EP1053007A2 - Methodes et compositions pour moduler l'activite de la leptine - Google Patents

Methodes et compositions pour moduler l'activite de la leptine

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Publication number
EP1053007A2
EP1053007A2 EP99906877A EP99906877A EP1053007A2 EP 1053007 A2 EP1053007 A2 EP 1053007A2 EP 99906877 A EP99906877 A EP 99906877A EP 99906877 A EP99906877 A EP 99906877A EP 1053007 A2 EP1053007 A2 EP 1053007A2
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European Patent Office
Prior art keywords
socs
leptin
activity
cells
cell
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EP99906877A
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German (de)
English (en)
Inventor
Jeffrey S. Flier
Christian Bjorbaek
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Beth Israel Deaconess Medical Center Inc
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Beth Israel Deaconess Medical Center Inc
Beth Israel Hospital Association
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Publication of EP1053007A2 publication Critical patent/EP1053007A2/fr
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    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • Leptin the ad ⁇ ocyte de ⁇ ved hormone, acts on specific regions of the brain to regulate food intake, energy expenditure and neuroendocnne function (Zhang Y, et al. Nature 572:425-432, 1994; Halaas JL et al, Science 269:543-546, 1995; Campfield LA, et al. Science 269:546-549,1995, and Pellymounter MA. et al. Science 269:540-543, 1995 ).
  • Leptin is structurally related to cytokines (Zhang F, et al. Nature 387:206-209, 1991) and acts on receptors that belong to the cytokine-receptor superfamily (Tartagha LA, et al. Cell 83.1263- 1271. Lee G-H. et al. Nature 579:632-635, 1996).
  • leptin-resistance which characterizes human obesity may be due to defects in leptin signal-transduction in the brain.
  • Two potential mechanisms for leptin resistance are defects at the level of the blood brain barrier, and defects in the pathway of leptin signal transduction in target cells.
  • the leptin receptor (OBR) (mutation of which causes obesity in dh/db mice and fa/fa rats), is most closely related to the gpl 30 and LIFR signal transducing subunits that are activated by cytokines such as IL-6, LIF and CNTF and hormone receptors for growth hormone such as erythropoietin (Tartaglia, 1995).
  • cytokines such as IL-6, LIF and CNTF
  • hormone receptors for growth hormone such as erythropoietin (Tartaglia, 1995).
  • Several isoforms of the leptin receptor exist including a long form that is predominantly expressed in specific cell bodies in the
  • cytokine-inducible inhibitors of signaling including CIS (cytokine-inducible sequence), SOCS-1 (suppressor of cytokine signaling), SOCS-2 and SOCS-3 (Starr R et al Nature 387:9 1-921, 1997; Endo TA, et al. Nature 387:921-924, 1997; Naka T et al, Nature, 387:924-929, 1997; Masuhara M et al, Biochem Biophys Res Commun, 239:439-446, 1997).
  • cytokines including IL-6, LIF, growth hormone (GH) and erythropoietin (EPO) induce transcriptional activation of one or more of the CIS or SOCS genes in vivo and in vitro, through activation of the JAK-STAT pathway (Starr, 1997; Endo, 1997; Naka, 1997; Yoshimura et al. Embo J. 14:2816-2826,
  • the present invention encompasses methods and compositions for altering, or modulating, leptin activity by altering, or modulating, cytokine inhibitor activity.
  • cytokine inhibitor e.g., IL-4, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12.
  • SOCS-3 cytokine inhibitor
  • SOCS-3 expression is rapidly induced by leptin treatment in regions of the hypothalamus that are known to be involved in the regulation of body weight.
  • SOCS-3-mediated leptin cell-signaling inhibitory pathway exists.
  • SOCS-3 is a negative regulator of leptin signal-transduction.
  • the present invention also relates to methods of treating delayed onset of puberty in mammals by increasing SOCS-3-mediated leptin cell signaling and to methods of treating reproductive dysfunction, or infertility, such as anovulation or decreased spermatogenesis associated with low serum/plasma levels of leptin, inactive leptin, leptin resistance or ineffective production of leptin.
