JP2006511237A - Methods for identifying compounds that modulate protein activity - Google Patents

Abstract

A method for identifying a compound that modulates the activity of a target polypeptide, wherein a test compound is combined with a target polypeptide and a substrate for the target polypeptide, and the test compound modulates the activity of the target polypeptide. To be measured. The test compound can be a small molecule drug used to treat obesity, diabetes, insulin resistance, or to promote insulin secretion. Also disclosed are target polypeptides and corresponding nucleic acids, and variants thereof.

Description

Detailed Description of the Invention

(Technical field)
The present invention relates to novel polypeptides that are targets for small molecule drugs and have properties related to the stimulation of biochemical or physiological responses of cells, tissues, organs or organisms. More specifically, the novel polypeptide is the gene product of a novel gene or a specific biologically active fragment or derivative thereof. Methods of use include screening, diagnostic and prognostic assay procedures and methods for treating a wide range of pathological conditions.

(Background technology)
Obesity and diabetes are major public health problems in the developed and developing world. Over half of adults in the United States are considered overweight. This includes those whose body mass index (BMI) value, defined as weight (Kg) / [height (m)] ² , exceeds the upper limit of normal values (25). Common consequences of overweight are hyperlipidemia and progression of insulin resistance. Thereafter, hyperglycemia, which is characteristic of type 2 diabetes, occurs. If left untreated, hyperglycemia causes microvascular disease and end organ damage including retinopathy, kidney disease, heart disease, peripheral neuropathy and peripheral vascular injury. Currently, more than 16 million adults in the United States suffer from type 2 diabetes, and today the condition is widespread among school-age children as a result of the prevalence of obesity.

  Diabetes is a disorder in which the blood level of glucose (monosaccharide) is abnormally high because the body does not release enough insulin or respond to insulin. Blood glucose (glucose) levels change throughout the day, rise after meals, and return to normal within 2 hours. Blood glucose levels are typically 70 to 110 milligrams / deciliter (mg / dL) blood in the morning after an overnight fast. Usually less than 120-140 mg / dL even after eating or drinking a food containing sugar or other carbohydrates.

  Insulin is a hormone secreted from the pancreas and is the first entity involved in maintaining an appropriate blood glucose level. Insulin transports glucose into the cell, allowing the cell to produce energy or store energy from glucose until it is needed. The pancreas is stimulated by an increase in blood glucose level after eating and drinking, and insulin is produced, thereby preventing a significant increase in blood glucose level and gradually lowering it. Since muscles use glucose as energy, blood glucose levels can also decrease during physical activity.

  Diabetes results when the body does not produce enough insulin to maintain normal blood glucose levels, or when cells do not respond appropriately to insulin. In type 2 diabetes, the pancreas continues to produce insulin, sometimes even higher than normal. However, the body becomes resistant to its effects and becomes relative insulin deficient.

  The main purpose of diabetes treatment is to keep blood glucose levels within the normal range as much as possible. Although it is difficult to maintain a completely normal value, it can be more strictly maintained within the normal range and less likely to be temporary or long-term complications.

  Accordingly, therapeutic agents that reduce insulin resistance and / or promote insulin secretion would be useful in the treatment of obesity and / or diabetes. Such therapeutic agents may also be useful in the treatment of conditions that cause insulin resistance, often the progression of diabetes.

  In order to treat diseases, pathologies and other abnormal conditions or situations where a mammal is diagnosed as being at or at risk for a normal condition or situation, the identification of a new therapeutic agent is required. is important.

  Eukaryotic cells feature biochemical and physiological methods that are delicately balanced to achieve cell maintenance and proliferation under normal conditions. When such cells are components of multicellular organisms such as vertebrates, or more particularly organisms such as mammals, the regulation of biochemical and physiological methods involves sophisticated signaling pathways. . Often such signaling pathways involve extracellular signaling proteins, cellular receptors that bind to signaling proteins, and signaling components that are localized within the cell.

  Signal transduction proteins can be classified as endocrine effectors, paracrine effectors or autocrine effectors. Endocrine effectors are signaling molecules that are secreted into the circulatory system by a particular organ, which is then transported to a remote target organ or tissue. Target cells contain receptors for endocrine effectors, and when the endocrine effectors bind, a signaling cascade is induced. Paracrine effectors involve secretory cells and recipient cells in close proximity to each other, for example, two different classes of cells within the same tissue or organ. One class of cells secretes a paracrine effector, which then reaches a second class of cells, for example by diffusion through the extracellular fluid. The second class of cells contains receptors for paracrine effectors, and effector binding induces a signaling cascade that triggers the corresponding biochemical or physiological action. Autocrine effectors have a high degree of similarity to paracrine effectors, except that the same type of cells that secrete autocrine effectors also contain receptors. Thus, autocrine effectors bind to receptors on the same cell and on the same adjacent cells. The binding process then causes a characteristic biochemical or physiological effect.

  Signal transduction processes for cells and tissues include, but are not limited to, induction of cell or tissue proliferation, inhibition of growth or proliferation, induction of cell or tissue differentiation or maturation, and cell or tissue differentiation or maturation. Can cause a variety of effects including suppression.

  Many pathological conditions involve dysregulation of the expression of important effector proteins. In certain classes of conditions, dysregulation manifests as a reduced or suppressed level of protein effector synthesis and secretion. In other types of pathology, unregulation is manifested as increased or upregulated levels of protein effector synthesis and secretion. In the clinical setting, a subject may be suspected of suffering from an abnormality caused by increased or excessive levels of protein effector of interest. Therefore, there is a need to assay the level of protein effector of interest in a biological sample from such a subject and compare that level to that characteristic of a non-pathological condition. There is also a need to provide protein effectors as products of manufacture. Administering an effector to a subject in need thereof is useful for treating a pathological condition. Thus, there is a need for methods of treatment of pathological conditions caused by reduced or suppressed levels of protein effectors of interest. In addition, there is a need for methods of treatment of pathological conditions caused by increased or effected up-regulation of protein effectors of interest.

  Small molecule targets are implicated in various disease states or pathologies. These targets can be proteins, particularly enzyme proteins on which small molecule drugs act to alter the function of the target and achieve the desired result. Cellular, animal and clinical studies can be conducted to elucidate the genetic involvement in pathogenesis and virulence of conditions involving small molecule targets in various physiological, pharmacological or natural states. These studies include differential gene expression, protein-protein interactions, extensive sequencing of expressed genes, and, for example, single nucleotide polymorphisms (SNPs) or splice variants in test and subject biological samples Core technology at CuraGen Corporation was used to observe the association of genetic variation in but not limited to the body. The purpose of such studies is to identify potential means of accomplishment for various therapeutic interventions to prevent, treat or cure the condition.

  In order to treat diseases, pathologies and other abnormal conditions or situations where a mammal is diagnosed as being at or at risk for a normal condition or situation, the identification of a new therapeutic agent is required. is important. Such a procedure includes at least the steps of identifying a target component in the affected tissue or organ and identifying candidate therapeutic agents that modulate the functional properties of the target. The target component may be any biopolymer involved in disease or pathology. In general, the target is a polypeptide or protein having specific functional properties. Other macromolecular classes are lipids such as nucleic acids, polysaccharides, complex lipids or glycolipids; and the target may be an intracellular or extracellular structure consisting of one or more of these classes of macromolecules . Once such a target is identified, it can be used in screening assays to identify preferred candidate therapeutic agents from a large population of substances or compounds.

  Often the purpose of such screening assays is to identify small molecule candidates; this is generally done through the use of combinatorial methods to construct a population of substances to be tested. Implementation of high-throughput screening methods is advantageous when performed with large combinatorial libraries of compounds.

(Summary of the Invention)
The present invention includes nucleic acid sequences and novel polypeptides that they encode. Novel nucleic acids and polypeptides are referred to herein as NOVX or NOV1, NOV2, NOV3, etc., nucleic acids and polypeptides. These nucleic acids and polypeptides, as well as derivatives, homologues, analogs and fragments thereof are hereinafter nucleotide sequences selected from the group consisting of SEQ ID NO: 2n-1 (n is an integer from 1 to 85). "NOVX" nucleic acid, or a polypeptide sequence selected from the group consisting of SEQ ID NO: 2n (n is an integer from 1 to 85).

  In one aspect, the present invention provides an isolated polypeptide comprising a mature form of NOVX amino acid. One example is a mature variant of the NOVX amino acid sequence, where any amino acid in the mature form has been changed to a different amino acid, but not more than 15% in the mature sequence. Amino acids have changed that way. An amino acid may be, for example, a NOVX amino acid sequence, or a variant of a NOVX amino acid sequence, wherein one of the amino acids in the selected sequence has been changed to another amino acid, but in the mature form No more than 15% of the amino acids in the sequence are so changed. The present invention includes any of these fragments. In another aspect, the invention also includes an isolated nucleic acid encoding a NOVX polypeptide, or a fragment, homologue, analog or derivative thereof.

The invention also includes polypeptides that are natural allelic variants. In one embodiment, these allelic variants comprise an amino acid sequence that is a translation of the nucleic acid sequence that differs by a single nucleotide from the NOVX nucleic acid sequence. In another embodiment, the NOVX polypeptide is a variant polypeptide described herein, where it changes to provide a conservative substitution if any amino acid in the selected sequence is changed. In other embodiments, the present invention discloses a method of determining the presence or amount of a NOVX polypeptide in a sample. The method provides a sample; introduces the sample to an antibody that immunospecifically binds to the polypeptide; and determines the presence or amount of antibody bound to the polypeptide, thereby the presence of the polypeptide in the sample Or a step of determining the amount. In another embodiment, the invention provides a method for determining the presence or predisposition of a disease associated with an altered level of NOVX polypeptide in a mammalian subject. This method measures the level of expression of a polypeptide in a sample from a first mammalian subject; and knows the amount of the polypeptide in the sample is not suffering from or predisposed to the disease. Comparing the amount of the polypeptide present in a control sample from a second mammalian subject to the expression level of the polypeptide of the first subject when compared to the control sample. Changes indicate the presence or predisposition of the disease.

  In a further embodiment, the invention includes a method of identifying an agent that modulates a NOVX polypeptide. The method includes introducing a polypeptide into the agent; and determining whether the agent binds to the polypeptide. In various embodiments, the agent is a cell receptor or a downstream effector.

  In other embodiments, the invention includes a method of identifying a potential therapeutic agent for use in the treatment of a pathology, wherein the pathology comprises abnormal expression of NOVX polypeptide or abnormal physiological interaction. Related to. The method provides a cell that expresses a polypeptide of the invention and has properties or functions attributable to the polypeptide; contacts the cell with a composition comprising a candidate substance; and the substance is attributable to the polypeptide. Determining whether the property or function is changed, so that if the change observed in the presence of the substance is not observed when the cell is contacted with the composition without the substance, the substance is a potential Identified as a therapeutic agent. In another aspect, the present invention describes screening methods for pathological activity or potential modulators, or predispositions associated with NOVX polypeptides. The method comprises the steps of: administering a test compound to a test animal at increased risk of pathology associated with the polypeptide of the invention, wherein the test animal recombinantly expresses the polypeptide of the invention Including; This method measures the activity of a polypeptide in a test animal after administering a test compound in the process; and compares the protein activity of the test animal with the activity of a polypeptide in a control animal that is not administered the polypeptide. Where a change in the activity of the polypeptide in the test animal relative to the control animal comprises indicating a modulator of the latent or predisposing pathology associated with the NOVX polypeptide. In one embodiment, the test animal is a recombinant test animal that expresses the test protein transgene or can express the transgene at an increased level relative to the wild-type test animal under the control of a promoter, and The promoter is not the promoter of the original gene of the transgene. In another aspect, the present invention provides a method of modulating the activity of a NOVX polypeptide, comprising modulating a cell sample expressing a NOVX polypeptide with a compound that binds to the polypeptide and the activity of the polypeptide. Including a method comprising introducing in a sufficient amount.

  In order to treat diseases, pathologies and other abnormal conditions or situations where a mammal is diagnosed as being at or at risk for a normal condition or situation, the identification of a new therapeutic agent is required. is important. Such a procedure includes at least the steps of identifying a target component of a diseased tissue or organ and identifying a candidate therapeutic agent that modulates the functional properties of the target. The target component may be any biopolymer involved in disease or pathology. Usually, the target is a polypeptide or protein having specific functional properties. Other classes of macromolecules are lipids such as nucleic acids, polysaccharides, complex lipids or glycolipids; and the target may be an intracellular or extracellular structure consisting of one or more of these classes of macromolecules . Once such a target is identified, it can be used in screening assays to identify preferred candidate therapeutic agents from a large population of substances or compounds.

  Often the purpose of such screening assays is to identify small molecule candidates; this is generally done through the use of combinatorial methods to construct a population of substances to be tested. Implementation of high-throughput screening methods is advantageous when performed with large combinatorial libraries of compounds.

  It is an object of the present invention to provide a cell line that recombinantly or endogenously expresses a target biopolymer or isolated target biopolymer for use as a macromolecular component in a screening assay for candidate drug identification. .

  Another object of the present invention is to provide a screening assay for unambiguously identifying candidate agents from a combinatorial library of low molecular weight substances or compounds.

  A further aspect of the invention uses candidate agents in any of a variety of in vitro, ex vivo and in vivo assays to identify agents that have advantageous therapeutic uses in the treatment of disease, pathology, abnormal conditions or situations. It is.

In another aspect, the invention uses a target polypeptide (NOVX) that has a special relationship with a disease to identify a test compound as a candidate therapeutic agent for the treatment of the disease, pathology, abnormal condition or situation. Provide a method. This method is:
(A) combining a test compound with a target polypeptide and a substrate for the target polypeptide; and (b) determining whether the test compound modulates the activity of the target polypeptide.

  In one embodiment of this method, the chemical is a constituent of a combinatorial library of compounds; the combination in step (a) is performed on one or more biopolymer replicate samples; and The replicate sample is contacted with at least one component of the combinatorial library. In yet a further embodiment of this method, the biopolymer is contained intracellularly and is functionally expressed therein. In further embodiments, the binding of the compound modulates the function of the biomacromolecule, which identifies the compound as a potential therapeutic agent. In yet a further embodiment of this method, the target biopolymer is a polypeptide.

  As used herein, “substrate” includes any compound capable of binding to or interacting with a target polypeptide, including peptides, polypeptides, nucleic acids, carbohydrate moieties, lipids, small molecules (eg, circular AMP, ATP), agonists, antagonists, and inhibitors.

In another aspect of the invention, a method for identifying a medicament for treating a disease, pathology, or abnormal condition or symptom is provided. The method comprises the following steps:
(1) identifying a candidate therapeutic agent for treating the disease, pathology, or abnormal condition or situation by the method described in the previous paragraph;
(2) contacting a biological sample associated with the disease, pathology, or abnormal condition or situation with the candidate therapeutic agent;
(3) determining whether the candidate induces an effect on the biological sample associated with a therapeutic response; and (4) identifying a candidate that exerts such an effect as a pharmaceutical.

  In important embodiments of the method, the biological sample comprises cells, tissues or organs, or is a non-human mammal.

  Several cell, animal and clinical studies have been conducted to elucidate the genetic involvement of these conditions in the pathogenesis and pathogenicity in various physiological, pharmacological or natural conditions. These studies include differential gene expression, protein-protein interactions, extensive sequencing of expressed genes, and, for example, single nucleotide polymorphisms (SNPs) or splice variants in test and subject biological samples The core technology at CuraGen Corporation was used to observe the association of genetic variations, but not limited to them. The purpose of such studies is to identify various therapeutic interventions to prevent, treat, or cure the outcome of obesity and / or diabetes.

  The present invention discloses novel associations of proteins and polypeptides, and nucleic acids encoding them, with various diseases or pathologies. Proteins and similar related proteins are encoded by cDNA and / or genomic DNA. In some cases, proteins, polypeptides and their homologous nucleic acids have been identified by the inventors. Furthermore, the present invention is recombinantly expressed and / or endogenously expressed in various screens for the identification of therapeutic antibodies and / or therapeutic small molecules that modulate the activity of the disclosed NOVX polypeptides. Includes the use of proteins.

  The invention also includes an isolated nucleic acid encoding a NOVX polypeptide, or a fragment, homologue, analog or derivative. In a preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence of a natural allelic nucleic acid variant. In another embodiment, the nucleic acid encodes a variant polypeptide having a polypeptide sequence of a natural polypeptide variant. In another embodiment, the nucleic acid molecule differs from the NOVX nucleic acid sequence by a single nucleotide. In one embodiment, the NOVX nucleic acid molecule is a nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-1 (n is an integer from 1 to 85), or the complement of the nucleotide sequence, under stringent conditions. Hybridize to the object. In another aspect, the present invention provides a vector or cell that expresses a NOVX nucleotide sequence.

  In one embodiment, the present invention discloses a method for modulating the activity of a NOVX polypeptide. The method includes introducing a cell sample expressing NOVX polypeptide in a quantity sufficient to modulate the polypeptide and a compound that binds to the polypeptide. In another embodiment, the invention includes an isolated NOVX nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising a NOVX amino acid sequence or a variant of the mature NOVX amino acid sequence, wherein the maturation of the selected sequence Any amino acid in the type changes to another amino acid, except that no more than 15% of the amino acid residues in the mature type sequence change. In another embodiment, the invention includes an amino acid sequence that is a variant of the NOVX amino acid sequence, wherein any amino acid identified in the selected sequence is changed to another amino acid, provided that As such, no more than 15% of the amino acid residues in the mature sequence are changed.

  In one embodiment, the invention discloses a NOVX nucleic acid fragment encoding a NOVX polypeptide or at least a portion of any variant of said polypeptide, wherein any amino acid of the selected sequence is separate However, no more than 10% of the amino acid residues in the sequence are so changed. In another embodiment, the invention includes the complement of either a NOVX nucleic acid molecule or a natural allelic nucleic acid variant. In another embodiment, the invention includes a NOVX nucleic acid molecule that encodes a variant polypeptide, wherein the variant polypeptide has a polypeptide sequence of a natural polypeptide variant. In another embodiment, the present invention discloses NOVX nucleic acids that differ from NOVX nucleic acid molecule sequences by a single nucleotide.

  In another embodiment, the invention includes a NOVX nucleic acid molecule, wherein one or more nucleotide sequences in the NOVX nucleotide sequence are changed to another nucleotide, provided that no more than 15% nucleotides Change. In one embodiment, the invention provides that the NOVX nucleic acid fragment and one or more nucleotides in the NOVX nucleic acid fragment are changed from one selected from the group consisting of a selected sequence to another nucleotide. However, nucleic acid fragments in which no more than 15% nucleotides are so altered are disclosed. In another embodiment, the invention includes a nucleic acid molecule that hybridizes under stringent conditions to a NOVX nucleotide sequence or a complement of a NOVX nucleotide sequence. In one embodiment, the invention includes nucleic acid molecules that have been altered in sequence such that no more than 15% of the nucleotides in the coding sequence differ from the NOVX nucleotide sequence or fragments thereof.

  In a further embodiment, the present invention includes a method for determining the presence or amount of a NOVX nucleic acid molecule in a sample. The method provides a sample; introduces the sample to a probe that binds to the nucleic acid molecule; and determines the presence or amount of the probe bound to the nucleic acid molecule, thereby determining the presence or amount of the nucleic acid molecule in the sample. The process of carrying out is included. In one embodiment, the presence or amount of a nucleic acid molecule is used as a cell or tissue type marker.

  In another embodiment, the present invention relates to a method for determining the presence or predisposition of a disease associated with an altered level of a NOVX nucleic acid molecule in a first mammalian subject. The method measures the amount of nucleic acid in the sample from the first mammalian subject; and knows the amount of nucleic acid in the sample of step (a) not suffering from or predisposed to the disease. Comparing the amount of nucleic acid present in a control sample from a second mammalian subject to be obtained; wherein the change in the level of nucleic acid present in the first subject when compared to the control sample is Point out the presence or predisposition of the disease.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative and not intended to be limiting.

  Other features and advantages of the invention will be apparent from the following detailed description and claims.

(Detailed description of the invention)
The present invention provides novel nucleotides and polypeptides encoded thereby. The present invention includes novel nucleic acid sequences, their encoded polypeptides, antibodies, and other related compounds. As used herein, sequences are collectively referred to as “NOVX nucleic acids” or “NOVX polynucleotides” and the corresponding encoded polypeptides are referred to as “NOVX polypeptides” or “NOVX proteins”. Unless otherwise stated, “NOVX” is meant to refer to any novel sequence disclosed herein. Table A shows a summary of NOVX nucleic acids and their encoded polypeptides.

Table 1: Sequences and corresponding sequence numbers

  Table 1 shows the homology of NOVX polypeptides with known protein families. Thus, nucleic acids, polynucleotides, antibodies and related compounds according to the present invention corresponding to NOVX identified in the first column of Table 1 are, for example, lesions associated with known protein families identified in the fifth column of Table 1. And useful in therapeutic and diagnostic applications related to disease.

  Pathologies, diseases, disorders and conditions associated with NOVX sequences include, but are not limited to, for example: cardiomyopathy, atherosclerosis, hypertension, congenital heart disease, aortic stenosis, atrial septal defect Disease (ASD), atrioventricular (AV) duct defect, arterial duct, pulmonary valve stenosis, aortic valve stenosis, ventricular septal defect (VSD), valve disease, tuberous sclerosis, scleroderma, obesity, Obstruction-related metabolic disturbances, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, diabetes, metabolic disorders, neoplasms; adenocarcinoma, lymphoma, uterine cancer, fertility, hemophilia, hypercoagulability, idiopathic Thrombocytopenic purpura, immunodeficiency, psoriasis, skin disease, graft-versus-host disease, AIDS, bronchial asthma, lupus, Crohn's disease; multiple sclerosis, Albright hereditary osteodystrophy, infectious disease, appetite Slump, cancer ream Cachexia, cancer, neurodegenerative disorders, Alzheimer's disease, Parkinson's disease, immune disorders, hematopoietic disorders, and various dyslipidemias, metabolic syndrome X and chronic diseases and various cancers, and conditions such as transplantation and fertility Associated debilitating disorder.

  NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and situations. Various NOVX nucleic acids and polypeptides according to the present invention are useful as novel members of the protein family according to the existence of domains and sequence relationships with the proteins. In addition, NOVX nucleic acids and polypeptides can be used to identify proteins that are members of the family to which the NOVX polypeptide belongs.

  Consistent with other known members of the protein family identified in column 5 of Table 1, the NOVX polypeptides of the present invention show homology to and characterize other members of such protein families. Contains the domain. Details of sequence relatedness analysis and domain analysis for each NOVX are shown in the examples for identification of human sequences in individual sections for each NOVX polypeptide.

  NOVX nucleic acids and polypeptides can be used to screen for molecules that inhibit or promote NOVX activity or function. In particular, the nucleic acids and polypeptides according to the invention can be used as targets for the identification of small molecules that modulate or inhibit diseases associated with the protein families listed in Table 1.

NOVX nucleic acids and polypeptides are also useful for detecting specific cell types. Details of expression analysis for each NOVX are in the examples showing the expression profiles in individual sections for each NOVX polypeptide. Accordingly, NOVX nucleic acids, polypeptides, antibodies and related compounds according to the present invention have diagnostic and therapeutic applications in the detection of various diseases having different expression in normal tissue versus diseased tissue, eg, various cancers. . SNP analysis for each NOVX is shown in the SNP examples in individual sections for each NOVX polypeptide, if applicable.
Additional uses for NOVX nucleic acids and polypeptides according to the present invention are disclosed herein.

NOVX clones NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and situations. Various NOVX nucleic acids and polypeptides according to the present invention are useful as novel members of a protein family according to the presence of domains and sequences associated with the protein. In addition, NOVX nucleic acids and polypeptides can be used to identify proteins that are members of the family to which the NOVX polypeptide belongs.

  NOVX genes and their corresponding encoded proteins are useful for preventing, treating or alleviating medical conditions, eg, by protein therapy or gene therapy. A disease state can be diagnosed by measuring the amount of a novel protein in a sample or by measuring the presence of a mutation in a novel gene. Based on the tissues in which the NOVX gene is most highly expressed, the specific use for each NOVX gene will be described. Use includes developing products for diagnosing or treating various diseases and disorders.

  The NOVX nucleic acids and proteins of the present invention are useful as potential diagnostic and therapeutic applications and research tools. These include serving as specific or selective nucleic acids, or proteins, diagnostic markers and / or prognostic markers. Here, the presence or amount of nucleic acids or proteins, as well as potential therapeutic applications such as: (i) protein therapeutics, (ii) small molecule drug targets, (iii) antibody targets (therapeutic, Diagnostic, drug targeting / cytotoxic antibodies), (iv) nucleic acids useful for gene therapy (gene delivery / gene surgery), and (v) compositions that promote tissue regeneration in vitro and in vivo, (vi) organisms Defense weapon.

  In one specific embodiment, the invention provides a mature form having an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 2n (n is an integer from 1 to 85); (b) SEQ ID NO: 2n ( n is an integer from 1 to 85) and is a mature variant having an amino acid sequence selected from the group consisting of 15% of amino acid residues in the mature sequence. (C) an amino acid sequence selected from the group consisting of SEQ ID NO: 2n (n is an integer from 1 to 85); (d) SEQ ID NO: 2n ( a variant having an amino acid sequence selected from the group consisting of n is an integer of 1 to 85, provided that any amino acid identified by the selected sequence has 15% or less of the amino acid residues in the sequence Change to a different amino acid under the condition It is; to and (e) (a) no comprising an isolated polypeptide comprising any of the fragments, the amino acid sequence selected from the group consisting of (d).

  In another specific embodiment, the invention provides a mature form having an amino acid sequence given (a) SEQ ID NO: 2n (n is an integer from 1 to 85); (b) SEQ ID NO: 2n (n is A mature variant having an amino acid sequence selected from the group consisting of 1 to 85, wherein any amino acid in the mature form of the selected sequence is an amino acid residue in the mature sequence (C) an amino acid sequence selected from the group consisting of SEQ ID NO: 2n (n is an integer from 1 to 85); d) a variant of an amino acid sequence selected from the group consisting of SEQ ID NO: 2n (n is an integer from 1 to 85), wherein the specified amino acid in the selected sequence is an amino acid residue in the sequence Different nets on condition that 15% or less is subject to change A variant that is altered to an acid; and (e) at least a portion of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2n (n is an integer from 1 to 85), or a selected sequence A fragment of a nucleic acid encoding any variant of a polypeptide, wherein any amino acid is altered to a different amino acid provided that less than 10% of the amino acid residues in the sequence are altered; and (f) a nucleic acid molecule An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of:

  In yet another particular embodiment, the present invention includes an isolated nucleic acid molecule. Wherein the nucleic acid molecule is (a) a nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-1 (n is an integer of 1 to 85); (b) SEQ ID NO: 2n-1 (n is 1 to 85) A condition wherein one or more nucleotides in a nucleotide sequence selected from the group consisting of: is selected from the group consisting of the selected sequence and no more than 15% of the nucleotides are altered (C) a nucleic acid fragment having a sequence selected from the group consisting of SEQ ID NO: 2n-1 (n is an integer from 1 to 85); and (d) SEQ ID NO: 2n Whether one or more nucleotides are selected from the group consisting of selected sequences in a nucleotide sequence selected from the group consisting of -1 (n is an integer from 1 to 85) Contain different nucleic acid fragments that are modified nucleotide, a nucleotide sequence selected from the group consisting of with the proviso that more than 15% of the nucleotides undergo changes.

NOVX Nucleic Acids and Polypeptides One aspect of the invention pertains to isolated nucleic acid molecules encoding NOVX polypeptides or biologically active portions thereof. The invention also provides nucleic acid fragments sufficient for use as hybridization probes to identify nucleic acids encoding NOVX (eg, mRNA of NOVX), and PCR primers for amplification and / or mutation of NOVX nucleic acid molecules Including fragments for use as. As used herein, the term “nucleic acid molecule” refers to a DNA molecule (eg, cDNA or genomic DNA), an RNA molecule (eg, mRNA), a DNA analog or RNA analog produced using nucleotide analogs, And their derivatives, fragments and homologues. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

  The NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a “mature” form of a polypeptide or protein disclosed in the present invention is a naturally occurring polypeptide, or precursor, or product of a proprotein. Naturally occurring polypeptides, precursors or proproteins include, by way of non-limiting example, full-length gene products encoded by the corresponding genes. Alternatively, it may be defined as a polypeptide, precursor or proprotein encoded by the ORFs described herein. A “mature” form of a product occurs, as a non-limiting example, as a result of one or more naturally occurring processing steps that can occur in a cell (eg, a host cell) that produces a gene product. Examples of such processing steps that lead to a “mature” form of a polypeptide or protein include cleavage of the N-terminal methionine residue encoded by the initiation codon of the ORF, or proteolytic cleavage of the signal peptide or leader sequence. Thus, a mature form resulting from a precursor polypeptide or protein having residues 1-N where residue 1 is the N-terminal methionine has residues 2-N remaining after removal of the N-terminal methionine. Alternatively, the mature form resulting from a precursor polypeptide or protein having residues 1-N that cleaves the N-terminal signal sequence from residues 1-residue M is from remaining residues M + 1-residue N. Has a residue. Further, as used herein, a “mature” form of a polypeptide or protein can result from post-translational steps other than proteolytic cleavage events. Such further methods include, but are not limited to glycosylation, myristoylation, or phosphorylation. In general, a mature polypeptide or protein can be produced by manipulation of only one of these methods, or any combination thereof.

  As used herein, the term “probe” refers to a variable length nucleic acid sequence, preferably between at least about 10 nucleotides (nt) to about 100 nt, or depending on the particular use, eg, as long as about 6000 nt. Point to. Probes can be used for the detection of identical, similar or complementary nucleic acid sequences. Longer probes are generally obtained from natural or recombinant sources. Longer probes are slower to hybridize than oligomer probes with higher specificity and shorter lengths. Probes can be single-stranded or double-stranded and can be designed to have specificity in PCR, membrane-based hybridization techniques, or ELISA-like techniques.

  As used herein, the term “isolated” nucleic acid molecule is a nucleic acid that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid does not have sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (ie, sequences located at the 5 ′ and 3 ′ ends of the nucleic acid). For example, in various embodiments, the isolated NOVX nucleic acid molecule is about 5 kb, 4 kb naturally adjacent to the nucleic acid molecule in the genomic DNA of the cell / tissue from which the nucleic acid is derived (eg, brain, heart, liver, spleen, etc.). It may contain nucleotide sequences of 3 kb, 2 kb, 1 kb, 0.5 kb, 0.1 kb or less, or less. Furthermore, an “isolated” nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material, media, or chemical precursors or other chemicals.

  A nucleic acid molecule of the present invention, eg, a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 2n-1 (where n is an integer from 1 to 85), or the complement of this nucleotide sequence, can be obtained using standard molecular biology techniques and It can be isolated using the sequence information provided in the specification. Using all or part of the nucleic acid sequence of SEQ ID NO: 2n-1 (where n is an integer from 1 to 85) as a hybridization probe, standard hybridization and cloning techniques (eg, Sambrook, et al., ( eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons , New York, NY, 1993) can be used to isolate NOVX molecules.

  The nucleic acids of the invention can be amplified with appropriate oligonucleotide primers according to standard PCR amplification techniques using cDNA, mRNA, or alternatively genomic DNA as a template. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. In addition, oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, such as the use of automated DNA synthesizers.

  The term “oligonucleotide” as used herein refers to a series of linking nucleotide residues. Short oligonucleotide sequences can be based on or designed from genomic or cDNA sequences. Short oligonucleotide sequences are then used to amplify, confirm, or reveal the presence of identical, similar, or complementary DNA or RNA in a particular cell or tissue. The oligonucleotide comprises a nucleic acid sequence of about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment of the invention, the oligonucleotide comprising a nucleic acid molecule of less than 100 nt in length is at least 6 consecutive nucleotides of SEQ ID NO: 2n-1 (n is an integer from 1 to 85), or It further includes a complement. Oligonucleotides can be chemically synthesized and used as probes.

  In another embodiment, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence set forth in SEQ ID NO: 2n-1 (where n is an integer from 1 to 85), or a portion of this nucleotide sequence (eg, NOVX polypeptide A nucleic acid molecule that is the complement of a fragment that can be used as a probe, primer, or fragment encoding a biologically active portion of A nucleic acid molecule that is complementary to a nucleotide sequence that represents SEQ ID NO: 2n-1 (n is an integer from 1 to 85) is a nucleotide sequence that represents SEQ ID NO: 2n-1 (n is an integer from 1 to 85). It is sufficiently complementary and can hydrogen bond with the nucleotide sequence shown in SEQ ID NO: 2n-1 (where n is an integer from 1 to 85) with little or no mismatch. Therefore, the nucleic acid molecule forms a stable double strand.

  The term “complementary” as used herein refers to Watson-Crick or Hoogsteen base pairing between nucleotide units of a nucleic acid molecule, and the term “binding” refers to two polypeptides or compounds, or related polypeptides. Or means a physical or chemical interaction between compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. The physical interaction can be direct or indirect. Indirect interactions may be through or due to the action of other polypeptides or compounds. Direct binding refers to an interaction that does not occur through or due to the action of another polypeptide or compound, but instead takes place without other substantial chemical mediators.

  “Fragments” as provided herein are at least 6 long enough to allow specific hybridization in the case of nucleic acids, and long enough for specific recognition of epitopes in the case of amino acids. Defined as a sequence of (contiguous) nucleic acids or a sequence of at least 4 (contiguous) amino acids and is some fraction of most shorter than the full length. Fragments can be derived from any contiguous portion of the selected nucleic acid or amino acid sequence.

A full-length NOVX clone is identified as containing an ATG translation start codon and an in-frame stop codon. Any disclosed NOVX nucleotide sequence lacking the ATG initiation codon therefore encodes a truncated C-terminal fragment of each NOVX polypeptide and extends in the 5 'direction of the sequence disclosed by the corresponding full length cDNA. Require to do. Any disclosed NOVX nucleotide sequence lacking an in-frame stop codon similarly encodes a truncated N-terminal fragment of the respective NOVX polypeptide, such that the corresponding full-length cDNA extends in the 3 'direction of the disclosed sequence. Request.
A derivative is a nucleic acid or amino acid sequence generated either directly from the original compound, either by modification or by partial substitution. Analogs are nucleic acid or amino acid sequences that have a structure that is similar but not identical to the original compound, eg, they differ from a natural compound with respect to a particular component or side chain. Analogs can be synthetic, can be derived from different evolutionary origins, and can have similar or opposite metabolic activities compared to wild-type. A homologue is the nucleic acid or amino acid sequence of a particular gene derived from a different species.

  Derivatives and analogs can be full length or other than full length. The nucleic acid or protein derivatives or analogs of the present invention, in various embodiments, when compared to alignment sequences aligned over nucleic acid or amino acid sequences of the same size or by computer homology programs known in the art, include: A molecule comprising a region that is substantially homologous to a nucleic acid or protein of the invention with at least about 70%, 80%, or 95% identity (preferably 80-95% identity), or encoding thereof Nucleic acids include, but are not limited to, molecules that are capable of hybridizing under stringent, moderately stringent, or low stringency conditions to the complement of a sequence encoding a protein of the invention. See, for example, Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993 and the following.

  “Homologous nucleic acid sequence” or “homologous amino acid sequence” or variants thereof refer to sequences characterized by homology at the nucleotide or amino acid level as described above. Homologous nucleotide sequences include those sequences that encode isoforms of the NOVX polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the present invention, a homologous nucleotide sequence includes a nucleotide sequence encoding a non-human species NOVX polypeptide, including but not limited to vertebrates. Thus, for example, include frogs, mice, rats, rabbits, dogs, cats, cows, horses, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variants and mutants of the nucleotide sequences set forth herein. However, homologous nucleotide sequences do not include the exact nucleotide sequence encoding human NOVX protein. Homologous nucleic acid sequences include nucleic acid sequences that encode conservative amino acid substitutions (see below) of SEQ ID NO: 2n-1 (where n is an integer from 1 to 85) as well as polypeptides having NOVX biological activity. Various biological activities of the NOVX protein are described below.

  NOVX polypeptides are encoded by the open reading frame (“ORF”) of NOVX nucleic acids. The ORF corresponds to a nucleotide that can potentially be translated into a polypeptide. A series of nucleic acids containing the ORF is not interrupted by a stop codon. The ORF representing the sequence encoding the full-length protein begins with an ATG “start” codon and ends with one of three “stop” codons, TAA, TAG or TGA. For the purposes of the present invention, the ORF can be any portion of the coding sequence that may or may not have a start codon, a stop codon, or both. In order to consider the ORF as a good candidate for encoding a genuine cellular protein, minimal requirements are often defined, for example a series of DNAs encoding 50 amino acids or more.

  Nucleotide sequences determined from cloning of the human NOVX gene are for use in identifying and / or cloning NOVX homologues from other cell types, eg, other tissues, and NOVX homologues from other vertebrates Allows creation of probes and primers to be designed. The probe / primer typically comprises a substantially purified oligonucleotide. The oligonucleotide is at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotides of SEQ ID NO: 2n-1 (n is an integer from 1 to 85) An antisense strand nucleotide sequence of SEQ ID NO: 2n-1 (n is an integer from 1 to 85); or a naturally occurring variant of SEQ ID NO: 2n-1 (n is an integer from 1 to 85) Typically includes regions of nucleotide sequences that hybridize under stringent conditions.

  Probes based on human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe is provided with a detectable label, for example, the label can be a radioisotope, a fluorescent compound, an enzyme, or a cofactor of the enzyme. Such probes are part of a diagnostic test kit for identifying cells or tissues that misexpress a NOVX protein, such as by measuring the level of a NOVX-encoding nucleic acid in a cell sample from a subject. For example, NOVX mRNA is detected or whether the genomic NOVX gene has been mutated or deleted.

  A “polypeptide having a biologically active portion of a NOVX polypeptide” includes mature forms as measured in a particular biological analysis and is similar, but not necessarily identical, to the activity of a polypeptide of the invention. It refers to a polypeptide that exerts no activity with or without dose dependency. A nucleic acid fragment encoding a “biologically active portion of NOVX” is represented by SEQ ID NO: 2n-1 (where n is a polypeptide encoding a polypeptide having a certain NOVX biological activity (the biological activity of the NOVX protein is described below)). Can be prepared by isolating a portion of 1 to 85), expressing the NOVX protein-encoding portion (eg, by in vitro recombinant expression), and evaluating the activity of the NOVX-encoding portion. .

NOVX nucleic acid and polypeptide variants The present invention further differs from the nucleotide sequence shown in SEQ ID NO: 2n-1 (where n is an integer from 1 to 85) due to the degeneracy of the genetic code, and thus SEQ ID NO: 2n-1 ( n is an integer from 1 to 85) and includes a nucleic acid molecule that encodes the same NOVX protein that is encoded by the nucleotide sequence shown. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence that encodes a protein having an amino acid sequence set forth in SEQ ID NO: 2n (n is an integer from 1 to 85).

  In addition to the human NOVX nucleotide sequence set forth in SEQ ID NO: 2n-1 (where n is an integer from 1 to 85), polymorphisms of the DNA sequence that result in a change in the amino acid sequence of the NOVX polypeptide are a population (eg, a human population). Those skilled in the art will appreciate that they can be present in Such genetic polymorphisms in the NOVX gene may exist among individuals in the population due to natural allelic variation. The terms “gene” and “recombinant gene” as used herein refer to a nucleic acid molecule comprising an open reading frame (ORF) encoding a NOVX protein, preferably a vertebrate NOVX protein. Such natural allelic variations typically result in 1-5% variation in the nucleotide sequence of the NOVX gene. Any and all such nucleotide variations that are the result of natural allelic variation and do not alter the functional activity of the NOVX polypeptide and the amino acid polymorphisms in the NOVX polypeptide obtained therefrom are intended to be within the scope of the invention. .

  Furthermore, nucleic acid molecules encoding NOVX proteins from other species, and thus nucleic acid molecules having a nucleotide sequence different from human SEQ ID NO: 2n-1 (n is an integer from 1 to 85) are within the scope of the present invention. It shall be in Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX cDNA of the present invention are obtained using human cDNA or a portion thereof as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. It can be isolated based on homology with the human NOVX nucleic acids disclosed herein.

  Thus, in another embodiment, an isolated nucleic acid molecule of the present invention is at least 6 nucleotides in length and is exact for a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2n-1 (n is an integer from 1 to 85). Hybridizes under conditions. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, 2000 or more nucleotides in length. In yet another embodiment, the isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term “hybridizes under stringent conditions” describes hybridization and washing conditions in which nucleotide sequences that are at least about 65% homologous to each other typically remain hybridized to each other. Intended.

  Homologues (i.e., nucleic acids encoding NOVX proteins from non-human species) or other related sequences (e.g., paralogs) can be converted to specific human sequences using methods well known in the art of nucleic acid hybridization and cloning. All or part of the probe is used as a probe, and can be obtained by low, medium, or highly stringent hybridization.

  As used herein, the term “stringent hybridization conditions” refers to conditions under which a probe, primer or oligonucleotide hybridizes to a target sequence but not to other sequences. The exact condition is sequence dependent and varies in different environments. Longer sequences hybridize specifically at higher temperatures than shorter sequences. In general, stringent conditions are selected to be about 5 ° C. lower than the melting temperature (Tm) for the specific sequence at a constant ionic strength and pH. Tm is the temperature (under a defined ionic strength, pH and nucleic acid concentration) at which 50% of the probe complementary to the target sequence hybridizes to the target sequence at equilibrium. Since the target sequence is generally present in excess at Tm, it accounts for 50% of the probe at equilibrium. Typically, the strict conditions are pH 7.0-8.3 and a salt concentration of less than about 1.0M sodium ion, typically about 0.01-1.0M sodium ion (or other salt), The temperature is at least about 30 ° C. for short probes, primers or oligonucleotides (eg, 10 nt to 50 nt) and at least about 60 ° C. for longer probes, primers and oligonucleotides. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.

  The exact conditions are known to those skilled in the art and can be found in Ausubel, et al., (Eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. it can. Preferably, the conditions are such that sequences that are at least 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. is there. Non-limiting examples of stringent hybridization include 6x SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA and 500 mg / ml denaturation. Hybridization is performed at 65 ° C. in a high salt buffer containing salmon sperm DNA, and then washed once or more at 50 ° C. in 0.2 × SSC, 0.01% BSA. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 2n-1 (n is an integer from 1 to 85) corresponds to a naturally occurring nucleic acid molecule. As used herein, the term “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleic acid sequence that occurs in nature (eg, encodes a natural protein).

  In a second embodiment, the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2n-1 (n is an integer from 1 to 85), or a fragment, analog or derivative thereof, hybridizes under moderately stringent conditions. Nucleic acid sequences for soy are provided. Non-limiting examples of moderately stringent hybridization conditions include hybridization at 55 ° C. in 6 × SSC, 5 × Reinhardt solution, 0.5% SDS and 100 mg / ml denatured salmon sperm DNA, followed by 1 X Washing once or more at 37 ° C in SSC, 0.1% SDS. Other moderately stringent conditions that can be used are well known in the art. See, for example, Ausubel, et al. (Eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Krieger, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY. .

