CN1242055A - Method for the detection of compounds that modulate the effects of the OB3se (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 response 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 detecting compounds that modulate the effect of an obesity (OB) gene

The present invention relates to a novel method, more particularly, to a method for detecting compounds that mimic, enhance or inhibit the physiological effects of ob protein.

The ob protein (or leptin) is a secreted hormone that acts as a signal from adipose tissue to other organs, regulating weight and energy balance (Zhang et al., Nature, 1994, 372, 425). The ob protein has also been shown to have other roles in hematopoietic and reproductive functions (Cioffi et al., Nature Medicine, 1996, 2(5), 585). Protein molecules containing a core of four α-helices forming a bundle of up-up-down-down topology comprise a family of cytokines and growth factors. Proteins of this family cause homo- and hetero-oligomerization of membrane receptors, which are known to activate kinase cascades leading to gene transcription. The oligomerization-activated receptors of this family fall into two broad categories: one class, such as the epidermal growth factor receptor, has integrated tyrosine kinase activity in their intracellular domain (A.Ullrich and J.Schlessinger, Cell , 1990, 61, 203-212); a class such as IL4 receptor and erythropoietin receptor, lack this activity, but mediate their response through a related protein tyrosine kinase (JNIhle et al. Al, TIBS, 1994, 19, 222-227). Both receptor subclasses are activated by cytokines, but 4-helix bundle proteins only activate a subclass of non-integrated tyrosine kinases. Receptors for non-integral protein tyrosine kinases generally function through a pathway involving Janus kinases (JAK) and signal transducers and activators of transcription (STAT) proteins associated with the receptors. When activated, STAT proteins bind DNA response elements to control gene transcription. Oligonucleotides comprising DNA regulatory elements with the general sequence TT(N) _nAA have been identified (Seidel et al., Proc Nat. Acad. Sci. USA, 1995, 92, 3041) as STAT response elements. These elements bind to STAT proteins in response to signaling molecules such as cytokines.

In co-pending UK Patent Application No. 9509164.1 we have stated our discovery that the ob protein is characterized by a tertiary structure of four helical bundles. We now believe that the ob protein interacts with a membrane-bound receptor, that this interaction activates the JAK-STAT kinase cascade, and thus constitutes an assay system for the detection of compounds that mimic, enhance or inhibit the physiological effects of the ob protein Foundation. Such an assay can be used to screen compounds for the treatment of ob protein regulated weight disorders, energy balance disorders, hematopoietic disorders, fertility disorders, and other conditions. This assay is particularly useful in screening compounds for the treatment of conditions associated with obesity, anorexia, cachexia, and diabetes.

Co-pending International Patent Application No. PCT/EP96/02291 relates to a new detection method using JAK-STAT technology. We have now discovered a particularly advantageous detection method that also utilizes this technique.

Therefore, the present invention provides a method for detecting compounds that simulate, enhance or inhibit the physiological effects of ob protein, said method comprising:

(a) for compounds that mimic the physiological effects of the ob protein, assessing the effect of the compound on the signal transducer and activator of transcription (STAT) DNA response element of the ob protein linked to a reporter gene; or

(b) for compounds that enhance or inhibit the physiological effects of the ob protein, assessing the effect of the compound on the response provided by the ob protein on an ob protein-activated STAT DNA response element linked to a reporter gene;

in

The response element and the reporter gene are expressed in an ob protein-responsive cell line selected from the group consisting of:

Hypothalamic-derived cell lines;

Pheochromocytoma-derived cell lines;

Hematopoietic-derived cell lines;

Pancreatic-derived cell lines;

Liver-derived cell lines;

Preadipocyte-derived cell lines;

Skeletal muscle-derived cell lines; and

Ovarian-derived cell lines; or

The response element and the reporter gene are expressed in a cell line (engineered cell line) that is also transfected with a polypeptide that mediates the ob protein and contains a suitable STAT protein Stimulation of STAT DNA response elements activated by ob proteins.

