JP2005532787A - MINR as a modifier of insulin receptor signaling and methods of use thereof - Google Patents

Abstract

Human MINR genes have been identified as modulators of INR signaling and thus are therapeutic targets for diseases associated with defective INR signaling. A method of identifying a modulator of MINR is provided, comprising screening to look for agents that modulate MINR activity.

Description

(Reference to related applications)
This application includes US Provisional Patent Application No. 60 / 354,824, filed February 6, 2002, 60 / 358,217, filed February 20, 2002, and filed February 20, 2002. No. 60 / 358,189, No. 60 / 358,126 filed on Feb. 20, 2002, No. 60 / 358,995 filed on Feb. 21, 2002, No. 60 / 358,995, Feb. 21, 2002 60 / 358,756, 60 / 358,765 filed on February 21, 2002, 60 / 359,531 filed on February 25, 2002, filed February 26, 2002 No. 60 / 360,222, No. 60 / 360,224 filed Feb. 26, 2002, No. 60 / 360,167 filed Feb. 26, 2002, and Feb. 26, 2002. No. 60 / 360,1 of Japanese application. Which claims the priority of No. 6. The contents of the earlier application are incorporated herein in their entirety.

(Background of the Invention)
Insulin is a central hormone that controls metabolism in vertebrates (reviewed in Steiner et al., 1989, Endocrinology, DeGroot, Philadelphia, Saunders: 1263-1289). In humans, insulin is secreted by pancreatic beta cells in response to elevated blood glucose levels, but it naturally occurs after a meal. The immediate effect of insulin secretion is to induce glucose uptake by muscle, adipose tissue, and liver. The long-term effect of insulin is to enhance the enzyme activity that synthesizes glycogen in the liver and triglycerides in adipose tissue. Insulin goes beyond the “classical” metabolic activity, including increased potassium transport in muscle, promoted adipocyte cell differentiation, increased sodium renal retention, and enhanced androgen production by the ovary The activity of can be demonstrated. Deficiencies in insulin secretion and / or response to insulin contribute to diabetes and have great economic significance. In the United States, diabetes is the fourth most common reason for patients to visit a doctor; it is the last stage of kidney disease, atraumatic limb amputation, and blindness in individuals in the working age group Main causes (Warram et al., 1995, In Joslin's Diabetes Mellitus, Kahn and Weir, eds., Philadelphia, Lea & Febiger, pp201-215; Kahn et al., 1996, Annu. Rev. Med. 47: 509- 531; Kahn, 1998, Cell 92: 593-596). Beyond its role in diabetes, the phenomenon of insulin resistance is associated with other pathogenic diseases including obesity, ovarian androgen hypertension, and hypertension.

The pharmaceutical industry is interested in understanding the molecular mechanisms that link lipid deficiency and insulin resistance. Hyperlipidemia and elevated free fatty acid levels are often associated with several diseases, including obesity and insulin resistance, which develop in the same patient and are the main risk factors for progression to type 2 diabetes and cardiovascular disease Correlates with “metabolic syndrome” as defined in Current studies suggest that in addition to blood glucose levels, control of lipid levels is required to treat symptoms of type 2 diabetes, heart disease, and other metabolic syndromes (Santomauro AT et al., Etc.). Diabetes (1999) 48: 1836-1841).
The ability to manipulate and screen the genomes of model organisms such as Drosophila and nematodes has a direct link to more complex vertebrate organisms due to significant evolutionary conservation of genes, pathways and cellular processes. Provides a powerful means of analyzing biochemical processes. Identification of novel functions of genes involved in specific pathways in such model organisms can directly contribute to an understanding of correlated pathways and how to regulate them in mammals (Dulubova I et al., J Neurochem 2001 Apr; 77 (1): 229-38; Cai T et al., Diabetologia 2001 Jan; 44 (1): 81-8; Pasquinelli AE et al., Nature. 2000 Nov 2; 408 (6808): 37-8; Ivanov IP et al., EMBO J 2000 Apr 17; 19 (8): 1907-17; Vajo Z et al., Mamm Genome 1999 Oct; 10 (10): 1000-4; Miklos GL and Rubin GM, Cell 1996,86: 521-529; Mechler BM et al., 1985 EMBO J 4: 1551-1557; Gateff E. 1982 adv. Cancer Res. 37: 33-74; Watson KL. Et al., 1994 J Cell Sci. 18: 19-33; Miklos GL, and Rubin GM. 1996 Cell 86 : 521-529; Wassarman DA et al., 1995 Curr Opin Gen Dev 5: 44-50; and Booth DR. 1999 Cancer Metastasis Rev. 18: 261-284). Drosophila and nematodes are rarely affected by human disease, but they can mimic pathological conditions in various experimental models. Correlation of the altered expression of the Drosophila or nematode gene with a pathological model can identify the association of human orthologs with human disease.

In one example, genetic screening is performed in an invertebrate model organism that exhibits a mutant (generally visible or selectable) phenotype due to misexpression of a known gene—generally reduced, enhanced or ectopic expression. Performed (“gene entry point”). Additional genes are mutated randomly or in a targeted manner. If further genetic mutations alter the original mutant phenotype, the gene is identified as a “modifier” that interacts directly or indirectly with the genetic entry point and its associated pathway. If the genetic entry point is an ortholog of a human gene related to human pathology, a modifier gene that is a candidate target for a novel therapeutic agent can be identified by screening.
Gene screening uses RNA interference (RNAi) technology to disrupt the activity of genes having homologous sequences by introducing exogenous double-stranded (ds) RNA, and has a specific reduced function phenotype (Fire et al., 1998, Nature 391: 806-811). Suitable methods for introducing dsRNA into animals include injection, feeding, and water bath (Tabara et al., 1998, Science 282: 430-431). RNAi has also been shown to produce specific gene disruptions in cultured Drosophila and mammalian cells (Paddison et al., Proc Natl Acad Sci USA published January 29, 2002, 10.1073 / pnas.032652399; Clemens et al. , 2000, Proc Natl Acad Sci USA 97: 6499-503; Wojcik and DeMartino, J Biol Chem, 10.1074 / jbc.M109996200 published December 5, 2001; Goto et al., 2001, Biochem J 360: 167-72; Elbashir Et al., 2001, Nature 411: 494-8).
The insulin receptor (INR) signaling pathway has been extensively studied in nematodes. Signal transduction by the nematode INR ortholog, daf-2, mediates a variety of events including reproductive growth and normal adult life (eg, US Pat. No. 6,225,120; Tissenbaum HA and Ruvkun G, 1998, Genetics 148: 703-). 17; see Ogg S and Ruvkun G, 1998, Mol Cell 2: 887-93; Lin K et al., 2001, Nat Genet 28: 139-45).

  NOT2 and S. Cerevisiae ortholog CDC36 are part of a complex of proteins that interact with polymerase II holoenzyme to regulate gene expression. The complex includes in particular CCR4, CAF and NOT family proteins. The NOT protein may limit the access of the TATA box protein to atypical TATAAs. NOT2 deficiency can lead to derepression of genes (Benson et al., 1998, EMBO 17: 6714-6722; Collart et al., 1994, Genes Dev. 8: 525-537; Liu et al., 2001, J. Biol Chem. 276: 7541-7548). The Drosophila Regena (Rga) gene is an ortholog of NOT2 and was first identified in a Drosophila screen for genes that regulate white eye gene expression. Regena has been shown to affect the expression of 4 of the 7 genes tested. This suggests that it is involved in the normal regulation of gene expression. RP49 ribosomal gene expression was not affected by Rga mutations. Based on sequence similarity and functional similarity, Rga was shown to be a homologue of the yeast gene CDC36 / NOT2 (Frolov et al., 1998, Genetics 148: 317-329).

  myotubularins (MYT) is a family of proteins conserved from multiple organisms including humans, Drosophila, and nematodes (Laporte et al., 1998, Hum. Molec. Genet. 7: 1703-1712; Laporte et al., 2001 Trends in Genetics 17: 221-228). The human family consists of at least 10 genes, and Drosophila and C. elegans each have 6 myotubularin related genes. Myotubularin has active site residues that are consistent with both protein and lipid phosphatase activities and has been shown to have these activities biochemically (Laporte et al., 1998, 2001). In addition, based on experimental evidence in yeast, it has been shown that myotubularin can down-regulate PI-3-kinase activity. In yeast, myotubularin strongly prefers PtdIns3P as a substrate (Taylor et al., 2000, Proc. Natl. Acad. Sci. USA 97: 8915). Residues conserved in the catalytic domain are in harmony with their activity as monophosphoinositide phosphatases, and mutations in these residues abolish lipid phosphatase activity in vitro (Taylor et al., 2000; Laporte et al., 2001) . In addition, mutant forms of human myotubularin were immunoprecipitated with yeast PI-3 kinase when introduced into yeast. This indicates that myotubularin can directly affect PI-3 kinase activity (Blondeau et al., 2000, Hum. Mol. Genet. 9: 2223-2229). The Drosophila myotubularin gene of GI17737395 is included in the human MTM1 / MTMR2 subgroup, and this is the only Drosophila gene included in this subgroup. MTM1 mutations are associated with disease X-linked tubule myopathy (Laporte et al., 1996, Nat. Genet. 13: 175-182), which leads to muscle fiber destruction. Mutations identified in the patient's MTM1 are often missense mutations that act on residues conserved between human and Drosophila proteins. Mutations in MTMR2 cause Charcot-Marie-Tooth disease and affect myelination of motor and sensory neurons (Bolino et al., 2000, Nat. Genet. 25: 17-19).

  DNMT1 is an enzyme that maintains mammalian DNA methylation and is a component of a repressible transcriptional complex. A protein related to DNMT (DMAP1) was identified by screening using a yeast two-hybrid method for proteins that interact with DNMT1. DMAP1 intrinsically has transcriptional repressive activity and binds to the tumor suppressor gene TSG101. TSG101 has been shown to act as a transcriptional corepressor involved in nuclear hormone-induced gene silencing and functions in late endosomal trafficking (Roundtree et al., 2000, Nature Genetics 25: 269-277).

