CA2222409A1 - Method for the detection of compounds that modulate the effects of the obese protein - Google Patents
Method for the detection of compounds that modulate the effects of the obese protein Download PDFInfo
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- CA2222409A1 CA2222409A1 CA002222409A CA2222409A CA2222409A1 CA 2222409 A1 CA2222409 A1 CA 2222409A1 CA 002222409 A CA002222409 A CA 002222409A CA 2222409 A CA2222409 A CA 2222409A CA 2222409 A1 CA2222409 A1 CA 2222409A1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat
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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; the response element and the reporter being expressed in an ob-protein responsive cell line; a kit of parts adapted for use in such method and a compound when identified by such method.
Description
W 096/38586 PCT~EP96/02291 Method for the detectlon of compounds that modulate the effects of the obese protein The invention relates to a novel method and more partieularly to a method for the detection of eompounds that mimie, potentiate or inhibit the physiologieal effeets 5 of the ob-protein The ob-protein (or leptin) is a seereted hormone that aets as signal from adipose tissue to other organs to regulate weight and energy balanee (Zhang et. al., Nature, 1994, 372, 425). Additional roles for the ob-protein in hematopoietie and reproduetive funetion have been suggested (Cioffi et. al. Nature Medicine, 1996, 2(5), 10 585). Protein moleeules that eontain a eore eomposed of four o~-heliees forming a bundle of up-up-down-down topology eomprise of a family of eytokines and growth factors. Proteins of this family cause homo- and hetero-oligermerisation of membrane receptors known to activate kinase cascades resulting in gene transcription. Receptors of the family whieh are aetivated by oligermt-ric~tion fall into two broad elasses; those 15 sueh as epidermal growth faetor, whieh possess integral tyrosine kinase activity in their intracellular ~ m~inc (A. Ullrich & J. Schleccing~r, Cell, 1990, 61, 203-212), and those such as IL4 and erythropoietin, which lack this activity and m~ te their response by way of an associated protein tyrosine kinase (J.N. Ihle et al., TIBS, 1994, 19, 222-227). Both receptor subtypes are activated by eytokines, but the 4-helix20 bundle proteins aetivate only the non-integral tyrosine kinase subtype. The non-integral protein tyrosine kinase reeeptors generally aet through a pathway involving Janus kinase (JAK) and their assoeiated signal transdueers and aetivators of transeription (STAT) proteins. On aetivation STAT proteins bind to DNA response elements thereby eontrolling gene transcription. Oligonucleotide sequences 25 cnmpricing DNA regulatory elements of the general sequence TT(N)nAA have beenidentified (Seidel et al., Proc. Nat. Acad. Sci. USA., 1995, 92, 3041) as STAT
response elem~nt~ These elements bind STAT proteins in response to sign~ling molecules such as eytokines.
In eopending United Kingdom patent applieation number 9509164.1 we have 30 deseribed our diseovery that the ob-protein is eharaeterised by a four helix bundle tertiary strueture. We now believe that the ob-protein interaets with a membranebound reeeptor that aetivates a JAK-STAT kinase easeade and henee forms the basis for an assay system for the deteetion of eompounds that mimie, potentiate or inhibit the physiologieal effeets of the ob-protein. Sueh an assay has utility in seleeting 35 eompounds for the treatment of weight, energy balance, hematopoietic, fertility and other disorders modulated by the "ob-protein". The assay is especially useful for selecting compounds for the treatment of those disorders related to obesity, anorexia, cachexia and diabetes.
Accordingly, the invention provides a method for the detection of a compound 40 that mimies, potenti~tes 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 CA 02222409 1997~ 16 W 096/38586 PCT~P96/02291 activator of transcription (STAT) DNA response element coupled to a reporter gene;
or (b) for a compound which pott-nti~tes or inhibits the physiologicalleffect of the ob-protein, ~3~se~ing the effect of the con~,ulld upon the response provided by ob 5 protein upon an ob-protein activated STAT DNA response element coupled to a reporter gene;
the response element and the l~p~lL~I being expressed in an ob-protein responsive cell line. ,, Suitably, the response element is coupled to a ~lullloL~I gene, preferably a minim~l ~lumoL~l.