One suitable cell line is a hypothalamus-derived cell line.

One suitable cell line is a pheochromocytoma-derived cell line.

A suitable cell line is a hematopoietic-derived cell line.

A suitable cell line is a pancreatic-derived cell line.

A suitable cell line is a liver-derived cell line.

One suitable cell line is a preadipocyte-derived cell line.

A suitable cell line is a skeletal muscle-derived cell line.

A suitable cell line is an ovarian-derived cell line.

The response element and the reporter gene are preferably expressed in a cell line (engineered cell line) which is also transfected with a polypeptide and contains a suitable STAT protein, wherein the polypeptide mediates Stimulation of Ob-activated STATDNA response elements induced by Ob.

An example of a hypothalamus-derived cell line is the neuronal GT1-7 cell line described by W.C. Wetsel in Cellular and Molecular Neurobiology, 1995, 15(1), 43.

An example of a pheochromocytoma-derived cell line is the adrenal pheochromocytoma rat PC12 cells (L.A. Greene and A.S. Tischler, Proc. Natl. Acad. Sci. USA. 1976, 73(7), 2424).

An example of a hematopoietic derived cell line is the HEL92.1.7 (ATCC TIB 180) cell line obtained from human erythroleukemia (P.Martin, Science.1982, 216(4551), 1233)

Another example of a hematopoietic derived cell line is the K562 (ATCC CCL-243) cell line derived from human chronic myelogenous leukemia (Proc Soc. Exp. Biol Med. 1981, 166, 546 and Blood, 1975, 45, 321) .

An example of a pancreatic-derived cell line is the βTC-3 cell line (T.J. Kieffer et al., Biochem. and Biophys. Res. Comm., 1996, 224, 552).

Examples of other pancreatic cell lines include RIN5mF cells, which were recently reported to be responsive to obesin (Md Shahidul Islam et al; Biochem Biophys Res Comm. 1997, 238, 851-855).

An example of a preadipocyte-derived cell line is the 30A5 cell line (Y. Bai et al., J Biol. Chem., 1996, 271(24), 13939).

Examples of other preadipocyte-derived cell lines include the differentiated 3T3-L1 and differentiated 3T3-F442A cell lines, both of which are now commercially available.

An example of a liver-derived cell line 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 the H35 cell line (Y Wang et al; J Biol Chem, 1997, 272, 16216-16223).

Examples of other liver-derived cell lines are WRL68 hepatocytes and Change hepatocytes, both of which are now commercially available.

An example of a skeletal muscle-derived cell line is the mouse myotube C2 C12 cell line (Nature, 1977, 270, 725).

An example of an ovarian-derived cell line is the SK-OV-3 cell line, ATCC No. HTB77, (J. Fogh and G Trempe in Human Tumor Cells In Vitro pp. 155-159, J. Fogh (ed.) Plenum Press, New York , 1975; J. Fogh and G Trempe J. Natl. Cancer Inst Bethesda, 1977, 587: 209-214)

A suitable polypeptide that mediates the stimulatory effect of ob protein on STAT DNA response elements activated by ob protein is a functional isoform of the ob gene receptor, such as Tartaglia et al. (Cell, 1995, 83, 1263 ) identified.

The response element is suitably linked to a promoter gene, preferably a minimized promoter.

One suitable response element is a nucleotide of formula TT(N) _nAA , where N is any nucleotide and n is 4, 5 or 6.

A preferred response element is selectively activated by intracellular events mediated by the interaction of the ob protein with its receptor. Such selective response elements can be determined by transfecting a range of response element-reporter gene constructs into an ob effector cell line and measuring the relative activation of the ob protein compared to other cytokines.

A preferred response element is a nucleotide of the formula TT(N) _nAA , where N is any nucleotide and n is 4 or 5.

Another suitable effector element is TTCCCGGAA.