  • the methods of the present invention can be used in either male or female mammals.
  • the present invention also relates to the methods of treating affective mood disorders in an individual associated with elevated leptin levels, such as atypical depression, wherein atypical depression is characterized by elevated leptin levels in an individual such that treatment resulting in decreased leptin-induced cell signaling would result in prevention or alleviation of symptoms of atypical depression.
  • the present invention also relates to methods of treating affective mood disorders associated with decreased leptin levels, such as melancholic depression, wherein melancholic depression is characterized by decreased leptin levels in an individual, such that treatment resulting in increased leptin-induced cell signaling would result in prevention or alleviation of symptoms of melancholic depression.
  • the biological activity of leptin is defined herein as the ability of leptin to activate one, or more signal transduction pathways in a cell as a result of interaction between (e.g., binding) leptin and a leptin receptor associated with the cell.
  • the signal transduction pathway includes for example, the activation of Janus Kinase 2 (referred to herein as JAK2), thereby activating other pathways, such as signal transducers and activators of transcription (STAT), phosphoinositide-3 kinase ras/ mitogen-activated protein kinase pathways ultimately leading to activation or inactivation of gene transcription as well as other, non-transcriptional effects.
  • leptin can bind its cognate receptor, which is associated with JAK2; JAK2 is activated, and phosphorylates the receptor, JAK2 and STAT3 proteins (among others). Phosphorylated STAT3 dimerizes and translocates to the nucleus, where it serves as a transcriptional activator. Therefore, leptin activity can be measured as the level of phosphorylation of the receptor, JAK2 or STAT3. Further, leptin activity can be measured by the amount of gene transcription from STAT3 responsive genes.
  • SOCS-3 activity is the inhibition or inactivation (completely or partially) of leptin induced cell signaling.
  • SOCS-3 mediates the down regulation of leptin signaling as measured by lack of phosphorylation of leptin receptor. JAK2 or STAT3, as well as by the association of JAK2 and SOCS-3.
  • SOCS-3 transcription is part of a negative feedback loop triggered by leptin activation of the leptin receptor.
  • SOCS-3 activity can be inhibited by inhibiting or reducing the amount of SOCS-3 protein expressed in a cell, or by introducing a polynucleotide encoding a modified SOCS-3 protein into a cell, wherein the modified SOCS-3 protein comprises a mutant, variant, derivative, or analog of the SOCS-3 protein.
  • SOCS-3 expression can be inhibited or reduced by transfecting a cell with a polynucleotide construct encoding SOCS-3 antisense DNA or RNA.
  • the antisense RNA can hybridize to the endogenous SOCS-3 mRNA and prevent translation of SOCS-3 mRNA, thereby inhibiting or reducing expression of SOCS-3 protein.
  • SOCS-3 expression can also be inhibited or reduced by transfecting the cell with a polynucleotide construct encoding a transcriptional inhibitor such that transcription of SOCS-3 is inhibited or reduced.
  • a transcriptional inhibitor would interact specifically with SOCS-3 promoter sequences, resulting in decreased transcription of SOCS-3, decreased SOCS-3 protein expression and thus decreased SOCS-3 activity.
  • SOCS-3 activity can also be inhibited by transfecting the cell with a polynucleotide construct encoding an altered, or modified SOCS-3 protein, polypeptide or peptide.
  • the modified SOCS-3 polypeptide is a competitive inhibitor (e.g., antagonist) of endogenous SOCS-3.
  • SOCS-3 can interact with a SOCS-3 target protein (e.g., JAK2), without interfering with the activity of the target protein. Because the modified SOCS-3 protein interacts with the intended SOCS-3 target, endogenous SOCS-3 could not interact with its intended target, thereby inhibiting or reducing the level of SOCS-3 mediated leptin cell signaling.
  • SOCS-3 activity can be inhibited or reduced by introducing a SOCS-3 inhibitor into the cell.
