WO1999023493A9 - Procedes d'identification d'agents modulant une activite de la leptine - Google Patents

Procedes d'identification d'agents modulant une activite de la leptine

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Publication number
WO1999023493A9
WO1999023493A9 PCT/US1998/022797 US9822797W WO9923493A9 WO 1999023493 A9 WO1999023493 A9 WO 1999023493A9 US 9822797 W US9822797 W US 9822797W WO 9923493 A9 WO9923493 A9 WO 9923493A9
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WIPO (PCT)
Prior art keywords
ptp
leptin
phosphorylation
phosphorylated
agent
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PCT/US1998/022797
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English (en)
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WO1999023493A1 (fr
Inventor
Cai Li
Jeffrey M Friedman
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Univ Rockefeller
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Application filed by Univ Rockefeller filed Critical Univ Rockefeller
Publication of WO1999023493A1 publication Critical patent/WO1999023493A1/fr
Publication of WO1999023493A9 publication Critical patent/WO1999023493A9/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Definitions

  • the present invention relates to methods for identifying agents that can modulate the effect of the signal transduction pathway initiated by the natural hormone, leptin. Such agents can be used to aid the modulation of mammalian body mass.
  • a new member of this signal transduction pathway has been identified.
  • This new member, PTP-ID has previously been identified as being a protein tyrosine phosphatase.
  • the present invention discloses that the modulation of the phosphorylation state of PTP-ID can have an important effect on the biological activity of leptin. Therefore, the present invention has important therapeutic implications in homeostasis of body weight and fat tissue mass.
  • Obesity defined as an excess of body fat relative to lean body mass, is associated with important psychological and medical morbidities, the latter including hypertension, elevated blood lipids, and Type II or non-insulin-dependent diabetes melitis
  • NIDDM Neurodeficiency dilutica dilutica dilutica dilutica dilutica dilutica dilutica dilutica dilutica dilutica dilutica dilutica dilutica dilutica dilutica dilutica dilutica dilutica dilutica dilutica diluticaally fibros, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma,
  • the assimilation, storage, and utilization of nutrient energy constitute a complex homeostatic system central to survival of metazoa.
  • storage in adipose tissue of large quantities of metabolic fuel as triglycerides is crucial for surviving periods of food deprivation.
  • the need to maintain a fixed level of energy stores without continual alterations in the size and shape of the organism requires the achievement of a balance between energy intake and expenditure.
  • the identification of the relevant factors in the complex homeostasis system and the identification of agents that can be administered to control these factors is therefore of great importance.
  • mice The most intensively studied mouse obesity mutations are the ob (obese) and db (diabetes) genes. Mice homozygous for either mutation are hyperphagic and hypometabolic, leading to an obese phenotype that is notable at one month of age. The weight of these animals tends to stabilize at 60-70 g (compared with 30-35 g in control mice).
  • Each of the rodent obesity models is accompanied by alterations in carbohydrate metabolism resembling those in Type II diabetes in man. In some cases, the severity of the diabetes depends in part on the background mouse strain [Leiter, Endocrinology, 124:912-922 (1989)].
  • congenic C57BL/Ks mice develop a severe diabetes with ultimate ⁇ cell necrosis and islet atrophy, resulting in a relative insulinopenia.
  • congenic C57BL/6J ob and db mice develop a transient insulin-resistant diabetes that is eventually compensated by ⁇ cell hypertrophy resembling human Type II diabetes.
  • mice resembles human obesity in ways other than the development of diabetes - the mutant mice eat more and expend less energy than do lean controls (as do obese humans). This phenotype is also quite similar to that seen in animals with lesions of the ventromedial hypothalamus, which suggests that both mutations may interfere with the ability to properly integrate or respond to nutritional information within the central nervous system.
  • the mouse obesity (ob) gene encodes an adipose tissue- derived signaling factor for body weight homeostasis [Zhang et al, Nature, 372:425 (1994); U.S. Patent Application No. 08/292,345 filed August 1, 1994; U.S. Patent Application No. 08/483,211, filed June 7, 1995, each of which is hereby incorporated by reference in its entirety].
  • Escherichia coli can correct the obesity related phenotypes in ob/ob mice when exogenously administered [Campfield et al, Science, 269:546 (1995); Pellymounter et al, Science, 269:540 (1995); Halaas et al, Science, 269:543 (1995); Stephens et al, Nature, 377:530 (1995)]. Weight-reducing effects of the recombinant leptin were also observed in normal mice and mice with diet-induced obesity. Another advance occurred with the determination that the db gene encoded the leptin receptor [U.S. Patent Application No. 08/586,594 filed January 16, 1996; U.S. Patent Application No.
  • Leptin is a 16kD hormone that is the afferent signal in a negative feedback loop regulating food intake and body weight.
  • the leptin receptor is a member of the cytokine family. Leptin' s anorexigenic effect is dependent on binding to the Ob-Rb isoform of this receptor which encodes a long intracytoplasmic domain that includes several motifs for protein-protein interaction. Ob-Rb is highly expressed in the hypothalamus suggesting that this brain region is an important site of leptin action. Signal transduction by this class of receptor generally depends on ligand induced phosphorylation of soluble tyrosine receptor kinases such as JAK1 , 2, 3, and tyk2.
  • kinases in turn phosphorylate tyrosine residues on the receptor which serve as docking sites for SH2 proteins. Phosphorylation of SH2 proteins after receptor binding initiates signal transduction. Leptin binds to a homodimer of the Ob-Rb isoform of its receptor thus activating JAK2. While the Stat3 transcription is activated by leptin in vivo, the identity of other components of this signal transduction pathway have not yet been identified.
  • the present invention provides a method of identifying a modulator of the binding of a phosphorylated leptin receptor with PTP-ID.
  • One such embodiment comprises contacting the phosphorylated leptin receptor or phosphorylated fragment thereof with PTP-ID or fragment thereof in the presence and absence of an agent under conditions in which in the absence of the agent, the binding of the phosphorylated leptin receptor or phosphorylated fragment with PTP- 1 D or fragment thereof can be detected. The binding of the phosphorylated leptin receptor or phosphorylated fragment thereof with PTP-ID or fragment thereof is then detected.
  • the agent When an increase in binding is detected in the presence of the agent, the agent is identified as a modulator that enhances the binding of the phosphorylated leptin receptor or phosphorylated fragment thereof with PTP-ID or fragment thereof.