  The human tuberous sclerosis (TCS) complex is a disease that results in the formation of benign tumors in many tissues (Cheadle et al., 2000, Hum. Genet. 107: 97-114). These tumors contain differentiated cells, but these cells are much larger than normal cells. This disease appears most severely in the central nervous system and can cause epilepsy, developmental delay, and autism and is caused by mutations in the TSC1 or TSC2 gene (Consortium TECTS, 1993, Cell 75: 1305-1315; van Slegtenhorst et al., 1997, Science 277: 805-808). TSC1 encodes hamartin and TSC2 encodes tuberin, and human proteins have been shown to interact in vitro (Plank et al., 1998, Cancer Res. 58: 4766-4770; van Slegtenhorst et al., 1998, Hum. Mol. Genet. 7: 1053-1057). Tuberin, the product of TSC2 protein, contains a GTP hydrolase activating protein (GAP) domain in addition to the coiled-coil domain and has GAP activity in vitro (Wienecke et al., 1995, J. Biol. Chem. 270: 16409- 16414). Rap / ran-GAP domains have also been found in GTP hydrolase-activating proteins responsible for the activities of the nuclear Ras-related regulatory proteins Rap1, Rsr1 and Ran in vitro, which affects cell cycle progression. Gigas (GIG) is the Drosophila ortholog of TCS2. GIG-reduced mutagenesis genes exhibit a range of phenotypes including larval lethality and various neuroanatomical and behavioral defects depending on the strength of the mutagenic gene (Meinertzhagen, 1994, J. Neurogenet 9: 157 -176; Canal et al., 1998, J. Neurosci 18: 999-1008; Acebes and Ferrus 2001, J. Neurosci 21: 6264-6273). In addition, GIG mutant cells differentiate normally, but their size is 2-3 times that of normal. Overexpression of the Drosophila TSC1 and TSC2 (GIG) genes results in a decrease in cell size, number and organ size (Potter et al., 2001, Cell 105: 357-368; Tapon et al. 2001). Genetic experiments using flies have shown that the combination of TSC1 and TSC2GIG genes antagonizes insulin receptor signaling (Gao et al., 2001, Genes and Dev. 15: 1383-1392; Potter et al., 2001; Tapon et al., 2001, Cell 105: 345-355). According to the genetic data, TSC1 and TSC2 (GIG) appear to function downstream of Akt and upstream of S6 kinase in the same pathway as these genes or in a parallel pathway.

  RAB5 is a member of the Ras superfamily of GTP hydrolases involved in vesicle trafficking (Somsel Rodman and Wandinger-Ness, 2000, J. Cell Sci. 113: 183-192). The endocytic pathway is important for nutrient uptake, cell surface receptor regulation, and recycling of proteins used in the secretory pathway. RAB5 is associated with clathrin-coated vesicles and early endosomes and has the function of regulating endocytosis internalization and early endosome fusion (Woodman, 2000, Traffic 9: 695-701). The FYVE domain protein Rabenosyn-5 has been shown to be an effector of Rab5 and Rab4 and physically connects early endosomes and receptor recycling to the cell surface (De Renzis et al., 2002, Nat. Cell Biol 4: 124-133). Insulin responsive tissues express multiple Rab isoforms, including Rab3b, Rab4, Rab5, and Rab8. Of these isoforms, only Rab4 has been shown to play a role in mediating intracellular insulin action, including insulin-stimulated GLUT4 translocation to the cell membrane (Knight et al., 2000, Endocrinology 141: 208- 218). There is some evidence that the membrane involvement of Rab5 changes in skeletal muscle isolated from type 2 diabetic patients with resistance to insulin (Bao et al., 1998, Horm. Metab. Res. 30: 656-662 ).

  Drosophila SNAP is an ortholog of the human α-soluble NSF gene (α-SNAP or aSNAP). In Drosophila, SNAP is known as part of the conserved SNARE complex required for secretory vesicle fusion with the plasma membrane (Ordway et al., 1994, PNAS USA 91: 5715-5719). No Drosophila mutations have been reported in Drosophila, but mutations in NSF are defective in motor behavior and paralysis because the major protein SNAP recruits (Littleton et al., 1998, Neuron 21: 401 -413). In vertebrates, SNAP has been shown to be involved in trans during vesicle binding in association with the SNARE complex (Xu et al., 1999, EMBO J. 18: 3293-3304). SNAP recruits and stimulates NSF, and ATPase degrades and recycles the SNARE complex (Sudlow et al., 1996, FEBS Lett 393: 185-188; Barnard et al., 1997, J. Cell Biology 139: 875 -883; Cheatham 2000, Trends in Endocrinol. Metab. 11: 356-361). The combination of SNAP and NSF dramatically increases the rate of exocytosis. Vertebrate βSNAP is similar to αSNAP, but αSNAP has been shown to increase exocytosis over βSNAP (Xu et al., 2002, J. Neurosci 22: 53-61). αSNAP variability analysis indicates a requirement for leucine 294. αSNAP (L294A) is normally associated with SNARE complex and NSF, but acted as a dominant mutation by inhibiting ATPase-dependent stimulation of exocytosis by exogenous αSNAP (Barnard et al., 1997, Supra).

  CAF-1 (catabolite repressor protein (CCR4) -related factor 1), also known as CCR4-NOT transcriptional complex subunit 7, is a protein complex that interacts with RNA polymerase II holoenzyme to regulate gene expression (Albert et al., 2000, Nucleic Acids Res. 28: 809-817). The complex also specifically includes CCR4 and NOT proteins. In addition to globally regulating RNA polymerase II transcription, CAF-1 also regulates gene expression by regulating early ribosome assembly (Schaper et al., 2001, Curr. Biol. 11: 1885-1890). CCR4 and CAF-1 are also components of the major cytoplasmic mRNA deadenylase in yeast and can function in the early stages of mRNA turnover by initiating poly (A) tail shortening ( Tucker et al., Cell 104: 377-386).

  VAMP is a member of the SNARE protein family, a protein that is important for membrane fusion in both regulatory and intrinsic vesicle trafficking. The VAP33 (33 kDa VAMP-related protein) protein binds VAMP and SNARE (Weir et al., 2001, Biochem Biophys Res Commun 286: 616-21). Mammalian VAP33 (VAP-A) is widely expressed in multiple tissues and appears to be associated with ER and microtubules and trafficking vesicles (Weir et al., 1998, Biochem. J. 333: 247-251). . Three types of human isoforms are known for VAP33. VAP-A and VAP-B are encoded by separate genes and about 60% are identical; VAP-C is a splice variant of VAP-B and lacks the C-terminal transmembrane domain ( Nishimura et al., 1999, Biochem. Biophys. Res. Commun. 254: 21-26). VAP33 has been found to play a central role in the migration of insulin-stimulated GLUT4 to the cell surface in L6 myoblasts and 3T3-L1 adipocytes (Foster et al., 2000, Traffic 6: 512- 521). It has also been demonstrated that the yeast homologue SCS2 is required for inositol metabolism (Kagiwada et al., 1998, J. Bacteriol. 180: 1700-1708).

  PP2 (also called PP2A) is a serine / threonine protein phosphatase associated with the dephosphorylation of proteins Akt and Gsk3β (Ivaska et al., 2002, Mol Cell Biol 22: 1352-1359); glycogen synthesis by dephosphorylation of Gsk Enzyme activity is increased. Accompanying reports indicate that ceramide-mediated insulin resistance induces PP2 activity and can be reduced by treatment with the PP2 inhibitor okadaic acid (Teruel et al., 2001, Diabetes 50: 2563-2571). Finally, it has been demonstrated to stimulate acetyl core carboxylase, an enzyme that can catalyze the production of long chain fatty acids and regulate insulin secretion (Kowluru et al., 2001, Diabetes 50: 1580-1587). PP2 is also thought to inhibit acyl core: cholesterol acyltransferase (ACAT) and cholesterol ester synthesis (Hernandez et al., 1997, Biochim Biophys Acta 1349: 233-41). Drosophila MTS (microtubule star) is an ortholog of PP2, plays an essential role in spindle formation, and has important implications for the attachment of microtubules to the centromere in the mitosis stage (Snaith et al. , 1996, J. Cell Sci. 109: 3001-3012). Mouse PP2 is also required for meiosis (Lu et al., 2002, Biol Reprod. 66 (1): 29-37). The need for MTS / PP2 is thought to be due to hyperphosphorylation and inactivation of τ protein, which is associated with microtubules and promotes their stabilization (Brandt and Lee 1993, J Neurochem. 61: 997 -1005; Planel et al. 2001, J. Biol. Chem. 276 (36): 34298-34306).

  CSNK1 is a serine / threonine protein kinase and belongs to the mammalian casein kinase I gene family and produces multiple isoforms. Family members include a highly conserved ˜290 residue N-terminal catalytic domain that binds to the variable C-terminal region. The C-terminal region has a role in promoting differential subcellular localization of individual isoforms and regulating enzyme activity (Mashhoon et al., 2000 J Biol Chem 275: 20052-20060). CSNK1 appears to contribute to the regulation of the rhythm of the approximately 24-hour cycle, intracellular trafficking, DNA repair, cell morphology, and protein stabilization (Liu et al., 2001, Proc Natl Acad Sci 98: 11062- 11068). CSNK1 has also been shown to be involved in the regulation of eIF2B in coordination with GSK3, which is part of the insulin signaling response (Wang et al., 2001, EMBO 20: 4349-4359). Drosophila GISH (Gilgamesh) is an ortholog of human CSNK1 and has been characterized as part of the mechanism of repulsive signaling that regulates glial migration and ocular neurodevelopment (Hummel et al., 2002, Neuron 33: 193-203).

  ERF1 (eukaryotic termination factor 1) terminates protein biosynthesis. Termination of protein biosynthesis and release of the unfinished polypeptide chain is signaled by the presence of an in-frame stop codon at the aminoacyl site of the ribosome. ERF1 identifies a stop codon and promotes hydrolysis of ester bonds that link the polypeptide chain to the peptidyl site tRNA (Frolova et al., 1994, Nature 372: 701-703). The crystal structure of the termination factor has been determined, the overall shape and size of ERF1 is similar to the tRNA molecule, and the dedicated domains 1, 2 (Song et al., 2000, Cell 100: 311-321).

  All references cited herein, including patents, patent applications, publications and referenced Genbank identification numbers, are incorporated herein in their entirety.

(Summary of Invention)
We have discovered a gene that regulates the INR pathway in Drosophila cells, and identified its human ortholog, referred to herein as the insulin receptor signaling modifier (MINR). The present invention uses these INR modifier genes and polypeptides to identify a MINR modulator that is a candidate therapeutic that can be used to treat a disease associated with a defective or damaged INR function and / or MINR function. provide. Preferred MINR modulators specifically bind to MINR polypeptides and restore INR function. Other preferred MINR modulators are nucleic acid modulators such as antisense oligomers, or RNAi that suppress MINR gene expression or product activity by binding to and inhibiting each nucleic acid (ie, DNA or mRNA), for example. is there.

