JP2002506004A - Methods and compositions for modulating leptin activity - Google Patents

Abstract

  (57) [Summary] Leptin administration affects food intake and body weight in animals and humans by mechanisms involving specific areas of the hypothalamus. The CIS-1, SOCS-1, SOCS-2 and SOCS-3 genes were examined for their ability to antagonize leptin action. In mammalian cell lines, SOCS-3 completely blocked leptin-induced signaling, whereas CIS, SOCS-1 and SOCS-2 had no effect. SOCS-3 is a major target for leptin action in leptin-responsive cells in the hypothalamus, and SOCS-3 is a potent inhibitor of leptin signaling. Increased SOCS-3 activity in leptin-responsive neurons is a strong mechanism of leptin resistance observed in syndromes such as obesity, depression syndrome and reproductive dysfunction.

Description

DETAILED DESCRIPTION OF THE INVENTION

Related Application This application is a continuation-in-part of 09 / 044,278 filed on March 19, 1998, claiming priority and filed on February 11, 1998. Claims priority of US Provisional Application No. 60 / 074,320, the teachings of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION [0002] Leptin, a hormone derived from fat cells, acts on specific areas of the brain to regulate food intake, energy expenditure and neuroendocrine function (Zhang Y et al., N
atature, 372: 425-432, 1994; Halaas JL et al., Sc.
Nature, 269: 543-546, 1995; Campfield LA et al., Science, 269: 546-549, 1995; and Pellymo.
unter MA et al., Science, 269: 540-543, 1995).
Leptin is structurally related to cytokines (Zhang F et al., Nature).
387: 206-209, 1997), which act on receptors belonging to the cytokine receptor superfamily (Tartaglia LA et al., Cell).
83: 1263-1271; Lee GH et al., Nature, 379: 63.
2-635, 1996).

[0003] In dietary-induced obesity in rodents and in most obese humans, resistance to peripheral leptin exists and needs to be further elucidated. Human obesity may be associated with low levels of functional circulating leptin, or reduced effects on target cells in the brain. The latter possibility indicates that a functional mutation in the leptin gene is present in humans (Montague CT et al., Natu.
re, 387: 903-908, 1997), supported by data demonstrating that such mutations are extremely rare (Maffei M et al., D
iabetes, 45: 679-682, 1996; Shigemoto M et al., Eur J Endocrinol. 137: 511-513, 1997; C
arlsson B et al., Obes Res, 5:30, 1997). Furthermore, serum leptin levels are increased in obese humans and are positively correlated with body weight (Maf
fei M et al., Nat Med, 1: 1155-1161, 1995; Cons.
idine RV et al., Engl J Med, 334: 292-295,199.
6). In addition, all studies of diet-induced obesity in rodents (V
an Heek M et al., J Clin Invest, 99: 385-390,
1997), but some studies (Widowson PS et al., Diab
etes, 46: 1782-1785, 1997), and these animals have been shown to develop both peripheral and central resistance to recombinant leptin. Taken together, these data are consistent with the possibility that the leptin resistance that characterizes human obesity may be due to a defect in leptin signaling in the brain. Two possible mechanisms for leptin resistance are defects at the level of the blood-brain barrier and defects in the leptin signaling pathway in target cells. Regarding the latter possibility, the leptin receptor (OBR), whose mutation causes obesity in db / db mice and fa / fa rats,
Gp activated by cytokines such as L-6, LIF and CNTF
130 and the signaling subunits of LIFR, and most closely related to hormone receptors for growth hormone such as erythropoietin (Tart
Aglia, 1995). There are several isoforms of the leptin receptor, including the long form that is predominantly expressed in certain cell bodies in the hypothalamus. Possible mechanisms for inhibition or enhancement of leptin signaling are of considerable interest.

Recently, CIS (cytokine-inducing sequence), SOCS-1 (cytokine signaling inhibitor), SOCS-2 and SOCS-3 (Starr R et al., N
Nature, 387: 917-921, 1997; Endo TA et al., Natu.
re, 387: 921-924, 1997; Naka T et al., Nature, 3
87: 924-929, 1997; Masuhara M et al., Biochem.
Biophys Res Commun, 239: 439-446, 1997)
A new family of cytokine-inducible inhibitors of signaling has been identified, including. IL-6, LIF, growth hormone (GH) and erythropoietin (E
A number of different cytokines, including PO), induce the in vivo and in vitro transcriptional activation of one or more CIS or SOCS genes through activation of the JAK-STAT pathway (Starr, 1997; Endo). , 199
7; Naka, 1997; Yoshimura et al., Embo J, 14: 281.
6-2826, 1995; Masuhara, 1997). The results suggest that the CIS and SOCS proteins can act in a typical negative feedback circuit by inhibiting JAK activity, thereby switching off cytokine signaling.

SUMMARY OF THE INVENTION [0005] The present invention includes methods and compositions for modifying or modulating leptin activity by modifying, ie, modulating, the activity of a cytokine inhibitor. Specifically, the present invention provides a cytokine inhibitory factor, SOCS-3.
Methods and compositions for altering the activity of are included. SOCS-3 expression is rapidly induced by leptin treatment in areas of the hypothalamus known to be involved in regulating body weight. As shown herein, this time SOCS-
It has been shown that there is an inhibitory pathway for 3-mediated leptin cell signaling. Thus, this suggests that SOCS-3 is a negative regulator of leptin-mediated signaling. Further, as described herein,
Excessive SOCS-3 activity is believed to be an important factor in leptin resistance, which characterizes most syndromes of obesity in rodents and humans. Therefore,
Inhibition of SOCS-3 expression and function is a potential target for developing drugs aimed at improving leptin sensitivity in mammals and thus reducing body weight. In addition, inappropriately increased SO in leptin-responsive neurons
CS-3 activity is a possible mechanism of leptin resistance observed in various syndromes of obesity. Thus, altering SOCS-3 activity, as described herein, provides a means for modulating leptin-induced cell signaling, and thus regulating body weight.

The present invention also provides a method of treating delayed onset of puberty (maturity) in mammals by increasing cellular signaling of leptin mediated by SOCS-3, and a method of treating leptin-low serum / It relates to a method of treating reproductive dysfunction or infertility such as reduced aovulation or reduced spermatogenesis associated with plasma levels, inactive leptin, leptin resistance or ineffective production of leptin. The method of the present invention comprises:
Either male or female mammals can be used.

[0007] The present invention is also a method of treating a depression syndrome in an individual associated with elevated leptin levels, such as atypical depression, wherein the atypical depression is elevated in the individual. A method characterized by leptin levels, wherein the treatment resulting in reduced leptin-induced cell signaling results in the prevention or alleviation of the symptoms of amorphous depression. The invention also provides a method of treating a depression syndrome in an individual associated with reduced leptin levels, such as melancholic depression, wherein melancholic depression is characterized by reduced leptin levels in the individual. A method that results in the prevention or alleviation of the symptoms of melancholic depression by a treatment that increases cell signaling induced by leptin.

Accordingly, the present invention relates to a method of modulating leptin cell signaling by altering SOCS-3 activity in a mammal. As defined herein, modulating, modifying, modulating, or controlling leptin activity refers to inhibiting or enhancing leptin or SOCS-3 biological activity. Inhibition of leptin activity includes partial inhibition of leptin activity as well as complete inhibition of leptin activity.

[0009] The bioactivity of leptin is defined herein as one or more of the intracellular levels of leptin as a result of the interaction (eg, binding) between leptin and a cell-associated leptin receptor. Defined as the ability to activate a signaling pathway.
As the signal transduction pathway, for example, Janus kinase 2 (J
AK2), thereby activating transcriptional signal transducers and activators (signal transducer and ac).
Activates other pathways, such as the pathways of transcription (STAT), the activation or inactivation of gene transcription as well as the phosphoinositide-3 kinase ras / mitogen-activated protein kinase pathway that ultimately results in other non-transcriptional effects Is included. For example, leptin is JAK
JAK2 is activated and phosphorylates the receptor (especially) the JAK2 and STAT3 proteins. Phosphorylated STAT3 dimerizes and translocates to the nucleus where it acts as a transcriptional activator. Thus, the activity of leptin is determined by the receptor, JAK2
Alternatively, it can be measured as the phosphorylation level of STAT3. Further, leptin activity can be measured by the amount of gene transcript from a STAT3 responsive gene.

[0010] As defined herein, SOCS-3 activity or SOCS-3-mediated leptin cell signaling is the inhibition or inactivation (complete or partial) of leptin-induced cell signaling. . As shown by the present invention, SO
CS-3 mediates the lack of phosphorylation of the leptin receptor JAK2 or STAT3 and downregulation of leptin signaling as measured by association of JAK2 and SOCS-3. As described herein, SOCS-3 transcription is part of a negative feedback circuit initiated by leptin activation of the leptin receptor.

[0011] The activity of SOCS-3 is inhibited by inhibiting or decreasing the amount of SOCS-3 protein expressed in the cell, or by introducing a polynucleotide encoding the modified SOCS-3 protein into the cell, Modified SOCS-
The three proteins include variants, variants, derivatives or analogs of the SOCS-3 protein.

[0012] SOCS-3 expression can be inhibited or reduced by transfecting cells with a polynucleotide construct encoding an antisense DNA or antisense RNA of SOCS-3. For example, the antisense RNA hybridizes to the endogenous SOCS-3 mRNA and produces SOCS-3 mRN.
A translation can be prevented, thereby inhibiting or reducing SOCS-3 protein expression. SOCS-3 expression also indicates that SOCS-3
The inhibition or reduction can be achieved by transfecting the cell with a polynucleotide construct that encodes a transcription inhibitor so as to inhibit or reduce transcription. Such transcription inhibitors specifically interact with the promoter sequence of SOCS-3, resulting in decreased transcription of SOCS-3, and reduced expression of SOCS-3 protein, and thus reduced SOCS-3 activity.