A suitable response element is a nucleotide of formula TT(N)n AA, where N is any nucleotide and n is 4, 5 or 6.
A favoured response el~m~nr is selectively activated by the intracellular eventsme~ ted the by the ob-protein interacting with its receptor. Such selective response el--ment~ can be determined by ~nilling the relative activation of a range of response element-reporter gene constructs when transfected into an ob-responsive cell line by the ob-protein versus other cytokines.
A favoured response elt-ment is a nucleotide of formula TT(N)n AA, where N
is any nucleotide and n is 5.
A further suitable response element is TTCCCGGAA.
A further suitable response element is that region of the promoter of a gene regulated by the ob-protein that is required for STAT interactions. This 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 minim~l promoter such as the herpes simplex virus thymidine kinase or SV40 promoter.
An example of an "ob-responsive" cell line is a liver or liver hepatoma derived cell line. Liver or liver hepatoma cell lines are available from either the American Type Culture Collection (ATCC) or the Eurpean Collection of Animal Cell Cultures (ECACC). An example of one such cell line is Hep G2 (hep~tocçll~ r carcinoma, human) which is available from the ATCC (EIB-8065). The Hep G2 cell line is disclosed and claimed by US patent 4393133.
A further example of an "ob-responsive" cell line is the rat-l fibroblast (Kroder et. al., Exp. Clin. Endocrinol. Diabetes, 1996, 104 (suppl 2), 66). A ratl fibroblast cell line is available from the ATCC (CRL-2210).
Other responsive cell lines can be identified using a displacement binding assay.
Although binding may not be to a functional long form of the receptor, which is the form that transmits a signal to the cytoplasm. Td.-ntific~tion of a functional long form of the receptor may be by PCR or Northern blot analysis (eg. Human ob-receptor:
Tartaglia et al., Cell, 1995, 83, 1263). Ultimately responsive cells are detected by monitoring cellular events in the presence of varying concentrations of leptin.
CA 02222409 l997-ll-l6 W 096/38S86 PCT~EP96/02291 Potential methods for identifying c~nfli~l~te cell lines or monitoring these cellular events include the following:-1. Microphysiometer: This method detects small changes in pH res-lltin~ from biochemical changes in the cell. Ob-protein responsive cells upon s*m~ tion may S undergo biochemical changes that cause a small change in the extracellular ~rlific~tion rate which can be detected by a silicon microphysiometer. The microphysiometer biosensor methodology has been reviewed by McConnell, Science, =i 1992, 257, 1906.
response elem~nt~ These elements bind STAT proteins in response to sign~ling molecules such as eytokines.
In eopending United Kingdom patent applieation number 9509164.1 we have 30 deseribed our diseovery that the ob-protein is eharaeterised by a four helix bundle tertiary strueture. We now believe that the ob-protein interaets with a membranebound reeeptor that aetivates a JAK-STAT kinase easeade and henee forms the basis for an assay system for the deteetion of eompounds that mimie, potentiate or inhibit the physiologieal effeets of the ob-protein. Sueh an assay has utility in seleeting 35 eompounds for the treatment of weight, energy balance, hematopoietic, fertility and other disorders modulated by the "ob-protein". The assay is especially useful for selecting compounds for the treatment of those disorders related to obesity, anorexia, cachexia and diabetes.
Accordingly, the invention provides a method for the detection of a compound 40 that mimies, potenti~tes 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 CA 02222409 1997~ 16 W 096/38586 PCT~P96/02291 activator of transcription (STAT) DNA response element coupled to a reporter gene;
or (b) for a compound which pott-nti~tes or inhibits the physiologicalleffect of the ob-protein, ~3~se~ing the effect of the con~,ulld upon the response provided by ob 5 protein upon an ob-protein activated STAT DNA response element coupled to a reporter gene;
the response element and the l~p~lL~I being expressed in an ob-protein responsive cell line. ,, Suitably, the response element is coupled to a ~lullloL~I gene, preferably a minim~l ~lumoL~l.