Another suitable response element is that region in the promoter of a gene regulated by the ob protein that is required for STAT interaction. This gene depends on the specific therapeutic application of the compound to be screened by the assay.

Suitable reporter genes are firefly luciferase or chloramphenicol acetyltransferase.

A suitable promoter is a minimal promoter such as the herpes simplex virus thymidine kinase promoter or the SV40 promoter.

Other effector cell lines can be identified using a displacement binding assay. However, binding may not be to the functional long form of the receptor, the form that transmits a signal to the cytoplasm. Functional long-term forms of receptors can be identified by PCR or Northern blot analysis (eg human ob receptor: Tartaglia et al., Cell, 1995, 83, 1263). Finally, effector cell lines were detected by monitoring cellular events that occurred in the presence of different concentrations of obesin. Possible approaches to identify candidate cell lines or monitor these cellular events include the following:

1. Microphysiometer: This method detects small changes in pH caused by biochemical changes in cells. Ob protein effector cells undergo biochemical changes when stimulated that cause small changes in the rate of extracellular acidification that can be detected with a silicon microphysiometer. Miniature biometer biosensor methodology has been reviewed by McConnell, Science, 1992, 257, 1906 .

2. Electrophoretic mobility shift assay (EMSA): nuclear extracts obtained from ob protein-treated cells are mixed with radiolabeled oligonucleotides containing a promiscuous or specific Sexual STAT response element DNA sequence. Cell extracts responsive to ob protein cause gel shift of oligonucleotides of STAT response elements.

References: Book "Recombinant DNA", 2nd Edition, Watson et al., 1992, p. 158; Lamb et al., Blood, 1994, 83, 2063.

3. Protein phosphorylation analysis method:

The coupling of receptor activation through tyrosine phosphorylation of intracellular proteins to the final response can be analyzed by using antibodies that recognize phosphorylated tyrosine. More specifically, since the obesin receptor can stimulate tyrosine phosphorylation of the JAK/STAT pathway, this approach provides a means to detect obesin-responsive cell lines. Specific JAK/STAT antibodies can be used together with anti-phosphotyrosine antibodies to detect obesin activation in obesin-responsive cell lines. Both inhibition and stimulation of protein phosphorylation can occur. In particular, inhibition of insulin-stimulated phosphorylation of the insulin receptor and insulin receptor substrate-1 by the ob protein has been shown in rat-1 fibroblasts overexpressing the insulin receptor (Kroder et al. 1996, Exp . Clin. Endocrinol. Diabete, 104, Suppl. 2, p. 66).

4. Displacement binding: After incubating cell lines with radioactively labeled obesin, such as [ ¹²⁵ I]-obesin, the relationship between non-specific binding and specific binding of obesin can be studied by adding unlabeled obesin . The high ratio of specific to non-specific binding suggests that the cell line likely contains the obesin receptor.

5. Detection of a functional form of the ob receptor, preferably a functional long form of the protein, by use of selective antibodies.

6. Detection of functional form of ob receptor, preferably functional long form mRNA, by Northern analysis, RT-PCR analysis or "slot blot" analysis.

7. Detection of increased c-fos mRNA after treatment with obesin. c-fos mRNA can be detected using Northern analysis, RT-PCR analysis, or "slot blot" analysis.

Cell lines that are known to be involved in the control of certain aspects of a particular disease state for which compounds are being sought to treat are preferred.

Cell lines and fibroblasts derived from liver, brain, or pancreatic tissue are particularly useful "ob effector" cells for analysis of compounds against obesity or diabetes. Certain regions of the brain are the focus of the weight-controlling and energy-balance-regulating effects of the ob protein. The liver controls many metabolic processes that regulate lipid and glucose levels. Cells derived from these organ specific regions containing appropriate endogenous JAK, STAT proteins and other intracellular proteins required to mediate the effects of obesin are preferred.

The response element, reporter gene, and preferably also the promoter are suitably inserted into a vector capable of transfecting ob effector cell lines.