[0013] SOCS-3 activity can also be inhibited by transfecting cells with a polynucleotide construct encoding an altered or modified SOCS-3 protein, polypeptide or peptide. In one embodiment, the modified S
OCS-3 polypeptides are competitive inhibitors (eg, antagonists) of endogenous SOCS-3. The modified SOCS-3 can interact with a SOCS-3 target protein (eg, JAK2) without interfering with the activity of the target protein. Because the modified SOCS-3 protein interacts with the target SOCS-3 target, endogenous SOCS-3 is unable to interact with the target of interest, thereby resulting in a SOCS-3-mediated leptin cell signal. Transmission levels can be inhibited or reduced. In another embodiment, the SOCS-3 activity is
The CS-3 inhibitor can be inhibited or reduced by introducing it into cells. Such inhibitors can be peptides or small organic molecules that interfere with SOCS-3 activity. Such inhibitors may inhibit SOCS-3 activity,
It can interact specifically with CS-3 or its intended target. For example, inhibitors can interact with targets downstream of SOCS-3, such as JAK2.

[0014] The invention further includes a method of increasing or enhancing SOCS-3 activity in a cell. Increased intracellular SOCS-3 activity can inhibit or reduce leptin-induced cell signaling. Reducing or inhibiting leptin-induced cell signaling may be useful for preventing, inhibiting or ameliorating amorphous depression in an individual, or promoting weight gain in an individual. The activity of SOCS-3 is determined by bioactive SOCS
-3 protein or a biologically active fragment thereof can be increased by transfecting the cell with a polynucleotide construct. In another embodiment, the activity of SOCS-3 is a modified SOC with increased bioactivity
It can be increased by transfecting the cells with a nucleic acid encoding the S-3 protein.

The present invention also provides cell lines that can be used to assess SOCS-3 mediated leptin activity and to screen candidate SOCS-3 inhibitors, antagonists and agonists for activity. is connected with. For example, a cell line that expresses SOCS-3, a cytokine receptor and a reporter gene construct, wherein the transcription of the reporter gene construct causes the SOCS-
Cell lines that are inhibited by 3 can be made. In one embodiment, the cytokine receptor is a leptin receptor. In another embodiment, the reporter gene construct is a leptin-responsive promoter linked to the reporter gene. The reporter gene can be a CAT gene, a luciferase gene, or a β-galactosidase gene. Another cell line suitable for use in the present invention is a cytokine-dependent cell line that stably expresses SOCS-3 and leptin receptor. In one embodiment, the cytokine receptor is an IL-3 receptor.

The cell lines of the 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. it can. In one embodiment, 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 with increased reporter gene activity are selected to identify organic molecules or cDNA. In another embodiment, I expressing leptin receptor and SOCS-3
L-3-dependent cells are removed from IL-3 and contacted with a member of an organic molecule library or transfected with one of the cDNA expression libraries. Cells that can grow in leptin are selected and organic molecules or cDNA are identified.

Accordingly, as a result of the findings described herein, we now modulate the activity of leptin, in particular, the activity of the cytokine inhibitory SOCS-3, and thereby leptin Methods and compositions that can be used to modulate leptin activity by effecting either increased or decreased induced cell signaling.

DETAILED DESCRIPTION OF THE INVENTION The present invention involves the control of leptin activity in the brain. In particular, the invention encompasses the control of leptin-induced cell signaling in the hypothalamus via the control of SOCS-3 activity.

Leptin is a hormone that has been shown to interact with its cognate receptor and thereby initiate its cell signaling pathway. The area of the hypothalamus,
It is anticipated that there will be several different isoforms of the leptin receptor, including the long form, which has the highest levels of expression in that particular area, including the arcuate nucleus and the dorsomedial hypothalamus. In vitro and in vivo studies have shown that leptin is a typical JAK-STAT via the long receptor isoform.
Show activation of cytokine-like signaling by stimulating the pathway (Ghilardi N et al., Proc Natl Acad Sci USA
, 93: 6231-6235, 1997; Baumann H et al., Proc N.
atl Acad Sci USA, 93: 8374-8378, 1996;
aise C et al., Nature Genetics, 14: 95-97,19.
96). The lack of functional leptin or the leptin receptor long form, respectively, in ob / ob and db / db mice causes severe obesity (Zhang et al., Nature, 372: 425-432,199).
4; Lee GH et al., Nature, 379: 632-635, 1996;
hen H et al., Cell, 84: 491-495, 1996).

Cloning of the gene encoding leptin (Zhang, 1994) and cloning of the gene encoding the leptin receptor (Tartaglia)
Et al., 1995, 61183: 1263-1271), and in vivo and in vitro studies of these proteins have dramatically revealed the importance of this ligand-receptor system in normal regulation of body weight and energy balance.

In addition to such a role, which has been proposed to be its major function, circulating leptin also appears to play an important role in the neuroendocrine axis, including the control of reproduction (Ahima, R., et al. S. et al., Nature, 382: 250-252, 1
996). Administration of exogenous leptin has been shown to induce the onset of puberty in mice (US patent application Ser. No. 08 / 749,534, the teachings of which are incorporated herein by reference in its entirety). Is). Treatment of freely fed female mice with leptin doses that did not significantly change body weight resulted in earlier onset of puberty.

Recently, a new family of cytokine inhibitors including CIS-1 and SOCS-1, 2 and 3 has been identified. CIS-1 is a signaling inhibitor of the cytokine receptor, and directly binds to the cytokine receptor.
It is presumed to block important phosphotyrosine residues of the receptor (Yoshimura, 1995). SOCS-1, 2, and 3 are cytokine-inducible inhibitors that have been found to be active in hematopoietic cells that are thought to interfere with cytokine-mediated cell proliferation (Starr, 1997).
Endo, 1997; and Naka, 1997). As first identified herein, leptin is expressed in SOCS-3 mRNA in the hypothalamic nucleus, where high levels of leptin receptor long form are known to be expressed.
Is specifically induced. No effect on CIS, SOCS-1 or SOCS-2 can be detected (see Example 1).

Leptin of SOCS-3 mRNA in the arcuate nucleus and dorsal medial hypothalamus by in situ hybridization experiments on brain sections of both ob / ob and normal mice using an antisense RNA probe of SOCS-3 Dependent specific increase was revealed (Example 2). These areas are
In the hypothalamus, the highest levels of the leptin receptor long form mRN
A is expressed, strongly suggesting that the effect of leptin on SOCS-3 mRNA levels is a direct effect on certain neurons expressing the leptin receptor long form. SOC after leptin treatment
The regions of the arcuate nucleus that show stimulation of S-3 mRNA are the regions that are known to express NPY, POMC and AGRP, all of which are regulated by leptin in vivo; It suggests that expressing cells may be direct targets for leptin.

Fos is often used as a marker for activated neurons. But,
Its use is limited due to the inability to distinguish between the direct and indirect effects of the administered drug. Furthermore, N in the arcuate nucleus that is negatively controlled by leptin
PY expressing neurons are not positive for activation of Fos after leptin treatment. However, as shown in Example 2 herein, SOCS-3 mRNA
Increased in the area of the arcuate nucleus expressing NPY, suggesting that SOCS-3 is a better marker than Fos for leptin-controlled neurons.

[0025] Lethal yellow (A ^y / a) mice have potent melanocortin receptor (MCR)
) Develop obesity by ectopic and uncontrolled overexpression of the agouti protein, an antagonist of (Dickie, J. Hered. 60: 20-2)
5, 1969; Bultman et al., Cell, 71: 1195-1204,19.
92; Miller et al., Genes Dev. 7: 454-467, 1993)
. Obesity in this model is characterized by hyperleptinemia and resistance to both central and peripheral leptin administration (Halaas, 1997). Ultimately, leptin resistance in this model must result from agonout-induced melanocortin antagonism, but the molecular basis for leptin resistance is unknown. It has been hypothesized that leptin resistance in A ^y / a mice may be due to blocking of the MC4 receptor downstream of leptin signaling in the brain (Seeley et al., Nature, 390: 3).
49, 1997). However, recently, A ^y / a mice also defective in leptin, such as A ^y / a, lep ^ob / lep ^ob , have been shown to respond normally to leptin (Boston et al., Science, 278: 164
1-1644, 1997). Subsequent observations indicate that leptin resistance in A ^y / a mice is a consequence of chronic exposure to high circulating levels of leptin in addition to blocking the MC4 receptor, or at least such chronic exposure. Is suggested to be necessary. As described in Example 4, i
n situ hybridization histochemistry indicates that SOCS-3 mRNA
Elevated in freely fed ^Ay / a mice compared to lean control littermates, and especially elevated in the dorsolateral hypothalamic nucleus, where leptin induces SOCS-3 gene expression It was revealed that. A similar area of the dorsomedial hypothalamus nucleus was obtained after intravenous leptin administration to normal rats (El
mquist et al., Endocrinology, 138: 839-842,19.
98) or after central leptin administration (Van Dijk et al., Am J Ph.
ysiol. 271: R1096-R1100, 1996). This site is also responsible for NPY mRNA in A ^y / a mice.
Levels were shown to be increasing (Kesterson et al., Mol
Endocrinol. 11: 360-637, 1997). Therefore, it is reasonable to assume that leptin resistance in this model is the result of hyperleptinemia; serum leptin levels in the A ^y / a mice used here were compared to 7 ng / ml in control mice. Was 40 ng / ml. In this model, increased leptin may govern SOCS-3 expression in key hypothalamic nuclei involved in weight control, thereby inhibiting leptin's weight loss effect.

In mammalian cell lines, SOC but not CIS-1 or SOCS-2
S-3 completely blocks leptin-induced signaling (described in Example 5), and leptin receptor signaling also in vivo SOCS-
3 indicates that it is negatively controlled.

