator (STST) of DNA response element coupled to a reporter gene; or (b) for a compound that potentiates or inhibits the physiological effect of the ob protein by evaluating the effect of the compound on the response provided by the ob protein in a STAT activated protein SEAT of the DNA response element coupled to a reporter gene; Wherein the response element and the reporter are expressed in a line of ob-response cells, whose cell line is selected from the list consisting of: a line of cells derived from the hypothalamus; a line of cells derived from ceocromocitoma; a line of cells derived from hematopoietic cells; a line of cells derived from pancreatic cells; a line of liver derived cells; a line of cells derived from preadipocyte; a line of cells derived from skeletal muscle; a line of ovarian derived cells; or the response element and the reporter are expressed in a cell line, whose cell line (a cell line created by genetic engineering) is also transferred with a polypeptide that is capable of adjusting the stimulation by the protein ob of a protein ob activated STAT of the DNA response element and contains the appropriate STAT proteins. A suitable cell line is a line of hypothalamic derived cells. A suitable cell line is a line of cells derived from feocreomocitoma. A suitable cell line is a cell line derived from hematopoietic cells. A suitable cell line is a line of cells derived from pancreatic cells. A suitable cell line is a line of liver-derived cells. A suitable cell line is a line of cells derived from preadipocytes.

A suitable cell line is a line of cells derived from skeletal muscle. A suitable cell line is a line of ovarian derived cells. Suitably, the response element and the reporter are expressed in a cell line, whose cell line (cell line created by genetic engineering) is also transferred with a polypeptide that is capable of adjusting the stimulation by the protein ob of an STAT activated protein STAT of the DNA response element and contains the appropriate STAT proteins. An example of a line of hypothalamic derived cells is the neuronal cell line GT1-7 described by W.C. Wetsel in Cellular and Molecular Neurobiology, 1995, 15 (1), 43. An example of a cell derived from pheochromocytoma are rat PCO cells from sotarenales phylochromocitoma (L: A. Greene and AS Tischier, Proc. Nati. Acad. Sci USA, 1976, 73 (7), 2424. An example of a cell line derived from hematopoietic cells is the HEL 92.1.7 cell line (ATCC TIB 180) derived from a human erythroleukemia (P. Martin. (4551), 1233) Another example of a cell line derived from hematopoietic cells is the K562 cell line (ATCC CCL-243) derived from human chronic myelogenous leukemia (Proc.Soc.Exp.Bíol.Med.1981, 166, 546 and Blood, 1975, 45, 321).

An example of a pancreatic derived cell line is the BetaTC-3 cell line (T.J. Kieffer et al., Biochem.and Biophys.Res.Comm., 1996, 224, 552). Other examples of pancreatic cell line include RIN5mF cells recently reported to respond to leptin (Md Shahidul Islam and others Biochem Biophys Res Comm, 1997, 238851-855). An example of a cell line derived from preadiposites is the 30A5 cell line (Y. Bail et al., J. Biol. Chem., 1996, 271 (24), 13939). Other examples of cell lines derived from preadiposites include differentiated 3T3-L1 and 3T3-F442A cell lines, which are commercially available. An example of a line of liver derived cells is the HepG2 cell line (B Cohen et al, Science, 1996, 274, 1185-1188 and Y Wang et al, J Biol Chem, 1997, 272, 16216-16223) or H35 cell line (Y Wang et al; J Bioi Chem, 1997,272,16216-16223) Other examples of liver derived cell lines are liver cells WRL68 and Change, which are commercially available. An example of a cell line derived from skeletal muscle is the C2 C12 cell line of mouse myotube (Nature, 1977, 270, 725). An example of a line of ovarian derived cells is the SK-OV-cell line. 3, ATCC number HTB77, (J. Fogh and G Trempe in Human Tu our Cells In Vitro p155-159, J. Fogh (ed.) Plenum Press, New York, 1975; J. Fogh and G Trempe J. Nati. Cancer Inst Bethesda, 1977, 578: 209-214) A suitable polypeptide that is capable of adjusting stimulation by the ob protein of a STAT activated protein or DNA response element is a functional isoform of the gene receptor., for example, the one identified in Tartaglia et al. Cell, 1995, 83, 1263. Suitably, the response element is coupled to a promoter gene as preferably a minimal promoter. A suitable response element is a nucleotide of the formula TT (N) pAA, where N is any nucleotide and n is 4.5 or 6. A preferred response element is activated selectively by the intracellular events adjusted by the protein or interacting with your receiver. Said selective response elements can be determined by examining the relative activation of a reporter element-reporter gene fabrication scale when it is transferred in a cell line of ob response by the ob protein to other cytosines. A preferred response element is a nucleotide of the formula TT (N) n AA, where N is any nucleotide and n is 4 or 5. Another suitable response element is TTCCCGGAA. Another suitable response element is the promoter region of a gene regulated by the ob protein that is required for STAT interactions. Said gene will depend on the particular therapeutic use of the compounds to be selected by the assay.

