WO2002055664A2 - Modulating insulin receptor signaling - Google Patents

Abstract

Human ISM genes are identified as modulators of INR signaling and thus are therapeutic targets for disorders associated with defective INR signaling. Methods for identifying modulators of ISM, comprising screening for agents that modulate the activity of ISM are provided.

Description

MODULATING INSULIN RECEPTOR SIGNALING

This application claims priority to U.S. Provisional Application Nos.: 60/261,335, 60/261,532, 60/261,590, 60/261,361, 60/261,531, 60/261,457, 60/261,694, 60/261,226, 60/261,304, 60/261,459, 60/261,456, 60/261,589, 60/261,461, 60/261,697, 60/261,458, 60/261,695, 60/261,336, 60/261,518, and 60/261,303, filed on January 12, 2001, which are incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Insulin is the central hormone governing metabolism in vertebrates (reviewed in Steiner et al., 1989, In Endocrinology, DeGroot, eds. Philadelphia, Saunders: 1263-1289). In humans, insulin is secreted by the beta cells of the pancreas in response to elevated blood glucose levels, which normally occur following a meal. The immediate effect of insulin secretion is to induce the uptake of glucose by muscle, adipose tissue, and the liver. A longer-term effect of insulin is to increase the activity of enzymes that synthesize glycogen in the liver and triglycerides in adipose tissue. Insulin can exert other actions beyond these "classic" metabolic activities, including increasing potassium transport in muscle, promoting cellular differentiation of adipocytes, increasing renal retention of sodium, and promoting production of androgens by the ovary. Defects in the secretion and/or response to insulin are responsible for the disease diabetes mellitus, which is of enormous economic significance. Within the United States, diabetes mellitus is the fourth most common reason for physician visits by patients; it is the leading cause of end-stage renal disease, non-traumatic limb amputations, and blindness in individuals of working age (Warram et al., 1995, In Joslin's Diabetes Mellitus, Kahn and Weir, eds., Philadelphia, Lea & Febiger, pp. 201-215; Kahn et al., 1996, Annu. Rev. Med. 47:509-531; Kahn, 1998, Cell 92:593-596). Beyond its role in diabetes mellitus, the phenomenon of insulin resistance has been linked to other pathogenic disorders including obesity, ovarian hyperandrogenism, and hypertension.

Within the pharmaceutical industry, there is interest in understanding the molecular mechanisms that connect lipid defects and insulin resistance. Hyperlipidemia and elevation of free fatty acid levels correlate with "Metabolic Syndrome," defined as the linkage between several diseases, including obesity and insulin resistance, which often occur in the same patients and which are major risk factors for development of Type 2 diabetes and cardiovascular disease. Current research suggests that the control of lipid levels, in addition to glucose levels, may be required to treat Type 2 Diabetes, heart disease, and other manifestations of Metabolic Syndrome (Santomauro AT et al, Diabetes (1999) 48:1836-1841).

The ability to manipulate and screen the genomes of model organisms such as Drosophila and C. elegans provides a powerful means to analyze biochemical processes that, due to significant evolutionary conservation of genes, pathways, and cellular processes, have direct relevance to more complex vertebrate organisms. Identification of novel functions of genes involved in particular pathways in such model organisms can directly contribute to the understanding of the correlative pathways in mammals and of methods of modulating them (see e.g., Miklos GL and Rubin GM, Cell 1996, 86:521-529). While Drosophila and C. elegans are not susceptible to human pathologies, various experimental models can mimic the pathological states. A correlation between the pathology model and the modified expression of a Drosophila or C. elegans gene can identify the association of the human ortholog with the human disease.

In one example, a genetic screen is performed in an invertebrate model organism displaying a mutant (generally visible or selectable) phenotype due to mis-expression - generally reduced, enhanced or ectopic expression - of a known gene (the "genetic entry point"). Additional genes are mutated in a random or targeted manner. When an additional gene mutation changes the original mutant phenotype, this gene is identified as a "modifier" that directly or indirectly interacts with the genetic entry point and its associated pathway. If the genetic entry point is an ortholog of a human gene associated with a human pathology, such as lipid metabolic disorders, the screen can identify modifier genes that are candidate targets for novel therapeutics.

Genetic screens may utilize RNA interference (RNAi) techniques, whereby introduction of exogenous double stranded (ds) RNA disrupts the activity of genes containing homologous sequences and induce specific loss-of-function phenotypes (Fire et al, 1998, Nature391:806-811). Suitable methods for introduction of dsRNA include injection, feeding, and bathing (Tabara et al, 1998, Science 282:430-431).

Genetic screens may also utilize mis-expression techniques to identify genes that, when over- or misexpressed in a pattern of interest, give a specific phenotype or modulate an existing mutant phenotype. An exemplary mis-expression screen is the Drosophila "EP" screen, which uses Drosophila lines carrying essentially random insertion of a P- element containing gene regulatory sequences oriented to act on flanking genomic sequences (Rorth P, 1996, Proc Natl Acad Sci U S A 93:12418-22; Rorth P et al, Development (1998) 125:1049-1057; WO0015843).

The insulin receptor (INR) signaling pathway has been extensively studied in C. elegans and Drosophila. For instance, it has been found that signaling through daf-2, the C. elegans INR ortholog, mediates various events, including reproductive growth and normal adult life span (see, e.g., US PAT NO 6,225,120; Tissenbaum HA and Ruvkun G, 1998, Genetics 148:703-17; Ogg S and Ruvkun G, 1998, Mol Cell 2:887-93; Lin K et al, 2001, Nat Genet 28:139-45).

Modifiers of INR signaling were identified in genetic screens using Drosophila or C. elegans. SUMMARY OF THE INVENTION

The invention provides a method of identifying candidate INR signaling modulating agents using an assay system comprising an ISM polypeptide or nucleic acid; contacting the assay system with a test agent under conditions whereby, but for the presence of the test agent, the system provides a reference activity; and detecting a test agent-biased activity of the assay system, wherein a difference between the test agent- biased activity and the reference activity identifies the test agent as a candidate INR signaling modulating agent. Candidate test agents include small molecule modulators, antibodies, and nucleic acid modulators such as antisense oligomers and PMOs, among others.

In one embodiment of the invention, the assay system comprises cultured cells or a non-human animal expressing ISM, and the assay system detects an agent-biased change in INR signaling.

In certain embodiments, candidate INR signaling modulating agents are identified in cell-free or cell-based assays, and a second assay system that detects an agent-biased change in an activity associated with INR signaling is used to confirm the INR signaling modulating activity of the candidate agent. In a preferred embodiment, the second assay detects an agent-biased change in an activity associated with INR signaling. Preferred second assay systems are carried out in cultured cells.

The invention further provides a method of modulating INR signaling in a mammalian cell comprising contacting the cell with an agent that specifically binds an ISM polypeptide or nucleic acid. In a preferred embodiment, the agent is administered to a mammalian animal predetermined to have a pathology associated with INR signaling. Preferred agents include small molecule modulators, nucleic acid modulators, or antibodies.

DETAILED DESCRIPTION OF THE INVENTION

We identified novel invertebrate modifiers of insulin receptor (INR) signaling and their human orthologs. The human orthologs of these genes (nucleic acids and polypeptides) are collectively refeιτed to as ISMs (Insulin receptor Signaling Modifiers).

A reverse genetics screen was performed using a C. elegans model for defective INR function, with the goal of identifying genes that when inactivated result in restoration of INR signaling. The C. elegans INR signaling pathway negatively regulates entry into a dauer state, an alternate developmental fate that normally occurs only in adverse conditions (Riddle DL and PS Albert. 1997. In C. elegans II, DL Riddle, T. Blumenthal, BJ Meyer, and JR Priess, eds., p. 739-768). When daf-2, the C. elegans insulin receptor, is inactivated by mutation, animals enter the dauer state regardless of the environmental conditions (Kimura KD, et al,. 1997, Science 277:942). We used an RNAi-based screen to identify modifiers (suppressors) of the dauer-formation phenotype of daf-2 loss-of- function mutations. The screen used two worm strains, each containing a missense mutation in the ligand-binding domain of daf-2, and involved RNAi treatment of these strains with dsRNA derived from cDNA or exon-rich genomic fragments of worm genes in order to cause reduction-of -function of these genes. Potential suppressors were identified as those genes that, when knocked down by RNAi treatment, allowed growth of the insulin-receptor mutant strains rather than larval arrest. Standard sequence analysis was used to determine the human (or mouse) orthologs of these genes, which are candidate targets for therapeutics designed to treat pathologies related to insulin signaling. Since many of the symptoms of diabetes are a result of decreased insulin signaling, candidate targets for antagonist therapies are those genes that, when mutated, restore INR signaling.