[0027] It is believed that the leptin receptor long form may exhibit signaling effects similar to those of granulocyte colony stimulating factor, leukemia inhibitory factor receptor and gp130. Ligand binding to these receptors leads to activation of receptor-bound JAK kinases that phosphorylate tyrosine in the cytoplasmic domain of the receptor as well as other cytoplasmic target proteins. Several pathways can be activated by JAK kinases, including the transcriptional signal transducer and activator (STAT) pathway, the ras / mitogen-activated protein kinase pathway, and the phosphoinositide-3 kinase pathway. As shown in Example 7, stimulation of leptin in vitro results in induction of SOCS-3 mRNA. Furthermore, the presence of SOCS-3 inhibits leptin-induced phosphorylation of STAT3, JAK2 and leptin receptors. Furthermore, leptin pretreatment of leptin receptor-expressing CHO cells results in prolonged inhibition of leptin-induced signaling (greater than 24 hours). For example,
As shown in Example 8, SOCS-3 mRNA was not induced and STA
Although it did not increase the DNA binding activity of T3, the leptin receptor was not phosphorylated in response to subsequent leptin treatment. This inhibition lasted at least 24 hours after leptin pretreatment.

Based on the results described herein, SOCS-3 interacts with JAK2 and competes with the binding between JAK2 and its substrate (leptin receptor or STAT), or It is reasonable to think that it competes with leptin-induced cell signaling by acting as a pseudosubstrate for JAK2, thereby preventing phosphorylation of the targeted target. SOCS-3 is JA
It can inhibit K2 kinase, thereby preventing phosphorylation of downstream elements. Thus, it has now been demonstrated that an inhibitory pathway for SOCS-3-mediated leptin cell signaling exists. Thus, SOCS-3 can negatively regulate cell signaling through the leptin receptor.

[0029] SOCS-3 can antagonize leptin-induced cell signaling by recruiting tyrosine phosphatases, which have been shown to be involved in cytokine receptor dephosphorylation. Two candidates are SHP-1 and SHP-2 (
(Also known as SYP). Both SHP-1 and SHP-2 have been shown to regulate cytokine signaling. The gradual increase of SHP-1 is J
Associated with dephosphorylation / inactivation of AK2 and subsequent termination of erythropoietin signaling (Klingmuller et al., Cell, 80: 729-
738, 1995). A similar role for SHP-1 in mediating JAK2 down-regulation following growth hormone stimulation of cells has been reported.

The present invention includes methods and compositions for modulating leptin activity that include the step of altering SOCS-3 activity in cells (eg, cells of the hypothalamus). The cells encompassed by the present invention can be found in all vertebrates, including mammals and humans. The cell may also be a cell maintained in a cell line: for example, a transformed cell suitable for use in determining leptin activity or SOCS-3 activity.

In one aspect of the invention, it is desirable to increase or up-regulate leptin activity through inhibition of SOCS-3 activity. Increased leptin activity results in increased leptin cell signaling pathways, which in particular results in weight loss, reproductive function restoration, and / or symptomatic relief of melancholic depression. In this aspect, SOCS-3 activity is inhibited by preventing SOCS-3 from interacting (eg, binding) to its target target. For example, a target of interest in SOCS-3 interacts with JAK2 (Example 6)
. SOCS-3 can act as a pseudo-substrate for JAK2, or it can recruit phosphatases to the JAK2-receptor complex. Therefore, Ge
an inhibitor of a polypeptide or peptide comprising the amino acid sequence of SOCS-3 of nBank accession number U88328, or the amino acid sequence of modified SOCS-3 /
The antagonist or active fragment thereof can interact competitively with SOCS-3 and / or its intended target, for example by binding to JAK2, resulting in increased or up-regulated leptin activity. .

As defined herein, modified SOCS-3 includes SOCS-3, including fragments, derivatives, analogs, variants and variants of SOCS-3 protein.
Includes three molecules. These modified SOCS-3 molecules have inhibitory / antagonist activity of SOCS-3, whereby endogenous SOCS-
Inhibits the activity of leptin 3 and consequently increases leptin activity. Another activity of the modified SOCS-3 molecule may be the antigenicity of the modified SOCS-3 molecule, including that the modified SOCS-3 may bind to an antibody specific for SOCS-3. The modified SOCS-3 molecule also indicates that the modified SOCS-3 molecule has an immunogenic response (eg, an endogenous (natural) S
Production of antibodies that specifically bind to OCS-3).

[0033] Fragments of SOCS-3 include polypeptides that contain only a portion of the full-length SOCS-3 protein and that inhibit the activity of endogenous SOCS-3. Such fragments can be made by amino / carboxyl terminal deletions as well as by internal deletions. Fragments can also be made by enzymatic digestion. Such modified SOCS-3 molecules can be tested for inhibitory activity as described herein.

“Derivatives” and “variants” of SOCS-3 can include truncated and hybrid forms of SOCS-3. The "truncated" form is a shortened form of SOCS-3, typically with the deletion of an internal region of the protein. "Hybrid" forms of SOCS-3 are SOCS-3 molecules that include a portion of the amino acid sequence of SOCS-3 and an amino acid sequence that is not SOCS-3, for example, a SOCS-1 or SOCS-2 sequence.

“Variants” and “variants” of SOCS-3 use in vitro and in vivo techniques (eg, site-directed mutagenesis and oligonucleotide mutagenesis) that are well known to those skilled in the art. Can be manufactured. SOCS-
Manipulation of the protein sequence of 3 can also be performed at the protein level as well. Any myriad chemical modifications can be made by known techniques. Such techniques include, but are not limited to, specific chemical cleavage with cyanogen bromide, trypsin and papain. SOCS-3 is also structurally modified or denatured, for example, by heat. Generally, the mutation may be a conservative or non-conservative amino acid substitution, insertion or deletion. Such a mutation is
It may be at or near the binding site of OCS-3.

For example, DNA encoding a SOCS-3 mutant is prepared by site-directed mutagenesis of DNA encoding endogenous SOCS-3. The site-directed mutagenesis method is sufficient to obtain the DNA sequence of the desired mutation, as well as a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides joining the deletion junction. The use of specific oligonucleotide sequences encoding a large number of contiguous nucleotides allows for the generation of SOCS-3 variants. Typically, between about 20 nucleotides and about 5 to 10 residue changes on either side of the junction of the sequence.
Primers 25 nucleotides in length are preferred. In general, the techniques of site-directed mutagenesis are well known in the art, as exemplified by publications such as Edelman et al., DNA 2: 183, 1983. The site-directed mutagenesis technique typically employs a phage vector that exists in both a single-stranded and double-stranded form. Representative vectors useful in the site-directed mutagenesis method include vectors such as M13 phage.
ng et al., "The 3rd Cleveland Symposium on Macromolecules and Recombinant DNA, disclosed by A. Walton, Elsevier, Amsterdam, 1981. This phage vector and other phage vectors are commercially available and their Use is well known to those skilled in the art.A useful and efficient procedure for the construction of site-directed mutations by oligonucleotides in DNA fragments using M13-derived vectors is described in Zoller, MJ and Smit.
h, M .; Nucleic Acids Res. 10: 6487-6500,
Published by 1982. Further, using a plasmid vector containing a single-strand phage origin of replication, single-stranded DNA can be obtained.
Meth. Enzymol. 152: 3, 1987.

Alternatively, nucleotide substitutions can be introduced by synthesizing a suitable DNA fragment in vitro and amplifying it by PCR techniques known in the art.

In general, a site-specific mutation according to the specification is a DNA encoding a related protein.
This can be done by first obtaining a single-stranded vector containing the A sequence within that sequence. Oligonucleotide primers having the desired mutated sequence are generally synthesized, for example, by Crea et al. (Proc Natl Acad Sci.
USA, 75: 5765, 1978). Next, the primer is annealed with a single-stranded protein sequence-containing vector and subjected to a DNA polymerase such as Klenow fragment of Escherichia coli polymerase I to complete the synthesis of a chain having a mutation. In this way, a heteroduplex is formed in which one strand encodes the original unmutated sequence and the other strand has the desired mutation. The heteroduplex vector can then be used to transform a suitable host cell, such as a JM101 cell, and a clone containing the recombinant vector having the mutant sequence arrangement can be selected. Thereafter, the mutated region can be removed and placed in an expression vector suitable for protein production.

[0039] The PCR technique can also be used when making SOCS-3 amino acid sequence variants. When a small amount of template DNA is used as a starting material for PCR, using a primer with a sequence that differs slightly in the corresponding region in the template DNA, a specific DNA fragment that differs from the template sequence only at positions that differ from the template Can be produced in a relatively large amount. To introduce the mutation into the plasmid DNA, one of the primers can be designed to overlap and contain the mutation location; the sequence of the other primer is preferably the opposite of the plasmid. Identical to the sequence region, but this sequence can be located anywhere along the plasmid DNA. However, the sequence of the second primer should be located within 500 nucleotides of the sequence of the first primer so that the entire amplified region of the DNA bounded by the primer at the end can be easily sequenced. Is preferred. By PCR amplification using primer pairs such as those just described,
A population of different DNA fragments is obtained at the terminal position of the mutation defined by the primer.

The generated DNA fragment with the desired mutation can be used to replace the corresponding region in the plasmid that serves as a PCR template using standard DNA techniques. Mutation at another position can be accomplished by using a mutated second primer or by performing a second PCR with a different mutated primer, and dividing the resulting two PCR fragments into three (or more) vector fragments. Can be introduced at the same time.

An alternative method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene, 34, 315, 1985. The starting material can be a plasmid (or vector) containing the SOCS-3 DNA to be mutated. Identify one or more codons in SOCS-3 to be mutated. On one side of the one or more identified mutation sites, a characteristic restriction endonuclease site must be present. If such restriction sites are not present, restriction sites may be created using the oligonucleotide-mediated mutagenesis method described above and
It can be introduced at an appropriate position in CS-3 DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to open circles. Double-stranded oligonucleotides that encode the DNA sequence between the restriction sites but contain one or more desired mutations are synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is called a cassette. Such a cassette,
It is designed to have 3 'and 5' ends that are compatible with both ends of the opened circular plasmid so that it can be directly ligated to the plasmid. Thus, the plasmid contains the DNA sequence of the mutated SOCS-3 that can be expressed to produce SOCS-3 with altered binding activity.

