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WO2008016995A2 - Procédés d'identification de modulateurs de la signalisation de l'insuline - Google Patents

Procédés d'identification de modulateurs de la signalisation de l'insuline Download PDF

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Publication number
WO2008016995A2
WO2008016995A2 PCT/US2007/075001 US2007075001W WO2008016995A2 WO 2008016995 A2 WO2008016995 A2 WO 2008016995A2 US 2007075001 W US2007075001 W US 2007075001W WO 2008016995 A2 WO2008016995 A2 WO 2008016995A2
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Prior art keywords
jnk
cdc42
sample
mammal
insulin sensitivity
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PCT/US2007/075001
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English (en)
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WO2008016995A3 (fr
Inventor
Cullen Taniguchi
Koji Ueki
C. Ronald Kahn
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Joslin Diabetes Center, Inc.
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Publication of WO2008016995A2 publication Critical patent/WO2008016995A2/fr
Publication of WO2008016995A3 publication Critical patent/WO2008016995A3/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
    • G01N33/68—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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • This invention relates to methods of identifying drugs for the treatment of insulin resistance and diabetes.
  • Type 2 diabetes is the leading cause of kidney failure, blindness, and amputations, and is a major risk factor for heart disease and stroke.
  • Hepatic steatosis is the second most common cause of liver failure in the U.S., and the metabolic syndrome is a major risk factor in as many as 60% of individuals suffering heart attack or stroke.
  • the metabolic syndrome is a major risk factor in as many as 60% of individuals suffering heart attack or stroke.
  • PI 3-kinase phosphatidylinositol 3-kinase
  • the present invention is based, at least in part, on novel mechanistic insights into the connection between PI3K/JNK signalling and improved insulin sensitivity.
  • Provided herein are a number of screening methods that use the proteins in this pathway as targets.
  • the invention provides methods for identifying candidate compounds for improving insulin sensitivity, e.g., in a mammal in need thereof.
  • the methods include providing a sample comprising p85 ⁇ and cdc42; contacting the sample with a test compound, and evaluating binding of p85 ⁇ to cdc42 in the sample.
  • a test compound that decreases binding of p85 ⁇ to cdc42 is a candidate compound for improving insulin sensitivity in a mammal.
  • the invention provides methods for identifying candidate compounds for improving insulin sensitivity, e.g., in a mammal in need thereof.
  • the methods include providing a sample comprising cdc42 and MLK3; contacting the sample with a test compound, and evaluating phosphorylation of MLK3 by cdc42 in the sample.
  • a test compound that decreases phosphorylation of MLK3 by cdc42 is a candidate compound for improving insulin sensitivity in a mammal.
  • the invention provides methods for identifying candidate compounds for improving insulin sensitivity, e.g., in a mammal in need thereof.
  • the methods include providing a sample comprising MLK3 and MKK4; contacting the sample with a test compound, and evaluating phosphorylation of MKK4 by MLK3 in the sample.
  • a test compound that decreases phosphorylation of MKK4 by MLK3 is a candidate compound for improving insulin sensitivity in a mammal.
  • the invention provides methods for identifying candidate compounds for improving insulin sensitivity, e.g., in a mammal in need thereof.
  • the methods include providing a sample comprising MKK4 and JNK; contacting the sample with a test compound, and evaluating phosphorylation of JNK by MKK4 in the sample.
  • a test compound that decreases phosphorylation of JNK by MKK4 is a candidate compound for improving insulin sensitivity in a mammal.
  • the invention provides methods for identifying candidate compounds for improving insulin sensitivity, e.g., in a mammal in need thereof.
  • the methods include providing a sample comprising PTEN and JNK; contacting the sample with a test compound, and evaluating phosphorylation of PTEN by JNK in the sample.
  • a test compound that decreases phosphorylation of PTEN by JNK is a candidate compound for improving insulin sensitivity in a mammal.
  • the invention provides methods for identifying candidate compounds for improving insulin sensitivity, e.g., in a mammal in need thereof.
  • the methods include providing a sample comprising one or more target proteins selected from the group consisting of cdc42, MLK3, or MKK4; contacting the sample with a test compound; evaluating binding of the test compounds to the target protein; and selecting the test compound as a candidate compound if it binds to the target protein.
  • the methods include providing a cell having a functional insulin signalling pathway comprising p85 ⁇ , cdc42, MLK3, MKK4, and JNK; contacting the cell with the candidate compound; contacting the cell with an amount of insulin sufficient to activate said pathway; evaluating activation of said pathway in the cell in the presence of the test compound; comparing activation of said pathway in the cell in the presence of the test compound to a reference representing activation of said pathway in the cell in the absence of the test compound, and selecting the candidate compound as a candidate therapeutic agent for improving insulin sensitivity in a mammal if activation of said pathway is reduced in the presence of the test compound as compared to activation of said pathway in the absence of the test compound.
  • activation of said pathway is determined by one or more of detecting cdc42 activation of MLK3; detecting binding of cdc42 to MLK3; detecting MLK3 kinase activity; detecting phosphorylation of MKK4; or detecting JNK activation.
  • the test compound can be, e.g., a small molecule, or a peptide or peptidomimetic.
  • small molecules refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons.
  • the sample is or includes a cell, e.g., a cell expressing the recited proteins, either endogenously or exogenously..
  • the methods described herein further include administering the candidate compound to a mammal, e.g., a mammal in need of increased insulin sensitivity, and evaluating whether the candidate compound increases insulin sensitivity in the mammal.
  • a mammal e.g., a mammal in need of increased insulin sensitivity
  • the methods further include selecting the compound if is increases insulin sensitivity and evaluating the compound in a clinical trial.
  • FIG. IA is a set of Western blots for P ⁇ kirl gene products with an antibody against the N-terminal SH2 domain (pan-p85) in tissues lysates, as indicated, from control, heterozygous KO and L-Pik3rlKO mice. Tissues were collected from mice after an overnight fast, and proteins were extracted and processed as described in the Methods section. Each lane represents lysates from a different mouse.
  • FIGs. IB are line graphs of fasted blood glucose (IB) and fasted serum insulin levels (1C) at 8, 16, and 24 weeks of age in lox/lox and KO mice. Open circles (O) — lox/lox; closed circles (•)— L-Pik3rlKO.
  • FIGs. ID and IE are bar graphs of serum triglycerides (ID) and serum non- esterified free fatty acid levels (IE) from lox/lox or KO mice in the fasted state.
  • 2A-2E are bar graphs illustrating the results of hyperinsulinemic- euglycemic clamp analyses and gene expression changes in L-Pik3rlKO mice.
  • Male mice (n l 1) of the indicated genotype at 10-12 weeks of age were subjected to hyperinsulinemic-euglycemic clamp analysis.