  A MINR modulator can be assessed by any convenient in vitro or in vivo assay for molecular interaction with a MINR polypeptide or nucleic acid. In one embodiment, candidate MINR modulating agents are tested using an assay system comprising MINR polypeptides or nucleic acids. Agents that cause a change in the activity of the assay system relative to the control are identified as candidate INR modulating agents. The assay system may be cell based or cell free. MINR modulators include MINR-related proteins (eg, dominant negative mutants and biotherapeutic agents); antibodies specific for MINR; antisense oligomers and other nucleic acid modulators specific for MINR; and specific binding or Chemical agents that interact or compete with the MINR binding pair (eg, by binding to the MINR binding pair) are included. In one particular embodiment, binding assays are used to identify small molecule modulators. In certain embodiments, the screening assay system is selected from a liver lipid accumulation assay, a plasma lipid accumulation assay, an adipocyte lipid accumulation assay, a plasma blood glucose level assay, a plasma insulin level assay, and an insulin sensitivity assay.

  In another embodiment, candidate INR pathway modulators are further tested using a second assay system that detects a change in activity associated with INR signaling. In the second assay system, cultured cells or non-human animals can be used. In certain embodiments, the secondary assay system uses non-human animals, including animals that have been previously determined to have a disease or disorder associated with the INR pathway.

  The invention further provides a method of modulating MINR function and / or INR pathway in a mammalian cell by contacting the mammalian cell with an agent that specifically binds to a MINR polypeptide or nucleic acid. The agent can be a small molecule modulator, a nucleic acid modulator, or an antibody and can be administered to a mammal that has been previously determined to have a pathology associated with the INR pathway.

(Detailed description of the invention)
Cellular RNAi screening was used to identify INR pathway modifiers and signaling activity. The modulators of the INR pathway were identified, and then their orthologs were identified. Thus, the insulin receptor signaling modifier (MINR) gene (ie, nucleic acids and polypeptides) is an attractive drug target for the treatment of diseases associated with INR signaling. In one example, the treatment includes increasing signaling through the INR to treat a pathological condition associated with diabetes and / or metabolic syndrome.

  The present invention provides in vitro and in vivo methods for assessing MINR function and methods for modulating (generally inhibiting or agonizing) MINR activity, which further elucidate INR signaling and pathological conditions associated with INR signaling Useful for developing diagnostic and therapeutic modalities. As used herein, pathological conditions associated with INR signaling include pathological conditions in which INR signaling contributes to maintaining a healthy state, as well as pathological conditions in which the process can be altered by modulation of INR signaling Is included.

MINR Nucleic Acids and Polypeptides Sequences related to MINR nucleic acids and polypeptides that can be used in the present invention are disclosed in Genbank (referred to by Genbank identification (GI) or reference number), and Table 1 Example 1).

  The term “MINR polypeptide” means a full-length MINR protein or a “functionally active” fragment or derivative thereof, “functionally active” means that the MINR protein fragment or derivative is a complete Means to exhibit one or more functional activities associated with a long wild type MINR protein. By way of example, a fragment or derivative may be antigenic so that it can be used for immunoassay, immunization, production of inhibitory antibodies, as discussed further below. Preferably, the functionally active MINR fragment or derivative exhibits one or more biological activities associated with the MINR protein, such as enzyme activity, signal transduction activity, ability to bind to natural cell substrates, etc. In one embodiment, the functionally active MINR polypeptide is a MINR derivative that can rescue defective endogenous MINR activity, eg, in a cell-based or animal assay; the rescue derivative is homologous or different From different species. When MINR fragments are used in the assay to identify modulators, the fragments preferably have a MINR domain, such as in particular the C or N-terminus or catalytic domain, preferably at least 10, preferably at least 20, of the MINR protein. More preferably it comprises at least 25, and most preferably at least 50 contiguous amino acids. Preferred MINR fragments contain a catalytic domain. Functional domains can be identified using the PFAM program (Bateman A. et al., 1999 Nucleic Acids Res 27: 260-262; website pfam.wustl.edu).

  The term “MINR nucleic acid” refers to a DNA or RNA molecule that encodes a MINR polypeptide. Preferably, the MINR polypeptide or nucleic acid or fragment thereof is of human origin but has at least 70% sequence identity with human MINR, preferably at least 80%, more preferably 85%, even more preferably 90%, most preferably It may be an ortholog or derivative thereof having at least 95% sequence identity. Methods for identifying human orthologs of these genes are known in the art. Usually, different species of orthologs retain the same function due to the presence and / or three-dimensional structure of one or more protein motifs. In general, orthologs are typically identified by sequence homology analysis, such as BLAST analysis, using protein bait sequences. When the best matching sequence of forward BLAST results retrieves the original query sequence of reverse BLAST, the sequence is designated as a potential ortholog (Huynen MA and Bork P, Proc Natl Acad Sci (1998) 95: 5849- 5856; Huynen MA et al., Genome Research (2000) 10: 1204-1210). Using a program for multiple sequence comparison, such as CLUSTAL (Thompson JD et al., 1994, Nucleic Acids Res 22: 4673-4680) to highlight conserved regions and / or residues of orthologous proteins and You may create it. In a phylogenetic tree representing multiple homologous sequences of various species (eg, those extracted by BLAST analysis), the two orthologous sequences appear as the closest in the phylogenetic tree with respect to all the other two sequences. . Potential orthologs may be identified by structural threading or other methods of protein folding (eg, using software from ProCeryon, Biosciences, Salzburg, Austria). In evolution, when gene replication occurs following speciation, a single species of a single species such as a nematode may correspond to multiple genes (paralogs) of another species such as a human. As used herein, the term “ortholog” includes paralogs. “Percent (%) sequence identity” as used herein with respect to an identified subject sequence or portion of a subject sequence refers to the program WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol., (1997) 215: 403-410; http://blast.wustl.edu/blast/README.html) to align sequences and obtain maximum percent sequence identity Therefore, after introducing gaps as necessary, it is defined as the percentage of nucleotides or amino acids in the sequence of candidate derivatives that are identical to the nucleotides or amino acids in the subject sequence (or a specific portion thereof). The HSP S and HSP S2 parameters are dynamic values and are determined by the program itself depending on the composition of the particular sequence and the composition of the particular database in which the target sequence is being compared and searched. A “% identity value” is determined by dividing the number of matching identical nucleotides or amino acids by the length of the sequence for which the percent identity is reported. “Percent (%) amino acid sequence similarity” is determined by performing the same calculation as determining% amino acid sequence identity but including conservative amino acid substitutions in addition to the same amino acid. A conservative amino acid substitution is a substitution in which one amino acid is replaced with another amino acid having similar properties so 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; compatible hydrophobic amino acids are leucine, isoleucine, methionine, and valine; compatible polar amino acids are glutamine and asparagine; The basic amino acids are arginine, lysine and histidine, the compatible acidic amino acids are aspartic acid and glutamic acid, and the compatible small amino acids are alanine, serine, threonine, cysteine and glycine.

  Alternatively, nucleic acid sequence alignments are provided by Smith and Waterman's local homology algorithm (Smith and Waterman, 1981, Advances in Applied Mathematics, 2: 482-489; 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” (website www.psc.edu) and references cited therein; WRPearson, 1991, Genomics, 11: 635-650). This algorithm was developed by Dayhoff (Dayhoff: Atlas of Protein Sequences and Structure, MODayhoff, 5th Appendix, 3: 353-358, National Biomedical Research Foundation, Washington, DC, USA) and normalized by Gribskov (Gribskov 1986, Nucl. Acids Res. 14 (6): 6745-6763) Can be applied to amino acid sequences by using a scoring matrix. The Smith-Waterman algorithm with default parameters can be used to score (eg gap start penalty 12, gap extension penalty 2). In the generated data, the “match” value reflects “sequence identity”.

  Derived nucleic acid molecules of the subject nucleic acid molecules include sequences that hybridize to MINR nucleic acid sequences. The stringency of hybridization can be adjusted by the presence of denaturing agents such as formamide during temperature, ionic strength, pH, and hybridization and washing. Conventionally used conditions are described in readily available procedures (eg, Current Protocol in Molecular Biology, Volume 1, Chapter 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 comprises 6 × unit strength citrate (SSC) (1 × SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5 X Prehybridization of the filter containing nucleic acid from 65 hours to overnight in a solution containing Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg / ml herring sperm DNA; 6 × SSC; Hybridization at 65 ° C. for 18-20 hours in a solution containing 1 × Denhart solution, 100 μg / ml yeast tRNA and 0.05% sodium pyrophosphate; and 0.1 × SSC and 0. Stringent hybridization including washing the filter with a solution containing 1% SDS (sodium dodecyl sulfate) at 65 ° C. for 1 hour. The nucleic acid molecule comprising the nucleotide sequence of MINR can be hybridized under the conditions. In other embodiments, 35% formamide, 5 × SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and Pretreat the filter containing nucleic acids for 6 hours at 40 ° C. in a solution containing 500 μg / ml denatured salmon sperm DNA; 35% formamide, 5 × SSC, 50 mM Tris-HCl (pH 7.5), In a solution containing 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, and 10% (weight / volume) dextran sulfate, Performing hybridization at 40 ° C. for 18-20 hours; followed by washing twice with a solution containing 2 × SSC and 0.1% SDS for 1 hour at 55 ° C. To use the stringent hybridization conditions. Alternatively, in a solution containing 20% formamide, 5 × SSC, 50 mM sodium phosphate (pH 7.6), 5 × Denhart solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA, Incubate from time to overnight at 37 ° C .; perform hybridization in the same buffer for 18-20 hours; and wash the filter with 1 × SSC at about 37 ° C. for 1 hour. Gent conditions can be used.

MINR Nucleic Acids and Polypeptides Isolation, Generation, Expression, and Misexpression MINR Nucleic Acids and Polypeptides are Useful for Identification and Testing of Agents that Modulate MINR Function and Other Applications Related to MINR Involvement in INR Signaling It is. MINR nucleic acids can be obtained using any available method. Techniques for isolating a cDNA or genomic DNA sequence of interest, for example, by screening a DNA library or by using polymerase chain reaction (PCR) are well known in the art.