The inhibitor compound of the present invention interacts with endogenous SOCS-3 or interacts with a SOCS-3 target molecule such as JAK2, and thereby interacts with the molecule. Sometimes the inhibitor is the SOC of leptin's cell signaling activity
Any molecule that results in S-3 mediated inhibition is included. The present invention includes inhibitor compounds that, when interacting with the binding or active site, mimic the structure and conformation of the substrate moiety. Inhibitor molecules of the invention typically have an inhibition constant (K _i ) of 10 μM or less. In particular, it includes organic molecules that mimic the structure and conformation of the SH2 binding domain, interact with SOCS-3, and thereby inhibit its activity. In one embodiment, the inhibitor contains or mimics phosphotyrosine.

Furthermore, the present invention also encompasses small organic molecules that mimic the structure of SOCS-3, or the binding site of the SOCS-3 target, and thus prevent the interaction of SOCS-3 with its intended target molecule. Is done.

[0044] Peptides suitable for use as SOCS-3 inhibitors can be produced in libraries. The peptides of the library can be immobilized on a surface, for example, the peptides can be immobilized on chips or beads.

A library of peptides contains a substantially equimolar mixture of peptides. In one embodiment, the library is a SOCS-3 target molecule (eg, J
AK2) can be designed. In another embodiment, the library comprises
Peptides or photyrosines that interact with the SH2 domain of SOCS-3, thereby inhibiting the ability of SOCS-3 to bind to the target molecule
in) containing peptide.

The inhibitors of the present invention can be synthesized using standard laboratory methods well known to those skilled in the art, including standard solid phase techniques. Inhibitors containing naturally-occurring amino acids can also be made by recombinant DNA techniques known to those skilled in the art and subsequently phosphorylated.

[0047] The inhibitors of the present invention can contain either the 20 naturally occurring amino acids or other synthetic amino acids. Synthetic amino acids encompassed by the present invention include, for example, naphthylalanine; L-hydroxypropylglycine; L-3,4-dihydroxyphenylalanyl; L-α-hydroxylysyl and D-α-methylalanyl, L-α-. Α-amino acids such as methyl-alanyl; β-alanine (β-
β-amino acids such as aniline); and isoquinolyl.

[0048] D-amino acids and other non-naturally occurring synthetic amino acids can also be incorporated into the inhibitors of the present invention. Such other non-naturally occurring synthetic amino acids include naturally occurring side chains of the 20 genetically encoded amino acids (or any L or D amino acids) with other side chains, eg, alkyl, lower alkyl. 4-, 5-, 6-, and 7-membered alkyl, amide, amide lower alkyl, amide di (lower alkyl)
, Lower alkoxy, hydroxy, carboxy and lower ester derivatives thereof, and 20 genetically encoded amino acids substituted with 4-, 5-, 6-, or 7-membered heterocycles) (Or any L or D amino acid) substituted with another side chain. In particular, a proline analog in which the ring size of a proline residue is changed from a 5-membered ring to a 4-, 6-, or 7-membered ring can be used.

“Lower alkyl” as used herein refers to methyl, ethyl, propyl (e
straight-chain having 1 to 6 carbon atoms such as
And a branched alkyl group. "Lower alkoxy" includes methoxy, ethoxy, etc.
And straight-chain and branched alkoxy groups having 1 to 6 carbon atoms.

A cyclic group can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic. Heterocyclic groups typically contain one or more nitrogen, oxygen and / or sulfur heteroatoms and include, for example, flazanyl, furyl, imidazolidinyl,
Imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl, pyrrolinyl, pyrrolyl, thiadiazolinyl, thiazolyl, thionyl, thioenyl, thioenyl, thionyl, thioenyl, thioenyl, thionyl For example, thiomorpholino), triazolyl, etc. The heterocyclic group is
It can be substituted or unsubstituted. When a group is substituted, the substituent may be an alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl (see US Pat. Nos. 5,654,276 and 5,643,873). See the specification, the teachings of which are incorporated herein by reference).

[0051] Peptidomimetics that mimic the SOCS-3 protein can also be designed to inhibit SOCS-3 activity, thereby resulting in increased leptin activity. These mimetics can be designed and made by techniques known to those skilled in the art (eg, US Pat. Nos. 4,612,132, 5,643,87).
3 and 5,654,276, the teachings of which are incorporated herein by reference). These mimetics, based on the sequence of SOCS-3, have an activity that antagonizes the biological activity of the corresponding peptide compound, but have one or more of the following properties: solubility, stability, and susceptibility to hydrolysis and proteolysis. It has “biological advantages” over the corresponding peptide inhibitors in this regard:

Methods for preparing peptidomimetics include modifying the N-terminal amino group, the C-terminal carboxyl group, and / or replacing one or more amino bonds in the peptide with non-amino bonds. . Two or more such modifications can be combined in one peptidomimetic inhibitor. Modified examples of peptides to make peptidomimetics are described in US Pat. Nos. 5,643,873 and 5,654,27.
No. 6, the teachings of which are incorporated herein by reference. Libraries of peptidomimetics can also be made as described above.

On the other hand, SOCS-3 inhibitors interact with SOCS-3, thereby
It may be an antibody or antibody fragment that prevents CS-3 from interacting with a downstream target molecule such as JAK2, or such that SOCS-3 interacts with JAK2 without interfering with JAK2 kinase activity. It can be an antibody or an antibody fragment. The term “antibody” refers to polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies (eg, humanized antibodies), and Fab, Fab ′,
It is meant to include biologically active antibody fragments that specifically bind to SOCS-3, such as F (ab ') 2 and Fv. Furthermore, single-chain antibodies (sFv
) Are also included. These antibody fragments lack the Fc portion of the intact antibody, are cleared more rapidly from the circulation, and may have less nonspecific tissue binding than the intact antibody. These fragments can be prepared in a manner well known in the art, for example, using papain (to make Fab fragments) or
Produced by proteolytic cleavage with an enzyme such as pepsin (to produce F (ab ') 2 fragments).

[0054] Polyclonal antibodies are a heterogeneous population of antibody molecules obtained from the sera of animals immunized with the antigen. Monoclonal antibodies (mAbs) comprise a substantially homogeneous population of antibodies specific for the antigen, and such populations contain substantially similar epitope binding sites. mAbs can be obtained by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature, 256: 49.
5-497, 1975; U.S. Pat. No. 4,376,110; Ausube
L et al., Current Protocols in Molecular B
iology, Green Publishing Assoc. and Wi
ley Interscience, N.M. Y. 1987, 1992; Harl.
ow and Lane, Antibodies: A Laboratory Ma
Nual, Cold Spring Harbor Laboratory, 1
988; Ed. Colligan et al., Current Protocols in.
Immunology, Green Publishing Assoc. a
nd Wiley Interscience, N.W. Y. , 1992, 1993
See, etc .: The contents of these references are incorporated herein by reference in their entirety. Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA and any subclass thereof. Hybridomas producing the mAbs of the invention can be cultured in vitro, in situ or in vivo. In vivo or in situ production of high titers of mAbs is a currently preferred method of production.

A chimeric antibody including a humanized antibody is a molecule in which various portions are derived from different animal species, such as an antibody having a variable region derived from a murine mAb and a human immunoglobulin constant region. is there. Chimeric antibodies are used primarily, for example, to reduce immunogenicity upon administration and / or to increase production. Chimeric antibodies and methods for their production are known in the art (Cab
illy et al., Proc Natl Acad Sci USA, 81: 2733.
-3277, 1984; Morrison et al., Proc Natl Acad.
Sci USA, 81: 6851-6855, 1984; Boulianne et al., Nature, 312: 643-646, 1984; Cabilly et al., European Patent Application No. 125023 [published Nov. 14, 1984 (publishes
hed)]; Neuberger et al., Nature, 314: 268-270.
1985; Taniguchi et al., European Patent Application 171496 (19)
Morrison et al., European Patent Application No. 1739494.
Neuberger et al., PCT Publication No. 86/01533, published on March 13, 1986; K.
Udos et al., European Patent Application No. 184187, issued June 11, 1986; Sahagan et al., J Immunol. 137: 1066-1074, 1
986; Robinson et al., International Patent Application PCT / US86 / 02269 (1
Published May 7, 987); Liu et al., Proc Natl Acad Sci.
USA, 84: 3439-3443, 1987; Sun et al., Proc Natl Acad Sci USA, 84: 214-218, 1987;
Harlow and Lane, Antibodies: A Laboratory Manua, et al., Science, 240: 1041-1043, 1988.
1, Cold Spring Harbor Laboratory, 1988
. These references are incorporated herein by reference in their entirety.

Typically, the antibodies of the present invention are high affinity anti-SOCS-3 antibodies and fragments or regions thereof with potent in vivo inhibitory and / or neutralizing activity against SOCS-3. Such antibodies may include antibodies raised by immunization with purified recombinant SOCS-3 or a peptide fragment thereof.

Methods for measuring antibody specificity and affinity are described in Harlow et al., Antibod
ies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring
Harbor, N .; Y. 1988; Ed. Colligan et al., Current.
Protocols in Immunology, Greene Publi
shing Assoc. and Wiley Interscience, N
. Y. , 1992, 1993; and Muller, Meth. Enzymol
. 92: 589-601, 1983, which references are incorporated herein by reference in their entirety.

In addition, inhibitors / antagonists of SOCS-3 may function at the genetic level. Such antagonists include agents that reduce, inhibit, block, or impair the expression, production, or activity of SOCS-3. Such an agent can be an antisense nucleic acid or a sequence-specific peptide nucleic acid. Further, such antagonists can interfere with SOCS-3 promoter activity. Further, such antagonists can be variants of SOCS-3, such as variants that function as competitive inhibitors that can be introduced and expressed in cells that are trying to reduce SOCS-3 activity. The mutant expresses the mutant in a cell,
It can be a full-length derivative of SOCS-3 that inhibits the activity of endogenous SOCS-3 or a fragment or derivative of SOCS-3 described above. Such antagonists can be introduced into cells by transfection, eg, calcium phosphate precipitation or lipofection; or by transfection with a virus or pseudovirus containing the desired construct, or by electroporation. Methods for introducing nucleic acids into cells are well known in the art.