  In a third embodiment, the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2n-1 (where n is an integer from 1 to 85), or a fragment, analog or derivative thereof, hybridizes under less stringent conditions. Nucleic acid sequences for soy are provided. Non-limiting examples of low stringency hybridization conditions include 35% formamide, 5 × SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.0%. Hybridization in 2% BSA, 100 mg / ml denatured salmon sperm DNA, 10% (wt / vol) dextran sulfate at 40 ° C., followed by 2 × SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, And wash one or more times at 50 ° C. in 0.1% SDS. Other less stringent conditions that can be used (eg, for interspecies hybridization) are well known in the art. For example, Ausubel, et al. (Eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY .; Shilo and Weinberg 1981. Proc Natl Acad Sci USA 78: 6789-6792.

Conservative mutations In addition to the naturally occurring allelic variation of the NOVX sequence that may be present in the population, a mutation is introduced into the nucleotide sequence of SEQ ID NO: 2n-1 (where n is an integer from 1 to 85). One skilled in the art further understands that changes in the amino acid sequence of the encoding NOVX protein can be made without altering the functional activity of the NOVX protein. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of SEQ ID NO: 2n-1 (n is an integer from 1 to 85). “Non-essential” amino acid residues are those residues that can change the wild-type sequence of NOVX proteins without altering their biological activity, while “essential” amino acid residues are such biological activity. Need to. For example, amino acid residues that are conserved in the NOVX protein of the present invention are not expected to be particularly resistant to conversion. Amino acids for which conservative substitutions can be made are well known in the art.

  Another aspect of the invention pertains to nucleic acid molecules encoding NOVX proteins that contain changes in amino acid residues that are not essential for activity. Such NOVX protein differs from SEQ ID NO: 2n-1 (n is an integer from 1 to 85) in amino acid sequence, but still retains biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence that encodes a protein, wherein the protein is at least about 50% homologous to the amino acid sequence of SEQ ID NO: 2n (n is an integer from 1 to 85). Contains amino acid sequence. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NO: 2n (n is an integer from 1 to 85); more preferably, the protein encoded by the nucleic acid molecule is SEQ ID NO: 2n ( n is an integer from 1 to 85); more preferably at least about 80% homologous to SEQ ID NO: 2n (n is an integer from 1 to 85); Preferably, it is at least about 90% homologous to SEQ ID NO: 2n (n is an integer from 1 to 85); most preferably, it is at least about 95% homologous to SEQ ID NO: 2n (n is an integer from 1 to 85). It is.

  An isolated nucleic acid molecule encoding a NOVX protein homologous to the protein of SEQ ID NO: 2n (n is an integer from 1 to 85) within the protein encoding one or more amino acid substitutions, additions or deletions Can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 2n-1 (where n is an integer from 1 to 85).

  Mutations can be introduced into SEQ ID NO: 2n-1 (n is an integer from 1 to 85) by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one of the amino acid residues that is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged side chains (e.g., glycine, Asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta side chains (e.g., Threonine, valine, isoleucine), as well as amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a NOVX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a NOVX coding sequence, such as saturation mutagenesis, and the resulting mutations The body can be screened for NOVX biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 2n-1 (n is an integer from 1 to 85), the encoded protein can be expressed by any recombinant technique known in the art and the activity of the protein can be measured. .

  Amino acid family relationships can also be determined based on side chain interactions. Substituted amino acids can be fully conserved “strong” residues or fully conserved “weak” residues. The “strong” group of conserved amino acid residues can be any one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, where the one letter code for amino acids Are grouped with amino acids that can be substituted for each other. Similarly, the “weak” group of conserved residues can be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEKK, NDEQHK, NEQHRK, HFY, where each Group letters represent the single letter code for amino acids.

In one embodiment, the mutant NOVX protein is (i) another NOVX protein, other cell surface protein or protein that interacts with their biological active site: the activity of forming the protein, (ii) the mutant NOVX protein May be assayed for complex formation between certain NOVX ligands, or (iii) the activity of mutant NOVX proteins that bind to intracellular target proteins or their biologically active sites (eg, avidin protein).
In yet another embodiment, mutant NOVX proteins can be assayed for activity that modulates a particular biological function (eg, modulation of insulin secretion).

Interfering RNA
In one embodiment of the invention, NOVX gene expression can be attenuated by RNA interference. One approach that is well known in the art is gene silencing mediated by short interfering RNA (siRNA), in which at least 19 to 25 nucleotides long segments of the NOVX gene transcript A specific double-stranded NOVX-derived siRNA nucleotide sequence that is complementary and includes a 5 ′ untranslated (UT) region, an ORF, or a 3 ′ UT region targets the NOVX gene expression product. See, for example, PCT applications WO00 / 44895, WO99 / 32619, WO01 / 75164, WO01 / 92513, WO01 / 29058, WO01 / 89304, WO02 / 16620, and WO02 / 29858, each incorporated herein by reference. . The targeted gene may be the NOVX gene or may be an upstream or downstream modulator of the NOVX gene. Non-limiting examples of upstream or downstream modulators of the NOVX gene are, for example, kinases or phosphatases that interact with the transcription factor NOVX polypeptide that binds to the NOVX gene promoter, and polypeptides included in the NOVX regulatory pathway.

  According to the methods of the present invention, NOVX gene expression is silenced using interfering RNA. The NOVX polynucleotides of the present invention include siRNA polynucleotides. Using the NOVX polynucleotide sequence, for example, by processing the NOVX polynucleotide sequence in a cell-free system such as, but not limited to, Drosophila extract, by transcription of recombinant double-stranded NOVX RNA, or by the NOVX sequence Such NOVX siRNA can be obtained by synthesizing a nucleotide sequence homologous to. See, for example, Tuschul, Zamire, Lehmann, Bartel and Sharp (1999), Genes & Dev. 13: 3191-3197 (incorporated into the system of the present invention by reference). When synthesized, typical 0.2 micromolar scale RNA synthesis yields about 1 milligram of siRNA, sufficient for 1000 transfection experiments using a 24-well tissue culture plate format.

  The most efficient silencing is generally observed using a siRNA duplex containing a 21 nucleotide sense strand and a 21 nucleotide antisense strand paired to have a 2 nucleotide 3 ′ overhang. The The sequence of the 2 nucleotide 3 'overhang makes a further small contribution to the specificity of siRNA target recognition. The contribution to specificity is localized to unpaired nucleotides adjacent to the first paired base. In one embodiment, the nucleotide in the 3 'overhang is a rib nucleotide. In another embodiment, the nucleotide in the 3 'overhang is a deoxyribonucleotide. Using 2'-deoxyribonucleotides in the 3 'overhang is effective as a use of ribonucleotides, but deoxyribonucleotides are often cheaper to synthesize and are most likely to be more nuclease resistant.

  The contemplated recombinant expression vector of the present invention is in an expression vector comprising proximity regulatory sequences operably linked to NOVX sequences in a manner that allows expression of both strands (by transcription of the DNA molecule). Contains integrated NOVX DNA molecules. An RNA molecule that is antisense to NOVX mRNA is transcribed by a first promoter (eg, the 3 ′ promoter sequence of the cloned DNA), and an RNA molecule that is the sense strand for NOVX mRNA is transcribed by a second promoter (clone Transcribed by the 5 'promoter sequence of the modified DNA). The sense and antisense strands hybridize in vivo to yield a siRNA construct for silencing the NOVX gene. Alternatively, two constructs can be used to create the sense and antisense strands of the siRNA construct. Eventually, the cloned DNA may encode a construct having a second structure, with a single transcript having sense and complementary antisense sequences from the target gene (s). In one example of this embodiment, the hairpin RNAi product is homologous to all or part of the target gene. In another example, the hairpin RNAi product is siRNA. The regulatory sequences adjacent to the NOVX sequence may be the same or different if their expression can be modulated independently or temporally or positionally.

  In particular embodiments, siRNAs are transcribed intracellularly, for example by incorporating a NOVX gene template into a vector comprising an RNA pol III transcription unit derived from smaller nuclear RNA (snRNA) U6 or human RNase P RNA H1. The One example of a vector system is the GeneSuppressor ™ RNA Interference kit (commercially available from Imgenex). The U6 and H1 promoters are members of the type III class of the pol III promoter. The +1 nucleotide of the U6-like promoter is always guanosine, whereas the +1 of the H1 promoter is adenosine. The termination signal for these promoters is defined by 5 consecutive thymidines. Typically, the transcript is cleaved after the second uridine. Cleavage at this position results in a 3'UU overhang in the expressed siRNA, which is similar to the 3 'overhang of synthetic siRNA. Sequences less than 400 nucleotides in length can be transcribed by these promoters, and therefore they are ideally suited for expressing about 21 nucleotides of siRNA, eg, in an RNA stem loop transcript of about 50 nucleotides Is.

  SiRNA vectors are considered advantageous over synthetic siRNA when long term expression knockdown is required. Cells transfected with siRNA expression vectors will be stable and will undergo prolonged mRNA inhibition. In contrast, cells transfected with exogenous synthetic siRNA typically recover from mRNA suppression within 7 days or during 10 rounds of cell division. The long-term gene silencing ability of siRNA expression vectors may allow application to gene therapy.

  In general, siRNA is cleaved from longer dsRNAs by an ATP-dependent ribonuclease called DICER. DICER is a member of the RNase III family of double-stranded RNA-specific endonucleases. siRNAs assemble with cellular proteins to form an endonuclease complex. In vitro studies in Drosophila suggest that the siRNA / protein complex (siRNP) is subsequently transferred to a second enzyme complex called an RNA-induced silencing complex (RISC) that contains an endoribonuclease different from DIECR. is doing. RISC uses the sequence encoded by the siRNA strand to find and destroy the complementary sequence of mRNA. Thus, the siRNA acts as a guide, limiting the ribonuclease to cleave only mRNA that is complementary to one of the two siRNA strands.

  The NOVX mRNA region to be targeted by siRNA is generally selected from the desired NOVX sequence starting from 50 to 100 nucleotides downstream of the start codon. Alternatively, the 5 'or 3' UTR and the region near the start codon can be used but are generally avoided. Because they are abundant in regulatory protein binding sites. The UTR binding protein and / or translation initiation complex may interfere with the binding of the siRNP or RISC endonuclease complex. An initial BLAST homology search for the selected siRNA sequence is performed on the available nucleotide sequence library to ensure that only one gene is targeted. The target recognition specificity by the siRNA duplex indicates that a single point mutation present in the paired region of the siRNA is sufficient to prevent degradation of the target mRNA. See Elbashir et al. 2001 EMBO J. 20 (23): 6877-88. Therefore, SNPs, polymorphisms, allelic variants or species-specific changes should be taken into account when targeting the desired gene.

  In one embodiment, a complete NOVX siRNA experiment uses the correct negative control. In general, negative control siRNA has the same nucleotide composition as NOVX siRNA, but lacks significant sequence homology to the genome. Typically, the nucleotide sequence of the NOVX siRNA is scrambled and a homology search is performed to confirm that it lacks homology with any other gene.

  Two independent NOVX siRNA duplexes can be used to knock down the target NOVX gene. This helps to control the specificity of the silencing effect. Furthermore, the expression of two independent genes can be knocked down simultaneously by using equal concentrations of different NOVX siRNA duplexes, eg, NOVX siRNA and siRNA for NOVX gene or polypeptide regulators. The availability of siRNA binding proteins appears to be more limited than the target mRNA accessibility.

  Typically, the targeted NOVX region is two adenines (AA) and two thymidines (TT) separated by a spacer region of 19 (N19) residues (eg, AA ( N19) TT). Desirable spacer regions have a G / C-content of about 30-70%, more preferably about 50%. If the sequence AA (N19) TT is not present in the target sequence, another target region would be AA (N21). The sequence of the NOVX sense siRNA corresponds to (N19) TT or N21, respectively. In the latter case, if such a sequence is not essentially present in the NOVX polynucleotide, conversion of the sense siRNA to the 3 'end TT can be performed. The logical interpretation of this sequence conversion is to produce a symmetric duplex with respect to the sequence composition of the sense and antisense 3 'overhangs. Symmetric 3 'overhangs can help ensure that siRNP is formed at the appropriate equal proportion of sense and antisense target RNA-cleavage siRNP. See, for example, Elbashir, Lendeckel and Tuschl (2001). Genes & Dev. 15: 188-200 (incorporated herein by reference). It is not believed that modification of the siRNA duplex sense sequence overhang affects the recognition of the targeted mRNA. This is because the antisense siRNA strand guides target recognition.

  Alternatively, if the NOVX target mRNA does not contain the appropriate AA (N21) sequence, the sequence NA (N21) may be searched. Furthermore, the sequence of the sense strand and the antisense strand may be synthesized as 5 '(N19) TT. This is because the sequence of most nucleotides on the 3 'side of the antisense siRNA does not contribute to specificity. Unlike antisense or ribozyme methods, the secondary structure of the target mRNA is not thought to strongly influence silencing. See Harborth, et al. (2001) J. Cell Science 114: 4557-4565 (incorporated herein by reference).

  Standard nucleic acid transfection methods such as OLIGOFECTAMINE Reagent (commercially available from Invitrogen) can be used to transfect NOVX siRNA duplexes. In general, assays for NOVX gene silencing are performed about 2 days after transfection. In the absence of transfection reagent, NOVX gene silencing was not observed, allowing comparative analysis of wild type and silencing NOVX phenotypes. In particular embodiments, about 0.84 μg siRNA duplex is usually sufficient for one well of a 24-well plate. Typically, cells are seeded the day before and transfected when approximately 50% confluent. The selection of cell culture medium and culture conditions is usually done by those skilled in the art and will vary with the choice of cell type. The efficiency of transfection depends not only on the cell type, but also on the cell passage number and confluence. The formation time and method (eg, inverted vortex) of the siRNA-liposome complex is also important. Low transfection efficiency is the biggest cause of unsuccessful NOVX silencing. It is necessary to carefully examine the transfection efficiency for the new cell line used. Preferred cells are derived from mammals, more preferably from rodents such as rats or mice, and most preferably from humans. When used for therapeutic treatment, preferably the cells are autologous, but non-autologous cell sources are also within the scope of the invention.

  For control experiments, transfection of 0.84 μg single-stranded sense NOVX siRNA did not affect NOVX silencing, and 0.84 μg antisense siRNA had a weaker silencing effect when compared to 0.84 μg double-stranded siRNA. Have Control experiments further allow comparative analysis of wild type and silent NOVX phenotypes. In order to control transfection efficiency, protein targeting is typically performed. For example, targeting Lamin A / C or transfection of CMV driven EGFP expression plasmid (eg, commercially available from Clontech). In the above example, fragments of lamin A / C knockdown in the cells are performed the next day by methods such as immunofluorescence, Western blot, Northern blot or other similar assays for protein expression or gene expression. Lamin A / C monoclonal antibody was obtained from Santa Cruz Biotechnology.

  Depending on the abundance and half-life (or turnover) of the targeted NOVX polynucleotide in the cell, a knockdown phenotype can be revealed after 1-3 days or thereafter. If a NOVX knockdown phenotype is not observed, it may be desirable to analyze whether the target mRNA (NOVX or NOVX upstream or downstream gene) was effectively destroyed by the transfected siRNA duplex. Two days after transfection, total RNA is prepared, reverse transcribed using target-specific primers, and PCR amplification is performed using primer pairs that convert at least one exon-exon junction to control pre-mRNA amplification. . RT / PCR of untargeted mRNA is also required as a control. A decrease in target protein that is an effective depletion of mRNA but cannot be detected may indicate that a large reservoir of stable NOVX protein is present in the cell. Multiple transfections with sufficiently long intervals may be required before the target protein can eventually be depleted and the phenotype can be revealed. If multiple transfection steps are required, the cells are split 2-3 days after transfection. The cells may be transfected immediately after dividing.

  The therapeutic methods of the invention contemplate administering a NOVX siRNA construct as a treatment to correct for increased or deviated NOVX expression or activity. As described above, NOVX ribopolynucleotides are obtained and processed into siRNA fragments or NOVX siRNAs are synthesized. As described above, NOVX siRNA is administered to cells or tissues using known nucleic acid transfection methods. NOVX siRNA specific for the NOVX gene reduces or knocks down NOVX transcripts, which reduces NOVX polypeptide production and reduces NOVX polypeptide activity in cells or tissues.

  The invention also encompasses a method of treating a disease or condition associated with the presence of NOVX protein in an individual, the method comprising an RNAi construct that targets the mRNA of the protein to be degraded (the mRNA encoding the protein). It is characterized by being administered to an individual. Special RNAi constructs include siRNA or double stranded gene transcripts that are processed into siRNA. The treatment results in no production of the target protein or a lower level of production than without treatment.

If NOVX gene function does not correlate with a known phenotype, a control sample of cells or tissue from a healthy individual provides a reference standard for determining NOVX expression levels. Expression levels are detected using the described assay, eg, RT-PCR, Northern blotting, ELISA, etc. A sample of cells or tissue is collected from a diseased mammal, preferably a human subject. These cells or tissues are treated by administering NOVX siRNA to the cells or tissues by the methods described for transfection of nucleic acids into cells or tissues, and NOVX polypeptides or polypeptides in the subject sample using the described assay. Observe the change in nucleotide expression and compare to that of the control sample. This NOVX gene knockdown method provides a rapid method for determining the NOVX minus (NOVX ⁻ ) phenotype in a sample to be treated. Thus, the NOVX ^- phenotype observed in the sample to be treated serves as a marker for follow-up of the disease state being treated.

  In a special embodiment, NOVX siRNA is used for therapy. Methods for producing and using NOVX siRNA are known to those skilled in the art. An example of the method is shown below.

Generation of RNA NOVX sense RNA (ssRNA) and antisense (asRNA) are obtained using known methods such as transcription in RNA expression vectors. In the first experiment, sense and antisense RNA are each about 500 bases in length. The resulting ssRNA and asRNA (0.5 μM) were heated in 10 mM Tris-HCl (pH 7.5) containing 20 mM NaCl for 1 minute at 95 ° C., then cooled and annealed at room temperature for 12-16 hours. Let RNA is precipitated and resuspended in lysis buffer (below). To monitor annealing, the RNA is electrophoresed in a 2% agarose gel in TBE buffer and stained with ethidium bromide. See, for example, Sambrook et al., Molecular Cloning. Cold Spring Harbor Laboratory Press, Plainview, NY (1989).

Lysate preparation Collect untreated rabbit reticulocyte lysate according to the manufacturer's instructions (Ambion). Incubate dsRNA in lysate at 30 ° C. for 10 minutes, then add mRNA. Thereafter, NOVX mRNA is added and the incubation is continued for another 60 minutes. The molar ratio of double stranded RNA to mRNA is about 200: 1. NOVX mRNA is radiolabeled (using known methods) and its stability is monitored by gel electrophoresis.

In experiments performed in parallel under the same conditions, double-stranded RNA is radiolabeled internally with ³² P-ATP. The reaction is stopped with 2X proteinase K buffer and deproteinized as previously described (Tuschl et al., Genes Dev., 13: 3191-3197). Products are analyzed by electrophoresis in 15% or 18% polyacrylamide sequencing gels using appropriate RNA standards. By monitoring the gel for radioactivity, the natural production of 10-25 nucleotide RNA from double stranded RNA can be examined.

  A band of double-stranded RNA of about 21 to 23 bp is eluted. The effectiveness of these 21-23 mer NOVX transcriptional repressions is assayed in vitro using the same rabbit reticulocytes as described above using 50 nanomolar double stranded 21-23 mers for each assay. These 21-23 mer sequences are then determined using standard nucleic acid sequencing methods.

Preparation of RNA Based on the sequence determined above, nucleotide RNA is chemically synthesized using Expedite RNA phosphoramidites and thymidine phosphoramidite (Proligo, Germany) 21. Synthetic oligonucleotides are deprotected and gel purified (Elbashir, Lendeckel, & Tuschl, Genes & Dev. 15, 188-200 (2001)), then Sep-Pak C18 cartridge (Waters, Milford, Mass., USA) (Tuschl, et al., Biochemistry, 32: 11658-11668 (1993)).
These RNA (20 μM) single strands are incubated in annealing buffer at 90 ° C. for 1 minute and then at 37 ° C. for 1 hour.

Cell culture Cell cultures known in the art for regular expression of NOVX are performed using standard conditions. 24 hours prior to transfection, cells are trypsinized at approximately 80% confluence, diluted 1: 5 in fresh medium without antibiotics (1-3 × 10 ⁵ cells / ml) and transferred to 24-well plates (500 ml / well). Transfection is performed using a commercial lipofection kit, and NOVX expression is monitored using standard methods with positive and negative controls. Positive controls are cells that naturally express NOVX, and negative controls are cells that do not express NOVX. Base-paired 21 and 22 nucleotide siRNAs with an overhang 3 'mediate effective sequence-specific mRNA degradation in lysates and cell cultures. Different concentrations of siRNA are used. The effective concentration for in vitro suppression in mammalian cell culture is a final concentration of 25 nM to 100 nM. This indicates that siRNA concentrations several orders of magnitude lower than those used in conventional antisense or ribozyme gene targeting experiments are effective.

  The above methods provide methods for both inference of NOVX siRNA sequences and use of such siRNA for ion vitro suppression. In vivo suppression may be performed using the same siRNA using well-known in vivo transfection or gene therapy transfection methods.

Antisense Nucleic Acid Another aspect of the present invention is capable of hybridizing to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2n-1 (where n is an integer from 1 to 82), or a fragment, homologue or derivative thereof. Or an isolated antisense nucleic acid molecule. An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid that encodes a protein (eg, complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In particular embodiments, antisense nucleic acid molecules comprising at least about 10, 25, 50, 100, 250, or 500 nucleotides or a sequence complementary to the entire NOVX coding strand, or only a portion thereof, are provided. A nucleic acid molecule encoding a fragment, homologue, derivative and analog of SEQ ID NO: 2n (n is an integer from 1 to 85), or SEQ ID NO: 2n-1 (n is an integer from 1 to 82) ) And an antisense nucleic acid complementary to the NOVX nucleic acid sequence provided.

  In one embodiment, the antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding a NOVX protein. The term “coding region” refers to a region of a nucleotide sequence that includes codons translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a “non-coding region” of the coding strand of a nucleotide sequence encoding a NOVX protein. The term “non-coding region” refers to 5 ′ and 3 ′ sequences that flank the coding region that are not translated into amino acids (ie, also referred to as 5 ′ and 3 ′ untranslated regions).

  Given the sequence of the coding strand encoding the NOVX protein disclosed herein, the antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or non-coding region of NOVX mRNA. . For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA. Antisense oligonucleotides can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) increases the biological stability of a naturally occurring nucleotide or molecule, or a double-stranded physical stability that forms between an antisense nucleic acid and a sense nucleic acid. Various modified nucleotides designed to increase can be chemically synthesized (eg, phosphorothioate derivatives and nucleotides substituted with acridine can be used).

  Examples of modified nucleotides that can be used to make antisense nucleic acids include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5 -Carboxymethylaminomethyl-2-thiouridine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosilk enosine, inosine, N6-isopentenyladenine, 1-methylguanine 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 5-methoxyuracil, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methyl Aminomethyl Lacil, 5-methoxyaminomethyl-2-thiouracil, 2-thiouracil, 4-thiouracil, beta-D-mannosyl eosin, 5′-methoxycarboxymethyluracil, 2-methylthio-N6-isopentenyladenine, uracil-5 Oxyacetic acid (v), wivetoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, uracil-5-oxy Acetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid is subcloned in the antisense orientation of the nucleic acid (ie, RNA transcribed from the inserted nucleic acid is the antisense orientation for the target nucleic acid of interest and is further described in the subsection below). Can be produced biologically using the expression vector.

  Protein expression by hybridizing or binding the antisense nucleic acid molecules of the invention to cellular mRNA and / or genomic DNA that encodes certain NOVX proteins thereby (eg, inhibiting transcription and / or translation) Is typically administered to a subject so as to inhibit or made in tissue. Hybridization can also be due to the complementarity of conventional nucleotides to form stable duplexes. Alternatively, for example, in the case of an antisense nucleic acid that binds to a DNA double strand, it can be through a specific interaction in the double-stranded major groove. An example of a route of administration of antisense nucleic acid molecules of the invention is direct injection into a tissue site. Alternatively, antisense nucleic acid molecules can be modified to selected cellular targets where they can be administered systemically. For example, for systemic administration, antisense molecules are specifically bound to receptors or antigens expressed on selected cell surfaces (e.g., linking antisense nucleic acid molecules to peptides or cell surfaces). May be modified (by antibodies that bind to receptors or antigens). Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. In order to deliver sufficient nucleic acid molecules, a vector structure that places the antisense nucleic acid molecule under the control of a strong pol II or pol III promoter is preferred.

  In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. α-anomeric nucleic acid molecules form specific double-stranded hybrids with complementary RNAs, which are opposite to normal β units and run parallel to each other. See, for example, Gaultier, et al., 1987. Nucl. Acids Res. 15: 6625-6641. Antisense nucleic acid molecules can also be 2′-o-methyl ribonucleotides (see, eg, Inoue, et al. 1987. Nucl. Acids Res. 15: 6131-6148) or chimeric RNA-DNA analogs (eg, Inoue, et al., 1987. FEBS Lett. 215: 327-330).

Ribozymes and PNA moieties Nucleic acid modifications include, by way of non-limiting example, modified bases and nucleic acids modified or derived from sugar phosphate backbones. These modifications are made at least in part to enhance the chemical stability of the modified nucleic acid so that it can be used, for example, as an antisense that binds to the nucleic acid in therapeutic applications to a subject.

  In one embodiment, the antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytically active RNA molecules with ribonuclease activity that are capable of cleaving single-stranded nucleic acids such as mRNA having a complementary region. Thus, ribozymes (eg, hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) are used to catalytically cleave NOVX mRNA transcripts, thereby resulting in NOVX mRNA. Translation may be inhibited. Ribosomes having specificity for a nucleic acid encoding NOVX can be designed based on the nucleotide sequence of certain NOVX cDNAs disclosed herein (ie, SEQ ID NO: 2n-1 (n is an integer from 1 to 82)). . For example, a derivative of Tetrahymena L-19IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to a nucleotide sequence that can be cleaved in mRNA encoding NOVX. See, for example, Cech, et al. U.S. Pat. No. 4,987,071 and Cech, et al. U.S. Pat. No. 5,116,742. NOVX mRNA can also be used to select catalytic RNAs with specific ribonuclease activity from a pool of RNA molecules. See, for example, Bartel et al., (1993) Science 261: 1411-1418.

  On the other hand, NOVX gene expression is inhibited by targeting a nucleotide sequence complementary to the regulatory region of NOVX nucleic acid (eg, NOVX promoter and / or enhancer), and triple helical that blocks transcription of NOVX gene in the target cell. A structure can be formed. See, for example, Helene, 1991. Anticancer Drug Des. 6: 569-84; Helene, et al. 1992. Ann. NY Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15. .

  In various embodiments, NOVX nucleic acids can be modified at the base moiety, sugar moiety, or phosphate backbone moiety to improve, for example, molecular stability, hybridization, or solubility. For example, the nucleic acid deoxyribose phosphate backbone can be modified to produce peptide nucleic acids. See, for example, Hyrup, et al., 1996. Bioorg Med Chem 4: 5-23. As used herein, the term “peptide nucleic acid” or “PNA” refers to a nucleic acid mimetic (eg, a DNA mimetic) that replaces the deoxyribose phosphate backbone with a pseudopeptide backbone and retains only four nucleobases. . The neutral backbone of PNA is shown to allow specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers is described in Hyrup, et al., 1996. supra; Perry-O'Keefe, et al., 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675 It can be performed using a standard solid phase peptide synthesis protocol.

NOVX PNA may be used for therapeutic and diagnostic applications. For example, PNA can be used as an antisense or antigenic agent for sequence specific modulation of gene expression, eg, by inducing transcription or translational arrest or inhibiting replication. NOVX PNAs also serve as artificial restriction enzymes that can be used in combination with, for example, single base pair mutations in genes (eg, PNAs that direct PCR fixation; other enzymes, such as S ₁ nuclease ( Hyrup, et al., 1996.supra); or as DNA sequence and hybridization probes or primers (Hyrup, et al., 1996, supra; see Perry-O'Keefe, et al., 1996. supra) )) Can also be used.

  In another embodiment, the NOVX PNA is modified, eg, by attaching a lipophilic or other auxiliary group to the PNA, by generation of a PNA-DNA chimera, or by liposomes or the art Can be used to enhance their stability or cellular uptake by use of other drug delivery techniques known in the art. For example, PNA-DNA chimeras of NOVX can be generated that can combine the beneficial properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (eg, RNase H and DNA polymerase) to interact with the DNA moiety, while the PNA moiety provides binding high affinity and binding specificity. PNA-DNA chimeras can be joined using linkers of appropriate lengths selected for base linkage, number of linkages between nucleotide bases and orientation (see Hyrup, et al., 1996. supra). Synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al., 1996. supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA strand can be synthesized on a solid support using standard phosphoramidite coupling chemistry. Modified nucleoside analogs, such as 5 ′-(4-methoxytrityl) amino-5′-deoxythymidine phosphoramidites, can then be used between the PNA and the 5 ′ end of the DNA. See, for example, Mag, et al., 1989. Nucl Acid Res 17: 5973-5988. The PNA monomer is then coupled stepwise to produce a chimeric molecule having a 5 ′ PNA segment and a 3 ′ DNA segment. See, for example, Finn, et al., 1996. supra. Alternatively, the chimeric molecule can be synthesized with a 5 ′ DNA segment and a 3 ′ PNA segment. See, for example, Petersen, et al., 1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.

  In other embodiments, the oligonucleotides are attached to groups such as peptides (e.g., to target host cell receptors in vivo), or cell membranes (e.g., Letsinger, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; see PCT publication number WO 88/09810) or blood brain barrier (eg, PCT publication number). Agents that facilitate transport across (see WO89 / 10134). In addition, oligonucleotides can be combined with hybridization-inducing cleavage agents (see, eg, Krol, et al., 1988. BioTechniques 6: 958-976) or intercalating agents (eg, Zon, 1988. Pharm. Res. 5: 539 -549). For this purpose, the oligonucleotide can be complexed with, for example, peptides, hybridization-inducing cross-linking agents, transport agents, hybridization-inducing cleavage agents, and the like.

NOVX polypeptides Polypeptides according to the present invention include polypeptides that contain the amino acid sequence of the NOVX polypeptide whose sequence is provided in SEQ ID NO: 2n (n is an integer from 1 to 85). The present invention also provides a protein that retains its NOVX activity and physiological function while any residue thereof can be converted from the corresponding residue shown in SEQ ID NO: 2n (n is an integer from 1 to 85). Or a mutant or variant protein that still encodes a functional fragment thereof.

  In general, one NOVX variant that retains a NOVX-like function includes any variant that substitutes a residue at a particular site in the sequence with another amino acid, with another residue between the two residues of the parent protein. Or further including the possibility of inserting multiple residues as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion or deletion is encompassed by the present invention. In preferred circumstances, the substitution is a conservative substitution as defined above.

  One aspect of the present invention relates to isolated NOVX proteins and biologically active portions thereof, or derivatives, fragments, analogs or homologues thereof. Polypeptide fragments suitable for use as an immunogen for production of anti-NOVX antibodies can also be provided. In one embodiment, native NOVX protein may be isolated from a cell or tissue source by a suitable purification scheme using standard protein purification techniques. In another embodiment, NOVX protein is produced by recombinant DNA technology. As an alternative to recombinant expression, certain NOVX proteins or polypeptides can be chemically synthesized using standard peptide synthesis techniques.

  An “isolated” or “purified” polypeptide or protein or biologically active portion thereof is substantially free of cellular or tissue source cellular material or other contaminating protein from which the NOVX protein is derived, or chemically When synthesized, it is substantially free of chemical precursors or other chemicals. The term “substantially free of cellular material” includes a sample of NOVX protein that has separated the protein from the cellular components of the original cell from which the protein was isolated or recombinantly produced. In one embodiment, the term “substantially free of cell source” means less than about 30% (dry weight ratio) of non-NOVX protein (also referred to herein as “contaminating protein”), more preferably about Samples of NOVX protein having less than 20% non-NOVX protein, even more preferably less than about 10% non-NOVX protein, and most preferably less than about 5% non-NOVX protein are included. In recombinantly producing NOVX proteins or biologically active portions thereof, it is also preferably substantially free of medium, ie, the medium is less than about 20% of the volume of the NOVX protein specimen, more preferably It represents less than about 10%, and most preferably less than 5%.

  The term “substantially free of chemical precursors or other chemicals” includes a sample of NOVX protein where proteins are separated from chemical precursors or other chemicals involved in protein synthesis. To do. In one embodiment, the term “substantially free of chemical precursors or other chemicals” refers to less than about 30% (dry weight ratio) chemical precursors or non-NOVX chemicals, more preferably Less than about 20% chemical precursor or non-NOVX chemical, even more preferably less than about 10% chemical precursor or non-NOVX chemical, and most preferably less than about 5% chemical precursor or non-NOVX Includes specimens of NOVX protein with chemicals.

The biologically active portion of the NOVX protein includes the amino acid sequence of the NOVX protein that contains fewer amino acids than the full-length NOVX protein and exhibits at least one activity of the NOVX protein (eg, SEQ ID NO: 2n, where n is 1 to 85 The amino acid sequence derived from the amino acid sequence sufficiently homologous to (the amino acid sequence shown in the amino acid sequence of the NOVX protein). Typically, the biologically active portion includes a domain or motif having at least one activity of the NOVX protein. A biologically active portion of a NOVX protein can be a polypeptide, eg, 10, 25, 50, 100 or more amino acid residues long.
In addition, other biologically active portions lacking other regions of the protein can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native NOVX protein.

  In one embodiment, the NOVX protein has an amino acid sequence set forth in SEQ ID NO: 2n (where n is an integer from 1 to 85). In other embodiments, the NOVX protein is substantially homologous to SEQ ID NO: 2n (where n is an integer from 1 to 85) and SEQ ID NO: 2n (where n is an integer from 1 to 85). Retains the functional activity of certain proteins, but still differs in amino acid sequence due to natural allelic variants or mutations as detailed below. Accordingly, in another embodiment, the NOVX protein comprises an amino acid sequence that is at least about 45% homologous to the amino acid sequence of SEQ ID NO: 2n (where n is an integer from 1 to 85), Wherein n is an integer of 1 to 85).

Measuring homology between two or more sequences To determine the percentage of homology between two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison purposes (eg, gaps 2 Can be placed within the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleic acid sites are then compared. When a site in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding site in the second sequence, both molecules are homologous at that site (ie, as used herein, amino acids or Nucleic acid “homology” is equivalent to amino acid or nucleic acid “identity”).

  Nucleic acid sequence homology can be measured as the degree of identity between two sequences. Homology can be measured using computer programs known in the art, such as GAP software provided in the GCG program package. See Needleman and Wunsch, 1970. J Mol Biol 48: 443-453. Using the GCG GAP software with the following settings: GAP generation penalty 5.0 or GAP extension penalty 0.3, the coding region of the above similar nucleic acid sequence is SEQ ID NO: 2n-1 (where n is Preferably at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with the CDS (encoding) portion of the DNA sequence shown in FIG. Indicates the degree.

  The term “sequence identity” refers to the extent to which two polynucleotide or polypeptide sequences are identical from residue to residue over a particular region of comparison. The term “percentage of sequence identity” is used to compare two sequences that are optimally aligned over the region of comparison and to determine the number of matching sites (for example, A, in the case of nucleic acids, A, T, C, G, U, or I) is measured in the number of sites present in both sequences, the number of matching sites is divided by the total number of sites in the comparison region (ie, window size), and the quotient is 100. Can be calculated by multiplying to obtain a percentage of sequence identity. As used herein, the term “substantial identity” means a property of a polynucleotide sequence. Here, the polynucleotide comprises at least 80% sequence identity, preferably at least 85% sequence identity and often 90-95% sequence identity compared to the standard sequence over the comparison region, more usually Includes sequences with at least 99% sequence identity.

Chimeric or fusion proteins The present invention also provides NOVX chimeric or fusion proteins. As used herein, certain NOVX “chimeric proteins” or “fusion proteins” include certain NOVX polypeptides operably linked to non-NOVX polypeptides. “NOVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to the NOVX protein of SEQ ID NO: 2n, where n is an integer from 1 to 85, while “non-NOVX polypeptide” A polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, for example, a protein that is the same or different from the NOVX protein. Within a NOVX fusion protein, a NOVX polypeptide may correspond to all or part of a NOVX protein. In one embodiment, a NOVX fusion protein includes at least one biologically active portion of a NOVX protein. In another embodiment, a NOVX fusion protein comprises at least two biologically active portions of a NOVX protein. In yet another embodiment, a NOVX fusion protein comprises at least three biologically active portions of a NOVX protein. Within the fusion protein, the term “operably linked” shall indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame to each other. The non-NOVX polypeptide can be fused to the N-terminus or C-terminus of the NOVX polypeptide.

  In one embodiment, the fusion protein is a GST-NOVX fusion protein. Here, the NOVX sequence is fused to the C-terminus of GST (glutathione S transferase). Such fusion proteins can facilitate the purification of recombinant NOVX polypeptides.

  In another embodiment, the fusion protein is a NOVX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (eg, mammalian host cells), NOVX expression and / or secretion may be enhanced through the use of heterologous signal sequences.

  In yet another embodiment, the fusion protein is a NOVX-immunoglobulin fusion protein. Here, the NOVX sequence is fused to a sequence derived from a member of the immunoglobulin protein family. A NOVX-immunoglobulin fusion protein of the invention is incorporated into a pharmaceutical composition and administered to a subject to inhibit the interaction of a NOVX ligand with a NOVX protein on a cell, thereby inhibiting NOVX-mediated in vivo. Information transmission can be suppressed. NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of certain NOVX-like ligands. Inhibition of the NOVX ligand / NOVX interaction may be therapeutically useful for the treatment of proliferation and differentiation disorders and for modulating (eg, enhancing or inhibiting) cell survival. In addition, the NOVX-immunoglobulin fusion protein of the present invention is used to produce anti-NOVX antibodies in a subject, purify NOVX ligand, and inhibit the interaction of NOVX with a NOVX ligand in a screening assay Can be identified.

  The NOVX chimeric or fusion proteins of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences may be used, for example, using blunt ends or staggered ends for ligation, cleavage with restriction enzymes to provide appropriate ends, filling appropriately sticky ends, desirably Ligated together in-frame according to conventional methods such as alkaline phosphatase treatment to prevent unbound and enzymatic coupling. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments is performed using anchor primers that produce complementary overhangs between two consecutive gene fragments, which are subsequently annealed and re-amplified to produce a chimeric gene sequence. (See, for example, Ausubel, et al. (Eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors (eg, GST polypeptides) that already encode a fusion moiety are commercially available. A nucleic acid encoding NOVX can be cloned into such an expression vector such that the fusion moiety binds in-frame to the NOVX protein.

NOVX Agonists and Antagonists The present invention also includes variants of the NOVX protein that function as either NOVX agonists (ie, mimetics) or NOVX antagonists. NOVX protein variants may be generated by mutation (eg, point mutation or truncation of the NOVX protein). NOVX protein agonists retain substantially the same biological activity, or a subset thereof, as the naturally occurring form of NOVX protein. An agonist of NOVX protein inhibits the activity of one or more naturally occurring forms of NOVX protein by, for example, antagonistic binding to downstream or upstream members of the cellular signaling cascade, including NOVX protein Can do. Thus, specific biological effects can be exerted by treatment with mutants having limited functions. In one embodiment, treatment of a subject with a variant having a subset of the biological activity of a naturally occurring form of the protein has fewer side effects in the subject than treatment with a naturally occurring form of NOVX protein.

  Variants of NOVX proteins that function as either NOVX agonists (ie, mimetics) or NOVX antagonists screen recombinant libraries of NOVX protein mutants (eg, truncation mutants) for NOVX protein agonist or antagonist activity Can be identified. In one embodiment, a NOVX variant-rich library is generated by recombinant (combinatorial) mutagenesis at the nucleic acid level and is encoded by a gene library that is rich in change. A library rich in variation of NOVX mutants can, for example, enzymatically link a mixture of synthetic oligonucleotides to a gene sequence so that a degenerate set of potential NOVX sequences can be expressed as individual polypeptides. Or alternatively, as a set of larger fusion proteins (eg, for phage display) containing a set of NOVX sequences therein. There are various methods that can be generated from a degenerate oligonucleotide sequence using a library of potential NOVX variants. Chemical synthesis of degenerate gene sequences can be performed with an automated DNA synthesizer, and then the synthetic gene can be ligated into an appropriate expression vector. The use of a degenerate set of genes allows the provision of a single mixture of all of the sequences that encode the desired set of potential NOVX sequences. Methods for synthesizing degenerate oligonucleotides are well known in the art. For example, Narang, 1983. Tetrahedron 39: 3; Itakura, et al., 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al., 1984. Science 198: 1056; Ike, et al., 1983. See Nucl. Acids Res. 11: 477.

Polypeptide libraries In addition, a fragment library of NOVX protein-encoding sequences can be used to generate populations rich in changes in NOVX fragments for screening and subsequent selection of NOVX protein variants. In one embodiment, a library of coding sequence fragments is subjected to nuclease treatment of a double stranded PCR fragment of a NOVX coding sequence under conditions where nicking occurs only once per molecule to denature the double stranded DNA. Forming a double-stranded DNA that can contain sense / antisense pairs from different nicking products, removing the single-stranded portion from the double helix regenerated by treatment with S ₁ nuclease, and the resulting fragment live Can be generated by linking the library to an expression vector. By this method, an expression library encoding various sized N-terminal or internal fragments of the NOVX protein can be derived.

  Various techniques are known in the art for screening gene products of recombinant (combinatorial) libraries generated by point mutations or truncations, and screening cDNA libraries for gene products having selected properties. . Such techniques are applicable to rapid screening of gene libraries generated by recombinant (combinatorial) mutagenesis of NOVX proteins. The most widely used technique applicable to high-throughput analysis to screen large gene libraries is to clone the gene library into a replicable expression vector and transform the appropriate cells with the resulting vector library, And the detection of the desired activity typically involves expressing the recombinant (combinatorial) gene under conditions that facilitate the isolation of the vector encoding the gene that detects the product. A new technique that increases the frequency of functional mutations in the library, repetitive ensemble mutagenesis (REM), can be used in combination with screening assays to identify NOVX mutants. See, for example, Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al., 1993. Protein Engineering 6: 327-331.

Anti-NOVX Antibodies Antibodies to NOVX proteins or fragments of NOVX proteins are included in the present invention. As used herein, the term “antibody” refers to an immunologically active portion of an immunoglobulin molecule and an immunoglobulin (Ig) molecule, ie, an antigen that specifically binds (and reacts immunologically) with an antigen. Refers to a molecule containing a binding site. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F _ab , F _{ab ′} and F _{(ab ′) 2} fragments and a F _ab expression library. In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from each other due to the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG ₁ , IgG ₂ and others. Furthermore, in humans, the L chain can be a k chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.

  Generate isolated antibodies that immunospecifically bind to antigens using standard techniques of polyclonal and monoclonal antibody preparation using the isolated protein of the present invention, or a portion or fragment thereof, intended for use as an antigen, as an immunogen Can do. The full-length protein may be used, or alternatively, the present invention provides an antigenic peptide fragment of an antigen for use as an immunogen. The antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full-length protein, such as the amino acid sequence set forth in SEQ ID NO: 2n (where n is an integer from 1 to 82), and for the peptide The raised antibody includes the epitope so as to form a specific immune complex with the full-length protein or any fragment containing the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by antigenic peptides are protein regions located on the surface; these are usually hydrophilic regions.