Suitable vectors are commercially available vectors such as pGL2-basic luciferase vector (Promega).

A logical configuration of the vector is to place the STAT DNA response element upstream of a promoter and a reporter gene. A more rational configuration of the vector is to insert multiple tandem repeats (2-10) of STAT DNA response elements upstream of a thymidine kinase promoter and a luciferase reporter gene.

Construction of vectors comprising a reporter gene linked to a minimal promoter such as the herpes simplex virus thymidine kinase promoter or the SV40 promoter such as the firefly firefly Luciferase or chloramphenicol acetyltransferase. The DNA fragment of the STAT response element was inserted into the vector using the appropriate restriction enzyme sites upstream of the minimized promoter.

Responsive elements, reporter genes, and promoters are incorporated into the vector as required using conventional expression techniques, such as inserting DNA fragments of the responsive elements into the vector using appropriate restriction enzyme sites upstream of the minimized promoter.

The STAT response element-luciferase reporter system 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.

Ob effector cells are transfected with a STAT response element-minimized promoter-luciferase reporter gene construct using standard methodology, such as the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456). To correct for differences in transfection efficiency, cells can be co-transfected with a reference plasmid expressing β-galactosidase activity. After a certain period of time (12-24 hours) after transfection, cells were treated with various concentrations of compounds, then harvested and lysed. Lysates were assayed for luciferase activity and, if appropriate, beta-galactosidase activity. Potentiation or antagonist activity can be assayed by adding an appropriate concentration of ob protein prior to or simultaneously with the compound being evaluated and measuring the increase or decrease in the luciferase response compared to ob protein alone. There are standard methods for assaying luciferase activity (eg Ow et al., Science, 1986, 234, 856 and de Wet et al., Mol. Cell Biol., 1987, 7, 725) as well as several commercial kits.

Stable cell lines can be generated by transfecting an "ob effector" cell line with a reporter gene construct and a selectable marker. Selectable markers are often used to generate stable cell lines as described in Recombinant DNA, Second Edition, J.D. Watson et al., 1992, p. 216. These stably transfected cell lines can be used for high throughput assays of compounds that mimic, enhance or block the physiological effects of the ob protein.

The invention also extends to compounds identified using the methods disclosed herein that mimic, enhance or inhibit the physiological effects of the ob protein.

The invention also extends to kits partially adapted to use the methods disclosed herein.

As used herein, a "compound that mimics the physiological effects of ob protein" refers to a compound that, in the absence of ob protein, either stimulates the ob protein receptor to provide substantially the same physiological effect as the ob protein, or activates this receptor downstream (responsive Post-body (post-receptor)) response.

A "compound that enhances the physiological effect of ob protein" as used herein refers to a compound that enhances the potency and/or maximal physiological effect of ob protein.

A "compound that inhibits the physiological effects of ob protein" as used herein refers to a compound that weakens or substantially blocks the physiological effects of ob protein.

A cDNA encoding a functional form of the polypeptide can be transfected and placed under the control of a constitutive promoter (such as a viral promoter) or a regulatable promoter, optimized for the identification of agonists or antagonists as desired. Expression of the polypeptide is optimized. Alternatively, the response element and reporter gene are expressed in a cell line in which a constitutive or regulatable promoter has been inserted by homologous recombination at a position upstream of the chromosomal encoding gene for the ob protein receptor. Such methods have been reviewed by Waldman, Critical Review in Oncology/Hematology, 1992, 12, 49, and a concrete example has been given in Riele et al., Proceeding of the National Academy of Sciences, 1992, 89, 5128.

The following examples illustrate the invention but do not limit it in any respect.