In another aspect, the invention involves introducing into a cell an expression construct for a nucleotide that encodes a modified form of SOCS-3. The modified SOCS-3 can include a dominant negative SOCS-3. Such molecules are known as SOC
It can competitively bind to a target molecule of S-3 without inactivating the target molecule (eg, dominant negative SOCS-3 binds to its target molecule, such as JAK2, and To remain phosphorylated, and / or
Or endogenous SOCS-3 binding so that JAK2 remains able to phosphorylate appropriate downstream molecules such as cytokine receptor molecules or STAT molecules).

Some vectors used in such constructs are well-known in the art. Furthermore, the mechanisms by which such constructs are delivered to an individual are well known in the art. For example, a recombinant expression vector comprising a synthetic or cDNA-derived DNA fragment encoding a modified SOCS-3 molecule can be engineered with appropriate transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. Contains DNA encoding a modified SOCS-3 protein operably linked. Such regulatory elements include a transcription promoter, any operator sequence that controls transcription, a sequence that encodes an appropriate mRNA ribosome binding site, and a sequence that controls termination of transcription and translation, as described in detail below. . Usually, a replication gene provided by the origin of replication and a selection gene which facilitates recognition of a transformant may be further incorporated. Operably linked is the DNA encoding the fusion protein.
Indicates that the components are linked in such a way that expression of is controlled by regulatory elements. Generally, operably linked means continuous.

[0062] Mammalian expression vectors contain non-transcribed elements, such as an origin of replication, an appropriate promoter and enhancer linked to the gene to be expressed, and other 5 'or 3' adjacent non-transcribed sequences. It may contain ribosome binding sites, polyadenylation sites, splice donor and splice acceptor sites, and 5 'or 3' (5 'to 3') untranslated sequences such as transcription termination sequences.

[0062] Transcriptional and translational control sequences in expression vectors that can be used when transforming vertebrate cells may be provided by viral sources. For example,
Widely used promoters and enhancers are derived from polyoma, adenovirus 2, simian virus 40 (SV40) and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, eg, SV40
Origins, early and late promoters, enhancers, splice sites and polyadenylation sites may be used to provide other genetic elements required to express heterologous DNA sequences. Both early and late promoters are particularly useful because both are readily obtained from the virus as a fragment that also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113, 1978). Virus origin of replication (vir
HindI located in al origin or replication)
Smaller or larger SV40 fragments may also be used provided that they include about 250 bp of sequence ranging from the II site to the BglII site. Illustrative vectors are described by Okayayama and Berg (Mol C
Cell Biol, 3: 280, 1983).

[0063] Preferred eukaryotic vectors for expression of mammalian DNA include pIXY3
21 and pIXY344, both of which include pBC102. K22 (ATC
C67, 255) and yeast derived vectors derived from yeast.

In a further aspect of the present invention, there is provided a method for increasing leptin-induced signaling that results in phosphorylation of a STAT molecule, thereby increasing the amount of gene transcript of a STAT responsive gene.

In another aspect of the invention, leptin activity is reduced or down-regulated through increasing the activity of the SOCS-3-mediated leptin cell signaling pathway, thereby gaining weight or amorphous depression It is desirable to be able to prevent / alleviate the symptoms. SOCS-3 activity can be increased by introducing a nucleic acid construct that expresses SOCS-3 or a bioactive fragment thereof into the cell. In this embodiment, the SOCS-3 protein, or bioactive fragment of SOCS-3, comprises a SOCS-3 protein or fragment having a bioactivity comparable to that of endogenous SOCS-3 that results in negative regulation of leptin activity. I do.

The present invention further provides methods for identifying molecules that modulate SOCS-3-mediated leptin cell signaling pathway. In particular, methods for identifying inhibitors / antagonists / agonists of SOCS-3 activity are encompassed by the present invention. Inhibitors of SOCS-3 activity can be identified and tested in in vitro assays and ex vivo cell-based assays as described herein. Candidates that exhibit the desired activity in vitro or ex vivo can be further evaluated in animal models valid in the art.

[0067] Candidate inhibitors, such as peptides, small organic molecules or derivatives of JAK2, are evaluated for their ability to specifically interact with SOCS-3 in standard binding or capture assays known in the art. be able to. For example, SOCS-
3 is contacted under physiological conditions with a peptide library, organic molecule library or JAK2 derivative that has been immobilized on a suitable surface (such as the wall or beads of a plastic microtiter plate) and subsequently labeled for detection. Can be. In another embodiment, a library of peptides or small organic molecules; antibodies or antibody fragments or target molecules or target molecule derivatives can be immobilized on a solid support and contacted with SOCS-3.

A directional peptide library (Z. Songyang et al., Cell, 72: 7).
67, 1993) can be screened for peptides that interact with SOCS-3. Peptide libraries and other small organic molecule libraries are also available in proximity assays or biospecific interaction assays (B
It can be screened using other assays known in the art, such as IA). Biospecific interaction analysis (BIA) can be performed in real time to evaluate candidate molecules for their ability to bind SOCS-3. BIA
Surface plasmon resonance (SPR), which is the basis for measurement, is an optical phenomenon that occurs in thin metal films under the condition of total internal reflection. This phenomenon causes a sharp decrease in reflected light intensity at a specific angle. The location of the resonance angle depends on several factors, including the refractive index of the medium near the non-irradiated side of the thin metal film. The refractive index is directly related to the concentration of the substance dissolved in the medium. By keeping other factors constant, SP
R is used to measure the change in polymer concentration in the surface layer of the solution in contact with the dextran-coated gold film. Pharmacia Biosensor (
The Biacore ^™ device of Pharmacia Biosensor AB can be used to measure the association and dissociation rate constants of peptides or organic molecules that bind to SOCS-3. Polypeptides exhibit greater association constant (K _a), a peptide, peptidomimetic or small organic molecules may interact with SOCS-3, have the greatest potency for the ability to inhibit SOCS-3 activity.

[0069] The present invention includes cell lines suitable for use in the screening methods described herein. In one embodiment, the cell line is a CHO cell, a Ba / F3 cell,
A mammalian cell line, such as HepG2 cells or H35 hepatoma cells,
Such cells stably express a reporter gene construct that is active in the absence of cytokine receptors and SOCS-3. The cell line is SOCS-3
The reporter gene construct is thereby inhibited by the expression of SOCS-3. In one embodiment, the cytokine receptor is a long form leptin receptor. In another embodiment, the reporter gene encodes luciferase. In another embodiment, the reporter gene encodes β-galactosidase. In a further aspect,
The reporter gene construct contains the SOCS-3 promoter element.

In a preferred embodiment, the cell line, cell signaling component (leptin receptor, JAK2, etc.), SOCS-3 is of human origin.

[0071] Candidate antagonists / agonists include reporter gene expression or SO
The ability of CS-3 expressing cells to allow cell proliferation can be assessed for their ability to inhibit / enhance SOCS-3 activity, and culturing the cells under conditions suitable for maintenance and growth The cell as a candidate molecule or S
Contacting with an organic molecule library containing an OCS-3 inhibitor, or transfecting cells with a cDNA expressing a candidate molecule or a cDNA expression library containing DNA encoding a candidate SOCS-3 inhibitor Contacting said cells with leptin; selecting cells with increased reporter gene activity and identifying organic molecules or cDNAs that have contacted the selected cells. Methods for measuring gene transcripts, and methods for enhancing or inhibiting them, are well known to those skilled in the art.

The present invention further includes cytokine-dependent cell lines that also stably express SOCS-3 and the leptin receptor long form. For example, the cytokine can be IL-3, IL-6, and other closely related cytokines. In one embodiment, the cytokine dependent cell line is a Ba / F3 cell. In another aspect, the cytokine is IL-3. The invention further comprises culturing said cytokine-dependent cell line in the presence of said cytokine under conditions suitable for maintenance and growth; the cells are cytokines (in the case of BA / F3, the cytokine is IL-3) ) And contacting the cells with a candidate organic molecule or with a library containing SOCS-3 inhibitor molecules, or by contacting the cells with a cDNA expressing a candidate SOCS-3 inhibitor. Or transfecting with a cDNA expression library containing DNA encoding a candidate SOCS-3 inhibitor; contacting the cells with leptin under conditions suitable for cell growth and maintenance; leptin A cell capable of growing in the presence of the organic molecule or the organic molecule in contact with the selected cell as described. Comprising the steps of identifying a DNA, it provides a method for the isolation and identification of SOCS-3 inhibitors. Methods for transfecting cells with a cDNA expression library and subsequently isolating the cDNA are well known in the art (Sambrook et al., Molecular Cloning).

[0073] Inhibitor / agonist candidates can be further evaluated in animal models. Animal models capable of assessing SOCS-3 activity are known in the art and are described, for example, in Leibel et al. Biol. Chem. 272: 31933
7-319340, 1997.

Inhibitors identified as described according to the present invention may be useful for treating obesity in a mammal or preventing weight gain in a mammal. Such molecules would also be useful for treating emotional depression, such as melancholic depression, inducing onset of puberty, or restoring reproductive dysfunction. On the other hand, some depression syndromes can be treated by reducing leptin activity. For example, a SOCS-3 agonist can be administered to an individual in need of such treatment.

The invention further encompasses a method of reducing food intake in a mammal, comprising the step of increasing leptin cell signaling comprising inhibiting SOCS-3 activity. In one embodiment, the mammal loses weight.

The invention further encompasses a method of inducing puberty in a mammal, comprising the step of increasing leptin cell signaling comprising inhibiting SOCS-3 activity.

The invention further encompasses a method of treating depression syndrome, comprising the step of inhibiting SOCS-3 activity.