  • Such an inhibitor can be a peptide or small organic molecule that interferes with SOCS-3 activity.
  • Such an inhibitor can interact specifically with SOCS-3, or to its intended target, to inhibit SOCS-3 activity.
  • the inhibitor can interact with downstream targets of SOCS-3 such as JAK2.
  • the present invention further encompasses methods of increasing or enhancing SOCS-3 activity in a cell.
  • Increased SOCS-3 activity in a cell can inhibit or reduce leptin-induced cell signaling.
  • a reduction or inhibition of leptin-induced cell signaling can be useful to prevent, inhibit or alleviate atypical depression in an individual, or to promote weight gain in an individual.
  • SOCS-3 activity can be increased by transfecting a cell with a polynucleotide construct encoding a biologically active form of SOCS-3 protein, or a biologically active fragment thereof.
  • SOCS-3 activity can be increased by transfecting a cell with a nucleic acid encoding a modified SOCS-3 protein that has increased biological activity.
  • the present invention also pertains to cell lines that can be used to evaluate SOCS-3 mediated leptin activity and to screen candidate SOCS-3 inhibitors, antagonists and agonists for activity.
  • a cell line can be produced that expresses SOCS-3, a cytokine receptor and a reporter gene construct wherein transcription of the reporter gene construct is inhibited by SOCS-3.
  • the cytokine receptor is the leptin receptor.
  • the reporter gene construct is a leptin responsive promoter attached to a reporter gene.
  • the reporter gene can be the CAT gene, the luciferase gene or the ⁇ -galactosidase gene.
  • Another cell line suitable for use in the present invention is a cytokine dependent cell line wherein SOCS-3 and the leptin receptor are stably expressed.
  • the cytokine receptor is the IL-3 receptor.
  • the cell lines of the present invention can be used to screen libraries such as organic molecule libraries or cDNA libraries to select and identify molecules that inhibit (or enhance) SOCS-3 activity.
  • cells expressing the leptin receptor, SOCS-3 and a reporter gene construct are contacted with an organic molecule library or transfected with a cDNA expression library. These cells are then stimulated with leptin. Cells having increased reporter gene activity are selected and the organic molecule or cDNA is identified.
  • IL-3 dependent cells expressing leptin receptor and SOCS-3 are removed from IL-3, contacted with a member of an organic molecule library or transfected with a member of a cDNA expression library. Cells capable of proliferating in leptin are selected and the organic molecule or cDNA is identified.
  • Figure 1 A shows the results of a 32 P-RT-PCR assay demonstrating the in vivo effects of leptin on CIS-1 , SOCS-1, SOCS-2, and SOCS-3 mRNA levels in the hypothalamus from ob/ob mice.
  • Figure IB is the quantification of the data in Figure 1A.
  • Figure 1C is a plot of quantitative 32 P-RT-PCR of SOCS-mRNA from hypothalami of db/db mice and lean (+/?) controls upon leptin treatment.
  • Figures 2 A and 2B show the results of in situ hybridization with 5 S-labeled antisense SOCS-3 probes to brain sections from normal rats treated with saline or leptin.
  • Figure 3 A is a graphic representation of leptin-induced erg-1 promoter activation by SOCS-3 in CHO cells.
  • Figure 3B shows a Western blot demonstrating inhibition of leptin induced leptin receptor tyrosine phosphorylation by SOCS-3 in COS-1 cells.
  • the present invention encompasses the regulation of leptin activity in the brain. Specifically encompassed by the present invention is the regulation of the leptin- induced cell signaling pathway in the hypothalamus via regulation of SOCS-3 activity.
  • the libraries of peptides comprise a mixture of substantially equimolar amounts of peptides.
  • the library can be designed to mimic SOCS-3 target molecules, e.g., JAK2.
  • the library comprises peptides or phostyrosin containing peptides that interact with the SH2 domain of SOCS-3, thereby inhibiting the ability of SOCS-3 to bind target molecules.