  • 2E is a bar graph showing the results of quantitative RT-PCR analysis of mRNA levels in lox/lox and L-Pik3r IKO mice of phosphoenolpyruvate carboxykinase (Pckl), glucose-6-phosphatase (G6Pc), fructose- 1,6-bisphosphatase (Fbpl), peroxisome proliferator-activated receptor (PPAR)- ⁇ coactivator-1 alpha (Ppargcl) and tribbles3 (trib3) and glucokinase (Gckl).
  • Pckl phosphoenolpyruvate carboxykinase
  • G6Pc glucose-6-phosphatase
  • Fbpl fructose- 1,6-bisphosphatase
  • PPAR peroxisome proliferator-activated receptor
  • trib3 tribbles3
  • glucokinase Gckl
  • 3A is a bar graph illustrating enhanced Akt activation in L-Pik3rlKO mice.
  • FIGs. 3B is a Western blot of pi 10a and p85 ⁇ from pi 10a immunoprecipitates.
  • FIG. 3 C is a par of blots of pTyr (upper panel) and insulin receptor (lower panel) in insulin receptor ( ⁇ -subunit) immunoprecipitates.
  • FIG. 3D is a blot showing Ser473 phosphorylation of Akt.
  • FIG. 4A is a set of six photomicrographs illustrating enhanced PIP3 levels in L-Pik3rlKO mice (bottom three panels) as compared to lox/lox mice (top three panels) due to decreased PTEN activity.
  • Immunofluorescent staining with a primary anti-PIP 3 antibody (IgM) and an anti-mouse secondary antibody conjugated to Alexafluor Red and counterstained with DAPI. Following an overnight fast, mice were injected with saline (time 0) or 5U of insulin for 5 or 15 minutes.
  • N six mice in each genotype/treatment group.
  • FIG. 4B is a bar graph of the results of quantification of the immunofluorescence from PIP3 staining shown in FIG. 4A. Representative slides were chosen from each mouse and the fluorescence intensity was measured and analyzed with VH-H 1A5 Analyzer software (KEYENCE, Osaka, Japan).
  • FIGs. 4C and 4D are bar graphs of insulin-stimulated pTyr-associated PI3K activity (4C) and PTEN activity (4D) in lox/lox or KO animals at the indicated timepoints.
  • FIG. 4E is a Western blot showing PTEN levels in lox/lox and KO mice at the indicated timepoints.
  • FIG. 5 A is a blot showing expression levels of hepatic p85 ⁇ and p50 in lox/lox and KO mice.
  • Six-week-old L-Pik3rl KO mice and lox/lox controls were fed a normal chow (NC) or a high fat diet (HFD) for a total of 8 weeks.
  • NC normal chow
  • HFD high fat diet
  • FIG. 5B is a line graph indicating body weight for each week on either diet. Open squares (D)- lox/lox, NC, Open circles (O)- L-Pik3r IKO, NC; Closed squares ( ⁇ )— lox/lox, HFD; closed circles (•)— L-Pik3rlK0, HFD.
  • FIG. 5C is a trio of Western blots performed against liver lysates of mice of indicated genotype and diet using the phosphoserine 473 Akt antibody, phospho-JNK antibody, phosphoserine307 IRS-I antibodies.
  • the phospho-specific antibody blots were stripped and re-probed with the antibody for total levels of the corresponding proteins, which in each case did not change and are therefore not shown.
  • FIG. 5D is a line graph of fasting blood glucose from mice of the indicated genotype and diet. *p ⁇ 0.05, **p ⁇ 0.01
  • FIG. 6 is a series of seven immunoblots of pJNK, JNK, pS307IRS-l, IRS-I, pAkt, Akt, and p85 in lox/lox and KO mice.
  • FIG. 7 A is a bar graph illustrating cdc42 activity as determined by PAKl pulldown assay from liver lysates after three minutes of insulin stimulation via the portal vein.
  • FIG. 7B is a quartet of Western blots against phosphor-MKK4 (pMM4),
  • MKK4 phospho-JNK (pJNK) and JNK from liver lysates of mice of indicated genotypes.
  • FIG. 7C is a series of five phosphoimmunoblots from primary hepatocytes against pJNK, pMKK4, pAkt, Myc tag, and p85.
  • FIG. 8A is an immunoblot of LacZ, p85 ⁇ , p55 ⁇ , and p50 ⁇ . Recombinant adenoviruses were injected via tail-vein into 10-12 week old male mice of the indicated genotype. Mice were injected with adenoviruses encoding control LacZ, or one of the Pik3rl gene products, p85 ⁇ , p55 ⁇ , and p50 ⁇ . An extra band of approximately 5OkD appears in the livers treated with p55 ⁇ adenovirus, and this likely represents a proteolytic breakdown product of p55 ⁇ .
  • FIG. 8B is a line graph of PBK activity from the mice injected with the indicated adenoviruses is 8A.
  • FIG. 8C is a trio of Western blots performed against liver lysates of mice treated with adenovirus, using phospho-JNK and phosphoserine307 antibodies. The phospho-specific antibody blots were stripped and re-probed with the antibody for total levels of the corresponding proteins (data not shown).
  • FIGs. 8D and 8E are bar graphs of fasted blood glucose (8D) and fasted serum o insulin (8E) in mice treated with the indicated adenoviruses.
  • FIG. 9A is trio of immunoblots of LacZ, p85 ⁇ , p55 ⁇ , and p50 ⁇ .
  • Recombinant5 adenoviruses were injected via tail-vein into 10-12 week old male mice of the indicated genotype. Mice were injected with adenoviruses encoding control LacZ, or one of the Pik3rl gene products, p85 ⁇ , p55 ⁇ , and p50 ⁇ .
  • An extra band of approximately 5OkD appears in the livers treated with p55 ⁇ adenovirus, and this likely represents a proteolytic breakdown product of p55 ⁇ .
  • FIGs. 9B and 9C are bar graphs of PDK activity (9B) and cdc42 activity (9C) in the mice injected with the indicated adenoviruses.
  • FIG. 1OA is an immunoblot of LacZ, p85 ⁇ , ⁇ SH3, ⁇ BH, and ⁇ expression,5 showing that the Activation of cdc42 Requires an Intact N-terminus of p85 ⁇ .
  • FIGs. 1OB and 1OC are bar graphs of PI3K activity (10B) and cdc42 activity
  • FIG. 11 is a schematic illustration of a hypothetical assignment of functions to the regions of p85 ⁇ .
  • This regulatory subunit of PI3K regulates insulin sensitivity through both positive and negative mechanisms.
  • p85 ⁇ regulates PIP3 levels through its traditional role as a regulator of PDK activity, but it also independently regulates PIP 3 levels via the activation of JNK via cdc42 and possibly through the activation of a lipid phosphatase or by the alteration of subcellular localization of PDK.
  • FIG. 12A is a gar graph illustrating the average weekly food intake of lox/lox (FLOX) or KO mice of either normal chow (NC) or high- fat diet HFD) over a week, expressed in grams. Genotypes of animals are indicated directly on the bars.