A suitable response element is a nucleotide of formula TT(N)n AA, where N is any nucleotide and n is 4, 5 or 6.
A favoured response el~m~nr is selectively activated by the intracellular eventsme~ ted the by the ob-protein interacting with its receptor. Such selective response el--ment~ can be determined by ~nilling the relative activation of a range of response element-reporter gene constructs when transfected into an ob-responsive cell line by the ob-protein versus other cytokines.
A favoured response elt-ment is a nucleotide of formula TT(N)n AA, where N
is any nucleotide and n is 5.
A further suitable response element is TTCCCGGAA.
A further suitable response element is that region of the promoter of a gene regulated by the ob-protein that is required for STAT interactions. This 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 minim~l promoter such as the herpes simplex virus thymidine kinase or SV40 promoter.
An example of an "ob-responsive" cell line is a liver or liver hepatoma derived cell line. Liver or liver hepatoma cell lines are available from either the American Type Culture Collection (ATCC) or the Eurpean Collection of Animal Cell Cultures (ECACC). An example of one such cell line is Hep G2 (hep~tocçll~ r carcinoma, human) which is available from the ATCC (EIB-8065). The Hep G2 cell line is disclosed and claimed by US patent 4393133.
A further example of an "ob-responsive" cell line is the rat-l fibroblast (Kroder et. al., Exp. Clin. Endocrinol. Diabetes, 1996, 104 (suppl 2), 66). A ratl fibroblast cell line is available from the ATCC (CRL-2210).
Other responsive cell lines can be identified using a displacement binding assay.
Although binding may not be to a functional long form of the receptor, which is the form that transmits a signal to the cytoplasm. Td.-ntific~tion of a functional long form of the receptor may be by PCR or Northern blot analysis (eg. Human ob-receptor:
Tartaglia et al., Cell, 1995, 83, 1263). Ultimately responsive cells are detected by monitoring cellular events in the presence of varying concentrations of leptin.
CA 02222409 l997-ll-l6 W 096/38S86 PCT~EP96/02291 Potential methods for identifying c~nfli~l~te cell lines or monitoring these cellular events include the following:-1. Microphysiometer: This method detects small changes in pH res-lltin~ from biochemical changes in the cell. Ob-protein responsive cells upon s*m~ tion may S undergo biochemical changes that cause a small change in the extracellular ~rlific~tion rate which can be detected by a silicon microphysiometer. The microphysiometer biosensor methodology has been reviewed by McConnell, Science, =i 1992, 257, 1906.
2. Electrophoretic mobility shift assay (EMSA): Nuclear extracts from cells 10 after tre~tmçnt with ob-protein are mixed with radiolabeled oligonucleotides cnnt~ining a promiscuous or specific STAT response element DNA sequence.
Extracts from cells that respond to the ob-protein may cause a gel shift of the oligonucleotide for the STAT response element.
References: Book "Recombinant DNA", 2nd Edition, Watson et al., 1992, Page 158;
Lamb et al., Blood, 1994, 83, 2063;
Extracts from cells that respond to the ob-protein may cause a gel shift of the oligonucleotide for the STAT response element.
References: 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 intr~ee~ r proteins may be assayed by the use of antibodies recognising phosphorylated tyrosines.
More specifically since the leptin receptor may srim~ te tyrosine phosphorylation of 20 the JAK/STAT pathway this method provides a method of detecting leptin response cell lines. Specific JAK/ STAT antibodies may be used alongside antibodies for tyrosine phosphorylation to detect leptin activation in a leptin responsive cell line.
Inhibition as well as stimlll~tion of protein phosphorylation may occur. In particular, inhibition by the ob-protein of insulin stim~ tell phosphorylation of the insulin 25 receptor and insulin receptor substrate- 1 has been shown in rat-l fibroblasts ov~lc~ ssing insulin receptors (Kroder et. al 1996, Exp. Clin. Endocrinol. Diabetes, 104, suppl 2, p66) 4. Displ~em~nt binding: After incubation of cell lines with radiolabelled leptin, for example [125Il-leptin, the non-specific binding versus specific binding of 30 leptin can studied by the addition of unlabelled leptin. A high specific to non-specific ratio binding suggests that the cell line may contain the leptin receptor.
