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WO1996038586A1 - Procede de detection de composes modulant les effets de la proteine ob - Google Patents

Procede de detection de composes modulant les effets de la proteine ob Download PDF

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Publication number
WO1996038586A1
WO1996038586A1 PCT/EP1996/002291 EP9602291W WO9638586A1 WO 1996038586 A1 WO1996038586 A1 WO 1996038586A1 EP 9602291 W EP9602291 W EP 9602291W WO 9638586 A1 WO9638586 A1 WO 9638586A1
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WO
WIPO (PCT)
Prior art keywords
protein
compound
response element
stat
promoter
Prior art date
Application number
PCT/EP1996/002291
Other languages
English (en)
Inventor
Lee James Beeley
Richard Anthony Godwin Smith
Original Assignee
Smithkline Beecham Plc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9510857.7A external-priority patent/GB9510857D0/en
Priority claimed from GBGB9511602.6A external-priority patent/GB9511602D0/en
Application filed by Smithkline Beecham Plc filed Critical Smithkline Beecham Plc
Priority to JP8536176A priority Critical patent/JPH11505725A/ja
Priority to EP96917454A priority patent/EP0832284A1/fr
Priority to NZ309777A priority patent/NZ309777A/xx
Priority to BR9608686A priority patent/BR9608686A/pt
Priority to MX9709360A priority patent/MX9709360A/es
Priority to AU60023/96A priority patent/AU715215B2/en
Publication of WO1996038586A1 publication Critical patent/WO1996038586A1/fr
Priority to NO975504A priority patent/NO975504L/no

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • 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