  • FIGs. 12B and 12C are bar graphs illustrating fasting blood glucose (12B)and fasting serum insulin (12C) in lox/lox or KO mice after an eight-week treatment with either normal chow or high-fat diet.
  • FIGs. 13A-B are schematic illustrations of natural variants (13A) or artificial mutants (13B) of p85 ⁇ , with their effect on PDK activity, JNK activity, or insulin sensitivity.
  • FIG. 14 is a schematic illustration of the structure of the PTEN (top) and a phosphoimmunoblot of PTEN showing phosphorylation by JNK (bottom).
  • the present invention describes methods of identifying novel modulators of the PI3-kinase/p85 ⁇ signalling pathway that eventually modulate JNK and PTEN activity, thereby regulating insulin action and sensitivity.
  • PI 3-kinase regulatory subunits as modulators of the stress kinases JNK and p38
  • the enzyme phosphatidylinositol 3-kinase is central to the metabolic actions of insulin.
  • the enzyme itself is comprised of a regulatory subunit and a catalytic subunit.
  • the catalytic subunit is either pi 10a (GenBank Ace. No. NM_006218.2) or pi lO ⁇ (GenBank Ace. No. BCl 14432.1).
  • the most common forms of regulatory subunit are p85 ⁇ (GenBank Ace. No. NM_181523.1) and p85 ⁇ (GenBank Ace. No. BC090249.1 or BC070082.1), which are products of separate genes.
  • GRBl the gene encoding p85 ⁇
  • GRBl also produces several alternatively spliced variants, p55 ⁇ , p50 ⁇ and forms with small additional inserted exons.
  • These different regulatory subunits are expressed to different levels in different tissues and also differentially regulated in disease states such as obesity.
  • US Pat. App. Pub. No. 2002-0051786-A1 the present inventors demonstrated that heterozygous deletion of p85 ⁇ improves insulin sensitivity and can protect mice with genetic and acquired forms of insulin resistance, including the insulin resistance associated with high fat diet, from developing diabetes. Furthermore, this effect can be mimicked by reducing expression of p85 ⁇ in liver only via tissue specific knockout (see Barbour et al, J. Biol. Chem. 280(45):37489-94 (2005). Epub 2005 Sep 8).
  • the work described herein demonstrates novel mechanistic insights into this connection between p85 ⁇ and improved insulin sensitivity.
  • One aspect of the improved sensitivity is the result of a decrease in serine phosphorylation of the insulin receptor substrate IRS-I .
  • Serine phosphorylation of IRS-I has been previously shown to reduce its tyrosine phosphorylation and reduce its ability to activate the PI 3-kinase pathway, thus reducing insulin's metabolic actions.
  • Several protein kinases have been shown to phosphorylate IRS-I on serine residues, but a key kinase in this pathway is JNK.
  • p85 ⁇ plays a novel role in JNK activation by binding to and activating the small GTPase cdc42, which leads to the activation of Mixed Lineage Kinase-3 (MLK3), which phosphorylates Mitogen-Activated Protein Kinase Kinase 4 (MKK4), leading to JNK activation.
  • MKK3 Mixed Lineage Kinase-3
  • MKK4 Mitogen-Activated Protein Kinase Kinase 4
  • this pathway is normally increased in obesity-related insulin resistance, and reducing the level of p85 improves insulin sensitivity, as least in part, by reducing activation of cdc42, MMK4 and JNK, leading to reduced serine phosphorylation of IRS-I . Therefore, modulation of this pathway provides a number of novel targets for drugs that improve insulin sensitivity.
  • the methods described herein can be used to identify and optimize small molecule inhibitors that reduce the interaction of cdc42 with p85 and/or reduce cdc42- mediated activation of MKK4, thereby reducing JNK activation and serine phosphorylation of IRS-I, and enhancing insulin sensitivity in insulin resistant states.
  • the methods include the use of a high throughput in vitro assay system to identify test compounds, e.g., small molecules, that have this property and can therefore serve as drugs for diabetes, metabolic syndrome and related disorders.
  • PI 3-kinase regulatory subunits as modulators of the lipid phosphatase PTEN
  • PI 3-kinase is central to the metabolic actions of insulin. This occurs via formation of its phospholipid products, in particular PIP3, which activates downstream enzymes like Akt and the atypical PKCs ⁇ and ⁇ . This process is antagonized or reverse by the action of lipid phosphatases, which break down PIP3, the most important of which is the enzyme "phosphatase and tensin homolog," or PTEN.
  • PTEN activity is multi-factorial and includes allosteric regulation of PTEN by its lipid products, subcellular targeting, cofactor interactions, as well as post-translational modifications (Gericke, Gene. 2006 Jun 7;374: l-9. Epub 2006 Mar 14).
  • PTEN phosphorylation occurs on serine residues (>90%), especially serine residues 370 and 385.
  • phosphorylation has been reported on other serines (e.g., serine 229, 360, 362, and/or 380) and threonines (e.g., threonine 223, 319, 321, 366, 382, 383, and/or 401), residues that map to the C2 lipid binding domain (amino acids (a. a.) 190-351) and a region proximal to the PDZ ligand sequence (a.a. 401-403) at the C-terminus.
  • serines e.g., serine 229, 360, 362, and/or 380
  • threonines e.g., threonine 223, 319, 321, 366, 382, 383, and/or 401
  • residues that map to the C2 lipid binding domain amino acids (a. a.) 190-351)
  • a region proximal to the PDZ ligand sequence a.a. 401-403
  • JNK is the critical PTEN Kinase.
  • K/RXXXXLXL conserved JNK-binding motif
  • JNK/MAP kinase family two potential phosphorylation sites (Ser 338 and Thr 366 ) for JNK/MAP kinase family exist in the C-terminal region of PTEN, and this region has been shown to be important for stability and/or activity of PTEN. Since JNK activity is decreased o in p85 ⁇ KO mice and cells, a decrease in JNK would also be expected to result in a change in serine phosphorylation and activity of PTEN.
  • sequences for the protein and nucleic acid targets useful in the methods described herein are known in the art. The following is a list of exemplary sequences5 that can be used. As one of skill in the art will appreciate, homologs of these sequences from other species can also be used. For example, nucleic acids that hybridize under stringent conditions to a sequence listed herein, or a polypeptide encoded by such a sequence, can equally be used. As used herein, the term “hybridizes under stringent conditions” describes conditions for hybridization and0 washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1- 6.3.6.
  • Aqueous and nonaqueous methods are described in that reference and either can be used.
  • stringency conditions are 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2 X SSC, 1% SDS at 65°C. 5
  • a sequence that is at least 80%, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical to a sequence listed herein can be used.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For purposes of the present methods, the percent identity between two amino acid sequences is 0 determined using the Needleman and Wunsch ((1970) J. MoI. Biol.