  A great variety of methods are available for obtaining MINR polypeptides. In general, the intended use of a polypeptide defines the details of expression, production, and purification methods. For example, generating polypeptides for use in screening for modulators may require a method that preserves the specific biological activity of these proteins, to generate polypeptides for antibody production. May require the structural integrity of a particular epitope. Expression of the polypeptide to be purified for screening or antibody production may require the addition of specific tags (eg, production of fusion proteins). To overexpress MINR polypeptides for cell-based assays used to assess MINR function, such as involvement in tubuleogenesis, in eukaryotic cell lines capable of these cellular activities. Expression may be necessary. Methods for expressing, producing and purifying proteins are well known in the art and any suitable means therefor can be used (eg, Higgins SJ and Hames BD (ed.) Protein Expression: A Practical Approach, Oxford University Press Inc., New York 1999; Stanbury PF, Principles of Fermentation Technology, 2nd Edition, Elsevier Science, New York, 1995; Doonan S, Protein Purification Protocols, Humana Press, New Jersey, 1996; Coligan JE, etc. Current Protocols in Protein Science, 1999, John Wiley & Sons, New York; US Pat. No. 6,165,992).

  The nucleotide sequence encoding the MINR polypeptide can be inserted into any suitable expression vector to express the inserted protein coding sequence. The necessary transcriptional and translational signals including the promoter / enhancer element may be derived from the native MINR gene and / or its flanking region or may be heterologous. Mammalian cell lines infected with viruses (eg, vaccinia virus, adenovirus, etc.); insect cell lines infected with viruses (eg, baculovirus); yeast containing yeast vectors or bacteriophage, plasmid, or cosmid DNA Various host-vector expression systems such as transformed microorganisms such as bacteria can be used. Host cell systems that modulate, modify, and / or specifically process the expression of the gene product can be used.

  A MINR polypeptide may optionally be expressed as a fusion or chimeric product linked to a heterologous protein sequence via a peptide bond. In one application, the heterologous sequence encodes a transcription reporter gene (eg, GFP or other fluorescent protein, luciferase, β-galactosidase, etc.). A chimeric product can be prepared by linking the appropriate nucleic acid sequences encoding the desired amino acid sequence together using standard methods within the appropriate coding frame and expressing the chimeric product. Chimeric products can also be prepared by protein synthesis techniques, such as using a peptide synthesizer (Hunkapiller et al., Nature (1984) 310: 105-111).

MINR polypeptides can be isolated and purified using standard methods (eg, ion exchange, affinity, and gel exclusion chromatography; centrifugation; difference in solubility; electrophoresis). Alternatively, native MINR protein can be purified from natural sources by standard methods (eg, immunoaffinity purification). Once the protein is obtained, it can be quantified and measured for its activity by an appropriate method such as immunoassay, bioassay, or measurement of other physical properties such as crystallography.
In the methods of the invention, cells that have been engineered to change (to be misexpressed) the expression of other genes associated with MINR or INR signaling may also be used. Misexpression as used herein includes ectopic expression, overexpression, underexpression, and no expression (eg, due to knockout of a gene or block of expression normally caused normally).

Genetically modified animals The methods of the invention may use non-human animals that have been genetically modified to modify the expression of other genes known to be involved in MINR and / or INR signaling. Preferred genetically modified animals are mammals, particularly mice or rats. Suitable non-mammalian species include zebrafish, nematodes, and drosophila. Preferably, the modified MINR or other gene expression is an altered level of INR signaling, an altered level of plasma glucose or insulin, for example when compared to a control animal having normal expression of the altered gene, Or produce a detectable phenotype, such as an altered lipid profile. The genetically modified animals can be used to further elucidate INR signaling in animal models of conditions associated with INR signaling and for in vivo testing of candidate therapeutics, as described below.
Suitable genetically modified animals are transgenic, and at least a portion of the cells have a non-natural nucleic acid present as a stable genomic insert or extrachromosomal element, typically a mosaic. Preferred transgenic animals have germline inserts that are stably transmitted to all progeny cells.

  Non-natural nucleic acids are introduced into the host animal by any convenient method. Methods for producing transgenic animals are well known in the art (for transgenic mice, see Brinster et al., Proc. Nat. Acad. Sci. USA 82: 4438-4442 (1985), both of which are US Pat. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 to Wagner et al., And Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1986); see Sandford et al., US Pat. No. 4,945,050 for particle bombardment; Rubin and Spradling, Science, 1982, 218: 348-53 and US Pat. See 4670388; for transgenic insects see Berghammer AJ et al., A Universal Marker for Transgenic Insects (1999) Nature 402: 370-371; (See Lin S., Transgenic Zebrafish, Methods Mol Biol. (2000); 136: 375-3830); for microinjection procedures in fish, amphibian eggs and birds, see Houdebine and Chourrout, Experientia (1991). ) 47: 897-905; for transgenic rats see Hammer et al., Cell (1990) 63: 1099-1112; embryonic stem (ES) cells are cultured, followed by electroporation, calcium phosphate / DNA precipitation See, for example, Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, edited by EJRobertson, IRL Press, (1987) for the production of transgenic animals by introducing DNA into ES cells using methods such as direct injection. ). Non-human transgenic animals can be cloned according to available methods (Wilmut, I et al. (1997) Nature 385: 810-813; see PCT International Publication Nos. WO 97/07668 and WO 97/07669) .

  In one embodiment, the transgenic animal has a heterozygous or homozygous change in the sequence of the endogenous MINR gene that results in reduced MINR function, preferably such that MINR expression is undetectable or negligible. A “knock-out” animal. Knockout animals are usually produced by homologous recombination using a vector containing a transgene having at least part of the gene to be knocked out. Usually, in order to functionally disrupt the transgene, a deletion, addition, or substitution is introduced into it. The transgene may be a human gene (eg, derived from a human genomic clone), but more preferably is an ortholog of a human gene derived from a transgenic host species. For example, the mouse MINR gene is used to construct a homologous recombination vector suitable for altering the endogenous MINR gene in the mouse genome. Detailed methods of homologous recombination in mice are available (see Capecchi, Science (1989) 244: 1288-1292; Joyner et al., Nature (1989) 338: 153-156). Procedures for generating non-rodent transgenic mammals and other animals are also available (Houdebine and Chourrout, supra; Pursel et al., Science (1989) 244: 1281-1288; Simms et al., Bio / Technology (1988). ) 6: 179-183). In a preferred embodiment, a knockout animal, such as a mouse in which a particular gene is knocked out, can be used to produce antibodies against the human counterpart of the knocked out gene (Claesson MH et al., (1994) Scan J Immunol 40: 257-264; Declerck PJ et al. (1995) J Biol Chem. 270: 8397-400).

  In another embodiment, the transgenic animal may express MINR gene expression, for example, by introducing additional copies of MINR or by operably inserting regulatory sequences that alter expression of an endogenous copy of MINR gene. A “knock-in” animal that has changes in its genome that result in changes in it (eg, increased expression (including increased ectopicity) and decreased). Such regulatory sequences include inducible, tissue-specific and constitutive promoter and enhancer elements. Knock-ins can be homozygous or heterozygous.

  Non-human transgenic animals can also be generated that contain selected systems that allow for controlled expression of the transgene. An example of such a system that can be made is the cre / loxP recombinase system of bacteriophage P1 (Lakso et al., PNAS (1992) 89: 6232-6236; US Pat. No. 4,959,317). When using the cre / loxP recombinase system to control transgene expression, an animal containing a transgene encoding both the Cre recombinase and the selected protein is required. Such animals can be produced, for example, by creating a “double” transgenic animal by crossing two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. Can be offered. Another example of a recombinase system is the Saccharomyces cerevisiae FLP recombinase system (O'Gorman et al. (1991) Science 251: 1351-1355; US Pat. No. 5,654,182). In a preferred embodiment, both Cre-LoxP and Flp-Frt are used in the same system to control transgene expression and so that vector sequences in the same cell are sequentially deleted (Sun X Et al. (2000) Nat Genet 25: 83-6).

  Genetically engineered animals are used for genetic research as an animal model of diseases and disorders associated with defects in INR function and for in vivo testing of candidate therapeutics such as those identified in the screening described below. It can be further elucidated. Candidate therapeutic agents are administered to genetically modified animals with altered MINR function, and phenotypic changes can be made in genetically modified animals that have been treated with placebo and / or animals in which MINR expression has not been altered that has been given candidate therapeutic agents, etc. Compare with appropriate control animals.

  In addition to the above-described genetically modified animals with altered MINR function, animal models with defective INR function (and otherwise normal MINR function) can be used in the methods of the invention. For example, INR knockout mice can be used to assess in vivo the activity of candidate INR modulators identified in one of the in vitro assays described below. Preferably, when a candidate INR modulator is administered to a model system having cells that are defective in INR function, it results in a detectable phenotypic change in the model system, thereby restoring INR function. Is shown.

MINR Modulators The present invention provides methods for identifying agents that interact with and / or modulate MINR function and / or INR signaling. Such agents are useful for various diagnostic and therapeutic applications associated with INR signaling and for a more detailed analysis of MINR proteins and their contribution to INR signaling. Accordingly, the present invention also provides a method of modulating INR signaling comprising the step of specifically modulating MINR activity by administering a MINR interacting or modulating agent.

  As used herein, a “MINR modulator” is any agent that modulates MINR function, such as interacting with MINR to inhibit or enhance MINR activity or otherwise affect normal MINR function. It is a drug to give. MINR function can be affected at any level including transcription, protein expression, protein localization, cellular activity or extracellular activity. In a preferred embodiment, the MINR modulating agent specifically modulates MINR function. Phrases such as “specific modulator”, “specifically modulate” and the like herein directly bind to a MINR polypeptide or nucleic acid, preferably inhibit, enhance, or otherwise inhibit MINR function. Used to mean a modifier that changes. These phrases also refer to the interaction of MINR with a binding partner or substrate, or cofactor (eg, by binding to the binding partner of MINR or by modifying the MINR function by binding to a protein / binding partner complex). Also included are modulators that vary. In further preferred embodiments, the MINR modulator is a modulator of the INR pathway (eg, restores and / or upregulates INR function) and is therefore also an INR modulator.

  Preferred MINR modulating agents include small molecule chemical agents; MINR interacting proteins including antibodies and other biotherapeutic agents; and nucleic acid modulators including antisense oligomers and RNA. The modulator may be formulated into the pharmaceutical composition as a composition that may include other active ingredients and / or suitable carriers and excipients, such as in combination therapy. Techniques for formulating or administering compounds are found in “Remington's Pharmaceutical Sciences”, Mack Publishing Co., Easton, PA, 19th edition.