  • Mammalian expression vectors may comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' to 3' nontranslated sequences, such as necessary ribosome binding sites, a poly- adenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' to 3' nontranslated sequences, such as necessary ribosome binding sites, a poly- adenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • the transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells may be provided by viral sources.
  • viral sources for example, commonly used promoters and enhancers are derived from Polyoma, Adenovirus 2. Simian Virus 40 (SV40), and human cytomegalovirus.
  • DNA sequence derived from the SV40 viral genome for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence.
  • the early and late promoters are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin or replication (Fiers et al, Nature 275:1 13, 1978.
  • SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgll site located in the viral origin or replication is included.
  • Exemplary vectors can be constructed as disclosed by Okayama and Berg (Mol Cell Biol 5:280, 1983.
  • Peptide libraries such as an oriented peptide library (Z. Songyang et al. Cell 72:161, 1993; can be screened for peptides that interact with SOCS-3.
  • Peptide libraries and other small organic molecule libraries can also be screened using other assays known in the art, such as proximity assays or Biospecific Interaction Analysis (BIA). Biospecific Interaction Analysis (BIA) in real time can be performed tc evaluate candidate molecules for their ability to bind SOCS-3.
  • SPR Surface plasmon resonance
  • This resonance angle depends on several factors, including the refractive index of the medium close to the non-illuminated side of the metal film. Refractive index is directly related to the concentration of dissolved material in the medium.
  • SPR is used to measure changes in the concentration of macromolecules in a surface layer of solution in contact with a dextran-coated gold film.
  • the association and dissociation rate constants for a peptide or organic molecule binding to SOCS-3 can be measured.
  • Polypeptides peptides, peptide mimics or small organic molecules exhibiting higher association constants (KJ have the greatest potential for ability to interact with SOCS-3 and inhibit SOCS-3 activity.
  • the present invention includes cell lines suitable for use in the screening methods described herein.
  • the cell line is a mammalian cell line such as CHO cells, Ba/F3 cells, HepG2 cells or H35-hepatoma cells, wherein said cells stably express a cytokine receptor and a reporter gene construct wherein the reporter gene construct is active in the absence of SOCS-3.
  • the cell line is further modified by the introduction of SOCS-3 whereby the reporter gene construct is inhibited by SOCS-3 expression.
  • the cytokine receptor is the leptin receptor long form.
  • the reporter gene encodes luciferase.
  • the reporter gene encodes ⁇ -galactosidase.
  • the reporter gene construct contains SOCS-3 promoter elements.
  • Candidate antagonists/agonists can be assessed for their ability to inhibit/enhance SOCS-3 activity, by their ability to allow reporter gene expression or cell proliferation of SOCS-3 expressing cells comprising the steps of: culturing the cells described above under conditions suitable for maintenance and growth; contacting said cells with the candidate molecule or an organic molecule library comprising SOCS-3 inhibitors or transfecting the cells with a cDNA expressing the candidate molecule with a cDNA expression library comprising DNA encoding candidate SOCS-3 inhibitors, contacting the cells with leptin.
  • the present invention further encompasses a cytokine dependent cell line wherein the cells also stably express SOCS-3 and the leptm receptor long form
  • the cytokine can be IL-3, IL-6 and other closely related cytokines
  • the cytokine dependent cell line is Ba/F3 cells
  • the cytokine is IL-3
  • the invention further provides a method of isolating and identifying inhibitors of SOCS-3, comprising the steps of cultu ⁇ ng the cytokine-dependent cells descnbed above in the presence of said cytokine under conditions suitable for maintenance and growth, removing said cells from the cytokine (in the case of BA/F3, the cytokine would be IL-3), contacting the cells with a candidate organic molecule or with
  • Inhibitors identified as descnbed by the present invention can be useful to treat obesity or prevent weight gain in a mammal Such molecules may also be useful to treat affective depression, such as melancholic depression, to induce the onset of puberty or to correct reproductive dysfunction Alternatively, some affective disorders can be treated by decreasing leptin activity. For example a SOCS-3 agonist can be administered to an individual in need of such treatment
  • the present invention further encompasses methods of reducing food intake in a mammal compnsing increasing leptin cell-signaling compnsing inhibiting SOCS-3 activity.