The C. elegans screen identified the genes whose amino acid sequences are provided in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22.

An overexpression screen was performed in Drosophila. Signaling through the Drosophila insulin receptor, designated Dinr (GI 1362614), has been implicated in controlling cell size and cell number (Fernandez R, et al, 1995, EMBO J 14:3373-3384; Brogiolo W et al., Stocker H and Hafen E, 2000, Curr Opin Genet Dev 10:529-35; Chen C et al, 1996, Endocrinology 137:846-56). Mis-expression of a dominant negative form of Dinr in the Drosophila eye results in small eye phenotype. The overexpression screen used a collection of Drosophila lines carrying insertions of a P-element vector ("EP element") comprising a GAL4-regulated promoter oriented to transcribe flanking genomic sequences (Rorth P, 1996, Proc Natl Acad Sci U S A 93:12418-22; Rorth P et al., 1998, Development 125:1049-57). Each EP line was crossed to lines expressing the dominant negative Dinr, as well as GAL4, in the eye. Resulting progeny were examined for a change in the small eye phenotype. Sequence information surrounding the P insertion site was used to identify the overexpressed genes.

The Drosophila screen identified the genes whose amino acid sequences are provided in SEQ ID NOS: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76

The invertebrate screens identified novel associations of ISM proteins and INR signaling. To applicants' knowledge, associations between diabetes (i.e., type II diabetes), glucose metabolism and/or INR signaling and the ISM proteins presented in SEQ ID NOs: 18, 30, 32, 34, 36, 38, 40, 50, 52, 54, 56, 58, 64, 66, 68, 70, 72, 74 and 76 have not previously been disclosed. For other ISM proteins, including those presented in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 24, 26, 28, 42, 44, 46, 48, 60, and 62, there have been previous reports correlating activity of these proteins to diabetes, glucose metabolism, or another output of INR signaling. However, to applicants' knowledge, the methods described herein present the first evidence that these proteins may directly influence and/or interact with INR signaling to regulate the metabolic effects of insulin.. ISM genes (i.e., nucleic acids and polypeptides) are attractive drug targets for the treatment of disorders related to INR signaling. In a preferred example, treatment involves increasing signaling through INR in order to treat pathologies related to diabetes and/or metabolic syndrome.

The invention provides in vitro and in vivo methods of assessing ISM function, and methods of modulating (generally inhibiting or agonizing) ISM activity, which are useful for further elucidating INR signaling and for developing diagnostic and therapeutic modalities for pathologies associated with INR signaling. As used herein, pathologies associated with INR signaling encompass pathologies where INR signaling contributes to maintaining the healthy state, as well as pathologies whose course may be altered by modulation of the INR signaling.

ISM nucleic acids and polypeptides

Human ISM nucleic acid (cDNA) sequences are provided in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75. Corresponding protein sequences are provided in SEQ ID NOs:. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76. ISM proteins of this invention include several categories of proteins with particular function domains. ISMs are identified as enzymes belonging to kinase (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 34, 36, 38, 40, 44, 46, 50, 60, and 62), protease (SEQ ID NOs: 30, 32, 74, and 76), desaturase (SEQ ID NO:48), and biosynthetic enzyme (SEQ ID NOs:42 and 52) classes. Other classes include mitochondrial transport proteins (SEQ ID NOs:54, 56 and 58), specific binding proteins (SEQ ID NOs:64 and 66), and transcriptional proteins (SEQ ID NOs: 68, 70, 72).

The term "ISM polypeptide" refers to a full-length ISM protein or a fragment or derivative thereof that is "functionally active," meaning that the ISM protein derivative or fragment exhibits one or more functional activities associated with a full-length, wild-type ISM protein. As one example, a fragment or derivative may have antigenicity such that it can be used in immunoassays, for immunization, for generation of inhibitory antibodies, etc, as discussed further below. Preferably, a functionally active ISM fragment or derivative displays one or more biological activities associated with ISM proteins such as enzymatic activity, signaling activity, ability to bind natural cellular substrates, etc. For those ISM proteins that are identified as enzymes, preferred ISM polypeptides display enzymatic (kinase, protease, desaturase, biosynthetic, etc.) activity. Other preferred ISM polypeptides display transport, binding, or transcriptional activity, according to the particular protein class. In one embodiment, a functionally active ISM polypeptide is an ISM derivative capable of rescuing defective endogenous ISM activity, such as in cell based or animal assays; the rescuing derivative may be from the same or a different species. If ISM fragments are used in assays to identify modulating agents, the fragments preferably comprise a ISM domain, such as a C- or N-terminal or catalytic domain, among others, and preferably comprise at least 10, preferably at least 20, more preferably at least 25, and most preferably at least 50 contiguous amino acids of a ISM protein. A preferred ISM fragment comprises a catalytic domain, such as a kinase domain, for those ISMs identified as kinases. Other preferred ISM fragments comprise specific binding domains. Functional domains can be identified using the PFAM program (Bateman A et al., 1999 Nucleic Acids Res 27:260-262; website at pfam.wustl.edu).

The term "ISM nucleic acid" refers to a DNA or RNA molecule that encodes a ISM polypeptide. Preferably, the ISM polypeptide or nucleic acid or fragment thereof is from a human, but it can be an ortholog or derivative thereof with at least 70%, preferably with at least 80%, preferably 85%, still more preferably 90%, and most preferably at least 95% sequence identity with a human ISM. As used herein, "percent (%) sequence identity" with respect to a specified subject sequence, or a specified portion thereof, is defined as the percentage of nucleotides or amino acids in the candidate derivative sequence identical with the nucleotides or amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU- BLAST-2.0al9 (Altschul et al, J. Mol. Biol. (1997) 215:403-410; http://blast.wustI.edu/blast/README.html) with search parameters set to default values. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. A "% identity value" is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported. "Percent (%) amino acid sequence similarity" is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation. A conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected. Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine and glycine.

Alternatively, an alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman (Smith and Waterman, 1981, Advances in Applied Mathematics 2:482-489; database: European Bioinformatics Institute wwwz.ebi.ac.uk/bic.sub.-- sw/; Smith and Waterman, 1981, J. of Molec.Biol., 147:195- 197; Nicholas et al, 1998, "A Tutorial on Searching Sequence Databases and Sequence Scoring Methods" (www.psc.edu) and references cited therein.; W.R. Pearson, 1991, Genomics 11:635-650). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff (Dayhof : Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA), and normalized by Gribskov (Gribskov 1986 Nucl. Acids Res. 14(6):6745-6763). The Smith- Waterman algorithm is used to search databases for sequences similar to a query sequence. Smith- Waterman uses dynamic programming to determine how an optimal alignment between the query sequence and a database sequence can be produced. This alignment is obtained by determining what transformations the query sequence would need to undergo to match the database sequence. Transformations include substituting one character for another and inserting or deleting a string of characters. A score is assigned for each character-to-character comparison— positive scores for exact matches and some substitutions, negative scores for other substitutions and insertions/deletions. The first character in an insertion or deletion gap is scored with a gap open penalty and subsequent characters are scored with a gap extension penalty. Scores are obtained from statistically-derived scoring matrices. The combination of transformations that results in the highest score is used to generate an alignment between the query sequence and database sequence. Smith- Waterman algorithm may be employed where default parameters are used for scoring (for example, gap open penalty of 12, gap extension penalty of two). From the data generated the "Match" value reflects "sequence identity."

Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that hybridize to an ISM nucleic acid sequence. The stringency of hybridization can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing. Conditions routinely used are set out in readily available procedure texts (e.g., Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al, Molecular Cloning, Cold Spring Harbor (1989)). In some embodiments, a nucleic acid molecule of the invention is capable of hybridizing to a nucleic acid molecule containing the an ISM nucleotide under stringent hybridization conditions that comprise: prehybridization of filters containing nucleic acid for 8 hours to overnight at 65° C in a solution comprising 6X single strength citrate (SSC) (IX SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5X Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C in a solution containing 6X SSC, IX Denhardt's solution, 100 μg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C for 1 h in a solution containing 0.2X SSC and 0.1% SDS (sodium dodecyl sulfate). In other embodiments, moderately stringent hybridization conditions are used that comprise: pretreatment of filters containing nucleic acid for 6 h at 40° C in a solution containing 35% foraiamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C in a solution containing 35% foraiamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C in a solution containing 2X SSC and 0.1% SDS. Alternatively, low stringency conditions can be used that comprise: incubation for 8 hours to overnight at 37° C in a solution comprising 20% foraiamide, 5 x SSC, 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1 x SSC at about 37° C for 1 hour.

In some embodiments, the ISM is an ortholog of human ISM. Methods of identifying the human orthologs of these genes are known in the art. Normally, orthologs in different species retain the same function, due to presence of one or more protein motifs and/or 3-dimensional structures. Orthologs are generally identified by sequence homology analysis, such as BLAST analysis, usually using protein bait sequences. Sequences are assigned as a potential ortholog if the best hit sequence from the forward BLAST result retrieves the original query sequence in the reverse BLAST (Huynen MA and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen MA et al, Genome Research (2000) 10:1204-1210). Programs for multiple sequence alignment, such as CLUSTAL (Thompson JD et al, 1994, Nucleic Acids Res 22:4673-4680) may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees. In a phylogenetic tree representing multiple homologous sequences from diverse species (e.g., retrieved through BLAST analysis), orthologous sequences from two species generally appear closest on the tree with respect to all other sequences from these two species. Structural threading or other analysis of protein folding (e.g., using software by ProCeryon, Biosciences, Salzburg, Austria) may also identify potential orthologs. In evolution, when a gene duplication event follows speciation, a single gene in one species, such as Drosophila, may correspond to multiple genes (paralogs) in another, such as human. As used herein, the term "orthologs" encompasses paralogs.

Isolation, Production, Expression, and Mis-expression of ISM Nucleic Acids and Polypeptides

ISM nucleic acids and polypeptides are useful for identifying and testing agents that modulate ISM function and for other applications related to the involvement of ISM in INR signaling. ISM nucleic acids may be obtained using any available method. For instance, techniques for isolating cDNA or genomic DNA sequences of interest by screening DNA libraries or by using polymerase chain reaction (PCR) are well known in the art.

A wide variety of methods are available for obtaining ISM polypeptides. In general, the intended use for the polypeptide will dictate the particulars of expression, production, and purification methods. For instance, production of polypeptides for use in screening for modulating agents may require methods that preserve specific biological activities of these proteins, whereas production of polypeptides for antibody generation may require structural integrity of particular epitopes. Expression of polypeptides to be purified for screening or antibody production may require the addition of specific tags (i.e., generation of fusion proteins). Overexpression of a ISM polypeptide for cell-based assays used to assess ISM function, such as involvement in tubulogenesis, may require expression in eukaryotic cell lines capable of these cellular activities. Techniques for the expression, production, and purification of proteins are well known in the art; any suitable means therefor may be used (e.g., Higgins SJ and Hames BD (eds.) Protein Expression: A Practical Approach, Oxford University Press Inc., New York 1999; Stanbury PF et al., Principles of Fermentation Technology, 2^nd edition, Elsevier Science, New York, 1995; Doonan S (ed.) Protein Purification Protocols, Humana Press, New Jersey, 1996; Coligan JE et al, Current Protocols in Protein Science (eds.), 1999, John Wiley & Sons, New York; U.S. Pat. No. 6,165,992).

The nucleotide sequence encoding an ISM polypeptide can be inserted into any appropriate vector for expression of the inserted protein-coding sequence. The necessary transcriptional and translational signals, including promoter/enhancer element, can derive from the native ISM gene and/or its flanking regions or can be heterologous. A variety of host-vector expression systems may be utilized, such as mammalian cell systems infected with virus (e.g. vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g. baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, plasmid, or cosmid DNA. A host cell strain that modulates the expression of, modifies, and/or specifically processes the gene product may be used.

The ISM polypeptide may be optionally expressed as a fusion or chimeric product, joined via a peptide bond to a heterologous protein sequence. In one application the heterologous sequence encodes a transcriptional reporter gene (e.g., GFP or other fluorescent proteins, luciferase, beta-galactosidase, etc.). A chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other in the proper coding frame using standard methods and expressing the chimeric product. A chimeric product may also be made by protein synthetic techniques, e.g. by use of a peptide synthesizer (Hunkapiller et al, Nature (1984) 310:105- 111). An ISM polypeptide can be isolated and purified using standard methods (e.g. ion exchange, affinity, and gel exclusion chromatography; centrifugation; differential solubility; electrophoresis). Alternatively, native ISM proteins can be purified from natural sources, by standard methods (e.g. immunoaffinity purification). Once a protein is obtained, it may be quantified and its activity measured by appropriate methods, such as immunoassay, bioassay, or other measurements of physical properties, such as crystallography.

The methods of this invention may also use cells that have been engineered for altered expression (mis-expression) of ISM or other genes associated with INR signaling. As used herein, mis-expression encompasses ectopic expression, over-expression, under- expression, and non-expression (e.g. by gene knock-out or blocking expression that would otherwise normally occur).

Genetically modified animals

The methods of this invention may use non-human animals that have been genetically modified to alter expression of ISM and/or other genes known to be involved in INR signaling. Preferred genetically modified animals are mammals, particularly mice or rats. Preferred non-mammalian species include Zebrafish, C. elegans, and Drosophila. Preferably, the altered ISM or other gene expression results in a detectable phenotype, such as modified levels of INR signaling, modified levels of plasma glucose or insulin, or modified lipid profile as compared to control animals having normal expression of the altered gene. The genetically modified animals can be used to further elucidate INR signaling, in animal models of pathologies associated with INR signaling, and for in vivo testing of candidate therapeutic agents, as described below.

Preferred genetically modified animals are transgenic, at least a portion of their cells harboring non-native nucleic acid that is present either as a stable genomic insertion or as an extra-chromosomal element, which is typically mosaic. Preferred transgenic animals have germ-line insertions that are stably transmitted to all cells of progeny animals.

Non-native nucleic acid is introduced into host animals by any expedient method. Methods of making transgenic non-human animals are well-known in the art (for mice see Brinster et al, Proc. Nat. Acad. Sci. USA 1985, 82:4438-42; U.S. Pat. Nos. 4,736,866, 4,870,009, 4,873,191, 6,127,598; Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); for homologous recombination see Capecchi, Sciencel989, 244:1288-1292; Joyner et al, Naturel989, 338:153-156; for particle bombardment see U.S. Pat. No., 4,945,050; for Drosophila see Rubin and Spradling, Science (1982) 218:348-53, U.S. Pat. No. 4,670,388; for transgenic insects see Berghammer A.J. et al., Nature 1999, 402:370-371; for Zebrafish see Lin S. Methods Mol Biol. (2000);136:375-3830; for fish, amphibians and birds see Houdebine and Chourrout, Experientia (1991) 47:897-905; for rats see Hammer et al., Cell (1990)63:1099- 1112; for embryonic stem (ES) cells see Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed., IRL Press (1987); for livestock see Pursel et al, Science (1989) 244:1281-1288; for nonhuman animal clones see Wilmut, I. et al. (1997) Nature 385:810-813, PCT Publication Nos. WO 97/07668 and WO 97/07669; for recombinase systems for regulated transgene expression see, Lakso et al., PNAS (1992) 89:6232-6236; U.S. Pat. No. 4,959,317 [for cre.loxP] and O'Gorman et al. (1991) Science 251:1351-1355; U.S. Pat. No. 5,654,182 [for FLP/FRT]).