Small molecule modulators Chemical agents referred to in the art as “small molecule” compounds are typically organic non-molecular having a molecular weight of less than 10,000, preferably less than 5,000, more preferably less than 1,000, and most preferably less than 500. It is a peptide molecule. This class of modulators includes chemically synthesized molecules such as compounds from combinatorial chemical libraries. Synthetic compounds can be rationally designed or identified based on known or putative properties of MINR proteins, or can be identified by screening compound libraries. Appropriate modulators of this class are natural products, particularly secondary metabolites from organisms such as plants and fungi that can also be identified by screening compound libraries to look for MINR modulating activity. . Methods for making 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 the screening assays described below can be used as lead compounds, from which candidate clinical compounds can be designed, optimized and synthesized. Such clinical compounds may be useful for treating conditions associated with INR signaling. The activity of candidate small molecule modulators may be improved several fold by interactive secondary functional verification, structure determination, and candidate modulator modification and testing as described further below. In addition, candidate clinical compounds are made with particular attention to clinical and pharmacological properties. For example, reagents can be derivatized and rescreened using in vitro and in vivo assays to optimize activity and minimize toxicity in pharmaceutical development.

Protein Modulators Specific MINR interacting proteins are useful in a variety of diagnostic and therapeutic applications associated with the INR pathway and related diseases, as well as validation assays for other MINR modulators. In preferred embodiments, MINR interacting proteins affect normal MINR function, including transcription, protein expression, protein localization, cellular activity or extracellular activity. In another embodiment, MINR interacting proteins are useful for detecting and providing information regarding MINR protein function (eg, for diagnostic purposes) as they are associated with diseases associated with INR, such as diabetes. ).

  MINR interacting proteins are endogenous, such as members of the MINR pathway that regulate MINR expression, localization, and / or activity, ie, those that interact genetically or biochemically with MINR. Good. MINR modulators include MINR interacting proteins and dominant negative forms of MINR proteins themselves. Yeast two hybrids and mutant screening provide a preferred method for identifying endogenous MINR interacting proteins (Finley, RL et al. (1996) DNA Cloning-Expression Systems: A Practical Approach, Glover D. and edited by Hames BD ( 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; US Pat. No. 5,928,868). Mass spectrometry is a preferred alternative method for elucidating protein complexes (eg, Pandley A and Mann M, Nature (2000) 405: 837-846; Yates JR 3rd, Trends Genet (2000) 16: 5-8 Is a review).

MINR interacting proteins can be exogenous proteins such as MINR-specific antibodies and T cell antigen receptors (eg, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory; Harlow and Lane (1999)). Using antibodies: a laboratory manual. See Cold Spring Harbor, NY: Cold Spring Harbor Loboratory Press). The MINR antibody is further described below.
In a preferred embodiment, the MINR interacting protein specifically binds to the MINR protein. In other preferred embodiments, the MINR modulating agent binds to a MINR substrate, binding partner, or cofactor.

Antibodies In another embodiment, the protein modulator is a MINR-specific antibody agonist or antagonist. This antibody has therapeutic and diagnostic uses and can be used in screening assays to identify MINR modulators. The antibody can also be used in the analysis of the portion of the MINR pathway responsible for various cellular responses and the general processing and maturation of MINR.

Antibodies that specifically bind to MINR polypeptides can be generated using well-known methods. Preferably, the antibody is specific for a mammalian ortholog of MINR polypeptide, more preferably human MINR. Antibodies include polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ′) ₂ fragments, fragments produced by FAb expression libraries, anti-idiotypes (anti-Id) ) Antibodies and any of the above epitope-binding fragments. Select epitopes of MINR that are particularly antigenic, for example, by routine screening of MINR polypeptides that exhibit antigenicity, or by applying a theoretical method to select the antigenic region of a protein against the amino acid sequence of MINR (Hopp and Wood, (1981) Proc. Nati. Acad. Sci. USA 78: 3824-28; Hopp and Wood, (1983) Mol. Immunol. 20: 483-89; Sutcliffe et al., (1983) Science 219: 660-66). Monoclonal antibodies with an affinity of 10 ⁸ M ⁻¹ , preferably 10 ⁹ M ⁻¹ to 10 ¹⁰ M ⁻¹ , or stronger can be made by the standard procedures described (Harlow, supra). And Lane; Goding (1986) Monoclonal Antibodies: Principles and Practice (2nd edition) Academic Press, New York; and US Pat. No. 4,381,292; US Pat. No. 4,451,570; and US Pat. No. 4,618,577). Antibodies against a crude cell extract of MINR or a substantially purified fragment thereof can be generated. When MINR fragments are used, these preferably comprise at least 10, more preferably at least 20 consecutive amino acids of the MINR protein. In certain embodiments, MINR-specific antigens and / or immunogens are bound to carrier proteins that stimulate the immune response. For example, the subject polypeptide is covalently linked to a keyhole limpet hemocyanin (KLH) carrier, and the conjugate is emulsified in Freund's complete adjuvant that enhances the immune response. Immunize an appropriate immune system such as experimental rabbits or mice according to conventional protocols.

The presence of MINR-specific antibodies was assayed by a suitable assay such as a solid phase enzyme linked immunosorbent assay (ELISA) using immobilized corresponding MINR polypeptide. Other assays such as radioimmunoassay and fluorescence assay can also be used.
Chimeric antibodies specific to MINR polypeptides can be made that contain different portions from different animal species. For example, a human immunoglobulin constant region may be linked to a variable region of a mouse mAb so that the biological activity of the antibody is derived from a human antibody and its binding specificity is derived from a mouse fragment. A chimeric antibody is produced by splicing together genes encoding 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). A humanized antibody, which is a form of a chimeric antibody, is obtained by recombinant DNA technology (Riechmann LM et al., (1988) Nature 323: 323-327). (Carlos, TM, JMHarlan (1994) Blood 84: 2068-2101). Humanized antibodies contain about 10% mouse sequence and about 90% human sequence, which further reduces or eliminates immunogenicity while retaining antibody specificity (Co MS and Queen C. 1991 Nature 351: 501-501; Morrison SL. 1992 Ann. Rev. Immun. 10: 239-265). Humanized antibodies and methods for producing them are well known in the art (US Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370). issue).

MINR-specific single-chain antibodies, which are recombinant single-chain polypeptides formed by linking heavy and light chain fragments of the Fv region by amino acid crosslinking, can be produced by methods well known in the art (US Patent 4,946,778; Bird, Science (1988) 242: 423-426; Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85: 5879-5883; Ward et al., Nature (1989) 334 : 544-546).
Other suitable techniques for producing antibodies include exposing lymphocytes in vitro to antigenic polypeptides, or alternatively to selected antibody libraries in phage or similar vectors (Huse et al., Science (1989)). 246: 1275-1281). As used herein, T cell antigen receptors are within the scope of antibody modulators (Harlow and Lane, 1988, supra).

  The polypeptides and antibodies of the present invention can be used with or without modification. In many cases, antibodies are labeled by either covalently or non-covalently linking a substrate that expresses a detectable signal or a target protein that is toxic to the cell (Menard S et al., Int J Biol Markers (1989) 4: 131-134). A wide variety of labels and conjugation techniques are known and widely reported in both scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, fluorescent lanthanide metals, chemiluminescent moieties, bioluminescent moieties, magnetic particles, and the like (US Pat. No. 3,817,837). No. 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; 4,366,241) . Recombinant immunoglobulins may also be produced (US Pat. No. 4,816,567). By conjugating with a transmembrane toxin protein, an antibody against the cytoplasmic polypeptide can be delivered and reached at its target (US Pat. No. 6,086,900).

  For therapeutic use in patients, the antibodies of the invention are administered to the target site, if possible, by parenteral or intravenous administration. Clinical studies will determine the therapeutically effective dose and dosing regimen. Usually, the amount of antibody administered is from about 0.1 mg / kg to about 10 mg / kg of the patient's body weight. For parenteral administration, the antibody is formulated in a unit dose injectable form (eg, solution, suspension, emulsion) containing a pharmaceutically acceptable vehicle. Such vehicles are essentially non-toxic and have no therapeutic effect. Examples are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as non-volatile oil, ethyl oleate, or liposomal carriers may also be used. The vehicle may contain minor amounts of additives such as buffers and preservatives that enhance isotonicity, chemical stability, or otherwise enhance the therapeutic potential. The antibody concentration in such vehicles is usually about 1 mg / ml to about 10 mg / ml. Immunotherapeutic methods are further described in the literature (US Pat. No. 5,859,206; International Publication No. WO0073469).

Nucleic Acid Modulators Other preferred MINR modulators include nucleic acid molecules such as antisense oligomers and double stranded RNA (dsRNA) that generally inhibit MINR activity. Preferred antisense oligomers interfere with MINR nucleic acid function such as DNA replication, transcription, MINR RNA translocation, protein translation from MINR RNA, RNA splicing, and any catalytic activity involving MINR RNA.
In one embodiment, the antisense oligomer is an oligonucleotide that is sufficiently complementary to bind to MINR mRNA, preferably by binding to the 5 ′ untranslated region, to prevent translation. Antisense oligonucleotides specific for MINR are preferably in the range of 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 single-stranded or double-stranded DNA or RNA, or a chimeric mixture, derivative or modified version of DNA and RNA. You may modify the base part, sugar part, or phosphate skeleton of this oligonucleotide. The oligonucleotide may contain other accessory groups such as peptides, agents that facilitate transport across the cell membrane, cleavage agents induced by hybridization, intercalation agents, and the like.

  In another embodiment, the antisense oligomer is a phosphothioate morpholino oligomer (PMO). PMOs are constructed from four different morpholino subunits, including one of the four gene bases (A, C, G, or T) each attached to a morpholine six-membered ring. . The polymers of these subunits are linked by linkages between nonionic phosphodiamidate subunits. Detailed methods for producing and using PMO and other antisense oligomers are well known in the art (eg, International Publication No. WO 99/18193; Summerton J and Weller D., Antisense Nucleic Acid Drug Dev 1997, 7: 187). Probst JC, Methods 2000, 22: 271-281; US Pat. No. 5,235,033; US Pat. No. 5,378,841).