More specifically since the leptin receptor may srim~ te tyrosine phosphorylation of 20 the JAK/STAT pathway this method provides a method of detecting leptin response cell lines. Specific JAK/ STAT antibodies may be used alongside antibodies for tyrosine phosphorylation to detect leptin activation in a leptin responsive cell line.
Inhibition as well as stimlll~tion of protein phosphorylation may occur. In particular, inhibition by the ob-protein of insulin stim~ tell phosphorylation of the insulin 25 receptor and insulin receptor substrate- 1 has been shown in rat-l fibroblasts ov~lc~ ssing insulin receptors (Kroder et. al 1996, Exp. Clin. Endocrinol. Diabetes, 104, suppl 2, p66) 4. Displ~em~nt binding: After incubation of cell lines with radiolabelled leptin, for example [125Il-leptin, the non-specific binding versus specific binding of 30 leptin can studied by the addition of unlabelled leptin. A high specific to non-specific ratio binding suggests that the cell line may contain the leptin receptor.
5. Detection of the protein for a functional form, preferably a functional long forrn, of the ob-receptor by use of selective antibodies.
6. Detection of mRNA for a functional form, preferably a functional long 35 form, of the ob-receptor by Northern, RT-PCR or "slot blot" analysis.
Cell lines known to be involved in controlling aspects of the particular diseasestate for which compounds are being sought are preferred.
Cells lines derived from liver, brain, or pancreatic tissue and fibroblasts are particularly useful for "ob-responsive" cells for the assaying of compounds directed at 40 obesity and diabetes. Certain areas of the brain are the focus of weight controlling and energy baIance regulating effects of the ob-protein. The liver controls many metabolic processes that modulate lipid and glucose levels. Cells derived from particular regions of these organs cont~ining the applu~liate endogenous JAKs, STAT proteins and I
other intracellular proteins which are required for m~ ing the effects of the leptin are ~lere~led.
The response element, the reporter, and preferably the promoter, are suitably incorporated into a vector capable of transfecting the ob-responsive cell line.
Suitable vectors are commercially available vectors, such as pGL2-basic luciferase vector (Promega). I
A suitable configuration of the vector is the STAT DNA response element upstream of a promoter and a reporter gene. A more suitable configuration of thevector is the STAT DNA response ele.mt-nt in multiple tandem repeats (2-10) upstream of a thymidine kinase promoter and a luciferase reporter gene Vectors are constructed containing a reporter gene for exam; ple firefly luciferase or chloramphenicol acetylLIdn~.r~;ldse enzyme linked to a;minim~l promoter for example the herpes simplex virus thymidine kinase or SV4Q promoter. The DNA
fragments for the STAT response element are inserted into the vector using apl-lv~liate restriction enzyme sites upstream of the minim~l promoter.
The response element, the reporter and the promoter, as required, are incorporated into the vector using conventional expression techniques, for example the DNA fragments for the response element may be inserted into the vector using a~lv~liate restriction enzyme sites upstream of the minim~l promoter.
STAT response elem~nt-luciferase enzyme reporter systems can be constructed as described by Lamb et al., Blood, 1994, 8, 2063 and Seidel et al., Proc. Nat. Acad.
Sci. usa., 199S, 92, 3041.
Ob-responsive cells are transfected with the STAT response element-minim~l promoter-luciferase reporter constructs using 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 co-transfected with a reference pl~cmi~l expressing ,B-galactosidase activity. After a perlod 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, and if a~ liate ~-galactosidase, activity. Potentiation or antagonist activity can be assayed by pre- or co-addition of an a~pl~ iate concentration of ob-protein to the compound under evaluation and measuring the potentiation or reduction in luciferase response relative to that of ob-protein alone. Standard methods exist for assaying lu, ciferase enzyme activity for example Ow et al., Science, 1986, 234, 856 and de Wçt et al., Mol. Cell Biol., 1987, 7, 725. as well as several comrnercial kits.
