WO2006052510A2 - Methods for identifying novel modulators of insulin signaling - Google Patents

Abstract

This invention provides novel cDNAs encoding G6PC expression regulators. The invention also provides methods of using the cDNAs to screen for compounds that modulate insulin signaling related activities such as gluconeogenesis and cell proliferation. The methods comprise first screening test compounds for modulators of a G6PC expression regulator that is encoded by the cDNAs, and then further screening the identified modulating compounds for modulators of insulin signaling related activities. The invention further provides methods and pharmaceutical compositions for treating diseases and conditions associated with abnormal insulin signaling.

Description

METHODS FOR IDENTIFYING NOVEL MODULATORS OF INSULIN

SIGNALING

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent

Application Serial No. 60/623,073 (filed October 28, 2004), the disclosure of which is incorporated herein by reference in its entirety and for all purposes.

FIELD OF THE INVENTION

[0002] The present invention generally relates to methods for identifying modulators of insulin signaling and gluconeogenesis, and to therapeutic applications of such modulators. More particularly, the invention pertains to cDNAs that modulate expression of glucose-6-phosphatase catalytic subunit, and to methods of using such cDNAs to identify novel compounds that regulate gluconeogenesis and insulin signaling.

BACKGROUND OF THE INVENTION

[0003] Insulin activates two major pathways, namely PtdIns-3 -kinase (PBK)-AKT and RAS-MAPK pathway. However, PI3 K-AKT signaling is thought to be the key pathway conferring the regulation of insulin on metabolic processes. Insulin inhibits the glucose-6-phosphatase (G6Pase) expression through the PI3K-AKT pathway. G6Pase catalyzes the terminal step in the gluconeogenic and glycogenolytic pathways, the hydrolysis of glucose-6-phosphate to glucose and phosphate. It plays a critical role in blood glucose homeostasis. Glucose-6-phosphatase activity is conferred by a set of proteins localized to the endoplasmic reticulum, including a glucose-6-phosphate translocase, a G6Pase phosphohydrolase or catalytic subunit (G6PC), and glucose and inorganic phosphate transporters in the endoplasmic reticulum membrane. [0004] G6PC expression is up-regulated in part by members of the forkhead transcription factor family, including FOXOlA (also termed FKHR) in the PI3K-AKT pathway. Stimulation of cells with insulin initiates a signaling cascade that ultimately leads to FOXOlA inactivation and marked down-regulation of G6PC expression levels. Overexpression of the G6PC alone is sufficient to activate flux through the glucose-6- phosphatase system in liver cells. Dysregulation of G6PC could contribute to insulin resistance in non-insulin dependent diabetes. See, Martin et al., J MoI Endocrinol. 29: 205- 22, 2002; Streeper et al., J Biol Chem. 276:19111-8, 2001; Ayala et al., Diabetes. 48: 1885- 9, 1999; and Seoane et al., J Biol Chem. 272(43): 26972-7, 1997. Compounds that modulate G6PC expression provide means of regulating gluconeogenesis and potential treatment of insulin resistance. Molecules that regulate G6PC expression through insulin- mediated inactivation of FOXOl A transcription factor also provide targets for medicinal intervention of the insulin signaling pathway.

[0005] There is a need in the art for better means for modulating insulin signaling related activities such as gluconeogenesis, and for treating diseases and conditions caused by or associated with aberrant insulin signaling and abnormal gluconeogenesis. The present invention addresses this and other needs.

SUMMARY OF THE INVENTION

[0006] In one aspect, the invention provides methods for identifying compounds that modulate insulin signaling-related activities. The methods involve (a) screening test compounds to identify one or more modulating compounds which modulate a glucose 6- phosphatase catalytic subunit (G6PC) expression regulator selected from the members listed in Table 3; and (b) testing the modulating compounds for ability to modulate insulin signaling. In some of these methods, (a) comprises screening the test compounds for ability to modulate expression level of the G6PC expression regulator. In some other methods, (a) comprises screening the test compounds for ability to modulate an enzymatic activity of the G6PC expression regulator.

[0007] In some methods, (b) comprises testing the modulating compounds for ability to modulate expression level of an insulin signaling pathway member. For example, the insulin signaling pathway member can be G6PC. In some of these methods, the modulating compounds identified in (b) up-regulate G6PC expression level. In some other methods, wherein the modulating compounds identified in (b) down-regulate G6PC expression level. In some methods, (b) comprises testing the modulating compounds for ability to modulate an FOXOlA transcription factor. In some of these methods, the modulating compounds are tested for ability to modulate FOXOlA in regulating expression of an insulin signaling-responsive gene. In some methods, the insulin signaling-responsive gene is G6PC. In some methods, the modulating compounds identified in (b) stimulate FOXOlA activity. In some methods, the modulating compounds identified in (b) inhibit FOXOlA activity.

[0008] In a related aspect, the invention provides methods for identifying agents that modulate expression level of glucose 6-phosphatase catalytic subunit (G6PC). These methods entail (a) assaying a biological activity of a G6PC expression-modulating polypeptide in the presence of a test agent to identify one or more modulating compounds that modulate the biological activity of the polypeptide; and (b) testing one or more of the modulating compounds for ability to modulate G6PC expression.

[0009] In some of these methods, the G6PC expression-modulating polypeptide is a kinase and the biological activity is its kinase activity. In some other methods, the G6PC expression-modulating polypeptide is a phosphatase and the biological activity is its phosphatase activity. In some methods, the modulating compounds enhance the biological activity of the G6PC-modulating polypeptide. In some methods, the modulating compounds inhibit the biological activity of the G6PC-modulating polypeptide. In some of the methods, the modulating compounds identified in (b) up-regulate G6PC expression. In some other methods, the modulating compounds identified in (b) down-regulate G6PC expression.

[0010] In some of these methods, (b) comprises testing the modulating compounds for ability to modulate expression of a second polynucleotide under the control of an insulin responsive element of G6PC. The second polynucleotide can encode a reporter polypeptide. In some of these methods, the testing comprises (a) providing a cell or cell lysate that comprises the second polynucleotide that is operably linked to the insulin responsive element; (b) contacting the cell or cell lysate with the test agent; and (c) detecting an increase or decrease in expression of the second polynucleotide in the presence of the test agent compared to expression of the second polynucleotide in the absence of the test agent.

[0011] In another aspect, the invention provides methods for identifying compounds that inhibit tumorigenesis. Such methods involve (a) screening test compounds to identify one or more modulating compounds which modulate a glucose 6-phosphatase catalytic subunit (G6PC) expression regulator selected from the members listed in Table 3; and (b) testing the modulating agent for ability to modulate tumorigenesis. In some methods, the modulating compounds enhance the biological activity of the G6PC- modulating polypeptide. In some of these methods, (a) comprises screening the test compounds for ability to modulate expression level or an enzymatic activity of the G6PC expression regulator. In some methods, (b) comprises testing the modulating compounds for ability to inhibit proliferation of a tumor cell in vitro. [0012] In another aspect, the invention provides methods of modulating gluconeogenesis in a subject. These methods entail administering to the subject a pharmaceutical composition comprising an effective amount of an insulin signaling modulator identified in accordance with the present invention. In some of these methods, the insulin signaling modulator inhibits gluconeogenesis. In some methods, the insulin signaling modulator down-regulates expression level of GP6C.

[0013] In a related aspect, methods for inhibiting tumorigenesis in a subject are provided. These methods entail administering to the subject a pharmaceutical composition comprising an effective amount of a tumorigenesis-inhibiting agent identified in accordance with methods of the invention. Some of the methods are directed to subjects who have been diagnosed to have tumor or are predisposed to developing tumor. [0014] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figures 1 A-IB show that paladin represses insulin-induced AKT phosphorylation: (A) expression of paladin resulted in diminished AKT phosphorylation at both S473 and T308 residues; and (B) mutation in the first but not the second phosphatase domain of paladin abolished its inhibitory effect on AKT phosphorylation. [0016] Figures 2A-2B show results from siRNA knock-down studies which indicate that paladin is a regulator of insulin-induced AKT phosphorylation: (A) transfection of mouse paladin siRNA into C2C12 cells resulted in the presence of reduced paladin transcripts; and (B) knocking-down paladin expression with siRNAs elevated insulin-induced AKT phosphorylation at both S473 and T308 were elevated.