  In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a NOVX region localized on the surface of the protein, eg, a hydrophilic region. Hydrophobic analysis of the human NOVX protein sequence indicates which regions of the NOVX polypeptide are particularly hydrophilic and are therefore likely to encode useful surface residues for targeting antibody production. As a means to target antibody production, hydropathy plots showing hydrophilic and hydrophobic regions are well known in the art including, for example, the Kyte Doolittle or Hopp Woods methods, both with or without Fourier transforms. It can be generated by any method. For example, Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142 (both are hereby incorporated by reference in their entirety) As a part of the book). Antibodies that are specific for one or more domains within an antigenic protein or derivative, fragment, analog or homologue thereof are also provided herein.

The term “epitope” includes all protein determinants (sites) that can specifically bind to an immunoglobulin or T cell receptor. Epitope-like determinants (sites) usually contain surface groups of chemically active molecules (eg amino acids or sugar side chains) and usually show specific charge characteristics in addition to specific three-dimensional structural characteristics . Certain NOVX polypeptides or fragments thereof have at least one antigenic epitope. Assay (e.g., to one skilled in the art known radioligand binding assays or similar assays) as measured by equilibrium binding constant _{K D} of, <1 [mu] M, preferably <100 nM, more preferably <10 nM, most preferably < 100 pM to about 1 pM, the anti-NOVX antibody of the present invention is said to specifically bind to the antigen NOVX.

  The proteins of the invention, or derivatives, fragments, analogs, homologues, or orthologs thereof can be used as an immunogen in the production of antibodies that immunospecifically bind to these protein components.

  Various methods known in the art can be used for the production of polyclonal or monoclonal antibodies against a protein of the invention, or derivatives, fragments, analogs, homologues, or orthologs thereof (eg, Antibodies: A Laboratory Manual). Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference). Some of these antibodies are discussed below.

Polyclonal antibodies For the production of polyclonal antibodies, various suitable host animals (eg rabbits, goats, mice, or other mammals) are injected with one or more natural proteins, synthetic variants thereof, or the aforementioned derivatives. And immunize. Suitable immunogenic formulations may include, for example, naturally occurring immunogenic proteins, chemically synthesized polypeptides that are representative of immunogenic proteins, or recombinantly expressed immunogenic proteins. Furthermore, the protein may be complexed with a second protein that is known to be immunogenic in the mammal to be immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The formulation may further contain an adjuvant. Various adjuvants used to increase the immune response include Freund's (complete and incomplete) adjuvants, mineral gels (e.g. aluminum hydroxide), surfactants (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions) , Dinitrophenol, and the like), adjuvants that can be used in humans, such as, but not limited to, Bacillus Calmette-Guerin and Corynebacterium parvum. Yet another example of an adjuvant that may be used may include MPL-TDM adjuvant (monophosphoryl lipid A, synthetic dicorynomycolic acid trehalose).

  Polyclonal antibodies against immunogenic proteins are isolated from mammals (eg, from blood) and are known in the art, such as affinity chromatography using protein A or protein G that provides a predominantly IgG fraction in immune serum Can be used for further purification. Subsequently, or alternatively, a specific antigen that is the target of the desired immunoglobulin, or an epitope thereof, can be immobilized on a column and the immunospecific antibody can be purified by immunoaffinity chromatography. The purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

Monoclonal antibody As used herein, the term “monoclonal antibody” (MAb) or “monoclonal antibody composition” refers to only one molecular species of an antibody molecule comprising a characteristic light chain gene product and a characteristic heavy chain gene product. Refers to the population of antibody molecules that it contains. In particular, the complementarity determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. Thus, MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of an antigen characterized by a characteristic binding activity thereto.

  Monoclonal antibodies can be prepared using hybridoma methods, as described by Kohler and Milstein, Nature, 256: 495 (1975). In the hybridoma method, lymphocytes that produce or are capable of producing antibodies that specifically bind to an immunizing agent are typically immunized by immunizing a mouse, hamster, or other suitable host animal with the immunizing agent. To trigger. Alternatively, lymphocytes can be immunized in vitro.

The immunizing agent typically includes protein antigens, fragments thereof or fusion proteins thereof. In general, either peripheral blood lymphocytes are used if cells derived from humans are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. . The lymphocytes are then fused with an immortal cell line using a suitable fusing agent such as polyethylene glycol to generate hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice , Academic Press, (1986) pp. 59 -103). Immortal cell lines are usually transformed mammalian cells, particularly myeloma cells from rodents, cattle or humans. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells may be cultured in a suitable medium that preferably contains one or more substances that inhibit the growth or survival of unfused, immortalized cells. For example, if the parent cell lacks the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas typically contains hypoxanthine, a substance that prevents the growth of HGPRT-deficient cells, Contains aminopterin and thymidine (“HAT medium”).

  Preferred immortal cell lines are those that fuse efficiently, support high level expression of antibodies by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Further preferred immortal cell lines are murine myeloma cell lines available from, for example, the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines are also described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

  The medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is measured by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA). Such techniques or assays are known in the art. The binding affinity of a monoclonal antibody can be measured, for example, by Scatchard analysis of Munson and Pollard, Anal. Biochem., 107: 220 (1980). Identifying antibodies with a high degree of specificity and high binding affinity for the target antigen is a particularly important objective in the therapeutic application of monoclonal antibodies.

  After identifying the desired hybridoma cells, clones can be subcloned by limiting dilution and grown by standard methods (Goding, 1986). Suitable culture media for this purpose can include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

  Monoclonal antibodies secreted by subclones are isolated or purified from the medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein-A sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Can do.

  Monoclonal antibodies can also be made by recombinant DNA methods as described in US Pat. No. 4,816,567. Using an oligonucleotide probe capable of specifically binding DNA encoding the monoclonal antibody of the present invention to genes encoding the heavy and light chains of murine antibodies using conventional procedures (eg. Can be easily isolated and sequenced. The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA is placed in an expression vector which is then host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that would otherwise not produce immunoglobulin proteins. It can be transfected into to obtain monoclonal antibody synthesis in recombinant host cells. DNA may also be replaced, for example, by replacing coding sequences for human heavy and light chain constant domains in place of homologous murine sequences (US Pat. No. 4,816,567; Morrison, Nature 368, 812- 13 (1994)), or all or part of the coding sequence for a non-immunoglobulin polypeptide may be modified by covalent linkage to the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be substituted for the constant domains of the antibodies of the invention, or can be substituted for the variable domains of one antigen binding site of the antibodies of the invention to produce chimeric 2 A titer antibody can be created.

Humanized antibodies Antibodies to protein antigens of the present invention further include humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without causing human immune responses to the administered immunoglobulins. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, etc.) that consist primarily of human immunoglobulin sequences and contain minimal sequence derived from non-human immunoglobulin. Fab ′, F (ab ′) ₂ or other antigen-binding subsequence of the antibody). Humanization has been described by Winter and co-workers (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239 : 1534-1536 (1988)) by replacing the rodent CDR or CDR sequence with the corresponding sequence of a human antibody. (See also US Pat. No. 5,225,539) In some cases, residues of the Fv framework of human immunoglobulins may be replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are not found in the recipient antibody or in the CDR or framework sequences. In general, a humanized antibody will contain at least one, and typically substantially all of the two variable domains. Here, all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulin, and all or substantially all of the framework regions are of human immunoglobulin consensus sequence. A humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta , Curr. Op. Struct. Biol., 2: 593-596 (1992)).

Human antibodies Completely human antibodies relate essentially to antibody molecules in which the entire L and H chain sequences, including the CDRs, originate from a human gene. Such antibodies are referred to herein as “human antibodies” or “fully human antibodies”. Human monoclonal antibodies can be prepared by trioma technology; human B cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4: 72) and EBV hybridoma technology to produce human monoclonal antibodies (Cole, et al. , 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Humanized monoclonal antibodies can be utilized in the practice of the invention and human hybridomas can be used (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or human B cells can be By transformation with Epstein Barr virus (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

  In addition, human antibodies can also be obtained from phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991)). Can be produced using further techniques including: Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, eg, mice. Here, the endogenous immunoglobulin genes are partially or completely inactivated. The challenge observes human antibody production very similar to that seen in humans in all aspects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in US Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016 And Marks et al. (Bio / Technology 10, 779-783 (1992)), Lonberg et al. (Nature 368 856-859 (1994)), Morrison (Nature 368, 812-13 (1994)), Fishwild et al, (Nature Biotechnology 14, 845-51 (1996)), Neuberger (Nature Biotechnology 14, 826 (1996)), Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)). .

  Human antibodies can additionally be produced using transgenic non-human animals that have been modified to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge with antigen (PCT Publication No. WO94 / 02602). The endogenous genes encoding immunoglobulin heavy and light chains in the non-human host are disabled, and active loci encoding human immunoglobulin heavy and light chains are inserted into the host genome. Human genes can be incorporated, for example, using yeast artificial chromosomes containing the necessary human DNA segments. Animals that provide all the desired modifications are then obtained as offspring by crossbreeding intermediate transgenic animals that contain less than the full complement of modifications. A preferred embodiment of such a non-human animal is a mouse, designated Xenomouse® as disclosed in PCT Publication Nos. WO96 / 33735 and WO96 / 34096. This animal produces B cells that secrete fully human immunoglobulins. The antibody can be obtained directly from the animal after immunization with the immunogen of interest, eg, as a polyclonal antibody specimen, or alternatively, an immortalized B derived from an animal, such as a hybridoma producing a monoclonal antibody. It can also be obtained from cells. In addition, genes encoding immunoglobulins with human variable regions can be recovered and expressed to obtain antibodies directly, or further modified, eg, for antibodies such as single chain Fv molecules. Analogues can be obtained.

  An example of a method of producing a non-human host, exemplified as a mouse, that lacks endogenous immunoglobulin heavy chain expression is disclosed in US Pat. No. 5,939,598. It is known from at least one endogenous heavy chain locus in embryonic stem cells to prevent loci rearrangement and to prevent transcript production of rearranged immunoglobulin heavy chain loci. A segment is deleted, this deletion being brought about by a targeting vector containing a gene encoding a selectable marker; and a transgenic mouse from which embryonic stem cells contain somatic cells or embryonic cells containing the gene encoding the selectable marker. It is obtained by a method that includes producing.

  Methods for producing antibodies of interest such as human antibodies are disclosed in US Pat. No. 5,916,771. It introduces an expression vector containing a nucleotide sequence encoding an H chain into one cultured mammalian host cell, and introduces an expression vector containing a nucleotide sequence encoding an L chain into yet another mammalian host cell. And fusing two cells to produce a hybrid cell. Hybrid cells express antibodies that contain heavy and light chains.

  In a further refinement of this procedure, a method for identifying clinically relevant epitopes on an immunogen and a correlated method for selecting antibodies that immunospecifically bind to the relevant epitope with high affinity are disclosed in PCT publication. No. WO99 / 53049.

_Fab fragments and single chain antibodies In accordance with the present invention, techniques can be adapted to the production of single chain antibodies specific for the antigenic proteins of the present invention (see, eg, US Pat. No. 4,946,778). In addition, the method is adapted to the construction of _Fab expression libraries (see, eg, Huse, et al., 1989 Science 246: 1275-1281), and to proteins or derivatives, fragments, analogs or homologues thereof. It may allow rapid and effective identification of monoclonal _Fab fragments with the desired specificity for. Antibody fragments containing idiotypes against protein antigens can be produced by techniques known in the art, which are (i) F _{(ab ′) 2} fragments produced by pepsin digestion of antibody molecules; (ii) F _{( ab ′) Fab} fragments obtained by reducing disulfide bridges of ₂ fragments; (iii) _Fab fragments generated by treating antibody molecules with papain and a reducing agent; and (iv) _Fv fragments, It is not limited to.

Bispecific Antibodies Bispecific antibodies are monoclonal antibodies, preferably human or humanized antibodies, that have binding specificities for at least two different antigens. As used herein, one of the binding specificities is for the antigenic protein of the present invention. The second binding target is any other antigen, preferably a cell surface protein or receptor or receptor subunit.

  Methods for making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs in which the two heavy chains have different specificities (Milstein and Cuello, Nature, 305 : 537-539 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually performed by affinity chromatography. Similar methods are disclosed in WO 93/088829 published on May 13, 1993 and Traunecker et al., EMBO J., 10: 3655-3659 (1991).

  Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion is preferably with the constant domain of an immunoglobulin heavy chain comprising at least part of the hinge, CH2 and CH3 regions. It is preferred to have a first heavy chain constant region (CH1) containing at least the site necessary for light chain binding present in one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and transfected into a suitable host organism. For further details of bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

  According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be modified to maximize the percentage of heterodimer recovered from the recombinant cell medium. A preferred interface includes at least a portion of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan). The interface of the second antibody molecule by replacing a compensatory “void” of the same or similar size as the large side chain (s) by replacing the large amino acid side chain with a smaller one (eg alanine or threonine). To generate. This provides a mechanism to increase the yield of heterodimers over other undesirable end products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg F (ab ′) ₂ bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibody fragments can be prepared using chemical linkage. Brennan et al., Science 229: 81 (1985) describes a procedure in which intact antibodies are cleaved with proteolytic enzymes to generate F (ab ′) ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The resulting Fab ′ fragment is then converted to a thionitrobenzoic acid (TNB) derivative. One of the Fab′-TNBs is then reconverted to a Fab′-thiol by reduction with mercaptoethylamine and mixed with an equal amount of other Fab′-TNB derivatives to produce bispecific antibodies. The resulting bispecific antibody can be used as an enzyme selective immobilization agent.

In addition, Fab ′ fragments can be recovered directly from E. coli and chemically coupled to generate bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describes the production of F (ab ′) ₂ molecules of fully humanized bispecific antibodies. Each Fab ′ fragment was secreted separately from E. coli and subjected to directed chemical coupling in vitro to form bispecific antibodies. Thus, the bispecific antibody formed can bind to cells that overexpress the ErbB2 receptor and normal human T cells, and can induce lytic activity of human cytotoxic lymphocytes against human breast tumor targets. did it.

Various techniques for making and isolating bispecific antibodies directly from recombinant cell media are also described. For example, bispecific antibodies are produced using leucine zippers. Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992). The leucine zipper peptide from Fos protein and Jun protein was linked to the Fab ′ portion of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provides yet another mechanism for bispecific antibody fragment production. The fragment contains a heavy chain variable domain (V _H ) linked to a light chain variable domain (V _L ) by a linker, but the short linker does not allow pairing between two domains on the same chain . Thus, the V _H and V _L domains of one fragment are forced to pair with the complementary _VL and V _H domains of another fragment, thereby forming two antigen binding sites. Another strategy for the generation of bispecific antibody fragments by the use of single chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994).

An antibody having a valence of 3 or more is also conceivable. For example, trispecific antibodies can be prepared. For example, Tutt et al., J. Immunol. 147: 60 (1991).
A typical bispecific antibody can bind to two different epitopes, at least one of which is derived from a protein antigen of the invention. Alternatively, the anti-antigen arm of an immunoglobulin molecule is linked to a T cell receptor molecule (e.g., CD2, CD3, CD28 or B7) or FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) on leukocytes. In combination with an arm that binds to an inducible molecule such as the Fc receptor for IgG (Fcγ), a cellular defense mechanism against cells expressing a particular antigen may be focused. Bispecific antibodies can also be used to direct cytotoxic agents to cells expressing a particular antigen. These antibodies possess an antigen binding arm and an arm that binds a cytotoxic agent or a radionuclide chelator such as EOTUBE, DPTA, DOTA or TETA. Another bispecific antibody of interest binds to the protein antigen described herein and further binds tissue factor (TF).

Heteroconjugate antibodies Heteroconjugate antibodies are also within the scope of the present invention. A heteroconjugate antibody consists of two antibodies linked by a covalent bond. Such antibodies are for example for targeting cells of the immune system to unwanted cells (US Pat. No. 4,676,980) and for the treatment of HIV infection (WO 91/00360; WO 92/003733; EP03089). ). It is believed that these antibodies can be prepared in vitro using known methods of protein synthesis chemistry, including those involving crosslinkers. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose may include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in US Pat. No. 4,676,980.

Effector Functional Engineering For example, to enhance the effectiveness of an antibody in the treatment of cancer, it is desirable to modify the antibodies of the present invention with respect to effector function. For example, cysteine residue (s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization ability and / or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linking agents as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody having a dual Fc region, thereby having enhanced complement lysis and ADCC ability, can be engineered. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

Immune complexes The present invention also includes chemotherapeutic agents, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof) or radioisotopes (i.e., radioconjugates). It relates to an immunoconjugate comprising an antibody conjugated with such a cytotoxic substance.

Chemotherapeutic agents useful for the generation of such immune complexes are described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, avirin A chain, modesin A chain, Alphasarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), momoridica charantia inhibitor, crucine, crotin, sapaonaria officinalis inhibitor, gelonin, mitogerin, Listristine, phenomycin, enomycin and trichothecene. A variety of radionuclides are available for the production of radioconjugated antibodies. As examples, ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re may be mentioned.

A complex of an antibody and a cytotoxic agent is converted into a bifunctional derivative of N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), an imide ester (such as dimethyl adipimidate HCL Active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazide compounds (such as bis- (p-azidobenzoyl) -hexanediamine), bisdiazonium derivatives (bis -(P-diazonium benzoyl) -ethylenediamine), diisocyanates (such as triene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) These are made using various bifunctional protein coupling agents. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238 : 1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a typical chelating agent for conjugating radionucleotides to antibodies. See WO94 / 11026.

  In another embodiment, the antibody can be conjugated to a “receptor” (such as streptavidin) for pretargeting the tumor, wherein the antibody-receptor complex is administered to the patient, Subsequently, unbound complex is removed from the blood circulation using a scavenger, and then a “ligand” (eg, avidin) conjugated to a cytotoxic agent is then administered.

Immunoliposomes The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies are described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); As well as in US Pat. Nos. 4,485,045 and 4,544,545, using methods known in the art. Liposomes with increased circulation time are disclosed in US Pat. No. 5,013,556.

  Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter with a defined pore size to obtain liposomes of the desired diameter. Fab ′ fragments of the antibodies of the present invention can be bound to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982). A chemotherapeutic agent (such as doxorubicin) is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst., 81 (19): 1484 (1989).

Diagnostic Applications of Antibodies Directed to the Proteins of the Invention In one embodiment, methods for screening for antibodies having the desired specificity are not limited, but include enzyme linked immunosorbent assay (ELISA) and other persons skilled in the art. Includes known immunological intervention techniques. In a specific embodiment, the selection of antibodies that are specific for a particular domain of NOVX protein is facilitated by the generation of hybridomas that bind to fragments of NOVX protein that possess such domain. Thus, also provided herein are antibodies specific for the desired domain within the NOVX protein, derivative, fragment, analog or homologue thereof.
Antibodies directed against the NOVX protein of the present invention may be used in methods known in the art for the localization and / or quantification of NOVX protein (eg, measuring the level of NOVX protein in an appropriate physiological sample) For use in diagnostic methods, for use in protein imaging, etc.). In certain embodiments, an antibody specific for a NOVX protein, derivative, fragment, analog or homolog thereof, comprising an antibody-derived antigen binding domain is utilized as a pharmacologically active compound (hereinafter “therapeutic” Referred to as "agent").

Antibodies specific for the NOVX proteins of the invention (eg, monoclonal or polyclonal antibodies) can be used to isolate NOVX polypeptides by standard techniques such as immunoaffinity chromatography or immunoprecipitation. Antibodies to NOVX polypeptides can facilitate the purification of naturally derived proteins from cells and recombinantly produced NOVX antigens expressed in host cells. Further, such anti-NOVX antibodies can be used to detect antigenic NOVX protein (eg, in cell lysates or cell supernatants) to assess the abundance or expression pattern of antigenic NOVX protein. it can. Antibodies directed against the NOVX protein can be used diagnostically to monitor protein levels in tissues as part of a clinical laboratory procedure, for example, to determine the effectiveness of a given treatment modality. Detection can be facilitated by coupling (ie, physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes may include horseradish peroxidase, alkaline phosphatase, β-galactosidase and acetylcholinesterase; examples of suitable prosthetic groups may include streptavidin / biotin and avidin / biotin; Examples of substances may include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent substances include luminol Examples of bioluminescent materials may include luciferase, luciferin and aequorin, and examples of suitable radioactive materials may include ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.

Antibody Therapy Methods The antibodies of the present invention, including polyclonal, monoclonal, humanized and fully human antibodies, can be used as therapeutic agents. Such agents are generally used for the treatment or prevention of the disease or lesion of interest. An antibody formulation, preferably one having high specificity and high affinity for its target antigen, is administered to a subject and generally has an effect due to binding to the target. Such an effect is one of two types depending on the specific nature of the interaction between a given antibody molecule and the target antigen in question. In the first case, administration of the antibody may prevent or inhibit binding of the target to the endogenous ligand to which it is bound. In this case, the antibody binds to the target and masks the naturally occurring ligand binding site where the ligand acts as an effector molecule. Thus, the receptor mediates the signaling pathway in which the ligand plays a role.

  Alternatively, the effect may be that the antibody elicits a physiological result by binding to an effector binding site on the target molecule. In this case, a target that is a receptor with an endogenous ligand that may not be present or defective in the disease or pathology, binds to the antibody as an alternative effector ligand and causes a receptor-based signaling event. Initiated by the receptor.

  A therapeutically effective amount of an antibody of the invention generally relates to the amount required to achieve a therapeutic purpose. As mentioned above, this may be a binding interaction between an antibody and its target antigen that in some cases inhibits the function of the target and in other cases promotes a physiological response. The amount required for administration will further depend on the binding affinity of the antibody for the particular antigen, and also on the rate at which the administered antibody can be removed from the free volume of the administered subject. The usual range of therapeutically effective doses of an antibody or antibody fragment of the invention can be, by way of non-limiting example, from about 0.1 mg / kg body weight to about 50 mg / kg body weight. The usual number of administrations can range, for example, from twice a day to once a week.

Antibody Pharmaceutical Compositions Antibodies that specifically bind to the proteins of the invention, as well as other molecules identified by the screening assays disclosed herein, are administered in the form of pharmaceutical compositions for the treatment of various disorders. obtain. Principles and considerations involved in the preparation of such compositions, as well as guidelines for component selection, are described, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., Editors) Mack Pub Co., Easton, Pa .: 1995, Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994, and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4 ), 1991, M. Dekker, New York.

  If the antigenic protein is intracellular and an intact antibody is used as an inhibitor, an internalizing antibody is preferred. However, liposomes can be used to deliver antibodies or antibody fragments into cells. When using antibody fragments, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules can be designed that retain the ability to bind to the sequence of the target protein based on the sequence of the variable region of the antibody. Such peptides can be synthesized by chemical and / or recombinant DNA techniques. See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993). The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively or in addition, the composition may contain an agent that enhances its function, eg, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. Such molecules are conveniently present in combination in amounts that are effective for the intended purpose.

  The active ingredient is prepared, for example, in microcapsules prepared by coacervation technology or interfacial polymerization, e.g. hydroxymethylcellulose or gelatin-microcapsules and poly- (methyl methacrylate) microcapsules, respectively. For example, they can be encapsulated in liposomes, albumin globules, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.

The formulation to be used for in vivo administration must be sterile. This is easily accomplished by filtration through a sterile filtration membrane.
Sustained release formulations can be prepared. Suitable examples of sustained release formulations include solid-phase hydrophobic polymer semipermeable matrices containing antibodies, which are in the form of shaped articles such as films or microcapsules, for example. Yes. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol), polylactide (US Pat. No. 3,773,919), L-glutamic acid and Degradable lactic acid- such as gamma ethyl-L glutamate copolymer, non-degradable ethylene-vinyl acetate, LUPRON DEPOT® (injectable microspheres comprising lactic acid-glycolic acid copolymer and leuprolide acetate) Glycolic acid copolymers include polymers such as ethylene vinyl acetate and lactic acid-glycolic acid, which can release molecules over 100 days, while certain hydrogels release proteins in a shorter period of time.

ELISA Assay The agent that detects the analyte protein is an antibody capable of binding to the analyte protein, preferably an antibody with a detectable label. The antibody may be polyclonal or more preferably monoclonal. An intact antibody, or a fragment thereof (eg, F _ab or F _{(ab) 2} ) can be used. The term “label” refers to a direct labeling of a probe or antibody by coupling (ie, physically linking) a detectable substance to the probe or antibody with respect to the probe or antibody, as well as another It is intended to include indirect labeling of probes or antibodies by reactivity with drugs. Examples of indirect labeling include detection of the first antibody using a fluorescently labeled second antibody and end labeling of the DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term “biological sample” is intended to include tissues, cells and fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Thus, the term “biological sample” can include blood fractions or components including blood and serum, plasma, or lymph. That is, the detection methods of the present invention can be used to detect analyte mRNA, protein or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for the detection of analyte mRNA may include Northern hybridization and in situ hybridization. Analytical protein in vitro detection techniques may include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation and immunofluorescence. A technique for in vitro detection of analyte DNA may include Southern hybridization. The procedure for performing an immunoassay is described, for example, in `` ELISA: Theory and Practice: Methods in Molecular Biology '', Vol. 42, JR Crowther (Ed.) Human Press, Totowa, NJ, 1995, `` Immunoassay '', E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996, and “Practice and Thory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. In addition, techniques for in vivo detection of analyte proteins include introducing labeled anti-analyte protein antibodies into a subject. For example, the antibody can be labeled with a radioactive marker whose presence or localization in a subject can be detected by standard imaging techniques.

NOVX Recombinant Expression Vectors and Host Cells Another aspect of the present invention relates to vectors, preferably expression vectors, containing nucleic acids encoding NOVX proteins or derivatives, fragments, analogs or homologues thereof. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid bound thereto. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop with additional DNA segments attached to it. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors can be autonomously amplified in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, mammalian non-episomal vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. In addition, certain vectors are capable of directing the expression of genes that are operably linked to them. Such vectors are referred to herein as “expression vectors”. In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors such as viral vectors with equivalent functions (eg, retroviruses, adenoviruses and adeno-associated viruses lacking replication ability).

  A recombinant expression vector of the invention comprises a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which expresses a recombinant expression virus selected based on the host cell used for expression. It is meant to include one or more regulatory sequences operably linked to the nucleic acid sequence of interest. Within a recombinant expression vector, “operably linked” means that the nucleic acid sequence of interest is capable of expressing the nucleic acid sequence (eg, in an in vitro transcription / translation system or by introducing the vector into a host cell). In this case, it is meant to be bound to a regulatory sequence (in the host cell).

  The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of nucleotide sequences in many types of host cells and those that direct expression of nucleotide sequences only in specific host cells (e.g., tissue-specific regulatory sequences). obtain. Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of host cells to transform, the expression level of the desired protein, and the like. A protein or peptide comprising a fusion protein or peptide encoded by a nucleic acid as described herein (e.g., NOVX protein, mutant form of NOVX protein, fusion) by introducing an expression vector of the invention into a host cell Protein and the like).

  The recombinant expression vectors of the invention can be designed for NOVX protein expression in prokaryotic or eukaryotic cells. For example, NOVX protein can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

  Protein expression in eukaryotic cells is most often performed using vectors containing constitutive or inducible promoters that direct the expression of either fusion or non-fusion proteins in Escherichia coli. Fusion vectors add a number of amino acids to the protein encoded therein, usually at the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of the recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) in affinity chromatography. To help purify recombinant protein by acting as a ligand. Often, in fusion expression vectors, proteolytic cleavage sites are introduced at the junction of the fusion moiety and the recombinant protein to allow purification of the fusion protein following separation of the recombinant protein from the fusion moiety. Such enzymes, and their homologous recognition sequences, may include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31) in which glutathione S-transferase (GST), maltose E-binding protein, or protein A is fused to a target recombinant protein, respectively. -40), pMAL (New England Biolabs, Beverly, Mass.) And pRIT5 (Pharmacia, Piscataway, NJ).

  Examples of suitable inducible non-fused E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

  One strategy to maximize protein expression in E. coli is to express the protein in a host cell that has impaired the ability to proteolytically cleave the recombinant protein. See Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid inserted into the expression vector so that individual codons for each amino acid are preferentially used in E. coli (eg, Wada, et al. al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of the nucleic acid sequences of the present invention can be made by standard DNA synthesis techniques.

  In another embodiment, the NOVX expression vector is a yeast expression vector. Examples of expression vectors in yeast Saccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), And picZ (InVitrogen Corp, San Diego, Calif.).

  Alternatively, NOVX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors that can be used for protein expression in cultured insect cells (eg, SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series. (Lucklow and Summers, 1989. Virology 170: 31-39).

  In yet another embodiment, the nucleic acids of the invention can be expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors may include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells, see, for example, Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor See Laboratory Press, Cold Spring Harbor, NY, 1989.

  In another embodiment, the recombinant mammalian expression vector is capable of preferentially directing the expression of a nucleic acid in a particular cell type (e.g., using tissue specific regulatory elements to express the nucleic acid). ). Tissue specific regulatory elements are known in the art. Non-limiting examples of suitable tissue specific promoters include albumin promoters (liver specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), especially T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729) -740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (eg, neurofilament promoters; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477 ), Pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (eg, whey promoter; US Pat. No. 4,873,316 and European Patent Publication No. 264). , 166). For example, the developmentally regulated promoters of the murine hox promoter (Kessel and Gruss, 1990. Science 249: 374-379) and the alpha-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546) Include.

  The present invention further provides a recombinant expression vector comprising a DNA molecule of the present invention cloned in an antisense orientation into the expression vector. That is, the DNA molecule is operably linked to regulatory sequences in a manner that allows expression of the RNA molecule that is antisense to NOVX mRNA (by transcription of the DNA molecule). Selecting regulatory sequences, such as viral promoters and / or enhancers, that are operably linked to the cloned nucleic acid in an antisense orientation that directs continuous expression of the antisense RNA molecule in various cell types Regulatory sequences can be selected that direct tissue-specific or cell type-specific constitutive expression of the antisense RNA. Antisense expression vectors can be in the form of recombinant plasmids, phagemids or attenuated viruses that produce antisense nucleic acids under the control of a highly efficient regulatory region whose activity is determined by the cell type into which the vector has been introduced. See, for example, Weintraub, et al., “Antisense RNA as a molecular tool for genetic analysis” Reviews-Trends in Genetics, Vol. 1 (1) 1986, for a discussion of the regulation of gene expression using antisense genes. I want.

  Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Such progeny may not actually be identical to the parent cell, as the mutations or environmental effects may cause certain modifications in later generations, but the scope of the terms used herein Still included.

  The host cell can be any prokaryotic or eukaryotic cell. For example, NOVX protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are well known to those skilled in the art.

  Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” refer to foreign nucleic acids (eg, DNA) including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran mediated transfection, lipofection, or electroporation. It shall refer to various techniques recognized in the art for introduction into a host cell. Suitable methods for transforming or transfecting host cells include MOLECULAR CLONING: A LABORATORY MANUAL.2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and others. Can be found in the laboratory manual.

  For stable transfection of mammalian cells, depending on the expression vector used and the transfection technique, it is known that a small fraction of cells can incorporate foreign DNA into its genome. Yes. In order to identify and select these integrants, a selectable marker (eg, resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NOVX or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (eg, cells that incorporate a selectable marker survive while other cells die).

  A host cell of the invention, such as a prokaryotic or eukaryotic cell in culture, can be used to produce (ie, express) NOVX protein. Therefore, the present invention further provides a method for producing NOVX protein using the host cell of the present invention. In one embodiment, the method comprises culturing a host cell of the invention (into which a recombinant expression vector encoding NOVX protein is introduced) in a suitable medium that produces NOVX protein. Including. In another embodiment, the method further comprises isolating NOVX protein from the medium or the host cell.

Transgenic NOVX Animals The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, the host cell of the invention is a fertilized oocyte or embryonic stem cell into which a NOVX protein coding sequence is introduced. A non-human transgenic animal that uses such a host cell to introduce an exogenous NOVX sequence into the genome, or a homologous recombinant animal that alters an endogenous NOVX sequence therein Can be created. Such animals are useful for studying NOVX protein function and / or activity, and for identifying and / or evaluating modulators of NOVX protein activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rat or mouse, in which one or more cells of the animal contain the transgene. It is a tooth. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. The transgene is incorporated into the genome of the cell from which the transgenic animal develops and remains in the genome of the mature animal, thereby coding in one or more cell types or tissues of the transgenic animal. This is an exogenous DNA that directs the expression of a modified gene product. As used herein, “homologous recombinant animal” refers to an endogenous NOVX gene introduced into an endogenous gene and animal cells, eg, animal embryo cells, prior to animal development. A non-human animal, preferably a mammal, more preferably a mouse, which has been altered by homologous recombination with an exogenous DNA molecule.

  The transgenic animal of the present invention introduces a nucleic acid encoding NOVX into the male pronucleus of a fertilized oocyte (eg, by microinjection, retroviral infection), and the oocyte is injected into a pseudopregnant female foster animal. It can be created by allowing it to occur in the body. The human NOVX cDNA sequence of SEQ ID NO: 2n-1 (where n is an integer from 1 to 85) can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue of the human NOVX gene, such as the mouse NOVX gene, can be isolated based on hybridization with human NOVX cDNA (detailed further above) and used as a transgene. Intron sequences and polyadenylation signals can also be included in the transgene to increase the expression efficiency of the transgene. Tissue specific regulatory sequences can be operably linked to the NOVX transgene to direct expression of the NOVX protein to specific cells. Methods for producing transgenic animals, particularly animals such as mice, by embryo manipulation and microinjection have become conventional in the art, for example, US Pat. No. 4,736,866; 4,870,009. And 4,873,191; and Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Similar methods can be used to produce other transgenic animals. Primary transgenic animals can be identified based on the presence of NOVX transgenes in the genome and / or expression of NOVX mRNA in animal tissues or cells. The primary transgenic animal can then be used to breed additional animals carrying the transgene. In addition, a transgenic animal carrying a transgene encoding a NOVX protein can be used to breed additional transgenic animals carrying other transgenes.

  A vector containing at least part of a NOVX gene into which deletions, additions or substitutions have been introduced into it in order to create homologous recombinant animals, thereby altering the NOVX gene, for example functionally disrupting To prepare. The NOVX gene can be a human gene (eg, cDNA of SEQ ID NO: 2n-1 (where n is an integer from 1 to 85)), but more preferably is a non-human homologue of the human NOVX gene. For example, a homologous set suitable for altering the endogenous NOVX gene in the mouse genome using the mouse homologue of the human NOVX gene of SEQ ID NO: 2n-1 (where n is an integer from 1 to 85). Replacement vectors can be constructed. In one embodiment, the vector is designed to functionally disrupt an endogenous gene (ie, no longer encodes a functional protein; also referred to as a “knockout” vector) by homologous recombination.

  Alternatively, the vector mutates or otherwise alters the endogenous NOVX gene by homologous recombination, but still encodes a functional protein (e.g., alters the upstream regulatory region thereby changing the endogenous To alter the expression of sex NOVX protein). In a homologous recombination vector, the altered protein of the NOVX gene is bound to the endogenous NOVX gene and embryonic stem cells carried by the vector adjacent to the 5 'end or 3' end by additional nucleic acids of the NOVX gene. Allows homologous recombination to occur between NOVX genes. Yet another flanking NOVX nucleic acid is of sufficient length to allow successful homologous recombination with the endogenous gene. Typically, a few kilobases of contiguous DNA (both 5 'and 3' ends) can be included in the vector. For a description of homologous recombination vectors, see, for example, Thomas, et al., 1987. Cell 51: 503. The vector is then introduced into an embryonic stem cell line (eg, by electroporation) and cells in which the introduced NOVX gene is homologously recombined with the endogenous NOVX gene are selected. See, for example, Li, et al., 1992. Cell 69: 915.

  The selected cells are then injected into an animal (eg, mouse) blastocyst to form an aggregated chimera. See, for example, Bradley, 1987. In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp. 113-152. The chimeric embryo is then implanted into a suitable pseudopregnant female foster animal, and the embryo is delivered at term. Using progeny holding DNA homologously recombined in germ cells, all cells of the animal will breed animals containing homologous recombinant DNA by germline transmission of the transgene be able to. Methods for constructing homologous recombination vectors and homologous recombination animals are described in Bradley, 1991. Curr. Opin. Biotechnol. 2: 823-829; PCT International Publication No .: WO90 / 11354; WO91 / 01140; WO92 / 0968. And further described in WO 93/04169.

  In another embodiment, non-human transgenic animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre / loxP recombinase system of bacteriophage P1. For a description of the cre / loxP recombinase system, see for example Lakso, et al., 1992. Proc. Natl. Acad. Sci. USA 89: 6232-6236. Another example of a recombinase system is the Saccharomyces cerevisiae FLP recombinase system. See O'Gorman, et al., 1991. Science 251: 1351-1355. If a cre / loxP recombinase system is used to regulate transgene expression, an animal containing a transgene encoding both the Cre recombinase and the selected protein is required. Such animals are, for example, “double” transgenic animals by mating two transgenic animals, one containing a transgene encoding the selected protein and the other containing a transgene encoding a recombinase. It can be provided by constructing.

Non-human transgenic animal clones described herein can also be produced according to the method described in Wilmut, et al., 1997. Nature 385: 810-813. Briefly, isolated cells from transgenic animals (e.g., somatic cells), induction, it is possible to enter the G ₀ phase exits from the growth cycle. The quiescent cells can then be fused with enucleated oocytes from the same type of animal from which the quiescent cells were isolated, for example, by use of electric pulses. The reconstituted oocyte is then cultured such that it develops into a morula or blastocyst and then introduced into a pseudopregnant female foster animal. The offspring born from this female foster animal is an animal clone that isolates cells (eg, somatic cells).

Pharmaceutical Compositions of pharmaceutical compositions suitable for administration of the NOVX nucleic acid molecules, NOVX proteins, anti-NOVX antibodies (also referred to herein as “active compounds”), and derivatives, fragments, analogs and homologs thereof of the present invention. Can be built in. Typically, such compositions comprise a nucleic acid molecule, protein or antibody and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. It is intended to include. Suitable carriers are described in the latest edition of Remington's pharmacology, a standard reference text in this field, which is hereby incorporated by reference. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, finger solutions, glucose solutions and 5% human serum albumin. Water-insoluble vehicles such as liposomes and oils are also used. Such media and agents for pharmaceutically active substances are well known in the art. Unless any conventional media or agents are compatible with the active compounds, their use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

  A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration may include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g. inhalation), transdermal (i.e. topical), transmucosal and rectal administration. . Solutions or suspensions used for parenteral, intradermal or subcutaneous applications may include the following components: sterile vehicles such as water for injections, saline solutions, fats and oils, polyethylene glycols, glycerin , Propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); acetates, citric acid Buffers such as salts or phosphates and agents that adjust isotonicity such as sodium chloride or glucose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple use vials made of glass or plastic.

  Pharmaceutical compositions suitable for injection may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for preparations prepared as necessary in sterile injectable solutions or dispersions. For intravenous administration, suitable carriers may include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Protection of microbial action is achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

  By incorporating the active compound (eg, a NOVX protein or anti-NOVX antibody) in the required amount, together with one or a combination of the above-listed ingredients, in a suitable solvent, and then sterile sterilizing, followed by filter sterilization Can be prepared. Generally, dispersions can be prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the method of preparation involves vacuum drying and freezing to produce a powder of the active ingredient plus any further desired ingredient powder from the pre-sterile filtered solution. It is dry.

  Oral compositions generally include an inert excipient or edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with additives and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash, where the compound in the liquid carrier is applied orally, quickly removed, and exhaled or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds with similar properties: binders such as microcrystalline cellulose, gum tragacanth or gelatin; additives such as starch or lactose, alginic acid Disintegrants such as primogel or corn starch; lubricants such as magnesium stearate or sterote; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or mint, methyl salicylate, A fragrance like orange fragrance.

  For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

  Systemic administration can also be obtained by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and may include detergents, bile salts, fusidic acid derivatives, for example, for transmucosal administration. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, ointments, gels, or creams as generally known in the art.

  The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

  In one embodiment, the active compounds are prepared with carriers that will prevent the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable and biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polyacetic acid. It will be clear to those skilled in the art how to prepare such formulations. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted from infected cells with monoclonal antibodies to viral antibodies) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

  It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to a physically discrete unit suitable as a single dosage for the subject being treated; each unit together with the required pharmaceutical carrier. Contains a predetermined amount of active compound calculated to produce the desired therapeutic effect. The unit dosage form specifications of the present invention are dictated by the unique features of the active compound and the particular therapeutic effect to be achieved, as well as limitations inherent in the art to formulate such active compounds for the treatment of individuals. And depends directly.

The nucleic acid molecule of the present invention can be inserted into a vector and used as a gene therapy vector. Gene therapy vectors can be obtained, for example, by intravenous injection, by local administration (see, eg, US Pat. No. 5,328,470) or by tactile injection (see, for example, Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). A pharmaceutical formulation of a gene therapy vector can include a gene therapy vector in an acceptable excipient, or can include a sustained matrix having the gene therapy vector embedded therein. Alternatively, where a complete gene therapy vector can be produced intact from a recombinant cell, such as a retrovirus vector, the pharmaceutical formulation can include one or more cells producing a gene delivery system.
The pharmaceutical formulation can be included in a container, pack or dispenser along with instructions for administration.

Screening and Detection Methods As detailed below, the isolated nucleic acid molecules of the invention are used to express NOVX protein (eg, via a recombinant expression vector in a host cell in gene therapy applications), Genetic defects in NOVX mRNA (eg, in a biological sample) or NOVX gene can be detected and NOVX activity can be modulated. In addition, NOVX protein is used to screen for drugs or compounds that modulate NOVX protein activity or expression, as well as poor or excessive production of NOVX protein, or reduced or abnormal compared to NOVX wild-type protein Diseases characterized by the production of NOVX protein types with active activity (eg; diabetes (modulating insulin release); obesity (lipid binding and transport); obesity-related metabolic disorders, metabolic syndrome X and chronic disorders and Anorexia and debilitating disorders associated with various cancers, as well as infectious diseases (having antimicrobial activity) and various dyslipidemias, in addition, using the anti-NOVX antibodies of the present invention In still further embodiments, the NOVX protein can be detected and isolated, and NOVX activity can be modulated. Akira can be used in such a way as to affect appetite, nutrient absorption and disposal of metabolic substrates in both positive and negative ways.
The invention further relates to novel agents identified in the screening assays described herein and their use for the treatments described above.

Screening assays The present invention relates to modulators, i.e. candidate or test compounds or agents (e.g. peptides, peptides) that bind to a NOVX protein or have a promoting or inhibitory effect on, e.g., NOVX protein expression or NOVX protein activity. Methods of identifying mimetics, small molecules or other drugs (also referred to herein as “screening assays”) are provided. The invention also includes compounds identified in the screening assays described herein.

  In one embodiment, the present invention provides an assay for screening candidate or test compounds that bind to or modulate the activity of a membrane-bound NOVX protein or polypeptide or biologically active portion thereof. To do. Test compounds of the present invention can be obtained from biological libraries; spatially addressable parallel solid or liquid phase libraries; synthetic library methods that require deconvolution; “one bead one compound” library Can be obtained using any of a number of approaches in combinatorial libraries well known in the art, including: methods; and synthetic library methods using affinity chromatography selection. While the biological library approach is limited to peptide libraries, the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, for example, Lam, 1997. Anticancer Drug Design 12: 145.