The antagonist / agonist of the present invention comprises an effective amount of an inhibitor /
It can be formulated into a composition having an antagonist / agonist. An effective amount of an inhibitor / antagonist of SOCS-3 is partially or completely SOCS-3
An amount effective to inhibit activity and cause an increase in leptin activity. Effective amount of S
An OCS-3 agonist is an amount effective to enhance SOCS-3 activity and cause a decrease in leptin activity. Methods for assessing leptin activity, such as monitoring food intake, energy expenditure, weight gain / loss, reproductive function, neuroendocrine function, are well known to those skilled in the art. The actual effective amount of the inhibitor / antagonist / agonist in a particular case will vary according to, for example, the particular compound utilized, the particular composition formulated, the mode of administration, and the age, weight and condition of the mammal. Dosages for a particular mammal can be determined by one of ordinary skill in the art using conventional considerations (eg, by appropriate conventional pharmacological protocols).

[0079] Such compositions may also include a pharmaceutically acceptable carrier, and are referred to herein as pharmaceutical compositions. The compositions of the present invention can be administered intravenously, parenterally, orally, or by transdermal patch, inhalation, or suppository. The inhibitor / antagonist / agonist composition is administered in a single dose or in two or more doses over a period of time when the level of the inhibitor / antagonist / agonist is sufficient to achieve the desired effect. be able to.

Suitable pharmaceutical carriers include water; salt solutions; alcohols; polyethylene glycols; gelatin; carbohydrates such as lactose, amylose or starch; magnesium stearate; talc; silicic acid; Hydroxymethylcellulose; polyvinylpyrrolidone and the like, but are not limited thereto. The pharmaceutical compositions can be sterilized and, if desired, auxiliaries which do not deleteriously react with the active compound (for example, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, affecting osmotic pressure). Salts, buffers, colorants, and / or fragrance materials). They can also be combined, if desired, with other active agents (eg, enzyme inhibitors) to reduce metabolic degradation.

For parenteral application, particularly suitable are sterile injectable solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions or implants, including suppositories. Ampules are convenient unit dosages.

The inhibitors / antagonists / agonists of the invention are administered to an individual mammal in need of such treatment, together with one or more agents that allow the inhibitor to cross the blood-brain barrier. can do. Such inhibitors / antagonists / agonists and agents can be administered simultaneously or sequentially. Such agents are known in the art, for example, US Pat.
112,596; 5,268,164; 5,686.
416; and 5,506,206, the teachings of which are incorporated herein by reference in their entirety.

The following examples are provided for the purpose of illustrating the invention and should not be construed as limiting the scope of the invention.

Examples Example 1: CIS and SOCSm in ob / ob mice after leptin administration
Quantification of RNA by RT-PCR. 7-8 week old male ob / ob mice (Jackson L.) fed ad libitum
laboratories, Bar Harbor, ME) were injected intraperitoneally with 100 μg of recombinant mouse leptin (Eli Lily, Indianapolis, Ind.) or saline. Two hours later the mice were decapitated and the hypothalamus was isolated by inverting the skull from the brain and immediately freezing in liquid nitrogen. Cerebellar, kidney and liver samples were also taken. Total RNA from various tissues was purchased from the manufacturer (TEL
-TEST, Inc. , Friendswood, TX) using RNA-STAT-60 reagent. Purification of total RNA and subsequent cDNA synthesis was performed in parallel from all tissue samples. cDNA is St
1.0 μg of total RNA using the dT-oligonucleotide of Ratagene (La Jolla, Calif.) and the Advantage RT-PCR kit.
Synthesized from The final volume of the cDNA sample was 100 μl. The following primers were used for the specific PCR amplification of mouse CIS-1, mouse SOCS-1, mouse SOCS-2 and mouse SOCS-3: CIS-1A: 5'-ctgggagctgcccggggccagcc-3 ', 4,
00bp (GenBank accession number D31943), SEQ ID NO: 1; CIS-1B: 5'-caaggctgaccatctgggg-3 ', SEQ ID NO: 2; SOCS-1A: 5'-ccactccgattaccggggcgcatc-3'
, 350 bp (GenBank accession number U88325), SEQ ID NO: 3; SOCS-1B: 5′-gctcctgcagcggccgcacg-3 ′, SEQ ID NO: 4;
00bp (GenBank access number U588327), SEQ ID NO: 5; SO
CS-2B: 5'-tctttgtggtaaaaggcagtccc-3 ', SEQ ID NO: 6; SOCS-3A: 5'-accagcgccacttctcacg-3', 4
50 bp (GenBank accession number U88328), SEQ ID NO: 7; SOCS-3B: 5′-gtgggagcatcatactgatcc-3 ′, SEQ ID NO: 8. Each 50 μl PCR reaction was performed using 5.0 μl cDNA as a template. Assay conditions are: 10 mM Tris-HCl (pH 8.8), 5
0 mM KCl, 1.5 mM MgCl, 0.01% gelatin, 0.2 mM each dNTP, 20 pmol each primer, 2.5 units Taq polymerase (Stratagene), and 1.0 μl ³² P-dCTP ( 29.6T
Bq / mmol, 370 MBq / ml) (NEN, Boston, MA). After overlaying the mixture with 25 μl of mineral oil and performing an initial denaturation at 96 ° C. for 3 minutes, the samples were subjected to 24-32 cycles of amplification: denaturation at 95 ° C. for 1 minute, annealing at 60 ° C. for 1 minute. And extension at 72 ° C. for 45 seconds. Then 1
0 μl of the reaction solution was added to 5 μg of the sequencing stop solution (Amersham Int.
blended with E. international, Buckinghamshire, UK)
Heat at 5 ° C. for 5 minutes, then 5 μl of 4% urea-acrylamide gel (38 × 3
1 × 0.03 cm). The electrophoresis was performed at a constant power of 60 W for 4 hours, after which the gel was transferred to filter paper, dried and finally subjected to ³² P quantification by Phosphorimager analysis (Molecular Dynamics).

Preliminary PCR experiments show that the amplification rate is linear for CIS-1, SOCS-1 and SOCS-3 when performing less than 30 PCR cycles. The amplification of SOCS-2 was linear for 27 cycles, but was non-linear thereafter. We have developed CIS-1, SOCS
-1, 25 cycles of PC to quantify SOCS-2 and SOCS-3
R amplification was chosen. ³² P-dCTP was added to the PCR reaction, and the PCR reaction
PCR on DNA in parallel with a limited number of cycles under the above conditions
It was subjected to amplification. The PCR products were then separated on a denaturing acrylamide gel and finally subjected to autoradiography.

Seven to eight week old male ob / ob mice fed ad libitum were injected intraperitoneally (ip) with 100 μg of recombinant mouse leptin or saline. 2 hours later, all R
NA was purified from the hypothalamus and quantitative RT-PCR of CIS, SOCS-1, SOCS-2 and SOCS-3 mRNA was performed. Leptin treatment increased SOCS-3 mRNA 2.0-fold, but no effect on CIS, SOCS-1 or SOCS-2 mRNA levels was detected (FIGUR).
e 1A and 1B). A similar effect on SOCS-3 mRNA was observed 1 or 3 hours after leptin administration (data not shown). CIS, SOCS
The effect of leptin on mRNA of -1, SOCS-2 or SOCS-3 is:
Not detected in cerebellum, kidney or liver (data not shown). S in the hypothalamus
To determine whether the effect of leptin on OCS-3 mRNA is mediated by the leptin receptor long form, a similar experiment was performed.
Performed on db / db mice and control littermates. Leptin was added to control mice (+
/?) Increased SOCS-3 mRNA 2.2-fold in the hypothalamus, but no leptin effect was detected in db / db mice.

Example 2: In situ in rodent brain after leptin administration in vivo
u Localization of SOCS-3 mRNA by hybridization. To reveal specific anatomical regions in the hypothalamus and other parts of the brain where leptin affects SOCS-3 mRNA levels, antisense probes of ³⁵ S-labeled RNA were generated. A 450 bp fragment of the mouse SOCS-3 cDNA was amplified using the SOCS-3A and SOCS-3B primers. The PCR product was converted to pCR2.1 (Invi) according to the manufacturer's instructions.
trogen, Carlsbad, CA). Cloned c
DNA orientation was confirmed by performing sequencing using standard double-stranded plasmid technology. To generate ³⁵ S-labeled sense RNA, the plasmid was opened circularly by digestion with BamHI and the manufacturer's protocol (Promega).
) For in vitro transcription with T7 polymerase. Histochemistry by in situ hybridization was performed according to methods well known in the art (Simons). Mouse and rat brain tissue sections were fixed on slides, air dried and stored in a dry box at -20 ° C. Prior to hybridization, slides were soaked in 10% neutral buffered formalin to give 0.001%
Incubate in Proteinase K (Boehringer Mannheim) for 30 minutes, then in 0.025% acetic anhydride for 10 minutes,
Dehydration was performed while increasing the ethanol concentration. The RNA probe was then
Formamide, 10 mM Tris-HCl, pH 8.0, 5 mg tRNA,
10 mM dithiothreitol, 10% dextran sulfate, 0.3 M NaCl,
Diluted to 10 ⁶ cpm / ml with a hybridization solution of 1 mM EDTA, pH 8 and 1 × Denhardt's solution (Sigma). The hybridization solution and glass coverslip were placed on each slide, and then the sections were cut at 56 ° C. for 1 hour.
Incubated for 2-16 hours. Remove slides and slide 4xSSC
And washed 4 times. The sections were then cut with 0.5 M NaCl, 10 mM Tris-
0.002% RNAa with HCl (pH 8.0) and 1 mM EDTA
Incubated with seA (Boehringer Mannheim) at 37 ° C. for 30 minutes. Sections were washed at reduced concentration with SSC containing 0.25% DTT: 2x for 1 hour at 50 ° C, 0.2x for 1 hour at 55 ° C, and 0.2x for 6 hours.
0 ° C for 1 hour. Sections were then stepwise increased in ethanol concentration (50%, 70%, 80% and 90%) with ethanol containing 0.3 M NH ₄ OAc, and then dehydrated with 100% ethanol. Air dry slides and use BMR-2 film (
Kodak) in an X-ray film cassette for 3-5 days. The slides were then dipped in NTB2 photographic emulsifier (Kodak), dried, wrapped in foil and stored in a slide box with desiccant at 4 ° C for 2-3 weeks. Slides were developed with D-19 developer (Kodak), counterstained with thionine, dehydrated with graded concentrations of ethanol, removed with xylene, and covered with Permaslip. Sections were analyzed on a Zeiss Axioplan optical microscope using brightfield and darkfield optics. Photomicrographs were taken with a digital camera (Kodak, DCS) attached directly to the microscope and Apple Macintosh Po
It was produced while capturing the image with a wer PC computer. Using a photo editing software (Adobe Photoshop), the micrographs were matched to the plates and the figures were printed on a dye sublimation printer (Kodak 8600). Only sharpness, contrast and brightness were adjusted. Result 2
Shown in Strong specificity was observed in brain sections of normal rats fed ad libitum and given a single intravenous injection (1 μg / g body weight) of recombinant leptin as compared to brain sections of rats injected with saline. Hybridization was detected in the arcuate nucleus (Arc) and the dorsomedial hypothalamus nucleus (DMH) (FIGURE 2B, respectively).
And 2A). No specific hybridization signal was detected in other areas of the brain, including the cerebellum.