DETAILED DESCRIPTION

I. Overview

[0017] The invention is predicated in part on the discoveries by the present inventors of genes that encode negative modulators of insulin signaling pathway. As detailed in the Example below, utilizing the promoter of glucose-6-phosphatase catalytic subunit (G6PC) as the insulin responsive reporter, the present inventors examined the impact of approximately 20,000 individually arrayed full-length human cDNAs on the insulin signaling pathway. A number of cDNA hits were found to positively regulate the expression of a reporter gene under the control of a G6PC promoter-containing sequence (Table 1). 75 of these genes were confirmed in various reconfirmation studies (Table T). The majority of these molecules apparently up-regulate G6PC expression through the insulin-mediated inactivation of FOXOl A transcription factor. Some of these genes were not previously known or implicated in regulating insulin signaling pathway. As listed in Table 3, these novel insulin-dependent modulators of G6PC expression are herein termed "G6PC-expression regulators."

[0018] The G6PC expression regulators were further subject to a collection of secondary assays. Results from these assays revealed interesting observations of how these molecules interact with insulin signaling network. In addition, to confirm their role as negative modulators of insulin signaling, an exemplary G6PC expression regulator, KIAA1274 (Accession No. XM_166125), was chosen for additional studies. The data generated from these analyses provided extensive biological evidence that KIAA 1274, a homologue of mouse paladin (Accession No. X99384), is indeed a negative modulator of insulin signaling.

[0019] In accordance with these discoveries, the present invention provides methods for identifying compounds that modulate insulin signaling pathway in general and gluconeogenesis in particular. Using compounds thus identified, the invention provides methods for treating various diseases or conditions mediated by or associated with abnormal insulin signaling, e.g., insulin resistance, in human or non-human subjects. Such compounds are also useful to modulate glucose level (e.g., to lower blood glucose level) in subjects with aberrant gluconeogenesis. Further, insulin is a growth factor that induces cell proliferation. Some of the cDNA hits which encode the G6PC expression regulators could be tumor suppressors. They could inhibit insulin-mediated cellular proliferation by interfering with the insulin signaling pathway. Therefore, the invention also provides methods for identifying compounds that can be useful to inhibit tumorigenesis in human and non-human subjects. The following sections provide guidance for making and using the compositions of the invention, and for carrying out the methods of the invention.

II. Definitions

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al, DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991). In addition, the following definitions are provided to assist the reader in the practice of the invention. [0021] The term "agent" or "test agent" or "test compound" includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms "agent", "substance", and "compound" can be used interchangeably. [0022] The term "analog" is used herein to refer to a molecule that structurally resembles a reference molecule but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screening of analogs, to identify variants of known compounds having improved traits (such as higher binding affinity for a target molecule) is an approach that is well known in pharmaceutical chemistry.

[0023] As used herein, "contacting" has its normal meaning and refers to combining two or more molecules (e.g., a test agent and a polypeptide) or combining molecules and cells (e.g., a test agent and a cell). Contacting can occur in vitro, e.g., combining two or more agents or combining a test agent and a cell or a cell lysate in a test tube or other container. Contacting can also occur in a cell or in situ, e.g., contacting two polypeptides in a cell by coexpression in the cell of recombinant polynucleotides encoding the two polypeptides, or in a cell lysate.

[0024] A "heterologous sequence" or a "heterologous nucleic acid," as used herein, is one that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that, although being endogenous to the particular host cell, has been modified. Modification of the heterologous sequence can occur, e.g., by treating the DNA with a restriction enzyme to generate a DNA fragment that is capable of being operably linked to the promoter. Techniques such as site-directed mutagenesis are also useful for modifying a heterologous nucleic acid.

[0025] The term "homologous" when referring to proteins and/or protein sequences indicates that they are derived, naturally or artificially, from a common ancestral protein or protein sequence. Similarly, nucleic acids and/or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of similarity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity is routinely used to establish homology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be used to establish homology.

[0026] A "host cell," as used herein, refers to a prokaryotic or eukaryotic cell to which a heterologous polynucleotide can be introduced. The polynucleotide can be introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, and/or the like. [0027] As used herein, the term "G6PC expression modulator" or "G6PC expression regulator" encompasses novel "G6PC expression-modulating genes" and "G6PC expression-modulating polypeptides." The term specifically refers to the polynucleotides shown in Table 3 and their encoded polypeptides that up-regulate G6PC expression as demonstrated in the Example below.

[0028] The term "sequence identity" in the context of two nucleic acid sequences or amino acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. A "comparison window" refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482; by the alignment algorithm of Needleman and Wunsch (1970) J. MoI. Biol.48:443; by the search for similarity method of Pearson and Lipman (1988) Proc. Nat. Acad. Sci U.S.A. 85:2444; by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC/Gene program by Intelligentics, Mountain View, CA; and GAP, BESTFIT, BLAST, FASTA, or TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.). [0029] Alignment can also be performed by inspection and manual alignment.

Typically, the polypeptides herein are at least 70%, generally at least 75%, optionally at least 80%, 85%, 90%, 95% or 99% or more identical to a reference polypeptide, e.g., a G6PC expression regulator encoded by a polynucleotide in Table 3, e.g., as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids can also be described with reference to a starting nucleic acid, e.g., they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to a reference nucleic acid, e.g., a polynucleotide in Table 3, e.g., as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. A "substantially identical" nucleic acid or amino acid sequence refers to a nucleic acid or amino acid sequence which comprises a sequence that has at least 90% sequence identity to a reference sequence using the programs described above (preferably BLAST) using standard parameters. The sequence identity is preferably at least 95%, more preferably at least 98%, and most preferably at least 99%.

[0030] As used herein, the term "insulin signaling related activity" encompasses any biochemical and physiological response caused or mediated by insulin signaling in regulating glucose homeostasis and regulating carbohydrate, lipid, and protein metabolism. Thus, it includes, e.g., insulin-stimulated receptor tyrosine kinase activity, insulin receptor substrate (IRS) phosphorylation or phosphoinositide (PI)-3 kinase activation, insulin- mediated activation or inactivation of transcription factors (e.g., FOXOlA), and modulation of other gluconeogenesis and glycogenolytic activities (e.g., regulation of G6PC expression). It also encompasses cell growth and proliferation in response to insulin signaling.

[0031] The term "modulate" with respect to a biological activity of a reference protein or its fragment refers to a change in the expression level or other biological activities of the protein. For example, modulation may cause an increase or a decrease in expression level of the reference protein, enzymatic modification (e.g., phosphorylation) of the protein, binding characteristics (e.g., binding to a target polynucleotide), or any other biological, functional, or immunological properties of the reference protein. The change in activity can arise from, for example, an increase or decrease in expression of one or more genes that encode the reference protein, the stability of an mRNA that encodes the protein, translation efficiency, or from a change in other biological activities of the reference protein. The change can also be due to the activity of another molecule that modulates the reference protein (e.g., a kinase which phosphorylates the reference protein). [0032] Modulation of a reference protein can be up-regulation (i.e., activation or stimulation) or down-regulation (i.e. inhibition or suppression). The mode of action of a modulator of the reference protein can be direct, e.g., through binding to the protein or to genes encoding the protein, or indirect, e.g., through binding to and/or modifying (e.g., enzymatically) another molecule which otherwise modulates the reference protein. [0033] The term "subject" includes mammals, especially humans. It also encompasses other non-human animals such as cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys. These subjects are all amenable for treatment with the insulin signaling-modulating compounds that can be identified in accordance with the present invention.

[0034] A "variant" of a reference molecule refers to a molecule substantially similar in structure and biological activity to either the entire reference molecule, or to a fragment thereof. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the sequence of amino acid residues is not identical.