  As used herein, “small molecule” refers to a composition having a molecular weight of less than about 5 kD, most preferably less than about 4 kD. Small molecules can be, for example, nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Biological mixtures, such as chemical and / or fungal, bacterial, or algae extracts are known in the art and can be screened using any assay of the present invention.

  Examples of molecular library synthesis methods are described in, for example, DeWitt, et al., 1993. Proc. Natl. Acad. Sci. USA 90: 6909, Erb, et al., 1994. Proc. Natl. Acad. Sci. : 11422, Zuckermann, et al., 1994. J. Med. Chem. 37: 2678, Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2059, Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061, and Gallop, et al., 1994. J. Med. Chem. 37: 1233 You can get a headline.

  A library of compounds can be obtained in solution (eg, Houghten, 1992. Biotechniques 13: 412-421) or on beads (Lam, 1991. Nature 354: 82-84) on a chip (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, US Pat. No. 5,223,409), spores (Ladner, US Pat. No. 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406, Cwirla, et al., 1990 Proc. Natl. Acad. Sci. USA 87: 6378-6382, Felici, 1991. J. Mol. Biol. 222: 301-310, Ladner, US Pat. No. 5,233,409).

In certain embodiments, the assay is a cell-based assay, wherein cells expressing membrane-bound NOVX protein or biologically active portion thereof on the cell surface are contacted with a test compound and the test compound is present The ability to bind to NOVX protein is measured. The cells can be, for example, mammalian or yeast cells. Measuring the ability of a test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzyme label to bind the test compound to the NOVX protein or biologically active portion thereof. The measurement is made possible by detecting the labeled compound in the complex. For example, the test compound is labeled directly or indirectly with ¹²⁵ I, ³⁵ S, ¹⁴ C or ³ H and the radioisotope is detected by direct counting of radiation emission or scintillation counting. Alternatively, the test compound is enzymatically labeled, eg, with horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzyme label is detected by measuring conversion of the appropriate substrate to product. In certain embodiments, the assay comprises contacting a cell expressing a membrane-bound NOVX protein or biologically active portion thereof on the cell surface with a known compound that binds to NOVX to form an assay mixture. Contacting the assay mixture with the test compound and measuring the ability of the test compound to interact with the NOVX protein, wherein measuring the ability of the test compound to interact with the NOVX protein is determined by the test Measuring the ability of a compound to bind preferentially to a NOVX protein or biologically active portion thereof compared to a known compound.

  In another embodiment, the assay is a cell-based assay, contacting a cell expressing a membrane-bound NOVX protein or biologically active portion thereof on the cell surface with a test compound, and the test compound is NOVX Measuring the ability to modulate (eg, promote or inhibit) the activity of a protein or biologically active portion thereof. Measuring the activity of a test compound that modulates the activity of NOVX or a biologically active portion thereof is, for example, by measuring the ability of a NOVX protein to bind to or interact with a NOVX target molecule. Can be achieved. As used herein, “target molecule” refers to a molecule to which a natural NOVX protein binds or interacts, for example, a cell surface molecule that expresses a protein that interacts with a certain NOVX, a second cell surface molecule A molecule in the extracellular environment, a molecule associated with the inner surface of the cell membrane or a cytoplasmic molecule. The target molecule of NOVX can be a non-NOVX molecule or some NOVX protein or polypeptide of the invention. In one embodiment, one NOVX target molecule is a signal transduction pathway that facilitates the transmission of extracellular signals (eg, signals generated by the binding of compounds to membrane-bound NOVX molecules) through and into the cell membrane. It is an ingredient. The target can be, for example, a second intracellular protein with catalytic activity or a protein that promotes the association of a downstream signal molecule with NOVX.

Measuring the ability of a NOVX protein to bind to or interact with a NOVX target molecule can be accomplished by one of the methods described above that measure direct binding. In one embodiment, measuring the ability of a NOVX protein to bind or interact with a NOVX target molecule can be accomplished by measuring the activity of the target molecule. For example, detecting the activity of the target molecule, detecting the induction of the target cellular second messenger (ie intracellular Ca ²⁺ , diacylglycerol, IP ₃ etc.), detecting the target catalytic / enzyme activity against the appropriate substrate Detecting the induction of a receptor gene (including a NOVX-responsive regulatory element operably linked to a nucleic acid encoding a detectable marker, such as luciferase), or a cellular response, such as a cell Can be measured by detecting cell survival, cell differentiation, or cell proliferation.

  In yet another embodiment, the assay of the present invention is a cell-free assay, wherein a NOVX protein or biologically active portion thereof is contacted with a test compound, and the test compound is NOVX protein or a biological thereof Measuring the ability to bind to an active moiety. Binding of the test compound to the NOVX protein can be measured either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the NOVX protein or biologically active portion thereof with a known compound that binds NOVX to form an assay mixture, contacting the assay mixture with the test compound. And measuring the ability of a test compound to interact with a NOVX protein, wherein measuring the ability of a test compound to interact with a NOVX protein includes determining that the test compound is a NOVX protein or organism thereof. Measuring the ability to bind preferentially to a chemically active moiety compared to a known compound.

  In yet another embodiment, the assay contacts a NOVX protein or biologically active protein thereof with a test compound, and the test compound modulates the activity of the NOVX protein or biologically active protein thereof ( A cell-free assay that involves measuring the ability to (eg promote or inhibit). Measuring the ability of a test compound to modulate the activity of NOVX can be achieved, for example, by one of the methods described above for measuring direct binding to the NOVX protein binding activity to a target molecule of NOVX. it can. In yet another embodiment, measuring the ability of a test compound to modulate the activity of a NOVX protein can be achieved by measuring the ability of the NOVX protein to modulate an additional NOVX target molecule. For example, the catalytic / enzymatic activity of the target molecule against the appropriate substrate can be measured as described above.

  In yet another embodiment, the cell-free assay comprises contacting the NOVX protein or biologically active portion thereof with a known compound that binds to the NOVX protein to form an assay mixture, wherein the assay mixture is a test compound. Measuring the ability of a test compound to interact with a NOVX protein, wherein measuring the ability of a test compound to interact with a NOVX protein is a target molecule of NOVX with a NOVX protein Measuring the ability to bind preferentially to or modulate its activity.

The cell-free assay of the present invention can be used for both soluble or membrane-bound NOVX proteins. In the case of cell-free assays involving membrane-bound NOVX protein, it is desirable to utilize a solubilizing agent so that the membrane-bound NOVX protein is maintained in solution. Examples of such solubilizers are n-octyl glucoside, n-dodecyl glucoside, n-dodecyl maltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X- 100, Triton® X-114, Thesit®, isotridecipoly (ethylene glycol ether) _n , N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, 3- (3-chloro Nonionic surfactants such as amidopropyl) dimethylaminol-1-propanesulfonate (CHAPS), or 3- (3-chloroamidopropyl) dimethylaminol-2-hydroxy-1-propanesulfonate (CHAPSO) Can be mentioned.

  In two or more embodiments of the above assay methods of the invention, the NOVX protein or its target molecule is immobilized to facilitate separation of complex forms from uncomplexed forms of one or both proteins, as well as assay automation. Can be adapted. Binding of the test compound to the NOVX protein or the interaction of the NOVX protein with the target molecule in the presence and absence of the candidate compound can be achieved in any container suitable for containing the reactants. Examples of such containers may include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both proteins to be bound to a matrix. For example, GST-NOVX fusion protein or GST-target fusion protein is adsorbed onto a microtiter plate derivatized with glutathione Sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione, which is then tested with a test compound or test The compound and either non-adsorbed target protein or NOVX protein are bound and the mixture is incubated under conditions that promote complex formation (eg, at physiological conditions with respect to salt and pH). After incubation, the beads or microtiter plate holes are washed to remove any unbound components, in the case of beads, the matrix is immobilized, and the complex is measured directly or indirectly, eg as described above. . Alternatively, the complex can be dissociated from the matrix and the level of NOVX protein binding or activity measured using standard techniques.

  Other techniques for immobilizing proteins on a matrix can also be used in the screening assays of the invention. For example, either the NOVX protein or its target molecule can be immobilized utilizing the binding of biotin and streptavidin. A biotinylated NOVX protein or target molecule is prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (eg, biotinylated kit, Pierce Chemicals, Rockford, Ill.) To produce streptavidin Can be immobilized in holes in a 96-well plate coated with (Pierce Chemical). Alternatively, an antibody that is reactive with the NOVX protein or target molecule but does not interfere with the binding of the NOVX protein to its target protein is derivatized into a hole in the plate to bind the unbound target or NOVX protein to the antibody. Can be captured in the hole. Methods for detecting such complexes include, in addition to those described above for GST-immobilized complexes, immunodetection of complexes using antibodies that react with NOVX proteins or target molecules, and NOVX proteins or target molecules. Mention may be made of enzyme binding assays that rely on detecting the relevant enzyme activity.

  In another embodiment, a modulator of NOVX protein expression is identified by a method of contacting a cell with a candidate compound and measuring expression of NOVX mRNA or protein in the cell. The expression level of NOVX mRNA or protein in the presence of the candidate compound is compared to the expression level of NOVX mRNA or protein in the absence of the candidate compound. Candidate compounds can then be identified as modulators of NOVX mRNA or protein based on this comparison. For example, when the expression of NOVX mRNA or protein is higher in the presence of the candidate compound (ie, statistically significantly higher) than in the absence, the candidate compound is used as a promoter of NOVX mRNA or protein expression. Identify. Alternatively, when the expression of NOVX mRNA or protein is less in the presence of the candidate compound (ie, statistically significantly less) in the presence of the candidate compound, the candidate compound is treated with NOVX mRNA or protein expression. Identify as an inhibitor. The level of expression of NOVX mRNA or protein in the cell can be measured by the methods described herein for detecting NOVX mRNA or protein.

  In yet another embodiment of the invention, the NOVX protein is a two-hybrid assay or a three-hybrid assay (eg, US Pat. No. 5,283,317, Zervos, et al., 1993. Cell 72: 223-232, Madura, et al., 1993). J. Biol. Chem. 268: 12046-12054, Bartel, et al., 1993. Biotechniques 14: 920-924, Iwabuchi, et al., 1993. Oncogene 8: 1693-1696, and Brent WO 94/10300. Can be used to identify other proteins that bind or interact with NOVX to modulate NOVX activity (“NOVX binding protein” or “NOVX-bp”). Such NOVX binding proteins are also involved, for example, in signal amplification by NOVX proteins as elements upstream or downstream of the NOVX pathway.

  The two-hybrid system is based on the modular nature of most transcription factors consisting of separable DNA binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene encoding NOVX is fused to a gene encoding the DNA binding domain of a known transcription factor (eg, GAL-4). In other constructs, DNA from a DNA sequence library encoding an unidentified protein ("prey" or "sample") is fused to a gene encoding the activation domain of a known transcription factor. If the “bait” and “prey” proteins can interact in vivo to form a NOVX-dependent complex, the DNA binding and activation domains of transcription factors can be drawn closer together. This proximity allows transcription of a reporter gene (eg, LacZ) operably linked to a transcriptional regulatory site responsive to the transcription factor. Reporter gene expression can be detected, cell colonies containing functional transcription factors can be isolated and used to obtain cloned genes encoding proteins that interact with NOVX.

  In yet another aspect of the present invention, a method of identifying a compound that modulates the activity of a target polypeptide (NOVX) is disclosed, wherein the method comprises the following: (a) a test compound comprising a target polypeptide and a target polypeptide Combining with a substrate of a peptide; and (b) determining whether the test compound modulates the activity of the target polypeptide, wherein the target polypeptide comprises SEQ ID NO: 2n, where n is from 1 to 85 An amino acid sequence selected from the group consisting of an integer), an amino acid sequence at least 95% identical to SEQ ID NO: 2n, an amino acid sequence that is at least one domain of SEQ ID NO: 2n, or at least 95% identical to at least one domain of SEQ ID NO: 2n Contains an amino acid sequence.

  The method further comprises identifying a compound that modulates the activity of the target polypeptide by modulating the activity of the target polypeptide as a modulator of the target polypeptide. Such modulators can be inhibitors, activators, antagonists or agonists of the NOVX target polypeptide.

  The method identifies compounds that modulate the activity of a target polypeptide by modulating the activity of the target polypeptide as a promoter of insulin secretion or as a therapeutic agent for the treatment of insulin resistance, obesity and / or diabetes. The method further includes the step of:

  In the foregoing method, the target polypeptide (NOVX) can be an isolated polypeptide.

  The target polypeptide can be produced by a method comprising culturing a recombinant host cell comprising a nucleic acid encoding the target polypeptide under conditions that promote expression of the target polypeptide. In such methods, the nucleic acid comprises the following: (a) SEQ ID NO: 2n-1 where n is an integer from 1 to 85; (b) a nucleotide encoding an amino acid sequence of at least one domain of SEQ ID NO: 2n; and (c ) An amino acid sequence selected from the group consisting of SEQ ID NO: 2n, an amino acid sequence that is at least 95% identical to SEQ ID NO: 2n, an amino acid sequence that is at least one domain of SEQ ID NO: 2n, or at least 95% with at least one domain of SEQ ID NO: 2n A nucleotide sequence selected from the group consisting of a nucleotide sequence encoding an amino acid sequence that is identical.

  Alternatively, the target polypeptide is an amino acid sequence selected from the group consisting of SEQ ID NO: 2n (n is an integer from 1 to 85), an amino acid sequence that is at least 95% identical to SEQ ID NO: 2n, and at least one domain of SEQ ID NO: 2n Or a recombinant vector comprising a nucleic acid encoding an amino acid sequence that is at least 95% identical to at least one domain of SEQ ID NO: 2n. Here, the test compound can be combined with the target polypeptide in cultured mammalian cells. Alternatively, the test compound can be combined with the target polypeptide in vitro. In this method, the nucleic acid comprises the following: (a) SEQ ID NO: 2n-1 where n is an integer from 1 to 85; (b) a nucleotide encoding an amino acid sequence of at least one domain of SEQ ID NO: 2n; and (c ) An amino acid sequence selected from the group consisting of SEQ ID NO: 2n, an amino acid sequence at least 95% identical to SEQ ID NO: 2n, an amino acid sequence that is at least one domain of SEQ ID NO: 2n, or at least 95% with at least one domain of SEQ ID NO: 2n A nucleotide sequence selected from the group consisting of a nucleotide sequence encoding an amino acid sequence that is identical.

  In yet another embodiment, the target polypeptide is generated by expression of an endogenous nucleic acid, wherein the endogenous nucleic acid is selected from the group consisting of SEQ ID NO: 2n (n is an integer from 1 to 85). An amino acid sequence that is at least 95% identical to SEQ ID NO: 2n, an amino acid sequence that is at least one domain of SEQ ID NO: 2n, or an amino acid sequence that is at least 95% identical to at least one domain of SEQ ID NO: 2n. Furthermore, the test compound can be combined with the target polypeptide in cultured mammalian cells. Alternatively, the test compound can be combined with the target polypeptide in vitro. In this method, the nucleic acid comprises: (a) SEQ ID NO: 2n-1 where n is an integer from 1 to 85; (b) a nucleotide encoding an amino acid sequence of at least one domain of SEQ ID NO: 2n; c) an amino acid sequence selected from the group consisting of SEQ ID NO: 2n, an amino acid sequence at least 95% identical to SEQ ID NO: 2n, an amino acid sequence that is at least one domain of SEQ ID NO: 2n, or at least one domain of SEQ ID NO: 2n and at least 95 Comprising a nucleotide sequence selected from the group consisting of: a nucleotide sequence encoding an amino acid sequence that is% identical.

  The invention further relates to novel agents identified by the aforementioned screening assays and their use in the treatments described herein.

Detection Assays A portion or fragment of the cDNA identified herein (and the corresponding complete gene sequence) can be used in various ways as a polynucleotide reagent. By way of example, and without limitation, these sequences: (i) map each gene on the chromosome; thus locate the gene region associated with the genetic disease; (ii) from a trace biological sample It can be used to help identify forensic identification of individuals (tissue typing); and (iii) biological samples. Some of these applications are described in the following subsections.

Chromosome mapping Once a sequence (or part of a sequence) of a gene is isolated, this sequence can be used to map the position of the gene on the chromosome. This method is called chromosome mapping. Thus, using a portion or fragment of the NOVX sequence of SEQ ID NO: 2n-1 (where n is an integer from 1 to 85), or a fragment or derivative thereof, the location of the NOVX gene on the chromosome, respectively Can be mapped. Mapping NOVX sequences to the chromosome is an important first step in correlating these sequences with genes associated with disease.

  Briefly, the NOVX gene can be mapped to a chromosome by preparing PCR primers (preferably 15-25 bp long) from the NOVX sequence. Computer analysis of NOVX sequences can be used to rapidly select primers that do not complicate the amplification process across two or more exons in genomic DNA. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the NOVX sequence will yield an amplified fragment.

  Somatic cell hybrids are prepared by fusing somatic cells from different mammals (eg, human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually delete human chromosomes in a random order, but retain mouse chromosomes. Mouse cells cannot grow because they lack specific enzymes, but human cells retain one human chromosome containing the gene encoding the required enzyme by using a medium that can grow. A panel of hybrid cell lines can be established by using various media. Each cell line in the panel contains either a single human chromosome or a small number of human chromosomes, and a complete set of mouse chromosomes, allowing individual genes to be easily mapped to specific human chromosomes To. See, for example, D'Eustachio, et al., 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.

  PCR mapping of somatic cell hybrids is a rapid procedure that maps a specific sequence to a specific chromosome. Using a single thermal cycler, it is possible to associate three or more sequences per day. By designing oligonucleotide primers with NOVX sequences, a detailed localization site specification can be achieved using a panel of fragments from specific chromosomes.

  Fluorescence in situ hybridization (FISH) of DNA sequences to metaphase chromosome spreads can further be used to provide precise chromosomal locations in one step. Chromosome spreads can be generated using cells that are blocked at metaphase by chemicals that disrupt the spindle, such as colcemid. Chromosomes can be briefly treated with trypsin and then Giemsa stained. A pattern of light and dark bands appears on each chromosome so that the chromosomes can be individually identified. FISH technology can be used with DNA sequences as short as 500 or 600 bases. However, clones larger than 1000 bases are more likely to bind to specific chromosomal locations with signal intensity sufficient for simple detection. Preferably 1000 bases, and more preferably 2000 bases are sufficient to obtain good results in a reasonable time. For a review of this technology, see Verma, et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).

  Use individual chromosome mapping reagents to label a single chromosome or a single site on that chromosome, or use a panel of reagents to label multiple sites and / or multiple chromosomes Can do. Reagents corresponding to non-coding regions of the gene are actually preferred for mapping purposes. The coding region is more likely to be conserved within the gene family, thus increasing the chance of cross-hybridization during chromosomal mapping.

  Once a sequence is mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data is found, for example, in McKusick, Mendelian Inheritance in Man (available online through Hopkins University Welch Medical Library). The relationship between the genes mapped to the same chromosomal site and the disease can then be determined using the association analysis described in, for example, Egeland, et al., 1987. Nature, 325: 783-787 (for physically adjacent genes). Can be identified by co-inheritance).

  In addition, DNA sequence differences between individuals suffering from and not affected by diseases associated with the NOVX gene can be measured. If the mutation is observed in some or all of the affected individuals but not in any of the unaffected individuals, the mutation is likely a causative agent for a particular disease. Comparison of affected and unaffected individuals is generally detected by first using a visible structural change in the chromosome, such as a visible deletion or translocation from the chromosome spread, or using PCR based on its DNA sequence With the search for possible structural changes. Finally, complete sequencing of genes from several individuals can be performed to confirm the presence of the mutation and to distinguish the mutation from the polymorphism.

Tissue typing The NOVX sequences of the invention can also be used to identify individuals from trace biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes and probed on a Southern blot to produce a unique band for identification. The sequences of the present invention are useful as yet another DNA marker for RFLP (restriction fragment length polymorphism, described in US Pat. No. 5,272,057).

  Furthermore, the sequences of the present invention may be used to provide yet another technique for measuring the actual base-by-base DNA sequence of a selected portion of an individual's genome. Thus, using the NOVX sequence described herein, two PCR primers can be prepared from the 5 'and 3' ends of the sequence. These primers can then be used to amplify the individual's DNA and subsequently sequence it.

  Since each individual has a characteristic set of such DNA due to allelic differences, a panel of corresponding DNA sequences from individuals prepared in this manner can provide identification of characteristic individuals. The sequences of the present invention can be used to obtain such identification sequences from individuals and tissues. The NOVX sequences of the present invention characteristically represent portions of the human genome. Allelic variation occurs to some extent in the coding regions of these sequences and to a greater extent in the non-coding regions. It is estimated that allelic variation between individual humans occurs at a frequency of about 1 in 500 bases. Many of the allelic variations are due to single nucleotide polymorphisms (SNPs), including restriction fragment length polymorphisms (RFLP).

  Each of the sequences described herein can be used to some extent as a standard by which DNA from individuals can be compared for identification purposes. Since more genetic polymorphisms occur in non-coding regions, fewer sequences are required to identify individuals. Non-coding sequences can reasonably provide an unambiguous identification of an individual with a panel of perhaps 10 to 1000 primers, each resulting in a 100 base non-coding amplified sequence. If a coding sequence such as that of SEQ ID NO: 2n-1 (where n is an integer from 1 to 85) is used, a more suitable number of primers for unambiguous individual identification is 500- 2000.

Predictive Medicine The present invention also relates to the field of predictive medicine, using diagnostic assays, prognostic assays, pharmacogenomics, and clinical trial monitoring for prognostic (predictive) purposes, thereby prophylactically treating an individual. Accordingly, one aspect of the present invention is to measure NOVX protein and / or nucleic acid expression and NOVX activity in the context of a biological sample (eg, blood, serum, cells, tissue), thereby causing an individual to become abnormal. Relates to a diagnostic assay for determining whether or not one is suffering from or at risk of developing a disorder associated with the expression or activity of NOVX. Disorders include metabolic disorders, diabetes, obesity, infectious diseases, anorexia, cancer-related cachexia, cancer, neurodegenerative disorders, Alzheimer's disease, Parkinson's disease, immune disorders, and hematopoietic disorders, and various different fats Mention may be made of blood disorders, metabolic disorders associated with obesity, metabolic syndrome X and debilitating diseases associated with chronic diseases and various cancers. The present invention also provides a prognostic (predictive) assay for determining whether an individual is at risk for developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, certain NOVX gene mutations can be assayed in a biological sample. Such assays are used for prognostic or predictive purposes, thereby prophylacticizing individuals prior to the development of a disorder characterized or associated with NOVX protein, nucleic acid expression or biological activity. Can be treated.

  Another aspect of the present invention is a method for measuring NOVX protein, nucleic acid expression or activity in an individual, thereby selecting an appropriate therapeutic or prophylactic agent for that individual (referred to herein as “pharmacogenomics”). Provided). Pharmacogenomics allows the selection of an agent (e.g., a drug) for therapeutic or prophylactic treatment of an individual based on the individual's genotype (e.g., examining an individual's genotype to identify an individual Measure ability to respond to).

Yet another aspect of the invention relates to monitoring the effect of a drug (eg, drug, compound) on NOVX expression or activity in clinical trials.
These and other drugs are described in detail in the following sections.

Diagnostic Assays An exemplary method for detecting the presence or absence of NOVX in a biological sample is to obtain a biological sample from a test subject, and to use the biological sample as a NOVX protein or a nucleic acid encoding NOVX protein. Involves contacting a compound or agent capable of detecting (eg, mRNA, genomic DNA) to allow the presence of NOVX to be detected in a biological sample. An agent for detection of NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA. The nucleic acid probe is, for example, a full-length NOVX nucleic acid such as a nucleic acid of SEQ ID NO: 2n-1 (where n is an integer from 1 to 85), or a part thereof, such as at least 15, 30, 50, 100, It may be 250 or 500 nucleotides long and sufficient oligonucleotides to specifically hybridize to NOVX mRNA or genomic DNA under stringent conditions. Other suitable probes for use in the diagnostic assays of the invention are described herein.

The agent for detecting NOVX protein is an antibody capable of binding to NOVX protein, preferably an antibody with a detectable label. The antibody may be polyclonal, or more preferably monoclonal. Intact antibodies, or fragments thereof (eg, Fab or F (ab ′) ₂ ) can be used. The term “label” for a probe or antibody refers to directly labeling a probe or antibody by coupling (ie, physically linking) a detectable substance to the probe or antibody, as well as another directly labeled Indirect labeling of the probe or antibody by reactivity with one reagent. Examples of indirect labeling may include detecting the first antibody using a fluorescently labeled second antibody and end-labeling the DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. . The term “biological sample” includes tissues, cells and fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection methods of the present invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro and in vivo. For example, in vitro techniques for detection of NOVX mRNA can include Northern hybridization and in situ hybridization. In vitro techniques for detecting NOVX protein may include enzyme linked immunoassay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting NOVX genomic DNA may include Southern hybridization. Further, in vivo techniques for detecting NOVX protein include introducing labeled anti-NOVX antibodies into the subject. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

  In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated from a subject by conventional means.

  In another embodiment, the method comprises obtaining a control biological sample from a control subject, using the control sample as a compound or agent capable of detecting NOVX protein, mRNA or genomic DNA, and NOVX protein, mRNA or genome. Further contacting to detect the presence of DNA in the biological sample and comparing the presence of NOVX protein, mRNA or genomic DNA in the control sample to NOVX protein, mRNA or genomic DNA in the test sample Accompany.

  The invention also includes a kit for detecting the presence of NOVX in a biological sample. For example, the kit: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; means for measuring the amount of NOVX in the sample; and means for comparing the amount of NOVX in the sample with a standard Can be included. The compound and drug can be packaged in a suitable container. The kit may further comprise instructions for using the kit to detect NOVX protein or nucleic acid.

Prognostic Assays Further, the diagnostic methods described herein can be used to identify subjects who have or are at risk of developing a disease or disorder associated with abnormal NOVX expression or activity. For example, using the assays described herein, such as the diagnostic assays described above and the following assays, to identify a subject having or at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity Can do. Alternatively, prognostic assays can be utilized to identify subjects having or at risk for developing a disease or disorder. Thus, the present invention provides a method of identifying a disease or disorder associated with abnormal NOVX expression or activity, wherein a test sample is obtained from a subject and abnormal NOVX protein or nucleic acid (eg, mRNA, genomic DNA) is detected. Wherein the presence of NOVX protein or nucleic acid is a diagnosis for a subject having or at risk of developing a disease or disorder associated with abnormal NOVX expression or activity. As used herein, “test sample” refers to a biological sample obtained from a subject of interest. For example, the test sample can be a body fluid (eg, serum), a cell sample, or a tissue.

  In addition, the prognostic assays described herein can be used to treat agents (eg, agonists, antagonists, peptidomimetics, proteins, peptides) to treat a disease or disorder associated with abnormal NOVX expression or activity. , Nucleic acids, small molecules, or other pharmaceutical candidates) can be determined. For example, such methods can be used to determine whether a subject's disease is effectively treated with a drug. Thus, the present invention provides a method for obtaining a test sample and determining whether an agent can effectively treat a disorder associated with abnormal NOVX expression or activity that detects NOVX protein or nucleic acid. (For example, where the presence of NOVX protein or nucleic acid becomes a diagnosis for a subject who can be administered an agent to treat a disorder associated with abnormal NOVX expression or activity).

  The method of the invention may also be used to detect genetic damage in certain NOVX genes, such that the subject with the damaged gene is at risk for a disorder characterized by abnormal cell growth and / or differentiation Can decide. In various embodiments, these methods are genetically characterized in at least one change that affects the integrity of a gene encoding a NOVX protein, or misexpression of a NOVX gene, in a cell sample from a subject. Includes detection of the presence or absence of damage. For example, such genetic damage may include (i) deletion of one or more nucleotides from a NOVX gene; (ii) addition of one or more nucleotides to a NOVX gene; (iii) Substitution of one or more nucleotides of a NOVX gene, (iv) chromosomal rearrangement of a NOVX gene, (v) changes in mRNA transcript level of a NOVX gene, (vi) methylation pattern of genomic DNA, etc. (Vii) presence of a non-wild type splicing pattern of an mRNA transcript of a NOVX gene, (viii) a non-wild type level of a NOVX protein, (ix) an allelic deletion of a NOVX gene , And (x) inappropriate post-translational modification of certain NOVX proteins. As described herein, there are a number of assay techniques known in the art that can be used to detect damage to a NOVX gene. A preferred biological sample is a peripheral blood leukocyte sample isolated from a subject by conventional means. However, any biological sample containing nucleated cells including, for example, buccal mucosa cells can be used.

  In certain embodiments, damage detection is in a polymerase chain reaction (PCR) such as anchor PCR or RACE PCR (see, eg, US Pat. Nos. 4,683,195 and 4,683,202) or Alternatively, Ligation Chain Reaction (LCR) (eg, Landegran, et al., 1988. Science 241: 1077-1080, and Nakazawa, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 360 With the use of probes / primers in (see -364), but the latter may be particularly useful for the detection of point mutations in the NOVX gene (Abravaya, et al., 1995. Nucl. Acids Res. 23: 675-682). See). This method involves collecting a cell sample from a patient, isolating nucleic acid (eg, genome, mRNA or both) from the sample cells, and under conditions such that hybridization and amplification of the NOVX gene (if present) occurs. Contacting the nucleic acid sample with one or more primers that specifically hybridize to a NOVX gene and detecting the presence or absence of the amplification product or detecting the size of the amplification product to lengthen the control sample A step of comparing the lengths. PCR and / or LCR are desirably used as a preliminary amplification step in conjunction with any technique used to detect mutations described herein.

  Further amplification methods include: self-retaining sequence replication (see Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcription amplification system (Kwoh, et al., 1989). Proc. Natl. Acad. Sci. USA 86: 1173-1177); Qβ replicase (see Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid amplification method followed by one skilled in the art May include detection of amplified molecules using well-known techniques. These detection schemes are particularly useful for the detection of such molecules if only a small number of nucleic acid molecules are present.

  In yet another embodiment, mutations in certain NOVX genes from sample cells can be identified by changes in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), treated with one or more restriction endonucleases, and fragment sizes are measured and compared by gel electrophoresis. Differences in fragment size between sample and control DNA indicate mutations in the sample DNA. In addition, sequence specific ribozymes (see, eg, US Pat. No. 5,493,531) can be used to assess the presence of specific mutations due to the generation or disappearance of ribozyme cleavage sites.

  In other embodiments, genetic mutations in NOVX are identified by hybridizing sample and control nucleic acids, eg, DNA or RNA, to a high density array containing hundreds or thousands of oligonucleotide probes. can do. See, for example, Cronin, et al., 1996. Human Mutation 7: 244-255, Kozal, et al., 1996. Nat. Med. 2: 753-759. For example, genetic variations in NOVX can be identified in a two-dimensional array containing DNA probes that generate light as described in Cronin, et al., Supra. Briefly, the first hybridization array of probes is used to scan a long stretch of DNA in a sample and a control to create a series of overlapping probe linear arrays to determine base changes between sequences. Can be identified. This step allows the identification of point mutations. This is followed by a second hybridization that allows the characterization of a particular mutation by using a smaller special probe array that is complementary to all the mutants or mutations detected. Each mutation array consists of a set of parallel probes, one complementary to the wild type gene and the other complementary to the mutant gene.

  In yet another embodiment, the NOVX gene is directly sequenced using any of a variety of sequencing reactions known in the art, and the NOVX sequence of the sample is compared with the corresponding wild type (control) sequence. Mutations can be detected by comparison. Examples of sequencing reactions are based on the technique developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463 You can list things. Sequencing by mass spectrometry (eg, PCT International Publication No. WO 94/16101; Cohen, et al., 1996. Adv. Chromatography 36: 127-162 and Griffin, et al., 1993. Appl. Biochem. Biotechnol. 38: It can also be envisaged that any of a variety of automated sequencing methods can be utilized in performing a diagnostic assay (see, eg, Naeve, et al., 1995. Biotechniques 19: 448).

Other methods for detecting mutations in the NOVX gene may include methods for detecting mismatched bases in RNA / RNA or RNA / DNA heteroduplexes using protection from cleaving agents. See, for example, Myers, et al., 1985. Science 230: 1242. In general, the art of “mismatch cleavage” is formed by hybridizing (labeled) RNA or DNA containing a wild-type NOVX sequence with RNA or DNA of a potential mutant obtained from a tissue sample. Begin by providing a heteroduplex. The duplex is treated with an agent that cleaves the single-stranded region of the duplex present due to base mismatches between the control and sample strands. For example, the RNA / DNA duplex is treated with RNase, DNA / DNA hybrids treated with _{S 1} nuclease may process the mismatched regions enzymatically. In other embodiments, either DNA / DNA or RNA / DNA duplexes can be treated with hydroxylamine or osmium tetroxide and piperidine to digest mismatched regions. After digestion of the mismatch region, the resulting material is then separated by size on a denaturing polyacrylamide gel to determine the site of mutation. See, for example, Cotton, et al., 1988. Proc. Natl. Acad. Sci. USA 85: 4397, Saleeba, et al., 1992. Methods Enzymol. 217: 286-295. In one embodiment, control DNA or RNA may be labeled for detection.

  In yet another embodiment, the mismatch cleavage reaction recognizes mismatched base pairs in double-stranded DNA in a defined system to detect and map point mutations in NOVX cDNA obtained from a sample of cells. One or more proteins (so-called “DNA mismatch repair” enzymes) are used. For example, E. coli mutY enzyme cleaves G / A mismatch A and thymidine DNA glycosidase from HeLa cells cleaves G / T mismatch T. See, for example, Hsu, et al., 1994. Carcinogenesis 15: 1657-1662. According to an exemplary embodiment, a probe based on a NOVX sequence, eg, a wild type NOVX sequence, is hybridized with cDNA or other DNA product from the test cell (s). This duplex is treated with a DNA mismatch repair enzyme, and if a cleavage product is present, it can be detected from an electrophoresis protocol or the like. See, for example, US Pat. No. 5,459,039.

  In other embodiments, changes in electrophoretic mobility are used to identify mutations in the NOVX gene. For example, single strand conformation polymorphism (SSCP) can be used to detect electrophoretic mobility differences between mutant and wild type nucleic acids. For example, Orita, et al., 1989. Proc. Natl. Acad. Sci. USA: 86: 2766, Cotton, 1993. Mutat. Res. 285: 125-144, Hayashi, 1992. Genet. Anal. Tech. Appl. 9: See 73-79. Single-stranded DNA fragments of sample and control NOVX nucleic acids can be denatured and renatured. The secondary structure of single-stranded nucleic acids varies from sequence to sequence, and the resulting change in electrophoretic mobility makes it possible to detect even single base changes. DNA fragments can be labeled or detected with a labeled probe. The sensitivity of the assay can be increased by using RNA (rather than DNA) whose secondary structure is more sensitive to sequence changes. In one embodiment, the subject method utilizes heteroduplex analysis to separate duplex helical duplex molecules based on changes in electrophoretic mobility. See, for example, Keen, et al., 1991. Trends Genet. 7: 5.

  In yet another embodiment, the migration of mutant or wild type fragments in a polyacrylamide gel containing a gradient of denaturing agent is assayed using denaturing concentration gradient gel electrophoresis (DGGE). See, for example, Myers, et al., 1985. Nature 313: 495. When using DGGE as an analytical method, DNA is modified to ensure that it is not completely denatured, for example by adding a GC clamp of high melting point GC-enriched DNA of approximately 40 bp by PCR. In a further embodiment, temperature gradients are used instead of denaturing gradients to identify differences in mobility of control and sample DNA. See, for example, Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.

  Examples of other techniques for detecting point mutations may include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, an oligonucleotide primer centered on a known mutation is prepared and then hybridized with the target DNA under conditions that allow hybridization only when a perfect match is found. See, for example, Saiki, et al., 1986. Nature 324: 163, Saiki, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 6230. When attaching oligonucleotides to a hybridizing membrane and hybridizing with labeled target DNA, such allele-specific oligonucleotides hybridize with PCR-amplified target DNA or several different mutants. .

  Alternatively, allele specific amplification techniques based on selective PCR amplification may be used in conjunction with the present invention. Oligonucleotides used as primers for specific amplification allow the mutation of interest to be centered in the molecule (as amplification depends on differential hybridization; see, eg, Gibbs, et al., 1989. Nucl. Acids Res. 17: 2437-2448) or, under appropriate conditions, mismatches can prevent or reduce polymerase extension (see, eg, Prossner, 1993. Tibtech. 11: 238) held at the 3 ′ extreme end of one primer. It may be. In addition, it is desirable to introduce new restriction sites in the region of the mutation in order to create detection based on cleavage. See, for example, Gasparini, et al., 1992. Mol. Cell Probes 6: 1. In certain embodiments, it is anticipated that amplification may also be performed using Taq ligase for amplification. See, for example, Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189. In such cases, ligation occurs only when there is a perfect match at the 3 'end of the 5' end sequence, and the presence of a known mutation at a particular site is detected by examining the presence or absence of amplification. Enable.

  The methods described herein can be performed, for example, by utilizing a prepacked diagnostic kit that includes at least one probe nucleic acid or antibody reagent described herein. This can be conveniently used, for example, in the clinical setting to diagnose a patient who exhibits symptoms or symptoms of a disease or disorder involving the NOVX gene or has a family history.

  In addition, any cell type or tissue that expresses NOVX, preferably peripheral blood leukocytes, may be utilized in the prognostic assays described herein. However, any biological sample containing, for example, nucleated cells of buccal mucosa cells may be used.

Pharmacogenomics An agent or modulator that has a promoting or inhibitory effect on NOVX activity (eg, NOVX gene expression) as identified by the screening assays described herein is administered to the individual to treat the disorder (prophylactically or therapeutically). Can be treated). Disorders include, but are not limited to, for example, the diseases, disorders and conditions described above, and in particular, diseases, disorders and conditions associated with homologues of the NOVX protein as summarized in Table 1.
Along with such treatment, an individual's pharmacogenomics (ie, a study of the relationship between an individual's genotype and the individual's response to a foreign compound or drug) may be considered. Differences in therapeutic drug metabolism can result in severe toxicity or treatment failure by altering the relationship between pharmacologically active drug dose and blood concentration. Thus, the pharmacogenomics of an individual allows the selection of an effective agent (eg, drug) for prophylactic or therapeutic treatment based on consideration of the individual's genotype. Such pharmacogenomics can further be used to determine the appropriate dose and method of treatment. Accordingly, the activity (s) of NOVX protein, the expression of NOVX nucleic acids, or the content of NOVX gene mutations in an individual can be measured, thereby selecting the agent (s) appropriate for the therapeutic or prophylactic treatment of the individual.

  Pharmacogenomics deals with clinically significant genetic variation in response to drugs due to altered drug distribution and abnormal effects in affected individuals. See, for example, Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985; Linder, 1997. Clin. Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be distinguished. A genetic state that conveys as a single factor that alters the way the drug acts on the body (altered drug action) or a genetic state that propagates as a single factor that alters the way the body acts on the drug (altered drug metabolism) There is. These pharmacogenetic conditions can occur as either rare defects or polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common hereditary enzyme disorder, in which the main clinical complications are oxidant drugs (antimalarials, sulfonamides) , Analgesics, nitrofurans) hemolysis after ingestion or after eating broad beans.

  In an exemplary embodiment, the activity of a drug's metabolic enzyme is a major determinant of both the intensity and duration of the drug's action. The discovery of genetic polymorphisms in drug metabolizing enzymes (eg, N-acetyltransferase 2 (NAT2) and cytochrome pregnancy band precursor protein enzymes CYP2D6 and CYP2C19) Provide an explanation of whether the expected effect of the drug is not achieved or if it shows excessive drug response and severe toxicity. These polymorphisms are expressed in the population population in two phenotypes: a high metabolic group (EM) and a low metabolic group (PM). The abundance of PM varies between different populations. For example, the gene encoding CYP2D6 is highly polymorphic and several mutations identify PMs that result in the absence of functional CYP2D6. The hypometabolic group of CYP2D6 and CYP2C19 very frequently experience excessive drug response and side effects when receiving standard doses. If the metabolite is an active therapeutic component, PM does not show a therapeutic response, as demonstrated by the analgesic action of codeine mediated by the metabolite morphine produced by CYP2D6. The exact opposite is the so-called ultrafast metabolic group that does not respond to standard doses. Recently, the molecular basis of the ultrafast metabolic group was confirmed to be due to CYP2D6 gene amplification.

  Thus, an individual's NOVX protein activity, NOVX nucleic acid expression, or mutation content of the NOVX gene can be measured, thereby selecting an appropriate drug (s) for therapeutic or prophylactic treatment of the individual . In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug metabolizing enzymes to the identification of individual drug responsiveness phenotypes. When this knowledge is applied to dosage and drug selection, adverse effects or treatment failures may occur when treating a subject with a NOVX modulator, such as a modulator identified by one of the exemplary screening assays described herein. Avoiding and thus enhancing therapeutic or prophylactic efficiency.

Monitoring the effects of clinical trials Monitoring the impact of a drug (eg, drug, compound) on NOVX expression or activity (eg, activity that modulates abnormal cell growth and / or differentiation) is a fundamental drug screen. It can be applied not only to clinical trials. For example, screening assays as described herein can reduce the effectiveness of agents determined to increase NOVX gene expression, increase protein levels, or upregulate NOVX activity, to reduce NOVX gene expression, protein levels, or lower It can be monitored in clinical trials of subjects exhibiting controlled NOVX activity. Alternatively, the efficacy of an agent that has been determined to decrease NOVX gene expression, protein levels, or down-regulate NOVX activity is compared to the clinical efficacy of a subject exhibiting increased NOVX gene expression, protein levels, or upregulated NOVX activity. Can be monitored in the test. In such clinical trials, expression or activity of NOVX, and preferably other genes associated with, for example, cell proliferation or immune disorders, may be used as a “readout” or marker of immune responsiveness of specific cells. .

  By way of non-limiting example, a gene comprising NOVX that is modulated in a cell by treatment with an agent (eg, a compound, drug or small molecule) that modulates NOVX activity (eg, identified in a screening assay described herein) Can be identified. Thus, to examine the effects of drugs on cell proliferation disorders, eg, in clinical trials, cells can be isolated and RNA prepared and the expression levels of NOVX and other genes suspected to be associated with the disorder can be analyzed . The amount of protein produced by gene expression levels (ie gene expression patterns) by Northern blot analysis or RT-PCR as described herein, or alternatively by one of the methods described herein. Or by measuring the activity level of NOVX or other genes. In this manner, gene expression patterns serve as markers that indicate the cellular physiological response to the drug. Thus, this response state can be measured before and during treatment of an individual with a drug at various times.

  In one embodiment, the present invention is an agent (e.g., agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified in the screening assays described herein). A method is provided for monitoring the effectiveness of treating a subject, comprising: (i) obtaining a pre-dose sample from a subject prior to drug administration; (ii) NOVX protein, mRNA, or in a pre-dose sample; Detecting the expression level of genomic DNA, (iii) obtaining one or more post-administration samples from the subject, and (iv) the level of expression or activity of NOVX protein, mRNA, or genomic DNA in the post-administration sample (V) one or more samples after administration of the level of expression or activity of NOVX protein, mRNA, or genomic DNA in the sample prior to administration The comparison with NOVX protein, mRNA or genomic DNA,, and comprising the step of changing the administration of the agent to a subject according to (vi) it. For example, to increase NOVX expression or activity to a higher level than detected, ie, to increase the effectiveness of the drug, increased administration of the drug is desirable. Alternatively, in order to reduce NOVX expression or activity to a lower level than detected, ie, reduce the effectiveness of the drug, reduced administration of the drug is desirable.