A suitable reporter gene is firefly luciferase or chloramphenicol acetyltransferase enzyme. A suitable promoter is a minimal promoter such as the herpes simplex virus of thymidine kinase or SV40 promoter. Another line of response cells can be identified using a displacement binding assay. However, the link may not be a long functional form of the receptor, which is in the form that transmits a signal to the cytoplasm. The identification of a long functional receptor form can be by PCR or Northern blot analysis (ie human ob receptor, Tartaglia et al., Cell, 1995, 83, 1263). Finally, the response cell is detected by monitoring cellular events in the presence of varying concentrations of leptin. Potential methods for identifying the candidate cell line or monitoring said cellular events include the following: 1. Microfiometer: This method detects small changes in pH that result from biochemical changes in the cell. The protein response cell or ob in the stimulation can undergo biochemical changes that cause a small change in the rate of extracellular acidification that can be detected by a silicon microphysiometer. The microphysiometer biosensor methodology has been reviewed by McConnell, Science, 1992, 257, 1906. 2.- Electrophoretic mobility shift assay (EMSA): The nuclear extracts of the cells after treatment with protein ob they mix with radiolabelled oligonucleotides containing a promiscuous or specific STAT response element DNA sequence. Extracts from the cell that respond to the ob protein may cause a change in the oligonucleotide gel for the STAT response element. Reference: Book "Recombinant DNA", 2nd Edition, Watson et al., 1992, page 158; Lamb et al., Blood, 1994, 83, 2063. 3.- Measurement of protein phosphorylation assay: The coupling of receptor activation to the final response through tyrosine phosphorylation of intracellular proteins can be assayed by the use of antibodies that recognize phosphorylated tyrosines. Very specifically, when the leptin receptor can stimulate the phosphorylation of the pathway JAK STAT, that method provides a method for detecting the cell line of the leptin response. JAK STAT specific antibodies can be used in antibodies to tyrosine phosphorylation to detect the activation of leptin in a leptin response cell line. Inhibition may occur, as well as stimulation of protein phosphorylation. In particular, inhibition by the insulin stimulated insulin phosphorylation insulin receptor-1 substrate and insulin receptor substrate 1 has been described in rat fibroblasts-1 to express insulin receptors (Kroder et al. 1996, Exp. Enedocrinol Clinic, Diabetes, 104. Supp 2, p66). 4 - Displacement link: After incubation of the cell line with radiolabeled leptin, for example [125l] -Laptin link not specific to the specific link of leptin can be studied by the addition of unlabeled leptin. A high to specific non-specific binding link suggests that the cell line may contain the leptin receptor.