III. Identification of Novel G6PC expression regulators

[0035] The present invention provides novel modulators which up-regulate G6PC expression through insulin mediated FOXOlA signaling pathway. Utilizing an expression vector which expresses a reporter gene under the control of G6PC promoter sequence, a number of cDNAs were identified which up-regulate expression of the reporter gene when the expression vector and the polynucleotides were co-transfected into a host cell. As detailed in the Example below, an arrayed and annotated cDNA library of human genome consisting of approximately 20,000 full length human cDNAs was employed by the present inventors to identify modulators of G6PC expression. These cDNAs were inserted into mammalian expression vectors and screened for hits that would induce expression of a luciferase reporter gene. The reporter gene was placed under the control of G6PC promoter. Because G6PC expression is regulated by the insulin signaling through the FOXOlA transcription factor, an FOXOl A-expression construct was also introduced into the host cell. [0036] After stimulation with insulin and addition of appropriate reagents, luciferase activity in each well was monitored hy quantifying luminescence with a luminescence plate reader. cDNAs exhibiting G6PC expression-modulating activity that is at least 1.5 fold above their respective plate medians were then identified from the library. As shown in Table 1, these genes include molecules that are known to play a role in insulin signaling, e.g., PTEN. However, the majority of the cDNAs identified from the screening are not previously implicated in regulating the insulin signaling pathway. In addition, the majority of the reconfirmed hits were found to regulate G6PC expression in an insulin dependent manner (hits 1-52 in Table 2). Table 3 lists genes which regulate G6PC expression through insulin signaling pathway and which are not previously known to be implicated in regulating insulin signaling. As shown in this Table, the novel G6PC- modulators include very diversified classes of proteins, including kinases, phosphatases, transcription factors, and etc.

Table 1. Primary screening hits which up-regulate G6PC expression

Table 2. Reconfirmed screening hits which modulate G6PC expression

Table 3. Insulin-dependent modulators of G6PC expression ("G6PC expression- regulators")

IV. Screening for Novel Modulators of Insulin Signaling and Gluconeogenesis

[0037] The G6PC expression regulators described above provide novel targets to screen for compounds that modulate insulin signaling and gluconeogenesis. Various biochemical and molecular biology techniques or assays well known in the art can be employed to practice the screening methods of the present invention. Such techniques are described in, e.g., Seethala et al., Handbook of Drug Screening, Marcel Dekker; 1st Ed. (2001); Janzen, High Throughput Screening: Methods and Protocols (Methods in Molecular Biology, 190), Humana Press; 1st Ed. (2002); Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N. Y., 3rd Ed. (2000); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1999). Further guidance to practice the screening methods of the present invention is provided below.

A. Screening scheme

[0038] Typically, test agents or compounds are first assayed for their ability to modulate a biological activity of a G6PC expression regulator encoded by the cDNAs shown in Table 1 ("the first assay step"). Modulating compounds thus identified are then subject to further screening for ability to modulate insulin signaling related activities, typically in the presence of the G6PC expression regulator ("the second testing step"). Depending on the G6PC expression regulator employed in the method, modulation of different biological activities of the G6PC expression regulator can be assayed in the first step. For example, the test agents can be screened for ability to modulate a known biochemical or enzymatic function of the G6PC expression regulator. The test agents can be assayed for activity to modulate expression or cellular level of the G6PC expression regulator, e.g., its transcription or translation. The test agents can also be screened for a specific binding activity to the G6PC expression regulator.

[0039] In some preferred embodiments, the G6PC expression regulator employed in the screening methods is an enzyme (e.g., a kinase or a protease). In these methods, the biological activity monitored in the first screening step is the specific enzymatic activity of the G6PC expression regulator. The substrate to be used in the screening can be a molecule known to be enzymatically modified by the enzyme (e.g., a kinase), or a molecule that can be easily identified from candidate substrates for a given class of enzymes. For example, many kinase substrates are available in the art. See, e.g., www.emdbiosciences.com; and www.proteinkinase.de. In addition, a suitable substrate of a kinase can be screened for in high throughput format. For example, substrates of a kinase may be identified using the Kinase-Glo® luminescent kinase assay (Promega) or other kinase substrate screening kits (e.g., kits developed by Cell Signaling Technology, Beverly, Massachusetts).

[0040] The test agents can be screened for ability to either up-regulate or down- regulate a biological activity of the G6PC expression regulator in the first assay step. Once test agents that modulate the G6PC expression regulator are identified, they are typically further tested for ability to modulate insulin signaling activities, e.g., G6PC expression or tumor suppressing activities. This further testing step is often needed to confirm that their modulatory effect on the G6PC expression regulator would indeed lead to modulation of insulin signaling related activities (e.g., gluconeogenesis or tumorigenesis). For example, a test agent which inhibits a biological activity of a G6PC expression regulator may be further tested in order to confirm that such modulation can result in enhanced or reduced expression of G6PC and gluconeogenesis. Similarly, a test agent which stimulates a biological activity of a G6PC expression regulator that is a tumor suppressor gene can be further tested to confirm that it can lead to suppression of tumorigenesis. [0041] In some embodiments, modulating compounds identified in the first screening step are examined in the second step to identify compounds that specifically inhibit G6PC expression. In some other embodiments, they are screened to identify compounds that enhance G6PC expression. In some of these applications, compounds that have been identified to modulate G6PC expression in the screening system are also examined for their impact on G6PC expression in a host that does not express FOXOlA. This step could confirm the compounds regulate G6PC expression in an FOXOlA- dependent manner.

[0042] • In both the first assaying step and the second testing step, either an intact G6PC expression regulator, or a fragment thereof, may be employed. Analogs or functional derivatives of the G6PC expression regulator could also be used in the screening. The fragments or analogs that can be employed in these assays usually retain one or more of the biological activities of the G6PC expression regulator (e.g., kinase activity if the G6PC expression regulator employed in the first assaying step is a kinase). Fusion proteins containing such fragments or analogs can also be used for the screening of test agents. Functional derivatives of a G6PC expression regulator usually have amino acid deletions and/or insertions and/or substitutions while maintaining one or more of the bioactivities and therefore can also be used in practicing the screening methods of the present invention. A functional derivative can be prepared from a G6PC expression regulator by proteolytic cleavage followed by conventional purification procedures known to those skilled in the art. Alternatively, the functional derivative can be produced by recombinant DNA technology by expressing only fragments of a G6PC expression regulator that retain one or more of their bioactivities.

B. Test compounds

[0043] Test agents or compounds that can be screened with methods of the present invention include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines, oligocarbamates, polypeptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Some test agents are synthetic molecules, and others natural molecules.

[0044] Test agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. Combinatorial libraries can be produced for many types of compound that can be synthesized in a step-by-step fashion. Large combinatorial libraries of compounds can be constructed by the encoded synthetic libraries (ESL) method described in WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503 and WO 95/30642. Peptide libraries can also be generated by phage display methods (see, e.g., WO 91/18980). Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained from commercial sources or collected in the field. Known pharmacological agents can be subject to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs. [0045] Combinatorial libraries of peptides or other compounds can be folly randomized, with no sequence preferences or constants at any position. Alternatively, the library can be biased, i.e., some positions within the sequence are either held constant, or are selected from a limited number of possibilities. For example, in some cases, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, or to purines. [0046] The test agents can be naturally occurring proteins or their fragments. Such test agents can be obtained from a natural source, e.g., a cell or tissue lysate. Libraries of polypeptide agents can also be prepared, e.g., from a cDNA library commercially available or generated with routine methods. The test agents can also be peptides, e.g., peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides can be digests of naturally occurring proteins, random peptides, or "biased" random peptides. In some methods, the test agents are polypeptides or proteins. The test agents can also be nucleic acids. Nucleic acid test agents can be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes can be similarly used as described above for proteins. In some embodiments, the test agents are inhibitory nucleic acids (e.g., siRNAs or antisense nucleic acids) that are directed to the G6PC expression regulators. For example, for a given G6PC expression regulator that is to be employed in the screening methods of the invention (Table 2), test compounds can include siRNAs that are produced to specifically inhibit expression of the gene encoding the G6PC expression regulator. In these embodiments, the siRNAs are first tested for ability to down-regulate expression of the gene encoding the G6PC expression regulator. siRNAs which have been shown to possess such activity are then further screened for ability to modulate G6PC expression or other activities in the insulin signaling pathway.

[0047] In some preferred methods, the test agents are small molecule organic compounds, e.g., chemical compounds with a molecular weight of not more than about 1,000 or 500. Preferably, high throughput assays are adapted and used to screen such small molecules. In some methods, combinatorial libraries of small molecule test agents as described above can be readily employed to screen for small molecule compound modulators of insulin signaling. A number of assays are available for such screening, e.g., as described in Schultz (1998) Bioorg Med Chem Lett 8:2409-2414; Weller (1997) MoI Divers. 3:61-70; Fernandes (1998) Curr Opin Chem Biol 2:597-603; and Sittampalam (1997) Curr Opin Chem Biol 1:384-91.