  • the mammal loses bodyweight.
  • the present invention further compnses a method of treating a mood affective disorder in a mammal comprising inhibiting SOCS-3 activity.
  • the antagonists/ agonists of the present invention can be formulated into compositions with an effective amount of the mhibitor/antagonist/agonist as the active ingredient.
  • An effective amount of a SOCS-3 inhibitor/antagonist is an amount effective to partially or completely inhibit SOCS-3 activity resulting in increased leptin activity
  • An effective amount of a SOCS-3 agonist is an amount effective to enhance SOCS-3 activity resulting in a decrease of leptin activity.
  • Suitable pharmaceutical carriers include, but are not limited to water, salt solutions, alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc.
  • the pharmaceutical preparations can be sterilized and desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active agents, e.g., enzyme inhibitors, to reduce metabolic degradation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers,
  • inhibitors/antagonists/agonists of the present invention can be administered to an individual mammal in need of such treatment, in conjunction with an agent or agents that allow the inhibitor to pass through the blood brain barrier.
  • the inhibitor/antagonist/agonist and the agent can be administered simultaneously or sequentia'ly.
  • agents are known in the art, such as those described in US Patents 5,112,596; 5,268,164; 5,686,416 and 5,506,206; the teachings of which are incorporated herein by reference in their entirety.
  • RNA-STAT-60 reagent as described by the manufacturer (TEL-TEST, Inc., Friendswood, TX). Total RNA purification and subsequent cDNA synthesis was done in parallel from all tissue samples.
  • the cDNA was synthesized from 1.0 ⁇ g of total RNA by using dT- oligonucleotides and the Advantage RT-PCR kit from Stratagene (La Jolla, CA). The final volume of the cDNA samples was 100 ⁇ l.
  • the following primers were used for specific PCR amplification of mouse CIS-1, mouse SOCS-1, mouse SOCS-2 and mouse SOCS-3:
  • CIS-1A 5'-ctggagctgcccgggccagcc-3', 400 bp (GenBank Ace. Number D31943),
  • CIS-IB 5'-caaggctgaccacatctggg-3', SEQ ID NO:2, SOCS-1A: 5'-ccactccgattaccggcgcatc-3', 350 bp (GenBank Accession Number
  • SOCS-1 B 5'-gctcctgcagcggccgcacg-3',SEQ ID NO:4;
  • SOCS-2A 5'-aagacgtcagctggaccgac-3 ⁇ 300 bp (GenBank Ace. Number U588327),
  • SOCS-3B 5'-gtggagcatcatactgatcc-3'.
  • SEQ ID NO:8 Each 50 ⁇ l PCR reaction was carried out with 5.0 ⁇ l of cDNA as template.
  • the assay conditions were: 10 mM Tris-HCl (pH 8.8), 50 mM KC1. 1.5 mM MgCl,. 0.Ol% gelatin 0.2 mM dNTPs. 20 pmol of each primer. 2.5 units of Taq polymerase (Stratagene) and 1.0 ⁇ l of 32 P-dCTP (29.6 TBq/mmol. 370 MBq/ml)(NEN, Boston, MA).
  • the mixture was overlaid with 25 ⁇ l of mineral oil, and after initial denaturation at 96°C for 3 min the samples were subjected to 24-32 cycles of amplification: denaturation at 95°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 45 seconds.
  • Ten ⁇ l of the reaction were then combined with 5 ⁇ g of sequencing stop solution (Amersham International, Buckinghamshire, UK) and heated to 85°C for five minutes before loading 5 ⁇ l onto a 4% urea-acrylamide gel (38 x 31 x 0.03 cm).
  • Electrophoresis was carried out at 60 W of constant power four hours, before the gels were transferred to filter paper, dried and finally subjected to 32 P quantification by Phosphorimager analysis (Molecular Dynamics).