. - Detection of the protein for a functional form, preferably a long functional form, of an ob receptor by the use of selective antibodies. 6. The detection of mRNA for a functional form, preferably a long functional form, of the receptor ob by means of Northern analysis, RT-PCR or "slot blot". 7.- Detection of increased c-fos mRNA after treatment with leptin. The c-fos mRNA can be detected by Northern analysis, RT-PCR or "slot blot". The known cell lines involved in the control aspects of the particular disease state for which the compounds are sought are preferred. The cell lines derived from liver, brain or pancreatic tissue and fibroblasts are particularly useful for "ob-response" cells for the assay of compounds targeting obesity and diabetes. Certain areas of the brain are the focus of the weight control and energy balance regulation effect of the ob protein. The liver controls several metabolic procedures that modulate lipid and glucose levels. The cells derived from the particular regions of said organs contain the Suitable JAKs proteins, endogenous STATs and other intracellular proteins that are required to adjust the effects of leptin are preferred. The response element, the reporter, and preferably the promoter, are suitably incorporated into a reactor capable of transferring the ob-response cell line. Suitable vectors are commercially available vectors, such as the basic luciferase vector pGL2 (Promega). An appropriate configuration of the vector is the response element DNA STAT 5 'of a promoter and a promoter and a reporter gene. A more suitable configuration of the vector is the STAT response element of DNA in multiple tandem repeats (2-10) at 5 'of the thymidine kinase promoter and a luciferase reporter gene. Vectors containing a reporter gene are constructed, for example, firefly luciferase or chloramphenicol acetyltransferase enzyme linked to a minimal promoter, for example, the herpes simplex virus of thymidine kinase or SV40 promoter. The DNA fragments for the STAT response element are inserted into the vector using the appropriate restriction enzyme sites at the 5 'of the minimal promoter. The response element, the report and the promoter, as required, are incorporated into the vector using conventional expression techniques, for example, the DNA fragments for the response element can be inserted into the vector using the enzyme sites of restriction appropriate to the 5 'of the minimal promoter.

The luciferase enzyme reporter systems of the STAT response element can be constructed as described by Lamb et al., Blood, 1994, 8, 2063 and Seidel et al., Proc. Nat. Acad. Sci. USA., 1995,92,3041. The ob response cells are transferred with minimal lucifer promoter reporter constructs of STAT response element using the standard methodology, for example, the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456). To correct differences in the efficiency of the tranfection, the cells can be cotransferred with a reference plasmid that expresses β-galactosidase activity. After a period of transfection (12-24 hours) the cells are treated with various concentrations of compound and then harvested and lysed. The lysates are tested for luciferase activity, and if β-galactosidase activity is suitable. The potentiation or antagonist activity can be assayed by pre-coadition of a suitable concentration of protein ob ai compound under evaluation and measurement of the enhancement or reduction in the luciferase response relative to that of the ob protein. Standard methods exist to test the activity of luciferase enzyme for example Ow et al., Science, 1986, 234, 856 and de Wet et al., Mol. Cell Biol., 1987, 7, 725, as well as several commercial teams. Stable cell lines can be generated by transferring a line of "ob answer" cells with the reporter constpjction and a selectable marker. Selectable markers are commonly used to generate stable cell lines as described in Recombinant DNA, 2nd eidition, J.D. Watson et al., 1992, page 216. Stably transfected cell lines can be used to generate high-pass assays for compounds that mimic, potentiate or block the physiological effects of the ob protein. The invention also extends to a compound that mimics, enhances or inhibits the physiological effect of the ob protein, when identified by the method described herein. The invention also extends to a set of parts adapted for use in the method described herein. When used herein "a compound that mimics the physiological effects of the ob protein" refers to a compound that is capable of acting in the absence of the ob protein or stimulating the ob protein receptor to provide substantially the same physiological effect as the ob protein or to activate a response towards the 3 'end of said receptor (post-receptor). When used herein "a compound that gives potency the physiological effect of the ob protein" refers to a compound that drives the potency and / or maximum physiological effect of the ob protein. When used herein "a compound that inhibits the physiological effect of the ob protein" refers to a compound that substantially reduces or blocks the physiological effect of the ob protein. The cDNA encoding the functional form of the polypeptide can be transfected under the control of a constitutive promoter (i.e., a viral promoter) or a promoter that can be regulated to optimize the expression of the polypeptide for the identification of agonists or antagonists as necessary. Alternatively, the response element and the reporter are expressed in a cell line, where a constitutive or regulatable promoter has been genetically engineered at a position 5 'of the chromosomally-conditional gene for the protein receptor or by the method of homologous recombination. These methods are reviewed by Waldman, Critical Reviews in Oncology / Hermatology, 1992, 12, 49 and a particular example is given in Riele et al., Proceedings of the Nationat Academy of Sciences, 1992, 89, 5128. The following examples illustrate the invention but they do not limit it in any way.