  • the agent when a decrease in binding is detected in the presence of the agent, the agent is identified as a modulator that inhibits the binding of the phosphorylated leptin receptor or phosphorylated fragment thereof with PTP-ID or fragment thereof.
  • the phosphorylated leptin receptor or phosphorylated fragment thereof contains a phosphorylated tyrosine-985, i.e., the leptin receptor or the fragment thereof contains a tyrosine which is analogous to tyrosine 985 of SEQ ID NO:2 which is phosphorylated and therefore is a binding partner of PTP-ID.
  • the phosphorylated leptin receptor or phosphorylated fragment thereof is bound to a solid support.
  • PTP-ID or fragment thereof is labeled.
  • the phosphorylated leptin receptor or phosphorylated fragment thereof is bound to a solid support and PTP-ID or fragment thereof is labeled.
  • the present invention also includes analogous embodiments in which the phosphorylated leptin receptor or phosphorylated fragment thereof is labeled; and/or the PTP-ID or fragment thereof is bound to a solid support.
  • the phosphorylated fragment can also be part of a fusion (or chimeric) protein.
  • the fusion protein comprises a phosphorylated fragment of the leptin receptor fused to a glutathione-S-transferase. In another preferred embodiment of this type the fusion protein comprises a phosphorylated fragment of the leptin receptor fused to green fluorescent protein.
  • Another aspect of the present invention includes methods of identifying a modulator for the phosphorylated leptin receptor-dependent PTP-ID phosphorylation.
  • One such embodiment comprises contacting a phosphorylated leptin receptor or phosphorylated fragment thereof with PTP-ID and JAK2 kinase in the presence and absence of an agent under conditions in which in the absence of the agent the phosphorylated leptin receptor or phosphorylated fragment thereof can stimulate the phosphorylation of PTP-ID.
  • the phosphorylated leptin receptor or phosphorylated fragment thereof contains a phosphorylated tyrosine-985.
  • the amount of phosphorylation of PTP-ID is determined.
  • the agent When either an increase or decrease in the phosphorylation is determined in the presence of the agent, relative to in its absence, the agent is identified as a potential modulator of the leptin receptor-dependent phosphorylation of PTP-ID.
  • This embodiment can include the subsequent step of contacting of the potential modulator with PTP-ID under the initial conditions of contacting (i.e., the first step) except a phosphorylated leptin receptor or phosphorylated fragment thereof containing the phosphorylated tyrosine-985 is not included.
  • the potential modulator When no significant change in phosphorylation is determined in the presence of the potential modulator relative to in its absence, the potential modulator is identified as a modulator of the leptin-dependent phosphorylation of PTP-ID.
  • the phosphorylated fragment is part of a fusion protein.
  • the fusion protein comprises the phosphorylated fragment fused to a glutathione-S-transferase. In another embodiment the fusion protein comprises the phosphorylated fragment fused to green fluorescent protein.
  • Another aspect of the present invention includes methods of identifying a potential modulator and/or modulator of the leptin-dependent PTP-ID phosphorylation in situ.
  • One such embodiment comprises contacting a cell with leptin in the presence and absence of an agent under conditions in which in the absence of the agent leptin induces the phosphorylation of PTP-ID.
  • the cell comprises PTP-ID, JAK2, and a leptin receptor which contains tyrosine-985.
  • the amount of phosphorylation of PTP-ID is then determined.
  • the agent is identified as a potential modulator of the leptin-dependent phosphorylation of PTP-ID.
  • the cell is transfected with a vector which encodes either PTP-ID, JAK2, and a leptin receptor which contains tyrosine-985.
  • the vector encodes two or more of the proteins.
  • nucleic acids encoding two or more the proteins are transfected into the cell in two or more vectors individually encoding a protein.
  • the cell is transfected with a vector encoding PTP-ID, a vector encoding JAK2, and a vector encoding a leptin receptor which contains tyrosine-985.
  • the method further comprises contacting a second cell with leptin and the potential modulator under the conditions of those of the initial step except in this case leptin cannot induce the phosphorylation of PTP-ID.
  • the second cell comprises PTP-ID, JAK2, and a leptin receptor that does not contain tyrosine- 985.
  • the amount of phosphorylation of PTP-ID is then determined. When no significant change in phosphorylation is determined in the presence of the potential modulator relative to in its absence the potential modulator is identified as modulator of the leptin-dependent phosphorylation of PTP-ID.
  • the leptin receptor that does not contain tyrosine-985 is the Ob-Ra form of the receptor. In another particular embodiment the leptin receptor that does not contain tyrosine-985 is the Ob-Rb containing a phenylalanine-985 as exemplified below.
  • the cell is transfected with a vector which encodes either PTP-ID, JAK2, and a leptin receptor which does not contains tyrosine-985.
  • the vector encodes two or more of the proteins.
  • nucleic acids encoding two or more the proteins are transfected into the cell in two or more vectors individually encoding a protein.
  • the cell is transfected with a vector encoding PTP-ID, a vector encoding JAK2, and a vector encoding a leptin receptor which does not contain tyrosine-985.
  • the modulator enhances the leptin receptor-dependent phosphorylation of PTP-ID. In another particular embodiment the modulator inhibits the leptin receptor-dependent phosphorylation of PTP-ID.
  • the present invention further provides a method of identifying a drug useful in a weight loss diet regimen.
  • One such embodiment comprises administering one or more doses, (preferably multiple doses) to a test animal of an agent found to be an inhibitory modulator of the leptin-dependent phosphorylation of PTP-ID and a identical number of doses of a placebo to a control animal over a prescribed time period. The change in weight of the test animal and the control animal during the prescribed time period is then determined. An inhibitory modulator that causes the test animal to lose weight relative to the control animal is selected as a drug that is useful in a weight loss diet regimen.
  • the present invention further provides a method of identifying a drug useful in a weight gain diet regimen.
  • One such embodiment comprises administering one or more doses, (preferably multiple doses) to a test animal of an agent found to be an enhancing modulator of the leptin-dependent phosphorylation of PTP-ID and a identical number of doses of a placebo to a control animal over a prescribed time period. The change in weight of the test animal and the control animal during the prescribed time period is then determined.
  • An enhancing modulator that causes the test animal to gain weight relative to the control animal is selected as a drug that is useful in a weight gain diet regimen.
  • the prescribed time period for laboratory animals being the test animal is preferably one week to two years, more preferably one month to one year, and even more preferably two months to six months.
  • Still another variation of the present invention includes a method of identifying a modulator of the leptin-dependent PTP-ID dephosphorylation of the JAK2 kinase in situ.