  A preferred alternative MINR nucleic acid modulator is a double-stranded RNA species that mediates RNA interference (RNAi). RNAi is a sequence-specific post-translational gene silencing process in animals and plants, initiated by double-stranded RNA (dsRNA) with a sequence homologous to the gene to be silenced. Methods relating to the use of RNAi to silence genes in nematodes, Drosophila, plants, and humans are well known in the art (Fire A et al., 1998 Nature 391: 806-811; Fire, A. Trends Genet. 15, 358-363 (1999); Sharp, PA RNA interference 2001. Genes Dev. 15,485-490 (2001); Hammond, SM 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, SM et al., Nature 404, 293-296 (2000); Zamore, PD et al., Cell 101, 25 Bernstein, E. et al., Nature 409, 363-366 (2001); Elbashir, SM et al., Genes Dev. 15, 188-200 (2001); International Publication No. WO0129058; International Publication No. WO9932619; Elbashir SM et al., 2001 Nature 411: 494-498).

  Nucleic acid modulators are commonly used as research reagents, diagnostic agents, and therapeutic agents. For example, antisense oligonucleotides that can specifically inhibit gene expression are often used to elucidate the function of a particular gene (see, eg, US Pat. No. 6,165,790). Nucleic acid modulators are also used, for example, to identify the function of various members of a biological pathway. For example, antisense oligomers have been utilized as therapeutic moieties in the treatment of diseased 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). Thus, in one aspect of the invention, MINR-specific antisense oligomers are used in assays to further elucidate the function of MINR in INR signaling. Zebrafish is a particularly useful model for the study of INR signaling using antisense oligomers. For example, PMO is used to selectively inactivate one or more genes in vivo in zebrafish embryos. By injecting PMO into zebrafish at the 1-16 cell stage, the effectiveness of candidate targets resulting from Drosophila screening is confirmed in this vertebrate model system. In another aspect of the invention, PMO is used to screen the zebrafish genome for the identification of other therapeutic modulators of INR signaling. In a further aspect of the invention, MINR-specific antisense oligomers are used as therapeutic agents for the treatment of metabolic pathological conditions.

Assay System The present invention provides an assay system and screening method for identifying specific modulators of MINR activity. As used herein, an “assay system” includes all components necessary to perform an assay that detects and / or measures a particular event and analyze the results. In general, primary assays are used to identify or confirm the specific biochemical or molecular effects of a modulator on MINR nucleic acids or proteins. In general, secondary assays will further assess the activity of MINR modulators identified by the primary assay and may confirm that this modulator affects MINR in a manner related to INR signaling. . In some cases, MINR modulators are tested directly in a “secondary assay” without being identified or confirmed in a “primary assay”.

  In a preferred embodiment, the assay system comprises a suitable assay comprising MINR polypeptide or nucleic acid under conditions that, in the absence of a candidate agent, provides a control activity based on the particular molecular event detected in the assay system. Contacting the system with a candidate agent. The method further comprises detecting the same type of activity in the presence of the candidate agent (“activity of the system affected by the agent”). The difference between the agent-affected activity and the control activity indicates that this candidate agent modulates MINR activity and thus INR signaling. A statistically significant difference between the agent-affected activity and the control activity indicates that this candidate agent modulates MINR activity and thus INR signaling. The MINR polypeptide or nucleic acid used in the assay can include any of the nucleic acids or polypeptides described above.

Primary Assay Generally, the type of primary assay is determined by the type of modulator being tested.

Primary assays for small molecule modulators For small molecule modulators, screening assays are used to identify candidate modulators. Screening assays may be cell-based and may use a cell-free system that reinitiates or retains the relevant biochemical response of this target protein (Sittampalam GS et al., Curr Opin Chem Biol (1997) 1: 384-91 and accompanying references are reviewed). As used herein, the term “cell-based” refers to assays using live cells, dead cells, or specific cell fractions such as membrane fraction, endoplasmic reticulum fraction, mitochondrial fraction, and the like. The term “cell-free” encompasses assays using substantially purified proteins (endogenous or recombinantly produced), partially purified cell extracts or crude cell extracts. Screening assays include protein-DNA interactions, protein-protein interactions (eg receptor-ligand binding), transcriptional activity (eg reporter genes), enzyme activity (eg via substrate characteristics), second messenger activity, immunity Various molecular events can be detected, including protogenicity and changes in cell morphology and other cellular characteristics. Appropriate screening assays can use a wide range of detection methods, including fluorescence, radioactivity, colorimetry, spectrophotometry, and amperometry, to provide a readout of the specific molecular events to be detected.

In a preferred embodiment, the screening assay uses fluorescence techniques including fluorescence polarization, time-resolved fluorescence, fluorescence resonance energy transfer. These systems provide a means to monitor protein-protein or DNA-protein interactions where the intensity of the signal emitted from the dye-labeled molecule depends on its interaction with its partner molecule (eg, 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).
Appropriate assay formats that can be adapted for screening of MINR modulators are well known in the art.

  Binding assay. Various assays are available to detect the activity of proteins with specific binding activity. Examples of such assays include those that use fluorescence polarization to measure the binding of fluorescently labeled proteins, peptides or other molecules, and those that use laser scanning techniques (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 a scintillation proximity assay (SPA) using a biotin-labeled peptide probe captured on streptavidin-coated SPA beads and a radiolabeled partner molecule. This assay detects radiolabeled protein bound specifically to peptide probes by scintillant immobilized on SPA beads (Sonatore LM et al., 1996, Anal Biochem 240: 289-297).

  Transcriptional activity assay. In one example, quantitative RT-PCR (eg TaqMan®, PE Applied Biosystems) is used to detect transcriptional activity. In another example, a transcriptional reporter (eg, luciferase, GFP, β-galactosidase, etc.) that is operably linked to a reactive gene regulatory sequence is used (eg, Berg M et al., 2000, J Biomol Screen, 5: 71-76). . Proteins that are part of a transcription complex can also be assayed for binding activity (ie, other members of the complex).

  Phosphatase assay. Protein phosphatases catalyze the removal of gamma phosphate, threonine or tyrosine residues from serine in protein substrates. Since phosphatases act oppositely to kinases, removal of phosphate from protein substrates can be monitored by appropriate assays. In one example, dephosphorylation of a fluorescently labeled peptide substrate allows trypsin cleavage of the substrate, which then significantly increases the fluorescence of the cleaved substrate (Nishikata M et al., Biochem J (1999) 343: 35-391). ). In another example, direct binding of phosphatase to target is monitored by fluorescence polarization; increasing the concentration of phosphatase increases the rate of dephosphorylation and changes polarization (Parker GJ et al., 2000. J Biomol Screen 5: 77-88). Lipid phosphatase activity can be monitored by another suitable assay and labeled removal of phosphate from the phosphatidylinositol substrate can be detected using a labeled substrate such as a fluorescent or radiolabel. In one example, in an assay using “FlashPlate” technology (US Pat. No. 5,972,595), a radiolabeled hydrophobic substrate is immobilized on a solid support within each well of a multiwell plate. The dephosphorylation of the substrate is measured by the reduction in bound radioactivity that can be detected by scintillant approximation. Other assays for detecting phosphoinositide phosphatase activity are known in the art (see, eg, US Pat. Nos. 6,001,354 and 6,238,903).

  GAP assay. GAP protein stimulates the hydrolysis of GTP to GDP. An exemplary assay can monitor GAP activity by, for example, GTP hydrolysis assay using labeled GTP (eg Jones S et al., Molec Biol Cell (1998) 9: 2819-2837). Another assay can detect GAP function in endosomal trafficking by monitoring the movement of cargo molecules. At this time, the molecules of the shipment may be labeled (Sonnichsen et al., 2000, J Cell Biol 149: 901-14).

Kinase assay. Some suitable assays detect kinase activity, transfer of gamma phosphate from adenosine triphosphate (ATP) to serine or threonine residues in protein substrates. Kinase activity can be assayed using a radioassay that monitors the transfer from [γ- ³² P or- ³³ P] ATP. Separation of the phosphate-labeled product from the remaining radiolabeled ATP is achieved by SDS-polyacrylamide gel electrophoresis, filtration using glass fiber filters and other substrates that bind to the peptide or protein, and the peptide or protein to a solid phase substrate. Can be achieved by various methods, such as allowing the removal of the remaining radiolabeled ATP by washing by absorption / binding of. In one example, scintillation assay monitors the transfer of gamma phosphate from [γ- ³³ P] ATP to a biotinylated peptide substrate. The substrate is captured on streptavidin-coated beads that transmit signals (Beveridge M et al., J Biomol Screen, (2000) 5: 205-212). This assay uses a scintillation proximity assay (SPA). In SPA, only the radioligand bound to the receptor bound to the surface of the SPA bead can be detected by the scintillant immobilized inside, thereby measuring binding without separating the conjugate from the free ligand. it can. In other assays of protein kinase activity, antibodies that specifically recognize phosphorylated substrates can be used. For example, the kinase receptor activation (KIRA) assay measures receptor tyrosine kinase activity with a ligand that stimulates intact receptor in cultured cells, then cultures the receptor solubilized with a specific antibody and phosphorylates with a phosphotyrosine ELISA. Is quantified (Sadick MD, Dev Biol Stand (1999) 97: 121-133). Another example of an antibody-based assay for protein kinase activity is TRF (time-resolved fluorometry). In this method, europium chelate labeled anti-phosphotyrosine antibodies are utilized to detect phosphate transfer to a polymeric substrate coated on microtiter plate wells. The amount of phosphorylation is then detected using time-resolved, detachment enhanced fluorescence (Braunwalder AF, et al., Anal Biochem 1996 Jul 1; 238 (2): 159-64). General assays can be established for protein kinases that rely on phosphorylation of substrates, such as myelin base protein, casein, histone, or synthetic peptides of polyglutamate / tyrosine and radiolabeled ATP.

Termination factor activity assay. Termination factor activity can be detected in vitro by appropriate assays (eg, Seit-Nebi et al., 2001, Nucleic Acids Res 29: 3982-7; Frolova et al., 1994, Nature 372: 701-3; Caskey et al., 1974, Methods Enzymol 30: 293-303).
Cell-based screening assays usually require a recombinant expression system for MINR and any auxiliary proteins required for the particular assay. Cell-free assays often use recombinantly produced and purified or substantially purified proteins. Appropriate methods for generating recombinant proteins retain sufficient biological activity and produce sufficient amounts of protein sufficient to optimize the activity and ensure assay reproducibility. Yeast two-hybrid screening, mutant screening, and mass spectrometry provide preferred methods for determining protein-protein interactions and elucidating protein complexes. For certain applications, when a MINR interacting protein is used in a screening assay, the binding specificity of the interacting protein for the MINR protein is determined by the binding equilibrium constant (usually at least about 10 ⁷ M ⁻¹ , preferably at least about 10 ⁸ M ⁻¹ , more preferably at least about 10 ⁹ M ⁻¹ ), and immunogenicity. For enzymes and receptors, binding can be assayed by treatment with substrate and ligand, respectively.