[0048] Libraries of test agents to be screened with the claimed methods can also be generated based on structural studies of the G6PC expression regulators discussed above or their fragments. Such structural studies allow the identification of test agents that are more likely to bind to the G6PC expression regulators. The three-dimensional structures of the G6PC expression regulators can be studied in a number of ways, e.g., crystal structure and molecular modeling. Methods of studying protein structures using x-ray crystallography are well known in the literature. See Physical Bio-chemistry, Van Holde, K. E. (Prentice- Hall, New Jersey 1971), pp. 221-239, and Physical Chemistry with Applications to the Life Sciences, D. Eisenberg & D. C. Crothers (Benjamin Cummings, Menlo Park 1979). Computer modeling of the G6PC expression regulators' structures provides another means for designing test agents to screen for modulators of insulin signaling. Methods of molecular modeling have been described in the literature, e.g., U.S. Patent No. 5,612,894 entitled "System and method for molecular modeling utilizing a sensitivity factor," and U.S. Patent No. 5,583,973 entitled "Molecular modeling method and system". In addition, protein structures can also be determined by neutron diffraction and nuclear magnetic resonance (NMR). See, e.g., Physical Chemistry, 4th Ed. Moore, W. J. (Prentice-Hall, New Jersey 1972), and NMR of Proteins and Nucleic Acids, K. Wuthrich (Wiley- Interscience, New York 1986).

[0049] Modulators of the present invention also include antibodies that specifically bind to a G6PC expression regulator in Table 1. Such antibodies can be monoclonal or polyclonal. Such antibodies can be generated using methods well known in the art. For example, the production of non-human monoclonal antibodies, e.g., murine or rat, can be accomplished by, for example, immunizing the animal with a G6PC expression regulator in Table 1 or its fragment (See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York). Such an immunogen can be obtained from a natural source, by peptides synthesis or by recombinant expression. [0050] Humanized forms of mouse antibodies can be generated by linking the

CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. See Queen et al., Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989) and WO 90/07861. Human antibodies can be obtained using phage-display methods. See, e.g., Dower et al., WO 91/17271 ; McCafferty et al., WO 92/01047. In these methods, libraries of phage are produced in which members display different antibodies on their outer surfaces. Antibodies are usually displayed as Fv or Fab fragments. Phage displaying antibodies with a desired specificity are selected by affinity enrichment to a G6PC expression regulator in Table 1.

[0051] Human antibodies against a G6PC expression regulator can also be produced from non-human transgenic mammals having transgenes encoding at least a segment of the human immunoglobulin locus and an inactivated endogenous immunoglobulin locus. See, e.g., Lonberg et al., WO93/12227 (1993); Kucherlapati, WO 91/10741 (1991). Human antibodies can be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. Such antibodies are particularly likely to share the useful functional properties of the mouse antibodies. Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be concentrated by affinity purification using a G6PC expression regulator or its fragment.

C. Screening for modulators of G6PC expression regulators

[0052] Typically, test agents are first screened for ability to modulate a biological activity of a G6PC expression regulator as shown in Table 1. A number of assay systems can be employed in this screening step. The screening can utilize an in vitro assay system or a cell-based assay system. The biological activities of a G6PC expression regulator to be monitored in this screening step include its specific binding to the test agents, its expression or cellular level, and other biochemical or enzymatic activities of the G6PC expression regulator. 1. modulating binding activities of G6PC expression regulators

[0053] In some methods, binding of a test agent to a G6PC expression regulator is determined in the first screening step. Binding of test agents to a G6PC expression regulator can be assayed by a number of methods including e.g., labeled in vitro protein- protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.), and the like. See, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168; and also Bevan et al., Trends in Biotechnology 13:115-122, 1995; Ecker et al., Bio/Technology 13:351-360, 1995; and Hodgson, Bio/Technology 10:973-980, 1992. The test agent can be identified by detecting a direct binding to the G6PC expression regulator, e.g., co-immunoprecipitation with the G6PC expression regulator by an antibody directed to the G6PC expression regulator. The test agent can also be identified by detecting a signal that indicates that the agent binds to the G6PC expression regulator, e.g., fluorescence quenching or FRET. [0054] Competition assays provide a suitable format for identifying test agents that specifically bind to a G6PC expression regulator. In such formats, test agents are screened in competition with a compound already known to bind to the G6PC expression regulator. The known binding compound can be a synthetic compound. It can also be an antibody, which specifically recognizes the G6PC expression regulator, e.g., a monoclonal antibody directed against the G6PC expression regulator. If the test agent inhibits binding of the compound known to bind the G6PC expression regulator, then the test agent also binds the G6PC expression regulator.

[0055] Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242-253, 1983); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619, 1986); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, Harlow and Lane, "Antibodies, A Laboratory Manual," Cold Spring Harbor Press, 3^rd ed., 2000); solid phase direct label RIA using ¹²⁵I label (see Morel et al., MoI. Immunol. 25(1):7-15, 1988); solid phase direct biotin-avidin EIA (Cheung et, al., Virology 176:546-552, 1990); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77-82, 1990). Typically, such an assay involves the use of purified polypeptide bound to a solid surface or cells bearing either of these, an unlabeled test agent and a labeled reference compound. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test agent. Usually the test agent is present in excess. Modulating compounds identified by competition assay include agents binding to the same epitope as the reference compound and agents binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference compound for steric hindrance to occur. Usually, when a competing agent is present in excess, it will inhibit specific binding of a reference compound to a common target polypeptide by at least 50 or 75%.

[0056] The screening assays can be either in insoluble or soluble formats. One example of the insoluble assays is to immobilize a G6PC expression regulator or its fragment onto a solid phase matrix. The solid phase matrix is then put in contact with test agents, for an interval sufficient to allow the test agents to bind. After washing away any unbound material from the solid phase matrix, the presence of the agent bound to the solid phase allows identification of the agent. The methods can further include the step of eluting the bound agent from the solid phase matrix, thereby isolating the agent. Alternatively, other than immobilizing the cellular regulator, the test agents are bound to the solid matrix and the G6PC expression regulator is then added. [0057] Soluble assays include some of the combinatory libraries screening methods described above. Under the soluble assay formats, neither the test agents nor the G6PC expression regulator are bound to a solid support. Binding of a G6PC expression regulator or fragment thereof to a test agent can be determined by, e.g., changes in fluorescence of either the G6PC expression regulator or the test agents, or both. Fluorescence may be intrinsic or conferred by labeling either component with a fluorophor. [0058] In some binding assays, either the G6PC expression regulator, the test agent, or a third molecule (e.g., an antibody against the G6PC expression regulator) can be provided as labeled entities, i.e., covalently attached or linked to a detectable label or group, or cross-linkable group, to facilitate identification, detection and quantification of the polypeptide in a given situation. These detectable groups can comprise a detectable polypeptide group, e.g., an assayable enzyme or antibody epitope. Alternatively, the detectable group can be selected from a variety of other detectable groups or labels, such as radiolabels (e.g., ¹²⁵1, ³²P, ³⁵S) or a chemiluminescent or fluorescent group. Similarly, the detectable group can be a substrate, cofactor, inhibitor or affinity ligand.

2. modulating other activities of G6PC expression regulators

[0059] Binding of a test compound to a G6PC expression regulator provides an indication that the agent can be a modulator of the G6PC expression regulator. It also suggests that the agent may modulate insulin signaling and gluconeogenesis through, e.g., binding to and modulating the G6PC expression regulator. Thus, a test compound that binds to a G6PC expression regulator can be further tested for ability to modulate G6PC expression and gluconeogenesis (i.e., in the second testing step outlined above). Alternatively, a test agent that binds to a G6PC expression regulator can be further examined to determine whether it modulates another biological activity (e.g., an enzymatic activity) of the G6PC expression regulator. The existence, nature, and extent of such modulation can be tested by an activity assay as detailed below. Such an activity assay can confirm that the test agent binding to the G6PC expression regulator indeed modulates the G6PC expression regulator. More often, such activity assays can be used independently to identify test agents that modulate activities of a G6PC expression regulator (i.e., without first assaying their ability to bind to the G6PC expression regulator). [0060] In general, the methods involve adding a test agent to a sample containing a

