CN1192248A - Method for detecting compounds that modulate the action of obesity proteins - Google Patents

Abstract

A method for detecting a compound which mimics, enhances or inhibits the physiological effect of leptin comprising assessing the effect of the compound on a leptin-activated signal transducer and a activator of transcription (STAT) DNA response element coupled to a reporter gene for a compound which mimics the physiological effect of leptin, or (b) assessing the effect of the compound on the response provided by the leptin to a STAT DNA response element activated by a leptin coupled to a reporter gene for a compound which enhances or inhibits the physiological effect of leptin, a response element and a reporter gene expressed in a leptin responsive cell line, a kit suitable for use in the method and a compound identified by the method.

Description

Method for detecting compounds that modulate the action of obesity proteins

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 obesity protein (ie leptine) is a secreted hormone that acts as a signal from adipose tissue to other organs to regulate body weight and energy balance (Zhang et al., Nature, 1994, 372, 425). It has been postulated that obesity proteins also play a role in hematopoiesis and reproductive functions (Cioffi et al., Nature Medicine, 1996, 2(5), 585). Protein molecules containing a core of four alpha helices forming a bundle with an up-up-down-down topology include a family of cytokines and growth factors. Proteins of this family cause homo- and hetero-oligomerization of membrane receptors known to activate kinase cascades leading to gene transcription. This family of receptors activated by oligomerization is divided into two categories; namely, epidermal growth factor, which has a complete tyrosine kinase activity in its intracellular functional domain (A.Ullrich & J.Schlessinger, Cell, 1990, 61, 203-212), and classes such as IL4 and erythropoietin, which lack this activity and mediate their response through a combined protein tyrosine kinase pathway (J.N.Ihle et al., TIBS, 1994, 19, 222 -227). Both receptor subtypes are activated by cytokines, but the 4-helix bundle protein activates only the nonintact tyrosine kinase subtype. Such nonintact protein tyrosine kinase receptors generally act through pathways involving Janus kinases (JAK) and signal transducer and activator of transcription (STAT) proteins that these receptors bind. During activation, STAT proteins bind to DNA response elements to control gene transcription. Oligonucleotide sequences of DNA regulatory elements including the general sequence TT(N)nAA have been identified as STAT response elements (Seidel et al., Proc. Nat. Acad. Sci. USA, 1995, 92, 3041). These elements bind to STAT proteins in response to signaling molecules such as cytokines.

In co-pending United Kingdom Patent Application No. 9509164.1 we have described our discovery that the obesity protein is characterized by a four-helix bundle tertiary structure. We now believe that obesity proteins interact with membrane-bound receptors that activate the JAK-STAT kinase cascade and thus form the basis of an assay system for detecting compounds that mimic, enhance or inhibit the physiological effects of obesity proteins. This assay is useful in selecting compounds for the treatment of body weight, energy balance, hematopoiesis, reproduction, and other diseases mediated by "obesity proteins." This assay is particularly useful for selecting compounds for the treatment of diseases associated with obesity, anorexia, extreme emaciation and diabetes.

Therefore, the present invention provides a method for detecting compounds that mimic, enhance or inhibit the physiological effects of obesity proteins, the method comprising:

(a) assessing the effect of a compound that mimics the physiological action of obesity protein on the signal transducer and activator of transcription (STAT) DNA response element of obesity protein coupled to a reporter gene; or

(b) for a compound that enhances or inhibits a physiological effect of an obesity protein, assessing the effect of the compound on the response provided by the obesity protein to a STATDNA response element activated by the obesity protein coupled to a reporter gene;

Response elements and reporter genes expressed in obesity protein responsive cell lines.

The response element is suitable for coupling to a reporter gene, preferably a small promoter.

One suitable response element is the nucleotide formula TT(N) _n AA, where N is any nucleotide and n is 4, 5 or 6.

Intracellular events mediated by obesity proteins interacting with their receptors selectively activate preferred response elements. This selective response element can be determined by examining the relative activation of obesity proteins relative to other cytokines when transfecting obesity-responsive cell lines with some of the response element-reporter constructs.