  • Preliminary PCR experiments showed that the rate of amplification was linear for CIS-1, SOCS-1 and SOCS-3 when applying less than 30 PCR-cycles.
  • the amplification rate of SOCS-2 was linear for 27 cycles, after which non-linear amplification appeared.
  • PCR reactions were spiked with 2 P-dCTP and assembled in parallel for each cDNA and subjected to PCR amplification under the above conditions of limiting number of cycles. PCR products were then separated on denaturing acrylamide gels and finally subjected to autoradiography.
  • Ad libitum fed male ob/ob mice aged 7-8 weeks were injected intraperitoneally (ip) with 100 ⁇ g recombinant mouse leptin, or saline. Two hours later, total RNA was purified from hypothalami, and quantitative RT-PCR for CIS, SOCS- 1 , SOCS-2 and SOCS-3 mRNAs was performed. Leptin treatment caused a 2.0 fold increase in SOCS-3 mRNA. while no effect on CIS, SOCS-1. or SOCS-2 mRNA levels were detected ( Figure 1 A and IB). A similar effect on SOCS-3 mRNA was seen 1 or 3 hours after leptin administration (data not shown).
  • Sections were rinsed in decreasing concentrations of SSC containing 0.25% DTT: 2x at 50°C for 1 hour, 0.2x at 55°C for 1 hour, and 0.2x for 1 hour at 60°C. Sections were next dehydrated in graded ethanol (50, 70, 80, and 90%) containing 0.3 M NH 4 OAc followed by 100% ethanol. Slides were air dried and placed in X-ray film cassettes with BMR-2 film (Kodak) for 3-5 days. Slides were then dipped in NTB2 photographic emulsion (Kodak), dried and stored with desiccant in foil-wrapped slide boxes at 4°C for 2-3 weeks.
  • graded ethanol 50, 70, 80, and 90%
  • Example 3 SOCS-3 inhibits leptin induced transcriptional activation in CHO cells.
  • SOCS-3 was tested for its ability to inhibit leptin induced transcriptional activation, erg-1 is an immediate early gene induced upon leptin stimulation.
  • erg-l-luc is a reporter construct expressing the promoter elements of erg-1 fused to the luciferase gene.
  • CHO cells were transiently transfected the leptin receptor erg-l- luc together with either alone or with CIS-1, SOCS-2 or SOCS-3.
  • SOCS-3 but not CIS-1 or SOCS-2 blocked leptin-induced activation of the erg-1 luciferase reporter construct while serum-induced erg-1 gene transcription was unaffected by expression of SOCS-3.
  • Example 4 Localization of SOCS-3 mRNA by in situ hybridization in the "Agouti" mouse.
  • the Agouti (or lethal yellow, A-7a) mouse is an autosomal dominant murine obesity model. Obesity in these mice is accompanied by increased linear growth and altered hair pigmentation.
  • Agouti mice Like other non ob or db mouse models of obesity, Agouti mice have elevated levels of leptin and are refactory to leptin treatment either intravenously or injected directly into brain tissue.
  • the disorder is caused by ectopic and unregulated expression of agouti, a protein normally restricted to hair follicles, where it affects pigmentation by antagonizing melanocyte stimulating hormone (-MSH).
  • agouti a protein normally restricted to hair follicles, where it affects pigmentation by antagonizing melanocyte stimulating hormone (-MSH).
  • Agouti also antagonizes MC4 receptors, whose expression is largely restricted to the brain.
  • SOCS-3 expression was localized in brain tissue of Agouti mice following the methods described in Example 2. Specific hybridization was detected in the arcuate nucleus, while no specific hybridization signals were detected in other regions of the brain. This data supports the theory that expression of SOCS-3 plays a role in the desensitization of these animals to leptin signaling and hence is an important factor in the loss of weight control in these animals.
  • Example 5 Suppression of leptin receptor signaling by SOCS-3 in mammalian cell lines.
  • COS-1 cells were grown in Dulbecco's modified Eagle's medium (DMEM. low glucose) supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin and 10 ⁇ g/ml streptomycin at 37°C in 5% CO 2 .