  • One such embodiment comprises contacting a cell with leptin in the presence and absence of an agent under conditions in which in the absence of the agent, leptin induces the PTP-ID dephosphorylation of JAK2.
  • the cell comprises PTP-ID, JAK2, and a leptin receptor which contains tyrosine-985.
  • the amount of phosphorylation of JAK2 is determined.
  • the agent When a decrease or increase in phosphorylation of JAK2 is determined in the presence of the agent relative to in its absence, the agent is identified as a potential modulator of the leptin-dependent PTP-ID dephosphorylation of JAK2. Then the cell is contacted with the potential modulator under the conditions of the initial step except in the absence of leptin. When no significant change in phosphorylation is determined in the presence of the potential modulator relative to in the absence of the potential modulator, the potential modulator is identified as a modulator of the leptin-dependent PTP-ID dephosphorylation of JAK2.
  • the cell is transfected with a vector which encodes either PTP-ID, JAK2, and a leptin receptor which contains tyrosine-985.
  • the vector encodes two or more of the proteins.
  • nucleic acids encoding two or more the proteins are transfected into the cell in two or more vectors individually encoding a protein.
  • the cell is transfected with a vector encoding PTP-ID, a vector encoding JAK2, and a vector encoding a leptin receptor which contains tyrosine-985.
  • the present invention further includes a method of identifying an inhibitor of the leptin-dependent PTP-ID phosphorylation in situ.
  • One such embodiment comprises (a)contacting a cell with leptin in the presence and absence of an agent under conditions in which in the absence of the agent, leptin induces the expression of a reporter gene operably under the control of a promoter containing a binding site for activated Stat3. (b) The level of expression of the reporter gene contained by a host cell is determined.
  • the binding of activated Stat3 protein to the binding site induces the expression of the reporter gene in the cell comprises PTP-ID,
  • step (c) a second cell is contacted with leptin in the presence and absence of the candidate inhibitor under conditions in which in the absence of the candidate inhibitor leptin induces the phosphorylation of PTP- ID and wherein the second cell comprises PTP-ID, JAK2, and a leptin receptor which contains tyrosine-985.
  • step (c) The amount of phosphorylation of PTP-ID of step (c) is then determined.
  • the candidate inhibitor When a decrease in phosphorylation of PTP-ID is determined in the presence of the candidate inhibitor relative to in the absence of the candidate inhibitor, the candidate inhibitor is identified as a potential inhibitor of the leptin-dependent phosphorylation of PTP-ID.
  • a third cell is then contacted with leptin and the inhibitor under the conditions of those of step (c) except in this case leptin cannot induce the phosphorylation of PTP-ID.
  • the third cell comprises PTP-ID, JAK2, and a leptin receptor that does not contain tyrosine-985.
  • the amount of phosphorylation of PTP-ID of step (e) then determined.
  • the potential inhibitor When no decrease in phosphorylation is determined in the presence of the potential inhibitor relative to in its absence, the potential inhibitor is identified as an inhibitor of leptin-dependent phosphorylation of PTP-ID.
  • the various proteins used in this assay can be native to the cell or preferably added by transfection of the cell with vectors encoding these proteins as described above.
  • the reporter gene encodes luciferase.
  • the reporter gene encodes green fluorescent protein.
  • One aspect of this method further includes identifying a drug useful in a weight loss diet regimen.
  • One such embodiment comprises administering one or more doses, (preferably multiple doses) to a test animal of an agent found to be an inhibitory modulator of the leptin-dependent phosphorylation of PTP-ID and a identical number of doses of a placebo to a control animal over a prescribed time period. The change in weight of the test animal and the control animal during the prescribed time period is then determined.
  • An inhibitory modulator that causes the test animal to lose weight relative to the control animal is selected as a drug that is useful in a weight loss diet regimen.
  • the present invention further provides a method of identifying a drug useful in a weight gain diet regimen.
  • One such embodiment comprises administering one or more doses, (preferably multiple doses) to a test animal of an agent found to be an enhancing modulator of the leptin-dependent phosphorylation of PTP-ID and a identical number of doses of a placebo to a control animal over a prescribed time period. The change in weight of the test animal and the control animal during the prescribed time period is then determined. An enhancing modulator that causes the test animal to gain weight relative to the control animal is selected as a drug that is useful in a weight gain diet regimen.
  • the prescribed time period for laboratory animals being the test animal is preferably one week to two years, more preferably one month to one year, and even more preferably two months to six months.
  • Figure 1 Binding of a 64kD protein to phosphotyrosine-985 of the Ob-Rb cytoplasmic region.
  • Figure 1A is a schematic representation of the full length leptin receptor (Ob-Rb) and shows the three segments used to make the three Glutathione-S- Transferase (GST) fusion fragments.
  • the three GST-fragments are ObRbTyrl, ObRbTyr2 and ObRbTyr3 which are named for the reversibly phosphorylatable tyrosines of this region: Tyrosine-985, Tyrosine- 1077, and Tyrosine-1138 respectively.
  • Figure IB shows the GST fusion fragments of the peptides spanning each of the three cytoplasmic tyrosines (Y 985, 1077, 1138) which were expressed in bacteria with or without co-expression of the elk tyrosine kinase. Co-expression of elk tyrosine kinase led to the specific phosphorylation of these three tyrosine residues.
  • the GST-Ob-Rb fragment 1 peptide (containing Y985) was incubated with protein extracts from bovine or mouse hypothalamus. After precipitation with anti-GST antibodies, the bound proteins were eluted, resolved on SDS PAGE, and stained with Coomassie Blue.
  • a 64kD protein was found in the precipitates of mouse and bovine hypothalamus.
  • the 64kD protein was not precipitated by the anti-GST antibody when tyrosine 985 was not phosphorylated.
  • the 64kD protein was sequenced and found to be identical to Phosphostyrosine Phosphotase ID.
  • Figure 2 Immunoblots of the protein precipitate of GST-ObRb fragments.
  • Figure 2 A depicts an immunoblot using anti-PTP-lD antibody.
  • Figure 2B depicts an immunoblot using anti-Stat3 antibody.
  • the phosphorylated and unphosphorylated GST-ObRb fusion fragments 1 and 3 were incubated with protein extracts of mouse hypothalamus.
  • PTP-ID was detected only in the material precipitated by the phosphorylated from of GST-ObRbTyrl.
  • Stat3 was detected only in the proteins precipitated by the phosphorylated form of GST-ObRbTyr3.