In screening assays, the ability of a candidate agent to specifically bind to or modulate the activity of a MINR polypeptide, a fusion protein thereof, or a cell or membrane containing the polypeptide or fusion protein can be measured. The MINR polypeptide can be full length or a fragment thereof that retains functional MINR activity. The MINR polypeptide may be fused to another polypeptide, such as a peptide tag for detection or immobilization or another tag. The MINR polypeptide is preferably human MINR, or an ortholog or derivative thereof as described above. In a preferred embodiment, the screening assay detects a modulation based on a candidate agent for interaction between MINR and an endogenous or exogenous protein, or a binding target such as a MINR specific binding activity, This can be used to evaluate normal MINR gene function.
Certain screening assays can also be used to test antibodies and nucleic acid modulators; for nucleic acid modulators, suitable assay systems include expression of MINR mRNA.

Primary assays for antibody modulators For antibody modulators, a suitable primary assay is a binding assay that tests the affinity and specificity of the antibody for MINR protein. Methods for testing antibody affinity and specificity are well known in the art (Harlow and Lane, supra, 1988, 1999). Enzyme-linked immunosorbent assay (ELISA) is the preferred method for detecting antibodies specific for MINR; other methods include FACS assays, radioimmunoassays, and fluorescence assays.

Primary assays for nucleic acid modulators For nucleic acid modulators, primary assays may test the ability of the nucleic acid modulator to inhibit MINR gene expression, preferably mRNA expression. In general, expression analysis compares MINR expression in similar populations of cells in the presence and absence of nucleic acid modulators (eg, two cell pools that endogenously or recombinantly express MINR). For example. Methods for analyzing mRNA and protein expression are well known in the art. For example, MINR in cells treated with nucleic acid modulators using Northern blotting, slot blotting, RNase protection, quantitative RT-PCR (eg, using TaqMan®, PE Applied Biosystems), or microarray analysis It can be confirmed that the expression of mRNA is reduced (for example, Current Protocols in Molecular Biology (1994) Ausubel FM et al., 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, A Curr Opin Biotechnol 2001, 12: 41-47). Protein expression can also be monitored. Proteins are most commonly detected using specific antibodies or antisera against either MINR proteins or specific peptides. Various means are available including Western blotting, ELISA, or in situ detection (Harlow E and Lane D, supra, 1988 and 1999).

Secondary Assay To further confirm the activity of MINR modulators identified by any of the methods described above using a secondary assay to confirm that the modulator affects MINR in a manner associated with INR signaling. be able to. As used herein, MINR modulators include candidate clinical compounds or other agents derived from previously identified modulators. Secondary assays can also be used to test the activity of modulators against specific genetic or biochemical pathways, or to test the specificity with which a modulator interacts with MINR.
Secondary assays generally compare similar populations of cells and animals (eg, two cell pools that express MINR endogenously or recombinantly) in the presence and absence of candidate modulators. Generally, in such assays, treating a cell or animal with a candidate MINR modulating agent results in a change in INR signaling compared to an untreated (or mock or placebo-treated) cell or animal. Test to see if it does. Changes in INR signaling can be detected as alterations in INR pathway components or changes in their expression or activity. The assay also includes normal outputs such as glucose uptake, or long-term effects such as normal progression of glycogen and triglyceride metabolism, adipocyte differentiation, or progression of diabetes or other INR-related pathologies. Or, a defective INR signaling output can be detected. In certain assays, it is used herein to represent cells or animals that have been engineered to alter expression of genes in the INR or interaction pathways, or pathways associated with INR signaling or the output of INR signaling. Use a sensitized genetic background.

Cell-based assays Cell-based assays use a variety of insulin-sensitive mammalian cells to detect endogenous INR signals or by recombinant expression of components of INR and / or other INR pathways. Exemplary insulin sensitive cells include adipocytes, hepatocytes, and pancreatic β cells. Suitable adipocytes include 3T3L1 cells most commonly used in insulin sensitivity assays, as well as primary cells from mouse or human biopsies. Suitable hepatocytes include the rat hepatoma H4-II-E cell line. Suitable β cells include rat INS-1 cells that perform optimized glucose-sensitive insulin secretion (clone 823-13, etc., Hohmeier et al., 2000, Diabetes 49: 424). Other suitable cells include myocytes, eg, L6 myotubes, CHO cells that have been engineered to overexpress INR. For certain assay systems, it is useful to treat cells with factors such as glucosamine, free fatty acids or TNFα that induce an insulin resistant state. Candidate modulators are typically added to the cell culture medium, but may be injected into the cells or delivered by any other effective means.
Cell-based assays generally test whether treatment of insulin-responsive cells with MINR modulators alters the INR signal in response to insulin stimulation (“insulin sensitivity”); such assays are known in the art. (See, for example, Sweeney et al., 1999, J Biol Chem 274: 10071). In a preferred embodiment, the assay is performed to determine whether MINR function inhibits an increase in insulin sensitivity.

In one example, INR signaling is assessed by measuring insulin responsive gene expression. Hepatocytes are preferred for these assays. Many insulin responsive genes are known (eg, p85 PI3 kinase, hexokinase II, glycogen synthase, lipoprotein lipase, etc .; PEPCK is specifically down-regulated in response to INR signaling). Any useful means for expression analysis, as already described, is used. Typically, mRNA expression is detected. In preferred applications, Taqman analysis is used to directly measure mRNA expression. Alternatively, the expression is a genetic recombination comprising a sequence encoding a reporter gene (luciferase, GFP or other fluorescent protein, β-galactosidase, etc.) under the control of an insulin response gene control sequence (eg, enhancer / promoter region). Monitored indirectly from the reporter construct. Methods for making and using reporter constructs are well known.
INR signaling is also measured by measuring the activity of components of the INR signaling pathway and is well known in the art (eg, Kahn and Weir, Joslin's Diabetes Mellitus, Williams & Wilkins, Baltimore, MD , 1994). Appropriate assays detect phosphorylation of members of pathways including IRS, PI3K, Akt, GSK3, etc., for example, using antibodies that specifically recognize phosphorylated proteins. The assay also detects changes in specific signaling activity of pathway components (eg, kinase activity such as PI3K, GSK3, Akt, etc.). Kinase assays and methods for detecting phosphorylated protein substrates are well known in the art (see, eg, Ueki K et al., 2000, Mol Cell Biol; 20: 8035-46).

In other examples, the assay measures glycogen synthesis in response to insulin stimulation, preferably using hepatocytes. Glycogen synthesis is assayed by a variety of means including measurement of glycogen content and quantification of glycogen synthesis using glucose labeled, such as with radiolabels (e.g., Aiston S and Agius L, 1999, Diabetes 48: 15-20; see Rother KI et al., 1998, J Biol Chem 273: 17491-7).
In other suitable assays, the cellular uptake of glucose (typically labeled glucose) in response to insulin stimulation is measured. Adipocytes are preferred for these assays. The assay also measures glucose transporter (GLUT) 4 translocation, which is the main mediator of insulin-induced glucose uptake primarily in muscle and adipocytes and specifically translocates to the cell surface after insulin stimulation. Is done. Such assays detect endogenous GLUT4 translocation using GLUT4-specific antibodies or detect exogenously introduced epitope-tagged GLUT4 using antibodies specific for a particular epitope. (See, eg, Sweeney, 1999, supra; Quon MJ et al., 1994, Proc Natl Acad Sci USA 91: 5587-91).
Another preferred assay detects insulin secretion from beta cells in response to glucose. Such assays typically use an ELISA (see, for example, Bergsten and Hellman, 1993, Diabetes 42: 670-4) or a radioimmunoassay (RIA; see, for example, Hohmeier et al., 2000, supra).

Animal Assays Various non-human animal models of metabolic diseases can be used to test candidate MINR modulators. Typically, such models include genetically modified animals that have been engineered such that genes involved in lipid metabolism, lipogenesis, and / or INR signaling pathways are misexpressed (eg, overexpressed or lack of expression). use. Also, certain feeding states, and / or administration or certain biologically active compounds contribute to or create animal models of lipid and / or metabolic diseases. Assays generally require systemic delivery of candidate modulators by oral administration, injection (intravenous, subcutaneous, intraperitoneal), bolus administration, and the like.
In one embodiment, the assay uses diabetes and / or insulin resistant model mice. Mice whose genes are knocked out in leptin pathways such as ob (leptin) or db (leptin receptor), or in INR signaling pathways such as INR or insulin receptor substrate (IRS), show symptoms of diabetes and hepatic lipid accumulation (Fatty liver) and often show 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 sensitive 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). Thus, a suitable assay using these model mice will test whether administration of a candidate modulator alters, and preferably reduces, lipid accumulation in the liver. Lipid levels in plasma and adipocytes are also tested. Methods for assaying lipid content typically include 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 scintillation measurement of incorporation of radiolabeled substrates (Horton JD et al., 1999, J Clin Invest 103: 1067-1076) is well known in the art. Other useful assays test blood glucose levels, insulin levels, and insulin sensitivity (eg, Michael MD, 2000, Molecular Cell 6:87). Insulin sensitivity is routinely tested, such as by a glucose tolerance test or an insulin tolerance test.

In other embodiments, the assay uses a mouse model of lipoprotein biology and cardiovascular disease. For example, apolipoprotein E (apoE) knockout mice show elevated plasma cholesterol and spontaneous arterial damage (Zhang SH, 1992, Science 258: 468-471). Transgenic mice overexpressing cholesterol ester transfer protein (CETP) also have increased plasma lipid levels (particularly very low density lipoprotein [VLDL] and low density lipoprotein [LDL] cholesterol levels) and plaques in arteries. The formation (Marotti KR et al., 1993, Nature 364: 73-75) is shown. In assays using these models, administration of candidate modulators reduces the levels of pro-atherogenic LDL and VLDL, increases HDL, or decreases total lipid (including triglycerides) levels. Tested to alter plasma lipid levels. Furthermore, histological analysis of arterial morphology and lesion formation (ie, number and size of lesions) indicates whether candidate modulators can reduce the progression and / or severity of atherosclerosis. It is. Many other mouse models for atherosclerosis are available, including knock-out of the scavenger receptor (SR) -B1 in Apo-A1, PPARγ, and LDLR or ApoE-null background (eg, Glass CK and (Reviewed in Witztum JL, 2001, Cell 104: 503-516).
In other embodiments, the ability of candidate modulators to alter plasma lipid levels and progression of atherosclerosis is tested in a mouse model for multiple lipid disorders. For example, mice in which both the leptin and LDL receptor genes are knocked out show hypercholesterolemia, hypertriglyceridemia and arterial damage, impaired fuel metabolism, increased plasma residual lipoproteins, diabetes, and atherosclerosis Provides a model of the relationship between atherosclerosis (Hasty AH et al., 2001, supra).