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • CHO cells were grown in HAM's F12 medium supplemented with 10% FCS, 100 units/ml penicillin, and 10 ⁇ g/ml streptomycin.
  • the amount of transfected JAK cDNA was 1/10 of the total amount of DNA transfected.
  • Triton X-100 0.5% Triton X-100, 10% glycerol, 150 mM NaCl, 2 mM Na 3 V0 4 , 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 5 ⁇ g/ml leupeptin, 5 ⁇ /ml aprotinin, 50 mM Tris-HCl, pH 7.4). Lysates were finally clarified by centrifugation at 23,000 g for 15 min. and the supernatant immunoprecipitated as described below.
  • Immunoprecipitations were performed at 4°C by incubating clarified cell extracts with the 12C A5 or OBR antibodies and protein A-agarose beads (1 :15 dilution of a 50% slurry in 1% Nonidet P-40, 0.5% Triton X-100, 10%, glycerol. 150 mM NaCl, 50 mM Tris-HCl, pH 7.4) on a rotating wheel overnight.
  • the agarose beads were pelleted by low speed centrifugation and washed 3 times with 1 ml of ice-cold lysis buffer B.
  • proteins were boiled for 5 min. and subjected to SDS-PAGE, followed by transfer of the resolved polypeptides to nitrocellulose membranes.
  • the membranes were blocked with 10% nonfat dried milk in Towbin buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) for 2 h at room temperature and then incubated with antibodies in 5% milk for 12-15 h at 4°C. After removal of unbound antibodies by three washes each for 20 min in Towbin buffer, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin (1 : 1000) in 2.5% milk for 1.5 h at room temperature and washed five times in Towbin buffer. The targeted proteins were detected using enhanced chemiluminescence (ECL) as described by the manufacturer (Amersham International, Buckinghamshire, UK).
  • ECL enhanced chemiluminescence
  • nitrocellulose membranes Stripping of nitrocellulose membranes was done by soaking membranes in 1% SDS, 70 mM Tris-HCl, pH 6.8 and 0.1% mercaptoethanol at 50°C for 30 minutes with slow agitation. Leptin Receptor and Stat3 Phosphorylation.
  • COS-1 cells were transiently co-transfected with expression vectors for mouse OBR1 and JAK2. together with either pcDNA3, or HA-tagged CIS-1, SOCS-2, SOCS-3 in PCDNA3 (Invitrogen).
  • the intact coding region of CIS was cloned by combining an EST (TIGR clone
  • SOCS-2 was cloned by combining an EST (IMAGE clone ID 131550, WashU-Merck EST project) with a 5'RACE product derived from human skeletal muscle Marathon cDNA (Clontech, Palo Alto, CA).
  • EST IMAGE clone ID 131550, WashU-Merck EST project
  • the intact coding region of SOCS-3 was cloned by PCR amplification from Balb/c mouse genomic DNA (Sigma).
  • HA hemagglutinin tag
  • JAK2 Phosphorylation CHO cells were transfected with OBR1 and JAK2 together with either empty- vector, CIS-HA. SOCS-2-HA, or SOCS-3-HA expression vectors. Forty-eight hours post transfection, including 15 hours of serum starvation, cells were either treated or not with 100 nM mouse leptin for 5 min. JAK2 immunoprecipitates (anti-JAK2 antibodies were from UBI) were subjected to SDS-PAGE and Western blotting with antiphosphotyrosine antibodies (4G10, UBI) or anti-JAK2 antibodies (Santa Cruz).
  • JAK2 was co-immunoprecipitated with SOCS-3, but not SOCS-2, in a leptin-dependent manner in transfected COS-1 cells. These data are consistent with results published earlier on SOCS-1 in transfected 293 cells (Endo et al., 1997).