Homozygous or heterozygous alterations in the genomes of transgenic animals may result in mis-expression of native genes, including ectopic expression, over- expression (e.g. by multiple gene copies), under-expression, and non-expression (e.g. by gene knock-out or blocking expression that would otherwise normally occur). In one application, a "knock-out" animal is generated, typically using homologous recombination, in which an alteration in an endogenous gene causes a decrease in that gene's function, preferably such that gene expression is undetectable or insignificant.

ISM Modulating agents

The invention provides methods to identify agents that interact with and/or modulate the function of ISM and/or INR signaling. Such agents are useful in a variety of diagnostic and therapeutic applications associated with INR signaling, as well as in further analysis of the ISM protein and its contribution to INR signaling. Accordingly, the invention also provides methods for modulating INR signaling comprising the step of specifically modulating ISM activity by administering an ISM-interacting or -modulating agent.

In a preferred embodiment, ISM-modulating agents inhibit or enhance ISM activity or otherwise affect normal ISM function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In a further preferred embodiment, the candidate INR signaling- modulating agent specifically modulates the function of the ISM. The phrases "specific modulating agent", "specifically modulates", etc., are used herein to refer to modulating agents that directly bind to the ISM polypeptide or nucleic acid, and preferably inhibit, enhance, or otherwise alter the function of the ISM. The term also encompasses modulating agents that alter the interaction of ISM with a binding partner or substrate (e.g. by binding to a binding partner of an ISM, or to a protein/binding partner complex, and inhibiting function).

Preferred ISM-modulating agents include small molecule chemical agents, ISM- interacting proteins, including antibodies and other biotherapeutics, and nucleic acid modulators, including antisense oligomers and RNA. The modulating agents may be formulated in pharmaceutical compositions, for example, as compositions that may comprise other active ingredients, as in combination therapy, and/or suitable carriers or excipients. Techniques for formulation and administration of the compounds may be ffoouunndd iinn "Remington's Pharmaceutical Sciences" Mack Publishing Co., Easton, PA, 19^l edition.

Small Molecule Modulators

Chemical agents, referred to in the art as "small molecule" compounds are typically organic, non-peptide molecules, having a molecular weight less than 10,000, preferably less than 5,000, more preferably less than 1,000, and most preferably less than 500. This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries. Synthetic compounds may be rationally designed or identified based on known or inferred properties of the ISM protein or may be identified by screening compound libraries. Alternative appropriate modulators of this class are natural products, particularly secondary metabolites from organisms such as plants or fungi, which can also be identified by screening compound libraries for ISM- modulating activity. Methods for generating and obtaining compounds are well known in the art (Schreiber SL, Science (2000) 151: 1964-1969; Radmann J and Gunther J, Science (2000) 151:1947-1948).

Small molecule modulators identified from screening assays, as described below, can be used as lead compounds from which candidate clinical compounds may be designed, optimized, and synthesized. Such clinical compounds may have utility in treating pathologies associated with INR signaling. The activity of candidate small molecule modulating agents may be improved several-fold through iterative secondary functional validation, as further described below, structure determination, and candidate modulator modification and testing. Additionally, candidate clinical compounds are generated with specific regard to clinical and pharmacological properties. For example, the reagents may be derivatized and re-screened using in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.

Protein Modulators

An ISM-interacting protein may be endogenous, i.e. one that normally interacts genetically or biochemically with an ISM, such as a member of the ISM pathway that modulates ISM expression, localization, and/or activity. ISM-modulators include dominant negative forms of ISM-interacting proteins and of ISM proteins themselves. Yeast two-hybrid and variant screens offer preferred methods for identifying endogenous ISM-interacting (Finley, R. L. et al. (1996) in DNA Cloning-Expression Systems: A Practical Approach, eds. Glover D. & Hames B. D (Oxford University Press, Oxford, England), pp. 169-203; Fashema SF et al., Gene (2000) 250:1-14; Drees BL Curr Opin Chem Biol (1999) 3:64-70; Vidal M and Legrain P Nucleic Acids Res (1999) 27:919-29; and U.S. Pat. No. 5,928,868). Mass spectrometry offers alternative preferred methods for the elucidation of protein complexes (reviewed in, e.g., Pandley A and Mann M, Nature (2000) 405:837-846; Yates JR 3^rd, Trends Genet (2000) 16:5-8).

Specific biotherapeutics

An ISM-interacting protein may have biotherapeutic applications. Biotherapeutic agents formulated in pharmaceutically acceptable carriers and dosages may be used to activate or inhibit signal transduction pathways. This modulation may be accomplished by binding a ligand, thus inhibiting the activity of the pathway; or by binding a receptor, either to inhibit activation of, or to activate, the receptor. Alternatively, the biotherapeutic may itself be a ligand capable of activating or inhibiting a receptor. Biotherapeutic agents and methods of producing them are described in detail in U.S. Pat. No. 6,146,628.

Antibody Modulators

In a preferred embodiment, the ISM-interacting protein is an antibody. In a further preferred embodiment, antibody agonists or antagonists are produced that have therapeutic utility.

Antibodies that specifically bind ISM polypeptides can be generated using known methods. Preferably the antibody is specific to a mammalian ISM polypeptide, and more preferably, a human ISM. Antibodies may be polyclonal, monoclonal (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab').sub.2 fragments, fragments produced by a FAb expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Monoclonal antibodies with affinities of 10⁸ M^"1 preferably 10⁹ M^"1 to 10¹⁰ M^"1, or stronger can be made by standard procedures as described (Harlow E and Lane D, 1988, Antibodies: A Laboratory Manual, CSH Laboratory Press; (Harlow E and Lane D, 1999 Using Antibodies: A Laboratory Manual, CSH Laboratory Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and 4,618,577). Antibodies may be generated against crude cell extracts of ISM or substantially purified fragments thereof. If ISM fragments are used, they preferably comprise at least 10, and more preferably, at least 20 contiguous amino acids of an ISM protein. In a particular embodiment, ISM-specific antigens and/or immunogens are coupled to carrier proteins that stimulate the immune response. For example, the subject polypeptides are covalently coupled to the keyhole limpet hemocyanin (KLH) carrier, and the conjugate is emulsified in Freund's complete adjuvant, which enhances the immune response. An appropriate immune system such as a laboratory rabbit or mouse is immunized according to conventional protocols.

Chimeric antibodies specific to ISM polypeptides can be made that contain different portions from different animal species. For instance, a human immunoglobulin constant region may be linked to a variable region of a murine mAb, such that the antibody derives its biological activity from the human antibody, and its binding specificity from the murine fragment. Chimeric antibodies are produced by splicing together genes that encode the appropriate regions from each species (Morrison et al., Proc. Natl. Acad. Sci. (1984) 81:6851-6855; Neuberger et al, Nature (1984) 312:604-608; Takeda et al., Nature (1985) 31:452-454). Humanized antibodies, which are a form of chimeric antibodies, can be generated by grafting complementary-determining regions (CDRs) (Carlos, T. M., J. M. Harlan. 1994. Blood 84:2068-2101) of mouse antibodies into a background of human framework regions and constant regions by recombinant DNA technology (Riechmann LM, et al, 1988 Nature 323: 323-327). Humanized antibodies contain ~10% murine sequences and -90% human sequences, and thus further reduce or eliminate immunogenicity, while retaining the antibody specificities (Co MS, and Queen C. 1991 Nature 351: 501-501; Morrison SL. 1992 Ann. Rev. hnmun. 10:239-265). Humanized antibodies and methods of their production are well-known in the art (US PAT NO: 5,530,101; US PAT NO:5,585,089; US PAT NO:5,693,762, and US PAT NO:6,180,370).

ISM-specific single chain antibodies, which are recombinant, single chain polypeptides formed by linking the heavy and light chain fragments of the Fv regions via an amino acid bridge, can be produced (U.S. Pat. No. 4,946,778; Bird, Science (1988) 242:423-426; Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85:5879-5883; and Ward et al., Nature (1989) 334:544-546).

Other suitable techniques for antibody production involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors (Huse et al., Science (1989) 246:1275-1281).