Stable cell lines can be generated by transfecting an "ob-responsive" cell line with the reporter construct and a selectable marker. Selectable markers are routinely used to generate stable cell lines as described in Recombinant DNA, 2nd edition, J.D.
Watson et. al., 1992, page 216. These stably transfected cell lines can be used to generate high throughput assays for compounds that mimic, potentiate or block the physiological effects of the ob-protein.
The invention also extends to a compound that mimics, potentiates or inhibits the physiological effect of the ob-protein, when i(lenhfie.l by the method disclosed herein.
W 096/38586 PCT~EP96/02291 The invention also extends to a kit of parts adapted for use in the method disclosed herein.
When used herein 'a compound which mimics the physiological effects of the o~protein' refers to a compound which is capable of acting in the absence of the ob-S protein to either stimnl~te the ob-protein receptor to provide substantially the same physiological effect as the ob protein or to activate a response down stream of this receptor (post-receptor).
When used herein 'a compound that potentiates the physiological effect of the o~protein' refers to a compound which enhances the potency and/or maximal 10 physiological effect of the ob-protein.
When used herein 'a compound that inhibits the physiological effect of the ob-protein' refers to a compound which reduces or subst~nti~lly blocks the physiological effect of the ob protein.
The following example illustrates the invention ~v ~: ;
W 096/38586 PCT~P96/02291 F,Y~mrle General Procedure:
Ob-responsive cells are transfected with a reporter plasmid cont~ining a STAT
response element, in multiple tandem copies upstream of a minim~l~ promoter for example herpes simplex thymidine kinase and a luciferase gene reporter constructusing standard methodology for example the calcium phosphate method (Graham and Van t Der Eb, Virology, 1973, 52, 456). To correct for differences in transfection efficiency, 10 the cells can be co-transfected with a reference pl~mi~l expressing ~-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 Iys tes are assayed for luciferase, and if appropriate ~3-galactosidase, activity. Antagonist ctivity can be assayed by pre- or co-addition of an a~lu~liate concentration of o~-protein to the 15 compound under evaluation and measuring the reduction in lucifer$e response relative to that of ob-protein alone. Standard methods exist for assaying luc,iferase enzyme activity for example Ow et al., Science, 1986, 234, 856 and de Wet et al., 1987, 7, 725. as well as several commercial kits.
Example A liver hepatoma derived cell line is transfected with a reporter plasmid, pGL2-basic luciferase vector (promega) cont:~ining an insert of an oligonucleotide corresponding to a four fold tandem repeat of the STAT response element, TTCCCGGAA, upstream of the minim~l promoter for herpes simplex thymidine kinase (-35 to +10) using standard methodologyfor 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 co-transfected with a reference pl~mi~l expressing ~-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 Iysates are assayed for luciferase, and if a~ liate ,13-galactosidase, activity. Antagonist activity can be assayed by pre- or co-addition of an ap~ liate concentration of ob-protein to the cc,.ll~oulld under evaluation and measuring the reduction in luciferase response relative to that of ob-protein alone. Standard methods exist for assaying luciferase enzyme activity 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 kits.
Cell lines known to be involved in controlling aspects of the particular diseasestate for which compounds are being sought are preferred.
Cells lines derived from liver, brain, or pancreatic tissue and fibroblasts are particularly useful for "ob-responsive" cells for the assaying of compounds directed at 40 obesity and diabetes. Certain areas of the brain are the focus of weight controlling and energy baIance regulating effects of the ob-protein. The liver controls many metabolic processes that modulate lipid and glucose levels. Cells derived from particular regions of these organs cont~ining the applu~liate endogenous JAKs, STAT proteins and I
other intracellular proteins which are required for m~ ing the effects of the leptin are ~lere~led.
The response element, the reporter, and preferably the promoter, are suitably incorporated into a vector capable of transfecting the ob-responsive cell line.