Methods of Treatment The present invention provides both prophylactic and therapeutic methods of treating subjects at risk (susceptibility) or having a disorder associated with abnormal NOVX expression or activity. Disorders include, but are not limited to, for example, the diseases, disorders and conditions described above, and in particular, diseases, disorders and conditions associated with homologues of the NOVX protein as summarized in Table 1.
These treatments are discussed in further detail below.

Diseases and Disorders Therapeutic agents that antagonize (i.e. reduce or inhibit) activity of diseases and disorders characterized by increased levels (as compared to subjects not suffering from the disease or disorder) or biological activity Can be treated with. A therapeutic that antagonizes activity may be administered in a therapeutic or prophylactic manner. The therapeutic agents that can be used include: (i) the aforementioned peptides, or analogs, derivatives, fragments or homologues thereof; (ii) antibodies to the aforementioned peptides; (iii) nucleic acids encoding the aforementioned peptides; (iv) Antisense nucleic acids and nucleic acids that are “dysfunctional” used to “knock out” the endogenous functions of the aforementioned peptides by homologous recombination (i.e. (See, eg, Capecchi, 1989. Science 244: 1288-1292); or (v) a modulator that alters the interaction of the aforementioned peptide and its binding partner (ie, of the present invention). Further inhibitors, agonists, antagonists, including but not limited to mimetics of other peptides or antibodies specific for the peptides of the present invention.

  Diseases and disorders characterized by decreased levels (as compared to subjects not afflicted with the disease or disorder) or biological activity can be treated with therapeutic agents that increase activity (ie, are agonists). A therapeutic agent that upregulates activity may be administered in a therapeutic or prophylactic manner. The therapeutic agents that can be utilized can include, but are not limited to, the aforementioned peptides, or analogs, derivatives, fragments, or homologues thereof; or agonists that increase bioavailability.

  Increased or decreased levels are obtained from patient tissue samples (e.g., from biopsy tissue) and assayed in vitro for RNA or peptide levels, expressed peptide (or mRNA for the aforementioned peptides) structure and / or activity By doing so, it can be easily detected by quantifying the peptide and / or RNA. Methods well known in the art include to detect immunoassays (eg Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and / or mRNA expression. Hybridization assays such as, but not limited to, Northern assays, dot blots, in situ hybridization, and the like.

Prophylactic methods In one aspect, by administering to a subject an agent that modulates NOVX expression or at least some NOVX activity, the present invention prevents a disease or disorder associated with abnormal NOVX expression or activity in the subject. Provide a method. Identifying a subject at risk for a disease caused or contributed to by aberrant NOVX expression or activity, for example, by either a diagnostic assay or a prognostic assay, or combinations thereof, as described herein Can do. A prophylactic agent may be administered prior to the onset of symptoms characteristic of NOVX abnormalities to prevent or alternatively delay the progression of the disease or disorder. Depending on the type of NOVX abnormality, for example, an agent that is a certain NOVX agonist or NOVX antagonist may be used to treat the subject. The appropriate agent can be determined based on the screening methods described herein. The prophylactic methods of the present invention are further discussed in the following subsections.

Therapeutic Methods Another aspect of the present invention relates to methods of modulating NOVX expression or activity for therapeutic purposes. The modulating method of the invention comprises contacting a cell with an agent that modulates one or more activities of NOVX protein activity associated with the cell. An agent that modulates NOVX protein activity can be an agent described herein, such as a nucleic acid or protein, a naturally-occurring ligand of a NOVX protein, a peptide, an NOVX peptide mimetic, or a small molecule. In one embodiment, the agent promotes one or more NOVX protein activities. Examples of such facilitating agents can include active NOVX proteins and nucleic acid molecules encoding NOVX introduced into cells. In another embodiment, the agent inhibits one or more NOVX protein activities. Examples of such inhibitory agents may include antisense NOVX nucleic acid molecules and anti-NOVX antibodies. These adjustment methods can be performed in vitro (eg, by culturing cells with the drug) or alternatively in vivo (eg, by administering the drug to a subject). Thus, the present invention provides a method of treating an individual suffering from a disease or disorder characterized by abnormal expression or activity of certain NOVX proteins or nucleic acid molecules. In one embodiment, the method comprises a combination of agents (e.g., agents identified in the screening assays described herein) or combinations of agents that modulate NOVX expression or activity (e.g., upregulate or downregulate). With administration. In another embodiment, the method involves administering a NOVX protein or nucleic acid molecule as a treatment to compensate for reduced or abnormal NOVX expression or activity.

  In situations where NOVX is abnormally down-regulated and / or increased NOVX activity appears to have a beneficial effect, it is desirable to promote NOVX activity. One example of such a situation is when the subject has a disorder characterized by abnormal cell proliferation and / or differentiation (eg, cancer or an immune related disease). Another example of such a situation is when the subject has a gestational disorder (eg, preeclampsia).

Measuring the biological effect of a therapeutic agent In various embodiments of the invention, appropriate in vitro or in vivo assays are performed to adapt the effect of a particular therapeutic agent and treatment of the tissue affected by its administration. Determine whether or not.

  In various specific embodiments, an in vitro assay is performed on a representative type (s) of cells involved in a patient's disorder to determine whether the administered therapeutic agent has the desired effect on the cell type. Can be measured. The compounds used for treatment can be tested in a suitable animal model system including, without limitation, rats, mice, chickens, cows, monkeys, rabbits, etc. prior to testing in human subjects. Similarly, for in vivo studies, any animal model system known in the art can be used prior to administration to a human subject.

Prophylactic and therapeutic uses of the compositions of the invention The NOVX nucleic acids and proteins of the invention are useful for potential prophylactic and therapeutic applications associated with various disorders. Disorders include, but are not limited to, for example, the diseases, disorders and conditions described above, and in particular, diseases, disorders and conditions associated with homologues of the NOVX protein as summarized in Table 1.

  By way of example, a cDNA encoding a NOVX protein of the invention can be useful for gene therapy, and the protein can be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the present invention have efficacy for the treatment of subjects suffering from diseases, disorders, conditions, etc., including but not limited to those listed herein. I will.

Both the novel nucleic acids encoding NOVX proteins and the NOVX proteins of the present invention, or fragments thereof, may also be useful in diagnostic applications to assess the presence or amount of nucleic acids or proteins. A further use may be as an antibacterial molecule (ie, certain peptides have been found to have antibacterial properties). These substances are further useful for the production of antibodies that immunospecifically bind to the novel substances of the invention for use in therapeutic or diagnostic methods.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

NOVX Polypeptides The following paragraphs describe in detail screening methods for NOVX polypeptides and modulators of NOVX polypeptides of the present invention.

A.NOV1-Cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK)
The cytoplasmic isoform of PEPCK regulates glyceroneogenesis in adipose tissue. Glycerogenesis is shortened gluconeogenesis, where glycerol-3-phosphate is produced from substrates such as pyruvate, lactate and alanine. The glycerol-3-phosphate thus produced is used for triglyceride synthesis. It has recently been elucidated that glycerol neogenesis plays a role in maintaining triglyceride accumulation in adipose tissue. Cytoplasmic PEPCK is also a rate-limiting enzyme for gluconeogenesis, which is the pathway through which glucose is produced from pyruvate, lactic acid and alanine in the liver. The hepatic gluconeogenesis process is up-regulated in type 2 diabetes and is therefore thought to contribute to the fasting hyperglycemia characteristic of this disease. Genetic and environmental factors for type 2 diabetes complications, including increased hepatic glucose production, are not fully understood.

  CuraGen's GeneCalling® study shows that cytoplasmic PEPCK is up-regulated in adipose tissue of obese AKR versus normal C57B1 mice. This result indicates that upregulation of PEPCK may contribute to the obesity phenotype. This hypothesis is the fact that genetically modified overexpression of cytoplasmic PEPCK in fat leads to increased glycerol neogenesis, adipocyte, fat cell size and fat mass, and weight gain. (Franckhauser S, Munoz S, Pujol A, Casellas A, Riu E, Otaegui P, Su B, Bosch F. Increased fatty acid re-esterification by PEPCK overexpression in adipose tissue leads to obesity without insulin resistance.Diabetes. 2002 Mar; 51 (3): 624-30. Supported by PMID: 11872659). Furthermore, mutations in the PPAR gamma binding site of the cytoplasmic PEPCK gene result in decreased PEPCK activity and increased mouse body fat accumulation and body weight (Olswang Y, Cohen H, Papo O, Cassuto H, Croniger CM, Hakimi P , Tilghman SM, Hanson RW, Reshef L. A mutation in the peroxisome proliferator-activated receptor γ-binding site in the gene for the cytosolic form of phosphoenolpyruvate carboxykinase reduces adipose tissue size and fat content in mice.Proc Natl Acad Sci US A. 2002 Jan 22; 99 (2): 625-30. PMID: 11792850). Thus, decreasing cytoplasmic PEPCK expression decreased triglyceride accumulation in adipose tissue, while increasing PEPCK expression in fat increased mouse body fat mass.

  Diet-induced obesity is a well-known model for studying pathways involved in the progression of weight gain due to increased caloric intake. Mice are raised on a high fat diet (˜30-45% fat-derived calories) for 12-16 weeks, after which tissues are analyzed for changes in metabolic pathways. This analysis is performed in comparison with control animals fed on a normal rodent diet. A study of adipose tissue from mice raised on a high fat diet versus mice fed on a normal chow diet with CuraGen's GeneCalling® has shown results inconsistent with those discussed above. Contrary to predictions, cytoplasmic PEPCK is down-regulated at a number of fat accumulation sites in high fat versus normal chow fed mice. Down-regulation of fatty PEPCK was recorded at a number of time points during the course of the diet-induced obesity protocol. For these results we interpret that PEPCK down-regulation may be a compensatory response to limit triglyceride accumulation. In support of this hypothesis, a number of other adipose biosynthesis genes are down-regulated in adipose tissue under diet-induced obesity conditions. This gene includes diacylglycerol transferase 2, monoglyceride lipase, lipoprotein lipase and aquaporin fat.

  The inconsistency in PEPCK gene expression in the diet-induced obesity versus genetic obesity model may be explained by the difference between the two models. In genetic obesity, the pathophysiology is static because the obesity is incorporated into the body. In contrast, in diet-induced obesity, the increase in body fat mass proceeds over 12-16 weeks, which is due to an increase in high fat calories. The response to caloric overload is dynamic (as observed in the CuraGen GeneCalling® study), and this response includes a compensatory response by the body to maintain energy homeostasis and body weight. Such compensatory responses to caloric overload are thought to include down-regulation of PEPCK in adipose tissue.

  CuraGen's GeneCalling® study also shows that cytoplasmic PEPCK is up-regulated in mouse liver in a diet-induced obesity model (data included below). This gene is up-regulated 1.8 times in obese mice undergoing transition from normoglycemia to hyperglycemia. This data strongly supports the pathogenic role of cytoplasmic PEPCK for increased hepatic glucose production and hyperglycemia in type 2 diabetes. An important note is that over 90% of patients with type 2 diabetes are obese. In the diet-induced obesity model, the obese group of mice also develops hyperglycemia and other metabolic disorders characteristic of type 2 diabetes. Thus, cytoplasmic PEPCK is also a therapeutic target for type 2 diabetes.

  The following summarizes the biochemistry for human cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) and potential assays that may be used for screening therapeutic antibodies or small molecule drugs to treat obesity and / or diabetes.

Cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) is an important enzyme for systemic energy homeostasis and catalyzes the following reactions:

GTP + oxaloacetate = GDP + phosphoenolpyruvate + CO ₂

This is the rate-limiting enzyme for gluconeogenesis in the liver and glycerol in adipose tissue and is involved in the glycerol's nascent pathway in adipocytes during glucose restriction. Glycerol neogenesis in adipose tissue produces glycerol-3-phosphate. This is a substrate for triglyceride accumulation. Phosphoenolpyruvate carboxykinase (PEPCK) activity is affected by numerous hormones that control this metabolic process, such as glucagon, insulin, and glucocorticoids.

  The data collectively indicate that modulators of human cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK), such as inhibitors / antagonists, would be beneficial in the treatment of obesity and / or diabetes.

  In addition, the inventors have found that modulators of cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) activity, such as inhibitors, activators, activators, antagonists or agonists of PEPCK, are obesity, diabetes And may be useful in the treatment of disorders such as insulin resistance and in enhancing insulin secretion.

Discovery process In the following paragraphs, cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) coding protein and any variants thereof, diagnostic markers for obesity and diabetes, targets for therapeutic antibodies And describes the study design (s) and techniques used to identify proteins and variants suitable as targets for small molecule drugs.

Example A1. Study of Genetic Obese Mice vs. Genetic Lean Mice A protocol for the study of genetic obese mice vs. genetically lean mice is disclosed in Example Q6.

  First, a fragment of the mouse cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) 1 gene (mouse strain AKR, C57BL) is up-regulated three-fold in adipose tissue of obese AKR mice compared to lean C57L / J mice I found out. Here, CuraGen's GeneCalling (registered trademark) method (described in Example Q7) showing a difference in gene expression was used. It was clearly identified that a mouse gene fragment showing an expression difference that moves to a position of about 254 nucleotides in length is a component of mouse cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) 1 cDNA. Competitive PCR was used to confirm this gene evaluation. The electropherographic peak corresponding to the mouse cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) 1 gene fragment is the gene-specific primer (shown in Table A1) during PCR amplification. If it competed with, it would be ablated. The peak at the 254 nt long position disappeared in samples from both obese AKR and normal C57L / J mice.

Example A2. Mouse Diet-Induced Obesity Study A protocol for mouse diet-induced obesity study is disclosed in Example Q1.

  A number of mouse strains that differ in weight and body composition have been identified. The AKR and NZB strains are obese, the SWR, C57BL and C57BL / 6 strains are average weight, while the SM / J and Cast / Ei strains are lean. Understanding the differences in gene expression in major metabolic tissues from these strains will elucidate the pathophysiological basis for obesity. These specific strains of rats were selected for analysis of gene expression differences. This selection was possible because published genetic studies have reported quantitative trait loci (QTLs) for body weight and related traits. Tissue included whole brain, skeletal muscle, visceral fat, and liver.

  The main cause of obesity in the clinical population is excessive caloric intake. This so-called diet-induced obesity (DIO) is mimicked in animal models by breeding on a high fat diet with a fat content greater than 40%. This DIO study was designed to identify changes in gene expression that contribute to the development and progression of diet-induced obesity. In addition, this study design sought to identify factors that induce resistance to the effects of high-fat diet in certain individuals, thereby preventing obesity. The study sample group had control body weights + 1 S.D., +4 S.D. and +7 S.D. Furthermore, the biochemical profile of +7 SD mice shows that these animals are further stratified into mice that maintain normal blood glucose profile despite obesity and mice that exhibit hyperglycemia. It was done. The test tissues were hypothalamus, brain stem, liver, retroperitoneal white adipose tissue (WAT), epididymal WAT, brown adipose tissue (BAT), gastrocnemius muscle (fast twitch skeletal muscle) and soleus muscle (slow twitch muscle) Skeletal muscle). Differences in gene expression profiles for these tissues indicated genes and pathways that could be used as therapeutic targets for obesity. Protocols relating to differential gene expression analysis, GeneCalling® are disclosed in Example Q7.

Results In a study of diet-induced obesity, a gene fragment of mouse cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) was found in Ngsd7 (normoglycemic, obese) versus epididymal fat bed (efp) in mice fed chow. It was found to be down-regulated 7 times. The study used CuraGen's GeneCalling® method, which shows differential gene expression. It was clearly identified that a mouse gene fragment showing an expression difference that moves to a position of about 349 nucleotides in length is a component of mouse cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) cDNA. Competitive PCR was used to confirm this gene evaluation. The electropherographic peak corresponding to the mouse cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) gene fragment is the result of the gene-specific primer (shown in Table A2) competing with the primer in the linker-adapter during PCR amplification. It disappeared. In the samples from both Ngsd7 and chow fed mice, the peak at 349 nt length disappeared. In addition, the mouse cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) gene is expressed in Ngsd7 (euglycemic, obese) and Hgsd7 (hyperglycemic, obese) versus Sd1 or chow fed mice retroperitoneal fatbed (rfp ) Is controlled downward. It should be noted that in the diet-induced obesity model, downregulation of this gene in fat may be a compensatory response to limit triglyceride accumulation and body fat accumulation.

  Our data also show that in studies of diet-induced obesity, cytosolic PEPCK is down-regulated 1.8-fold in the liver of Ngsd7 (normoglycemic and obese) vs. Hgsd7 (hyperglycemic and obese) mice Showed that. The identity of PEPCK as a down-regulated gene was then confirmed by TrapPing technology (disclosed in Example Q7). Thus, PEPCK is upregulated in Hgsd7 liver versus Ngsd7 liver. Thus, upregulation of PEPCK occurs at the transition phase between pathological changes from normoglycemia to hyperglycemia. Since PEPCK is a rate-controlling stage of the hepatic gluconeogenesis process (glucose production from non-glucose substrates), this data indicates that PEPCK up-regulation is a critical step in the progression to hyperglycemia and type 2 diabetes Indicates.

Example A3. Identification of human PEPCK sequence The sequence of human PEPCK (accession number CG101190-01) was obtained by cloning a cDNA fragment at the laboratory level. This cloning was performed by in silico prediction of the sequence. A group of cDNA fragments containing the full length of this DNA sequence, or part of the sequence, or both were cloned. In silico prediction was based on sequences available in CuraGen's private sequence database or public human sequence database to obtain full-length DNA sequences, or any portion thereof. A protocol for identifying human sequence (s) is disclosed in Example Q8.

  Table A3 shows the protein sequence alignment (ClustalW) of cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) for human (CG101190-01), mouse (PPCC_MOUSE = Q9Z2V4) and rat (PPCC_RAT = P07379) versions. Table A4 shows the sequence of the cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) version of the mouse (PPCC_MOUSE = Q9Z2V4; SEQ ID NO: 174) and rat (PPCC_RAT = P07379; SEQ ID NO: 173).

  Laboratory cloning was performed using one or more methods outlined in Example Q8. The NOV1 clone was analyzed to obtain the nucleotide sequence shown in Table A6 and the encoded polypeptide sequence.

  In the ClustalW comparison of the above protein sequences, the sequence alignment shown in Table A7 below is obtained.

  The NOV1a protein was further analyzed to obtain the properties shown in Table A8 below.

  The NOV1a protein was searched against the Geneseq database and several homologous proteins shown in Table A9 were obtained. This database is a private database containing sequences published in patents and patent publications.

  In a BLAST search of public sequence databases, the NOV1a protein was found to have homology with the proteins shown in the BLASTP data of Table A10.

  As predicted by PFam analysis, the NOV1a protein contains the domains shown in Table A11.

Example A4. Mutant and SNP of human cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) gene
Variant sequences are included in this application. Variant sequences can include single nucleotide (nucleotide) polymorphisms (SNPs). In some cases, the SNP can be referred to as “cSNP”, which indicates that the nucleotide sequence containing this SNP is originally cDNA. SNPs can occur by several routes. For example, SNPs can arise from the substitution of one nucleotide with another at the polymorphic site. Such a substitution can be a base transition or a transversion. SNPs can also result from deletion of nucleotides or insertion of nucleotides relative to a reference allele (allele). In this case, the polymorphic site is a site where one allele holds a gap with respect to a specific nucleotide in another allele. When SNP is present in a gene, the amino acid encoded by the gene may change at the position of this SNP. However, intragenic SNPs may be silent, when the codon containing the SNP encodes an invariant amino acid due to the degeneracy of the genetic code. When SNP is present outside the gene region or in an intron within the gene, the amino acid sequence of the protein does not change, but the control of the expression pattern may change. This change is, for example, a change in temporal expression, physiological response control, cell type expression control, expression intensity, or stability of the transcribed message.

Novel SNP Identification Method: SNPs are identified by analyzing sequence assembly using CuraGen's private SNPTool algorithm. SNPTool uses the following criteria to identify mutations in the assembly: SNPs within 10 base pairs at both ends of the alignment are not analyzed; the window size (number of bases in the field of view) is 10; Is 2; minimum SNP base quality (PHRED score) is 23; the smallest change to score SNP is 2 / assembly position. SNPTool analyzes the assembly and displays the SNP position in the assembly, the associated individual mutant sequence, the assembly depth at this default position, the estimated assembly allele frequency, and the SNP sequence variation. The sequence trace is then selected and brought into the field for manual verification. Import consensus assembly sequences along with mutant sequence changes into CuraTools to identify candidate amino acid changes due to SNP sequence mutations. The comprehensive SNP data analysis is then exported to the SNP Calling database.

New SNP confirmation method: SNP is confirmed using a proven method known as pyrosequencing. A detailed protocol for Pyrosequencing can be found in: Alderborn et al. Determination of Single Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing. (2000). Genome Research. 10, Issue 8, August. 1249-1265.

  Simply put, pyrosequencing is a genotyping real-time primer extension process. In this protocol, double-stranded biotin-labeled PCR products are collected from a genomic DNA sample and bound to streptavidin beads. These beads are then denatured to obtain single-stranded bound DNA. The characterization of SNP is performed using a technique based on assaying pyrophosphate (PPi) released from each dNTP during DNA strand elongation by indirect bioluminescence measurement. When PPi is released following Klenow polymerase-mediated base uptake, it is used with adenosine 5′-phosphosulfate (APS) as a substrate for ATP sulfurylase to form ATP. Subsequently, luciferase acts by this ATP, and luciferin is converted to its oxy derivative. Subsequently, light is emitted, and the amount of light emitted is proportional to the number of bases added up to about 4 bases. Excess dNTPs are degraded by apyrase to allow the process to proceed. Apyrase is also included in the starting reaction mixture so that only a small amount of dNTP is added to the template during sequencing. This process is fully automated and tailored to a 96-well format, so it is possible to rapidly screen large SNP panels.

Results The DNA and protein sequences for the novel single nucleotide polymorphic variant of phosphoenolpyruvate carboxykinase-like gene CuraGen accession number CG101190-01 are reported in Table A12. Variants are reported individually, but any combination of all or a selected subset of variants is also included. In Table A12, the positions of the mutant base and the mutant amino acid residue are underlined. Overall, there are 16 variants reported in Table A12. Mutant 13374203 is a SNP in which the 187 bp position of the nucleotide sequence is changed from G to A, and this mutation does not cause a change in the protein sequence (silent). Mutant 13374204 is a SNP in which the 322 bp position of the nucleotide sequence is changed from G to A, and this mutation does not cause a change in the protein sequence (silent). Mutant 13374205 is a SNP in which the 400 bp position of the nucleotide sequence is changed from C to T, and this mutation does not cause a change in the protein sequence (silent). Mutant 13374206 is an SNP in which the 668 bp position of the nucleotide sequence is changed from C to G, and this mutation changes amino acid position 184 of the protein sequence from Leu to Val. Mutant 13378862 is an SNP in which the 868 bp position of the nucleotide sequence is changed from G to A, and this mutation changes amino acid position 250 of the protein sequence from Met to Ile. Mutant 13380271 is an SNP in which the 917 bp position of the nucleotide sequence is changed from A to G, and this mutation changes amino acid position 267 of the protein sequence from Ile to Val. Mutant 13378861 is an SNP in which the 944 bp position of the nucleotide sequence is changed from G to A, and this mutation changes the amino acid position 276 of the protein sequence from Glu to Lys. Mutant 13379542 is a SNP in which the 1258 bp position of the nucleotide sequence is changed from C to T, and this mutation does not cause a change in the protein sequence (silent). Mutant 13378352 is an SNP in which the 1339 bp position of the nucleotide sequence is changed from C to A, and this mutation changes amino acid position 407 of the protein sequence from Cys to a stop codon. Mutant 13375322 is an SNP in which the 1680 bp position of the nucleotide sequence is changed from A to G, and this mutation changes the amino acid position 521 of the protein sequence from Lys to Arg. Mutant 13375321 is an SNP in which the 1731 bp position of the nucleotide sequence is changed from A to G, and this mutation changes the amino acid position 538 of the protein sequence from Glu to Gly. Mutant 13375320 is an SNP in which the 1773 bp position of the nucleotide sequence is changed from T to C, and this mutation changes amino acid position 552 of the protein sequence from Leu to Pro. Mutant 13375319 is a SNP in which the 1848 bp position of the nucleotide sequence is changed from T to C, and this mutation changes amino acid position 577 of the protein sequence from Leu to Pro. Mutant 13375318 is an SNP in which the 1857 bp position of the nucleotide sequence is changed from T to C, and this mutation changes the amino acid position 580 of the protein sequence from Ile to Thr. Mutant 13377375 is a SNP in which the 1887 bp position of the nucleotide sequence is changed from A to G, and this mutation changes amino acid position 590 of the protein sequence from Glu to Gly. Mutant 13377374 is a SNP in which the 1929 bp position of the nucleotide sequence is changed from T to C, and this mutation changes amino acid position 604 of the protein sequence from Leu to Pro.

Table A12. Accession number CG101190-01 nucleotide sequence (SEQ ID NO: 1) variants

Example A5. Expression profile of human cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) gene A protocol for quantitative expression analysis is disclosed in Example Q9.

  Expression of the gene CG101190-01 was evaluated using the primer-probe set Ag1769 described in Table A14. The results of performing RTQ-PCR are shown in Tables A15, A16, and A17.

Table A14. Probe name Ag1769

Table A15 . Panel 1.3D

Table A16 . Panel 5 Island

Table A17. General_Screening_Panel_v1.7

Panel 1.3D Summary: Human cytoplasmic PEPCK gene expression was highest in the liver (CT = 24.6). Cytoplasmic PEPCK was expressed at moderate to high levels in fat, mammary gland, kidney, large intestine, small intestine, heart, and adrenal gland. This expression pattern is consistent with GeneCalling® results and publication reports.

Panel 5 Island Overview: Among the samples on this panel, human cytoplasmic PEPCK gene expression was highest in the small intestine (CT = 28.3). Low levels of expression were also detected in the islets of Langerhans in the liver, fat and pancreas.

General_Screening_Panel_v1.7 Summary: Ag1769 The maximum expression of this gene is liver (CT = 23.4), kidney (CT = 24.6), fat (CT = 26.31), large intestine (CT = 25.7), lymph node ( CT = 26.7) and pituitary (CT = 26.83). This gene encodes phosphoenolpyruvate carboxykinase (PEPCK). The cytoplasmic isoform of PEPCK regulates glycerol neogenesis in adipose tissue. Glycerol-3-phosphate, a nascent product of glycerol, is used for triglyceride synthesis. Cytoplasmic PEPCK may be contributed to this obesity phenotype because it is up-regulated in adipose tissue of obese AKR versus normal C57B1 fat. This hypothesis is the fact that genetically modified overexpression of cytoplasmic PEPCK in fat leads to increased glycerol neogenesis, increased fat cell size and fat mass, and increased body weight (Sun Y, Liu S, Ferguson S, Wang L, Klepcyk P, Yun JS, Friedman JE. Phosphoenolpyruvate carboxykinase over-expression selectively attenuates insulin signaling and hepatic insulin sensitivity in transgenic mice. J Biol Chem. 2002). The therapeutic regulation of this gene, expressed protein and / or the use of antibodies or small molecule drugs that target this gene or gene product are useful in the treatment of endocrine / metabolic related diseases such as obesity and diabetes.

Example A6. Pathways Associated with Obesity and / or Diabetes Pathogenesis and Pathogenicity
PathCalling screening identified significant protein-protein interactions for cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) and stathmin-like 4 and heme-regulated initiation factor 2 alpha kinase (HRI). The PathCalling protocol is disclosed in Example Q10.

  Stasmin is a ubiquitous phosphorylated protein and is thought to act as an intracellular relay for diverse regulatory pathways (Sobel A. Stathmin: a relay phosphoprotein for multiple signal transduction? Trends Biochem Sci 1991 Aug; 16 (8) : 301-5. PMID: 1957351). HRI is also widely expressed and has significant transcription levels in fat, muscle and liver. HRI is activated by oxidative stress (Hwang SY, Kim MK, Kim JC. Cloning of hHRI, human heme-regulated eukaryotic initiation factor 2α kinase: down-regulated in epithelial ovarian cancers. Mol Cell 2000 Oct 31; 10 ( 5): 584-91 PMID: 11101152). Oxidative stress is associated with diabetes. Under oxidative stress conditions, HRI may phosphorylate and activate cytoplasmic phosphoenolpyruvate carboxykinase.

  Inhibiting the action of the human cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) gene potentially results in glycerol formation, triglyceride accumulation in adipose tissue, fat cell size and fat mass. , And will lose weight. Inhibition of cytoplasmic PEPCK will also reduce hepatic gluconeogenesis and improve fasting hyperglycemia in type 2 diabetes.

Example A7. Screening assay for modulators of phosphoenolpyruvate carboxykinase An incomplete list of cell lines expressing the phosphoenolpyruvate carboxykinase (PEPCK) gene is obtained from the RTQ-PCR results presented herein. be able to. These and other phosphoenolpyruvate carboxykinase (PEPCK) expressing cell lines could be used for screening purposes.

  Screening for PEPCK inhibitors / antagonists would be feasible using an in vitro glucose production assay. If H4IIE cells are transfected with recombinant cytoplasmic PEPCK, they can be tested for glucose production. This study was performed in Wang JC, Stafford JM, Scott DK, Sutherland C, Granner DK.The molecular physiology of hepatic nuclear factor 3 in the regulation of gluconeogenesis.J Biol Chem 275: 14717-21, 2000, PMID: 10799560 Perform as described in Methods.

  Our results indicate that modulators of cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) activity, such as inhibitors, activators, antagonists or agonists of PEPCK, are obesity, diabetes, and insulin resistance. It may be useful in the treatment of disorders such as) as well as in enhancing insulin secretion.

B.NOV2-Transketolase Transketolase is a thiamine-dependent enzyme that links the pentose phosphate pathway to glycolysis. The pentose phosphate pathway is active in most tissues and provides a sugar phosphate for intermediate biosynthesis, particularly nucleotide metabolism. The pentose pathway also generates reducing power through cellular biosynthesis in the form of NADPH. Transketolases are directly involved in branching the pathway that directs excess sugar phosphate to glycolysis, allowing the production of NADPH to be maintained under various metabolic conditions. The pentose phosphate pathway is particularly active at sites where fatty acid production and steroid synthesis occur, because they require the reducing power of NADPH (Lewandowski PA, Cameron-Smith D, Jackson CJ, Kultys ER , Collier GR. The role of lipogenesis in the development of obesity and diabetes in Israeli sand rats (Psammomys obesus) .J Nutr 1998 Nov; 128 (11): 1984-8. PMID: 9808653; Parks EJ, Krauss RM, Christiansen MP , Neese RA, Hellerstein MK. Effects of a low-fat, high-carbohydrate diet on VLDL-triglyceride assembly, production, and clearance.J Clin Invest. 1999 Oct; 104 (8): 1087-96. PMID: 10525047; Marques -Lopes I, Ansorena D, Astiasaran I, Forga L, Martinez JA. Postprandial de novo lipogenesis and metabolic changes induced by a high-carbohydrate, low-fat meal in lean and overweight men. Am J Clin Nutr. 2001 Feb; 73 ( 2): 253-61. PMID: 11157321).

  The inventors have discovered that transketolase is down-regulated in brown adipose tissue from mice with various body weights. Mice with various body weights range from obesity (sd4 for chow-fed mice) to severe obesity (ngsd7 for chow-fed mice) and hyperglycemic, severe obese mice (for chow-fed mice). Vs. hgsd7 +) on a high fat diet; transketolase remained unchanged in white fat from the same group of mice. This down-regulation of transketolase works with down-regulation of several enzymes in the fatty acid synthesis and recruitment pathways, including ATP citrate lyase, fatty acid elongation enzyme, and malate enzyme, and SREBP It is. This fact indicates that fatty acid synthesis and lipid biosynthesis are down-regulated in brown fat, a compensatory mechanism for high fat diets. Such a compensatory mechanism does not exist in white fat (Swierczynski J, Goyke E, Wach L, Pankiewicz A, Kochan Z, Adamonis W, Sledzinski Z, Aleksandrowicz Z. Comparative study of the lipogenic potential of human and rat adipose tissue Metabolism. 2000 May; 49 (5): 594-9. PMID: 10831168; Hellerstein MK. De novo lipogenesis in humans: metabolic and regulatory aspects. Eur J Clin Nutr. 1999 Apr; 53 Suppl 1: S53-65. Review PMID: 10365981).

FIG. 1 is an overview of a potential assay that may be used for biochemistry for human transketolase and for screening for therapeutic antibodies or small molecule drugs to treat obesity and / or diabetes. FIG. 1 shows the pentose phosphate pathway that produces NADPH for fatty acid and steroid biosynthesis. This pathway has an oxidative first stage and a non-oxidative second stage. The non-oxidative step is the linkage step between the pentose phosphate pathway and glycolysis. The response of this pathway is cytoplasmic. Transketolase cofactors are thiamine pyrophosphate and Mg ²⁺ (Frank T, Bitsch R, Maiwald J, Stein G. Alteration of thiamine pharmacokinetics by end-stage renal disease (ESRD). Int J Clin Pharmacol Ther 1999 Sep; 37 (9): 449-55; Pietrzak I, Baczyk K. Erythrocyte transketolase activity and guanidino compounds in hemodialysis patients. Kidney Int Suppl 2001 Feb; 78: S97-101).

  FIG. 2 shows how changes in human transketolase expression and associated gene products function in relation to obesity and / or diabetes pathogenesis and pathogenicity. This scheme includes the new findings of these discovery studies, along with what is reported in the publication. Inhibiting the action of human transketolase will result in a decrease in insulin resistance, a major problem of obesity and / or diabetes.

  Therefore, mimicking brown fat by inhibiting transketolase with white fat may reduce the amount of NADPH and may lack the necessary amount for fatty acid synthesis and lipid biosynthesis. If NADPH available for fatty acid synthesis and lipid biosynthesis decreases, conserved fat may be forced to be used. Thus, transketolase modulators, such as antagonists or inhibitors for transketolases, may be beneficial in the treatment of obesity and / or diabetes. Furthermore, inhibition of fructose-6-phosphate production through inhibition of transketolase may reduce the hexosamine pathway and may have a beneficial effect on insulin resistance. Transketolase-expressing cell lines can be obtained from the RTQ-PCR results disclosed herein. These and other transketolase expressing cell lines could be used for screening purposes.

  Furthermore, the inventors have found that modulators of transketolase activity, such as inhibitors, activators, antagonists or agonists of transketolase, treat disorders such as obesity, diabetes, and insulin resistance, and It may be useful for enhancing insulin secretion.

Discovery Process In the following paragraphs, transketolase-encoded proteins and any variants thereof, proteins and mutations suitable as diagnostic markers for obesity and diabetes, therapeutic antibody targets and small molecule drug targets Describe the study design (s) and techniques used to identify the body.

Obesity and diabetes are major public health challenges in developed and developing worlds. Estimates assume that more than half of the US adult population is overweight and has a body mass index (BMI) above the normal upper limit (25). Here, the definition of BMI is weight (Kg) / [height (m)] ² . Overweight generally leads to the development of hyperlipidemia and insulin resistance, followed by the development of hyperglycemia, a characteristic of type II diabetes. If left untreated, hyperglycemia leads to microvascular disease and end-organ damage. This includes retinopathy, kidney disease, heart disease, peripheral neuropathy and peripheral vascular compromise. Currently, over 16 million adults in the United States are affected by type 2 diabetes, and as a result of the prevalence of obesity among school-aged children, the symptoms now prevail in this age group It has become.

  Diabetes mellitus is a disorder in which the blood level of glucose (monosaccharide) is abnormally high, because the body does not release insulin properly or does not respond properly to insulin. Blood glucose (glucose) levels change throughout the day, rise after a meal, and return to normal within 2 hours. Normal blood glucose levels range from 70 to 110 milligrams per deciliter of blood (mg / dL) the next morning after an overnight fast. Blood glucose levels are usually less than 120-140 mg / dL 2 hours after eating foods containing sugar or other carbohydrates or drinking liquids.

  Insulin is a hormone released from the pancreas and is the main substance responsible for maintaining proper blood sugar levels. Insulin transports glucose into the cell, allowing the cell to produce energy or store energy from glucose until needed. When the blood glucose level rises after eating or drinking, pancreatic insulin production is promoted, further blood glucose level rise is prevented, and blood glucose level gradually decreases. Because muscle uses glucose as energy, blood glucose levels can also decrease during exercise.

  Diabetes occurs when the body does not produce enough insulin to maintain normal blood glucose levels or when cells do not respond properly to insulin. In type II diabetes mellitus, the pancreas continuously produces insulin, which can even be above normal levels. However, the body creates resistance to its action, resulting in relative insulin deficiency.

  The main goal of diabetes treatment is to keep blood glucose levels within the normal range as much as possible. Although it is difficult to maintain a perfectly normal concentration, the closer it is to maintain blood glucose levels within the normal range, the less likely it is to develop temporary or long-term complications.

  Thus, a therapy that decreases insulin resistance and / or enhances insulin secretion would be beneficial for the treatment of obesity and / or diabetes. Furthermore, such therapies would be beneficial for the treatment of insulin resistance, a condition that often leads to the development of diabetes.

Example B1. Mouse Diet-Induced Obesity A protocol for studying mouse diet-induced obesity is disclosed in Example Q1.

  The main cause of obesity in the clinical population is excessive caloric intake. This so-called diet-induced obesity (DIO) is mimicked in an animal model (mouse strain C57BL / 6) by breeding on a high fat diet with a fat content greater than 40%. This DIO study was designed to identify changes in gene expression that contribute to the development and progression of diet-induced obesity. In addition, this study design sought to identify factors that induced resistance to the effects of high-fat diet in certain individuals, thereby preventing obesity. The study sample group had control body weights +1 S.D., +4 S.D. and +7 S.D. fed on chow (below). Furthermore, the biochemical profile of +7 SD mice shows that these animals are further stratified into mice that maintain normal blood glucose profile despite obesity and mice that exhibit hyperglycemia. It was done. The test tissues were hypothalamus, brain stem, liver, retroperitoneal white adipose tissue (WAT), epididymal WAT, brown adipose tissue (BAT), gastrocnemius muscle (fast twitch skeletal muscle) and soleus muscle (slow twitch muscle) Skeletal muscle). Differences in gene expression profiles for these tissues indicated genes and pathways that could be used as therapeutic targets for obesity and / or diabetes. Protocols relating to differential gene expression analysis, GeneCalling® are disclosed in Example Q7.

Results First, it was found that the transketolase gene fragment of the mouse (mouse strain C57BL / 6) was down-regulated 1.7 times in the brown adipose tissue of mice fed a high fat diet compared to the brown fat of mice fed a chow diet . This high-fat diet-fed mouse reaches 4 standard deviations of body weight compared to chow-fed mice. Here, CuraGen's GeneCalling (registered trademark) method showing a difference in gene expression was used. It was clearly identified that a human gene fragment showing an expression difference that moves to a position of about 385 nucleotides in length is a component of human transketolase cDNA. Competitive PCR was used to confirm this gene evaluation. The electroferrograph peak corresponding to the human transketolase gene fragment disappeared when the gene-specific primer (shown in Table B1) competed with the primer in the linker-adapter during PCR amplification. The peak at the 385 nt long position disappeared in the samples from both the chow fed mice and the high fat fed mice.

Example B2. Identification of human transketolase sequence The sequence of human transketolase (accession number CG175387-01) was obtained by cloning a cDNA fragment at the laboratory level. This cloning was performed by in silico prediction of the sequence. A group of cDNA fragments containing the full length of this DNA sequence, or part of the sequence, or both were cloned. In silico prediction was based on sequences available in CuraGen's private sequence database or public human sequence database to obtain full-length DNA sequences, or any portion thereof. A protocol for identifying human sequence (s) is disclosed in Example Q8.

  Table B2 shows the protein alignment (ClustalW) of human transketolase sequence (CG175387-01; SEQ ID NO: 8) and mouse transketolase sequence (SEQ ID NO: 179). Table B3 shows the murine transketolase sequence (SEQ ID NO: 179).

  Laboratory cloning was performed using one or more methods outlined in Example Q8. The NOV2 clone was analyzed to obtain the nucleotide sequence shown in Table B4 and the encoded polypeptide sequence.

  In the ClustalW comparison of the protein sequences, the sequence alignment shown in Table B5 below is obtained.

  The NOV2a protein was further analyzed and the properties shown in Table B6 below were obtained.

  The NOV2a protein was searched against the Geneseq database and several homologous proteins shown in Table B7 were obtained. This database is a private database containing sequences published in patents and patent publications.

  In a BLAST search of public sequence databases, the NOV2a protein was found to have homology with the proteins shown in the BLASTP data in Table B8.

  As predicted by PFam analysis, the NOV2a protein contains the domains shown in Table B9.

Example B3. Mutant and SNP of transketolase gene
Variant sequences are included in this application. Variant sequences can include single nucleotide polymorphisms (SNPs). In some cases, the SNP can be referred to as “cSNP”, which indicates that the nucleotide sequence containing this SNP is originally cDNA. SNPs can occur by several routes. For example, SNPs can arise from the substitution of one nucleotide with another at the polymorphic site. Such a substitution can be a base rearrangement or base conversion. SNPs can also result from deletion of nucleotides or insertion of nucleotides relative to a reference allele. In this case, the polymorphic site is a site where one allele holds a gap with respect to a specific nucleotide in another allele. When SNP is present in a gene, the amino acid encoded by the gene may change at the position of this SNP. However, intragenic SNPs may be silent, when the codon containing the SNP encodes an invariant amino acid due to the degeneracy of the genetic code. When SNP is present outside the gene region or in an intron within the gene, the amino acid sequence of the protein does not change, but the control of the expression pattern may change. This change is, for example, changes in temporal expression, physiological response control, cell type expression control, expression intensity, and stability of the transcribed message.

New SNP identification method:
SNPs are identified by analyzing sequence assembly using CuraGen's private SNPTool algorithm. SNPTool uses the following criteria to identify mutations in the assembly: SNPs within 10 base pairs at both ends of the alignment are not analyzed; the window size (number of bases in the field of view) is 10; Is 2; the minimum SNP base quality (PHRED score) is 23; the smallest change to score SNP is 2 / assembly position. SNPTool analyzes the assembly and displays the SNP position in the assembly, the associated individual variant sequence, the degree of assembly at this default position, the estimated assembly allele frequency, and the SNP sequence variation. The sequence trace is then selected and brought into the field for manual verification. Import consensus assembly sequences along with mutant sequence changes into CuraTools to identify candidate amino acid changes due to SNP sequence mutations. The comprehensive SNP data analysis is then exported to the SNP Calling database.

New SNP confirmation method: SNP is confirmed using a proven method known as pyrosequencing. A detailed protocol for Pyrosequencing can be found in: Alderborn et al. Determination of Single Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing. (2000). Genome Research. 10, Issue 8, August. 1249-1265.