  • mouse PTEN sequences are available at:
  • JNKl also known as mitogen-activated protein kinase 8
  • JNK2 also known as mitogen activated protein kinase 9
  • MLK3 also known as Mitogen- Activated Protein Kinase Kinase Kinase 11 (MAPKKl 1) can be found at:
  • MKK4 mitogen-activated protein kinase kinase 4
  • PIK3R1 phosphoinositide-3-kinase, regulatory subunit 1 (PIK3R1)
  • the invention includes methods for screening of test compounds, to identify compounds that modulate a pathway described herein, e.g., compounds that (i) reduce the interaction of cdc42 with p85, thereby reducing cdc42 activity, or (ii) reduce the activation of MLK3 by cdc42, thereby reducing MLK3 activity, or (iii) reduce the phosphorylation of MKK4 by MLK3, thereby reducing MKK4 activity, thus reducing JNK activation and serine phosphorylation of IRS-I, and enhancing insulin action and sensitivity.
  • each of cdc42, MLK3, and MKK4 are novel targets for inhibition, and can be used as targets for a screening method described herein.
  • the methods described herein can be used to identify compounds that bind to p85, cdc42, MLK3, or MKK4.
  • the effect of a test compound on the p85 signalling pathway can be determined.
  • the methods can be used to identify compounds that demonstrate (i) binding to p85 and/or cdc42, and/or decrease p85-mediated activation of cdc42, (ii) binding to cdc42 and/or MLK3, and/or decrease csc42-mediated activation of MLK3, or (iii) binding to MLK3 and/or MKK4, and/or decrease MLK3 -mediated phosphorylation of MKK4.
  • the methods will include providing a sample that includes one or more of p85, cdc42, MLK3, and MKK4.
  • the sample can include p85 ⁇ and cdc42, e.g., isolated and purified p85 ⁇ and cdc42 proteins, and the methods include identifying a compound that affects binding between p85 and cdc42.
  • Screening methods suitable for use in these embodiments are known in the art and include, but are not limited to, yeast or mammalian 2-hybrid systems, tagged protein assays, immunoprecipitation assays, and proteomics assays. These methods can be used to identify natural (i.e., endogenous) regulators of cdc42, for example.
  • the sample includes isolated and purified cdc42 and MLK3, with or without MKK4, and the methods include detecting identifying compounds that affect activation of MLK3 by cdc42, and/or phosphorylation of MKK4. In some embodiments, the sample includes isolated and purified MLK3 and
  • MKK4 and the methods include detecting identifying compounds that affect phosphorylation of MKK4.
  • MKK7 can be used in place of MKK4.
  • the methods include identifying isoform-specific inhibitors of JNK, i.e., inhibitors that substantially reduce the activity of JNKl while not substantially affecting the activity of JNK2, or vice-versa.
  • the different isoforms have different activities depending on the tissues, therefore, it may be desirable to affect only the isoform that is active in the particular cell type.
  • the methods can include adding the compound to cells that express all of these proteins, and exhibit p85 signalling via cdc42 and MKK4 that results in JNK activation and serine phosphorylation of IRS- 1 and/or PTEN.
  • the methods can then include contacting the cells with the compound, and evaluating an effect of the compound on JNK activity (e.g., serine phosphorylation of IRS-I or PTEN).
  • the methods further include determining whether the compound has an effect on phosphorylation of other substrates by JNK, and selecting a test compound if it selectively inhibits JNK phosphorylation of PTEN.
  • such a compounds might be designed such that it binds a JNK-recognition site, or a JNK-phosphorylation site, on PTEN.
  • a number of suitable assays are known in the art, see, e.g., Methods in Enzymology. Volume 201 : Protein Phosphorylation. Part B: Analysis of Protein Phosphorylation. Protein Kinase Inhibitors, and Protein (Methods in Enzymology) by John N. Abelson, Melvin I. Simon, Tony Hunter, and Bartholomew M. Sefton (Hardcover - Jan 15, 1991), Protein Phosphorylation. Part A: Protein Kinases: Assays. Purification.
  • volume 200 Volume 200, Part A (Methods in Enzymology) by John N. Abelson, Melvin I. Simon, Tony Hunter, and Bartholomew M. Sefton (Hardcover - JuI 28, 1991).
  • test compounds can be, e.g., natural products or members of a combinatorial chemistry library.
  • the test compounds are initially members of a library, e.g., an inorganic or organic chemical library, peptide library, oligonucleotide library, or mixed-molecule library.
  • the methods include screening small molecules, e.g., natural products or members of a combinatorial chemistry library. These methods can also be used, for example, to screen a library of proteins or fragments thereof, e.g., proteins that are expressed in liver or pancreatic cells.
  • a given library can comprise a set of structurally related or unrelated test compounds.
  • a set of diverse molecules should be used to cover a variety of functions such as charge, aromaticity, hydrogen bonding, flexibility, size, length of side chain, hydrophobicity, and rigidity.
  • Combinatorial techniques suitable for creating libraries are known in the art, e.g., methods for synthesizing libraries of small molecules, e.g., as exemplified by Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular- Weight Compound Libraries. Pergamon-Elsevier Science Limited (1998). Such methods include the "split and pool” or “parallel” synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, Curr. Opin. Chem. Bio. 1 :60-6 (1997)). In addition, a number of libraries, including small molecule libraries, are commercially available.
  • the test compounds are peptide or peptidomimetic molecules, e.g., peptide analogs including peptides comprising non-naturally occurring amino acids or having non-peptide linkages; peptidomimetics (e.g., peptoid oligomers, e.g., peptoid amide or ester analogues, ⁇ -peptides, D-peptides, L-peptides, oligourea or oligocarbamate); small peptides (e.g., pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, or larger, e.g., 20-mers or more); cyclic peptides; other non-natural or unnatural peptide-like structures; and inorganic molecules (e.g., heterocyclic ring molecules).
  • the test compounds are nucleic acids,
  • test compounds and libraries thereof can be obtained by systematically altering the structure of a first test compound.
  • a small molecule e.g., a first small molecule is selected that is, e.g., structurally similar to a known phosphorylation or protein recognition site.
  • a general library of small molecules is screened, e.g., using the methods described herein, to select a fist test small molecule.
  • the structure of that small molecule is identified if necessary and correlated to a resulting biological activity, e.g., by a structure-activity relationship study.
  • test compounds identified as "hits" e.g., test compounds that demonstrate activity in a method described herein
  • a first screen is selected and optimized by being systematically altered, e.g., using rational design, to optimize binding affinity, avidity, specificity, or other parameter.
  • Such potentially optimized structures can also be screened using the methods described herein.
  • the invention includes screening a first library of test compounds using a method described herein, identifying one or more hits in that library, subjecting those hits to systematic structural alteration to create one or more second generation compounds structurally related to the hit, and screening the second generation compound. Additional rounds of optimization can be used to identify a test compound with a desirable therapeutic profile.
  • Test compounds identified as hits can be considered candidate therapeutic compounds, useful in treating disorders described herein.