As used herein, T-cell antigen receptors are included within the scope of antibody modulators (Harlow and Lane, 1988, supra).

The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance that provides for a detectable signal, or that is toxic to cells that express the targeted protein (Menard S, et al., Int J. Biol Markers (1989) 4:131-134). A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, fluorescent emitting lanthanide metals, chemiluminescent moieties, bioluminescent moieties, magnetic particles, and the like (U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241). Also, recombinant immunoglobulins may be produced (U.S. Pat. No. 4,816,567). Antibodies to cytoplasmic proteins may be delivered and reach their targets by conjugation with membrane-penetrating toxin proteins (U.S. Pat. NO. 6,086,900). When used therapeutically in a patient, the antibodies of the subject invention are typically administered parenterally, when possible at the target site, or intravenously. The therapeutically effective dose and dosage regimen is determined by clinical studies. Typically, the amount of antibody administered is in the range of about 0.1 mg/kg -to about 10 mg/kg of patient weight. For parenteral administration, the antibodies are formulated in a unit dosage injectable form (e.g., solution, suspension, emulsion) in association with a pharmaceutically acceptable vehicle. Such vehicles are inherently nontoxic and non-therapeutic. Examples are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils, ethyl oleate, or liposome carriers may also be used. The vehicle may contain minor amounts of additives, such as buffers and preservatives, which enhance isotonicity and chemical stability or otherwise enhance therapeutic potential. The antibodies' concentrations in such vehicles are typically in the range of about 1 mg/ml-to aboutlO mg/ml. Immunotherapeutic methods are further described in the literature (US Pat. No. 5,859,206; WO0073469).

Nucleic Acid Modulators

Other preferred ISM-modulating agents comprise nucleic acid molecules, such as antisense oligomers or double stranded RNA (dsRNA), which generally inhibit ISM activity.

Preferred antisense oligomers interfere with the function of ISM nucleic acids, such as DNA replication, transcription, ISM RNA translocation, translation of protein from the ISM RNA, RNA splicing, and any catalytic activity in which the ISM RNA participates. In one embodiment, the antisense oligomer is an oligonucleotide that is sufficiently complementary to an ISM mRNA to bind to and prevent translation from the ISM mRNA, preferably by binding to the 5' untranslated region. ISM-specific antisense oligonucleotides preferably range from at least 6 to about 200 nucleotides. In some embodiments the oligonucleotide is preferably at least 10, 15, or 20 nucleotides in length. In other embodiments, the oligonucleotide is preferably less than 50, 40, or 30 nucleotides in length. The oligonucleotide can be DNA or RNA, a chimeric mixture of DNA and RNA, derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide may include other appending groups such as peptides, agents that facilitate transport across the cell membrane, hybridization-triggered cleavage agents, and intercalating agents.

In another embodiment, the antisense oligomer is a phosphorothioate morpholino oligomer (PMO). PMOs are assembled from four different morpholino subunits, each of which containing one of four genetic bases (A, C, G, or T) linked to a six-membered morpholine ring. Polymers of these subunits are joined by non-ionic phosphodiamidate inter-subunit linkages. Methods of producing and using PMOs and other antisense oligonucleotides are well known in the art (e.g. see W099/18193; Summerton J, and Weller D, Antisense Nucleic Acid Drug Dev 1997, 7:187-95; Probst JC, Methods 2000, 22:271-281; US PAT NO: 5,325,033; US PAT NO: 5,378,841).

Antisense oligomers are commonly used as research reagents, diagnostics, and therapeutics. For example, antisense oligonucleotides, which are able to specifically inhibit gene expression, are often used to elucidate the function of particular genes (see, e.g., US PAT NO 6,165,790). Antisense oligomers are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense oligomers have been employed as therapeutic moieties in the treatment of disease states in animals and humans and have been demonstrated in numerous clinical trials to be safe and effective (Milligan JF et al, 1993, J Med Chem 36:1923-1937; Tonkinson JL et al, 1996, Cancer Invest 14:54-65). Accordingly, in one aspect of the invention, an ISM-specific antisense oligomer is used in an assay to further elucidate the function of ISM in F R signaling. Zebrafish is a particularly useful model for the study of INR signaling using antisense oligomers. For example, PMOs are used to selectively inactive one or more genes in vivo in the Zebrafish embryo. By injecting PMOs into Zebrafish at the 1-16 cell stage candidate targets emerging from the Drosophila screens are validated in this vertebrate model system. In another aspect of the invention, PMOs are used to screen the Zebrafish genome for identification of other therapeutic modulators of INR signaling. In a further aspect of the invention, an ISM-specific antisense oligomer is used as a therapeutic agent for treatment of metabolic pathologies.

Alternative preferred ISM-modulating agents are double-stranded RNA species mediating RNA interference (RNAi). RNAi is the process of sequence-specific, post- transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. Methods relating to the use of RNAi to silence genes in C. elegans, Drosophila, plants, and mammals are known in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363 (1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490 (2001); Hammond, S. M., et al., Nature Rev Genet 2, 110-1119 (2001); Tuschl, T. Chem Biochem. 2, 239-245 (2001); Hamilton, A. et al., Science 286, 950-952 (1999); Hammond, S. M., et al., Nature 404, 293-296 (2000); Zamore, P. D., et al, Cell 101, 25-33 (2000); Bernstein, E„ et al., Nature 409, 363-366 (2001); Elbashir, S. M., et al., Genes Dev. 15, 188-200 (2001); WO0129058; W09932619, and Elbashir SM, et al., 2001, Nature 411:494-498).

Assay Systems

The invention provides assay systems for identifying specific modulators of ISM activity. As used herein, an "assay system" encompasses all the components required for performing and analyzing results of an assay that detects and/or measures a particular event or events. In general, primary assays are used to identify or confirm a modulator's specific biochemical or molecular effect with respect to the ISM nucleic acid or protein. In general, secondary assays further assess the activity of an ISM-modulating agent identified by a primary assay and may confirm that the modulating agent affects ISM in a manner relevant to INR signaling. In some cases, ISM-modulators will be directly tested in a "secondary assay," without having been identified or confirmed in a "primary assay." In a preferred embodiment, the assay system comprises contacting a suitable assay system comprising an ISM polypeptide or nucleic acid with a candidate agent under conditions whereby, but for the presence of the agent, the system provides a reference activity, which is based on the particular molecular event the assay system detects. The method further comprises detecting the same type of activity in the presence of a candidate agent (" the agent-biased activity of the system"). A difference between the agent-biased activity and the reference activity indicates that the candidate agent modulates ISM activity, and hence INR signaling. A difference, as used herein, is statistically significant. The assay systems generally include positive and/or negative controls, as are well known in the art.

Primary Assays

The type of modulator tested generally determines the type of primary assay.

Primary assays for small molecule modulators

For small molecule modulators, screening assays are used to identify candidate modulators. Screening assays may be cell-based or may use a cell-free system that recreates or retains the relevant biochemical reaction of the target protein (reviewed in Sittampalam GS et al., Curr Opin Chem Biol (1997) 1:384-91 and accompanying references). As used herein the term "cell-based" refers to assays using live cells, dead cells, or a particular cellular fraction, such as a membrane, endoplasmic reticulum, or mitochondrial fraction. The term "cell free" encompasses assays using substantially • purified protein (either endogenous or recombinantly produced), partially purified cellular extracts, or crude cellular extracts. Screening assays may detect a variety of molecular events, including protein-DNA interactions, protein-protein interactions (e.g., receptor- Iigand binding), transcriptional activity (e.g., using a reporter gene), enzymatic activity (e.g., via a property of the substrate), activity of second messengers, immunogenicty and changes in cellular morphology or other cellular characteristics. Appropriate screening assays may use a wide range of detection methods including fluorescent, radioactive, colorimetric, spectrophotometric, and amperometric methods, to provide a read-out for the particular molecular event detected.

In a preferred embodiment, screening assays use fluorescence technologies, including fluorescence polarization, time-resolved fluorescence, and fluorescence resonance energy transfer. These systems offer means to monitor protein-protein or DNA- protein interactions in which the intensity of the signal emitted from dye-labeled molecules depends upon their interactions with partner molecules (e.g., Selvin PR, Nat Struct Biol (2000) 7:730-4; Fernandes PB, Curr Opin Chem Biol (1998) 2:597-603; Hertzberg RP and Pope AJ, Curr Opin Chem Biol (2000) 4:445-451).