  • FIG. 3 Binding of Leptin to Ob-Rb leads to the Inducible Phosphorylation of PTP- ID.
  • 293 cells were transfected using combinations of Ob-R, JAK2 and PTP-ID as indicated.
  • the state of PTP-ID phosphorylation was assayed with and without leptin treatment.
  • PTP-ID was first assayed by immunoprecipitation using an anti PTP-ID antibody followed by immunobloting using an anti-phosphotyrosine antibody. In separate studies the total cell lysate was immunobloted with the anti-phosphotyrosine antibody.
  • Treatment of cells with leptin transfected with Ob-Rb, JAK2 and PTP-ID led to an increase PTP-ID phosphorylation.
  • FIG. 4 PTP-ID Phosphorylation by Ob-Rb is Dependent on Tyr 985. 293 cells were transfected with wild type Ob-Rb or a Y — >F 985 (i.e., tyrosine to the phenylalanine substitution) mutant leptin receptor together with JAK2 and PTP-ID. The level of PTP-ID phosphorylation was scored at various times after leptin treatment. Leptin increased the level of PTP-ID phosphorylation only in the cells that received the wild type leptin receptor. PTP-ID was not phosphorylated in cells transfected with the Y — >F 985 mutant.
  • FIG. 5 Phosphorylated PTP-ID Dephosphorylates JAK2.
  • the level of phosphorylation of JAK2 was assayed after leptin treatment in cells transfected with the wild type and Y — >F 985 mutant. After leptin treatment, the level of JAK2 phosphorylation was five-fold greater in cells that received the wild type leptin receptor. When PTP-ID was cotransfected, the level of JAK2 phosphorylation was ten-fold greater in the cells that were transfected with the Y — >F 985 mutant leptin receptor.
  • the present invention provides novel assays for agents which can potentially serve as drugs in the modulation of mammalian body mass.
  • the present invention is based in part on the discovery that Protein Tyrosine Phosphatase ID, PTP-ID, described in
  • PTP-ID has important implications for the treatment of nutritional disorders including, but not limited to, obesity, weight loss associated with cancer and AIDS and the treatment of diseases associated with obesity such as hypertension, heart disease, and Type II diabetes.
  • diseases associated with obesity such as hypertension, heart disease, and Type II diabetes.
  • agents identified by the methods described herein in cases where one might wish to modulate the body weight of domestic animals.
  • physiological obesity and “physiologically obese” refer to excessive adipose tissue that is due at least in part to abnormalities in the endogenous leptin pathway, including abnormalities in the effective signaling initiated by the binding of leptin to the leptin receptor.
  • Abnormalities in the endogenous leptin pathway may be manifested in a number of ways including an abnormal food intake, an abnormal activity level, or an abnormal body temperature.
  • the present invention allows drugs to be identified which can modulate body mass completely independently of any inherent abnormality in the endogenous leptin pathway per se by augmenting or diminishing the natural effect of leptin.
  • Leptin encompasses biologically active variants of naturally occurring leptin, as well as biologically active fragments of naturally occurring leptin and variants thereof, and combinations of the preceding.
  • Leptin is the polypeptide product of the ob gene as described in the International Patent Publication No. WO 96/05309, and the US Patent Application No. 08/483,211 to which it claims priority, each of which is incorporated herein by reference in its entirety.
  • Putative analogs and fragments of leptin are reported in US Patent 5,521,283, US Patent 5,532,336 and International Patent Publication No. PCT 96/22308 for International Application No. PCT/US96/ 01471, each of which is incorporated herein by reference in its entirety.
  • bound or “binds” or “associates” or “associated” are meant to include all such specific interactions that result in two or more molecules showing a preference for one another relative to some third molecule. This includes processes such as covalent, ionic, hydrophobic and hydrogen bonding but does not include nonspecific associations such solvent preferences.
  • condition related to abnormalities of the endogenous leptin pathway encompasses conditions and diseases due, at least in part, to abnormalities involving leptin as detailed above.
  • the term "medically assisting" is used herein as a manner of attending to the health care needs of a subject who has a particular problem (e.g., an abnormality in the endogenous leptin pathway) which encompasses either diagnosing or treating that problem, and all combinations thereof.
  • the invention provides for medically assisting a mammalian subject suffering from an abnormality in the endogenous leptin pathway resulting in decreased leptin activity.
  • a mammalian subject may be suffering from an abnormality resulting in increased leptin activity.
  • the decreased or increased leptin activity may be manifested as a pathological state, such as obesity (decreased leptin activity) or anorexia (increased leptin activity).
  • detectable is used broadly herein to include a factor that directly emits the detectable signal (such as a fluorescent molecule) an entity bound directly to that factor (such as an antibody carrying a fluorescent molecule) as well as a detectable target of the factor or the entity (such as an epitope of an antigen that reacts with the antibody carrying a fluorescent molecule).
  • a target may be termed detectable due to either its potential or realized association with either a factor that functions as a detectable label or a factor that is bound to a detectable label.
  • Detectable labels include but not limited to an enzyme, a radioactive element, a biochemiluminescent, a chromophore that absorbs in the ultraviolet and/or visible and/or infrared region of the electromagnetic spectrum; and a fluorophore.
  • a "quantitative relationship" between two or more determinations of one or more substances includes the relative absolute amounts, a relative percentage, a relative ratio, the difference, sum, multiple and/or quotient for two or more determinations; and can further include appropriate first or higher order equations that express the relationship between two or more determinations in a manner that can be understood by a person skilled in the art to which the present invention pertains.
  • a “determination” or “determining” as used herein is the result of an assay that includes an attempt to detect and/or the quantifying of the substance detected such as that expressed in a quantity or an amount of that substance.
  • mono-specific antibody describes an antibody to a protein raised against a particular peptide sequence in that protein and is meant to imply that the antibody is specific for an epitope that includes that particular sequence.
  • a monospecific antibody may be either polyclonal or monoclonal.
  • a phosphorylated leptin receptor or phosphorylated fragment thereof contains a phosphorylated "tyrosine-985" or "Y985" when the receptor or fragment thereof contains the amino acid sequence of Amino Acid 983 to Amino Acid 990 of SEQ ID NO:2 and tyrosine-985 is phosphorylated.
  • the receptor or fragment thereof contains the amino acid sequence of Amino Acid 971 to Amino Acid 1000 of SEQ ID NO:2 and tyrosine-985 is phosphorylated.