EXAMPLE General procedure: Ob response cells are transfected with a reporter plasmid containing a STAT response element, in 5 'tandem multiple copies and a minimal promoter, eg, simplex herpes timirin kinase and a reporter gene construct of luciferase using the standard methodology, for example, the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456). To correct for differences in transfection efficiency, the cells can be contrasted with a reference plasmid that expresses β-galactosidase activity. After a transfection period (12-24 hours) the cells are treated with various concentrations of compound and then they are harvested and lysed. The lysates are tested for luciferase activity, and if they are suitable for β-galactosidase activity. The antagonist activity can be assayed by pre-or coalition of an appropriate concentration of protein ob to the compound under evaluation and measurement of the reduction in luciferase response relative to that of the ob protein. There are standard methods for testing the activity of the luciferase enzyme for example, Ow et al., Science, 1986, 234, 856 and de Wet et al., 1987, 7, 725, as well as various commercial kits.

EXAMPLE 1 Gt1-5 cells (WC Wetsel, Cellular and Molecular Neurobioiogy, 1995, 15 (1), 43) are transfected with a pGL2-basic luciferase reporter vector (Promega) containing an insert of an oligonucleotide corresponding to a 4-fold tandem repeat of the STAT response element, TTCCCGGAA, 5 'of the minimal promoter for herpes simplex thymidine kinase (-35 to +10) using the standard methodology, eg, the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456). To correct the differences in transfection efficiency, the cells can be contrasted with a reference plasmid that expresses the activity of β-galactosidase. After a transfection period (12-24 hours), the cells are treated with varying concentrations of the compound and then harvested and lysed. The lysates are tested for luciferase activity, and are suitable for β-galactosidase activity. The antagonist activity can be tested by pre-coaddition and a suitable concentration of protein ob to the compound under evaluation and measurement of the reduction in the luciferase response relative to the ob protein. Standard methods exist to test luciferase enzyme activity, for example Ow et al., Science, 1986, 234, 856 and Wt et al., Mol. Cell Biol., 1987, 7, 725, as well as several commercial teams.

EXAMPLE 2 Rat PC12 cells (LA Greene and AS Tischier, Proc. Nati, Acad. Sci. USA, 1976, 73 (7), 2424) are transfected with a reporter plasmid, the pGL2-basic luciferase vector (Promega) contains a insert of an oligonucleotide corresponding to a 4-fold tandem repeat of the STAT response element, TTCCCGGAA, 5 'of the minimal promoter for the simplex herpes thymidine kinase (-35 to +10) using the standard methodology, for example, the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456). To correct the differences in the efficacy of trapsfection, the cells can be counteracted with reference plasmid expressing β-galactosidase activity. After a transfection period (12-24 hours) the cells are treated with varying concentrations of the compound and then harvested and used. The lysates are tested for luciferase activity, and if they are suitable for the β-galactosidase activity. The antagonist and luciferase enzyme activity can be assayed as described above.

EXAMPLE 3 HEL 92..1.7 cells (ATCC TIB 180) derived from a human erythroleukemia (P. Martin, Sccccience, 1982, 216 (4551), 1233) were transfected with a reporter plasmid, pGL2-basic luciferase vector (Promega) which contains an insert of an oligonucleotide corresponding to a 4-fold tandem repeat of the STAT response element, TTCCCGGAA, 5 'of the minimal promoter for herpes simplex thymidine kinase (-35 to +10) using the standard methodology, example, the calcium phosphate method (Graham and Van Der Eb, Viralogy, 1973, 52, 456). To correct for differences in transfection efficiency, cells can be counteracted with a reference plasmid expressing β-galactosidase activity. After a transfection period (12-24 hours) the cells are treated with varying concentrations of the compound and then cooked and lysed. The lysates are tested for luciferase activity, and if β-galactosidase activity is adequate. The antagonist and luciferase enzyme activity can be assayed as described above.

EXAMPLE 4 K562 cells (ATCC CCL-243) derived from chronic myelogenous leukemin (Proc. Soc. Exp. Med. 1981, 166, 546 and Blood, 1975, 45, 321) are transfected with a reporter plasmid, the luciferase vector pGL2 -báciso (Promega) contains an insert of an oligonucleotide corresponding to a 4-fold tandem repeat of the STAT response element, TTCCCGGAA, at 5 'of the minimal promoter for herpes simplex thymidine kinase (-35 to +10) using the standard methodology, for example, the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456). To correct the difference in transfection efficiency, cells can be contrasted with a reference plasmid that expresses β-galactosidase activity. After a transfection period (12-24 hours) the cells are treated with varying concentrations of compounds and then harvested and lysed. The lysates are tested for luciferase activity, and if β-galactosidase activity is adequate. The antagonist and enzyme activity of luciferase can be tested as described above.