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 early gene induced upon leptin stimulation. e
rg-1-luc is a reporter construct that expresses the erg-1 promoter element fused to the luciferase gene. Transient transfection of CHO cells was performed with leptin reporter erg-1-luc alone or with C
Performed with IS, SOCS-2 or SOCS-3. Figure 3A
As shown in Figure 3, SOCS-3 blocked leptin-induced activation of the erg-1 luciferase reporter construct, but not CIS-1 or SOCS-2, while serum-induced erg-1 gene transcription was It was not affected by SOCS-3 expression.

Example 4: Localization of SOCS-3 mRNA by in situ hybridization in "Agouti" mice. Agouti (ie, lethal yellow, A ^y / a) mice are an autosomal dominant murine obesity model. Obesity in these mice is accompanied by increased linear growth and altered hair pigmentation. Like other non-ob or non-db mice obesity models, Agouti mice have elevated leptin levels and are resistant to leptin treatment by either intravenous injection or direct injection into brain tissue . The disease is due to ectopic, uncontrolled expression of agouti, a protein normally restricted to the hair follicle, and agouti in the hair follicle by antagonizing melanocyte stimulating hormone (MSH). Affects pigmentation. Agouti is MC4, whose expression is largely restricted in the brain.
It also antagonizes the receptor. SOCS-3 expression was localized to the brain tissue of Agouti mice according to the method described in Example 2. Specific hybridization was detected in the arcuate nucleus, but no specific hybridization signal was detected in other areas of the brain. This result supports the theory that SOCS-3 expression plays a role in the desensitization of these animals to leptin signaling and therefore is a key factor in loss of weight control in these animals. I have.

Example 5: Inhibition of leptin receptor signaling by SOCS-3 in a mammalian cell line. SOCS-3 was examined for its effect on leptin receptor signaling in mammalian cells. COS-1 cells were transformed with 10% fetal calf serum (FCS),
The cells were grown in Dulbecco's modified Eagle's medium (DMEM, low glucose) supplemented with 100 units / ml penicillin and 10 μg / ml streptomycin at 37 ° C. under 5% CO ₂ . CHO cells were treated with 10% FCS, 100 units /
HAM supplemented with ml penicillin and 10 μg / ml streptomycin
Propagated in F12 medium. In all experiments involving JAK cDNA, the amount of transfected JAK cDNA was 1/10 of the total amount of transfected DNA. For Western blotting experiments, cells were grown in 10 cm dishes and transfected with 80 μl lipofectamine and 20 μg plasmid DNA. Cells were hormone stimulated after serum starvation for 12-15 hours. Cells were harvested 48 hours after transfection. For Western blotting experiments, cells were washed with ice-cold phosphate buffered saline and 1000 μl of ice-cold lysis buffer B (1% Nonide
tP-40, 0.5% Triton X-100, 10% glycerol,
150 mM NaCl, 2 mM Na ₃ VO ₄ , 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 5 μg / ml leupeptin, 5 μ / ml
Aprotinin, 50 mM Tris-HCl, pH 7.4). The lysate was finally clarified by centrifugation at 23,000 g for 15 minutes and the supernatant was immunoprecipitated as described below. Immunoprecipitation was performed and the clarified cell extract was
5 antibody or OBR antibody and protein A-agarose beads [1% Non
idet P-40, 0.5% Triton X-100, 10% glycerol, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4)
0% slurry diluted 1:15] at 4 ° C. Agarose beads were pelleted by low speed centrifugation and washed three times with 1 ml of ice-cold lysis buffer B. For immunoblotting, proteins were boiled for 5 minutes and subjected to SDS-PAGE, after which the separated polypeptide was transferred to a nitrocellulose membrane. This film is
bin buffer [20 mM Tris-HCl (pH 7.4), 150 mM Na
Cl, 0.05% Tween 20], blocked for 2 hours at room temperature with 10% nonfat dry milk, and then with antibodies dissolved in 5% milk for 12 hours at 4 ° C.
Incubated for 5 hours. After removing unbound antibody by three washes of 20 minutes each in Towbin buffer, the membrane was washed with horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin (2.5% milk). 1: 1000) at room temperature for 1.5 hours, and
Washed 5 times with wbin buffer. The targeting protein was purchased from the manufacturer (Amersha).
m International, Buckinghamshire, UK) as described using enhanced chemiluminescence (ECL). The nitrocellulose membrane was peeled off with 1% SDS, 70 mM Tri-H while gently stirring.
The membrane was placed in Cl (pH 6.8) and 0.1% mercaptoethanol at 50 ° C. for 30 minutes.
Performed by soaking for minutes.

Phosphorylation of Leptin Receptor and Stat3 COS-1 cells were transformed with pcDNA3 or HA-labeled CIS-1, SOCS-2, SOCS using expression vectors for mouse OBR1 and JAK2.
Cotransfected with any of PCDNA3 (Invitrogen) containing -3.

The complete coding region of the CIS was replaced with EST (TIGR clone ID 10484).
4. Damien Dunnington, SmithKline Beech
am Pharmaceuticals) provided human skeletal muscle mRNA (C
(Rontech, Palo Alto, Calif.). The complete coding region of SOCS-2 was obtained by combining EST (IMAGE clone ID 131550, WashU-Merck EST project) and Marathon cDNA of human skeletal muscle (Clon).
tech, Palo Alto, CA). The complete coding region of SOCS-3 is Ba
Cloning was performed by PCR amplification from genomic DNA of lb / c mice (Sigma). A tandem hemagglutinin tag (HA) was fused to the C-terminus of all clones, followed by the mammalian expression vector pcDNA3 (Invitrogen,
Carlsbad, CA). All clones were confirmed by sequencing.

48 hours after transfection, including 15 hours of serum starvation, cells were harvested for 1 hour.
Treatment with lOOnM leptin for 10 minutes or no treatment. Western blotting of leptin receptor immunoprecipitates with anti-pY antibodies was performed using SOCS
-3 completely blocked leptin-induced tyrosine phosphorylation of the leptin receptor, whereas CIS-1 or SOCS-2 showed no effect (FIGURE 3B, upper panel). SOCS-3 was tested for its ability to block downstream signaling by measuring tyrosine phosphorylation of STAT3 in transfected COS-1 cells.
SOCS-3 completely blocked leptin-induced STAT3 phosphorylation, whereas CIS and SOCS-3 had no effect.

Phosphorylation of JAK2 CHO cells were transformed with the empty vector, CIS-HA, using OBR1 and JAK2.
, SOCS-2-HA or SOCS-3-HA. 48 hours after transfection, including 15 hours of serum starvation, cells were treated with 100 nM mouse leptin for 5 minutes or not. JAK2 immunoprecipitates (anti-JAK2 antibodies obtained from UBI) were purified by SDS-PA
It was subjected to GE, and Western blotting was performed using an anti-phosphotyrosine antibody (4G10, UBI) or an anti-JAK2 antibody (Santa Cruz).

SOCS-3 inhibited the phosphorylation of JAK2 on leptin tyrosine,
S or SOCS-2 did not inhibit. CIS-HA, SOCS-2-HA
Alternatively, SOCS-3-HA expression has no effect on JAK2 protein expression in these cells.

Example 6 Leptin-dependent co-immunoprecipitation of JAK2 with SOCS-3. COS-1 cells were isolated from OBR1 and JAK as described in Example 5.
2 was transfected with a SOCS-2-HA expression vector or a SOCS-3-HA expression vector. 48 hours after transfection, including 15 hours of serum starvation, cells were stimulated with lOnM leptin for 5 minutes or not. The HA immunoprecipitate (anti-HA antibody obtained from Babco) was subjected to SDS-PAGE, and Western blotting was performed using an anti-JAK2 antibody (Santa Cruz).

[0097] JAK2 co-immunoprecipitated with SOCS-3 but not with SOCS-2 in transfected COS-1 cells in a leptin-dependent manner.
These data are consistent with previously published results for SOCS-1 in transfected 293 cells (Endo et al., 1997). These results
Along with the results from above (FIGURE 3), SOCS-3 was used by Endo et al.
Consistent with the possibility of inhibiting leptin receptor signaling by interacting with JAK2 and thus inhibiting its tyrosine kinase activity, as proposed for SOCS-1 by 1997.

Example 7: Activation of SOCS-3 mRNA by leptin in CHO cells stably expressing the leptin receptor. CHO cells stably expressing either the long OBRl or the short form of the leptin receptor (OBRs) were grown and serum starved as described in Example 4. Total RNA was isolated from confluent cells grown on a 10 cm dish using the RNA-STAT method described in Example 1. Northern blotting was performed using standard techniques (Sambrook et al., 1).
989), and examined using a ³² P-labeled DNA probe (Gibco-BRL random labeling kit) containing the coding region of the mouse SOCS-3 gene.