Suitable vectors are commercially available vectors, such as pGL2-basic luciferase vector (Promega). I
A suitable configuration of the vector is the STAT DNA response element upstream of a promoter and a reporter gene. A more suitable configuration of thevector is the STAT DNA response ele.mt-nt in multiple tandem repeats (2-10) upstream of a thymidine kinase promoter and a luciferase reporter gene Vectors are constructed containing a reporter gene for exam; ple firefly luciferase or chloramphenicol acetylLIdn~.r~;ldse enzyme linked to a;minim~l promoter for example the herpes simplex virus thymidine kinase or SV4Q promoter. The DNA
fragments for the STAT response element are inserted into the vector using apl-lv~liate restriction enzyme sites upstream of the minim~l promoter.
The response element, the reporter and the promoter, as required, are incorporated into the vector using conventional expression techniques, for example the DNA fragments for the response element may be inserted into the vector using a~lv~liate restriction enzyme sites upstream of the minim~l promoter.
STAT response elem~nt-luciferase enzyme reporter systems can be constructed as described by Lamb et al., Blood, 1994, 8, 2063 and Seidel et al., Proc. Nat. Acad.
Sci. usa., 199S, 92, 3041.
Ob-responsive cells are transfected with the STAT response element-minim~l promoter-luciferase reporter constructs using 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 co-transfected with a reference pl~cmi~l expressing ,B-galactosidase activity. After a perlod 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, and if a~ liate ~-galactosidase, activity. Potentiation or antagonist activity can be assayed by pre- or co-addition of an a~pl~ iate concentration of ob-protein to the compound under evaluation and measuring the potentiation or reduction in luciferase response relative to that of ob-protein alone. Standard methods exist for assaying lu, ciferase enzyme activity for example Ow et al., Science, 1986, 234, 856 and de Wçt et al., Mol. Cell Biol., 1987, 7, 725. as well as several comrnercial kits.
Stable cell lines can be generated by transfecting an "ob-responsive" cell line with the reporter construct and a selectable marker. Selectable markers are routinely used to generate stable cell lines as described in Recombinant DNA, 2nd edition, J.D.
Watson et. al., 1992, page 216. These stably transfected cell lines can be used to generate high throughput assays for compounds that mimic, potentiate or block the physiological effects of the ob-protein.
The invention also extends to a compound that mimics, potentiates or inhibits the physiological effect of the ob-protein, when i(lenhfie.l by the method disclosed herein.
W 096/38586 PCT~EP96/02291 The invention also extends to a kit of parts adapted for use in the method disclosed herein.
When used herein 'a compound which mimics the physiological effects of the o~protein' refers to a compound which is capable of acting in the absence of the ob-S protein to either stimnl~te the ob-protein receptor to provide substantially the same physiological effect as the ob protein or to activate a response down stream of this receptor (post-receptor).
When used herein 'a compound that potentiates the physiological effect of the o~protein' refers to a compound which enhances the potency and/or maximal 10 physiological effect of the ob-protein.
When used herein 'a compound that inhibits the physiological effect of the ob-protein' refers to a compound which reduces or subst~nti~lly blocks the physiological effect of the ob protein.
The following example illustrates the invention ~v ~: ;
W 096/38586 PCT~P96/02291 F,Y~mrle General Procedure:
Ob-responsive cells are transfected with a reporter plasmid cont~ining a STAT
response element, in multiple tandem copies upstream of a minim~l~ promoter for example herpes simplex thymidine kinase and a luciferase gene reporter constructusing standard methodology for example the calcium phosphate method (Graham and Van t Der Eb, Virology, 1973, 52, 456). To correct for differences in transfection efficiency, 10 the cells can be co-transfected with a reference pl~mi~l expressing ~-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 Iys tes are assayed for luciferase, and if appropriate ~3-galactosidase, activity. Antagonist ctivity can be assayed by pre- or co-addition of an a~lu~liate concentration of o~-protein to the 15 compound under evaluation and measuring the reduction in lucifer$e response relative to that of ob-protein alone. Standard methods exist for assaying luc,iferase enzyme activity for example Ow et al., Science, 1986, 234, 856 and de Wet et al., 1987, 7, 725. as well as several commercial kits.