General method of embodiment:

Using standard methodology, such as the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456), ob effector cells are transfected with a reporter plasmid containing a minimal promoter (e.g., herpes simplex virus Thymidine kinase) and multiple tandem repeat copies of the STAT response element upstream of a luciferase reporter gene construct. To correct for differences in transfection efficiency, cells can be co-transfected with a reference plasmid expressing β-galactosidase activity. After a certain period of time (12-24 hours) after transfection, cells were treated with various concentrations of compounds, then harvested and lysed. Lysates were assayed for luciferase activity and, if appropriate, beta-galactosidase activity. Antagonist activity can be assayed by adding an appropriate concentration of ob protein to the compound being evaluated, either in advance or simultaneously, and measuring the attenuation of the luciferase response compared to ob protein alone. There are standard methods for assaying luciferase activity (eg Ow et al., Science, 1986, 234, 856 and de Wet et al., Mol. Cell Biol., 1987, 7, 725) as well as several commercial kits. Example 1

Gt1-7 cells (W.C. Wetsel, Cellular and Molecular Neurobiology, 1995, 15(1), 43), wherein the plasmid contains STAT corresponding to four tandem repeats upstream of a minimal promoter such as herpes simplex virus thymidine kinase (-35 to +10) Oligonucleotide insert of the response element TTCCCGGAA. To correct for differences in transfection efficiency, cells can be co-transfected with a reference plasmid expressing β-galactosidase activity. After a certain period of time (12-24 hours) after transfection, cells were treated with various concentrations of compounds, then harvested and lysed. Lysates were assayed for luciferase activity and, if appropriate, beta-galactosidase activity. Antagonist activity can be assayed by adding an appropriate concentration of ob protein to the compound being evaluated, either in advance or simultaneously, and measuring the attenuation of the luciferase response compared to ob protein alone. There are standard methods for assaying luciferase activity (eg Ow et al., Science, 1986, 234, 856 and de Wet et al., Mol. Cell Biol., 1987, 7, 725) as well as several commercial kits. Example 2

Rat PC12 cells (L.A. Greene and A.S. Tischler) were transfected with the reporter plasmid pGL2-basic luciferase vector (Promega) using standard methodology, such as the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456). , Proc.Natl.Acad.Sci.USA.1976,73(7),2424), wherein said plasmid contains a minimal promoter (such as herpes simplex virus thymidine kinase (-35 to +10)) upstream Oligonucleotide inserts corresponding to four tandem repeats of the STAT response element TTCCCGGAA. To correct for differences in transfection efficiency, cells can be co-transfected with a reference plasmid expressing β-galactosidase activity. After a certain period of time (12-24 hours) after transfection, cells were treated with various concentrations of compounds, then harvested and lysed. Lysates were assayed for luciferase activity and, if appropriate, beta-galactosidase activity. Antagonist and luciferase activity can be assayed as described above. Example 3

HEL 92.1.7 from human erythroleukemia ( ATCC TIB 180) cells (P.Martin, Science, 1982, 216(4551), 1233), wherein the plasmid contains the expression of a minimal promoter (such as herpes simplex virus thymidine kinase (-35 to +10)) The upstream oligonucleotide insert corresponds to four tandem repeats of the STAT response element TTCCCGGAA. To correct for differences in transfection efficiency, cells can be co-transfected with a reference plasmid expressing β-galactosidase activity. After a certain period of time (12-24 hours) after transfection, cells were treated with various concentrations of compounds, then harvested and lysed. Lysates were assayed for luciferase activity and, if appropriate, beta-galactosidase activity. Antagonist and luciferase activity can be assayed as described above. Example 4

K562 obtained from human chronic myelogenous leukemia was transfected with the reporter plasmid pGL2-basic luciferase vector (Promega) using standard methodology, such as the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456). (ATCC CCL-243) cell line (Proc. Soc. Exp. Biol. Med. 1981, 166, 546 and Blood, 1975, 45, 321), wherein the plasmid contains Upstream of thymidine kinase (-35 to +10)) corresponds to an oligonucleotide insert of four tandem repeats of the STAT response element TTCCCGGAA. To correct for differences in transfection efficiency, cells can be co-transfected with a reference plasmid expressing β-galactosidase activity. After a certain period of time (12-24 hours) after transfection, cells were treated with various concentrations of compounds, then harvested and lysed. Lysates were assayed for luciferase activity and, if appropriate, beta-galactosidase activity. Antagonist and luciferase activity can be assayed as described above. Example 5