EXAMPLE 5 BetaTC-3 insulinoma cells (T.J. Kieffer et al., Biochem, and Biophys, Res.Comm, 1996, 224, 552) are transfected with a reporter plasmid, and the pGL2-basic luciferase vector (Promega) contains an insert of a oligonucleotide corresponding to a 4-fold tandem repeat of the STA response element, TTCCCGGAA at 5 'of the minimal promoter for herpes simplex thymidine kinase (-35 to +10) using the standard methodology, for example, the phosphate method of calcium (Graham and Van Der Eb. Virology, 1973, 52, 456). To correct the differences in transfection efficiency, the cells can be contrasted with a reference plasmid expressing β-gaiactosidase activity. After a transfection period (12-24 hours) the cells are treated with varying concentrations of compound and then harvested and lysed. The lysates are tested for luciferase activity, and if β-galactosidase activity is suitable. The antagonist and luciferase enzyme activity can be tested as described above.

EXAMPLE 6 A5 cells derived from preadipocyte (Y. Bai et al., J. Biol.

Chem, 1996, 271 (24), 13939) is transfected with a reporter plasmid, the pGL2-basic luciferase vector (Promega) contains an insert of an oligopucieotide corresponding to a 4-fold tandem repeat of the STAT response element, TTCCCGGAA, the 5 'of the minimal promoter for the tlmidlna kinase simplex herpes (-35 to +10) using the standard methodology, for example, the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456) . To correct the differences in transfection efficiency, the cells can be contrasted with a reference plasmid that expresses the β-galactosidase activity. After a transfection period (12-24 hours) the cells are treated with varying compound considerations then harvested and lysed. The lysates are tested for luciferase activity, and if they are suitable for β-galactosidase activity. The antagonist and luciferase activity can be assayed as described above.

EXAMPLE 7 The C2 C12 cells of mouse myotube (Nature, 1977, 270, 725) and transfected with a reporter plasmid, the pGL2-basic luciferase vector (Promega) contains an insert of an oligonucleotide corresponding to a 4-fold tandem repeat of the STAT response element, TTCCCGGAA, 5 'from the minimal promoter for the simplex herpes thymidine kinase (-35 to +10) using the standard methodology, for example, the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456 ). To correct for differences in transfection efficiency, the cells can be contrasted with a reference plasmid that expresses β-galactosidase activity.

After a period of transfection (12-24 hours) the cells are treated with varying concentrations of compound and then harvested and lysed. The lysates are assayed for luciferase activity, and if β-galactosidase activity is adequate. The antagonist and luciferase enzyme activity can be assayed as described above.

EXAMPLE 8 Ovarian SK-OV-3 cells, ATCC number HTB77, (J. Fogh and G Trempe in Human Tummmour Cells In Vitrro p155-159, J. Fogh (ed) Plenum Press, New York, 1975, J. Fogh and G Trempe J. Nati, Cancer Inst Bethesda, 1977, 587: 209-214) are transfected with a reporter plasmid, pGL2-basic luciferase vector (Promega) containing an insert of an oligonucleotide corresponding to a 4-fold tandem repeat. of the response element STAT, TTCCGGAA, at the 5 'of the minimal promoter for thymidine kinases simplex hefes (-35 to +10) using the standard methodology. For example, the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456). To correct the differences in transfection efficiency of the cells they can be counteracted with a reference plasmid that expresses the β-galactosidase activity. After a transfection period (12-24 hours) the cells are treated with varying concentrations of compound and then harvested and lysed. The lysates are tested for luciferase activity, and if β-galactosidase activity is suitable. The antagonist and luciferase enzyme activity can be assayed as described above.

EXAMPLE 9 Cell lines derived from hepatoma transfected with the functional form of the leptin receptor (Baummmann et al., Proc. Nat. Acad.