  • the receptor or fragment thereof contains the amino acid sequence of Amino Acid 961 to Amino Acid 1010 of SEQ ID NO:2 and tyrosine-985 is phosphorylated.
  • a leptin receptor can contain a dephosphorylated tyrosine- 985 (i.e., a tyrosine that is not phosphorylated at position 985 of SEQ ID NO:2) or a phenylalanine at position 985 of SEQ ID NO:2 in place of the tyrosine.
  • the receptor containing such a substitution is referred to "F985" or the "F985 mutant receptor.”
  • the OB receptor contains three important structural domains: an extracellular (or extracytoplasmic) domain, a transmembrane domain, and a cytoplasmic domain.
  • the extracellular domain binds leptin.
  • the transmembrane domain comprises a stretch of highly non-polar amino acid residues that localize to the hydrophobic region of the cell membrane.
  • the term transmembrane domain has its ordinary meaning in molecular and cellular biology.
  • the cytoplasmic domain of an OB receptor of the invention may contain none, one, or two JAK-binding concensus sequences, termed "Box 1" and "Box 2". A receptor having "Box 1" and "Box 2" is believed competent for signal transduction via the JAK-Stat pathway upon binding of leptin.
  • the OB-R polypeptide from one species is closely related (homologous) to the OB-R in another species.
  • the human OB-R polypeptide is highly homologous to murine OB-R polypeptide. This observation is consistent with the data showing that human leptin is active in mice: for the hormone to be active interspecies, one would expect a high degree of similarity or homology between the receptors from different species as well.
  • the present invention is directed to the identification of agents that function as modulators of mammalian body weight.
  • substantially similar means at least 50% sequence similarity, preferably at least 60% sequence similarity, more preferably at least 70% sequence similarity, even more preferably at least 80% sequence similarity, and most preferably at least 90% sequence similarity.
  • gene refers to a nucleic acid, such as DNA, which codes on expression for a protein. Unless stated otherwise, gene may include mRNA, cDNA, or genomic DNA.
  • a composition comprising "A” (where "A” is a single protein, DNA molecule, vector, recombinant host cell, etc.) is substantially free of “B” (where “B” comprises one or more contaminating proteins, DNA molecules, vectors, etc., but excluding racemic forms of A) when at least about 75% by weight of the proteins, DNA, vectors (depending on the category of species to which A and B belong) in the composition is "A".
  • "A” comprises at least about 90% by weight of the A+B species in the composition, most preferably at least about 99% by weight. It is also preferred that a composition, which is substantially free of contamination, contain only a single molecular weight species having the activity or characteristic of the species of interest.
  • Contemplated by the present invention are analogs comprising conservative amino acid substitutions.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • one polar amino acid may be substituted with another to preserve local hydrophilicity; more likely, a substitution that conserves charge, or at least does not introduce the opposite charge, is required. Such alterations will not be expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis, or isoelectric point. Particularly preferred substitutions are:
  • Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property.
  • a Cys may be introduced a potential site for disulfide bridges with another Cys.
  • a His may be introduced as a particularly "catalytic" site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis).
  • Pro may be introduced because of its particularly planar structure, which induces ⁇ -turns in the protein's structure.
  • homologous in all its grammatical forms refers to the relationship between proteins that possess a "common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al., 1987, Cell
  • Such proteins have sequence homology as reflected by their high degree of sequence similarity.
  • sequence similarity in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., supra).
  • sequence similarity when modified with an adverb such as “highly,” may refer to sequence similarity and not a common evolutionary origin.
  • corresponding to is used herein to refer similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured.
  • corresponding to refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.
  • a gene encoding the leptin receptor, leptin, PTP-ID or JAK2, whether genomic DNA or cDNA, can be isolated from any animal source, particularly from a mammal. Methods for obtaining the Stat protein gene are well known in the art, as described above (see, e.g., Sambrook et al, 1989, supra).
  • heterologous nucleotide sequence is a nucleotide sequence that is added to a nucleotide sequence of the present invention by recombinant methods to form a nucleic acid which is not naturally formed in nature.
  • Such nucleic acids can encode chimeric and/or fusion proteins.
  • the heterologous nucleotide sequence can encode peptides and/or proteins which contain regulatory and/or structural properties.
  • the heterologous nucleotide can encode a protein or peptide that functions as a means of detecting the protein or peptide encoded by the nucleotide sequence of the present invention after the recombinant nucleic acid is expressed.
  • heterologous nucleotide can function as a means of detecting a nucleotide sequence of the present invention.
  • a heterologous nucleotide sequence can comprise non-coding sequences including restriction sites, regulatory sites, promoters and the like.
  • Nucleic Acids in accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); Glover ed., DNA Cloning: A Practical Approach, Volumes I and II, MRL Press, Ltd., Oxford, U.K.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control.
  • a "vector” is a replicon, such as a plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a "cassette” refers to a segment of DNA that can be inserted into a vector at specific restriction sites.
  • the segment of DNA encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.
  • a cell has been "transfected" by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”) in either single-stranded form, or a double- stranded helix. Double-stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary or quaternary forms.
  • this term includes double- stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al, 1989, supra). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a T m of 55 °C, can be used, e.g. ,
  • Moderate stringency hybridization conditions correspond to a higher T m , e.g., 40% formamide, with 5x or 6x SCC.
  • High stringency hybridization conditions correspond to the highest T m , e.g., 50% formamide, 5x or 6x SCC.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art.
  • T m The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
  • the relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • equations for calculating T m have been derived (see Sambrook et al, 1989, supra, 9.50-0.51).
  • a minimum length for a hybridizable nucleic acid is at least about 18 nucleotides; more preferably at least about 27 nucleotides; most preferably the length is at least about 36 nucleotides.
  • standard hybridization conditions refers to a Tm of 55°C, using conditions as set forth above.
  • the Tm is 60°C, in a more preferred embodiment, the Tm is 60°C.
  • Homologous recombination refers to the insertion of a foreign DNA sequence of a vector in a chromosome.
  • the vector targets a specific chromosomal site for homologous recombination.
  • the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3 ' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3 ' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • polyadenylation signals are control sequences.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.
  • a DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • the term "operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted upstream (5 ') of and in reading frame with the gene.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • leptin receptor, PTP-ID, or the various proteins used in the fusion fragments described herein can be produced recombinantly or by chemical synthesis, and may be used as an immunogen to generate antibodies that recognize the proteins.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
  • a molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor.
  • An antigenic polypeptide contains at least about 5, and preferably at least about 10, amino acids.