Example A liver hepatoma derived cell line is transfected with a reporter plasmid, pGL2-basic luciferase vector (promega) cont:~ining an insert of an oligonucleotide corresponding to a four fold tandem repeat of the STAT response element, TTCCCGGAA, upstream of the minim~l promoter for herpes simplex thymidine kinase (-35 to +10) using standard methodologyfor 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 co-transfected with a reference pl~mi~l expressing ~-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 Iysates are assayed for luciferase, and if a~ liate ,13-galactosidase, activity. Antagonist activity can be assayed by pre- or co-addition of an ap~ liate concentration of ob-protein to the cc,.ll~oulld under evaluation and measuring the reduction in luciferase response relative to that of ob-protein alone. Standard methods exist for assaying luciferase enzyme activity 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 kits.
Claims (11)
1. A method for the detection of a compound that mimics, potentiates or inhibitsthe 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 theob-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;
the response element and the reporter being expressed in an ob-protein responsive cell line.
(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 theob-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;
the response element and the reporter being expressed in an ob-protein responsive cell line.
2. A method according to claim 1, wherein the response element is coupled to a promoter gene, preferably a minimal promoter.
3. A method according to claim 2, wherein the response element is a nucleotide of formula TT(N)n AA, where N is any nucleotide and n is 4, 5 or 6, preferably 5.
4. A method according to claim 2, wherein the response element is a nucleotide of formula TTCCCGGAA.
5. A method according to claim 1, wherein the reporter gene is firefly luciferase or chloramphenicol acetyltransferase enzyme.
6. A method according to claim 1, wherein the promoter is the herpes simplex virus thymidine kinase or SV40 promoter.
7. A method according to claim 1, wherein the ob-responsive cell line is a liver or liver hepatoma derived cell line.
8. A method according to claim 1, wherein the response element, the reporter, and the promoter, are incorporated into a vector capable of transfecting the ob-responsive cell line.
9. A method according to claim 8, wherein the vectors pGL2-basic luciferase vector (Promega).
10. A method according to claim 8 or claim 9, wherein the configuration of the vector is such that the STAT DNA response element is upstream of the promoter and reporter gene.
11. A kit of parts adapted for use in the method for the detection of a compoundthat 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 theob-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;
the response element and the reporter being expressed in an ob-protein responsive cell line.
(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 theob-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;
the response element and the reporter being expressed in an ob-protein responsive cell line.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9510857.7 | 1995-05-30 | ||
| GBGB9510857.7A GB9510857D0 (en) | 1995-05-30 | 1995-05-30 | Novel assay |
| GBGB9511602.6A GB9511602D0 (en) | 1995-06-08 | 1995-06-08 | Novel assay |
| GB9511602.6 | 1995-06-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2222409A1 true CA2222409A1 (en) | 1996-12-05 |
Family
ID=26307107
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002222409A Abandoned CA2222409A1 (en) | 1995-05-30 | 1996-05-28 | Method for the detection of compounds that modulate the effects of the obese protein |
Country Status (15)
| Country | Link |
|---|---|
| EP (1) | EP0832284A1 (en) |
| JP (1) | JPH11505725A (en) |
| KR (1) | KR19990022134A (en) |