Suitable assay formats that may be adapted to screen for ISM modulators are known in the art. Examples of assays useful for the various ISM protein classes are provided below.

Protein kinases, key signal transduction proteins that may be either membrane- associated or intracellular, catalyze the transfer of gamma phosphate from adenosine triphosphate (ATP) to a serine, threonine or tyrosine residue in a protein substrate. Radioassays, which monitor the transfer from [gamma-³²P or -³³P]ATP, are frequently used to assay kinase activity. Separation of the phospho-labeled product from the remaining radio-labeled ATP can be accomplished by various methods including SDS- polyacrylamide gel electrophoresis, filtration using glass fiber filters or other matrices which bind peptides or proteins, and adsorption/binding of peptide or protein substrates to solid-phase matrices allowing removal of remaining radiolabeled ATP by washing. In one example, a scintillation assay monitors the transfer of the gamma phosphate from [gamma -³³PJ ATP to a biotinylated peptide substrate. The substrate is captured on a streptavidin coated bead that transmits the signal (Beveridge M et al, J Biomol Screen (2000) 5:205- 212). This assay uses the scintillation proximity assay (SPA), in which only radio-ligand bound to receptors tethered to the surface of an SPA bead are detected by the scintillant immobilized within it, allowing binding to be measured without separation of bound from free ligand. Other assays for protein kinase activity may use antibodies that specifically recognize phosphorylated substrates. For instance, the kinase receptor activation (KIRA) assay measures receptor tyrosine kinase activity by ligand stimulating the intact receptor in cultured cells, then capturing solubilized receptor with specific antibodies and quantifying phosphorylation via phosphotyrosine ELISA (Sadick MD, Dev Biol Stand (1999) 97: 121-133). Another example of antibody based assays for protein kinase activity is TRF (time-resolved fluorometry). This method utilizes europium chelate-labeled anti- phosphotyrosine antibodies to detect phosphate transfer to a polymeric substrate coated onto microtiter plate wells. The amount of phosphorylation is then detected using time- resolved, dissociation-enhanced fluorescence (Braunwalder AF, et al., Anal Biochem 1996 Jul 1;238(2): 159-64). Generic assays may be established for protein kinases that rely upon the phosphorylation of substrates such as myelein basic protein, casein, histone, or synthetic peptides such as polyGlutamate/Tyrosine and radiolabeled ATP.

Phosphoinositide kinases catalyze the phosphorylation of phosphatidylinositol substrates. Assays for lipid kinase activity may use labeled, such as radio-labeled substrates to detect transfer of a phosphate to lipid substrates. In one example, assays may use chromatography techniques to detect phosphorylation (Sbrissa D et al. , 1999, J Biol Chem 274:21589-21597). In another example, an assay uses "FlashPlate" technology (U.S. Patent No. 5,972,595), in which the hydrophobic substrate is immobilized on a solid support in each well of a multi-well plate. Phosphorylation of the substrate with a radio- labeled phosphate is measured as an increase in bound radioactivity, which is detected by the close proximity of the scintillant.

Fatty acid CoA ligases catalyze the elongation of fatty acid chains. Assays may detect elongation of fatty acid substrates, for instance using chromatographic (e.g., HPLC) analysis of labeled elongation products. Long chain fatty acids may be labeled with 14-C (Moon YA et al., 2001, J Biol Chem 276:45358-66).

Fatty acid desaturases catalyze the insertion of double bonds into saturated fatty acid molecules. In one application, radioassays for inhibitors of fatty acid desaturase activity use thin layer chromatography to detect conversion of fatty acid substrates (Obukowicz et al., Biochem Pharmacol (1998) 55:1045-1058).

Mitochondrial adenine transporters mediate the passage of adenosine from the mitochondrial compartment to the cytoplasm. The mitochondrial aspartate/glutamate carrier catalyzes an important step in both the urea cycle and the aspartate/malate NADH shuttle. Mitochondrial transport assays may directly measure transported molecules (e.g., Ruck A et al., 1998, FEBS Lett 426:97-101) and may detect transport activity in intact mitochondria or reconstituted vesicles (e.g., Haucke V and Schatz G, 1997, EMBO J 16:4560-7; Genchi G et al., 1996 Plant Physiol 112:845-51). In one example, vesicles are reconstituted in the presence of the labeled substrate (Palmieri F et al, 1995, Methods Enzymol 260:349-69). Assays for mitochondrial transport may alternatively detect electrogenic or fluorogenic properties of the mitochondrial proteins (e.g., Burstovetsky N et al, 1996, Proc Natl Acad Sci USA 93:664-8; Streicher-Scott J, 1994, Arch Biochem Biophys 15:548-54; U.S. Pat. No. 6,183,948). p-Hydroxybenzoate polyprenyl transferase (COQ2) is involved in the biosynthesis of ubiquinone, an essential component of the electron transfer system (Ashby MN et al., 1992, J Biol Chem 267:4128-4136). Assays for COQ2 activity may use chromatographic methods to detect ubiquinone biosynthesis (e.g., Uchida N et al., 2000 J Bacteriol 182:6933-6939). Alternatively, assays may directly detect the COQ2 polyprenyltransferase activity (condensation of p-hydroxybenzoate and polyprenyl diphosphate). A variety of assays are available to detect the activity of proteins that have specific binding activity. Exemplary assays use fluorescence polarization, fluorescence polarization, and laser scanning techniques to measure binding of fluorescently labeled proteins, peptides, or other molecules (Lynch BA et al., 1999, Anal Biochem 275:62-73; Li HY, 2001, J Cell Biochem 80:293-303; Zuck P et al., Proc Natl Acad Sci USA 1999, 96: 11122-11127). In another example, binding activity is detected using the scintillation proximity assay (SPA), which uses a biotinylated peptide probe captured on a streptavidin coated SPA bead and a radio-labeled partner molecule. The assay specifically detects the radio-labeled protein bound to the peptide probe via scintillant immobilized within the SPA bead (Sonatore LM et al., 1996, Anal Biochem 240:289-297).

Proteins that are part of a transcriptional complex may be assayed for binding activity, as described above. Other suitable assays may measure transcriptional activity. In one example, transcriptional activity is detected using quantitative RT-PCR (e.g., using the TaqMan®, PE Applied Biosystems). In another example, a transcriptional reporter (e.g., luciferase, GFP, beta-galactosidase, etc.) operably linked to a responsive gene regulatory sequence is used (e.g., Berg M et al, 2000, J Biomol Screen, 5:71-76).

Preferred screening assays are high throughput or ultra high throughput and thus provide automated, cost-effective means of screening compound libraries for lead compounds (Fernandes PB, 1998, supra; Sundberg SA, Curr Opin Biotechnol 2000, 11:47-53).

Cell-based screening assays usually require systems for recombinant expression of ISM and any auxiliary proteins demanded by the particular assay. Cell-free assays often use recombinantly produced purified or substantially purified proteins. Appropriate methods for generating recombinant proteins produce sufficient quantities of proteins that retain their relevant biological activities and are of sufficient purity to optimize activity and assure assay reproducibility. Yeast two-hybrid and variant screens, and mass spectrometry provide preferred methods for determining protein-protein interactions and elucidation of protein complexes. In certain applications when ISM-interacting proteins are used in screening assays, the binding specificity of the interacting protein to the ISM protein may be assayed by various known methods, including binding equilibrium constants (usually at least about 10⁷M^_1, preferably at least about 10⁸ M^"1, more preferably at least about 10 M^"1), and immunogenic properties. For enzymes and receptors, binding may be assayed by, respectively, substrate and ligand processing.