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

A further suitable response element is TTCCCGGAA.

Further suitable response elements are the promoter regions of genes regulated by obesity proteins, which are required for STAT interaction. This gene depends on the specific therapeutic use of the compound selected by this test.

Suitable reporter genes are firefly luciferase or chloramphenicol acetyltransferase.

Suitable promoters are small promoters such as the herpes simplex virus thymidine kinase or SV40 promoter.

An example of an "obesity responsive" cell line is a liver or liver cancer derived cell line. Liver or liver cancer cell lines were obtained from American Type Culture Collection (ATCC) or Eurpean Collection of Animal Cell Cultures (ECACC). An example of such a cell line is Hep G2 (human hepatocellular carcinoma) obtained from ATCC (HB8065). The Hep G2 cell line is disclosed and claimed by US Patent 4393133.

A further example of an "obesity-responsive" cell line is rat-1 fibroblasts (Kroder et al., Exp. Clin. Endocrinol. Diabetes, 1996, 104(suppl2), 66). The rat 1 fibroblast cell line was obtained from ATTCC (CRL-2210).

Other responsive cell lines can be identified using displacement binding assays. While binding may not be the persistent form of receptor function, it is the one that transmits the signal to the cytoplasm. Persistent forms of receptor function can be identified by PCR or Northern blot analysis (eg human obesity protein: Tartaglia et al., Cell, 1995, 83, 1263). Responsive cells were finally detected by monitoring cellular activity in the presence of different concentrations of leptin. Possible methods for identifying candidate cell lines or monitoring the activity of these cells include the following:

1. Microphysiometer: This method detects small changes in pH caused by changes in intracellular biochemistry. Obesity protein-responsive cells undergo biochemical changes when stimulated resulting in small changes in the rate of extracellular acidification, which can be detected by a silicone microphysiometer. The microphysiometer biosensor approach has been reviewed in McConnell, Science, 1992, 257, 1906 .

2. Electrophoretic mobility shift assay (EMSA): Nuclear extracts from cells treated with obesity protein were mixed with DNA sequences containing promiscuous or specific STAT response elements. Extracts from cells responsive to obesity proteins cause gel shift of SATA response element oligonucleotides.

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

3. Protein Phosphorylation Assay Measurement: By using antibodies that recognize phosphorylated tyrosine, the coupling of the receptor activation response through intracellular protein tyrosine phosphorylation to the final reaction can be assayed. More specifically, since leptin receptors stimulate tyrosine phosphorylation of the JAK/STAT pathway, this method provides a means to examine leptin-responsive cell lines. Specific JAK/STAT antibodies can be used in parallel with phosphotyrosine antibodies to detect leptin activation in leptin responsive cell lines. Inhibition as well as stimulation of protein phosphorylation may occur. In particular, insulin-stimulated phosphorylation of the insulin receptor and insulin receptor substrate-1 by the obesity protein has been shown in rat-1 fibroblasts that overpress the insulin receptor Inhibition in the process of oxidization (Kroder et al., 1996, Exp. Clin. Endocrinol. Diabetes, 104, suppl 2, p66).

4. Displacement binding: After incubating the cell line with radiolabeled leptin, such as [ ¹²⁵ I]-leptin, specific binding to leptin relative to non-specific binding can be studied by adding non-labeled leptin. The high ratio of specific/nonspecific binding suggested that this cell line may contain leptin receptors.

5. By using selective antibodies to detect the protein functional form of the obesity protein receptor, preferably a functional persistent form of the protein.

6. Detection of the mRNA functional form of the obesity protein receptor, preferably the functional persistent form of the mRNA, by Northen, RT-PCR or "slot blot" analysis.

Preferred are cell lines known to be involved in the control of the particular disease state for which the compound is being sought.