| CN (1) | CN1192248A (en) |
| AU (1) | AU715215B2 (en) |
| BR (1) | BR9608686A (en) |
| CA (1) | CA2222409A1 (en) |
| CZ (1) | CZ379097A3 (en) |
| HU (1) | HUP9801744A3 (en) |
| MX (1) | MX9709360A (en) |
| NO (1) | NO975504L (en) |
| NZ (1) | NZ309777A (en) |
| PL (1) | PL323638A1 (en) |
| TR (1) | TR199701469T1 (en) |
| WO (1) | WO1996038586A1 (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6001968A (en) | 1994-08-17 | 1999-12-14 | The Rockefeller University | OB polypeptides, modified forms and compositions |
| US6429290B1 (en) | 1994-08-17 | 2002-08-06 | The Rockefeller University | OB polypeptides, modified forms and derivatives |
| US6007998A (en) * | 1996-04-22 | 1999-12-28 | Merck & Co., Inc. | Leptin assay |
| WO1997040380A1 (en) * | 1996-04-22 | 1997-10-30 | Merck & Co., Inc. | Leptin assay |
| US6001816A (en) * | 1996-06-20 | 1999-12-14 | Merck & Co., Inc. | Gene therapy for leptin deficiency |
| JP2001503982A (en) * | 1996-11-01 | 2001-03-27 | スミスクライン・ビーチャム・パブリック・リミテッド・カンパニー | Method for detecting a compound that modulates the effect of obesity (OB) protein |
| IT1293533B1 (en) * | 1997-07-14 | 1999-03-01 | Angeletti P Ist Richerche Bio | METHOD FOR THE SELECTION OF MOLECULES ABLE TO MIMIC, INHIBIT OR ENHANCE THE EFFECTS OF THE INTERACTION BETWEEN LEPTIN AND CELLS THAT |
| WO1999023493A1 (en) * | 1997-10-31 | 1999-05-14 | The Rockefeller University | Methods of identifying agents that modulate leptin activity |
| AU2668699A (en) * | 1998-02-11 | 1999-08-30 | Beth Israel Deaconess Medical Center | Methods and compositions for modulating leptin activity |
| GB9814199D0 (en) * | 1998-06-30 | 1998-08-26 | Univ Buckingham | Novel method |
| GB9828087D0 (en) * | 1998-12-18 | 1999-02-17 | Smithkline Beecham Plc | Novel method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU7795094A (en) * | 1993-09-15 | 1995-04-03 | New York University | Dna sequence which binds transcriptional regulatory proteins activated in response to various cytokines and uses thereof |
| WO1995028482A2 (en) * | 1994-04-14 | 1995-10-26 | Ligand Pharmaceuticals Incorporated | Dna spacer regulatory elements responsive to cytokines and methods for their use |
-
1996
- 1996-05-28 PL PL96323638A patent/PL323638A1/en unknown
- 1996-05-28 KR KR1019970708613A patent/KR19990022134A/en not_active Application Discontinuation
- 1996-05-28 NZ NZ309777A patent/NZ309777A/en unknown
- 1996-05-28 MX MX9709360A patent/MX9709360A/en unknown
- 1996-05-28 HU HU9801744A patent/HUP9801744A3/en unknown
- 1996-05-28 BR BR9608686A patent/BR9608686A/en active Search and Examination
- 1996-05-28 CN CN96195983A patent/CN1192248A/en active Pending
- 1996-05-28 CZ CZ973790A patent/CZ379097A3/en unknown
- 1996-05-28 EP EP96917454A patent/EP0832284A1/en not_active Withdrawn
- 1996-05-28 AU AU60023/96A patent/AU715215B2/en not_active Ceased
- 1996-05-28 JP JP8536176A patent/JPH11505725A/en active Pending
- 1996-05-28 TR TR97/01469T patent/TR199701469T1/en unknown
- 1996-05-28 CA CA002222409A patent/CA2222409A1/en not_active Abandoned
- 1996-05-28 WO PCT/EP1996/002291 patent/WO1996038586A1/en not_active Application Discontinuation
-
1997
- 1997-11-28 NO NO975504A patent/NO975504L/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| PL323638A1 (en) | 1998-04-14 |
| TR199701469T1 (en) | 1998-03-21 |
| BR9608686A (en) | 1999-07-06 |
| NO975504D0 (en) | 1997-11-28 |
| EP0832284A1 (en) | 1998-04-01 |
| AU6002396A (en) | 1996-12-18 |
| HUP9801744A3 (en) | 2000-06-28 |
| NZ309777A (en) | 2000-01-28 |
| CN1192248A (en) | 1998-09-02 |
| CZ379097A3 (en) | 1998-05-13 |
| NO975504L (en) | 1997-11-28 |
| WO1996038586A1 (en) | 1996-12-05 |
| HUP9801744A2 (en) | 1998-10-28 |
| AU715215B2 (en) | 2000-01-20 |
| JPH11505725A (en) | 1999-05-25 |
| KR19990022134A (en) | 1999-03-25 |
| MX9709360A (en) | 1998-02-28 |
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