The insulinoma βTC-3 cell line (T.J. Kieffer et al., Biochem and Biophys. Res. Comm., 1996, 224, 552), wherein the plasmid contains a minimal promoter (such as herpes simplex virus thymidine kinase (-35 to +10)) corresponding to Oligonucleotide insert of four tandem repeats of the STAT response element TTCCCGGAA. To correct for differences in transfection efficiency, cells can be co-transfected with a reference plasmid expressing β-galactosidase activity. After a certain period of time (12-24 hours) after transfection, cells were treated with various concentrations of compounds, then harvested and lysed. Lysates were assayed for luciferase activity and, if appropriate, beta-galactosidase activity. Antagonist and luciferase activity can be assayed as described above. Example 6

The preadipocyte-derived 30A5 cell line (Y .Bai et al., J.Biol.Chem., 1996, 271 (24), 13939), wherein the plasmid contains a minimal promoter (such as herpes simplex virus thymidine kinase (-35 to +10)) The upstream oligonucleotide insert corresponds to four tandem repeats of the STAT response element TTCCCGGAA. To correct for differences in transfection efficiency, cells can be co-transfected with a reference plasmid expressing β-galactosidase activity. After a certain period of time (12-24 hours) after transfection, cells were treated with various concentrations of compounds, then harvested and lysed. Lysates were assayed for luciferase activity and, if appropriate, beta-galactosidase activity. Antagonist and luciferase activity can be assayed as described above. Example 7

The mouse myotube C2 C12 cell line (Nature , 1977, 270, 725), wherein the plasmid contains an oligonucleotide corresponding to four tandem repeats of the STAT response element TTCCCGGAA upstream of a minimized promoter such as herpes simplex virus thymidine kinase (-35 to +10) Nucleotide inserts. To correct for differences in transfection efficiency, cells can be co-transfected with a reference plasmid expressing β-galactosidase activity. After a certain period of time (12-24 hours) after transfection, cells were treated with various concentrations of compounds, then harvested and lysed. Lysates were assayed for luciferase activity and, if appropriate, beta-galactosidase activity. Antagonist and luciferase activity can be assayed as described above. Example 8

Ovarian SK-OV-3 cells were transfected with reporter plasmid pGL2-basic luciferase vector (Promega) using standard methodology, such as the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456), ATCC No. HTB77, (J. Fogh and G Trempe, Human Tumor Cells In Vitro pp. 155-159, J. Fogh (ed.) Plenum Press, NewYork, 1975; J. Fogh and G Trempe J. Natl. Cancer Inst Bethesda, 1977, 587 :209-214), wherein the plasmid contains oligonucleotides corresponding to four tandem repeats of the STAT response element TTCCCGGAA upstream of a minimal promoter such as herpes simplex virus thymidine kinase (-35 to +10) acid insert. To correct for differences in transfection efficiency, cells can be co-transfected with a reference plasmid expressing β-galactosidase activity. After a certain period of time (12-24 hours) after transfection, cells were treated with various concentrations of compounds, then harvested and lysed. Lysates were assayed for luciferase activity and, if appropriate, beta-galactosidase activity. Antagonist and luciferase activity can be assayed as described above. Example 9

A functional form of the obesin receptor ( Baumann et al., Proc.Nat.Acad.Sci., 1996,8374-8378) transfected hepatoma-derived cells, wherein the plasmid contains a minimal promoter (such as herpes simplex virus thymidine kinase (-35 Upstream to +10)) corresponds to the oligonucleotide insert of four tandem repeats of the STAT response element TTCCCGGAA. To correct for differences in transfection efficiency, cells can be co-transfected with a reference plasmid expressing β-galactosidase activity. After a certain period of time (12-24 hours) after transfection, cells were treated with various concentrations of compounds, then harvested and lysed. Lysates were assayed for luciferase activity and, if appropriate, beta-galactosidase activity. Antagonist and luciferase activity can be assayed as described above. Example 10