Sci., 1996, 93, 8374-8378) are contrasted with a reporter plasmid, the pGL2-basic luciferase vector (Promega) containing an insert of an oligonucleotide corresponding to a 4-fold tandem repeat of the STAT response element, TTCCCGGAA, at 5 'of the minimal promoter for the thiimidine kinase of hefes simplex (-35 to +10) using the standard methodology, for example, the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456). To correct for differences in transfection efficiency, the cells can be contrasted with a reference plasmid that expresses β-galactosidase activity. After a transfection period (12-24 hours) the cells are treated with varying concentrations of compound and then harvested and lysed. The lysates are tested for luciferase activity, and if β-galactosidase activity is suitable. The antagonist and luciferase enzyme activity can be tested as described above.

EXAMPLE 10 WRL68 cells were cultured to agglutinate in their standard culture medium in the simultaneous presence of 10% fetal calf serum. In binding the cells to a serum-free medium for a minimum of 18 hours (reducing the serum concentration can also produce similar results). Cells lacking serum are then treated with leptin or with serum (as a positive control for the higher regulation c-fos) during varying periods of 5 minutes to 2 hours. The RNA was extracted from the cells and subjected to electofóresis gel and northern blot. The RNA subjected to northern blot was then tested with a test labeled c-fos to analyze the expression of c-fos, the information was normalized when analyzing the expression of beta-actin. The leptin treatment significantly boosted the expression of c-fos in the WRL68 cells (see figure 1) as if it were the positive control serum, and therefore STATs are expected to be activated. Therefore, it is expected that WRL68 cells are suitable for the identification of leptin mimics. Other cell lines could also be identified by analyzing c-fos expression in response to leptin.

Claims (13)

NOVELTY OF THE INVENTION CLAIMS

1. A method for the detection of a compound that enhances or inhibits the physiological effect of the ob protein, which method comprises: a) a compound that mimics the physiological effect of the ob protein, evaluating the effect of the compound in the activated protein signal transducer and transcription activator (STAT) of the DNA response element coupled to a reporter gene; or b) a compound that potentiates or inhibits the physiological effect of the ob protein, by evaluating the effect of a compound on the response provided by the ob protein in a STAT activated protein or STAT of the DNA response element coupled to a reporter gene; wherein the response element and the reporter are expressed in a line of response cells of protein ob, whose cell line is selected from the list consisting of: a line of cells derived from the hlpotálamo; a line of cells derived from pheochromocytoma; a line of hematopoietic cells; a line of cells derived from the pancreatic cell; a line of liver derived cells; a line of cells derived from preadipocytes; a line of cells derived from skeletal muscle; and a line of cells derived from the ovarian cell; or the response element and the reporter are expressed in a cell line, whose cell line (the cell line created by genetic engineering) is also transfected with a polypeptide that is capable of stimulating an activated ob protein.

STAT of the DNA response element and contains the appropriate STAT proteins. 2. A method according to claim 1. further characterized because the line of hypothalamic derived cells is the neuronal cell line GT1-7.

3. A method according to claim 1. further characterized in that the cell derived from pheochromocytoma is renal pheochromocytoma of rat PC12 cells.

4. A method according to claim 1. further characterized in that the hematopoietic derived cell line is the HEL 92.1.7 cell line (ATCC TIB 180) derived from a human erythroleukemia or the K562 cell line (ATCC CCL- 243) derived from chronic myelogenous leukemia.

5. A method according to claim 1. further characterized in that the line of pancreatic derived cells is the BetaTC-3 cell line or the RIN5mF cells.

6. A method according to claim 1. further characterized in that the cell line derived from predipocytes is the 30A5 cell line or the differentiated cell lines 3T3-L1 or 3T3-F442A.

7. A method according to claim 1. further characterized in that the line of liver derived cells is the HepG2 cell line or the liver cells WRL68 and Change.

8. - A method according to claim 1. further characterized in that the line of cells derived from skeletal muscle is the C2 C12 cell line of mouse myotube.

9. A method according to claim 1. further characterized in that the line of theozoic derivative cells is the cell line SK-OV-3, ATCC number HTB77.

10. A method according to claim 1, further characterized in that the polypeptide capable of stimulating a STAT activated protein or STAT of the DNA response element is a functional isoform of the leptin receptor.

11. A method according to claim 1, further characterized in that the response element is coupled to a promoter gene, preferably a minimal promoter.

12. A method according to claim 1, further characterized in that the response element is a nucleotide of the formula TT (N) n AA, where N is any pucleotide and n is 4, 5 or 6. 13.- A method according to claim 1, further characterized in that the response element is TTCCCGGAA