  • the invention also includes compounds identified as "hits" by a method described herein, and methods for their administration and use in the treatment, prevention, or delay of development or progression of a disease described herein.
  • Example 1 Phosphoinositide 3-Kinase Regulatory Subunit p85 ⁇ Suppresses Insulin Action via Positive Regulation of PTEN
  • Insulin action on the liver is required for the proper maintenance of metabolic homeostasis. Under normal conditions, insulin inhibits gluconeogenesis and activates lipogenesis to promote proper fuel utilization in the fed state. The failure of insulin to regulate hepatic function can lead to unfettered hepatic glucose output and elevated lipogenesis in the liver, which are two key features of the metabolic syndrome (Shimomura, et al., (2000) MoI. Cell, 6(l):77-86). Not surprisingly, an overwhelming amount of epidemiological (Tripathy et al., (2004) Diabetologia, 47(5):782-93) and physiological (Fisher and Kahn, (2003) J. Clin. Invest., 111(4):463-468) evidence links hepatic insulin resistance to the development of type 2 diabetes.
  • PI3K phosphoinositide 3-kinase pathway
  • Saltiel and Kahn, (2001) Nature, 414(6865):799-806 PI3K is an obligate heterodimer, with an SH2-containing regulatory subunit (p85) and a catalytic subunit (pi 10).
  • the regulatory subunit mediates the binding, activation and localization of the PI3K enzyme (Virkamaki, et al., (1999) J. Clin. Invest.
  • the negative effects of p85 ⁇ on insulin action may have important consequences in the pathophysiology of insulin resistance and diabetes.
  • the increased expression of p85 ⁇ in mouse models of gestational diabetes (Barbour et al., (2004) Endocrinology 145(3): 1144-50) and in obese humans (Bandyopadhyay et al., (2005) Diabetes 54(8):2351-9) is strongly linked with insulin resistance.
  • heterozygosity for P ⁇ kirl prevents the onset of diabetes in genetically insulin resistant mice (Mauvais-Jarvis et al, (2002) J. Clin. Invest. 109, 141-149).
  • mice with a liver-specific deletion oiPik3rl were created.
  • This conditional knockout oiPik3rl (p85 ⁇ , p55 ⁇ , and p50 ⁇ ) in hepatocytes circumvents the perinatal lethality observed in the corresponding germline knockout mice and furthermore allows the investigation of the specific role of the liver in the physiological actions of p85 ⁇ .
  • L-Pik3rlKO mice have enhanced hepatic and whole body insulin sensitivity as well as increased Akt activity in liver, despite decreased total hepatic PBK activity.
  • p85 ⁇ mediates its negative effect on insulin sensitivity at least in part via the activation of PTEN lipid phosphatase activity.
  • mice used in this study were on a 129Sv-C57BL/6-FVB mixed genetic background. Metabolic studies.
  • GTT glucose tolerance testing
  • blood samples were obtained at 0, 15, 30, 60, and 120 minutes after intraperitoneal injection of 2 g/kg dextrose. Insulin tolerance tests were performed by injecting 1 U/kg insulin (Novolin, Novo Nordisk, Denmark) intraperitoneally, followed by blood collection at 0, 15, 30 and 60 minutes after injection. Blood glucose values were determined using a One Touch II glucose monitor (Lifescan Inc., Milipitas, CA).
  • Plasma insulin levels were measured by ELISA using mouse insulin as a standard (Crystal Chem Inc., Chicago, IL). Non-esterified free fatty acid levels were measured from random fed mice using a kit from Wako Diagnostics, while serum triglycerides were measured by Anilytics (Gaithersburg, MD).
  • Hyperinsulinemic-euglycemic clamp Mice were anaesthetized with a 1.2% solution of 2,2,2-tribromoethanol in normal saline, followed by the microsurgical insertion of a catheter into the right jugular vein. Approximately 7 days of recovery, mice were fasted for 5 hours and were infused with a constant (2.5 mU/kg/min) dose of insulin and a variable glucose infusion rate to maintain euglycemia and assess whole-body insulin sensitivity.
  • mice were anesthetized with Avertin (2,2,2-tribromoethanol in PBS), and injected with 5 U of regular human insulin (Novolin, Novo Nordisk, Denmark) via the inferior vena cava. Five minutes after the insulin bolus, tissues were removed and frozen in liquid nitrogen. Immunoprecipitation and immunoblot analysis of insulin signaling molecules was performed as previously described (Taniguchi et al, (2005) J. Clin. Invest. 115(3):718-27).
  • TATA box binding protein TBP
  • Antibodies Rabbit polyclonal anti-IRS-1 antibody (IRS-I), anti-IRS-2 antibody (IRS-2), anti-IR antibody (IR) and pan-p85 ⁇ antibody were generated as described previously (Ueki et al., (2002) Proc. Natl. Acad. Sci. U.S.A., 99(l):419-24).
  • the anti-TRB3 antibody was a gift from Marc Montminy (Du et al., (2003) Science 300, 1574-7).
  • Rabbit polyclonal anti-Akt, anti-phospho Akt (S473) anti-PTEN were purchased from Cell Signaling Technology (Beverly, MA).
  • the phosphotyrosine (pTyr) antibody, 4G10 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).
  • Goat polyclonal anti-Aktl/2 antibody (Akt) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
  • Phosphatidylinositol (Avanti Polar Lipids) was in vitro phosphorylated to form phosphatidylinositol 3 -phosphate (PI-3P) by recombinant PI3K (Upstate) with [ 32 P] ⁇ -ATP.
  • the PI-3P was re- extracted with methanol/choloroform and run on a TLC plate with a n-propanaol:2M acetic acid (65:35). The intensity of PI-3P spots were quantitated with NIH Image. PTEN activity was determined relative to IgG immunoprecipiates from lox/lox livers. Relative PTEN activity was compared with lox/lox without insulin stimulation. Statistics. Data are presented as ⁇ s.e.m. Student's t-test was used for statistical analysis between two groups, while statistical significance between multiple treatment groups was determined by analysis of variance (ANOVA) and Tukey's t- test.
  • ANOVA analysis of variance
  • mice with a liver-specific deletion oiPik3rl were generated via the Cre-loxP system using animals carrying a floxed exon 7, which encodes the N-terminal SH2 domain (Luo, et al., (2005) MoI. Cell. Biol. 25:491-502) and mice carrying the Cre transgene driven by the albumin promoter (Postic and Magnuson, (2000) Genesis 26(2): 149-50).
  • the presence of loxP sites in the Pik3rl gene did not affect expression of p85 ⁇ as compared to WT littermate controls (Luo et al., (2005) MoI. Cell. Biol. 25:491-502) nor did the presence of albumin-Cre (Michael et al., (2000) MoI Cell
  • L-Pik3rl KO mice were born in a normal Mendelian distribution and exhibited normal postnatal growth (data not shown).