  • An antigenic portion of a molecule can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the antigenic portion to a carrier molecule for immunization.
  • a molecule that is antigenic need not be itself immunogenic, i.e., capable of eliciting an immune response without a carrier.
  • an “antibody” is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope.
  • the term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos.
  • antibody molecule in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule containing the antibody combining site.
  • antibody combining site is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab',
  • Fab and F(ab') 2 portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous et al.
  • Fab' antibody molecule portions are also well-known and are produced from F(ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.
  • An antibody containing intact antibody molecules is preferred herein.
  • the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen.
  • a monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
  • adjuvant refers to a compound or mixture that enhances the immune response to an antigen.
  • An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response [Hood et al., in Immunology, p. 384, Second Ed., Benjamin/Cummings, Menlo Park, California (1984)].
  • a primary challenge with an antigen alone, in the absence of an adjuvant will fail to elicit a humoral or cellular immune response.
  • Adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, dinifrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette- Guerin) and Corynebacterium parvum.
  • the adjuvant is pharmaceutically acceptable.
  • Various procedures known in the art may be used for the production of polyclonal antibodies for use in the invention.
  • various host animals can be immunized by injection with any protein used in the present invention, including but not limited to rabbits, mice, rats, sheep, goats, etc.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinifrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Gueri ⁇ ) and Corynebacterium parvum .
  • BCG Bacille Calmette-Gueri ⁇
  • Corynebacterium parvum bacille Calmette-Gueri ⁇
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler et al, Nature, 256:495-497 (1975), as well as the trioma technique, the human B-cell hybridoma technique
  • Immortal, antibody- producing cell lines can be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • radioimmunoassay e.g., ELISA (enzyme-linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoa
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
  • the foregoing antibodies can be used in methods known in the art, e.g., for Western blotting, imaging.
  • a solid support for use in the present invention will be inert to the reaction conditions for binding.
  • a solid support for use in the present invention may have reactive groups in order to attach a binding partner, such as an antibody to the leptin receptor.
  • the solid support may be a useful chromatographic support, such as the carbohydrate polymers SEPHAROSE, SEPHADEX, and agarose.
  • a solid support is not limited to a specific type of support. Rather, a large number of supports are available and are known to one of ordinary skill in the art.
  • Solid supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, magnetic beads, membranes (including but not limited to nitrocellulose, cellulose, nylon, and glass wool filters), plastic and glass dishes or wells, etc.
  • solid supports used for peptide or oligonucleotide synthesis can be used, such as polystyrene resin (e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), POLYHLPE® resin (obtained from Aminotech,
  • the solid support can be formulated as a chromatography support, e.g., in a column; it can be used in suspension followed by filtration, sedimentation, magnetic association, or centrifugation; or by washing, as in a membrane, well, plastic film, etc.
  • Phage libraries have been constructed which when infected into host E. coli produce random peptide sequences of approximately 10 to 15 amino acids [Parmley and
  • the phage library can be mixed in low dilutions with permissive E. coli in low melting point LB agar which is then poured on top of LB agar plates. After incubating the plates at 37°C for a period of time, small clear plaques in a lawn of E. coli will form which represents active phage growth and lysis of the E. coli.
  • a representative of these phages can be absorbed to nylon filters by placing dry filters onto the agar plates. The filters can be marked for orientation, removed, and placed in washing solutions to block any remaining absorbent sites.
  • the filters can then be placed in a solution containing, for example, PTP-ID and the leptin receptor containing a phosphorylated tyrosine-985. After a specified incubation period, the filters can be thoroughly washed and developed for autoradiography. Plagues containing the phage that interfere with the PTP-ID leptin receptor binding can then be identified. These phages can be further cloned and then retested. Once the phages have been purified, the binding sequence contained within the phage can be determined by standard DNA sequencing techniques. Once the DNA sequence is known, synthetic peptides can be generated which represents these sequences.
  • Suitable labels include enzymes, fluorophores (e.g., fluorescene isothiocyanate (FITC), phycoerythrin (P ⁇ ), Texas red (TR), rhodamine, free or chelated lanthanide series salts, especially ⁇ u 3+ , to name a few fluorophores), chromophores, radioisotopes, chelating agents, dyes, colloidal gold, latex particles, ligands (e.g., biotin), and chemiluminescent agents.
  • fluorophores e.g., fluorescene isothiocyanate (FITC), phycoerythrin (P ⁇ ), Texas red (TR), rhodamine, free or chelated lanthanide series salts, especially ⁇ u 3+ , to name a few fluorophores
  • chromophores e.g., radioisotopes, chelating agents, dyes, colloidal gold, latex particles, lig
  • radioactive label such as the isotopes H, 14 C, 32 P, 35 S, 36 C1, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 1, 13 T, and 186 Re
  • known currently available counting procedures may be utilized.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
  • Direct labels are one example of labels which can be used according to the present invention.
  • a direct label has been defined as an entity, which in its natural state, is readily visible, either to the naked eye, or with the aid of an optical filter and/or applied stimulation, e.g. UN. light to promote fluorescence.
  • colored labels include metallic sol particles, for example, gold sol particles such as those described by Leuvering (U.S. Patent 4,313,734); dye sole particles such as described by Gribnau et al. (U.S. Patent 4,373,932) and May et al.
  • direct labels include a radionucleotide, a fluorescent moiety or a luminescent moiety.
  • indirect labels comprising enzymes can also be used according to the present invention.
  • enzyme linked immunoassays are well known in the art, for example, alkaline phosphatase and horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease, these and others have been discussed in detail by Eva Engvall in Enzyme Immunoassay ELISA and EMIT in Methods in Enzymology, 70. 419-439, 1980 and in U.S. Patent 4,857,453.
  • Suitable enzymes include, but are not limited to, alkaline phosphatase and horseradish peroxidase.
  • labels for use in the invention include magnetic beads or magnetic resonance imaging labels.
  • a phosphorylation site can be created on an antibody of the invention for labeling with 32 P, e.g., as described in European Patent No. 0372707 (application No. 89311108.8) by Sidney Pestka, or U.S. Patent No. 5,459,240, issued October 17, 1995 to Foxwell et al.
  • proteins can be labeled by metabolic labeling.
  • Metabolic labeling occurs during in vitro incubation of the cells that express the protein in the presence of culture medium supplemented with a metabolic label, such as [ 35 S]-methionine or [ 32 P]-orthophosphate.