Serum was expressed in SOC in both CHO-OBR and CHO-OBRs cells.
Induces S-3 mRNA. Leptin is one of the treatments in CHO-OBR1
After hours, SOCS-3 mRNA levels were induced, but not CHO-OBRs. At 2 and 4 hours after leptin treatment, SOCS-3 mRNA levels return to baseline in CHO-OBR1 cells. Therefore,
Leptin has the ability to activate endogenous SOCS-3 gene expression in cells expressing the leptin receptor long form, consistent with leptin, which directly activates SOCS-3 mRNA in OBR1-expressing neurons. CIS
And SOCS-3 mRNA was not induced by leptin in the two cell lines.

Example 8 Leptin Pretreatment of CHO-OBRl Causes Leptin Resistance in Proximal Leptin-Receptor Signaling CHO-OBR1 cells were tested for leptin resistance under conditions of increasing endogenous SOCS-3 protein levels. CHO-OBR1 cells were stimulated with leptin for 1 hour and then washed to remove leptin from the medium. At various times after leptin treatment, freshly added leptin was examined for its ability to induce intracellular signaling. As shown by Northern blotting
Leptin was unable to induce SOCS-3 mRNA up to 24 hours after leptin pretreatment. On the other hand, in leptin pretreated cells, fetal calf serum retains the ability to induce SOCS-3 mRNA, and leptin pretreatment of CHO-OBR1 cells results in leptin resistance signaling upstream of the socs-3 gene. Suggests that it is occurring at a stage.

Since it has been reported that induction of the socs gene by cytokines requires activation of STATs, STN induced by leptin in CHO-OBR1 cells.
The DNA binding activity of the AT can be determined using STA using methods well known in the art.
Measured using an electrophoretic mobility shift assay (EMSA) specific for T1 and STAT3. Leptin rapidly induced the DNA binding activity of STATs, with the highest levels detected about 5 minutes after leptin treatment. However, as shown by EMSA, leptin was unable to activate STAT until 24 hours after leptin pretreatment. Nevertheless, in cells treated with the same leptin pretreatment, TNF retains its full ability to activate STAT, and leptin pretreatment of CHO cells blocks leptin signaling upstream of STAT activation. It is suggested that it occurs at the stage of.

Since proximal leptin signaling involves the phosphorylation of tyrosine by the JAK kinase of the leptin receptor, leptin pretreatment of CHO-OBR1 cells requires
The ability to inhibit subsequent stimulation of leptin receptor phosphorylation was examined. 3
Pretreatment with lM or 100 nM leptin for 1 hour blocked the ability of the new leptin to induce receptor phosphorylation. Leptin binding of the tracer was not significantly affected by previous leptin treatment, as measured 1.5 to 24 hours after leptin pretreatment; thus, reduced levels of leptin receptor phosphorylation were But not for down regulation of the leptin receptor itself. In summary, these data are CHO-OB
Figure 4 shows that leptin pretreatment of Rl cells blocks proximal leptin signaling without affecting surface leptin receptor expression.

Equivalents While the invention has been particularly shown and described with reference to the preferred embodiments thereof, without departing from the spirit and scope of the invention, which is defined by the appended claims, It will be understood by those skilled in the art that various modifications in form and detail can be made to the present invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed by the following claims.

[Brief description of the drawings]

FIG. 1. FIG. 1A shows CIS-1 in the hypothalamus from ob / ob mice.
3 shows the results of a ³² P-RT-PCR assay showing the in vitro effects of leptin on mRNA levels of SOCS-1, SOCS-2 and SOCS-3.

FIG. 2 is a quantification of the data of FIG. 1A.

FIG. 3. FIG. 1C shows db / db mice and leptin treated leptin.
Of SOCS-mRNA from hypothalamus of lean and lean control (+ /?)^{Three Two} It is a plot of P-RT-PCR.

FIGS. 4A and 4B show the results of in situ hybridization using a ³⁵ S-labeled antisense SOCS-3 probe to brain sections from normal rats treated with saline or leptin.

FIG. 5 is a graph showing SOCE-3 activation of the leptin-induced erg-1 promoter in CHO cells.

FIG. 6 shows a Western blot showing SOCS-3 inhibition of leptin-induced tyrosine phosphorylation of leptin receptor in COS-1 cells.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 48/00 A61P 3/04 4C085 A61P 3/04 15/00 4C086 15/00 25/24 25/24 43 / 00 111 43/00 111 C12Q 1/02 C12N 5/10 G01N 33/15 Z 15/09 ZNA 33/50 Z C12Q 1/02 A61K 37/02 G01N 33/15 C12N 5/00 B 33/50 15 / 00 ZNAA (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, W), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC , LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZWF terms (reference) 2G045 AA25 BB20 CB01 CB17 DA12 DA13 DA14 DA36 DA54 FB02 FB03 FB07 4B024 AA01 AA11 BA01 BA63 BA80 CA04 CA12 DA02 FA02 FA10 GA11 GA18 HA17 4B063 QA05 QA18 QQ08 QQ13 QQ43 QQ61 QQ79 QQ96 QR48 QR51 QR69 QR77 QR80 QR82 QS 11 QS24 QS38 QX01 4B065 AA90X AA90Y AA91X AA93X AB01 AC14 BA02 BB19 CA24 CA44 CA46 4C084 AA02 AA13 AA17 BA44 MA01 NA14 ZA122 ZA702 ZA812 ZB222 4C085 AA13 AA14 EE01 GG01 A01A01 ZA02A12A01A16A12A16A12A

Claims (20)

[Claims] 1. A method of modulating leptin cell signaling activity, comprising the step of modulating SOCS-3 activity. 2. The method of claim 1, which inhibits SOCS-3 activity and results in increased leptin cell signaling activity. 3. A step of introducing a nucleotide construct containing a polynucleotide that inhibits the transcription of SOCS-3 DNA, or a nucleotide construct containing a polynucleotide encoding a SOCS-3 antisense mRNA, and Introducing a nucleotide construct into the cell where the sense SOCS-3 mRNA binds to endogenous SOCS-3 mRNA in the cell, thereby inhibiting the expression of the SOCS-3 protein, or comprising a modified SOCS-3 Claims 1. A nucleotide construct comprising a polynucleotide encoding a polypeptide, wherein the modified SOCS-3 polypeptide comprises an endogenous S
Introducing a nucleotide construct that is a competitive inhibitor of OCS-3 into the cell, thereby inhibiting SOCS-3 activity, or SOCS-
3. The method of claim 2, wherein the expression of SOCS-3 protein is inhibited, comprising the step of: introducing a 3 inhibitor into the cell. 4. The method according to claim 3, wherein the SOCS-3 target protein is JAK2. 5. The SOCS-3 inhibitor is selected from the group consisting of polypeptides, peptides, peptidomimetics, organic molecules, antibodies and antibody fragments, wherein the inhibitor is SOCS-3 or SOCS-3. Interacts with the SOCS-3 target protein, thereby inhibiting the interaction between the SOCS-3 and the SOCS-3 target protein, resulting in inhibition of SOCS-3 activity and increased leptin-induced cell signaling activity The method of claim 3. 6. The method of claim 1, which increases SOCS-3 activity and results in a decrease in leptin cell signaling activity. 7. The method of claim 6, which increases the expression of the SOCS-3 protein, resulting in increased SOCS-3 activity and decreased leptin cell signaling activity. 8. The method of claim 7, comprising introducing a nucleotide construct containing a polynucleotide encoding a SOCS-3 polypeptide or a modified SOCS-3 polypeptide into the cell. 9. An SOCS comprising a molecule selected from the group consisting of polypeptides, peptides, antibodies, antibody fragments, peptidomimetics, small organic molecules and nucleic acids.
-3 inhibitor. 10. A cell line expressing SOCS-3, a cytokine receptor and a reporter gene construct, wherein the reporter gene is transcribed by SOCS.
-3 A cell line that is inhibited by mRNA induction. 11. The cell line according to claim 10, wherein the reporter gene construct contains a SOCS-3 promoter element. 12. The cell line according to claim 10, wherein the cytokine receptor is a leptin receptor long form. 13. a) culturing the cell of claim 10 under conditions suitable for growth; b) contacting the cell of step a) with an organic molecule library containing a SOCS-3 inhibitor candidate. C) transfecting said cells with a cDNA expression library containing DNA encoding a SOCS-3 inhibitor candidate; c) contacting the cells of step b) with leptin; d) a reporter gene Identifying an inhibitor of SOCS-3 activity, comprising the steps of: selecting cells of step c) exhibiting increased activity; and e) identifying an organic molecule or cDNA contacted with the cells selected in step d). Method. 14. Stably leptin receptor long form and SOC
A cytokine-dependent cell line expressing S-3. 15. The cell line according to claim 14, wherein the cytokine is IL-3. 16. The method according to claim 15, wherein a) under conditions suitable for growth in the presence of IL-3.
Culturing the cells as described above; b) removing the cells of step a) from the presence of IL-3; c) combining the cells of step b) with an organic molecule library containing SOCS-3 inhibitor candidates Or transfecting the cells with a cDNA expression library containing the SOCS-3 inhibitor candidate; d) contacting the cells with leptin in step c); e) in the presence of leptin Selecting a cell capable of growing in d); and f) identifying an organic molecule or cDNA contacted with the cell selected in e). 17. An SOCS-3 inhibitor identified by the method according to claim 13 or 16. 18. Use of a leptin cell signaling enhancer for the manufacture of a medicament or therapeutic agent in an amount effective to cause weight loss or reduced food intake in a mammal. 19. Use of a SOCS-3 inhibitor for the manufacture of a medicament or therapeutic agent in an amount effective to cause a decrease in body weight or food intake in a mammal. 20. Use of a SOCS-3 inhibitor for the manufacture of a medicament or therapeutic agent in an amount effective to ameliorate a depression syndrome condition in a mammal.