The screening assay may measure a candidate agent's ability to specifically bind to or modulate activity of an ISM polypeptide, a fusion protein thereof, or to cells or membranes bearing the polypeptide or fusion protein. The ISM polypeptide can be full length or a fragment thereof that retains functional ISM activity. The ISM polypeptide may be fused to another polypeptide, such as a peptide tag for detection or anchoring, or to another tag. The ISM polypeptide is preferably human ISM, or is an ortholog or derivative thereof as described above. In a preferred embodiment, the screening assay detects candidate agent-based modulation of ISM interaction with a binding target, such as an endogenous or exogenous protein or other substrate that has ISM -specific binding activity, and can be used to assess normal ISM gene function.

Certain screening assays may also be used to test antibody and nucleic acid modulators; for nucleic acid modulators, appropriate assay systems involve ISM mRNA expression.

Primary assays for antibody modulators

For antibody modulators, appropriate primary assays are binding assays that test the antibody's affinity to and specificity for the ISM protein. Methods for testing antibody affinity and specificity are well known in the art (Harlow and Lane, 1988, 1999, supra). The enzyme-linked immunosorbant assay (ELISA) is a preferred method for detecting ISM-specific antibodies; others include FACS assays, radioimmunoassays, and fluorescent assays.

Primary assays for nucleic acid modulators

For nucleic acid modulators, primary assays may test the ability of the nucleic acid modulator to inhibit ISM gene expression, preferably mRNA expression. In general, expression analysis comprises comparing ISM expression in like populations of cells (e.g., two pools of cells that endogenously or recombinantly express ISM) in the presence and absence of the nucleic acid modulator. Methods for analyzing mRNA and protein expression are well known in the art. For instance, Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR (e.g., using the TaqMan®, PE Applied Biosystems), or microarray analysis may be used to confirm that ISM mRNA expression is reduced in cells treated with the nucleic acid modulator (e.g., Current Protocols in Molecular Biology (1994) Ausubel FM et al, eds., John Wiley & Sons, Inc., chapter 4; Freeman WM et al, Biotechniques (1999) 26:112-125; Kallioniemi OP, Ann Med 2001, 33:142-147; Blohm DH and Guiseppi-Elie, ACurr Opin Biotechnol 2001, 12:41-47). Protein expression may also be monitored. Proteins are most commonly detected with specific antibodies or antisera directed against either the ISM protein or specific peptides. A variety of means including Western blotting, ELISA, or in situ detection, are available (Harlow E and Lane D, 1988 and 1999, supra).

Secondary Assays

Secondary assays may be used to further assess the activity of an ISM-modulating agent identified by any of the above methods to confirm that the modulating agent affects ISM in a manner relevant to INR signaling. As used herein, ISM-modulating agents encompass candidate clinical compounds or other agents derived from previously identified modulating agent. Secondary assays can also be used to test the activity of a modulator on a particular genetic or biochemical pathway or to test the specificity of the modulator's interaction with ISM.

Secondary assays generally compare like populations of cells or animals (e.g., two pools of cells or animals that endogenously or recombinantly express ISM) in the presence and absence of the candidate modulator. In general, such assays test whether treatment of cells or animals with a candidate ISM-modulating agent results in changes in INR signaling, in comparison to untreated (or mock- or placebo-treated) cells or animals. Changes in INR signaling may be detected as modifications to INR pathway components, or changes in their expression or activity. Assays may also detect an output of normal or defective INR signaling, used herein to encompass immediate outputs, such as glucose uptake, or longer-term effects, such as changes in glycogen and triglycerides metabolism, adipocyte differentiation, or development of diabetes or other INR-related pathologies. Certain assays use sensitized genetic backgrounds, used herein to describe cells or animals engineered for altered expression of genes in the INR or interacting pathways, or pathways associated with INR signaling or an output of INR signaling.

Cell-based assays

Cell-based assays may use a variety of insulin-sensitive mammalian cells and may detect endogenous INR signaling or may rely on recombinant expression of INR and/or other INR pathway components. Exemplary insulin-sensitive cells include adipocytes, hepatocytes, and pancreatic beta cells. Suitable adipocytes include 3T3 LI cells, which are most commonly used for insulin sensitivity assays, as well as primary cells from mice or human biopsy. Suitable hepatocytes include the rat hepatoma H4-II-E cell line. Suitable beta cells include rat INS-1 cells with optimized glucose-sensitive insulin secretion (such as clone 823-13, Hohmeier et al., 2000, Diabetes 49:424). Other suitable cells include muscle cells, such as L6 myotubes, and CHO cells engineered to over- express INR. For certain assay systems it may be useful to treat cells with factors such as glucosamine, free fatty acids or TNF alpha, which induce an insulin resistant state. Candidate modulators are typically added to the cell media but may also be injected into cells or delivered by any other efficacious means.

Cell based assays generally test whether treatment of insulin responsive cells with the ISM - modulating agent alters INR signaling in response to insulin stimulation ("insulin sensitivity"); such assays are well-known in the art (see, e.g., Sweeney et al., 1999, J Biol Chem 274: 10071). In a preferred embodiment, assays are performed to determine whether inhibition of ISM function increases insulin sensitivity.

In one example, INR signaling is assessed by measuring expression of insulin- responsive genes. Hepatocytes are preferred for these assays. Many insulin responsive genes are known (e.g., p85 PI3 kinase, hexokinase II, glycogen synthetase, lipoprotein lipase, etc; PEPCK is specifically down-regulated in response to INR signaling). Any available means for expression analysis, as previously described, may be used. Typically, mRNA expression is detected. In a preferred application, Taqman analysis is used to directly measure mRNA expression. Alternatively, expression is indirectly monitored from a transgenic reporter construct comprising sequences encoding a reporter gene (such as luciferase, GFP or other fluorescent proteins, beta-galactosidase, etc.) under control of regulatory sequences (e.g., enhancer/promoter regions) of an insulin responsive gene. Methods for making and using reporter constructs are well known.

INR signaling may also be detected by measuring the activity of components of the INR-signaling pathway, which are well-known in the art (see, e.g., Kahn and Weir, Eds., Joslin's Diabetes Mellitus, Williams & Wilkins, Baltimore, MD, 1994). Suitable assays may detect phosphorylation of pathway members, including IRS, PI3K, Akt, GSK3 etc., for instance, using an antibody that specifically recognizes a phosphorylated protein. Assays may also detect a change in the specific signaling activity of pathway components (e.g., kinase activity of PI3K, GSK3, Akt, etc.). Kinase assays, as well as methods for detecting phosphorylated protein substrates, are well known in the art (see, e.g., Ueki K et al, 2000, Mol Cell Biol;20:8035-46).

In another example, assays measure glycogen synthesis in response to insulin stimulation, preferably using hepatocytes. Glycogen synthesis may be assayed by various means, including measurement of glycogen content, and determination of glycogen synthase activity using labeled, such as radio-labeled, glucose (see, e.g., Aiston S and Agius L, 1999, Diabetes 48:15-20; Rother KI et al., 1998, J Biol Chem 273:17491-7).

Other suitable assays measure cellular uptake of glucose (typically labeled glucose) in response to insulin stimulation. Adipocytes are preferred for these assays. Assays also measure translocation of glucose transporter (GLUT) 4, which is a primary mediator of insulin-induced glucose uptake, primarily in muscle and adipocytes, and which specifically translocates to the cell surface following insulin stimulation. Such assays may detect endogenous GLUT4 translocation using GLUT4-specific antibodies or may detect exogenously introduced, epitope-tagged GLUT4 using an antibody specific to the particular epitope (see, e.g., Sweeney, 1999, supra; Quon MJ et al., 1994, Proc Natl Acad Sci U S A 91:5587-91).

Other preferred assays detect insulin secretion from beta cells in response to glucose. Such assays typically use ELISA (see, e.g., Bergsten and Hellman, 1993, Diabetes 42:670-4) or radioimmunoassay (RIA; see, e.g., Hohmeier et al., 2000, supra).

Animal Assays

A variety of non-human animal models of metabolic disorders may be used to test candidate ISM modulators. Such models typically use genetically modified animals that have been engineered to mis-express (e.g., over-express or lack expression in) genes involved in lipid metabolism, adipogenesis, and/or the INR signaling pathway. Additionally, particular feeding conditions, and/or administration or certain biologically active compounds, may contribute to or create animal models of lipid and/or metabolic disorders. Assays generally required systemic delivery of the candidate modulators, such as by oral administration, injection (intravenous, subcutaneous, intraperitoneous), bolus administration, etc.