Diagnostic Methods The discovery that MINR is involved in INR signaling can be used to diagnose and prognose diseases and disorders involved in INR signaling and to identify subjects with a predisposition to such diseases and disorders. Methods are provided. Any method for assessing the expression of MINR in a sample, as previously described, can be used. Such methods include (1) detection of the presence of a MINR gene mutation, or detection of either over- or under-expression of MINR mRNA compared to a non-disease state, (2) MINR gene product compared to a non-disease state Reagents such as MINR oligonucleotides, as described above, for detection of either excess or under-abundance of RNA and (3) detection of perturbations or abnormalities in biological pathways mediated by MINR And antibodies against MINR may be utilized.
Accordingly, in certain embodiments, the present invention provides: a) obtaining a biological sample from a patient; b) contacting the sample with a probe for MINR expression; c) comparing the results from step (b) with a control. And (d) a method of diagnosing a patient's disease or disorder associated with altered expression of MINR, comprising determining whether step (c) indicates a possible disease or disorder. . The probe may be either DNA or a protein including an antibody.

  The following experimental sections and examples are provided for purposes of illustration and not limitation.

I. Drosophila Cell RNAi Screening Cell RNAi screening was used to identify INR pathway modifiers. Briefly, this screen affects the destruction of these cells by treating Dmel strain-derived cells, a Drosophila S2 cell line derivative grown in serum-free medium, with a dsRNA corresponding to the predicted Drosophila gene. (Adams et al., 2000, Science 287: 2185-95). Copy walls of cells in multiwell plates were treated with dsRNA corresponding to individual Drosophila genes (basically following the method described by Clemens et al., 2000, supra). Lactate dehydrogenase ("LDH", GI1519714; Abu-Shumays, which has been previously shown to be correlated with INR pathway activity by the applicant using quantitative RT-PCR using TaqMan® (PE Applied Biosystems) And Fristrom, 1997, Dev Genet 20: 11-22). Specifically, when the INR pathway activity was increased by RNAi-based knockdown of negative effectors of INR signaling (eg, PTEN, GSK3β, and AFX), the expression of LDH increased regardless of the presence or absence of insulin. LDH expression was reduced when INR pathway activity was reduced by RNAi-based knockdown of positive effectors of INR signaling (eg, INR, IRS, AKT). Therefore, lactate dehydrogenase expression activity was used instead of INR pathway activity. This screening identified gene “modifiers” where knockdown by RNAi resulted in changes in LDH expression. Genes that disruption by RNAi resulted in increased LDH expression were identified as candidate negative effectors of INR pathway activity, while genes that resulted in decreased LDH expression were identified as candidate positive effectors. In an approval test using LDHRT-PCR analysis, potential modifiers were retested three times. Moreover, the sodium / phosphate cotransporter (“CG4726”, GI107727399; GI7296119 amino acid sequence) in which the expression after destruction based on RNAi of INR was decreased was also the same. The dsRNA used for the approval test was produced from a PCR product generated using a different primer than the candidate modifier gene used to produce the initial results. Table 1 shows the modifiers and their orthologs.
Table 1

II. High Throughput In Vitro Fluorescence Polarization Assay Fluorescently labeled MINR peptide / substrate is 96 well microtiter plate with selected test compound in test buffer (10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6) Of each well. A change in fluorescence polarization relative to the control value determined using Fluorolite FPM-2 Fluorescence Polarization Microsystem System (Dynatech Laboratories, Inc) indicates that the test compound is a candidate modifier for MINR activity.

III. High-throughput in vitro binding assay
³³ P-labeled MINR peptide was assayed with the compound of interest in assay buffer (100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl ₂ , 1% glycerol, 0.5% NP-40, 50 mM β- Mercaptoethanol, 1 mg / ml BSA, protease inhibitor cocktail) was added to the wells of Neutralite-avidin coated assay plates and incubated at 25 ° C. for 1 hour. Subsequently, biotin-labeled substrate was added to each well and incubated for 1 hour. The reaction was stopped by washing with PBS and counted with a scintillation counter.

IV. Immunoprecipitation and immunoblotting For co-precipitation of transfected proteins, 3 × 10 ⁶ appropriate cells were seeded in 10 cm dishes and transfected the next day using expression constructs. The total DNA amount was kept constant at each transfection by adding empty vector. After 24 hours, cells were harvested, washed once with phosphate buffered saline, 50 mM Hepes, pH 7.9, 250 mM NaCl, 20 mM glycerophosphate, 1 mM sodium orthovanadate, 5 mM phosphate p. -Nitrophenyl, 2 mM dithiothreitol, protease inhibitors (complete, Roche Molecular Biochemicals), and 1 ml nonidet P-40 in 1 ml lysis buffer for 20 minutes on ice. Cell debris was removed by two centrifugations at 15000 xg for 15 minutes. Cell lysates were incubated with 25 μl of M2 beads (Sigma) for 2 hours at 4 ° C. with gentle rocking.

  After washing well with lysis buffer, the protein bound to the beads is dissolved by boiling in SDS sample buffer, fractionated by SDS polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and the indicator antibody Were blotted. Reactive bands were visualized by horseradish peroxidase conjugated to the appropriate secondary antibody and sensitive chemiluminescence (ECL) Western blotting detection system (Amersham Pharmacia Biotech).

VI. Kinase Assay Purified or partially purified MINR is added to an appropriate reaction buffer (eg, magnesium chloride or manganese chloride (1-20 mM), and a peptide or polypeptide substrate such as myelin basic protein or casein (1- Dilute to Hepes 50 mM) with pH 7.5 containing 10 μg / ml). The final concentration of kinase is 1-20 nM. Enzymatic reactions are performed in microtiter plates to facilitate optimization of reaction conditions by increasing assay throughput. Use a 96-well microtiter plate and bring the final volume to 30-100 μl. ³³ P gamma ATP (0.5 μCi / ml) is added to initiate the reaction and incubated at room temperature for 0.5 to 3 hours. A negative control is performed by addition of EDTA, thereby chelating divalent cations (Mg ²⁺ or Mn ²⁺ ) required for enzyme activity. After incubation, the enzyme reaction is quenched using EDTA. Transfer reaction samples to 96-well glass fiber filtering plates (MultiScreen, Millipore). The filter is then washed with phosphate buffered saline, diluted phosphoric acid (0.5%), or other suitable medium to remove excess radiolabeled ATP. Scintillation cocktail is added to the filtering plate and the radioactivity incorporated by scintillation counting is quantified (Wallac / Perkin Elmer). Activity is defined as the amount of radioactivity detected after subtraction of the negative control response value (EDTA quench).

Claims (22)

(A) providing an assay system comprising a MINR polypeptide or nucleic acid;
(B) contacting the assay system with the test agent under conditions such that the system provides control activity in the absence of the test agent;
(C) detecting the activity of the assay system affected by the test agent and identifying the test agent as a candidate INR signaling modulator by the difference between the activity affected by the test agent and the control activity;
A method of identifying a candidate INR signaling modulator comprising a step.   2. The method of claim 1, wherein the assay system comprises a screening assay comprising MINR polypeptide and the candidate test agent is a small molecule modulator.   The method of claim 2, wherein the screening assay is a binding assay.   2. The method of claim 1, wherein the assay system comprises a binding assay comprising a MINR polypeptide and the candidate test agent is an antibody.   The method of claim 1, wherein the assay system comprises an expression assay comprising MINR 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 PMO. The assay system comprises cultured cells or non-human animals that express MINR;
The method of claim 1, wherein the assay system comprises an assay that detects a change influenced by a test agent in INR signaling or the output of INR signaling.   9. The method of claim 8, wherein the assay system comprises cultured cells.   The assay comprises an event selected from the group consisting of insulin responsive gene expression, INR signaling pathway component phosphorylation, INR signaling pathway component kinase activity, glycogen synthesis, glucose uptake, GLUT4 translocation, and insulin secretion. The method according to claim 9, which detects.   9. 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 for diabetes and / or insulin resistance.   The assay system detects an event selected from the group consisting of lipid accumulation in the liver, lipid accumulation in plasma, lipid accumulation in fat, sugar levels in plasma, insulin levels in plasma, and insulin sensitivity 13. The method of claim 12, comprising an assay that comprises: (D) providing a second assay system comprising cultured cells or non-human animals expressing MINR;
(E) contacting the test agent of (b) or an agent derived therefrom with a second assay system under conditions such that in the absence of the test agent or agent derived therefrom, the system provides a control activity;
(F) detecting the activity of a second assay system affected by the test agent;
Comprising further steps,
Confirming the test agent or a drug derived therefrom as a candidate INR signaling modulator by the difference between the activity affected by the test agent and the control activity of the second assay system;
The method of claim 1, wherein the second assay system comprises a second assay that detects a change in activity associated with INR signaling or an output of the INR signaling affected by a test agent.   15. The method of claim 14, wherein the second assay system comprises cultured cells.   The second assay is selected from the group consisting of insulin responsive gene expression, phosphorylation of INR signaling pathway components, kinase activity of INR signaling pathway components, glycogen synthesis, glucose uptake, GLUT4 translocation, and insulin secretion 16. The method of claim 15, wherein an event is detected.   The method of claim 14, wherein the second assay system comprises a non-human animal.   18. The method of claim 17, wherein the non-human animal is a mouse that provides a model for diabetes and / or insulin resistance.   The second assay system is selected from the group consisting of lipid accumulation in liver, lipid accumulation in plasma, lipid accumulation in fat, sugar level in plasma, insulin level in plasma, and insulin sensitivity 19. The method of claim 18, comprising an assay that detects the event.   A method of modulating INR signaling in a mammalian cell, comprising contacting the cell with an agent that specifically binds to a MINR polypeptide or nucleic acid.   21. The method of claim 20, wherein the agent is administered to a mammal that has been previously determined to have a pathology associated with INR signaling.   21. The method of claim 20, wherein the agent is a small molecule modulator, nucleic acid modulator or antibody.