  • CHO-OBR1 cells were tested for leptin resistance under conditions where endogenous SOCS-3 protein levels are elevated. CHO-OBR1 cells were stimulated with leptin for 1 hour and then washed to remove leptin from the medium. At different times after the leptin pretreatment, freshly applied leptin was tested for the ability to induce intracellular signaling. As demonstrated by Northern blotting, leptin was unable to induce SOCS-3 mRNA for up to 24 hours after leptin pretreatment.
  • leptin pretreatment of CHO-OBRl cells was tested for the ability to inhibit subsequent stimulation of leptin receptor phosphorylation.

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Abstract

L'administration de leptine agit sur l'apport alimentaire et le poids corporel chez les humains et les animaux grâce à un mécanisme qui agit sur des zones spécifiques de l'hypothalamus. On a découvert que les gènes CIS-1, SOCS-1, SOCS-2, ET SOCS-3 produisent des effets antagonistes puissants sur l'action de la leptine. Dans des lignées cellulaires mammifères, le SOCS-3 bloque complètement la transduction de signal induite par la leptine, alors que les CIS, SOCS-1, et SOCS-2 restent sans effet. Le SOCS-3 constitue une cible importante de l'action de la leptine dans les cellules de l'hypothalamus sensibles à la leptine, ainsi qu'un inhibiteur puissant de la signalisation de la leptine. La hausse de l'activité du SOCS-3 dans des neurones sensibles à la leptine constitue un mécanisme potentiel de résistance à la leptine, observée dans les syndromes de l'obésité, les troubles de l'humeur, et les dysfonctionnements des fonctions reproductrices.
EP99906877A 1998-02-11 1999-02-10 Methodes et compositions pour moduler l'activite de la leptine Withdrawn EP1053007A2 (fr)

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ATE278952T1 (de) 1998-07-28 2004-10-15 Vlaams Interuniv Inst Biotech Leptin vermittelte geninduktion
WO2000037636A1 (fr) * 1998-12-21 2000-06-29 The Walter And Eliza Hall Institute Of Medical Research Boite socs contenant des peptides
AU4651900A (en) * 1999-04-20 2000-11-02 Beth Israel Deaconess Medical Center Methods and compositions for modulating ciliary neurotrophic factor activity
SE9903953D0 (sv) * 1999-11-01 1999-11-01 Sahltech Ab New use of the jak-stat system
AUPQ614700A0 (en) * 2000-03-09 2000-03-30 Walter And Eliza Hall Institute Of Medical Research, The A method and agents useful for same
AU4034601A (en) * 2000-03-09 2001-09-17 Inst Medical W & E Hall Methods of regulating cytokine signalling
AU8178401A (en) 2000-05-22 2001-12-03 Vlaams Interuniv Inst Biotech Receptor-based interaction trap
ATE374213T1 (de) * 2000-11-14 2007-10-15 Vlaams Interuniv Inst Biotech Funktionelles fragment des leptinrezeptors
US7235629B2 (en) 2000-11-14 2007-06-26 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Functional fragment of the leptin receptor
AU2002950745A0 (en) * 2002-08-13 2002-09-12 The Walter And Eliza Hall Institute Of Medical Research A method and agents useful for same
US7423113B2 (en) 2004-08-25 2008-09-09 Vib Vzw Leptin antagonist
US7863240B2 (en) 2004-10-08 2011-01-04 Enzo Therapeutics, Inc. Methods and uses of leptin in hepatocellular carcinoma
US8969291B2 (en) 2004-10-08 2015-03-03 Enzo Therapeutics, Inc. Methods for decreasing leptin levels or activity for treating inflammation
CA2585549A1 (fr) 2004-11-18 2006-05-26 Vib Vzw Domaine de fibronectine iii servant d'antagonistes de recepteurs de la leptine

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DK0948522T3 (da) * 1996-11-01 2008-09-22 Inst Medical W & E Hall Terapeutiske og diagnostiske midler, der kan modulere cellulær fölsomhed over for cytokiner
CA2276685A1 (fr) * 1997-11-03 1999-05-14 Incyte Pharmaceuticals, Inc. Suppresseur de la signalisation cytokinaire

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