Definitions

  • the invention relates to a novel method and more particularly to a method for the detection of compounds that mimic, potentiate or inhibit the physiological effects of the ob-protein
  • the ob-protein (or leptin) is a secreted hormone that acts as signal from adipose tissue to other organs to regulate weight and energy balance (Zhang et. al., Nature, 1994, 372, 425). Additional roles for the ob-protein in hematopoietic and reproductive function have been suggested (Cioffi et. al. Nature Medicine, 1996, 2(5), 585). Protein molecules that contain a core composed of four ⁇ -helices forming a bundle of up-up-down-down topology comprise of a family of cytokines 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 which are activated by oligermerisation fall into two broad classes; those such as epidermal growth factor, which possess integral tyrosine kinase activity in their intracellular domains (A. Ullrich & J. Schlessinger, Cell, 1990, 61, 203-212), and those such as IL4 and erythropoietin, which lack this activity and mediate 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 cytokines, but the 4-helix bundle proteins activate only the non-integral tyrosine kinase subtype.
  • the non- integral protein tyrosine kinase receptors generally act through a pathway involving Janus kinase (JAK) and their associated signal transducers and activators of transcription (STAT) proteins.
  • STAT proteins On activation STAT proteins bind to DNA response elements thereby controlling gene transcription.
  • Oligonucleotide sequences comprising DNA regulatory elements of 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 STAT proteins in response to signaling molecules such as cytokines.
  • ob-protein is characterised by a four helix bundle tertiary structure.
  • the ob-protein interacts with a membrane bound receptor that activates a JAK-STAT kinase cascade and hence forms the basis for an assay system for the detection of compounds that mimic, potentiate or inhibit the physiological effects of the ob-protein.
  • Such an assay has utility in selecting compounds 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.
  • the invention provides a method for the detection of a compound that mimics, potentiates or inhibits the physiological effect of the ob-protein, which method comprises:
  • the response element is coupled to a promoter gene, preferably a minimal promoter.
  • 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 element is selectively activated by the intracellular events mediated the by the ob-protein interacting with its receptor.
  • Such selective response elements can be determined by examining 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 element 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 minimal promoter such as the herpes simplex virus thymidine kinase or SV40 promoter.
  • 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 (hepatocellular carcinoma, human) which is available from the ATCC (HB-8065).
  • the Hep G2 ceU line is disclosed and claimed by US patent 4393133.
  • a further example of an "ob-responsive" cell line is the rat-1 fibroblast (Kroder et. al., Exp. Clin. Endoc ⁇ nol. Diabetes, 1996, 104 (suppl 2), 66).
  • a ratl fibroblast cell line is available from the ATCC (CRL-2210).
  • 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. Identification 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. Potential methods for identifying candidate cell lines or monitoring these cellular events include the following:-
  • Microphysiometer This method detects small changes in pH resulting from biochemical changes in the cell. Ob-protein responsive cells upon stimulation may undergo biochemical changes that cause a small change in the extracellular acidification rate which can be detected by a silicon microphysiometer.
  • the microphysiometer biosensor methodology has been reviewed by McConnell, Science, 1992, 257, 1906.
  • Electrophoretic mobility shift assay Nuclear extracts from cells after treatment with ob-protein are mixed with radiolabeled oligonucleo tides containing 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.
  • the coupling of receptor activation to the final response through tyrosine phosphorylation of intracellular proteins may be assayed by the use of antibodies recognising phosphorylated tyrosines. More specifically since the leptin receptor may stimulate tyrosine phosphorylation of 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 stimulation of protein phosphorylation may occur.
  • Displacement binding After incubation of cell lines with radiolabelled leptin, for example [ ⁇ I]-leptin, the non-specific binding versus specific binding of 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.
  • radiolabelled leptin for example [ ⁇ I]-leptin
  • mRNA for a functional form preferably a functional long form, of the ob-receptor by Northern, RT-PCR or "slot blot" analysis.
  • 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 obesity and diabetes. Certain areas of the brain are the focus of weight controlling and energy balance 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 containing the appropriate endogenous JAKs, STAT proteins and other intracellular proteins which are required for mediating the effects of the leptin are preferred.
  • 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 lucif erase vector (Promega).
  • 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 the vector is the STAT DNA response element 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 example firefly luciferase or chloramphenicol acetyltransferase enzyme linked to a minimal promoter for example the herpes simplex virus thymidine kinase or S V40 promoter.
  • a reporter gene for example firefly luciferase or chloramphenicol acetyltransferase enzyme linked to a minimal promoter for example the herpes simplex virus thymidine kinase or S V40 promoter.
  • the DNA fragments for the STAT response element are inserted into the vector using appropriate restriction enzyme sites upstream of the minimal promoter.
  • the response element, the reporter and the promoter 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 appropriate restriction enzyme sites upstream of the minimal promoter.
  • STAT response element-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., 1995, 92, 3041.
  • Ob-responsive cells are transfected with the STAT response element-minimal 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 plasmid 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 lysates are assayed for luciferase, and if appropriate ⁇ - galactosidase, activity.
  • Potentiation or antagonist activity can be assayed by pre- or co-addition of an appropriate 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 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.
  • 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 identified by the method disclosed herein.
  • the invention also extends to a kit of parts adapted for use in the method disclosed herein.
  • a compound which mimics the physiological effects of the ob-protein' refers to a compound which is capable of acting in the absence of the ob- protein to either stimulate 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).
  • a compound that potentiates the physiological effect of the ob-protein' refers to a compound which enhances the potency and/or maximal physiological effect of the ob-protein.
  • a compound that inhibits the physiological effect of the ob- protein 1 refers to a compound which reduces or substantially blocks the physiological effect of the ob protein.
  • Ob-responsive cells are transfected with a reporter plasmid containing a STAT response element, in multiple tandem copies upstream of a minimal promoter for example herpes simplex thymidine kinase and a luciferase gene reporter constructusing standard methodology for example the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456).
  • a minimal promoter for example herpes simplex thymidine kinase and a luciferase gene reporter construct using standard methodology for example the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456).
  • the cells can be co-transfected with a reference plasmid 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 lysates are assayed for luciferase, and if appropriate ⁇ -galactosidase, activity.
  • Antagonist activity can be assayed by pre- or co-addition of an appropriate concentration of ob-protein to the compound 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., 1987, 7, 725. as well as several commercial kits.
  • a liver hepatoma derived cell line is transfected with a reporter plasmid, pGL2-basic luciferase vector (promega) containing an insert of an oligonucleotide corresponding to a four fold tandem repeat of the STAT response element, TTCCCGGAA, upstream of the minimal promoter for herpes simplex thymidine kinase (-35 to +10) 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 plasmid expressing ⁇ -galactosidase activity.
  • the cells are treated with varying concentrations of compound and then harvested and lysed.
  • the lysates are assayed for luciferase, and if appropriate ⁇ -galactosidase, activity.
  • Antagonist activity can be assayed by pre- or co-addition of an appropriate concentration of ob-protein to the compound 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.