G6PC expression regulator in the presence or absence of other molecules or reagents which are necessary to test a biological activity of the G6PC expression regulator (e.g., enzymatic activity if the G6PC expression regulator is an enzyme), and determining an alteration in the biological activity of the G6PC expression regulator. Preferably, G6PC expression modulators that are kinases or phosphatases are employed in the screening methods. Examples of kinases include those encoded by polynucleotides with the following accession numbers in Table 1: NM_005027, NM_014216, XM_001416, XM_039010, NM_002953, NM_030662, NM_003646, NM_021135, NM_OOO788, NM_016542, and NM_012408. Polynucleotides encoding protein tyrosine phosphatases in Table 1 include those with accession numbers XMJ 66125 (KIAA1274), NM_130435, NM_130846, NM_006504, NM_030670, NM_002848, NM_002849, NM_O80840, and NM_002836. Also included are dual specificity phosphatases encoded by polynucleotides with accession numbers XM_037430, NM_001394, NM_001946, XM_039625, NM_004417, NM_007207, NM_022652, NM_144728, and NM_004419. Additional preferred G6PC expression regulators to be used in the screening methods include some kinase-associated proteins such as those encoded by NM_003581, NMJ324702, XM_039010, XMJ)44015, and NM_003974, as well as deacetylase encoded by NM_030593. [0061] Many of these preferred G6PC expression modulators are well known and characterized in the art, e.g., kinases (e.g., encoded by NM_014216, NM_021135 or NM_030662 in Table 1) or a phosphatases (e.g., encoded by XMJ 66125, NM_007207, NM_001946, NM_006504 in Table 1). Methods for assaying the enzymatic activities of these G6PC expression regulators are all routinely practiced in the art. For example, phosphatase activity of DUSP6 (encoded by NM_001946 in Table 1) can be assayed as described in, e.g., Kim et al., J. Biol. Chem., 278:37497-37510, 2003; Muda et al., J. Biol. Chem., 271(8):4319-26, 1996; and Groom et al., EMBO J., 15:3621-3632, 1996. Similarly, kinase activity of ITPKl (encoded by NM_014216 in Table 1) can be monitored using methods reported in Wilson et al., J. Biol. Chem., 271(20): 11904-10, 1996; and Yang et al., Biochem. J., 351:551-5, 2000. Activities of the other enzymes in Table 1 can also be examined using assays that are known in the art.

[0062] In addition to assays for screening agents that modulate enzymatic or other biological activities of a G6PC expression regulator, the activity assays also encompass in vitro screening and in vivo screening for alterations in expression level of the G6PC expression regulator. These assays can be performed using methods well known and routinely practiced in the art, e.g., Samrbook et al., supra; and Ausubel et al., supra.

D. Testing modulating compounds for effect on insulin signaling related activities

[0063] Once a modulating agent has been identified to bind to a G6PC expression regulator and/or to modulate a biological activity (including expression level) of the G6PC expression regulator, it can be further tested for activities in modulating insulin signaling related activities (e.g., gluconeogenesis). These include assaying activities or expression levels of insulin signaling pathway members. For example, in some embodiments, the modulating compounds are tested for ability to stimulate or inhibit FOXOlA activation. In some other embodiments, they are tested for ability to modulate G6PC expression level. Typically, this screening step is performed in the presence of the G6PC expression regulator on which the modulating agent acts. Preferably, this screening step is performed in vivo using cells that endogenously express the G6PC expression regulator. As a control, effect of the modulating compounds on insulin signaling or gluconeogenesis of a cell that does not express the G6PC expression regulator can also be examined. When the screening methods are directed to identifying compounds that inhibit or enhance G6PC expression through regulating FOXOlA, FOXOlA is typically also present in the cell. FOXOlA can be expressed either endogenously by the host cell or from a separate expressed vector that has been introduced into the host cell. For example, FOXOlA can be expressed from a commercially obtainable vector that has been introduced in to the cell, as exemplified in the Example below.

[0064] To monitor activity of the modulating compounds on G6PC expression, typically a vector bearing a G6PC transcription regulatory element operably linked to a reporter gene (e.g., a luciferase gene) is employed. The vector can also contain other elements necessary for propagation or maintenance in the host cell, and elements such as polyadenylation sequences and transcriptional terminators to increase expression of reporter genes or prevent cryptic transcriptional initiation elsewhere in the vector. Such vectors can be prepared using only routinely practiced techniques and methods of molecular biology (see, e.g., Sambrook et al. and Ausubel et al., supra). Alternatively, expression vectors containing a reporter gene under the control of a transcription regulatory element of a G6PC gene can also be obtained commercially (e.g., from OriGene as described in the Example below).

[0065] Various G6PC transcription regulatory elements can be employed in the present invention, e.g., a G6PC promoter sequence or a G6PC enhancer element such as an insulin responsive sequence. Preferably, transcription regulatory elements from a human G6PC gene are used. However, G6PC genes or their promoter sequences from other species may also be used to monitor test agents' activity in regulating G6PC expression. Many G6PC genes from various species are known and well characterized (see, e.g., accessionnumbers NM_138387, NM_021176, NM_000151, NM_008061, NM_021331, NM_013098, and D78592). The insulin responsive elements of the G6PC gene have been identified (see, e.g., Vander Kooi et al., J. Biol. Chem. 278:11782-93, 2003). [0066] The expression vector can be transfected into any mammalian cell line (e.g.,

CHO-Kl cell line as described in the Example). Preferably, the host cell does not express the reporter gene endogenously. The G6PC expression regulator with which the modulating compounds are identified in the first screening step can be either expressed endogenously by the cell or expressed from second expression vector. General methods of cell culture, transfection, and reporter gene assay have been described in the art, e.g., Ausubel, supra; and Transfection Guide, Promega Corporation, Madison, WI (1998). Other readily transferable mammalian cell line can also be employed in this screening step, e.g., HEK 293, MCF-7, and HepG2 cell lines.

[0067] When inserted into the appropriate host cell, the G6PC promoter or enhancer sequence (e.g., an insulin responsive sequence) induces transcription of the reporter gene by host RNA polymerases. Reporter genes typically encode polypeptides with an easily assayed enzymatic activity that is naturally absent from the host cell. Typical reporter polypeptides for eukaryotic promoters include firefly or Renilla luciferase, chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, alkaline phosphatase, and green fluorescent protein (GFP).

[0068] Transcription driven by G6PC promoter or enhancer sequences may also be detected by directly measuring the amount of RNA transcribed from the reporter gene. In these embodiments, the reporter gene may be any transcribable nucleic acid of known sequence that is not otherwise expressed by the host cell. RNA expressed from constructs containing a G6PC promoter or enhancer may be analyzed by techniques known in the art, e.g., reverse transcription and amplification of mRNA, isolation of total RNA or poly A⁺ RNA, northern blotting, dot blotting, in situ hybridization, RNase protection, primer extension, high density polynucleotide array technology and the like. These techniques are all well known and routinely practiced in the art.

[0069] Other than identifying compounds that modulate G6PC expression or activities of other insulin signaling pathway members (e.g., activation of FOXOlA), some of the screening methods are directed to identifying anti-tumor compounds. In such methods, the G6PC expression modulator employed in the first screening step is a tumor suppressor which can inhibit insulin-mediated cellular proliferation by interfering with insulin signaling pathway. In these methods, test compounds are first screened to identify compounds which modulate (e.g., stimulate) a biological activity (e.g., an enzymatic activity) of the G6PC expression modulator as described above. The modulating compounds thus identified are then examined in the second screening step for antitumor activities. Typically, the compounds are examined for ability to inhibit proliferation of a tumor cell in vitro. Preferably, this screening step is performed using cells that endogenously express the G6PC expression modulator. As a control, cytotoxicity of the modulating compounds on cells that do not express the cellular regulator can also be examined.

[0070] A variety of human tumor cell lines can be employed in this screening step, e.g., osteosarcoma cell line U2OS or glioblastoma cell line U373. Other tumor cell lines are available in the art, e.g., from American Type Culture Collection (Manassas, VA). Antitumor cytotoxicity of the compounds can be monitored by measuring the IC₅₀ value (i.e., the concentration of a compound which causes 50% cell growth inhibition) of each of the modulating compounds. Preferably, an antitumor agent identified from this screening step will have an IC₅₀ value less than lμM on one or more of the tumor cell lines. More preferably, the IC₅0 value of antitumor agents identified in accordance with the present invention is less than 25OnM. Some of the antitumor agents have an IC₅₀ value of less than 5OnM, less than 1OnM on at least one tumor cell line. Most preferably, the antitumor agents obtained from this screening step will have an IC5₀ value that is less than InM.