  • a metabolic label such as [ 35 S]-methionine or [ 32 P]-orthophosphate.
  • the invention further contemplates labeling with [ 14 C]-amino acids and [ 3 H]-amino acids (with the tritium substituted at non-labile positions).
  • Chimeric and/or fusion proteins containing luciferase or green fluorescent protein [U.S. Patent No. 5625,048 issued April 29, 1997 and WO 97/26333 published July 24, 1997 are hereby incorporated by reference] can also be used. Similarly these proteins can be encoded by reporter genes under the control of a promoter having an activated Stat3 binding site.
  • Vectors containing the reporter gene as well as nucleic acids encoding the leptin receptor, JAK2, and/or PTP-ID can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dexfran, calcium phosphate precipitation, lipofection
  • Potential drugs can be obtained from any source including the large chemical libraries compiled by large chemical companies such as Merck, Eli Lilly, Hoffman La Roche, Glaxo Burroughs Welcome, etc. or alternatively can be obtained from phage libraries as detailed above.
  • the drug assays of the present invention may use any of a large number of protocols known to those having skill in the art.
  • Such assays can measure protein-protein binding and/or stability, protein phosphorylation and/or dephosphorylation and protein activation.
  • protein phosphorylation can be measured by 32 P, with antibodies specific for phosphotyrosine as exemplified below or indirectly by the activation of expression of a reporter gene as described above.
  • the reporter gene is under the control of a promoter having a promoter element which recognizes activated Stat3. Examples of such binding elements include the promoter of acute phase proteins [Zhang et al, Biochem. Biophys. Res.
  • Drug screening assays may be performed in cells that naturally encode the proteins involved in the signal transduction pathway initiated by leptin, preferably a cell is used that is transfected with a plasmid encoding the proteins of interest.
  • transient transfections can be performed with 50% confluent U3A cells using the calcium phosphate method as instructed by the manufacturer (Stratagene).
  • 293 cells can be used.
  • the cells can also be modified to contain one or more reporter genes, a heterologous gene encoding a reporter such as luciferase, green fluorescent protein or derivative thereof, chloramphenicol acetyl transferase, ⁇ -galactosidase, etc.
  • reporter genes can be operable linked to a promoter comprising a Stat3 binding site.
  • Assays for detecting the reporter gene products are readily available in literature for example, luciferase assays can be performed according to the manufacturer's protocol (Promega), and ⁇ - galactosidase assays can be performed as described by Ausubel et al. [in Current Protocols in Molecular Biology, J.
  • the transfection reaction can comprise the transfection of a cell with plasmids modified to contain PTP-ID, the leptin receptor, and the JAK2 kinase, such as a pcDNA3 plasmid (Invitrogen).
  • plasmids modified to contain PTP-ID, the leptin receptor, and the JAK2 kinase such as a pcDNA3 plasmid (Invitrogen).
  • a reporter plasmid that contains a reporter gene under the control of a Stat3 responding promoter can also be included.
  • preparation of such plasmids is now routine in the art, many appropriate plasmids are commercially available, e.g., a plasmid with ⁇ -galactosidase is available from Stratagene.
  • the drugs identified in the present invention have significant therapeutic potential.
  • a therapeutically effective amount of such an agent is administered in a pharmaceutically acceptable carrier, diluent, or excipient.
  • phrases “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similarly untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • the term “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similarly untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • the term “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similarly untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • “pharmaceutically acceptable” may mean approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in Martin, Remington 's Pharmaceutical Sciences, 18th Ed., Mack
  • therapeutically effective amount is used herein to mean an amount sufficient to reduce by at least about 15%, preferably by at least 50%, more preferably by at least 90%, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host. Modulation of leptin activity can be useful for reducing body weight (by increasing its activity) or increasing body weight (by decreasing its activity).
  • Reduction of leptin activity should result in weight gain as might be desirable for the treatment of the weight loss associated with cancer, AIDS or anorexia nervosa.
  • compositions of the drugs identified are envisioned. Such pharmaceutical compositions may be for administration by injection, or for oral, pulmonary, nasal or other forms of administration.
  • pharmaceutical compositions comprising effective amounts of protein or derivative products of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes. Hylauronic acid or other anionic polymers may also be used.
  • Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives [See, e.g., Martin, Remington's
  • compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form.
  • Oral Delivery Contemplated for use herein are oral solid dosage forms, which are described generally in Martin, Remington's Pharmaceutical Sciences, 18th Ed. (1990 Mack Publishing Co. Easton PA 18042) at Chapter 89, which is herein incorporated by reference.
  • Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets.
  • liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S.
  • Patent No. 4,925,673 Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556).
  • U.S. Patent No. 5,013,556 A description of possible solid dosage forms for the therapeutic is given by Marshall, in Modern Pharmaceutics, Chapter 10, Banker and Rhodes ed., (1979), herein incorporated by reference.
  • PTP-ID Binding of leptin to its receptor, Ob-Rb, leads to JAK2 dependent tyrosine phosphorylation of the cytoplasmic domain of receptor and SH2 binding proteins including the STAT3 transcription factor.
  • the phosphotyrosine phosphatase PTP-ID was isolated from bovine and mouse hypothalamus by affinity chromatography using fragments from the Ob-Rb carboxyterminus and shown to be another component of the leptin signal transduction pathway. In vitro binding studies indicated that PTP-ID binds to a phosphotyrosine at position 985 of the Ob-Rb isoform of the leptin receptor.
  • Leptin is a 16kD hormone that has potent weight reducing effects in vivo.
  • leptin is the afferent signal in a negative feedback loop regulating food intake and body weight.
  • the leptin receptor is a member of the cytokine family. Leptins anorexigenic effect are dependent on binding to the Ob-Rb isoform of its receptor.
  • the Ob-Rb form of this receptor encodes a long intracytoplasmic domain that includes several motifs for protein-protein interaction. The other forms of this receptor have short cytoplasmic regions and are not capable of initiating signal transduction by themselves.
  • Ob-Rb is highly expressed in the hypothalamus suggesting that this region of the brain is an important site of leptin action.
  • a mutation that specifically ablates Ob-Rb expression in mutant diabetic mice results in obesity and complete leptin resistance.
  • Signal transduction by this class of receptor generally depends on ligand induced phosphorylation of soluble tyrosine receptor kinases such as JAK1, 2, 3, and tyk2. These kinases in turn phosphorylate tyrosine residues on the receptor which serve as docking sites for SH2 proteins. Phosphorylation of SH2 proteins after receptor binding initiates signal transduction.