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  • Chemical & Material Sciences (AREA)
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Abstract

Cette invention concerne un procédé de détection d'un composé qui imite, potentialise ou inhibe l'effet physiologique de la protéine ob, lequel procédé comprend les étapes suivantes: (a) pour un composé imitant l'effet physiologique de la protéine ob, déterminer l'effet du composé sur un élément de réponse d'ADN transducteur de signal et activateur de transcription (STAT) activé par protéine ob, couplé à un gène reporter; ou (b) pour un composé potentialisant ou inhibant l'effet physiologique de la protéine ob, déterminer l'effet du composé lors de la réponse donnée par une protéine ob sur un élément de réponse d'ADN STAT activé par protéine ob et couplé à un gène marqueur. L'élément sensible et le marqueur sont exprimés dans une lignée cellulaire réagissant à la protéine ob. Cette invention concerne également un nécessaire d'instruments pouvant être utilisés dans ce procédé, ainsi qu'un composé identifié à l'aide dudit procédé.
PCT/EP1996/002291 1995-05-30 1996-05-28 Procede de detection de composes modulant les effets de la proteine ob WO1996038586A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
JP8536176A JPH11505725A (ja) 1995-05-30 1996-05-28 肥満タンパク質の効果を調節する化合物の検出方法
EP96917454A EP0832284A1 (fr) 1995-05-30 1996-05-28 Procede de detection de composes modulant les effets de la proteine ob
NZ309777A NZ309777A (en) 1995-05-30 1996-05-28 Method for the detection of compounds that modulate the effects of the obese protein
BR9608686A BR9608686A (pt) 1995-05-30 1996-05-28 Método para a detecção de compostos que modulam os efeitos da obesoproteína
MX9709360A MX9709360A (es) 1995-05-30 1996-05-28 Metodo para la deteccion de compuestos que modulan los efectos de la proteina de la obesidad.
AU60023/96A AU715215B2 (en) 1995-05-30 1996-05-28 Method for the detection of compounds that modulate the effects of the obese protein
NO975504A NO975504L (no) 1995-05-30 1997-11-28 Fremgangsmåte for påvisning av forbindelser som modulerer virkningene av fettproteinet

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GBGB9510857.7A GB9510857D0 (en) 1995-05-30 1995-05-30 Novel assay
GB9510857.7 1995-05-30
GB9511602.6 1995-06-08
GBGB9511602.6A GB9511602D0 (en) 1995-06-08 1995-06-08 Novel assay

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US08952909 A-371-Of-International 1998-09-08
US10/036,956 Continuation US20020068300A1 (en) 1995-05-30 2001-12-21 Method for the detection of compounds that modulate the effects of the obese protein

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EP (1) EP0832284A1 (fr)
JP (1) JPH11505725A (fr)
KR (1) KR19990022134A (fr)
CN (1) CN1192248A (fr)
AU (1) AU715215B2 (fr)
BR (1) BR9608686A (fr)
CA (1) CA2222409A1 (fr)
CZ (1) CZ379097A3 (fr)
HU (1) HUP9801744A3 (fr)
MX (1) MX9709360A (fr)
NO (1) NO975504L (fr)
NZ (1) NZ309777A (fr)
PL (1) PL323638A1 (fr)
TR (1) TR199701469T1 (fr)
WO (1) WO1996038586A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997040380A1 (fr) * 1996-04-22 1997-10-30 Merck & Co., Inc. Dosage de la leptine
WO1998020158A1 (fr) * 1996-11-01 1998-05-14 Smithkline Beecham Plc Procede pour la detection de composes modulant les effets de la proteine de l'obesite (ob)
WO1999004264A1 (fr) * 1997-07-14 1999-01-28 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Procede d'identification de modificateurs de la leptine: interaction du recepteur de la leptine
WO1999023493A1 (fr) * 1997-10-31 1999-05-14 The Rockefeller University Procedes d'identification d'agents modulant une activite de la leptine
US5935810A (en) * 1994-08-17 1999-08-10 The Rockefeller University Mammalian ob polypeptides capable of modulating body weight, corresponding nucleic acids, and diagnostic and therapeutic uses thereof
WO1999040946A3 (fr) * 1998-02-11 1999-10-28 Beth Israel Hospital Methodes et compositions pour moduler l'activite de la leptine
US6001816A (en) * 1996-06-20 1999-12-14 Merck & Co., Inc. Gene therapy for leptin deficiency
US6007998A (en) * 1996-04-22 1999-12-28 Merck & Co., Inc. Leptin assay
WO2000000639A3 (fr) * 1998-06-30 2000-08-17 Univ Buckingham Procede de detection d'un compose simulant, renforçant ou inhibant l'effet physiologique de la leptine
WO2000037675A3 (fr) * 1998-12-18 2000-09-14 Smithkline Beecham Plc Nouveau procede
US6429290B1 (en) 1994-08-17 2002-08-06 The Rockefeller University OB polypeptides, modified forms and derivatives