V. Therapeutic Applications

[0071] The present invention provides novel methods and compositions for modulating insulin signaling related activities, e.g., gluconeogenesis and cell proliferation.

These methods can be used either in vitro or in vivo to modulate (e.g., to increase) insulin sensitivity and/or to modulate glucose output by the liver cells. The methods also find application in treating a disease characterized by dysfunctional insulin signaling (e.g., resistance, inactivity or deficiency) and/or excessive glucose production. Modulation of insulin signaling related activities with the novel compounds of the present invention is also useful for preventing or modulating the development of such diseases or disorders in a subject.

[0072] A great number of diseases and conditions are amenable to treatment with methods and compositions of the present invention. Such diseases include, but are not limited to diabetes, hyperglycemia, obesity, and glycogen storage disease. For example, compounds that modulate gluconeogenesis through regulating G6PC expression can also be employed to treat insulin resistance in type II diabetes. Type II diafcetes is caused by faulty regulation of glucose metabolism and characterized by the initial development of insulin resistance, i.e. diminution in the ability of the cells to respond adequately to insulin. Elevated G6Pase activity is implicated in type II diabetes. Compounds which down- regulate G6PC expression level are useful to treat or prevent the development of type II diabetes and hyperglycemia in a subject.

[0073] Obesity in humans and rodents is also commonly associated with insulin resistance. Before the development of diabetes, many obese patients develop a peripheral resistance to the actions of insulin. It was suggested that increased activities of key enzymes of pathways normally depressed by insulin contributes to insulin-resistance in obesity (Belfiore et al., Int J Obes 3:301-23, 1979). This failure of insulin to depress enzymes of catabolic pathways manifests itself in enhanced basal lipolysis in adipose tissue, increased amino acid release from muscle, and elevation in the activity of key gluconeogenic enzymes in the liver. Compounds which modulate (e.g., inhibit) gluconeogenesis can be employed to treat or prevent such disorders and conditions. [0074] Similarly, novel compounds that modulate gluconeogenesis are also useful to treat glycogen storage diseases. Glycogen metabolism in the liver plays a major role in the homeostatic regulation of blood glucose levels. Glycogen storage diseases are known to be the result of genetic defects within the group of enzymes and transport proteins required by glycogen metabolism. Glycogen storage disease Type Ice (GSD, also known as yon Gierke disease) is defined as the deficiency of glucose-6-phosphatase which is normally present in liver, kidney, and intestine. Thus, compounds which modulate (e.g., enhance) G6PC expression can be employed to treat subjects with these diseases. [0075] In some therapeutic applications of the present invention, therapeutic effects are monitored by measuring circulating glucose level in the subject before and/or after administering a compound that modulate insulin signaling pathway. Glucose level in the subject can be measured with methods well known in the art. For example, blood glucose levels can be measured very simply and quickly with many commercially available blood glucose testing kits.

[0076] The insulin signaling-modulating compounds of the present invention can be directly administered under sterile conditions to the subject to be treated. The modulators can be administered alone or as the active ingredient of a pharmaceutical composition. Therapeutic composition of the present invention can be combined with or used in association with other therapeutic agents. For example, a subj ect may be treated with a compound along with other conventional anti-diabetes drugs. Examples of such known anti-diabetes drugs include Actos (pioglitizone, Takeda, Eli Lilly ), Avandia (rosiglitazone, Smithkline Beacham), Amaryl (glimepiride, Aventis), Glipizide Sulfonlyurea (Generic) or Glucotrol (Pfizer), Glucophage (metformin., Bristol Meyers Squibb), Glucovance (glyburide/metformin, Bristol Meyers Squibb), Glucotrol XL (glipizide extended release, Pfizer), Glyburide (Micronase; Upjohn, Glynase; Upjohn, Diabeta; Aventis), Glyset (miglitol, Pharmacia & Upjohn), Metaglip (glipizide + metformin; fixed combination tablet), Prandin (repaglinide, NOVO), Precose (acarbose, Bayer), Rezulin (troglitazone, Parke Davis), and Starlix (nateglinide, Novartis). [0077] Further, some of the G6PC expression modulators disclosed in the present invention are tumor suppressors. Compounds that modulate (e.g., stimulate) a biological activity of these tumor suppressors and inhibit tumorigenesis can be used to treat subjects with tumors. Examples of tumors that can be treated with methods and compositions of the present invention include various forms of tumors. The antitumor compounds of the present invention can be used alone or used in association with other therapeutic agents. For example, a subject may be treated concurrently with conventional chemotherapeutic agents, particularly those used for tumor and cancer treatment. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamme, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycofbrmycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5- FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., pp. 1206-1228, Berkow et al., eds., Rahay, N.J., 1987).

[0078] Pharmaceutical compositions of the present invention typically comprise at least one active ingredient together with one or more acceptable carriers thereof. Pharmaceutically carriers enhance or stabilize the composition, or to facilitate preparation of the composition. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered (e.g., nucleic acid, protein, modulatory compounds or transduced cell), as well as by the particular method used to administer the composition. They should also be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. This carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral, sublingual, rectal, nasal, or parenteral. For example, the antitumor compound can be complexed with carrier proteins such as ovalbumin or serum albumin prior to their administration in order to enhance stability or pharmacological properties.

[0079] There are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20^th ed., 2000). Without limitation, they include syrup, water, isotonic saline solution, 5% dextrose in water or buffered sodium or ammonium acetate solution, oils, glycerin, alcohols, flavoring agents, preservatives, coloring agents starches, sugars, diluents, granulating agents, lubricants, and binders, among others. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

[0080] The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100% by weight. Therapeutic formulations are prepared by any methods well known in the art of pharmacy. See, e.g., Gilman et al., eds., Goodman and Gilman's: The Pharmacological Bases of Therapeutics , 8th ed., Pergamon Press, 1990; Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20^th ed., 2000; Avis et al., eds., Pharmaceutical Dosage Forms: Parenteral Medications, published by Marcel Dekker, Inc., N.Y., 1993; Lieberman et al., eds., Pharmaceutical Dosage Forms: Tablets, published by Marcel Dekker, Inc., N. Y., 1990; and Lieberman et al., eds., Pharmaceutical Dosage Forms: Disperse Systems, published by Marcel Dekker, Inc., N.Y., 1990. [0081] The therapeutic formulations can be delivered by any effective means that can be used for treatment. Depending on the specific antitumor agent to be administered, the suitable means include oral, rectal, vaginal, nasal, pulmonary administration, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) infusion into the bloodstream. For parenteral administration, antitumor agents of the present invention may be formulated in a variety of ways. Aqueous solutions of the modulators may be encapsulated in polymeric beads, liposomes, nanoparticles or other injectable depot formulations known to those of skill in the art. Additionally, the compounds of the present invention may also be administered encapsulated in liposomes. The compositions, depending upon its solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension. The hydrophobic layer, generally but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such a diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature. [0082] The therapeutic formulations can conveniently be presented in unit dosage form and administered in a suitable therapeutic dose. A suitable therapeutic dose can be determined by any of the well known methods such as clinical studies on mammalian species to determine maximum tolerable dose and on normal human subjects to determine safe dosage. Except under certain circumstances when higher dosages may be required, the preferred dosage of an antitumor agent of the present invention usually lies within the range of from about 0.001 to about 1000 mg, more usually from about 0.01 to about 500 mg per day. The preferred dosage and mode of administration of an antitumor agent can vary for different subjects, depending upon factors that can be individually reviewed by the treating physician, such as the condition or conditions to be treated, the choice of composition to be administered, including the particular antitumor agent, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the chosen route of administration. As a general rule, the quantity of an antitumor agent administered is the smallest dosage which effectively and reliably prevents or minimizes the conditions of the subjects. Therefore, the above dosage ranges are intended to provide general guidance and support for the teachings herein, but are not intended to limit the scope of the invention.

EXAMPLES

[0083] The following examples are offered to illustrate, but not to limit the present invention.