  • Glutathione-S- Transferase GST was fused to three polypeptide fragments spanning each of the three tyrosine residues encoded in the cytoplasmic region of Ob-Rb. The amino acids on the receptor included in each of these three constructs are as indicated ( Figure 1 A).
  • the three GST Ob-Rb fusion fragments were introduced into bacteria also expressing of the elk tyrosine kinase.
  • Precipitation of the 64kD protein was dependent on the presence of a phosphotyrosine at position 985 as the fusion fragment without a phosphotyrosine was not active.
  • the 64 kD protein was sequenced and the sequence was found to match PTP-ID.
  • PTP-ID is a phosphotyrosine phosphatase.
  • the relative efficiency of binding of PTP-ID to the GST Ob-Rb fragment 1 was compared to that of Stat3 for the GST fusion protein spanning fragment 3 (GST-ObRbTyr3).
  • Stat3 is known to be activated by Ob-Rb in vivo and a Stat3 binding motif, YXXL, is found in the region of tyrosine 1138.
  • Ob-Rb fragments 1 and 3 were incubated with hypothalamic extracts and immunoblotted using anti-Stat3 and anti PTP-ID specific antisera.
  • PTP-ID immunoactivity was found specifically in the precipitate using the GST Ob-Rb fusion fragment 1 ( Figure 2A) while Stat3 immunoactivity was found only in the material precipitated after incubation with GST Ob-Rb fragment 3 ( Figure 2B). The intensity of the PTP-ID signal was much greater than that of Stat3.
  • Ob-Ra is a short form of the leptin receptor that does not encode any tyrosine residues in its 34 amino acid carboxyterminus.
  • the transfected cells were treated with either PBS or leptin.
  • the cellular proteins were immunoprecipitated using a specific anti-PTP-lD antibody and detected using an anti-phosphotyrosine antibody.
  • a total cell lysate was immunoblotted using the anti-phosphotyrosine antibody.
  • PTP-ID in signal transduction was further assessed by following the time course and level of phosphorylation of the various components of the leptin signal transduction pathway.
  • Cells were transfected with either the wild type or Y — >F 985 mutant receptor in the presence or absence of PTP-ID ( Figure 5).
  • PTP-ID JAK2 was highly phosphorylated in response to leptin after 30 and 60 minutes of treatment.
  • the level of phosphorylation of the wild type receptor was greater than that of the Y — >F 985 mutant receptor.
  • PTP-ID was added, this relationship was reversed and the level of JAK2 phosphorylation was much greater in the cells that were transfected with the mutant form of the receptor.
  • PTP-ID is a component of the leptin signal transduction pathway.
  • PTP-ID can dephosphorylate JAK2 and thereby inhibit the leptin signal transduction pathway. This role is analogous to that played by PTP-1C, another phosphotyrosine phosphotase, which dephosphorylates JAK2 in hematopoietic cells. Previous studies have suggested that PTP-ID acts as an activator of signal transduction.
  • corkscrew the Drosophila homologue of PTP-ID
  • DOS sevenless receptor
  • corkscrew has been shown to lead to the dephosphorylation of daughter of sevenless leading to R7 differentiation.
  • PTP-ID has also been suggested to play a positive role in signal transduction pathways activated by prolaction, IL3, PDGF and other peptide hormones.
  • the data presented here indicate that in the setting of leptin signal transduction, PTP-ID acts as an inhibitor of signal transduction by decreasing the level of JAK2 phosphorylation.
  • PTP-ID has also been suggested to reduce signal transduction in T cells that are inhibited by CTL-4.
  • PTP-ID function is not likely to be limited to leptin action as it is expressed ubiquitously.
  • mice with induced mutations in PTP-ID are not viable and die after about 10.5 days of development.
  • PTP-ID has also been shown to associate with the prolaction PDGF, 1L3, 1L4 and receptors. Nevertheless, PTP-ID appears to modulate leptin action in vivo.

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Abstract

La présente invention concerne des nouveaux procédés d'identification de médicaments pouvant aider à réguler chez l'animal l'adiposité et la teneur en graisse, notamment chez les mammifères. La découverte que PTP-ID se fixe sur le récepteur phosphorylé de la leptine, lorsque ce récepteur contient une tyrosine-985 phosphorylée, a permis d'obtenir de nouveaux moyens d'identification d'agents aidant à la régulation de la surcharge pondérale et de l'adiposité. Ainsi, la présente invention exploite ce rôle jusqu'alors inconnu de la PTP-1D, en fournissant des moyens pour traiter et guérir potentiellement des anomalies du mécanisme de la leptine endogène, et permettre en même temps une modification choisie de la masse corporelle.
PCT/US1998/022797 1997-10-31 1998-10-27 Procedes d'identification d'agents modulant une activite de la leptine WO1999023493A1 (fr)

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US96180997A 1997-10-31 1997-10-31
US08/961,809 1997-10-31
US17869198A 1998-10-26 1998-10-26
US09/178,691 1998-10-26

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WO1999023493A9 true WO1999023493A9 (fr) 1999-09-30

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US6777388B1 (en) 1998-08-21 2004-08-17 Clf Medical Technology Acceleration Program, Inc. Leptin-related peptides
WO2001025797A2 (fr) * 1999-10-06 2001-04-12 Sumitomo Chemical Company, Limited Procedes evaluant si un agent d'essai affecte ou non un recepteur de la leptine
CA2405548A1 (fr) * 2000-04-17 2001-10-25 Sa Majeste La Reine Du Chef Du Canada Agriculture Et Agroalimentaire Canada Facteurs de modulation de la steatose et leur utilisation
CA2326952A1 (fr) * 2000-11-27 2002-05-27 The Hospital For Sick Children Tyrosine-phosphatase de proteines de cellules t
EP1448587B1 (fr) * 2001-10-01 2009-09-02 Mount Sinai School of Medicine Gene du syndrome de noonan

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US5589375A (en) * 1992-10-06 1996-12-31 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E. V. PTP 1D: a novel protein tyrosine phosphatase
US5827734A (en) * 1995-01-20 1998-10-27 University Of Washington Materials and methods for determining ob protein in a biological sample
CA2222409A1 (fr) * 1995-05-30 1996-12-05 Smithkline Beecham P.L.C. Procede de detection de composes modulant les effets de la proteine ob
AU1833197A (en) * 1996-01-18 1997-08-11 Progenitor, Inc. Detection of a leptin receptor variant and methods for regulating obesity

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