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995008001A1 (fr) * 1993-09-15 1995-03-23 New York University Sequence d'adn fixant des proteines regulatrices transcriptionnelles activees en reponse a diverses cytokines, et leurs utilisations
WO1995028482A2 (fr) * 1994-04-14 1995-10-26 Ligand Pharmaceuticals Incorporated Elements de regulation du segment espaceur d'adn sensibles aux cytokines et procedes d'utilisation de ces derniers

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1995008001A1 (fr) * 1993-09-15 1995-03-23 New York University Sequence d'adn fixant des proteines regulatrices transcriptionnelles activees en reponse a diverses cytokines, et leurs utilisations
WO1995028482A2 (fr) * 1994-04-14 1995-10-26 Ligand Pharmaceuticals Incorporated Elements de regulation du segment espaceur d'adn sensibles aux cytokines et procedes d'utilisation de ces derniers

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
H.M. SEIDEL ET AL.: "Spacing of palindromic half sites as a determinant of selective STAT (signal transducers and activators of transcription) DNA binding and transcriptional activity", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 92, March 1995 (1995-03-01), WASHINGTON US, pages 3041 - 3045, XP002013478 *
J.E. DARNELL JR.: "Reflections on STAT3, STAT5, and STAT6 as fat STATs", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 93, June 1996 (1996-06-01), WASHINGTON US, pages 6221 - 6224, XP002013479 *
L.A. TARTAGLIA: "Identification and expression cloning of a leptin receptor, OB-R", CELL, vol. 83, no. 7, 29 December 1995 (1995-12-29), BALTIMORE US, pages 1263 - 1271, XP000602068 *
T. MADEJ ET AL: "Threading analysis suggests that the obese gene product may be a helical cytokine", FEBS LETTERS, vol. 373, no. 1, 2 October 1995 (1995-10-02), AMSTERDAM NL, pages 13 - 18, XP000602067 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6429290B1 (en) 1994-08-17 2002-08-06 The Rockefeller University OB polypeptides, modified forms and derivatives
US5935810A (en) * 1994-08-17 1999-08-10 The Rockefeller University Mammalian ob polypeptides capable of modulating body weight, corresponding nucleic acids, and diagnostic and therapeutic uses thereof
US7544492B1 (en) 1994-08-17 2009-06-09 The Rockefeller University OB polypeptides, modified forms and derivatives
US7521258B2 (en) 1994-08-17 2009-04-21 The Rockefeller University Methods of detecting, measuring, and evaluating modulators of body weight in biological samples, and diagnostic, monitoring, and therapeutic uses thereof
WO1997040380A1 (fr) * 1996-04-22 1997-10-30 Merck & Co., Inc. Dosage de la leptine
US6007998A (en) * 1996-04-22 1999-12-28 Merck & Co., Inc. Leptin assay
US6001816A (en) * 1996-06-20 1999-12-14 Merck & Co., Inc. Gene therapy for leptin deficiency
WO1998020158A1 (fr) * 1996-11-01 1998-05-14 Smithkline Beecham Plc Procede pour la detection de composes modulant les effets de la proteine de l'obesite (ob)
WO1999004264A1 (fr) * 1997-07-14 1999-01-28 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Procede d'identification de modificateurs de la leptine: interaction du recepteur de la leptine
WO1999023493A1 (fr) * 1997-10-31 1999-05-14 The Rockefeller University Procedes d'identification d'agents modulant une activite de la leptine
WO1999040946A3 (fr) * 1998-02-11 1999-10-28 Beth Israel Hospital Methodes et compositions pour moduler l'activite de la leptine
WO2000000639A3 (fr) * 1998-06-30 2000-08-17 Univ Buckingham Procede de detection d'un compose simulant, renforçant ou inhibant l'effet physiologique de la leptine
WO2000037675A3 (fr) * 1998-12-18 2000-09-14 Smithkline Beecham Plc Nouveau procede

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CA2222409A1 (fr) 1996-12-05
PL323638A1 (en) 1998-04-14
AU6002396A (en) 1996-12-18
CN1192248A (zh) 1998-09-02
BR9608686A (pt) 1999-07-06
MX9709360A (es) 1998-02-28
NZ309777A (en) 2000-01-28
NO975504D0 (no) 1997-11-28
NO975504L (no) 1997-11-28
TR199701469T1 (xx) 1998-03-21
EP0832284A1 (fr) 1998-04-01
JPH11505725A (ja) 1999-05-25
AU715215B2 (en) 2000-01-20
CZ379097A3 (cs) 1998-05-13
HUP9801744A3 (en) 2000-06-28
KR19990022134A (ko) 1999-03-25
HUP9801744A2 (hu) 1998-10-28

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