  Simply put, pyrosequencing is a genotyping real-time primer extension process. In this protocol, double-stranded biotin-labeled PCR products are collected from a genomic DNA sample and bound to streptavidin beads. These beads are then denatured to obtain single-stranded bound DNA. The characterization of SNP is performed using a technique based on assaying pyrophosphate (PPi) released from each dNTP during DNA strand elongation by indirect bioluminescence measurement. When PPi is released following Klenow polymerase-mediated base uptake, it is used with adenosine 5′-phosphosulfate (APS) as a substrate for ATP sulfurylase to form ATP. Subsequently, luciferase acts by this ATP, and luciferin is converted to its oxy derivative. Subsequently, light is emitted, and the amount of light emitted is proportional to the number of bases added up to about 4 bases. Excess dNTPs are degraded by apyrase to allow the process to proceed. Apyrase is also included in the starting reaction mixture so that only a small amount of dNTP is added to the template during sequencing. This process is fully automated and tailored to a 96-well format, so it is possible to rapidly screen large SNP panels.

Results The DNA and protein sequences for the novel single nucleotide polymorphic variant of the transketolase-like gene CuraGen accession number CG175387-01 are reported in Table B10. Variants are reported individually, but any combination of all or a selected subset of variants is also included. In Table B10, the position of the mutant base and the mutant amino acid residue are underlined. Overall, there are 22 variants reported in Table B10. Mutant 13377687 is an SNP in which the 134 bp position of the nucleotide sequence is changed from G to A, and this mutation changes amino acid position 19 of the protein sequence from Ala to Thr. Mutant 13377688 is an SNP in which the 287 bp position of the nucleotide sequence is changed from C to T, and this mutation changes the amino acid position 70 of the protein sequence from Arg to Cys. Mutant 13377684 is a SNP in which the 566 bp position of the nucleotide sequence is changed from G to T, and this mutation changes the amino acid position 163 of the protein sequence from Val to Leu. Mutant 13377689 is a SNP in which the 651 bp position of the nucleotide sequence is changed from A to G, and this mutation changes the amino acid position 191 of the protein sequence from Asp to Gly. Mutant 13380050 is an SNP in which the 699 bp position of the nucleotide sequence is changed from A to T, and this mutation changes the amino acid position 207 of the protein sequence from Glu to Val. Mutant 13380051 is an SNP in which the 741 bp position of the nucleotide sequence is changed from T to C, and this mutation changes the amino acid position 221 of the protein sequence from Val to Ala. Mutant 13377683 is a SNP in which the 747 bp position of the nucleotide sequence is changed from A to G, and this mutation changes the amino acid position 223 of the protein sequence from Glu to Gly. Mutant 13377690 is an SNP in which the 894 bp position of the nucleotide sequence is changed from A to G, and this mutation changes the amino acid position 272 of the protein sequence from Gln to Arg. Mutant 13377682 is an SNP in which the 1253 bp position of the nucleotide sequence is changed from T to C, and this mutation changes the amino acid position 392 of the protein sequence from Phe to Leu. Mutant 13380092 is an SNP in which the 1307 bp position of the nucleotide sequence is changed from A to G, and this mutation changes the amino acid position 410 of the protein sequence from Ile to Val. Mutant 13380073 is a SNP in which the 1441 bp position of the nucleotide sequence is changed from A to G, and this mutation does not cause a change in the protein sequence (silent). Mutant 13380072 is a SNP in which the 1519 bp position of the nucleotide sequence is changed from C to T, and this mutation does not cause a change in the protein sequence (silent). Mutant 13380093 is an SNP in which the 1529 bp position of the nucleotide sequence is changed from A to G, and this mutation changes the amino acid position 484 of the protein sequence from Asn to Asp. Mutant 13380094 is an SNP in which the 1550 bp position of the nucleotide sequence is changed from C to T, and this mutation changes amino acid position 491 of the protein sequence from Gln to a stop codon. Mutant 13380095 is an SNP in which the 1603 bp position of the nucleotide sequence is changed from T to C, and this mutation does not cause a change in the protein sequence (silent). Mutant 13380052 is a SNP in which the 1645 bp position of the nucleotide sequence is changed from G to T, and this mutation does not cause a change in the protein sequence (silent). Mutant 13377691 is an SNP in which the 1683 bp position of the nucleotide sequence is changed from T to C, and this mutation changes the amino acid position 535 of the protein sequence from Phe to Ser. Mutant 13380053 is a SNP in which the 1783 bp position of the nucleotide sequence is changed from T to C, and this mutation does not cause a change in the protein sequence (silent). Mutant 13377686 is an SNP in which the 1802 bp position of the nucleotide sequence is changed from G to A, and this mutation changes amino acid position 575 of the protein sequence from Ala to Thr. Mutant 13380055 is a SNP in which the 1813 bp position of the nucleotide sequence is changed from C to T, and this mutation does not cause a change in the protein sequence (silent). Mutant 13377685 is an SNP in which the 1832 bp position of the nucleotide sequence is changed from A to G, and this mutation changes amino acid position 585 of the protein sequence from Thr to Ala. Mutant 13380056 is a SNP in which the 1862 bp position of the nucleotide sequence is changed from A to G, and this mutation changes the amino acid position 595 of the protein sequence from Ser to Gly.

Table B10. Nucleotide sequence of accession number CG175387-01 (SEQ ID NO: 7)

Example B4. Transketolase Expression Profile A protocol for quantitative expression analysis is disclosed in Example Q9.

  The expression of genes CG175387-01 and G175387-03 was evaluated using the primer-probe set Ag6328 described in Table B12. The results of performing RTQ-PCR are shown in Tables B13, B14 and B15.

Table B12. Probe name Ag6328

Table B13. General Screening Panel v1.5

Table B14. Panel 5 Islands

Table B15. General_Screening_Panel_v1.6

General Screening Panel v1.5 Summary: Transketolase is abundantly expressed in all tissues. Transketolase is highly expressed in fat (CT = 27.42), which is the target tissue.

Panel 5 Island Overview: Panel 5I shows maximum expression in the placenta and is well expressed in adipose tissue. This supports the data in panel 1.5.

General Screening Panel v1.6 Summary: (Ag6328) High expression of this gene was ubiquitously detected in all tissues (CT = 26-30). This ubiquitous expression pattern indicates that the product of this gene is involved in homeostatic processes for these and other cell types and tissues.

  The expression of this gene was increased in all cancer cell lines (melanoma, ovarian cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, and CNS cancer), CT = 22-26. Therapeutic regulation of this gene, expressed protein and / or the use of antibodies or small molecule drugs that target this gene or gene product may include melanoma, ovarian cancer, breast cancer, lung cancer (lung cancer), renal cancer, pancreatic cancer, and CNS cancer (CNS cancer). Furthermore, the expression of this gene can be used as a detection marker for these cancers.

  This gene was highly expressed in adipose tissue (CT27.42). The therapeutic regulation of this gene, expressed protein and / or the use of antibodies or small molecule drugs that target this gene or gene product are useful in the treatment of obesity and diabetes.

Example B5. PathCalling®
PathCalling® screening showed that transketolase (TKT) interacts with transcription factor (TIF1) and the extracellular domain of semaphorin (CG51896-04, see Table EB5). Both interactions have been detected many times in the screening process using different libraries. The protocol for PathCalling® technology is disclosed in Example Q10.

Example B6. Screening Assay for Transketolase Modulators An incomplete list of cell lines expressing the transketolase gene can be obtained from the RTQ-PCR results presented herein. These and other transketolase expressing cell lines could be used for screening purposes.

  Screening for modulators of transketolase may be performed by measuring NADPH produced from reactions involving transketolase as outlined above. Furthermore, transketolase activity was reviewed in Frank et al, High thiamine diphosphate concentrations in erythrocytes can be achieved in dialysis patients by oral administration of benfontiamine, Eur J Clin Pharmacol. 2000 Jun; 56 (3): 251-7). Will be measurable by the method.

  Functional / mechanistic assays for the effectiveness of transketolase modulators can be performed by measuring the effect of transketolase 1 inhibitors on TG synthesis / accumulation in 3T3-L1 mouse adipocytes, human adipocytes or hepatocytes It is. This measurement is performed by Oil Red O staining. Alternatively, the measurement could consist of post-treatment TG accumulation in cells transfected with transketolase 1.

  Our results indicate that modulators of transketolase activity, such as inhibitors, activators, antagonists or agonists of transketolase, treat disorders such as obesity, diabetes, and insulin resistance, and insulin. It may be useful for enhancing secretion.

C. NOV3-Long-Chain Acyl Elongase Enzyme Long-Chain Acyl Elongase (LCE) uses malonyl-CoA as a two-carbon donor and converts palmitic acid, a 16-carbon fatty acid, into 18-carbon fatty acid. An enzyme that extends to some stearic acid. This extension of fatty acids occurs in the endoplasmic reticulum, in which case every two carbon additions requires four sequential reactions: (1) condensation between fatty acyl-CoA and malonyl-CoA, thereby producing 3-ketoacyl- CoA is formed, (2) Reduction of 3-ketoacyl-CoA with NADPH, thereby forming 3-hydroxyacyl-CoA, (3) Dehydration of 3-hydroxyacyl-CoA, thereby trans-2 -Enoyl-CoA is obtained, and (4) reduction of trans-2-enoyl-CoA, which gives saturated acyl-CoA. LCE has been shown to be essential for the condensation step of fatty acid extension, which is the rate limiting step. LCE is controlled by SREBP, which is similar to many enzymes involved in fatty acid synthesis and lipid biosynthesis (Moon YA, Shah NA, Mohapatra S, Warrington JA, Horton JD. Identification of a mammalian long chain fatty acyl elongase regulated by sterol regulatory element-binding protein J Biol Chem 2001 Nov 30; 276 (48): 45358-66. PMID: 11567032).

  The inventors have discovered that LCE is down-regulated in brown adipose tissue from high fat diet-fed mice with various body weights. These mice with different body weights are obese (sd4 for chow fed mice), severely obese (sd7 for chow fed mice), and hyperglycemic, severely obese mice (for chow fed mice). For sd7 +). However, LCE remained unchanged in white fat from the same group of mice. This downregulation of LCE is linked to the downregulation of several enzymes in the fatty acid synthesis and recruitment pathways, including ATP citrate lyase, transketolase, malate enzyme, and SREBP. This fact indicates that fatty acid synthesis and lipid biosynthesis are down-regulated in brown fat, which is a compensation for a high fat diet. Such a compensatory mechanism does not exist in white fat. In publications, long chain fatty acids are less susceptible to oxidation than short chain fatty acids (DeLany JP, Windhauser MM, Champagne CM, Bray GA. Differential oxidation of individual dietary fatty acids in humans. Am J Clin Nutr. 2000 Oct; 72 (4 ): 905-11. PMID: 11010930) and long chain fatty acids are more likely to be retained in muscle and liver and may contribute to insulin resistance (Bessesen DH, Vensor SH, Jackman MR. Trafficking of dietary oleic , linolenic, and stearic acids in fasted or fed lean rats. Am J Physiol Endocrinol Metab. 2000 Jun; 278 (6): E1124-32. PMID: 10827016). Thus, mimicking brown fat by inhibiting LCE in white fat may reduce the amount of long chain fatty acids and thus promote fatty acid oxidation of short chain fatty acids.

  Thus, modulators of LCE, such as antagonists or inhibitors for LCE, may be beneficial in the treatment of obesity and / or diabetes. Potential assays are used to screen for human long-chain fatty acyl acylase-related therapeutic antibodies or small molecule drugs useful for the treatment of obesity and / or diabetes. In screening assays for inhibitors of LCE, radiolabeled malonyl-CoA is added to palmitate to yield radiolabeled stearate, which can be detected by a partition assay. An incomplete list of cell lines that express long chain fatty acyl extenders can be obtained from the differential gene expression (RTQ-PCR) results presented herein. These and other long chain fatty acid acyl extender expressing cell lines could be used for screening purposes.

  In addition, the inventors' results indicate that modulators of long chain fatty acyl acylase activity, such as inhibitors, activators, antagonists or agonists of long chain fatty acyl extender enzymes, are obese, diabetic, and insulin resistant. It may be useful in the treatment of such disorders, as well as in enhancing insulin secretion.

Discovery process In the following paragraphs, proteins encoded by long-chain fatty acyl-extending enzymes and any variants thereof, suitable as diagnostic markers for obesity and / or diabetes, therapeutic antibody targets and small molecule drug targets Describe the study design and techniques used to identify existing proteins and variants.

Example C1. Mouse diet-induced obesity
A protocol for studying mouse diet-induced obesity is disclosed in Example Q1.

  The main cause of obesity in the clinical population is excessive caloric intake. This so-called diet-induced obesity (DIO) is mimicked in animal models by breeding on a high fat diet with a fat content greater than 40%. This DIO study was designed to identify changes in gene expression that contribute to the development and progression of diet-induced obesity. In addition, this study design sought to identify factors that induce resistance to the effects of high-fat diet in certain individuals, thereby preventing obesity. The study sample group had control body weights + 1 S.D., +4 S.D. and +7 S.D. Furthermore, the biochemical profile of +7 SD mice shows that these animals are further stratified into mice that maintain normal blood glucose profile despite obesity and mice that exhibit hyperglycemia. It was done. The test tissues were hypothalamus, brain stem, liver, retroperitoneal white adipose tissue (WAT), epididymal WAT, brown adipose tissue (BAT), gastrocnemius muscle (fast twitch skeletal muscle) and soleus muscle (slow twitch muscle) Skeletal muscle). Differences in gene expression profiles for these tissues indicated genes and pathways that could be used as therapeutic targets for obesity. Protocols relating to differential gene expression analysis, GeneCalling® are disclosed in Example Q7.

ResultsFirst of all, obese mice (sd4) in which the mouse (mouse strain C57BL / 6J) long-chain fatty acyl-enzyme (LCE) gene fragment was bred on a high-fat diet compared to normal-weight mice (bred on chow) It was found that brown adipose tissue was down-regulated twice. Here, CuraGen's GeneCalling (registered trademark) method showing a difference in gene expression was used. It was clearly identified that a mouse gene fragment showing an expression difference that moves to a position of about 285 nucleotides in length is a component of mouse long-chain fatty acid acyl-extension enzyme cDNA. Competitive PCR was used to confirm this gene evaluation. The electropherographic peak corresponding to the gene fragment of this mouse long-chain fatty acid acylase disappears when the gene-specific primer (shown in Table C1) competes with the primer in the linker-adapter during PCR amplification. It was. The peak at 285 nt length disappeared in samples derived from both brown adipose tissues of obese mice (sd4) and normal weight mice (bred with chow) bred on high fat diet. Furthermore, LCE is down-regulated in the brown adipose tissue of hyperinsulinous (ngsd7) and hyperglycemic obese mice fed on a high fat diet compared to normal weight mice (bred on chow) I understood.

Example C2. Identification of human long-chain fatty acyl extender sequence The human long-chain fatty acyl extender sequence (accession number CG180320-01) was obtained by cloning a cDNA fragment at the laboratory level. This cloning was performed by in silico prediction of the sequence. A group of cDNA fragments containing the full length of this DNA sequence, or part of the sequence, or both were cloned. In silico prediction was based on sequences available in CuraGen's private sequence database or public human sequence database to obtain full-length DNA sequences, or any portion thereof. A protocol for identifying human sequence (s) is disclosed in Example Q8.

  Table C2 shows the protein sequence of human long chain fatty acyl extender (CG180320-01; SEQ ID NO: 18), mouse long chain fatty acyl extender (Q920L5; SEQ ID NO: 184) and rat long chain fatty acyl extender (Q920L6; The protein alignment (ClustalW) with respect to sequence number 185) is shown. Table C3 shows the sequences of mouse long-chain fatty acyl extender (Q920L5; SEQ ID NO: 184) and rat long-chain fatty acyl extender (Q920L6; SEQ ID NO: 185).

  Laboratory cloning was performed using one or more methods outlined in Example Q8. The NOV3 clone was analyzed to obtain the nucleotide sequence shown in Table C4 and the encoded polypeptide sequence.

  In the ClustalW comparison of the protein sequences, the sequence alignment shown in Table C5 below is obtained.

  The NOV3a protein was further analyzed and the properties shown in Table C6 below were obtained.

  The NOV3a protein was searched against the Geneseq database and several homologous proteins shown in Table C7 were obtained. This database is a private database containing sequences published in patents and patent publications.

  In a BLAST search of public sequence databases, the NOV3a protein was found to have homology with the proteins shown in the BLASTP data in Table C8.

  As predicted by PFam analysis, the NOV3a protein contains the domains shown in Table C9.

Example C3. Expression Profile of Human Long-Chain Fatty Acyl Elongase Gene A protocol for quantitative expression analysis is disclosed in Example Q9.

  Expression of the genes CG180320-01, CG180320-02, CG180320-03, and CG180320-04 was evaluated using the primer-probe set Ag6596 described in Table C10. The results of performing RTQ-PCR are shown in Tables C11 and C12.

Table C10. Probe name Ag6596

Table C11. General Screening Panel v1.6

Table C12. Panel 5 Islands

General Screening Panel v1.6 Summary: (Ag6596) Moderate expression of this gene was ubiquitously detected in all tissues (CT = 28-32). This ubiquitous expression pattern indicates that the product of this gene is involved in homeostatic processes for these and other cell types and tissues.

  Expression of this gene was increased in all cancer cell lines (melanoma, ovarian cancer, breast cancer, lung cancer, kidney cancer, colon cancer, pancreatic cancer, and CNS cancer), CT = 25.5-30 . Therapeutic modulation of this gene, expressed protein and / or the use of antibodies or small molecule drugs that target this gene or gene product can treat melanoma, ovarian cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, and CNS cancer May be useful. Furthermore, the expression of this gene can be used as a detection marker for these cancers.

  This gene was expressed in adipose tissue (CT = 30.98). The therapeutic regulation of this gene, expressed protein and / or the use of antibodies or small molecule drugs that target this gene or gene product may be useful in the treatment of obesity and diabetes.

Panel 5 Island Overview: (Ag6596) Expression of this gene was induced in fat differentiated from mesenchymal stem cells (compare donor-derived AD sample with U sample; donor 2 (CT = 30.46-30.86 vs. CT = 32.18) -32.24) and donor 3 (CT = 29.01-30.7 vs. CT = 32.84-33.07)). Thus, therapeutic regulation of this gene, expressed protein and / or the use of antibodies or small molecule drugs that target this gene or gene product may be useful in the treatment of obesity and diabetes.

Example C4. Screening Assay for Modulators of Long-Chain Fatty Acyl Elongase An incomplete list of cell lines that express long-chain fatty acyl extenders is derived from the differential gene expression (RTQ-PCR) results presented herein. Obtainable.

  An effective method for measuring fatty acyl elongation in microsomes is Nagi et al., Evidence for two separate beta-ketoacyl CoA reductase components of the hepatic microsomal fatty acid chain elongation system in the rat, Biochem Biophys Res Commun 1989 Dec 29; 165 (3): 1428-34 and Moon et al., Identification of a mammalian long chain fatty acyl elongase regulated by sterol regulatory element-binding protein, J Biol Chem. 2001 Nov 30; 276 (48): 45358-66. Epub 2001 Sep 20, after which the radiolabeled fatty acids are separated by HPLC.

  Functional / mechanistic assays for the effectiveness of modulator administration of fatty acyl extenders may be feasible by observing TG synthesis / accumulation in 3T3-L1 mouse adipocytes, human adipocytes or hepatocytes. This observation is performed by oil red O staining. Alternatively, the measurement could consist of post-treatment TG accumulation in cells transfected with fatty acyl extension enzymes.

  Our results indicate that modulators of long chain fatty acyl extender activity, such as inhibitors, activators, antagonists or agonists of long chain fatty acyl extender enzymes, such as obesity, diabetes, and insulin resistance Shows that it may be useful in treating disorders, as well as enhancing insulin secretion.

D. NOV4-Acetyl Coenzyme A Acyltransferase 2
Acetyl coenzyme A acyltransferase 2 (ACAA2) is the last enzyme involved in mitochondrial fatty acid beta-oxidation. Fatty acid oxidation may interfere with glucose utilization in skeletal muscle and liver, and thus may cause insulin resistance and hyperglycemia (Randle PJ. Regulatory interactions between lipids and carbohydrates: the glucose fatty acid cycle after 35 years. Diabetes Metab. Rev. 1998 14 (4), 263-83 PMID: 10095997; Deems RO, Anderson RC, Foley JE. Hypoglycemic effects of a novel fatty acid oxidation inhibitor in rats and monkeys. Am. J. Physiol. 1998 274 (2 Pt 2): R524-8; PMID: 9486313). A study by GeneCalling® showed that ACAA2 was significantly upregulated in diabetic muscle. ACAA2 was also a fast twitch versus slow twitch diabetic skeletal muscle that was down-regulated in response to vanadate, metformin and AICAR. These are compounds that ameliorate hypoglycemia and diabetes. GeneCalling® data indicate that ACAA2 may be a good target for promoting skeletal muscle glucose utilization and improving diabetes.

The reaction catalyzed by human acetyl coenzyme A acyltransferase 2 is shown below.

3-oxoacyl CoA + CoA ← → acyl CoA + acetyl CoA
Acetyl CoA → TCA circuit

  A potential assay that may be used for screening for therapeutic antibodies or small molecule drugs is the measurement of NAD + / NADH production in the reactions outlined above, from 3-hydroxyacyl CoA to 3-oxoacyl CoA by adjacent mitochondrial enzymes. It is coupled with the conversion reaction. A general inhibitor for mitochondrial thiolase (4-bromocrotonic acid) has been described in the publication (Schulz et al., Life Sci. (1987) 40, 1443). A cell line expressing acetyl coenzyme A acyltransferase 2 can be obtained from the RTQ-PCR results shown herein. These and other acetyl coenzyme A acyltransferase 2 expressing cell lines could be used for screening purposes.

  ACAA2 catalyzes the final stage of acetyl-CoA production. Regarding the accumulation of ACAA2 substrate, no toxicity has been reported for 3-oxoacyl-CoA, and the accumulation results in the inhibition of two other enzymes involved in the fatty acid oxidation cycle. Inhibiting the action of human acetyl coenzyme A acyltransferase 2 will result in inhibition of fatty acid oxidation in skeletal muscle and liver and promote glucose utilization.

  Acetyl coenzyme A acyltransferase 2 (ACAA2) (3-oxoacyl-CoA thiolase) is upregulated in obese / diabetic skeletal muscle. The level of expression of this enzyme is down-regulated in gastrocnemius versus soleus muscle in response to vanadate, AICAR and metformin treatment, all of which are known to improve muscle glucose utilization. ACAA2 is the last enzyme involved in fatty acid beta oxidation. Inhibiting ACAA2 will inhibit fatty acid oxidation in skeletal muscle and liver and promote glucose utilization. Thus, inhibitors / antagonists of ACAA2 may be useful as antiglycemic agents for the treatment of obesity and / or diabetes.

  In addition, the inventors have shown that acetyl coenzyme A acyltransferase 2 (ACAA2) is down-regulated in diabetic skeletal muscle of animals that have a positive effect on rosiglitazone treatment and improved glycemic control. discovered. However, it was found that the expression of acetyl coenzyme A acyltransferase 2 did not respond to rosiglitazone treatment and was not down-regulated in diabetic animals that did not show a detectable improvement in hyperglycemia. Since the expression of acetyl coenzyme A acyltransferase 2 is positively correlated with hyperglycemia, its down-regulation may be the principle of the anti-diabetic effect of rosiglitazone. Therefore, if the total enzyme activity of acetyl coenzyme A acyltransferase 2 is attenuated, it produces a good clinical effect equivalent to that of rosiglitazone on hyperglycemia and skeletal muscle insulin sensitivity. It may be enough to get. Our data support the development of antagonists of acetyl coenzyme A acyltransferase 2 as therapeutic agents for the treatment of insulin resistance and diabetes.

  Furthermore, our results indicate that modulators of ACAA2 activity, such as inhibitors, activators, antagonists or agonists of ACAA2, treat disorders such as obesity, diabetes, and insulin resistance, as well as insulin secretion. Indicates that it may be useful for enhancement.

Discovery Process In the following paragraphs, the protein encoded by acetyl coenzyme A acyltransferase 2 and any variants thereof are suitable as diagnostic markers for obesity and diabetes, therapeutic antibody targets and small molecule drug targets. Describe the study design (s) and techniques used to identify existing proteins and variants.

Example D1. Mouse diet-induced obesity
A protocol for studying mouse diet-induced obesity is disclosed in Example Q1.

  The main cause of obesity in the clinical population is excessive caloric intake. This so-called diet-induced obesity (DIO) is mimicked in an animal model (mouse strain C57BL) by breeding on a high fat diet with a fat content greater than 40%. This DIO study was designed to identify changes in gene expression that contribute to the development and progression of diet-induced obesity. In addition, this study design sought to identify factors that induce resistance to the effects of high-fat diet in certain individuals, thereby preventing obesity. The study sample group had control body weights + 1 S.D., +4 S.D. and +7 S.D. Furthermore, the biochemical profile of +7 SD mice shows that these animals are further stratified into mice that maintain normal blood glucose profile despite obesity and mice that exhibit hyperglycemia. It was done. The test tissues were hypothalamus, brain stem, liver, retroperitoneal white adipose tissue (WAT), epididymal WAT, brown adipose tissue (BAT), gastrocnemius muscle (fast twitch skeletal muscle) and soleus muscle (slow twitch muscle) Skeletal muscle). Differences in gene expression profiles for these tissues indicated genes and pathways that could be used as therapeutic targets for obesity and / or diabetes. Protocols relating to differential gene expression analysis, GeneCalling® are disclosed in Example Q7.

Results First, the mouse acetyl coenzyme A acyltransferase 2 gene fragment is 1.6 times higher in the muscles of obese and diabetic mice (hgsd7) fed on a high fat diet compared to mice fed on a normal diet (solid feed) It turns out that it is controlled. Here, CuraGen's GeneCalling (registered trademark) method showing a difference in gene expression was used. It was clearly identified that a mouse gene fragment showing an expression difference that moves to a position of about 380.4 nucleotides in length is a component of mouse acetyl coenzyme A acyltransferase 2 cDNA. Competitive PCR was used to confirm this gene evaluation. The electropherographic peak corresponding to the mouse acetyl coenzyme A acyltransferase 2 gene fragment disappears when the gene-specific primer (shown in Table D1) competes with the primer in the linker-adapter during PCR amplification. there were. The 380.4 nt long position disappeared in samples from both obese / diabetic mice and normal chow mice.

Example D2. Insulin sensitivity in rat skeletal muscle A protocol for rat insulin sensitivity studies is disclosed in Example Q3.

  ZDF rats were treated with various substances known to alter insulin sensitivity. Metformin, vanadate, and AICAR enhance the tissue response to insulin, while free fatty acids produced by Liposyn (intravenous lipid infusion). ZDF rats or their lean littermates were treated with various substances known to alter insulin sensitivity. Metformin, vanadate, and AICAR enhance the tissue response to insulin, while the free fatty acids produced by Liposyn (intravenous lipid infusion) treatment reduce this response. Various tissues were collected including: gastrocnemius and soleus, liver, retroperitoneum and epididymis WAT, and IBAT. Protocols relating to differential gene expression analysis, GeneCalling® are disclosed in Example Q7.

Results The gene fragment of rat acetyl coenzyme A acyltransferase 2 was found to be down-regulated 2.5-fold in the gastrocnemius muscle of vanadate, AICAR and metformin-treated ZDF rats compared to the soleus muscle. Here, CuraGen's GeneCalling (registered trademark) method showing a difference in gene expression was used. It was clearly identified that the rat gene fragment showing an expression difference that moves to a position of about 353 nucleotides in length is a component of rat acetyl coenzyme A acyltransferase 2 cDNA. Competitive PCR was used to confirm this gene evaluation. The electroferrograph peak corresponding to the rat acetyl coenzyme A acyltransferase 2 gene fragment disappears when the gene-specific primer (shown in Table D2) competes with the primer in the linker-adapter during PCR amplification. there were. In the sample from both gastrocnemius and soleus muscles, the peak at about 353 nt length disappeared.

Example D3. Mouse TZD Response Study A protocol for mouse TZD response study is disclosed in Example Q4.

  Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor subfamily of transcription factors and plays a major role in metabolic control (Lee CH, Olson P, Evans RM. Minireview: lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors.Endocrinology. 2003 Jun; 144 (6): 2201-7. PMID: 12746275). Thiazolidinedione (TZD) drugs, such as rosiglitazone, are synthetic agonists of the PPARg receptor. Rosiglitazone can normalize elevated plasma glucose levels in obese and diabetic rodents and is a highly therapeutic agent for the treatment of human non-insulin-dependent diabetes mellitus. Often effective (Doggrell S. Do peroxisome proliferation receptor-γ antagonists have clinical potential as combined antiobesity and antidiabetic drugs? Expert Opin Investig Drugs. 2003 Apr; 12 (4): 713-6., PMID: 12665425; Gurnell M, Savage DB, Chatterjee VK, O'Rahilly S. The metabolic syndrome: peroxisome proliferator-activated receptor gamma and its therapeutic modulation. J Clin Endocrinol Metab. 2003 Jun; 88 (6): 2412-21 PMID: 12788836). Diabetic animals show a unique response to TZD treatment. To understand the principle of this unique response, we compared changes in gene expression between diabetic animals that showed the effect of TZD treatment and diabetic animals that did not respond to TZD treatment. Female db / db mice were treated daily with 10 mg / kg body weight rosiglitazone for 7 days. On the 8th day, blood was collected from this mouse and blood glucose level was measured. Treated mice were classified into either a “responding animal group” that showed a significant reduction in hyperglycemia or a “non-responding animal group” that showed no change in blood glucose level. Gene expression in skeletal muscle and adipose tissue was compared between untreated diabetic mice and 2 subgroups of TZD treated mice. Protocols relating to differential gene expression analysis, GeneCalling® are disclosed in Example Q7.

Results First, using CuraGen's GeneCalling (registered trademark) method showing differential gene expression, a fragment of the mouse acetyl coenzyme A acyltransferase 2 gene was found in skeletal muscle of rosiglitazone-treated diabetic mice that showed improvement in hyperglycemia It was found that the blood glucose level was down-regulated 1.7 times compared to rosiglitazone-treated mice. The same fragment is known to be down-regulated 1.9-fold compared to diabetic controls in skeletal muscle of TZD-treated mice with improved hyperglycemia. It was clearly identified that a mouse gene fragment showing a difference in expression that migrates to about 252 nucleotides by capillary gel electrophoresis is a component of mouse acetyl coenzyme A acyltransferase 2 cDNA. Competitive PCR was used to confirm this gene evaluation. The electroferrograph peak corresponding to the mouse acetyl coenzyme A acyltransferase 2 gene fragment disappears when the gene-specific primer (shown in Table D3) competes with the primer in the linker-adapter during PCR amplification. there were. These data indicate that acetyl coenzyme A acyltransferase 2 is involved in skeletal muscle insulin resistance and diabetes progression.

Example D4. Identification of human acetyl coenzyme A acyltransferase 2 gene sequence The sequence of the human acetyl coenzyme A acyltransferase 2 gene (accession number CG181387-01) was obtained by cloning a cDNA fragment at the laboratory level. This cloning was performed by in silico prediction of the sequence. A group of cDNA fragments containing the full length of this DNA sequence, or part of the sequence, or both were cloned. In silico prediction was based on sequences available in CuraGen's private sequence database or public human sequence database to obtain full-length DNA sequences, or any portion thereof. A protocol for identifying human sequence (s) is disclosed in Example Q8.

  Table D4 shows the protein sequence alignment (ClustalW) of acetyl coenzyme A acyltransferase 2 for human (CG181387-01; SEQ ID NO: 30), rat (P13437; SEQ ID NO: 192) and mouse (BC028901; SEQ ID NO: 193) versions. Show. Table D5 shows the protein sequences of rat (P13437; SEQ ID NO: 192) and mouse (BC028901; SEQ ID NO: 193) versions of acetyl coenzyme A acyltransferase 2.

  Laboratory cloning was performed using one or more methods outlined in Example Q8. The NOV4 clone was analyzed to obtain the nucleotide sequence shown in Table D6 and the encoded polypeptide sequence.

  In the ClustalW comparison of the protein sequences, the sequence alignment shown in Table D7 below is obtained.

  The NOV4a protein was further analyzed and the properties shown in Table D8 below were obtained.

  The NOV4a protein was searched against the Geneseq database and several homologous proteins shown in Table D9 were obtained. This database is a private database containing sequences published in patents and patent publications.

  In a BLAST search of public sequence databases, the NOV4a protein was found to have homology with the proteins shown in the BLASTP data of Table D10.

  As predicted by PFam analysis, the NOV4a protein contains the domains shown in Table D11.

Example D5. Mutant of human acetyl coenzyme A acyltransferase 2 gene and SNP
Variant sequences are included in this application. Variant sequences can include single nucleotide polymorphisms (SNPs). In some cases, the SNP can be referred to as “cSNP”, which indicates that the nucleotide sequence containing this SNP is originally cDNA. SNPs can occur by several routes. For example, SNPs can arise from the substitution of one nucleotide with another at the polymorphic site. Such a substitution can be a base rearrangement or base conversion. SNPs can also result from deletion of nucleotides or insertion of nucleotides relative to a reference allele. In this case, the polymorphic site is a site where one allele holds a gap with respect to a specific nucleotide in another allele. When SNP is present in a gene, the amino acid encoded by the gene may change at the position of this SNP. However, intragenic SNPs may be silent, when the codon containing the SNP encodes an invariant amino acid due to the degeneracy of the genetic code. When SNP is present outside the gene region or in an intron within the gene, the amino acid sequence of the protein does not change, but the control of the expression pattern may change. This change is, for example, changes in temporal expression, physiological response control, cell type expression control, expression intensity, and stability of the transcribed message.

Novel SNP Identification Method: SNPs are identified by analyzing sequence assembly using CuraGen's private SNPTool algorithm. SNPTool uses the following criteria to identify mutations in the assembly: SNPs within 10 base pairs at both ends of the alignment are not analyzed; the window size (number of bases in the field of view) is 10; Is 2; minimum SNP base quality (PHRED score) is 23; the smallest change to score SNP is 2 / assembly position. SNPTool analyzes the assembly and displays the SNP position in the assembly, the associated individual variant sequence, the degree of assembly at this default position, the estimated assembly allele frequency, and the SNP sequence variation. The sequence trace is then selected and brought into the field for manual verification. Import consensus assembly sequences along with mutant sequence changes into CuraTools to identify candidate amino acid changes due to SNP sequence mutations. The comprehensive SNP data analysis is then exported to the SNP Calling database.

New SNP confirmation method: SNP is confirmed using a proven method known as pyrosequencing. A detailed protocol for Pyrosequencing can be found in: Alderborn et al. Determination of Single Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing. (2000). Genome Research. 10, Issue 8, August. 1249-1265.

  Simply put, pyrosequencing is a genotyping real-time primer extension process. In this protocol, double-stranded biotin-labeled PCR products are collected from a genomic DNA sample and bound to streptavidin beads. These beads are then denatured to obtain single-stranded bound DNA. The characterization of SNP is performed using a technique based on assaying pyrophosphate (PPi) released from each dNTP during DNA strand elongation by indirect bioluminescence measurement. When PPi is released following Klenow polymerase-mediated base uptake, it is used with adenosine 5′-phosphosulfate (APS) as a substrate for ATP sulfurylase to form ATP. Subsequently, luciferase acts by this ATP, and luciferin is converted to its oxy derivative. Subsequently, light is emitted, and the amount of light emitted is proportional to the number of bases added up to about 4 bases. Excess dNTPs are degraded by apyrase to allow the process to proceed. Apyrase is also included in the starting reaction mixture so that only a small amount of dNTP is added to the template during sequencing. This process is fully automated and tailored to a 96-well format, so it is possible to rapidly screen large SNP panels.

Results The DNA and protein sequences of the novel single nucleotide polymorphism variant of acetyl coenzyme A acyltransferase 2-like gene CuraGen accession number CG181387-01 are reported in Table D10. Variants are reported individually, but any combination of all or a selected subset of variants is also included. In Table D10, the positions of the mutant base and the mutant amino acid residue are underlined. Overall, there are 6 variants reported in Table D10. Mutant 13380063 is an SNP in which the 697 bp position of the nucleotide sequence is changed from A to G, and this mutation changes the amino acid position 217 of the protein sequence from Met to Val. Mutant 13380064 is a SNP in which the 1250 bp position of the nucleotide sequence is changed from A to G, and this SNP is not in the amino acid coding region, and therefore does not change the protein sequence. Mutant 13380065 is an SNP in which the 1451 bp position of the nucleotide sequence is changed from A to G, and this SNP is not in the amino acid coding region, and therefore does not change the protein sequence. Mutant 13380066 is an SNP in which the 1466 bp position of the nucleotide sequence is changed from C to T, and since this SNP is not in the amino acid coding region, the protein sequence does not change. Mutant 13380067 is an SNP in which the 1525 bp position of the nucleotide sequence is changed from A to C, and this SNP is not in the amino acid coding region, and therefore does not cause a change in the protein sequence. Also, variant 13380068 is an SNP in which the 1550 bp position of the nucleotide sequence is changed from T to C, and since this SNP is not in the amino acid coding region, it does not cause a change in the protein sequence.

Table D10. Nucleotide sequence accession numbers CG181387-01 (SEQ ID NO: 29) variants

Example D6. Expression profile of human acetyl coenzyme A acyltransferase 2 gene A protocol for quantitative expression analysis is disclosed in Example Q9.

  The expression of genes CG181387-01 and CG181387-02 was evaluated using the primer-probe set Ag6643 described in Table D13. The results of performing RTQ-PCR are shown in Tables D14 and D15.

Table D13. Probe name Ag6643

Table D14. General Screening Panel v1.6

Table D15. Panel 5 Islands

General Screening Panel v1.6 Summary: Ag6643 Maximum expression of this gene was detected in gastric cancer KATO III cell line (CT = 24.2). In addition, in the cluster of cancer cell lines derived from pancreatic cancer, stomach cancer, colon cancer, lung cancer, liver cancer, kidney cancer, breast cancer, ovarian cancer, prostate cancer (prostate cancer), melanoma and brain cancer, Medium to high levels of expression were observed. Thus, the expression of this gene could be used as a marker to detect the presence of these cancers. Therapeutic regulation of this gene, expressed protein and / or the use of antibodies or small molecule drugs targeting this gene or gene product can be used for pancreatic cancer, stomach cancer, colon cancer, lung cancer, liver cancer, kidney cancer, breast cancer, ovarian cancer, prostate It may be useful for the treatment of cancer, squamous cell carcinoma, melanoma and brain cancer.

  This gene was expressed at medium to high levels in pancreas, fat, adrenal gland, thyroid, pituitary, skeletal muscle, heart, liver and gastrointestinal tract among tissues having metabolic or endocrine functions. The therapeutic regulation of this gene, expressed protein and / or the use of antibodies or small molecule drugs that target this gene or gene product may be useful in the treatment of endocrine / metabolic related diseases such as obesity and diabetes. is there.

  In addition, this gene was expressed at moderate levels in all areas of the central nervous system examined, such as the amygdala, hippocampus, substantia nigra, thalamus, cerebellum, cerebral cortex and spinal cord. Therapeutic regulation of this gene, expressed protein and / or the use of antibodies or small molecule drugs that target this gene or gene product may lead to central nervous system disorders such as Alzheimer's disease, Parkinson's disease, It may be useful in the treatment of epilepsy, multiple sclerosis, schizophrenia and depression.

Panel 5 Island Overview: Ag6643 Maximum expression of this gene was detected in differentiated fat (CT = 28.2). High expression of this gene was observed in differentiating and fully differentiated adipose tissue, and in addition, medium level expression of this gene was detected in islet cells, skeletal muscle, small intestine and heart.

Example D7. Assay for Modulators of Acetyl-Coenzyme A Acyltransferase 2 One potential assay that may be used for screening for modulators of acetyl-coenzyme A acyltransferase 2 follows the reduction of enol and chelating acetoacetyl-CoA. Measure acetoacetyl-CoA degradation. This measurement is performed by optical assay with absorbance measurement at 305 nm as described in the publication (Berndt H, Schlegel HG. Kinetics and properties of β-ketothiolase from Clostridium pasteurianum. Arch Microbiol. 1975 Mar 12; 103 (1 ): 21-30, PMID: 240336).

  Another potential assay that may be used to screen for modulators of acetyl coenzyme A acyltransferase 2 is NAD + / NADH production in a reaction coupled with the conversion of 3-hydroxyacyl CoA to 3-oxoacyl CoA by adjacent mitochondrial enzymes (Barycki JJ, O'Brien LK, Bratt JM, Zhang R, Sanishvili R, Strauss AW, Banaszak LJ. Biochemical characterization and crystal structure determination of human heart short chain L-3-hydroxyacyl-CoA dehydrogenase provide insights into Bio mechanism. 1999 May 4; 38 (18): 5786-98. PMID: 10231530).

  Our results indicate that modulators of ACAA2 activity, such as inhibitors, activators, antagonists or agonists of ACAA2, are useful for treating disorders such as obesity, diabetes, and insulin resistance, and for enhancing insulin secretion. Indicates that it may be useful.

E. NOV5-Phosphoglycerate Mutase 2
Phosphoglycerate mutase is a dimeric enzyme and contains various proportions of slowly migrating (PGM2) and fast migrating brain (PGM1) isozymes in different tissues. These isozymes are encoded by different genes. PGM1 is a ubiquitously expressed isozyme and is most highly expressed in the brain. In contrast, PGM2 is specifically expressed in skeletal muscle. Phosphoglycerate mutase is an enzyme involved in the second stage of glycolysis (conversion from glyceraldehyde 3-phosphate to pyruvate). Complete deficiency of PGM2 results in a muscle phenotype with mild myopathy and exercise intolerance (Tsujino S, Shanske S, Sakoda S, Fenichel G, DiMauro S. The molecular genetic basis of muscle phosphoglycerate mutase (PGAM) deficiency. (1993) Am. J. Hum. Genet. 52 (3): 472-7 PMID: 8447317). This phenotype may promote energy expenditure in diabetic / obese symptoms.

  The inventors have found that most enzymes involved in the second stage of glycolysis are upregulated in diabetic skeletal muscle. This finding is consistent with published data showing that glycolytic enzyme activity increases and oxidative enzyme activity decreases in skeletal muscle from diabetic / obese patients (Simoneau JA, Kelley DE. Altered glycolytic and oxidative capacities of skeletal muscle contribute to insulin resistance in NIDDM. (1997) J. Appl. Physiol. 83 (1): 166-71 PMID: 9216960). When the glycolytic / oxidative capacity balance is disrupted, the lipid content increases, resulting in the development of insulin resistance in skeletal muscle.

  Data indicating the inventors' differential expression (GeneCalling®) data show that PGM1 is up-regulated in the soleus of diabetic animals compared to non-diabetic animals. PGM1 was found to be more highly expressed in glycolytic muscle fibers than in oxidized muscle fibers. RTQ-PCR results indicate that PGM2 is specifically expressed in muscle tissue as well as being upregulated in insulin resistant skeletal muscle from Gestational Diabetes patients. Upregulation of both PGM1 and PGM2 detected in our studies will contribute to the increased glycolytic muscle activity reported for diabetic symptoms.

  FIG. 3 shows how altered expression of human phosphoglycerate mutase 2 and the associated gene product function in relation to obesity and / or diabetes pathogenesis and pathogenicity. This scheme includes the novel findings of the inventors' research, as well as the content reported in the publication.

FIG. 4 is an overview of potential assays that may be used for biochemistry of human phosphoglycerate mutase 2 and screening for therapeutic antibodies or small molecule drugs to treat obesity and / or diabetes. In the presence of 2,3-bisphosphoglycerate, phosphoglycerate mutase interacts between 3-phosphoglycerate and 2-phosphoglycerate, ie glyceraldehyde-3-phosphate The third stage of conversion from pyruvate to:
It catalyzes 3-phosphoglyceric acid ← → 2-phosphoglyceric acid.