Cells and fibroblasts obtained from liver, brain or pancreatic tissue are particularly useful as "obesity response" cells suitable for testing compounds against obesity and diabetes. Certain regions of the brain are centers of weight control and energy balance that mediate the actions of obesity proteins. The liver controls many metabolic processes that regulate fat and sugar levels. Cells derived from specific regions of those organs containing appropriate endogenous JAK, STAT proteins and other intracellular proteins necessary to mediate leptin function are preferred.

The response element, reporter gene and preferably the promoter are suitably incorporated into a vector capable of transfecting obesity-responsive cell lines.

A suitable vector is a commercially available vector such as the pGL-2 based luciferase vector (Promega).

A suitable configuration for this vector is a promoter and a STATDNA response element upstream of the reporter gene. A more suitable configuration of the vector is multiple tandem repeats (2-10) of the STAT DNA response element upstream of the thymidine kinase promoter and luciferase reporter gene.

Vectors are constructed containing a reporter gene such as firefly luciferase or chloramphenicol acetyltransferase linked to a small promoter such as the herpes simplex virus thymidine kinase or SV40 promoter. A DNA fragment of a STAT response element can be inserted into the vector using appropriate restriction enzyme sites located upstream of the small promoter.

Response elements, reporter genes and promoters are incorporated into the vector as required using conventional expression techniques, for example DNA fragments of the response elements can be inserted into the vector using appropriate restriction enzyme sites located upstream of the small promoter.

A STAT response element-luciferase reporter gene system can be constructed as described in Lamb et al. Blood, 1994, 8, 2963 and Seidel et al., Proc.

Obesity responsive cells are transfected with a STAT response element-small promoter-luciferase reporter construct using standard methods such as the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456). To correct for differences in transfection efficiency, the cells can be co-transfected with a reference plasmid expressing β-galactosidase activity. After the transfection period (12-24 hours), the cells were treated with various concentrations of compounds and then harvested and lysed. Lysates were assayed for luciferase activity and, if appropriate, beta-galactosidase activity. Enhancer or antagonist activity can be identified by pre-addition or co-addition of appropriate concentrations of obesity protein to the compound when evaluating and measuring the enhancement and reduction of the luciferase response relative to obesity protein alone. Standard methods for identifying luciferase activity are available such as Ow et al., "Science, 1986, 234, 856 and Wet et al., Mol. Cell Biol. 1987, 7, 725 as well as several commercially available kits.

Stable cell lines can be generated by transfecting "obesity responsive cell lines" with reporter gene constructs and selectable markers. Routinely used selectable markers were used to generate stable cell lines as described in Recombinant DNA, Second Edition, J.D. Watson et al. 1992, p. 216. These stable transfected cell lines can be used for high yield assays to produce compounds that mimic, enhance or block the physiological effects of obesity proteins.

The present invention also provides compounds that mimic, enhance or inhibit the physiological effects of obesity proteins when assayed by the methods disclosed herein.

The invention also provides a kit suitable for use in the methods disclosed herein.

As used herein, a 'compound that mimics the physiological effect of obesity protein' means that in the absence of said obesity protein, it can act to stimulate the obesity protein receptor to provide substantially the same physiological effect as obesity protein or to activate the receptor Compounds that act on downstream reactions (post-receptor).

As used herein, a 'compound that enhances the physiological effect of an obesity protein' refers to a compound that enhances the potential and/or maximal physiological effect of an obesity protein.

As used herein, a 'compound that inhibits the physiological effects of an obesity protein' refers to a compound that reduces or substantially blocks the physiological effects of an obesity protein.