  • Western blots of liver extracts of L-Pik3rl KO mice revealed an 80-90% decrease in p85 ⁇ and a complete loss of p50 ⁇ (FIG. IA), consistent with complete ablation in hepatocytes.
  • the expression of p85 ⁇ was unaltered in brain, skeletal muscle and fat.
  • L-Pik3rl KO mice exhibited significant improvements in serum metabolic chemistries.
  • L-Pik3rl KO mice displayed lower fasted blood glucose and fasted serum insulin levels at 8, 16, and 24 weeks of age (FIG. IB and 1C). In addition, L-Pik3rlKO mice exhibited decreased levels of serum triglycerides and circulating free fatty acids (FIGs. ID and IE). At 16 weeks of age, L-Pik3rl KO mice were significantly more glucose tolerant to an intraperitoneal glucose challenge (FIG. IF), but this was observed as early as eight weeks of age and was maintained through 24 weeks of age (data not shown).
  • FIG. IF intraperitoneal glucose challenge
  • L-Pik3rl KO mice were subjected to a hyperinsulinemic- euglycemic clamp. This revealed increased hepatic insulin sensitivity such that hepatic glucose production was suppressed by 25% more in the L-Pik3rl KO mice compared to controls (FIG. 2A).
  • L-Pik3rl KO mice displayed improved peripheral insulin sensitivity, as determined by a two-fold increase in glucose infusion rates (FIG. 2B). This correlated with a 1.5-fold increase in glucose uptake in muscle (FIG. 2C) and a three-fold increase in glucose uptake in fat (FIG. 2D).
  • FIG. 2B shows improved peripheral insulin sensitivity
  • the physiologic data indicating increased hepatic insulin sensitivity corresponded with decreased expression of several key gluconeogenic genes.
  • Quantitative RT-PCR analysis found a decrease in phosphoenolpyruvate carboxykinase (Pckl), glucose-6 phosphatase (G6pc) and fructose- 1,6-bisphosphatase (Fbpl) mRNAs in liver by 30%, 70%, and 60%, respectively (FIG. 2e).
  • PI3K function was characterized in L-Pik3rlKO mice.
  • a 50% decrease in IRS-I -associated PBK activity was observed, as was a 60% decrease in IRS-2- associated PDK activity, which together resulted in a 50% reduction in total PBK activity, as measured from pTyr-immunoprecipitates (FIG. 3A).
  • These decreases in PBK were concordant with a 70% decrease in the expression of the pi 10a catalytic subunit (FIG. 3B), but were not associated with decreased insulin receptor activation in the L-Pik3rl KO mice (FIG. 3C).
  • the parallel decrease in expression of pi 10a was expected, since the regulatory subunits are known to stabilize the catalytic subunit (Yu et al., (1998) MoI. Cell. Biol. 18(3): 1379-87).
  • PBK activity or decreased turnover, total PBK activity and the activity of the lipid phosphatase, PTEN, which degrades the PIP3 formed by PBK were directly assessed.
  • pTyr-associated PBK activity was not changed in the livers of either the lox/lox or L-Pik3rlKO mice, though the insulin-stimulated PI3K activity of the knockout mice remained at only half the level of the controls (FIG. 4C).
  • the lipid phosphatase activity of the negative regulator PTEN was consistently decreased by 40% in L-Pik3rlKO at all timepoints (FIG. 4D).
  • insulin had no effect on PTEN activation in either the control or knockout mice. This decrease in PTEN activity is not due to decreased PTEN expression (FIG. 4E), but must be due to some other aspect of the actions of the regulatory subunit of PI3K.
  • Example 2 The p85 ⁇ Regulatory Subunit of Phosphoinositide 3 -Kinase activates JNK via a cd c42/MKK4 pathway
  • Insulin resistance is an underlying feature of type 2 diabetes and the metabolic syndrome (Reaven, (2005) Cell. Metab. 1 :9-14). Physiologic and epidemiologic studies have demonstrated strong links between obesity and the development of insulin resistance (Hu et al., (2001) N. Engl. J. Med. 345:790-7; Sinha et al., (2002) N. Engl. J. Med. 346:802-10). Not surprisingly, the rise of type 2 diabetes in the United States over the last decade has paralleled the rapid rise in obesity (Mokdad et al., (2003) JAMA 289:76-9).
  • JNK stress kinase
  • c-Jun-N-terminal kinase JNK
  • cytokines Baud et al., (1999) Genes Dev. 13: 1297-308
  • JNK c-Jun-N-terminal kinase
  • JNK phosphoinositide 3-kinase
  • PI3K is an obligate heterodimer, with an SH2 -containing regulatory subunit (p85) and a catalytic subunit (pi 10).
  • the regulatory subunit mediates the binding, activation and localization of the PBK enzyme (Backer et al., (1992) EMBO J.
  • mice carrying germline deletion of the P ⁇ kirl gene that encodes p85 ⁇ and its shorter isoforms p55 ⁇ and p50 ⁇ die perinatally (Fruman et al., (2000) Nat Genet 26:379-82).
  • mice with a liver-specific deletion oiPik3rl gene (L-Pik3rlKO) were created.
  • mice lacking p85 ⁇ in liver have diminished hepatic activation of JNK and improved whole body insulin sensitivity.
  • p85 ⁇ activates JNK via the cdc42-MKK4 pathway and that this interaction requires both an intact N-terminus and functional SH2 domains in the C-terminus of the p85a regulatory subunit.
  • p85 ⁇ may regulate insulin sensitivity in both lean and obese mice via crosstalk with the stress kinase pathway.
  • mice and high fat diet Animals and high fat diet. All animals were housed on a 12-h light-dark cycle and fed a standard rodent chow (Purina). High fat chow (45% kcal from fat) was purchased from Research Diets. All mice in this study were on a 129Sv- C57BL/6-FVB mixed genetic background, and littermates of the same mixed genetic background were used as controls.
  • Metabolic studies were performed as described above in Example 1.
  • the p50 ⁇ and p55 ⁇ adenoviruses were constructed as previously described (Ueki et al., (2000) MoI. Cell. Biol. 20:8035-46).
  • the constitutively active MKK4 adenovirus was purchased from CellBio Labs (San Diego, CA), and the constitutively active cdc42 adenovirus was a gift from James Bamburg (Kuhn et al., (2000) J. Neurobiol. 44: 126-144).
  • the wild type (WT) p85 ⁇ , ⁇ SH3, ⁇ BH, ⁇ iSH2, RARA and ⁇ p85 constructs were made as follows. Cloning ofp85 constructs
  • the p85 constructs were cloned from a cDNA of human p85 ⁇ using the following PCR primers:
  • the PCR fragments from the WT, and ⁇ SH3 reactions were digested with Xbal/Sall then ligated into pBluescript (pBS).
  • the 5' portion of the ⁇ BH reaction was digested with Xbal/BamHI and the 3' fragment was digested with BamHI/Sall.
  • the two fragments were ligated together in pBS.