In one embodiment, assays use mouse models of diabetes and/or insulin resistance. Mice carrying knockouts of genes in the leptin pathway, such as oh (leptin) or db (leptin receptor), or the INR signaling pathway, such as INR or the insulin receptor substrate (IRS), develop symptoms of diabetes, and show hepatic lipid accumulation (fatty liver) and, frequently, increased plasma lipid levels (Nishina et al., 1994, Metabolism 43:549- 553; Michael et al., 2000, Mol Cell 6:87-97; Bruning JC et al., 1998, Mol Cell 2:559-569). Certain susceptible wild type mice, such as C57BL/6, exhibit similar symptoms when fed a high fat diet (Linton and Fazio, 2001, Current Opinion in Lipidology 12:489-495). Accordingly, appropriate assays using these models test whether administration of a candidate modulator alters, preferably decreases lipid accumulation in the liver. Lipid levels in plasma and adipose tissue may also be tested. Methods for assaying lipid content, typically by FPLC or colorimetric assays (Shimano H et al, 1996, J Clin Invest 98:1575-1584; Hasty et al., 2001, J Biol Chem 276:37402-37408), and lipid synthesis, such as by scintillation measurement of incorporation of radio-labeled substrates (Horton JD et al., 1999, J Clin Invest 103:1067-1076), are well known in the art. Other useful assays test blood glucose levels, insulin levels, and insulin sensitivity (e.g., Michael MD, 2000, Molecular Cell 6: 87). Insulin sensitivity is routinely tested by a glucose tolerance test or an insulin tolerance test.

In another embodiment, assays use mouse models of lipoprotein biology and cardiovascular disease. For instance, mouse knockouts of apolipoprotein E (apoE) display elevated plasma cholesterol and spontaneous arterial lesions (Zhang SH, 1992, Science 258:468-471). Transgenic mice over-expressing cholesterol ester transfer protein (CETP) also display increased plasma lipid levels (specifically, very-low-density lipoprotein [VLDL] and low-density lipoprotein [LDL] cholesterol levels) and plaque formation in arteries (Marotti KR et al., 1993, Nature 364:73-75). Assays using these models may test whether administration of candidate modulators alters plasma lipid levels, such as by decreasing levels of the pro-atherogenic LDL and VLDL, increasing HDL, or by decreasing overall lipid (including trigyceride) levels. Additionally histological analysis of arterial morphology and lesion formation (i.e., lesion number and size) may indicate whether a candidate modulator can reduce progression and/or severity of atherosclerosis. Numerous other mouse models for atherosclerosis are available, including knockouts of Apo-Al, PPARgamma, and scavenger receptor (SR)-Bl in LDLR- or ApoE-null background (reviewed in, e.g., Glass CK and Witztum JL, 2001, Cell 104:503-516). In another embodiment, the ability of candidate modulators to alter plasma lipid levels and artherosclerotic progression are tested in mouse models for multiple lipid disorders. For instance, mice with knockouts in both leptin and LDL receptor genes display hypercholesterolemia, hypertriglyceridemia and arterial lesions and provide a model for the relationship between impaired fuel metabolism, increased plasma remnant lipoproteins, diabetes, and atherosclerosis (Hasty AH et al, 2001, supra.).

Diagnostic Methods

The discovery that ISM is implicated in INR signaling provides for a variety of methods that can be employed for the diagnostic and prognostic evaluation of diseases and disorders associated with INR signaling and for the identification of subjects having a predisposition to such diseases and disorders. Any method for assessing ISM expression in a sample, as previously described, may be used. Such methods may, for example, utilize reagents such as the ISM oligonucleotides and antibodies directed against ISM, as described above for: (1) the detection of the presence of ISM gene mutations, or the detection of either over- or under-expression of ISM mRNA relative to the non-disorder state; (2) the detection of either an over- or an under-abundance of ISM gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in a biological pathway mediated by ISM.

All references cited herein, including patents, patent applications, publications, and gene and sequence data accessible through the Genbank identifier numbers and websites provided, are incorporated in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A method of identifying a candidate INR signaling modulating agent, said method comprising the steps of:

(a) providing an assay system comprising an ISM polypeptide or nucleic acid;

(b) contacting the assay system with a test agent under conditions whereby, but for the presence of the test agent, the system provides a reference activity; and

(c) detecting a test agent-biased activity of the assay system, wherein a difference between the test agent-biased activity and the reference activity identifies the test agent as a candidate INR signaling modulating agent.

2. The method of Claim 1 wherein the assay system includes a screening assay comprising an ISM polypeptide, and the candidate test agent is a small molecule modulator.

3. The method of Claim 2 wherein the screening assay detect an activity selected from the group consisting of kinase activity, fatty acid elongation, fatty acid desaturation, mitochondrial transport, upbiquinone biosynthesis, protein binding, and transcription.

4. The method of Claim 1 wherein the assay system includes a binding assay comprising an ISM polypeptide and the candidate test agent is an antibody.

5. The method of Claim 1 wherein the assay system includes an expression assay comprising an ISM nucleic acid and the candidate test agent is a nucleic acid modulator.

6. The method of Claim 5 wherein the nucleic acid modulator is an antisense oligomer.

7. The method of Claim 6 wherein the nucleic acid modulator is a PMO.

8. The method of Claim 1 wherein the assay system comprises cultured cells or a non- human animal expressing an ISM, and wherein the assay system includes an assay that detects an agent-biased change in INR signaling or an output of INR signaling.

9. The method of Claim 8 wherein the assay system comprises cultured cells.

10. The method of Claim 9 wherein the assay detects an event selected from the group consisting of expression of insulin-responsive genes, phosphorylation of an INR signaling pathway component, kinase activity of an INR signaling pathway component, glycogen synthesis, glucose uptake, GLUT4 translocation, and insulin secretion.

11. The method of Claim 8 wherein the assay system comprises a non-human animal.

12. The method of Claim 11 wherein the non-human animal is a mouse providing a model of diabetes and/or insulin resistance.

13. The method of Claim 12 wherein the assay system includes an assay that detects an event selected from the group consisting of hepatic lipid accumulation, plasma lipid accumulation, adipose lipid accumulation, plasma glucose level, plasma insulin level, and insulin sensitivity.

14. The method of Claim 1, comprising the additional steps of:

(d) providing a second assay system comprising cultured cells or a non-human animal expressing an ISM ,

(e) contacting the second assay system with the test agent of (b) or an agent derived therefrom under conditions whereby, but for the presence of the test agent or agent derived therefrom, the system provides a reference activity; and

(f) detecting an agent-biased activity of the second assay system, wherein a difference between the agent-biased activity and the reference activity of the second assay system confirms the test agent or agent derived therefrom as a candidate INR signaling modulating agent, and wherein the second assay system includes a second assay that detects an agent-biased change in an activity associated with INR signaling or an output of INR signaling.

15. The method of Claim 14 wherein the second assay system comprises cultured cells.

16. The method of Claim 15 wherein the second assay detects an event selected from the group consisting of expression of insulin-responsive genes, phosphorylation of an INR signaling pathway component, kinase activity of an INR signaling pathway component, glycogen synthesis, glucose uptake, GLUT4 translocation, and insulin secretion.

17. The method of Claim 17 wherein the second assay system comprises a non-human animal.

18. The method of Claim 18 wherein the non-human animal is a mouse providing a model of diabetes and/or insulin resistance.

19. The method of Claim 18 wherein the second assay system includes an assay that detects an event selected from the group consisting of hepatic lipid accumulation, plasma lipid accumulation, adipose lipid accumulation, plasma glucose level, plasma insulin level, and insulin sensitivity.

20. A method of modulating INR signaling in a mammalian cell comprising contacting the cell with an agent that specifically binds an ISM polypeptide or nucleic acid.

21. The method of Claim 20 wherein the agent is administered to a mammalian animal predetermined to have a pathology associated with INR signaling.

22. The method of Claim 20 wherein the agent is a small molecule modulator, a nucleic acid modulator, or an antibody.