Example 1. Screening human cDNAs for G6PC expression modulators [0084] This Example describes identification of various insulin signaling- modulating polypeptides that regulate expression of a reporter gene under the control of an insulin-responsive sequence element derived from the promoter sequence of the Glucose 6- phosphatase catalytic subunit (G6PC). G6PC encodes for an enzyme that is required for hepatic glucose production and whose expression is up-regulated in part by members of the forkhead (FOXOlA) family of transcription factors. Stimulation of cells with insulin initiates a signaling cascade that ultimately leads to FOXOlA inactivation and marked down-regulation of G6PC expression levels. Utilization of a G6PC promoter-containing reporter provides an effective method for detecting this insulin response at the transcriptional level. The assay we employed examines the ability of a negative modulator of insulin signaling, upon forced expression, to restore the GόPase promoter activity in the presence of insulin. Subsequently, the assay was optimized in Chinese Hamster Ovary cells (CHO-Kl), which is responsive to insulin stimulation as demonstrated by AKT phosphorylation. Importantly, CHO-Kl cells express insignificant amount of FOXOl and the G6Pase promoter cannot be activated without exogenous expression of FOXOl (see supplementary data). Therefore, the screen is biased towards FOXO pathway. [0085] Specifically, an arrayed and annotated cDNA library in a mammalian expression vector was interrogated for modulators of FOXOl A-mediated insulin signaling as follows. The library, consisting of approximately 20,000 full-length human cDNAs (Human Full-Length "Clone Collection", OriGene Technologies, Rockville, MD), was spotted in 384-well micro assay plates such that each well contained an individual cDNA with known identity. The insulin-responsive luciferase reporter was constructed by inserting the promoter sequence of human G6PC (GenBank accession # NM_000151) upstream of the luciferase coding sequence in the pGL3 -Basic vector (Promega, Madison, WI). In a semi-automated process, a co-transfection mixture comprised of G6PC promoter-containing reporter, a vector expressing human FOXOlA (GenBank accession # NM_002015) obtained from Origene (a pCMV6-XL4 based vector), and FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN) were applied to each well containing a single, pre-spotted human cDNA clone. CHO-Kl (Chinese Hamser Ovary) cells (American Type Culture Collection, Manassas, VA) were then introduced into each well to complete the transfection procedure. The FOXOlA expression construct was introduced into the host cell because endogenous expression level of FOXOlA in the CHO-Kl cell is very low.

[0086] After 20 hours of incubation at 37 C, 5% CO2, cells were serum starved for an additional 8 hours before being stimulated by human recombinant insulin (500 nM, Sigma-Aldrich, St. Louis, MO) and dibutyryl-cAMP (2 mM, Roche Applied Science, Indianapolis, IN). Approximately 24 hours after stimulation, 40 μl of Bright-Glo reagent (Promega, Madison, WI) was added to each well and relative luminescence was quantitated using an Acquest (LJL Biosystems, Sunnyvale, CA) plate reader. After executing the assay, 161 cDNAs exhibiting insulin signaling-modulatory activity values > 1.5 fold above their respective plate medians were identified from the library (Table 1). [0087] Validity of this screen is authenticated by the presence in the screen hits of genes known to down-regulate insulin signaling. For example, phosphatase and tensin homolog (PTEN) has been demonstrated to negatively modulate insulin signaling by both gain-of-function and loss-of-function studies. In addition, both PI3K p85 alpha and PI3K p85 beta knock-out mice have shown improvement in insulin sensitivity. Recently, it has been demonstrated that the concentration of p85 subunit is essential for regulating the activity of PBK. It was observed that the forced expression of PI3K p85 reduces the insulin induced AKT phosphorylation. Furthermore, it was published that the expression of phosphoprotein enriched in astrocytes 15 (PEA 15) was up-regulated in type 2 diabetes mellitus and the transgenic animal expressing PEAl 5 causes diabetes by affecting both insulin sensitivity and insulin secretion. Moreover, genes such as NCK2 and D0K2 have been shown to interact with insulin receptor substrate- 1 (IRS-I) and insulin receptor (IR) respectively.

Example 2. Additional assays of the G6PC expression modulators [0088] A number of genes from the above described screening have not been known or implicated in insulin signaling. These include phosphatases, kinases, adaptor proteins, GTPase activating proteins (GAPs), transcription regulators/and novel genes. Interestingly, several members of two distinct phosphatase families, namely protein tyrosine phosphatase receptor type (Ptpr) family and MAP kinase phosphatase family (MKP), showed potential of inhibiting insulin signaling. To this end, we included other members of the two families in the collection that were picked from the primary screen hit according to the screen activity, novelty, and the presence of distinct domains. The final collection consists of 75 genes which are shown in Table 2. These novel G6PC expression modulators were subjected to further analysis.

[0089] It is possible that some of these G6PC expression modulators may re¬ activate GόPase promoter independent of insulin signaling. To address this concern, we first examined the disruption of insulin signaling by measuring the degree of GόPase promoter repression upon insulin stimulation. The results indicate that, when tubulin (a protein which is required for insulin release) was expressed, insulin was able to exert approximately 7 fold of repression on the GόPase promoter activity. However, the expression of GrblO or PTEN, both known inhibitors of insulin signaling, inhibited insulin dependent repression of GόPase promoter activity and resulted in only 1.5 fold and 3 fold of insulin induced repression respectively. With this approach, we were able to identify novel modulators of G6PC expression which act through an insulin dependent mechanism after the statistical analysis. These genes are listed in Table 3. [0090] Next, we aimed to evaluate at which part of insulin signaling do these targets act on. Since the primary screen is biased towards FOXOl pathway, we suspected that most of the novel G6PC expression modulators should affect the insulin regulated FOXOl transcriptional activity. To test this hypothesis, we generated a FOXO dependent reporter (IRE-4U) which contains four FOXO binding motifs of GδPase promoter. It was found that, when tubulin was expressed, insulin induced 3 fold of repression on IRE-4U. However, while the expression of GrblO completely abrogated insulin's repression on FOXO transcription activity, the expression of PTEN was able to limit insulin induced repression at 1.5 fold. To this end, we were able to determine that most of these genes down regulate insulin signaling were acting through the FOXOl dependent mechanism. [0091] It is well documented that insulin regulates FOXOl transcriptional activity predominately through AKT, we therefore investigated the perturbation of insulin induced AKT phosphorylation upon cDNA expression. Utilizing high throughput micro-ELSA technology, which detects the total AKT and phospho-AKT (S473) simultaneously, we were able to quantify the kinetics of AKT phosphorylation and monitor the effects of expressed cDNAs over a period of 24 hours. We observed that the expression of PTEN, GrblO, PI3Kp85α, PI3Kp85β, and Ptpra, which were shown to perturb insulin signaling, inhibited AKT phosphorylation as expected at all time points examined. Interestingly, we found several genes that down-regulated insulin induced AKT phosphorylation but have not been implicated in insulin signaling (see supplementary material for complete data). For example, the forced expression of KIAAl 274, a putative tyrosine phosphatase with unknown biological function, exhibited a comparative potency in hampering AKT phosphorylation as that of GrblO. Similarly, the expression of active BCR-related gene (ABR), a GTPase activating protein, also repressed the insulin induced AKT phosphorylation. However, there are genes that affect insulin signaling without inhibiting AKT phosphorylation. The dual specificity phosphatase 10 (DUSPlO), for example, did not interfere with the AKT phosphorylation at S473 but overcame insulin's repressive effect on FOXOl transcriptional activity.

[0092] From these studies, it is clear that numerous genes were able to override insulin regulated FOXOl transcription activity bypassing AKT phophorylation at S473. Interestingly, several members of DUSPs (DUSPl, 4, 6, 7, 10) are included in this category. These DUSPs have been characterized as MAP kinase phosphatases and exhibit different binding affinity to various MAP kinases. Importantly, DUSP6 and DUSP7 were both shown to overcome insulin's repression on PEPCK promoter. Knowing that the phosphorylation of S473 precedes and is crucial to the subsequent phosphorylation of T308, it is likely that similar results will be observed with T308 phosphorylation.