  • the ⁇ iSH2 construct was made by cutting the above 1.5 kb PCR fragment with EcoRV and Sail, then fusing it with the Xbal-EcoRV fragment of the WTp85 ⁇ .
  • the ⁇ p85 construct was made by ligating the Xbal-EcoRV fragment of WTp85 ⁇ FLAG construct (N-terminus) to the EcoRV- SaII ⁇ iSH2 fragment (C-terminal half).
  • adenoviruses were then produced according to the standard AdEasy protocol (He et al, (1998) Proc. Natl. Acad. Sci. U.S.A. 95:2509-14).
  • Adenovirus-mediated gene transfer and in vivo insulin stimulation Prior to use on primary hepatocytes or in vivo, all adenoviruses were purified on sequential cesium chloride gradients then dialyzed into PBS containing 10% glycerol. 10-12 week-old male mice were injected via tail vein with an adenoviral dose of 5xl ⁇ 8 pfu/g body weight as described previously (Taniguchi et al., (2005) J. Clin. Invest. 1 15:718- 27).
  • mice On the fifth day after injection, following an overnight fast, the mice were anesthetized with Avertin (1.2% 2,2,2-tribromoethanol in PBS), and injected with 5 U of regular human insulin (Novolin, Novo Nordisk, Denmark) via the inferior vena cava. Five minutes after the insulin bolus, tissues were removed and frozen in liquid nitrogen.
  • Avertin (1.2% 2,2,2-tribromoethanol in PBS
  • regular human insulin Novolin, Novo Nordisk, Denmark
  • tissue homogenates prepared in a tissue homogenization buffer that contained 25 mM Tris-HCl (pH 7.4), 10 mM Na 3 VO 4 , 100 mM NaF, 50 mM Na 4 P 2 O 7 , 10 mM EGTA, 10 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 1% Nonidet-P40 supplemented with the Complete protease inhibitor cocktail (Roche). All protein expression data were quantified by densitometry using NIH Image software. Antibodies.
  • Rabbit polyclonal anti-IRS-1 antibody (IRS-I), anti-IRS-2 antibody (IRS-2), anti-IR antibody (IR) and pan-p85 ⁇ antibody were generated as described previously (Ueki et al., (2002) Proc. Natl. Acad. Sci. U.S.A. 99:419-24).
  • mice were anesthetized and injected with 5U of insulin via the portal vein. Three minutes after the injection, the right lobe of the liver was quickly dissected and snap frozen directly into liquid nitrogen.
  • cdc42 activity in the livers was measured with a PAKl pulldown assay (Upstate).
  • the kit was used essentially as directed, but with the addition of 10 mM ortho vanadate to the reaction buffer.
  • Hepatocytes were isolated as described previously (Block et al, (1996). J. Cell. Biol. 132: 1133-49). Briefly, the mice were anesthetized with a 1.2% solution of 2,2,2-tribromoethanol and the portal vein was cannulated with a 24.5 G catheter, and the liver perfused for 15 minutes at a rate of 7 mL/min with calcium-free perfusion buffer. The blanched liver is perfused with collagenase solution (200U/mL) for 10 minutes at 7 mL/min to release hepatocytes from the extracellular matrix.
  • collagenase solution 200U/mL
  • the digested liver was excised and placed in preservation buffer, where the digested cells were gently scraped from the liver sac, washed and purified with Percoll to remove dead cells and enrich the hepatocyte fraction.
  • Typical viabilities are between 85-90%, with cell yields of 1.0-1.5 x 10 6 cells/g mouse.
  • the isolated hepatocytes are then grown on collagen-coated plates in Advanced DMEM (Gibco) supplemented with glutamine, antibiotic cocktail and 10% FBS.
  • mice with a liver-specific deletion oiPik3rl via the Cre-loxP system were generated as described previously (Postic and Magnuson. (2000) Genesis 26: 149-50).
  • Mice carrying a floxed exon 7, which encodes the N-terminal SH2 domain common to all three transcripts, p85 ⁇ , p55 ⁇ and p50 ⁇ were crossed with mice carrying the Cre transgene driven by the albumin promoter.
  • L-Pik3rlKO mice maintained lower fasting blood glucose and fasting serum insulin levels when fed either a high- fat diet or normal chow (FIGs. 12B and 12C).
  • obese lox/lox mice were severely glucose intolerant
  • obese L-Pik3rlKO mice exhibited normal to improved glucose tolerance even when compared against control mice on normal chow (FIG. 5C).
  • FIG. 5C the loss of p85 ⁇ expression in liver protected against obesity-induced insulin resistance and diabetes.
  • L-Pik3rlKO Hepatocytes are Resistant to JNK-induced insulin resistance.
  • JNKl was overexpressed in primary hepatocytes isolated from lox/lox or L- Pik3rlKO mice using adenovirus-mediate gene transfer.
  • the JNKl isoform was chosen because it is the only one of the three JNK isoforms that has been shown to have a significant role in mediating obesity -related insulin resistance in the liver
  • JNKl was overexpressed by six-fold in both p85 ⁇ knockout and control primary hepatocytes (FIG. 6). This forced expression of JNKl led to significant increases in JNK phosphorylation and serine phosphorylation of IRS-I in lox/lox hepatocytes. By contrast, an equal level of JNKl overexpression resulted in only a 2-fold increase in cells derived from L-Pik3rl mice (FIG. 6) (p ⁇ 0.05 knockout vs. control cells).
  • the p85 ⁇ regulatory subunit activates JNK via a cdc42/MKK4 pathway
  • One candidate effector is the small GTPase cdc42, which is known to activate both SEK1/MKK4 and JNK (Gallo and Johnson, (2002) Nat. Rev. MoI. Cell. Biol. 3:663-72), and has been shown to interact with p85 ⁇ , but it was unknown whether this interaction had any functional consequences in vivo (Zheng et al, (1994) J. Biol. Chem.
  • cdc42/MKK4/JNK signaling pathways is an intrinsic property of hepatic insulin signaling
  • primary hepatocytes from L-Pik3rlKO livers and lox/lox controls were infected with constitutively active forms of MKK4 and cdc42 and stimulated with either insulin or saline control (FIG. 7C).
  • Expression of activated MKK4 or cdc42 enhanced JNK phosphorylation, indicating that these enzymes were upstream of this pathway in hepatocytes.
  • Akt activation was differentially affected by the expression of p85 ⁇ .
  • control LacZ, p55 ⁇ , or p50 ⁇ maintained the elevated Akt activation observed in L-Pik3rlKO mice
  • the expression of p85 ⁇ caused a relative decrease in Akt phosphorylation to a level similar to lox/lox controls.
  • re-expression of p85 ⁇ specifically restored several mechanisms of negative regulation to L-Pik3rlKO animals.
  • the expression of p85 ⁇ restored insulin- stimulated JNK activation and levels of IRS-I serine phosphorylation back to levels comparable to lox/lox controls (FIG. 8C).