  Activation of glycolysis can unbalancely increase glycolytic activity in diabetic skeletal muscle and contribute to the development of insulin resistance. Since PGM2 is specifically expressed in skeletal muscle, inhibition of this may restore energy balance and promote the oxidized muscle phenotype with minimal impact on glycolysis in other tissues. is there. Inhibiting the action of human phosphoglycerate mutase 2 will result in a decrease in insulin resistance, a major problem of obesity and / or diabetes. Thus, antagonists or inhibitors of PGM2 may be used to treat obesity and / or diabetes.

  Cell lines that express phosphoglycerate mutase 2 are those described in the RTQ-PCR results described herein. These and other phosphoglycerate mutase 2 expressing cell lines could be used for screening purposes. Assays for measuring phosphoglycerate mutase activity and selective inhibitors for PGM2 are described in publications (e.g. White MF, Fothergill-Gilmore LA. Development of a mutagenesis, expression and purification system for yeast phosphoglycerate mutase. Investigation of the role of active-site His181.Eur J Biochem. 1992 Jul 15; 207 (2): 709-14.PMID: 1386023, Rigden DJ, Walter RA, Phillips SE, Fothergill-Gilmore LA. Polyanionic inhibitors of phosphoglycerate mutase: combined structural and biochemical analysis. See J Mol Biol. 1999 Jun 18; 289 (4): 691-9. PMID: 10369755).

  Furthermore, our results indicate that modulators of phosphoglycerate mutase 2 activity, such as inhibitors, activators, antagonists or agonists of phosphoglycerate mutase 2, such as obesity, diabetes, and insulin resistance Shows that it may be useful in treating disorders, as well as enhancing insulin secretion.

Discovery process In the diet-induced obesity study, dysregulation of PGM1 isoforms indicates that increased phosphoglycerate mutase activity in skeletal muscle may contribute to the development of insulin resistance and diabetes . Based on the expression data, the inventors have identified PGM2, a protein encoded by the major skeletal muscle isoform CG186640-02 of phosphoglycerate mutase and any variants thereof, a diagnostic marker for obesity and diabetes, Proteins and variants that are suitable as therapeutic antibody targets and small molecule drug targets are proposed. The dysregulation of the PGM1 gene in the following GeneCalling® study supports the disease principle for phosphoglycerate mutase isoform 2.

  In the following paragraphs, phosphoglycerate mutase-2 encoded protein and any variants thereof, proteins and mutations suitable as diagnostic markers for obesity and diabetes, therapeutic antibody targets and small molecule drug targets Describe the study design (s) and techniques used to identify the body.

Example E1. Mouse Diet-Induced Obesity A protocol for studying mouse diet-induced obesity is disclosed in Example Q1.

  The main cause of obesity in the clinical population is excessive caloric intake. This so-called diet-induced obesity (DIO) is mimicked in an animal model (mouse strain C57BL / 6J) by breeding on a high fat diet with a fat content greater than 40%. This DIO study was designed to identify changes in gene expression that contribute to the development and progression of diet-induced obesity. In addition, this study design sought to identify factors that induce resistance to the effects of high-fat diet in certain individuals, thereby preventing obesity. The study sample group had control body weights + 1 S.D., +4 S.D. and +7 S.D. Furthermore, the biochemical profile of +7 SD mice shows that these animals are further stratified into mice that maintain normal blood glucose profile despite obesity and mice that exhibit hyperglycemia. It was done. The test tissues were hypothalamus, brain stem, liver, retroperitoneal white adipose tissue (WAT), epididymal WAT, brown adipose tissue (BAT), gastrocnemius muscle (fast twitch skeletal muscle) and soleus muscle (slow twitch muscle) Skeletal muscle). Differences in gene expression profiles for these tissues indicated genes and pathways that could be used as therapeutic targets for obesity. Protocols relating to differential gene expression analysis, GeneCalling® are disclosed in Example Q7.

Results First, we found that the mouse phosphoglycerate mutase 1 gene fragment was upregulated 2.7 times in the soleus muscle of hyperglycemic (hgsd7, diabetic) mice compared to normoglycemic (sd1, normal control) mice It was. Here, CuraGen's GeneCalling® method showing gene expression differences was used (Table E1 shows the alignment of PGM1 and PGM2 protein sequences, and Table E2 shows the PGM1 protein sequences separately). It was clearly identified that the mouse gene fragment showing an expression difference that moves to a position of about 153 nucleotides in length is a component of mouse phosphoglycerate mutase 1 cDNA. Competitive PCR was used to confirm this gene evaluation. The electropherographic peak corresponding to the mouse phosphoglycerate mutase 1 gene fragment disappeared when the gene-specific primer (shown in Table E3) competed with the primer in the linker-adapter during PCR amplification. The peak at the 153 nt long position disappeared in samples from both hyperglycemic and normoglycemic mice. The mouse phosphoglycerate mutase 1 gene fragment is also found to be up-regulated approximately 2-fold in (glycolytic) gastrocnemius muscle fibers of obese hyperglycemic mice compared to slow twitch (oxidized) muscle fibers. It was. These data indicate that phosphoglycerate mutase may be involved in the development of diabetes / obesity and that its modulation, eg inhibition, may be beneficial in the treatment of these diseases.

Example E2. Identification of the human sequence of phosphoglycerate mutase 2 The sequence of human phosphoglycerate mutase 2 (accession number CG186640-02) was obtained by cloning a cDNA fragment at the laboratory level. This cloning was performed by in silico prediction of the sequence. A group of cDNA fragments containing the full length of this DNA sequence, or part of the sequence, or both were cloned. In silico prediction was based on sequences available in CuraGen's private sequence database or public human sequence database to obtain full-length DNA sequences, or any portion thereof.

  Table E4 shows protein sequence alignment (ClustalW) of phosphoglycerate mutase 2 for human (CG186640-02; SEQ ID NO: 46), rat (M31835; SEQ ID NO: 199) and mouse (AF029843; BC010750; SEQ ID NO: 200) versions. Show. Table E5 shows the sequences of the rat (M31835; SEQ ID NO: 199) and mouse (AF029843; BC010750; SEQ ID NO: 200) versions of phosphoglycerate mutase 2.

  Laboratory cloning was performed using one or more methods outlined in Example Q8. The NOV5 clone was analyzed and the nucleotide sequence shown in Table E6 and the encoded polypeptide sequence were obtained.

  In the ClustalW comparison of the protein sequences, the sequence alignment shown in Table E7 below is obtained.

  The NOV5a protein was further analyzed and the properties shown in Table E8 below were obtained.

  The NOV5a protein was searched against the Geneseq database and several homologous proteins shown in Table E9 were obtained. This database is a private database containing sequences published in patents and patent publications.

  In a BLAST search of public sequence databases, the NOV5a protein was found to have homology with the proteins shown in the BLASTP data in Table E10.

  As predicted by PFam analysis, the NOV5a protein contains the domains shown in Table E11.

Example E3. Expression profiles of human phosphoglycerate mutase 2 (PGM2) and human phosphoglycerate mutase 1 (PGM1) genes A protocol for quantitative expression analysis is disclosed in Example Q9.

  The expression of genes CG186640-02 (PGM2) and CG115294-01 (PGM1) was evaluated using the primer-probe sets Ag2379 and Ag6474 described in Tables E12 and E13. The results of performing RTQ-PCR are shown in Tables E14 and E15. Ag2379 and Ag6474 are specific for CG115294-01 and CG186640-02, respectively.

Table E12. Probe name Ag2379

Table E13. Probe name Ag6474

Table E14. General_Screening_Panel_v1.6

Table E15. Panel 5 Islands

General Screening Panel v1.6 Summary: PGM2 is specifically expressed in skeletal muscle, which is consistent with published data. PGM1 is ubiquitously expressed and its expression is higher in the cerebellum than PGM2 (CT = 26.8 vs 29.5), in contrast to skeletal muscle, which is lower than PGM2 (CT = 32.2 vs 26.6). Because PGM2 is specifically expressed in skeletal muscle, the drug will target skeletal muscle in advance and will not significantly interfere with glycolysis in other tissues, especially brain and islets To come up with.

Panel 5 Island Overview: In panel 5I, PGM2 is expressed only in skeletal muscle, which is consistent with the expression pattern in panel 1.4. PGM2 is also significantly upregulated in diabetic muscle (patient 12) compared to non-diabetic skeletal muscle (patient 7, patient 9, patient 11). These results further support the hypothesis that increased phosphoglycerate mutase levels / activity may contribute to the development of diabetes. Therefore, PGM2 inhibition may be beneficial for the treatment of diabetes. PGM1 is more widely expressed and is highly expressed in islet cells (Bayer patient 1).

Example E4. Assay for modulators of phosphoglycerate mutase 2 One potential assay that may be used for screening for modulators of phosphoglycerate mutase 2 is to measure the production of glycerate-3-phosphate, This glycerate-3-phosphate is formed by the following reaction catalyzed by phosphoglycerate mutase:

2-phospho-D-glyceric acid + 2,3-diphosphoglyceric acid <=> 3-phospho-D-glyceric acid + 2,3-diphosphoglyceric acid

  The production of glycerate-3-phosphate could be measured by a reaction coupled with phosphoglycerate kinase and glyceraldehyde phosphate dehydrogenase. This measurement was performed by Rosa R, Blouquit Y, Calvin MC, Prome D, Prome JC, Rosa J. Isolation, characterization, and structure of a mutant 89 Arg ---- Cys bisphosphoglycerate mutase.Implication of the active site in the mutation. J Biol Chem. 1989 May 15; 264 (14): 7837-43; PMID: 2542247.

  Our results indicate that modulators of phosphoglycerate mutase 2 activity, such as inhibitors, activators, antagonists or agonists of phosphoglycerate mutase 2, have been found to be effective in disorders such as obesity, diabetes, and insulin resistance. It indicates that it may be useful for treatment as well as enhancing insulin secretion.

F. NOV6-adenosine A1 receptor Adenosine A1 receptor (Adora1) is a G protein-coupled receptor found in the plasma membrane of many cell types. Adora1 is thought to be involved in cardiac contractility, adipose tissue lipolysis, glomerular filtration and renal glomerular feedback in the kidney, and sympathetic and parasympathetic activity of the nervous system (Fredholm BB, IJzerman AP, Jacobson KA, Klotz KN, Linden J, International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev. 2001 Dec; 53 (4): 527-52. Review. PMID: 11734617). This receptor is a member of the adenosine receptor family consisting of the adenosine A1, A2A, A2B and A3 receptors. Adenosine is the preferred endogenous ligand for these receptors. However, these adenosine receptors are not highly homologous and have the greatest percent identity (56% sequence identity at the protein level) seen between the A1 and A2B receptors. The low level of sequence identity is explained by the fact that selective agonists and antagonists have been identified for all adenosine receptors except A2B. A2B lacks a selective antagonist (Fredholm BB, IJzerman AP, Jacobson KA, Klotz KN, Linden J, International Union of Pharmacology.XXV.Nomenclature and classification of adenosine receptors.Pharmacol Rev. 2001 Dec; 53 (4) : 527-52. Review. PMID: 11734617).

  Adenosine is a major agonist of this receptor family. Under normal conditions, adenosine is continuously formed within cells of various cell types as well as extracellular of various tissue compartments. In any cell type or tissue compartment, the continued synthesis and decay of adenosine varies depending on the physiological environment, and of course, this difference naturally complicates the biology of adenosine receptors, and tissue and cell types. It is specific. Adenosine A1 receptor knockout mice that do not respond to Adora1 agonists have been reported (Johansson B, Halldner L, Dunwiddie TV, Masino SA, Poelchen W, Gimenez-Llort L, Escorihuela RM, Fernandez-Teruel A, Wiesenfeld-Hallin Z, Xu XJ, Hardemark A, Betsholtz C, Herlenius E, Fredholm BB.Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the adenosine A1 receptor.Proc Natl Acad Sci US A. 2001 Jul 31; 98 (16): 9407-12 PMID: 11470917). The mice were viable, fertile and did not have any severe damage. Hypothesis suggests that Adora1 is important for cardiovascular function, but arterial pressure and heart rate were indistinguishable between A1 receptor knockout and wild-type mice (Johansson et. Al.). To date, knowledge about the role of adenosine A1 receptors in normal physiological functions is incomplete.

  The tissue distribution of A1 receptors has been studied using selective radioligands (Fredholm et.al.). Its high expression has been reported in the brain (cortex, cerebellum, hippocampus), dorsal horn of the spinal cord, eyes and adrenal glands (Fredholm et.al.). Intermediate levels of Adora1 have been reported in other brain regions, skeletal muscle, liver, kidney, adipose tissue, salivary gland, esophagus, large intestine and testis. Currently, the clear role of Adora1 for all these cell types is unknown.

  Insulin-secreting beta cells are located on the islets of Langerhans in the pancreas. Insulin secretion can be measured while the isolated pancreas is perfused with glucose (physiological signal). Perfusion of isolated rat pancreas with a structural analog of adenosine results in decreased insulin secretion (Hillaire-Buys D, Chapal J, Bertrand G, Petit P, Loubatieres-Mariani MM. Purinergic receptors on insulin-secreting cell. Fundam Clin Pharmacol 1994 8 (2): 117-27. PMID: 8020870). However, this result did not clarify Adora1 specificity (because adenosine acts on all adenosine receptor subtypes) and was not specific for human islet cells.

  Aminophylline is a non-selective but potent antagonist of the adenosine receptor family. Intravenous aminophylline stimulated insulin secretion in patients with type 2 diabetes. This indicates that adenosine receptor family antagonists may be involved in insulin secretion (Arias AM, Bisschop PH, Ackerman MT, Nijpels G, Endert E, Romijn JA, Sauerwein HP.Aminophylline stimulates insulin secretion in Patients with Type 2 diabetes mellitus. Metabolism 2001 Sep; 50 (9): 1030-5. PMID: 11555834). The present invention relates to the use of specific Adora1 antagonists that enhance insulin secretion and reduce blood glucose. US 6,407,076 describes specific compounds. This compound is an agonist of the adenosine A1 receptor, functions as a lipolysis inhibitor, and may have an increased ability to lower blood sugar.

The adenosine A1 receptor couples with the Gα _i family of G protein effector systems and inhibits cAMP production (Fredholm et. Al.). Cyclic AMP has long been known as an enhancer of neurotransmitter-induced insulin secretion (Harndahl L, Jing XJ, Ivarsson R, Degerman E, Ahren B, Manganiello VC, Renstrom E, Holst LS. Important role of phosphodiesterase 3B for the stimulatory action of cAMP on pancreatic β-cell exocytosis and release of insulin. J Biol Chem. 2002 Oct 4; 277 (40): 37446-55. PMID: 12169692). The present invention relates to the use of specific Adora1 antagonists that enhance glucose-stimulated insulin secretion (as opposed to neurotransmitter-stimulated insulin secretion).

  The inventors have shown that Adora1 is expressed in a wide variety of human tissues, with the highest levels of expression observed in the brain and testis. The inventors have also recorded that Adora1 is expressed in human islet cells.

  Human type 2 diabetes is characterized by increased circulating free fatty acids during fasting and after eating, and impaired insulin secretion. The hypothesis is that increased free fatty acid levels are a major contributor to impaired insulin secretion. The pancreatic islets of Langerhans have beta cells that secrete insulin in response to increased blood glucose. In vitro culture of islet cells with oleate, one of the three most abundant circulating fatty acids in humans, provides a means to study beta cell secretion disorders associated with type 2 diabetes. The inventors have found that islet cells cultured in vitro with oleic acid for 5 days are significantly deficient in glucose-stimulated insulin secretion and upregulated Adora1 mRNA by a factor of 1.9. Thus, increased Adora1 expression in oleic acid-treated islets results in increased receptor activity in islet cells, decreased cAMP levels, and consequent decreased insulin secretion. A preferred method of the invention is to use the adenosine A1 receptor to identify antagonists that would be beneficial for the treatment of type 2 diabetes. Thus, the present invention embodies the use of recombinantly expressed proteins and / or endogenously expressed proteins for various screens that identify such therapeutic antibodies and / or small therapeutic molecules.

  In one embodiment, the present invention describes that Adora1 mRNA is specifically upregulated in rat islet cells cultured in oleic acid. Insulin cultured in oleic acid is significantly deficient in insulin secretion. Upregulation and activation of Adora1 in oleic acid cultured islands may be responsible for insulin secretion suppression.

  In particular, the invention relates to the use of Adora1 protein as a diagnostic and / or small molecule drug and therapeutic antibody target. The inventors have recorded that Adora1 is expressed in a wide variety of human tissues, with the highest expression in the brain and testis. The inventors have also newly discovered that Adora1 is expressed in human islet cells. Furthermore, the inventors have discovered that adenosine A1 receptor mRNA is up-regulated 1.9-fold on oleic acid-treated islands. In this oleic acid-treated island, it has been identified in the art that glucose-stimulated insulin secretion is suppressed as compared to the control island. Increased Adora1 expression on oleic acid-treated islets results in increased receptor activation in islet cells, decreased cAMP levels, and consequently decreased insulin secretion. In one aspect, it is preferred that Adora1 is activated by an agonist to reduce cellular cAMP accumulation. Thus, the present invention describes the role that Adora1 activation plays in suppressing glucose-stimulated insulin secretion (rather than neurotransmitter-stimulatory).

  Without being limited to a specific mechanism of action, the inventors have discovered that Adora1 inhibition may have a beneficial effect in the treatment of type 2 diabetes. Specifically, Adora1 is expressed in several metabolic tissues, including pancreatic islets of Langerhans. Thus, the inventors have shown that when islets are cultured in 2 mM oleic acid for 5 days, increased expression of islet cells Adora1 occurs. Increased Adora1 expression and / or activation may contribute to impaired insulin secretion in oleic acid-treated islands and type 2 diabetic patients. The finding that inhibition of glucose-stimulated insulin secretion in islet cells correlates with upregulation of adenosine A1 receptor mRNA in islet cells indicates a role for Adora1 in insulin secretion and glucose homeostasis. Therefore, Adora1 antagonists are useful in the treatment of diabetes. In a specific embodiment of the invention, Adora1 is a target for screening for antagonists of Adora1 expression or activity. That is, the present invention relates to the use of recombinantly expressed protein and / or endogenously expressed protein for various screens identifying therapeutic antibodies and / or small therapeutic molecules useful for the treatment of type 2 diabetes. It is to be embodied.

  Furthermore, the inventors' results indicate that modulators of Adora1 activity, such as inhibitors, activators, antagonists or agonists of Adora1, have been shown to treat disorders such as obesity, diabetes, and insulin resistance, and insulin secretion. Indicates that it may be useful for enhancement.

Discovery Process In the following paragraphs, adenosine A1 receptor-encoded protein and any variants thereof, which are suitable as diagnostic markers for obesity and diabetes, therapeutic antibody targets and small molecule drug targets and Describe the study design (s) and techniques used to identify the variants.

Example F1. Rat islet study:
The protocol for studying rat islets is disclosed in Example Q2.

  Over 80% of human type 2 diabetes is associated with obesity. An important clinical goal for early type 2 diabetes is to increase insulin secretion from pancreatic beta cells. A number of substances capable of modulating insulin secretion have been identified experimentally as well as under therapeutic conditions. When applied to isolated rat islets, changes in gene expression can correlate with insulin secretion. In this study, acute and chronic gene expression changes were examined on islands treated with drug substance after short-term (4 hours) and long-term (5 days) exposure, respectively, and compared to ground state (11 mM glucose). This substance contains high concentrations (25 mM) glucose, glucose (11 mM) and exendin-4 (1 nM), glucose (11 mM) and glibenclamide (50 uM), and glucose (11 mM) and oleate (oleate ) (2 mM). Obesity-related type 2 diabetes is characterized by an increase in both free and postprandial circulating free fatty acids. The hypothesis is that increased free fatty acid levels are a major contributor to the impaired insulin secretion observed in this disease. Islet cells are cultured in vitro with oleic acid, one of the three most abundant circulating fatty acids in humans, to provide a means to study beta cell secretion disorders associated with type 2 diabetes. A protocol for differential gene expression analysis, GeneCalling® is disclosed in Example Q7.

Results First, rat adenosine A1 receptor gene fragments were compared to islet cells treated with glucose alone in samples derived from oleic acid-treated islet cells 5 days after exposure to oleic acid, a known insulin secretion inhibitor. It was found to be up-regulated 1.9 times. Here, CuraGen's GeneCalling (registered trademark) method showing a difference in gene expression was used. It was clearly identified that the rat gene fragment showing a differential expression that moves to a position of about 370 nucleotides in length is a component of the rat adenosine A1 receptor cDNA. Competitive PCR was used to confirm this gene evaluation. The electropherographic peak corresponding to the rat adenosine A1 receptor gene fragment disappeared when the gene-specific primer (shown in Table F1) competed with the primer in the linker-adapter during PCR amplification. In the sample derived from oleic acid-treated and basal islet cells, the peak at the 370 nt long position disappeared.

  Furthermore, it was found that the second fragment of the rat adenosine A1 receptor gene was up-regulated 1.8-fold in the same oleic acid-treated islet cell sample (5 days after exposure to oleic acid). A rat gene fragment exhibiting this differential expression that migrates to approximately 383.4 nucleotides in length was also clearly identified as a component of the rat adenosine A1 receptor cDNA. This gene identification was performed by competitive PCR using gene specific primers (shown in Table F2) that compete with primers in the linker-adapter during PCR amplification. In samples derived from oleic acid-treated and basal islet cells, the electropherographic peak corresponding to the gene fragment at the 383 nt long position disappeared.

Example F2. Identification of human adenosine A1 receptor gene sequence The sequence of the human adenosine A1 receptor gene (accession number CG58655-01) was obtained by cloning a cDNA fragment at the laboratory level. This cloning was performed by in silico prediction of the sequence. A group of cDNA fragments containing the full length of this DNA sequence, or part of the sequence, or both were cloned. In silico prediction was based on sequences available in CuraGen's private sequence database or public human sequence database to obtain full-length DNA sequences, or any portion thereof. A protocol for identifying human sequence (s) is disclosed in Example Q8.

  Table F3 shows the protein alignment (ClustalW) of CG58655-01 (SEQ ID NO: 56) and a rat homologue of adenosine A1 receptor (scb_gb-m64299_1; SEQ ID NO: 209). Table F4 shows the protein sequence of the rat homologue of the adenosine A1 receptor (scb_gb-m64299_1; SEQ ID NO: 209).

  Laboratory cloning was performed using one or more methods outlined in Example Q8. The NOV6 clone was analyzed to obtain the nucleotide sequence shown in Table F5 and the encoded polypeptide sequence.

  In the ClustalW comparison of the protein sequences, the sequence alignment shown in Table F6 below is obtained.

  The NOV6a protein was further analyzed and the properties shown in Table F7 below were obtained.

  The NOV6a protein was searched against the Geneseq database and several homologous proteins shown in Table F8 were obtained. This database is a private database containing sequences published in patents and patent publications.

  In a BLAST search of public sequence databases, the NOV6a protein was found to have homology with the proteins shown in the BLASTP data of Table F9.

  As predicted by PFam analysis, the NOV6a protein contains the domains shown in Table F10.

Example F3. Human adenosine A1 receptor gene variant and SNP
Variant sequences are included in this application. Variant sequences can include single nucleotide polymorphisms (SNPs). In some cases, the SNP can be referred to as “cSNP”, which indicates that the nucleotide sequence containing this SNP is originally cDNA. SNPs can occur by several routes. For example, SNPs can arise from the substitution of one nucleotide with another at the polymorphic site. Such a substitution can be a base rearrangement or base conversion. SNPs can also result from deletion of nucleotides or insertion of nucleotides relative to a reference allele. In this case, the polymorphic site is a site where one allele holds a gap with respect to a specific nucleotide in another allele. When SNP is present in a gene, the amino acid encoded by the gene may change at the position of this SNP. However, intragenic SNPs may be silent, when the codon containing the SNP encodes an invariant amino acid due to the degeneracy of the genetic code. When SNP is present outside the gene region or in an intron within the gene, the amino acid sequence of the protein does not change, but the control of the expression pattern may change. This change is, for example, changes in temporal expression, physiological response control, cell type expression control, expression intensity, and stability of the transcribed message.

Novel SNP Identification Method: SNPs are identified by analyzing sequence assembly using CuraGen's private SNPTool algorithm. SNPTool uses the following criteria to identify mutations in the assembly: SNPs within 10 base pairs at both ends of the alignment are not analyzed; the window size (number of bases in the field of view) is 10; Is 2; minimum SNP base quality (PHRED score) is 23; the smallest change to score SNP is 2 / assembly position. SNPTool analyzes the assembly and displays the SNP position in the assembly, the associated individual variant sequence, the degree of assembly at this default position, the estimated assembly allele frequency, and the SNP sequence variation. The sequence trace is then selected and brought into the field for manual verification. Import consensus assembly sequences along with mutant sequence changes into CuraTools to identify candidate amino acid changes due to SNP sequence mutations. The comprehensive SNP data analysis is then exported to the SNP Calling database.

New SNP confirmation method: SNP is confirmed using a proven method known as pyrosequencing. A detailed protocol for Pyrosequencing can be found in: Alderborn et al. Determination of Single Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing. (2000). Genome Research. 10, Issue 8, August. 1249-1265.

  Simply put, pyrosequencing is a genotyping real-time primer extension process. In this protocol, double-stranded biotin-labeled PCR products are collected from a genomic DNA sample and bound to streptavidin beads. These beads are then denatured to obtain single-stranded bound DNA. The characterization of SNP is performed using a technique based on assaying pyrophosphate (PPi) released from each dNTP during DNA strand elongation by indirect bioluminescence measurement. When PPi is released following Klenow polymerase-mediated base uptake, it is used with adenosine 5′-phosphosulfate (APS) as a substrate for ATP sulfurylase to form ATP. Subsequently, luciferase acts by this ATP, and luciferin is converted to its oxy derivative. Subsequently, light is emitted, and the amount of light emitted is proportional to the number of bases added up to about 4 bases. Excess dNTPs are degraded by apyrase to allow the process to proceed. Apyrase is also included in the starting reaction mixture so that only a small amount of dNTP is added to the template during sequencing. This process is fully automated and tailored to a 96-well format, so it is possible to rapidly screen large SNP panels.

Results The DNA and protein sequences of the novel single nucleotide polymorphism variant of the adenosine A1 receptor-like gene CuraGen accession number CG58655-01 are reported in Table F11. Variants are reported individually, but any combination of all or a selected subset of variants is also included. In Table F11, the position of the mutant base and the mutant amino acid residue are underlined. Overall, there is one variant reported in Table F11. Mutant 13381538 is an SNP in which the 717 bp position of the nucleotide sequence is changed from T to C, and this mutation changes amino acid position 238 of the protein sequence from Leu to Pro.

Table F11. Variants of nucleotide sequence accession number CG58655-01 (SEQ ID NO: 55)

Example F4. Expression profile of human adenosine A1 receptor gene (CG58655-01) A protocol for quantitative expression analysis is disclosed in Example Q9.

  The expression of the gene CG58655-01 was evaluated using the primer-probe set Ag6342 described in Table F13. The results of performing RTQ-PCR are shown in Tables F14, F15, F16, and F17.

Table F13. Probe name Ag6342

Table F14. General Screening Panel v1.5

Table F15. General Screening Panel v1.7

Table F16. Panel 5 islands

Table F17. Human metabolic system

General Screening Panel v1.7 Summary: (Ag6342) Maximum expression of this gene was detected in the ovarian cancer cell line IGROV-1 (CT = 24). This gene is overexpressed in some ovarian cancer cell lines compared to normal tissues. This gene was overexpressed in some renal cancer cell lines compared to normal tissues. The therapeutic regulation of this gene, expressed protein and / or the use of antibodies or small molecule drugs targeting this gene or gene product may be useful in the treatment of renal and ovarian cancer.

  Among tissues having metabolic or endocrine functions, this gene was expressed at significantly high to medium levels in pancreas, fat, adrenal gland, thyroid, pituitary, skeletal muscle, heart, liver and gastrointestinal tract. The therapeutic regulation of this gene, expressed protein and / or the use of antibodies or small molecule drugs that target this gene or gene product are useful in the treatment of endocrine / metabolic related diseases such as obesity and diabetes.

Human Metabolic System Summary: (Ag6342) Maximum expression of this gene was detected in the hypothalamus of diabetic patients. This gene is expressed at high levels in the hypothalamus of diabetic patients. This gene was expressed at low levels in visceral fat, skeletal muscle and pancreas. The therapeutic regulation of this gene, expressed protein and / or the use of antibodies or small molecule drugs that target this gene or gene product are useful in the treatment of endocrine / metabolic related diseases such as obesity and diabetes.

Panel 5 Island Overview: (Ag6342) Expression of this gene was down-regulated in differentiating and differentiated adipocytes. The therapeutic regulation of this gene, expressed protein and / or the use of antibodies or small molecule drugs that target this gene or gene product is useful in the treatment of obesity. In accordance with the present invention, this data is the first to show that this receptor subtype is expressed in human islet cells (Bayer patient 1).

Example F5. Activation of adenosine A1 receptor modulators relates to screening assays adenosine A1 receptors mediate the inhibition of cAMP formation via a signaling G _i protein. This activation is associated with insulin secretion, since an increase in intracellular cAMP enhances insulin secretion. Type 2 diabetes is caused by very little insulin secretion. Thus, if there is an antagonist of Adora1, its action will increase insulin secretion, ie this antagonist would be suitable for the treatment of type 2 diabetes.

  Although the disclosed adenosine A1 receptor sequence is the preferred isoform, any other isoform may be used for similar purposes. Also, under various assay conditions, the isoform listed may be replaced by another isoform depending on the conditions. The CG58655-01 gene described herein, which encodes the human adenosine A1 receptor, is representative of a full-length physical clone and is used directly for expression and screening purposes. It's okay.

  Assays for screening for modulators of human Adora1 can be constructed using an incomplete list of cell lines that express Adora1. This list is derived from the RTQ-PCR results presented herein.

Activation of the adenosine A1 receptors mediate the inhibition of adenylate cyclase and cAMP formation via a signaling G _i protein. Measurement of intracellular cAMP can be used to assay inhibition of adenosine A1 receptor. Cyclic AMP measurements can be configured using the Biotrak cAMP assay system (Amersham Biosciences, Piscataway, NJ, USA) (Harndahl L, Jing XJ, Ivarsson R, Degerman E, Ahren B, Manganiello VC, Renstrom E, Holst LS. Important role of phosphodiesterase 3B for the stimulatory action of cAMP on pancreatic β-cell exocytosis and release of insulin. J Biol Chem. 2002 Oct 4; 277 (40): 37446-55. PMID: 12169692). An increase in cAMP will indicate a positive screening. In order to assess the efficacy of the compound (s) thus identified, its effect on glucose-stimulated insulin secretion can be assayed in vitro using the dispersed rat islets used herein, Alternatively, an in vivo assay can be performed based on the effect on blood glucose in a known rodent type 2 diabetes model.

  Our results show that modulators of Adora1 activity, such as inhibitors, activators, antagonists or agonists of Adora1, are useful for treating disorders such as obesity, diabetes, and insulin resistance, and for enhancing insulin secretion. Indicates that it may be useful.

G. NOV7-3-hydroxy-3-methylglutaryl coenzyme A lyase (HMG-CoA lyase or HMG-CoA or HL)
3-Hydroxy-3-methylglutaryl coenzyme A lyase catalyzes the final step of ketone body formation. This ketone body production is an important pathway of mammalian energy metabolism. HMG-CoA lyase deficiency, known as hydroxymethylglutaric aciduria, is an autosomal recessive congenital deficiency in humans that leads to the development of hypoglycemia and coma.

HMG-CoA lyase has the following catalytic activity:

(s) -3-Hydroxy-3-methylglutaryl-CoA = acetyl-CoA + acetoacetate

  HMG-CoA affects biochemical pathways related to the pathogenesis and pathogenicity of obesity and / or diabetes. This scheme includes the new findings of these discovery studies, along with what is reported in the publication. Inhibiting the action of human HMG CoA lyase will result in a decrease in insulin resistance, a major problem of obesity and / or diabetes. HMG-CoA lyase produces acetoacetate and acetyl-CoA using HMG-CoA as a substrate. This is the final stage of ketone body formation and leucine metabolism. Importantly, the acetyl-CoA derived from this reaction can be fed back not only to the TCA cycle but also to the lipid biosynthesis pathway.

  The inventors have found that mitochondrial 3-hydroxy-3methylglutaryl coenzyme A lyase (mHMG-CoA lyase) is upregulated in the liver of SHR and WKY rats after treatment with triglitazone. mHMG-CoA lyase is involved in the final steps of ketone body formation and leucine catabolism, and produces acetoacetate (ketone body) and acetyl-CoA using 3-hydroxy-methylglutaryl-CoA as its substrate. This process occurs in the liver, especially during weight loss, and the amount of acetyl-CoA produced during both fatty acid oxidation and ketone body formation often exceeds the capacity of the TCA cycle. In addition, excess citrate pushes acetyl-CoA back into the cytoplasm, where acetyl-CoA is used for cholesterol and fatty acid biosynthesis. Thus, inhibition of this enzyme during weight loss may slow ketone body formation and acetyl-CoA production and thus prevent saturation of the TCA cycle.

  The above formula outlines the biochemistry for human HMG-CoA lyase and potential assays that may be used for screening therapeutic antibodies or small molecule drugs to treat obesity and / or diabetes. Cell lines that express HMG-CoA lyase can be obtained from the RTQ-PCR results presented herein. These and other HMG-CoA lyase expressing cell lines could be used for screening purposes.

  Furthermore, our results show that modulators of HMG CoA lyase activity, such as inhibitors, activators, antagonists or agonists of HMG CoA lyase, treat disorders such as obesity, diabetes, and insulin resistance, As well as potential enhancement of insulin secretion.

Discovery Process In the following paragraphs, the HMG-CoA lyase-encoded protein and any variants thereof, proteins suitable as diagnostic markers for obesity and diabetes, therapeutic antibody targets and small molecule drug targets, and Describe the study design (s) and techniques used to identify the variants.

Example G1. Insulin Resistance Study A protocol for insulin resistance study is disclosed in Example Q5.

  Spontaneously hypertensive rats (SHR) are strains exhibiting characteristics of human metabolic syndrome X (Metabolic Syndrome X). Phenotypic characteristics include obesity, hyperglycemia, hypertension, dyslipidemia and dysfibrinolysis. Tissues were removed from adult male rats and control strains (Wistar-Kyoto) and the differences in gene expression, the principle of their pathology, were identified for animals treated with this SHR and various anti-hyperglycemic agents such as troglitizone. Tissues including subcutaneous fat, visceral fat and liver were studied.

  First, it was found that the gene fragment of rat HMG-COA lyase was up-regulated 1.6 times in the liver of WKY rats treated with troglitazone LD10 compared to WKY rats treated with 0.02% DMSO as a control. Here, CuraGen's GeneCalling (registered trademark) method showing a difference in gene expression was used. For troglitazone-treated and untreated WKY control rats, it was clearly identified that the rat gene fragment showing differential expression that migrates to approximately 426.4 nucleotides in length is a component of rat HMG CoA lyase cDNA. Competitive PCR was used to confirm this gene evaluation. The chromatographic peak corresponding to the rat HMG CoA lyase gene fragment disappeared when the gene-specific primer (shown in Table G1) competed with the primer in the linker-adapter during PCR amplification. The 426.4 nt long peak disappeared in samples from both troglitazone treated and untreated WKY control rats. Altered expression of these genes in animal models supports the role of HMG CoA lyase in obesity and / or diabetes pathogenesis.

  First, it was found that the gene fragment of rat HMG CoA lyase was upregulated 2.6 times in the liver of SHR rats treated with troglitazone LD10 compared to SHR rats treated with 0.02% DMSO as a control. Here, CuraGen's GeneCalling (registered trademark) method showing a difference in gene expression was used. For troglitazone-treated and untreated SHR control rats, it was clearly identified that the rat gene fragment showing differential expression that migrates to about 48.2 nucleotides in length is a component of rat HMG CoA lyase cDNA. Competitive PCR was used to confirm this gene evaluation. The chromatographic peak corresponding to the rat HMG CoA lyase gene fragment disappeared when the gene-specific primer (shown in Table G2) competed with the primer in the linker-adapter during PCR amplification. The 48.2 nt long peak disappeared in samples from both troglitazone treated and untreated WKY control rats. Altered expression of these genes in animal models supports the role of HMG CoA lyase in obesity and / or diabetes pathogenesis.

Example G2. Identification of human HMG CoA lyase sequence The sequence of human HMG CoA lyase (Acc. No CG96859-03) was obtained by screening a cDNA library at the laboratory level. A two-hybrid approach was used for this screening. Groups of cDNA fragments containing the full-length DNA sequence, or part of the sequence, or both were sequenced. In silico prediction was performed based on sequences available in CuraGen Corporation's private sequence database or public human sequence database to obtain the full length DNA sequence, or any portion thereof. A protocol for identifying human sequence (s) is disclosed in Example Q8.

  Table G3 shows CG96859-03 (SEQ ID NO: 60), HMG CoA lyase with different published 1 aa (amino acid) (P35914; SEQ ID NO: 215), novel spliced HMG CoA lyase (CG96859-02; SEQ ID NO: 66) and protein sequence alignment (ClustalW) of HMG CoA lyase mouse (S65036.1; SEQ ID NO: 216) and rat (Y10054; SEQ ID NO: 217) orthologs. Table G4 shows the protein sequence of the public HMG CoA lyase (P35914; SEQ ID NO: 215), HMG CoA lyase mouse (S65036.1; SEQ ID NO: 216) and rat (Y10054; SEQ ID NO: 217) orthologs.

  In addition to the human version of HMG CoA lyase that has been shown to show differential expression in experimental studies, other variants were identified by direct sequencing of cDNA from various human tissues and public database sequences. ing. Two splicing (type) variants have been identified in CuraGen. The two alternative splicing types are CG96859-02 and CG96859-05. Of all the mutants identified, the preferred mutant to be used for screening purposes is CG96859-03.

  Laboratory cloning was performed using one or more methods outlined in Example Q8. The NOV7 clone was analyzed to obtain the nucleotide sequence shown in Table G5 and the encoded polypeptide sequence.

  In the ClustalW comparison of the protein sequences, the sequence alignment shown in Table G6 below is obtained.

  The NOV7a protein was further analyzed and the properties shown in Table G7 below were obtained.

  The NOV7a protein was searched against the Geneseq database and several homologous proteins shown in Table G8 were obtained. This database is a private database containing sequences published in patents and patent publications.

  In a BLAST search of public sequence databases, the NOV7a protein was found to have homology with the proteins shown in the BLASTP data in Table G9.

  As predicted by PFam analysis, the NOV7a protein contains the domains shown in Table G10.

Example G3. Single nucleotide polymorphism (SNP) of CG96859-03
Variant sequences are included in this application. Variant sequences can include single nucleotide polymorphisms (SNPs). In some cases, the SNP can be referred to as “cSNP”, which indicates that the nucleotide sequence containing this SNP is originally cDNA. SNPs can occur by several routes. For example, SNPs can arise from the substitution of one nucleotide with another at the polymorphic site. Such a substitution can be a base rearrangement or base conversion. SNPs can also result from deletion of nucleotides or insertion of nucleotides relative to a reference allele. In this case, the polymorphic site is a site where one allele holds a gap with respect to a specific nucleotide in another allele. When SNP is present in a gene, the amino acid encoded by the gene may change at the position of this SNP. However, intragenic SNPs may be silent, when the codon containing the SNP encodes an invariant amino acid due to the degeneracy of the genetic code. When SNP is present outside the gene region or in an intron within the gene, the amino acid sequence of the protein does not change, but the control of the expression pattern may change. This change is, for example, changes in temporal expression, physiological response control, cell type expression control, expression intensity, and stability of the transcribed message.

Novel SNP Identification Method: SNPs are identified by analyzing sequence assembly using CuraGen's private SNPTool algorithm. SNPTool uses the following criteria to identify mutations in the assembly: SNPs within 10 base pairs at both ends of the alignment are not analyzed; the window size (number of bases in the field of view) is 10; Is 2; minimum SNP base quality (PHRED score) is 23; the smallest change to score SNP is 2 / assembly position. SNPTool analyzes the assembly and displays the SNP position in the assembly, the associated individual variant sequence, the degree of assembly at this default position, the estimated assembly allele frequency, and the SNP sequence variation. The sequence trace is then selected and brought into the field for manual verification. Import consensus assembly sequences along with mutant sequence changes into CuraTools to identify candidate amino acid changes due to SNP sequence mutations. The comprehensive SNP data analysis is then exported to the SNP Calling database.

New SNP confirmation method: SNP is confirmed using a proven method known as pyrosequencing. A detailed protocol for Pyrosequencing can be found in: Alderborn et al. Determination of Single Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing. (2000). Genome Research. 10, Issue 8, August. 1249-1265.

  Simply put, pyrosequencing is a genotyping real-time primer extension process. In this protocol, double-stranded biotin-labeled PCR products are collected from a genomic DNA sample and bound to streptavidin beads. These beads are then denatured to obtain single-stranded bound DNA. The characterization of SNP is performed using a technique based on assaying pyrophosphate (PPi) released from each dNTP during DNA strand elongation by indirect bioluminescence measurement. When PPi is released following Klenow polymerase-mediated base uptake, it is used with adenosine 5′-phosphosulfate (APS) as a substrate for ATP sulfurylase to form ATP. Subsequently, luciferase acts by this ATP, and luciferin is converted to its oxy derivative. Subsequently, light is emitted, and the amount of light emitted is proportional to the number of bases added up to about 4 bases. Excess dNTPs are degraded by apyrase to allow the process to proceed. Apyrase is also included in the starting reaction mixture so that only a small amount of dNTP is added to the template during sequencing. This process is fully automated and tailored to a 96-well format, so it is possible to rapidly screen large SNP panels.

Results The DNA and protein sequences of the novel single nucleotide polymorphism variant of the hydroxymethylglutaryl-COA lyase-like gene CuraGen accession number CG96859-03 are reported in Table G11. Variants are reported individually, but any combination of all or a selected subset of variants is also included. In Table G11, the positions of the mutant base and the mutant amino acid residue are underlined. Overall, there is one variant reported in Table G11. Mutant 13379476 is a SNP in which the 725 bp position of the nucleotide sequence is changed from C to T, and this mutation does not cause a change in the protein sequence (silent).

Table G11. Nucleotide sequence accession number CG96859-03 (SEQ ID NO: 59) variants

Example G4. Expression profile of HMG CoA lyase (CG96859-03) -like protein A protocol for quantitative expression analysis is disclosed in Example Q9.

  The expression of gene CG96859-03 was evaluated using the primer-probe set Ag8471 described in Table G13. The results of performing RTQ-PCR are shown in Table G14.