The following examples illustrate the invention

Example

General method:

Obesity-responsive cells were transfected with a reporter plasmid containing multiple tandem copies of the STAT response element upstream of a small promoter such as the herpes simplex virus thymidine kinase and luciferase gene reporter genes, constructed using standard methods such as phospho Calcium method (Grahamand Van Der Eb, Virology, 1973, 53, 456). To correct for differences in transfection efficiency, the cells can be co-transfected with a reference plasmid expressing -galactosidase activity. Some time after transfection (12-24 hours), cells were treated with different concentrations of compounds, then harvested and lysed. Lysates were assayed for luciferase activity and, if appropriate, beta-galactosidase activity. Enhancer or antagonist activity can be identified by pre-addition or co-addition of appropriate concentrations of obesity protein to the compound under conditions that assess and determine the enhancement and reduction of the luciferase response relative to obesity protein alone. Standard methods for identifying luciferase activity are available such as Ow et al., "Science, 1986, 234, 856 and de Wet et al., 1987, 7, 725, as well as several commercially available kits.

Example

Transfection with a reporter gene plasmid, pGL2-based luciferase vector (promega) containing an inserted oligonucleotide, using standard methods such as the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456) From a hepatocarcinoma-derived cell line, this oligonucleotide corresponds to the STAT response element upstream of the herpes simplex virus thymidine kinase (-35 to +10) small promoter, a quadruple tandem repeat of TTCCCGGAA. To correct for differences in transfection efficiency, the cells can be co-transfected with a reference plasmid expressing galactosidase activity. Some time after transfection (12-24 hours) cells were treated with different concentrations of compounds, then harvested and lysed. Lysates were assayed for luciferase activity and, if appropriate, beta-galactosidase activity. Enhancer or antagonist activity can be identified by pre-addition or co-addition of appropriate concentrations of obesity protein to the compound in assessing and measuring the enhancement and reduction of the luciferase response relative to obesity protein alone. There are standard methods for assaying luciferase activity such as Ow et al., "Science, 1986, 234, 856 and de Wet et al., Mol. cell biol, 1987, 7, 725, as well as several commercially available kits.

Claims (11)

1. method that detects simulation, strengthens or suppress the compound of the proteic physiological action of obesity, this method comprises:

(a) at the compound of simulation obesity albumen physiological action, assess this compound to obesity albumen activated signal transducer and with the effect of reporter gene link coupled transcriptional activation agent (STAT) DNA response element; Or

(b) to strengthening or suppress the compound of the proteic physiological action of obesity, assess this compound to provide by obesity albumen, for the effect of the reaction of reporter gene link coupled obesity albumen activated STATDNA response element;

Response element and reporter gene are expressed in obesity albumen test clone.

2. one kind meets and the process of claim 1 wherein response element and promoter gene coupling, preferably minimal promoter.

3. method that meets claim 2, wherein response element is formula TT (N) _nThe Nucleotide of AA, wherein N is that arbitrary Nucleotide and n are 4,5 or 6, preferably 5.

4. method that meets claim 2, wherein said response element is a formula TTCCCGGAA Nucleotide.

5. one kind meets and the process of claim 1 wherein that reporter gene is Photinus pyralis LUC or E.C. 2.3.1.28.

6. one kind meets and the process of claim 1 wherein that described promotor is herpes simplex virus thymidine kinase or SV40 promotor.

7. one kind meets and the process of claim 1 wherein that described obesity reacting cells system is liver or liver cancer deutero-clone.

8. one kind meets and the process of claim 1 wherein described response element, reporter gene and promotor participated in can the carrier of transfection obesity reacting cells system in.

9. method that meets claim 8, wherein said carrier are that pGL2 is the luciferase carrier (Promega) on basis.

10. method that meets claim 8 or claim 9, the configuration of wherein said carrier is such, promptly STAT DNA response element is in the upstream of promotor and reporter gene.

11. be adapted at detecting simulation, strengthen or suppress the cover reagent that uses in the method for compound of obesity albumen physiological action,

Described method comprises:

(a) at the compound of simulation obesity albumen physiological action, assess this compound to obesity albumen activation signal transducer and with the effect of reporter gene link coupled transcriptional activation agent (STAT) DNA response element; Or

(b) to strengthening or suppress the compound of the proteic physiological action of obesity, assess this compound to provide by obesity albumen, for the effect of the reaction of reporter gene link coupled obesity albumen activated STATDNA response element;

Response element and reporter gene are expressed in obesity albumen test clone.