Example 3. Characterization of an exemplary G6PC expression modulator in regulating insulin signaling pathway

[0093] This Example describes results from additional studies of an exemplary

G6PC expression modulator, paladin, in regulating insulin signaling pathway. The secondary assays described above indicate that human homologue of mouse paladin, KIAA1274 (a putative tyrosine phosphatase), inhibits insulin induced AKT phosphorylation and thereby reactivates FOXOl dependent transcription. The subsequent analysis of protein sequence revealed two conserved phosphatase motifs that consist of CXXGXGR and an N-terminal myristylation site. In addition, we found that both phosphatase domains are highly conserved among species. [0094] To determine whether the expression of paladin also affects AKT phosphorylation at T308, we repeated the high throughput AKT phosphorylation experiments and subjected cell lysate to western blot analysis. As shown in Fig. IA, the expression of paladin resulted in diminished AKT phosphorylation at both S473 and T308 and at every time point tested. Next, we were curious about the role of paladin phosphatase domains in inhibiting AKT phosphorylation. To this end, we converted the cysteine residue in each phosphatase domain to serine and created defective phosphatase domains. These constructs were then introduced into COS7 cells, and the extent of AKT phosphorylation was monitored by phospho-AKT micro-ELISA mentioned earlier. Interestingly, while the wild type paladin represses AKT phosphorylation, the mutation in the first (PALD Ml) or both phosphatase domains (PALD M 1+2) restored AKT phosphorylation, though not completely. On the contrary, PALD M2 exhibited the same degree of repression on AKT phosphorylation as the PALD WT (Fig. IB). Therefore, we concluded that the first phosphatase domain is important in paladin's activity. [0095] To clarify the biological relevance of paladin in regulating insulin induced

AKT phosphorylation, we proceed to knock down paladin expression by siRNA in C2C12 cells. As shown in the left panel of Fig. 2 A, the transfection of mouse paladin siRNA Smartpool and two individual siRNAs from the pool into C2C12 cells resulted in 60-70% reduction of paladin transcripts as measure by quantitative PCR (qPCR). These transfected cells were then treated with insulin at various time points. After western blot analysis, we found that by knocking down paladin expression with all presented siRNAs, insulin induced AKT phosphorylation at both S473 and T308 were elevated (Fig. 2B). The data indicated that paladin is indeed a regulator of insulin induced AKT phosphorylation. [0096] Since overexpression of paladin resulted in the inhibition of both S473 and

T308 phosphorylation on AKT, it is likely that paladin is targeting an upstream component in insulin signaling. It is possible that paladin could be modulating insulin receptor's activity since it is a putative tyrosine phosphatase. To test this possibility, we stripped the membrane used in Fig. IB and reprobed with phospho-tyrosine specific antibody targeting Yl 162/Yl 163 of insulin receptor (IR) to assay changes in insulin receptor tyrosine phosphorylation. Here, the results clearly demonstrated that the forced expression of paladin inhibits IR phosphorylation at Yl 162/Yl 163. Moreover, we also observed a faster migration of IR upon paladin expression. Interestingly, in paladin transfected samples, the protein level of IR is lower and decreased at a faster rate than that of the controls upon insulin treatment. This observation is unlikely to result from the inconsistent loading, since the level of total AKT is fairly constant (Fig. IB).

[0097] Observing that Cys→Ser mutations in phosphatase domains of paladin exhibit differential capacity to inhibit AKT phosphorylation, we were curious if the same trend could be observed with IR phosphorylation. To this end, we transfected wild type and mutant constructs of paladin into COS 7 cells and monitor the insulin receptor phosphorylation. Interestingly, we found that degree of the IR phosphorylation corresponds well to that of AKT phosphorylation in that both PALD-WT and PALD-M2 inhibit IR phosphorylation but not PALD-Ml and PALD-M1+2. Furthermore, we also observed that the abundance of IR is consistent with the level of IR phosphorylation in that PALD-Ml and PALD-M 1+2 transfected samples exhibit similar abundance of IR as that of the control, while PALD-WT and PALD-M2 transfected cells display decreased level of IR. To determine whether this observation is an experimental artifact due to overexpression, we proceed to knock-down paladin with siRNA in C2C12 cells. Interestingly, we found that IR phosphorylation is increased after knocking-down paladin, and that the increment in phosphorylation correlates with the increased IR protein level. [0098] To assess whether paladin and IR exist in the same protein complex, we expressed myc tagged paladin and ABR, both were found to inhibit AKT phosphorylation, and proceed with the co-immunoprecipitation experiments. Cell extracts were immunoprecipitated with antibody against IR β subunit (α-IRβ) then immunoblotted with either α-Myc antibody or α-IRβ. The results show that α-IR β precipitated similar amount of IR from the ABR and paladin transfected samples. However, immunoblotting with α- Myc revealed that paladin interacts with IR complex but not ABR. Finally, we devised a system to measure changes in insulin sensitivity when paladin is knocked down. To this end, we established H4IIE cell line stably integrated with G6Pase reporter (H4IIE- GόPAse). The sensitivity to insulin can be represented by the fold of repression that insulin exerts on the G6Pase promoter activity. Subsequently, three siRNAs targeting rat paladin were designed and transfected individually into H4IIE-G6Pase by electroporation. The degree of knock-down by each siRNA was assayed by qPCR. The data indicate that the only siRNA exhibiting effective knock-down is PALD-3. Interestingly, it was found that the efficiency of siRNA knock-down corresponded well with the improved insulin sensitivity. In conclusion, the data strongly indicated that the absence of paladin can indeed increase insulin sensitivity.

[0099] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. US2005/039304

[00100] AU publications, GenBank sequences, ATCC deposits, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes as if each is individually so denoted.

Claims

WE CLAIM:

1. A method for identifying an agent that modulates insulin signaling, the method comprising:

(a) screening test compounds to identify one or more modulating compounds which modulate a glucose 6-phosphatase catalytic subunit (G6PC) expression regulator selected from the members listed in Table 3; and

(b) testing the modulating compounds for ability to modulate insulin signaling.

2. The method of claim 1, wherein (a) comprises screening the test compounds for ability to modulate expression level of the G6PC expression regulator.

3. The method of claim 1 , wherein (a) comprises screening the test compounds for ability to modulate an enzymatic activity of the G6PC expression regulator.

4. The method of claim 1, wherein the identified modulating compounds up- regulate expression level or an enzymatic activity of the G6PC expression regulator.

5. The method of claim 1 , wherein the identified modulating compounds down-regulate expression level or an enzymatic activity of the G6PC expression regulator.

6. The method of claim 1 , wherein the G6PC expression regulator is

KIAA1274.

7. The method of claim 1, wherein (b) comprises testing the modulating compounds for ability to modulate expression level of an insulin signaling pathway member.

8. The method of claim 7, wherein the insulin signaling pathway member is G6PC.

9. The method of claim 8, wherein the modulating compounds are tested for ability to up-regulate G6PC expression level.

10. The method of claim 8, wherein the modulating compounds are tested for ability to down-regulate G6PC expression level.

11. The method of claim 1 , wherein (b) comprises testing the modulating compounds for ability to modulate an FOXOlA transcription factor.

12. The method of claim 11, wherein the modulating compounds are tested for ability to modulate FOXOlA in regulating expression of an insulin signaling-responsive gene.

13. The method of claim 12, wherein the insulin signaling-responsive gene is G6PC.

14. The method of claim 12, wherein the modulating compounds identified in (b) stimulate FOXOlA activity.

15. The method of claim 12, wherein the modulating compounds identified in (b) inhibit FOXOlA activity.

16. A method for identifying an agent that modulates expression level of glucose 6-phosphatase catalytic subunit (G6PC), the method comprising:

(a) screening test compounds to identify one or more modulating compounds which modulate a glucose 6-phosphatase catalytic subunit (G6PC) expression regulator selected from the members listed in Table 3, or a non-human homolog of said G6PC expression regulator; and

(b) testing the modulating compounds for ability to modulate G6PC expression.

17. The method of claim 16, wherein the identified modulating compounds modulate expression level of the G6PC expression regulator.

18. The method of claim 16, wherein the identified modulating compounds modulate an enzymatic activity of the G6PC expression regulator.

19. The method of claim 16, wherein the G6PC expression regulator is KIAAl 274 or its mouse homolog paladin.

20. The method of claim 16, wherein the G6PC expression regulator is a kinase and the biological activity is its kinase activity.

21. The method of claim 16, wherein the G6PC expression-modulating polypeptide is a phosphatase and the biological activity is its phosphatase activity.

22. The method of claim 16, wherein (b) comprises testing the modulating compounds for ability to modulate expression of a second polynucleotide under the control of an insulin responsive element of G6PC.

23. The method of claim 22, wherein the second polynucleotide encodes a reporter polypeptide.

24. The method of claim 22, wherein the testing comprises: providing a cell or cell lysate that comprises the second polynucleotide that is operably linked to the insulin responsive element; contacting the cell or cell lysate with a modulating compound; and detecting an increase or decrease in expression of the second polynucleotide in the presence of the modulating compound compared to expression of the second polynucleotide in the absence of the modulating compound.