  • the ⁇ iSH2 construct When overexpressed in cells or in mouse livers, the ⁇ iSH2 construct has a dominant negative effect (Miyake et al., (2002) J. Clin. Invest. 110: 1483-91).
  • the other p85 mutant contains arginine to alanine substitutions in critical residues in both SH2 domains in the C-terminus (RARA); this mutant is able to bind pi 10, but cannot bind to phosphorylated IRS proteins, which is required for the proper activation and localization of the PI3K holoenzyme (Hill et al., (2001) J. Biol. Chem. 276: 16374-8).
  • the ⁇ iSH2 mutant caused significant glucose intolerance consistent with diabetes, probably due to the inhibition of the positive effects of PBK in addition to the negative effects of JNK activation. Consistent with the cdc42/JNK data, the RARA p85a mutant had negligible effects on insulin sensitivity.
  • the N-terminus of p85 ⁇ is 339 amino acids long and contains an SH3 domain, two proline rich regions, and a domain homologous to a portion of breakpoint cluster region (bcr) gene product (BH domain).
  • adenoviral p85 constructs were created which substitute a FLAG tag for either the -80 amino acid SH3 domain ( ⁇ SH3) or the -170 amindacid BH domain ( ⁇ BH), which effectively deleted the domain while providing an epitope tag for easy detection by Western.
  • ⁇ SH3 domain -80 amino acid SH3 domain
  • ⁇ BH -170 amindacid BH domain
  • One construct with a combination deletion of both the SH3 domain and inter-SH2 domain ( ⁇ p85) was also created to serve as a control for PI3K activity (FIG. 10A).
  • JNK The activation of JNK, particularly in the liver, has been shown previously to be a major mediator of the insulin resistance that occurs in obesity (Ozcan et al., (2004) Science 306:457-61). Consequently, one of the mechanisms by which p85 ⁇ suppresses insulin action in vivo may occur through the JNK-mediated negative feedback on insulin signaling.
  • deletion of either the SH3 domain or BH domain from the N-terminus is sufficient to ablate the ability of p85 to activate cdc42, while fully maintaining PI3K activity (FIG. 1OB and Beeton et al, (1999) MoI. Cell Biol. Res. Commun. 1 : 153-7).
  • the functional inactivation of the SH2 domains in p85 also rendered it unable to activate either PI3K or cdc42/JNK, suggesting that the proper localization in the cell or within a complex may be required for JNK activation.
  • the data define the minimal requirements for the activation of JNK by p85 as a fully intact N- terminus of p85 and functional SH2 domains.
  • p85 has been demonstrated as an essential activator of small GTPases such as cdc42 or Rac (reviewed in Burridge and Wennerberg, (2004) Cell 116: 167-79) that mediate PDGF or EGF-induced cytoskeletal changes, such as membrane ruffling or stress fiber disassembly (Brachmann et al., (2005) MoI. Cell. Biol. 25:2593-606; Hill et al., (2001) J. Biol. Chem. 276: 16374-8).
  • small GTPases such as cdc42 or Rac (reviewed in Burridge and Wennerberg, (2004) Cell 116: 167-79) that mediate PDGF or EGF-induced cytoskeletal changes, such as membrane ruffling or stress fiber disassembly (Brachmann et al., (2005) MoI. Cell. Biol. 25:2593-606; Hill et al., (2001) J. Biol. Chem. 276: 16374
  • the BH domain is similar in structure to the Rho-GTPase activating protein (GAP) domain of the breakpoint cluster region (bcr) protein (Musacchio et al., (1996) Proc. Natl. Acad. Sci. U.S.A.
  • these domains might be responsible for the proper intracellular localization of the cdc42/JNK-activating complex.
  • the p85 subunit has been found to form insulin-dependent protein aggregates that do not generate PIP 3 (Luo et al., (2005) J. Cell. Biol. 170(3):455-64). These aggregates were proposed as sequestration complexes, but an alternate interpretation is that these complexes could be active negative regulatory complexes that activate cdc42 and JNK.
  • p85 is an essential part of the PDK heterodimer, it also plays a novel role in regulating a cdc42/MKK4/JNK pathway that suppresses insulin action in both lean and obese mice.
  • These mechanisms not only provide a level of internal negative feedback on this critical node (Taniguchi et al, (2006) Nat. Rev. MoI. Cell. Biol. 7:85-96) in insulin and growth factor signaling, but also allow crosstalk between the PBK signaling pathway and the stress or inflammatory responses, thus creating an important connection that could have broad impact in the basic understanding of cell growth and metabolism.
  • This powerful link between p85 ⁇ and JNK activation might also represent an exciting new therapeutic intervention into type 2 diabetes.
  • Example 3 JNK phosphorylates PTEN Since JNK activity is decreased in p85 ⁇ KO mice and cells, it is possible that a decrease in JNK could also result in a change in serine phosphorylation and activity of PTEN.
  • PTEN Activity Assay Phosphatidylinositol (Avanti Polar Lipids) was in vitro- phosphorylated to form phosphatidylinositol 3 -phosphate (PI-3-P) by recombinant PI3K (Upstate) with [gamma-32P]ATP.
  • the phosphorylated lipid was extracted with 1: 1 methanolxhloroform, dried under nitrogen gas, reconstituted into PTEN assay buffer (10mMTris-HCl/25mMNaCl, pH 7.5), and incubated with PTEN immunoprecipitates from lox/lox or L-Pik3rlKO liver lysates (Miller et al., FEBS Lett. 528:145-153 (2005)).
  • the PI-3-P was re-extracted with methanokchloroform and run on a TLC plate with n-propanol:2 M acetic acid (65:35).
  • PTEN activity was determined by decreased intensity of the PI-3-P spot relative to IgG immunoprecipitates from lox/lox livers. Relative PTEN activity was determined by normalizing PTEN activity to lox/lox livers without insulin stimulation. SDS-PAGE and autoradiography revealed that JNK was able to phosphorylate PTEN in vitro (FIG. 14). To further assess this phosphorylation of PTEN by JNK, mutants of PTEN were generated with substitution of the two potential JNK phosphorylation sites (PTEN-S338A, and PTEN-T366A).
  • mutants as well as wild-type PTEN, were then subcloned into pCMV-Tag2 vector that introduced an N- terminal FLAG tag. 5 ⁇ g of each construct was transfected into COS7 cells and the activities of Akt and p70S6 kinase following IGF-I stimulation was evaluated. In cells expressing these mutants, two downstream targets of the PI 3-kinase pathway (Akt and p70S6 kinase) were maintained at higher levels of activity than observed in wild-type control cells.
  • Akt and p70S6 kinase two downstream targets of the PI 3-kinase pathway

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Abstract

Cette invention concerne des procédés d'identification de médicaments pour le traitement de la résistance à l'insuline et du diabète.
PCT/US2007/075001 2006-08-01 2007-08-01 Procédés d'identification de modulateurs de la signalisation de l'insuline WO2008016995A2 (fr)

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