WRL68 cells were grown to confluency in their standard medium in the presence of 10% fetal bovine serum. After the cells have grown to confluence, transfer the cells to serum-free medium for at least 18 hours (lower serum concentrations can also have a similar effect). Serum-starved cells were then treated with obesin or serum (as a positive control for c-fos upregulation) for varying lengths of time from 5 minutes to 2 hours. RNA was extracted from cells and subjected to gel electrophoresis and Northern blotting. Blotted RNA was probed with a c-fos-labeled probe to analyze c-fos expression, and data were normalized by analysis for β-actin expression. Obesin treatment, like positive control serum treatment, significantly enhanced c-fos expression in WRL68 cells (Table 1), and thus was expected to activate STAT. It is therefore expected that WRL68 cells would be suitable for the identification of obesity protein mimetics. Other cell lines can also be identified by analyzing c-fos expression in response to obesin.

Claims (13)

1. A method for detecting a compound that simulates, enhances or inhibits the physiological effects of ob protein, the method comprising: (a) for compounds that mimic the physiological effects of the ob protein, assessing the effect of the compound on the ob protein-activated Signal Transducer and Activator of Transcription (STAT) DNA response element linked to a reporter gene; or (b) for compounds that enhance or inhibit the physiological effects of the ob protein, assessing the effect of the compound on the response provided by the ob protein on an ob protein-activated STAT DNA response element linked to a reporter gene; in The response element and the reporter gene are expressed in an ob protein-responsive cell line selected from the group consisting of: Hypothalamic-derived cell lines; Pheochromocytoma-derived cell lines; Hematopoietic-derived cell lines; Pancreatic-derived cell lines; Liver-derived cell lines; Preadipocyte-derived cell lines; Skeletal muscle-derived cell lines; and Ovarian-derived cell lines; or The response element and the reporter gene are expressed in a cell line (engineered cell line) that is also transfected with a polypeptide that stimulates the ob protein and contains a suitable STAT protein Activated STAT DNA response elements. 2. The method according to claim 1, wherein said hypothalamus-derived cell line is a neuronal GT1-7 cell line. 3. The method according to claim 1, wherein said pheochromocytoma-derived cell line is adrenal pheochromocytoma rat PC12 cells. 4. The method according to claim 1, wherein said hematopoietic derived cell line is the HEL 92.1.7 (ATCC TIB 180) cell line obtained from human erythroleukemia or the K562 (ATCC CCL-243) cell line obtained from human chronic myelogenous leukemia ) cell line. 5. The method according to claim 1, wherein said pancreatic-derived cell line is Beta TC-3 cell line or RIN5mF cells. 6. The method according to claim 1, wherein said preadipocyte-derived cell line is 30A5 cell line or differentiated 3T3-L1 or differentiated 3T3-F442A cell line. 7. The method according to claim 1, wherein said liver-derived cell line is a HepG2 cell line or WRL68 hepatocytes and Change hepatocytes. 8. The method according to claim 1, wherein said skeletal muscle-derived cell line is a mouse myotube C2 C12 cell line. 9. The method according to claim 1, wherein said ovarian-derived cell line is SK-OV-3 cell line, ATCC number HTB77. 10. The method according to claim 1, wherein said polypeptide capable of stimulating the ob protein-activated STAT DNA response element is a functional isoform of obesin. 11. The method according to claim 1, wherein said response element is linked to a promoter gene, preferably a minimal promoter. 12. The method according to claim 1, wherein said response element is a nucleotide of formula TT(N) _nAA , wherein N is any nucleotide and n is 4, 5 or 6. 13. The method according to claim 12, wherein said effector element is TTCCCGGAA.

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