Table G13. Probe name Ag8471

Table G14. General_Screening_Panel_v1.7

Panels 1, 1.1, 1.2, and 1.3D
Panels 1, 1.1, 1.2 and 1.3D included 2 control wells (genomic DNA control and chemical control) and 94 wells of cultured cell lines and primary normal tissue derived cDNA samples. The cell lines were derived from carcinomas (ca) including: lung cancer, small cell carcinoma (s cell var), non small cell carcinoma (non-s or non-sm); Breast cancer; melanoma; colon cancer; prostate cancer; glioma (glio), astrocytoma (astro) and neuroblastoma (neuro); squamous cell carcinoma (squam); ovary Cancer; liver cancer; kidney cancer; gastric cancer and pancreatic cancer. These were obtained from the American Type Culture Collection (ATCC, Bethesda, MD). Normal tissue was obtained from individual adults or fetuses including: adult and fetal skeletal muscle, adult and fetal heart, adult and fetal kidney, adult and fetal liver, adult and fetal lung, brain, spleen, Bone marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, large intestine, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and fat. The following abbreviations are used in the results report: metastasis (met); pleural effusion (pl. Eff or pl effusion). And * indicates that it was established from metastasis.

General_Screening_Panels v1.4, v1.5, v1.6 and v1.7
Panels 1.4, 1.5, 1.6 and 1.7 are as described for panels 1, 1.1, 1.2 and 1.3D above, except that normal tissue samples were pooled from 2 to 5 different adults or fetuses. It is.

General_Screening_Panel_v1.7 Summary: Ag8471 Maximum expression of this gene was detected in lung cancer samples (CT = 26). This gene is widely expressed. Among tissues with metabolic or endocrine function, this gene is expressed at high to medium levels in pancreas, fat, adrenal gland, thyroid, pituitary, skeletal muscle, heart, liver and gastrointestinal tract. The therapeutic regulation of this gene, expressed protein and / or the use of antibodies or small molecule drugs that target this gene or gene product are useful in the treatment of endocrine / metabolic related diseases such as obesity and diabetes.

Example G5. Screening assay for modulators of HMG CoA lyase
An incomplete list of cell lines that express HMG CoA lyase can be obtained from the differential gene expression (RTQ-PCR) results presented herein.

  An effective method for measuring the enzymatic activity of HMG-CoA lyase is the Stegink and Coon citrate synthase coupled assay (Stereospecificity and other properties of highly purified β-hydroxy-β-methylglutaryl coenzyme A cleavage enzyme from bovine liver, J Biol Chem. 1968 Oct 25; 243 (20): 5272-9), which is further modified by Kramer and Miziorko (Purification and characterization of avian liver 3-hydroxy-3-methylglutaryl coenzyme A lyase, J Biol Chem. 1980 Nov 25; 255 (22): 11023-8).

  Our results show that modulators of HMG CoA lyase activity, such as inhibitors, activators, antagonists or agonists of HMG CoA lyase, treat disorders such as obesity, diabetes, and insulin resistance, and insulin. It may be useful for enhancing secretion.

Protocols The following examples illustrate the procedures, protocols and techniques described herein.

Example Q1. Study of mouse diet-induced obesity (DIO) (BP24.02)
Overview The main cause of obesity in the clinical population is excessive caloric intake. This so-called diet-induced obesity (DIO) is mimicked in animal models by breeding on a high fat diet with a fat content greater than 40%. This DIO study was designed to identify changes in gene expression that contribute to the development and progression of diet-induced obesity. In addition, this study design sought to identify factors that induce resistance to the effects of high-fat diet in certain individuals, thereby preventing obesity.

  The study sample group usually had control body weights + 1 S.D., +4 S.D. and +7 S.D. Furthermore, according to the biochemical profile of +7 SD mice, these animals are usually further stratified into mice that maintain a normal blood glucose profile despite obesity and mice that exhibit hyperglycemia It has been shown. The test tissues were hypothalamus, brain stem, liver, retroperitoneal white adipose tissue (WAT), epididymal WAT, brown adipose tissue (BAT), gastrocnemius muscle (fast twitch skeletal muscle) and soleus muscle (slow twitch muscle) Skeletal muscle). Differences in gene expression profiles for these tissues indicated genes and pathways that could be used as therapeutic targets for obesity and / or diabetes.

Protocol Five groups of mice, 3 per group, were used. In some cases, more than 3 mice were used in a single group to maintain accurate parameters for the study. Even in this case, only three mice are sacrificed for tissue use. Groups were classified based on the following parameters:
Group 1. Solid feed mice.
Group 2. High fat diet-fed mice. This mouse exceeded the standard chow fed mouse by 1 standard deviation in body weight.
Group 3. High fat diet-fed mice. These mice exceeded the chow fed mice by 4 standard deviations in body weight.
Group 4. High fat diet fed mice. These mice exceeded the chow fed mice by 7 standard deviations in body weight and had normal glucose concentrations.
Group 5. High fat diet-fed mice. The mice exceeded the standard chow fed mice by 7 standard deviations in body weight and were hyperglycemic.

  Three mice in each group were sacrificed to obtain research tissue.

Example Q2. Rat islet study (BP24.03)
Summary An important clinical goal for early type 2 diabetes is to increase insulin secretion from pancreatic beta cells. A number of substances capable of modulating insulin secretion have been identified experimentally as well as under therapeutic conditions. When applied to isolated rat islets, changes in gene expression can correlate with insulin secretion. In this study, acute and chronic gene expression changes were examined on islands treated with drug substance after short-term (4 hours) and long-term (5 days) exposure, respectively, and compared to ground state (11 mM glucose). This substance contains high concentrations (25 mM) glucose, glucose (11 mM) and exendin-4 (1 nM), glucose (11 mM) and glibenclamide (50 uM), and glucose (11 mM) and oleic acid (2 mM).

Protocol All samples were isolated rat islets. These samples differed only in the treatment method to be tested. The following samples were used in the 4 hour treatment group:
1) 11 mM glucose
2) 25 mM glucose
3) 11 mM glucose and JTT 608
4) 11 mM glucose and carbachol
5) 11 mM glucose and exendin-4

  Isolated rat islets were treated with either 11 mM glucose (ground state) or 25 mM glucose (increased glucose). Three additional sets of rat islets were then treated with 11 mM glucose and one of three substances: JTT 608, carbachol, or exendin-4.

The following samples were used in the 5-day treatment group:
1) 11 mM glucose
2) 25 mM glucose
3) 11 mM glucose and 1 nM exendin
4) 11 mM glucose and 50 uM glibenclamide
5) 11 mM glucose and 2 mM oleic acid

  Isolated rat islets were treated with either 11 mM glucose (ground state) or 25 mM glucose (increased glucose). Three additional sets of rat islets were then treated with 11 mM glucose and one of three substances: exendin, glibenclamide, or oleic acid.

  All 10 samples were divided into 2 replicate groups each. These two replication groups were subjected to gene expression difference analysis (GeneCalling (registered trademark)).

Example Q3. Rat insulin sensitivity study (BP24.05)
Protocol ZDF rats or their lean littermates were treated with various substances known to alter insulin sensitivity. Metformin, vanadate, and AICAR enhance the tissue response to insulin, while the free fatty acids produced by Liposyn (intravenous lipid infusion) treatment reduce this response. Various tissues were collected including: gastrocnemius and soleus, liver, retroperitoneum and epididymis WAT, and IBAT.

  Only the gastrocnemius and soleus muscles were subjected to gene expression difference analysis (GeneCalling (registered trademark)) treatment.

The following 5 groups of samples were used:
1) Metformin vehicle (vehicle M)
2) Metformin-treated rats
3) AICAR and Vanadate Vehicle (Vehicle AV)
4) AICAR-treated rats
5) Vanadate-treated rats

  Treatment was performed for 4 hours to obtain glucose values before and after treatment. Each sample was tested in triplicate (5 groups X 3 rats X 2 tissues).

  In the second phase of the study, rats were given intravenous lipid infusions. This treatment should reduce the tissue response to insulin in the subject rats.

The following two groups were used in the intravenous lipid infusion phase of this study:
1) Rats treated with lipid infusion vehicle.
2) Rats with lipid infusion treatment.

  Each group included 3 rats (performed in 3 ways) and the soleus and gastrocnemius muscle samples were subjected to differential gene expression analysis (GeneCalling®) treatment (2 groups X 3 samples X 2 tissues).

Example Q4. Mouse TZD response study (BP24.07)
Overview and Protocol Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor subfamily of transcription factors and plays a major role in metabolic control. Thiazolidinedione (TZD) drugs, such as rosiglitazone, are synthetic agonists of the PPARg receptor. Rosiglitazone can normalize elevated plasma glucose levels in obese, diabetic rodents and is often very effective as a therapeutic agent for the treatment of human non-insulin dependent diabetes mellitus. Diabetic animals show a unique response to TZD treatment. To understand the principle of this unique response, we compared changes in gene expression between diabetic animals that showed the effect of TZD treatment and diabetic animals that did not respond to TZD treatment. Female db / db mice were treated daily with 10 mg / kg body weight rosiglitazone for 7 days. On the 8th day, blood was collected from this mouse and blood glucose level was measured. Treated mice were classified into either a responding animal group that showed a significant reduction in hyperglycemia and a non-responding animal group that did not show a change in blood glucose level. Gene expression in skeletal muscle and adipose tissue was compared between untreated diabetic mice and 2 subgroups of TZD treated mice.

  The following three tissues were collected for differential gene expression analysis (GeneCalling®): liver, thigh muscle, and uterine white fat.

Three groups of samples were used:
1) Vehicle treatment
2) Rosiglitazone responsive animals
3) Rosiglitazone non-responsive animals

  Each group had 3 mice. A total of 27 samples were subjected to gene expression difference analysis (GeneCalling (registered trademark)) processing.

Example Q5. Insulin resistance study (MB.01)
Spontaneously hypertensive rats (SHR) are strains exhibiting characteristics of human metabolic syndrome X. Phenotypic characteristics include obesity, hyperglycemia, hypertension, dyslipidemia and fibrinolysis failure. Tissues were removed from adult male rats and control strains (Wistar_Kyoto) and the differences in gene expression, the principle of their pathology, were identified for animals treated with this SHR and various antiglycemic agents such as troglitisone. Tissues including subcutaneous fat, visceral fat, brain, muscle, and liver were studied. Each tissue was collected in triplicate for gene expression difference analysis (GeneCalling (registered trademark)).

Example Q6. Genetic obese mice versus genetic lean mice study (MB.04)
Summary Numerous genetic models have been studied for obesity, with the most notable in mice and rats. However, only a few causative genes have been identified. Here we studied a set of mouse genetic models. Its purpose is to identify genes related to obesity in mice by positional expression cloning. This gene may be associated with human obesity.

protocol
Seven strains of mice were used. These included mice exhibiting a lean phenotype (skinned), normal weight mice, and genetically obese mice.

In this study, the following strains of mice were used:
1) CAST / EI -lean phenotype
2) SM / J black-Skinny
3) SWR / J-normal phenotype
4) C57L / J-Normal type
5) C57BL / 6J -Normal type
6) AKR / J-obese phenotype
7) NZB / BINJ-Obese type

  The following tissues were subjected to differential gene expression analysis (GeneCalling®) treatment: brain, liver, muscle, and fat.

  Samples were processed in triplicate (ie, 3 brain samples from 3 CAST / EI mice). A total of 7 lines X 4 tissues X 3 samples = 84 samples were used.

Example Q7. Gene and gene product identification method showing differential expression (GeneCalling (registered trademark))
GeneCalling (registered trademark) technology is a method developed by CuraGen for profiling differential gene expression between two or more samples. Shimkets, et al., "Gene expression analysis by transcript profiling coupled to a gene database query "Nature Biotechnology 17: 198-803 (1999). GeneCalling® technology is also disclosed in US Pat. No. 5,871,697. cDNA was obtained from a variety of samples representative of numerous tissue types, normal and disease states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue culture primary cells or cell lines. Cells and cell lines may be treated with biological or chemical substances that control gene expression, such as growth factors, chemokines or steroids. The cDNA thus obtained was then digested with a number of restriction enzymes up to 120 pairs and linker-adapter pairs specific for each restriction enzyme pair were ligated to the appropriate ends. This restriction enzyme digestion creates a mixture of unique cDNA gene fragments. Limited PCR amplification is performed using primers homologous to the linker-adapter sequence, where one primer is labeled with biotin and the other is fluorescently labeled. The double-labeled material was isolated, and the fluorescently labeled single strand was separated by capillary gel electrophoresis. A computer algorithm was used to compare electropherograms from experimental and control groups for each restriction enzyme digestion. This information and additional sequence-derived information are used to predict the identity of each gene fragment that exhibits differential expression. Various gene databases are used for this prediction. Three methods commonly used for confirmation of identity are described below with respect to gene fragments found to show altered expression in obesity and / or diabetes models or their patients.

A). Direct sequencing A gene fragment exhibiting differential expression is isolated, cloned into a plasmid, and sequenced. Thereafter, using this sequence information, an oligonucleotide corresponding to one or both ends of the gene fragment is designed. When this oligonucleotide is used in a competitive PCR reaction, the electroferrographic band from which this sequence is derived is removed.

B). Competitive PCR
In competitive PCR, the electropherographic peak corresponding to the gene fragment of the gene of interest is the primer in the linker-adapter when the PCR-amplified gene-specific primer (designed from a sequenced band or available database) It disappears when competing with.

C). PCR with complete or mismatched 3 'nucleotides (TraPping)
This method utilizes a competitive PCR approach, in which a degenerate set of primers is used that extends one or two nucleotides beyond the flanking restriction enzyme region and into the gene specific region of this fragment. Similar to the competitive PCR approach, primers that cause electropherographic band extinction provide additional sequence information. This additional sequence data, along with the size of the gene fragment and the sequence derived from the 12 nucleotide restriction site, allows one gene to be limited according to database analysis. TraPping is disclosed in published PCT application publication number WO 01/49886.

Example Q8. Identification of human sequences Laboratory level cloning was performed using one or more methods outlined below:

SeqCalling ™ technology: cDNA was obtained from a variety of human samples that were representative of a number of tissue types, normal and disease states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue culture primary cells or cell lines. Cells and cell lines may be treated with biological or chemical substances that control gene expression, such as growth factors, chemokines or steroids. The cDNA thus obtained was then sequenced using CuraGen Corporation's SeqCalling technology. This technique is fully disclosed in US application Ser. No. 09 / 417,386 filed Oct. 13, 1999 (and PCT application publication number: WO 00/40757) and 09 / 614,505 filed Jul. 11, 2000. Yes. Sequence traces were evaluated manually and edited and corrected if appropriate. The cDNA sequences from all samples were assembled together, which could include public human sequences, where a bioinformatics program was used to create a consensus sequence for each assembly. Each assembly is added to the CuraGen Corporation database. A sequence was added as a component for assembly if the degree of identity with another component was at least 95% over 50 bp. Each assembly is representative of the gene or portion thereof and includes information regarding variants such as splicing single nucleotide polymorphisms (SNPs), insertions, deletions and other sequence variations.

  Variant sequences are also included in this application. Variant sequences can include single nucleotide polymorphisms (SNPs). In some cases, the SNP can be referred to as “cSNP”, which indicates that the nucleotide sequence containing this SNP is originally cDNA. SNPs can occur by several routes. For example, SNPs can arise from the substitution of one nucleotide with another at the polymorphic site. Such a substitution can be a base rearrangement or base conversion. SNPs can also result from deletion of nucleotides or insertion of nucleotides relative to a reference allele. In this case, the polymorphic site is a site where one allele holds a gap with respect to a specific nucleotide in another allele. When SNP is present in a gene, the amino acid encoded by the gene may change at the position of this SNP. Intragenic SNPs may be silent, when the codon containing the SNP encodes an invariant amino acid due to the degeneracy of the genetic code. When SNP is present outside the gene region or in an intron within the gene, the amino acid sequence of the protein does not change, but the control of the expression pattern may change. This change is, for example, a change in temporal expression, physiological response control, cell type expression control, expression intensity, and stability of the transcribed message.

Information related to the shared sequence identity of genomic clones, public genes and ESTs with the disclosed sequences and Electronic Northern bioinformatics tools from CuraGen Corporation.
RACE: The predicted sequence of the cDNA of the present invention was isolated or completed using techniques based on the polymerase chain reaction, such as rapid amplification of cDNA ends (RACE). Usually, multiple clones were sequenced from one or more human samples to obtain the sequence for the fragments. Different human tissue samples from different donors were used for the RACE reaction. The sequences obtained by these procedures were added to the SeqCalling assembly process described in the paragraph above.

Exon Linking: cDNA coding for the CG101190-01 sequence was cloned by polymerase chain reaction (PCR). This PCR used primers designed based on known cDNA sequences or in silico predictions of the full length or any part (one or more exons) of the cDNA / protein sequence of the invention. These primers were used to amplify cDNA from pools containing expressive human sequences from the following tissues: adrenal gland, bone marrow, brain-amygdala, brain-cerebellum, brain-hippocampus, brain-substantia nigra, brain-thalamus. , Whole brain, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma-Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis , Thyroid, trachea and uterus.

Physical clone: A PCR product containing the entire open reading frame obtained by exon ligation was cloned into the pCR2.1 vector from Invitrogen to obtain a clone used for expression and screening purposes.

Example Q9. Quantitative expression analysis (RTQ-PCR) of clones in various cells and tissues
Quantitative expression of various NOV genes is assessed using real-time quantitative PCR (RTQ-PCR) using microtiter plates containing RNA samples from various normal and pathological cells, cell lines and tissues did. This RTQ-PCR was performed on an Applied Biosystems (Foster City, CA) ABI PRISM® 7700 or ABI PRISM® 7900 HT Sequence Detection System.

  RNA integrity of all samples was measured by visual assessment of agarose gel electrophoresis. In this visual evaluation, 28S and 18S ribosomal RNA staining intensity ratios (2: 1 to 2.5: 1 28s: 18s) and the absence of low molecular weight RNA (degradation products) were used as guidelines. Control samples that detect genomic DNA contamination include running RTQ-PCR reactions in the absence of reverse transcriptase, which includes probes designed to amplify beyond a single exon. A primer set was used.

  RNA samples were normalized relative to the constitutively expressed genes encoded by the nucleic acids (ie β-actin and GAPDH). Alternatively, non-standardized RNA samples were converted to single-stranded cDNA (sscDNA) using Superscript II (Invitrogen Corporation, Carlsbad, CA, Catalog No. 18064-147) and random hexamers as per manufacturer's instructions. . Reactions or scales containing up to 10 μg total RNA in a 20 μl volume were scaled up to contain 50 μg total RNA in a 100 μl volume and incubated at 42 ° C. for 60 minutes. Thereafter, sscDNA samples were standardized based on the nucleic acid.

  Probes and primers were designed according to the Applied Biosystems Primer Express Software package (version I, for Apple Computer Macintosh Power PC) or similar algorithms, where the target sequence was used as input. Before selecting primers, the default reaction conditions and the following parameters were set: Primer concentration 250 nM; Primer melting temperature (Tm) range 58 ° C-60 ° C; Primer optimal Tm59 ° C; Maximum primer difference 2 ° C (probe) Probe Tm must be 10 ° C. higher than primer Tm), and amplicon size 75 bp to 100 bp. Selection probes and primers were synthesized by Synthegen (Houston, TX). The probe was double purified by HPLC to remove unbound dye and evaluated by mass spectrometry to verify that the reporter and quencher dye were bound to the 5 'and 3' ends of the probe, respectively. These final concentrations were: 900 nM forward and reverse primers, and 200 nM probe.

  Normalized RNA was spotted in individual wells of 96 or 384 well PCR plates (Applied Biosystems, Foster City, CA). PCR cocktails included single gene specific probes and primer sets or two multiplex probes and primer sets. The PCR reaction was performed using TaqMan® One-Step RT-PCR Master Mix (Applied Biosystems, Catalog No. 4313803) according to the manufacturer's instructions. Reverse transcription was performed at 48 ° C. for 30 minutes, then amplified / PCR cycle: 95 ° C. for 10 minutes, then 40 cycles of 95 ° C. for 15 seconds, then 60 ° C. for 1 minute. Results were recorded as CT values (cycles where a given sample crosses the threshold level of fluorescence) and plotted using a log scale. The difference in RNA concentration between a given sample and the sample with the lowest CT value is expressed as 2 to the power of delta CT. The relative expression percentage was the reciprocal of this RNA difference multiplied by 100. CT values less than 28 indicate high expression, 28-32 range indicates moderate expression, 32-35 range indicates low expression, and greater than 35 expression Indicates that the level is too low to be measured accurately.

  Standardized sscDNA was analyzed by RTQ-PCR, where 1X TaqMan® Universal Master mix (Applied Biosystems; catalog No. 4324020) was used according to the manufacturer's instructions. PCR amplification and analysis was performed as described above.

Panels 1, 1.1, 1.2, and 1.3D
Panels 1, 1.1, 1.2 and 1.3D included 2 control wells (genomic DNA control and chemical control) and 94 wells of cultured cell lines and primary normal tissue derived cDNA samples. The cell lines were derived from carcinomas (ca) including: lung cancer, small cell carcinoma (s cell var), non-small cell carcinoma (non-s or non-sm); breast cancer; melanoma; colon cancer; prostate Cancer: glioma (glio), astrocytoma (astro) and neuroblastoma (neuro); squamous cell carcinoma (squam); ovarian cancer; liver cancer; kidney cancer; gastric cancer and pancreatic cancer. These were obtained from the American Type Culture Collection (ATCC, Bethesda, MD). Normal tissue was obtained from individual adults or fetuses including: adult and fetal skeletal muscle, adult and fetal heart, adult and fetal kidney, adult and fetal liver, adult and fetal lung, brain, spleen, Bone marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, large intestine, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and fat. The following abbreviations are used in the results report: metastasis (met); pleural effusion (pl. Eff or pl effusion). And * indicates that it was established from metastasis.

General_Screening_Panels v1.4, v1.5, v1.6 and v1.7
Panels 1.4, 1.5, 1.6 and 1.7 are as described for panels 1, 1.1, 1.2 and 1.3D above, except that normal tissue samples were pooled from 2 to 5 different adults or fetuses. It is.

Panel 2D, 2.2, 2.3 and 2.4
Panels 2D, 2.2, 2.3 and 2.4 show two control wells and the National Cancer Institute Cooperative Human Tissue Network (CHTN) or the National Disease Research Initiative (NDRI), Ardais (Lexington, MA) or Clinomics BioSciences (Frederick, MD ) Containing 94 wells containing RNA or cDNA from human surgical specimens obtained from. The tissues included human malignant and possibly matched adjacent normal tissues (NAT). Information regarding tumor differentiation grade, as well as histopathological assessment of the clinical stage of the patient from which the acquired sample was derived, was generally available. Normal tissue RNA and cDNA samples were purchased from various commercial sources such as Clontech (Palo Alto, CA), Research Genetics and Invitrogen (Carlsbad, CA).

HASS panel v 1.0
The HASS panel v1.0 included 93 cDNA samples and 2 controls including: 81 samples of cultured human cancer cell lines, which were subjected to various conditions, serum starvation, acidic conditions (acidosis) according to established procedures. ) And anaerobic conditions; 3 human primary cells; 9 malignant brain cancers (4 medulloblastomas and 5 neuroblastomas); and 2 controls. Cancer cell lines (ATCC) were cultured using the recommended conditions. This cancer cell line includes: breast cancer, prostate cancer, bladder cancer, pancreatic cancer and CNS cancer. Primary human cells were obtained from Clonetics (Walkersville, MD). Malignant brain samples were provided by the Henry Ford Cancer Center.

ARDAIS panel v1.0 and v1.1
ARDAIS panels v1.0 and v1.1 included 2 control and 22 test samples including: human lung adenocarcinomas, lung squamous cell carcinomas, and Optionally compatible neighboring normal tissue (NAT). This was obtained from Ardais (Lexington, MA). Lung-derived unmatched malignant and non-malignant RNA samples were obtained from Ardais with a total histopathological assessment of tumor differentiation grade and stage and the patient's clinical status.

ARDAIS prostate v1.0
The ARDAIS prostate v1.0 panel included two controls and 68 trial samples of human prostate malignant and optionally compatible adjacent normal tissue (NAT). This was obtained from Ardais (Lexington, MA). RNA from incompatible malignant and non-malignant prostate samples was also obtained from Ardais, with a total histopathological assessment of tumor differentiation grade and stage and the patient's clinical status.

ARDAIS kidney v1.0
The ARDAIS kidney v1.0 panel included 2 control wells and 44 test samples of human renal cell carcinoma and optionally compatible adjacent normal tissue (NAT). This was obtained from Ardais (Lexington, MA). Incompatible renal cell carcinoma and normal tissue-derived RNA were also obtained from Ardais, with a total histopathological assessment of tumor differentiation grade and stage and the clinical status of the patient.

ARDAIS breast v1.0
The ARDAIS breast v1.0 panel included 2 control wells and 71 test samples of human breast malignant and optionally compatible adjacent normal cells (NAT). This was obtained from Ardais (Lexington, MA). RNA from incompatible malignant and non-malignant breast samples was also obtained from Ardais, with a total histopathological assessment of tumor differentiation grade and stage and the clinical status of the patient.

Panel 3D, 3.1 and 3.2
Panels 3D, 3.1, and 3.2 contained 2 controls, 92 cDNA samples of cultured human cancer cell lines and 2 samples of human primary cerebellum. Cell lines derived from the following (ATCC, National Cancer Institute (NCI), German tumor cell bank) were cultured as recommended: squamous cell carcinoma of the tongue, melanoma, sarcoma, leukemia, lymphoma (lymphoma), and epidermoid carcinomas, bladder, pancreas, kidney, breast, prostate, ovary, uterus, cervix, stomach, colon, lung and CNS cancer.

Panels 4D, 4R, and 4.1D
Panels 4D, 4R, and 4.1D contain 2 controls and 94 test samples of RNA (panel 4R) or cDNA (panels 4D and 4.1D) from human cell lines or tissues associated with inflammatory conditions. there were. Controls included total RNA from normal tissues such as large intestine, lung (Stratagene, La Jolla, CA), thymus and kidney (Clontech, Palo Alto, CA). Total RNA from cirrhotic and lupus kidneys was obtained from BioChain Institute, Inc., (Hayward, CA). Crohn's intestinal and ulcerative colitis samples were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA). Cells were purchased from Clonetics (Walkersville, MD) including: astrocytes, lung fibroblasts, dermal fibroblasts, coronary artery smooth muscle cells, peripheral airway epithelium, bronchial epithelium, dermal microvascular endothelial cells, pulmonary microvascular endothelium Cells, human pulmonary aortic endothelial cells, and human umbilical vein endothelium. These primary cell types include various cytokines (IL-1 beta -1-5 ng / ml, TNF alpha -5-10 ng / ml, IFN gamma -20-50 ng / ml, IL-4 -5-10 (ng / ml, IL-9 to 5-10 ng / ml, IL-13 5-10 ng / ml) or a combination of the above cytokines. Starved endothelial cells were cultured in basal medium with 0.1% serum (Clonetics, Walkersville, MD).

Mononuclear cells were prepared from blood donations using Ficoll. LAK cells are medium (DMEM, 5% FCS (Hyclone, Logan, UT), 100 mM non-essential amino acids (Gibco / Life Technologies, Rockville, MD), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10 ^-5 M (Gibco), and 10 mM Hepes (Gibco)] and interleukin 2 for 4-6 days. Cells are activated for 6 hours with 10-20 ng / ml PMA and 1-2 μg / ml ionomycin, 5-10 ng / ml IL-12, 20-50 ng / ml IFN gamma or 5-10 ng / ml IL-18 did. Optionally, mononuclear cells were cultured for 4-5 days in medium containing ˜5 mg / ml PHA (phytohaemagglutinin) or PWM (American pokeweed mitogen; Sigma-Aldrich Corp., St. Louis, MO). Samples for RNA preparation were taken at 24, 48 and 72 hours. MLR (mixed lymphocyte culture reaction) samples are drawn from 2 donors, mononuclear cells are isolated using Ficoll, and these are mixed 1: 1 in media at a final concentration of ~ 2x10 ⁶ cells / ml Was obtained by MLR samples for RNA preparation were collected at various time points from 1-7 days.

  Monocytes were isolated from mononuclear cells. For this isolation, CD14 Miltenyi beads, + ve VS selection column and Vario Magnet (Miltenyi Biotec, Auburn, CA) were used according to the manufacturer's instructions. Monocytes were differentiated into dendritic cells by culturing for 5-7 days in a medium containing 50 ng / ml GMCSF and 5 ng / ml IL-4. Macrophages are cultured on monocytes for 5-7 days in medium containing ~ 50 ng / ml 10% AB human serum (Life technologies, Rockville, MD) or MCSF (Macrophage colony stimulating factor; R & D, Minneapolis, MN) Prepared. Monocytes, macrophages and dendritic cells were stimulated with 100 ng / ml lipopolysaccharide (LPS) for 6 or 12-14 hours. Dendritic cells were also stimulated with 10 μg / ml anti-CD40 monoclonal antibody (Pharmingen, San Diego, Calif.) For 6 or 12-14 hours.

CD4 + lymphocytes, CD8 + lymphocytes and NK cells were also isolated from mononuclear cells. For this isolation, CD4, CD8 and CD56 Miltenyi beads, positive VS selection columns and Vario Magnet (Miltenyi Biotec, Auburn, CA) were used according to the manufacturer's instructions. CD45 + RA and CD45 + RO CD4 + lymphocytes were isolated by depleting CD8 +, CD56 +, CD14 + and CD19 + cells from mononuclear cells using CD8, CD56, CD14 and CD19 Miltenyi beads and positive selection . CD45 + RO CD4 + lymphocytes were then separated from CD45 + RA CD4 + lymphocytes using CD45RO Miltenyi beads. CD45 + RA CD4 +, CD45 + RO CD4 + and CD8 + lymphocytes were cultured at 10 ⁶ cells / ml in the medium. This culture was performed on culture plates precoated overnight with 0.5 mg / ml anti-CD28 (Pharmingen, San Diego, Calif.) And 3 μg / ml anti-CD3 (OKT3, ATCC) in PBS. After 6 and 24 hours, the cells were harvested for RNA preparation. Chronically activated To prepare CD8 + lymphocytes, isolated CD8 + lymphocytes are activated on anti-CD28, anti-CD3 coated plates for 4 days, then harvested and IL-2 (1 ng / ml ). These CD8 + cells were again activated with anti-CD3 and anti-CD28 binding plates for 4 days and grown as described above. RNA was isolated 6 and 24 hours after secondary activation and after 4 days of secondary growth culture. The isolated NK cells were cultured in a medium containing 1 ng / ml IL-2 for 4-6 days, after which RNA was prepared.

B cells were prepared from tonsil tissue (NDRI) that was subdivided and passed through a sieve. Tonsil cells were pelleted and resuspended at 10 ⁶ cells / ml in medium. Activate cells with 5 μg / ml PWM (Sigma-Aldrich Corp., St. Louis, MO) or ~ 10 μg / ml anti-CD40 (Pharmingen, San Diego, CA) and 5-10 ng / ml IL-4 did. Cells were harvested for RNA preparation after 24, 48 and 72 hours.

To prepare primary and secondary Th1 / Th2 and Tr1 cells, cord blood CD4 + lymphocytes (Poietic at 10 ⁵ -10 ⁶ cells / ml in medium containing IL-2 (4 ng / ml) in 6-well Falcon plates Systems, German Town, MD) (This Falcon plate is precoated overnight with 10 μg / ml anti-CD28 (Pharmingen) and 2 μg / ml anti-CD3 (OKT3; ATCC), then washed twice with PBS Was).

  IL-12 (5 ng / ml) and anti-IL4 (1 μg / ml) were used to promote Th1 phenotypic differentiation; IL-4 (5 ng / ml) and anti-IL for Th2 phenotypic differentiation. -IFN gamma (1 μg / ml) was used; and IL-10 (5 ng / ml) was used for Tr1 phenotypic differentiation. After 4-5 days, the activated Th1, Th2 and Tr1 lymphocytes were washed once with DMEM and grown in a medium containing IL-2 (1 ng / ml) for 4-7 days. Activated Th1, Th2 and Tr1 lymphocytes were restimulated with anti-CD28 / CD3 and the above cytokines for 5 days, in which case anti-CD95L (1 μg / ml) was added to prevent apoptosis. After 4-5 days, the Th1, Th2 and Tr1 lymphocytes were washed and grown in a medium containing IL-2 for 4-7 days. Activated Th1 and Th2 lymphocytes were maintained for up to 3 cycles. RNA was prepared from primary and secondary Th1, Th2 and Tr1 after 6 and 24 hours secondary and tertiary activation with plate-bound anti-CD3 and anti-CD28 mAb and after 4 days of secondary and tertiary growth cultures .

Leukocyte cell lines Ramos, EOL-1, and KU-812 were obtained from ATCC. EOL-1 cells should be cultured at 5 x 10 ⁵ cells / ml in medium containing 0.1 mM dbcAMP for 8 days, changing the medium every 3 days and adjusting the cell concentration to 5 x 10 ⁵ cells / ml. Were further differentiated by. RNA was prepared from resting cells or cells activated with PMA (10 ng / ml) and ionomycin (1 μg / ml) for 6 and 14 hours. RNA was prepared from resting CCD 1106 keratinocyte cell line (ATCC) or cells activated with ~ 5 ng / ml TNF alpha and 1 ng / ml IL-1 beta. In medium containing resting NCI-H292, airway epithelial tumor cell line (ATCC) or 5 ng / ml IL-4, 5 ng / ml IL-9, 5 ng / ml IL-13, and 25 ng / ml IFN gamma RNA was prepared from cells that were activated for 6 and 14 hours.

RNA was prepared by lysing approximately 10 ⁷ cells / ml using Trizol (Gibco BRL), then adding 1/10 volume of bromochloropropane (Molecular Research Corporation, Cincinnati, OH), vortexing, and 10 minutes at room temperature. Incubation was followed by centrifugation at 14,000 rpm in a Sorvall SS34 rotor. This aqueous phase was placed in a 15 ml Falcon tube, an equal volume of isopropanol was added and placed at -20 ° C overnight. The precipitated RNA was spun down at 9,000 rpm for 15 minutes and washed with 70% ethanol. This pellet is redissolved in 300 μl RNAse-free water containing 35 ml buffer (Promega, Madison, WI), 5 μl DTT, 7 μl RNAsin and 8 μl DNAse and incubated at 37 ° C. for 30 minutes to remove contaminating genomic DNA. Extracted once with phenol / chloroform and reprecipitated with 1/10 volume of 3 M sodium acetate and 2 volumes of 100% ethanol. This RNA was spun down, placed in RNAse free water and stored at -80 ° C.

AI_Comprehensive panel_v1.0
The autoimmune (AI) comprehensive panel v1.0 included 2 controls and 89 cDNA test samples isolated from male (M) and female (F) surgical and postmortem human tissues. The human tissue was obtained from the Backus Hospital and Clinomics (Frederick, MD). Tissue samples are: normal, adjacency (Adj); compatible normal adjacency (adapted control); indirect tissue (Synovial fluid, synovium, bone and cartilage, osteoarthritis (OA), Rheumatoid arthritis (RA)); psoriatic; ulcerative colitis colon; Crohns disease colon; and emphysmatic, asthmatic, allergic ) And chronic obstructive pulmonary disease (COPD).

Lung and General Inflammation (PGI) panel v1.0
The Lung and General Inflammation (PGI) panel v1.0 included 2 controls and 39 test samples isolated as surgical or postmortem samples. Tissue samples are: 5 normal lung samples from Maryland Brain and Tissue Bank, University of Maryland (Baltimore, MD), International Bioresource systems, IBS (Tuscon, AZ), and Asterand (Detroit, MI), Ardais (Lexington, MA) 5 normal adjacent intestinal tissue (NAT), ulcerative colitis sample (UC) obtained from Ardais (Lexington, MA); Crohn's disease colon obtained from NDRI, National Disease Research Interchange (Philadelphia, PA); Ardais Emphysema tissue samples obtained from (Lexington, MA) and Genomic Collaborative Inc. (Cambridge, MA); asthma from Maryland Brain and Tissue Bank, University of Maryland (Baltimore, MD) and Genomic Collaborative Inc (Cambridge, MA) Tissues and fiber tissues obtained from Ardais (Lexinton, MA) and Genomic Collaborative (Cambridge, MA) were included.

Cellular OA / RA Panel The cellular OA.RA panel contains 2 control wells and 35 test samples of cDNA, which is derived from human cell lines or primary cells that are typical of human joints and their inflammatory symptoms. It was made from isolated total RNA. Cell types include normal human osteoblasts (Nhost) (obtained from Clonetics (Cambrex, East Rutherford, NJ)), human chondrosarcoma SW1353 cells (obtained from ATCC (Manossas, VA)), human fibroblast-like synoviocytes (Acquired from Cell Applications, Inc. (San Diego, CA)) and MH7A cell line (Rheumatoid fibroblast-like synoviocytes transformed with SV40 T antigen) (Riken Cell bank (Tsukuba Science City, Japan)) Obtained). These cell types are different cytokines (IL-1 beta -1-10 ng / ml, TNF alpha -5-50 ng / ml, or prostaglandin E2 (for Nhost cells)) and 1, 6, 18 or Activated by incubating for 24 hours. All these cells were starved for at least 5 hours and cultured in their corresponding basal medium containing ˜0.1-1% FBS.

Microstructure OA / RA panel
The OA / RA minipanel contains 2 control wells and 31 test samples consisting of cDNA, which is the University of Calgary (Alberta, Canada), NDRI (Philadelphia, PA), and Ardais Corporation (Lexington, MA) From total RNA isolated from surgical and post-mortem human tissue. Joint tissue samples include synovium, bone and cartilage, and normal synovial samples (RNA and tissue) from osteoarthritic and rheumatoid arthritis patients who have undergone knee reconstruction surgery. Visceral normal tissues were pooled from 2-5 adults including: adrenal gland, heart, kidney, brain, large intestine, lung, stomach, small intestine, skeletal muscle, and ovary.

AI.05 Chondrosarcoma
AI.05 chondrosarcoma plates contained SW1353 cells (ATCC). The cells were serum starved and treated with cytokines known to induce MMP (1, 3, and 13) synthesis (eg, IL1 beta) for 6 and 18 hours. These treatments included: IL-1 beta (10 ng / ml), IL-1 beta + TNF-alpha (50 ng / ml), IL-1 beta + oncostatin (50 ng / ml) ) And PMA (100 ng / ml). The supernatant was collected and analyzed for MMP 1, 3 and 13 production. RNA was prepared from these samples using standard procedures.

Panel 5D and 5I
Panels 5D and 5I show two controls and human tissues, human islet cells, cell lines, metabolic tissues from patients enrolled in the Gestational Diabetes Study (below), and stages of adipocyte differentiation, e.g. differentiated types ( It contained cDNA isolated from cells derived from (AD), developing (AM), and undifferentiated (U; human mesenchymal stem cells).

  Subjects in the gestational diabetes study were young (18-40 years) gestational diabetics and non-patients who had undergone normal (optional) cesarean section, but otherwise healthy women. Uterine wall smooth muscle (UT), visceral (Vis) fat, skeletal muscle (SK), placenta (Pl) omental fat (GO fat) and subcutaneous (SubQ) fat samples (less than 1 cc) are collected in sterile saline Rinse, blot, and snap frozen in liquid nitrogen. Included: patient 2, overweight diabetic Hispanic, insulin independent; patient 7-9, obese non-diabetic Caucasian, with body mass index (BMI) greater than 30; patient 10, overweight diabetic Hispanic, insulin dependent; patient 11, overweight non-diabetic African American; and patient 12, diabetic Hispanic, insulin dependent.

  Differentiated adipocytes were obtained from inducible donor progenitor cells (Clonetics, Walkersville, MD). Differentiated human mesenchymal stem cells (HuMSC) were prepared as described in Mark F. Pittenger, et al., Multilineage Potential of Adult Human Mesenchymal Stem Cell Science Apr 2 1999: 143-147. MRNA was isolated from Trizol lysate or frozen pellets to generate sscDNA. Contained the following human cell lines (ATCC, NCI or German tumor cell bank): renal proximal convoluted tubules, uterine smooth muscle cells, small intestine, liver HepG2 cancer cells, cardiac primary stromal cells and adrenal cortex Adenoma cells. Cells were cultured using standard procedures, RNA extracted and sscDNA generated.

  Panel 5I also contains islets (Diabetes Research Institute at the University of Miami School of Medicine).

Human Metabolic RTQ-PCR Panel The human metabolic RTQ-PCR panel contains 2 controls (genomic DNA control and chemical control) and 211 cDNA isolated from human tissues and cell lines associated with metabolic disease. there were. This panel identifies genes that play a role in the pathogenesis and virulence of obesity and / or diabetes. From the Gestational Diabetes Study (above), metabolic tissues including placenta (Pl), uterine wall smooth muscle (Ut), visceral fat, skeletal muscle (Sk) and subcutaneous (SubQ) fat were obtained. This panel includes: patients 7 and 8, obese non-diabetic Caucasian; patient 12, diabetic Caucasian, BMI unknown, insulin dependent (treatment); patient 13, overweight diabetic Caucasian, insulin independent ( Patient 15, obese, untreated, diabetic Caucasian; patients 17 and 25, untreated diabetic Caucasian, normal weight; patient 18, obese, untreated, diabetic Hispanic; patient 19, non-diabetic Caucasian, normal weight; patient 20, overweight, treated diabetic Caucasian; patients 21 and 23, overweight non-diabetic Caucasian; patient 22, treated diabetic Caucasian, normal weight; patient 23, overweight Non-diabetic Caucasian; and patients 26 and 27, obese, treated, diabetic Caucasian.

  Total RNA was isolated from metabolic tissues including: hypothalamus, liver, pancreas, islet, small intestine, lumbar muscle, diaphragm muscle, visceral (Vis) fat, subcutaneous (SubQ) fat and omentum (Go), at necropsy From 12 type II diabetic (Diab) patients and 12 non-diabetic subjects (Norm). Control diabetic and non-diabetic subjects were comparable, if possible, in terms of age: gender, male (M); female (F); ethnicity, Caucasian (CC); Hispanic (HI) African American (AA); Asian (AS); and BMI, 20-25 (Low BM), 26-30 (Med BM) or overweight, over 30 BMI (Hi BMI) (obese ( obese)).

  Using standard methods, RNA was extracted from cell lines (ATCC) to generate ss cDNA.

CNS panel
CNS panel CNSD.01, CNS neurodegenerative V1.0 and CNS neurodegenerative V2.0 included 2 controls and 46-94 test cDNA samples isolated from postmortem human brain tissue. This brain tissue was obtained from the Harvard Brain Tissue Resource Center (McLean Hospital). The brain was removed from the donor calvaria between 4-24 hours post-mortem and frozen at -80 ° C in liquid nitrogen vapor.

Panel CNSD.01
Panel CNSD.01 included: 2 samples each from Alzheimer's disease, Parkinson's disease, Huntington's disease, Progressive Supernuclear Palsy (PSP), depression and normal control. Tissues were collected including: Cing Gyr, Temp Pole, Globus palladus (Glob palladus), Black nib (Sub Nigra), Primary motor strip (Brodman Area 4), parietal area (7 Broadman areas), prefrontal area (9 Broadman areas), and occipital area (17 Broadman areas). Not every case is representative of all brain regions.

Panel CNS neurodegeneration V1.0
The CNS neurodegenerative V1.0 panel included: 6 Alzheimer's disease (AD) brains and 8 normal brains. It includes no brain with dementia and no Alzheimer-like pathology (control), or no dementia, but manifests a severe Alzheimer-like pathology (control pathology), with severe Alzheimer-like pathology In particular, senile plaque deposition was rated level 3 in the following 0-3 grades; 0 indicates no plaque symptoms and 3 indicates severe AD senile plaque deposition. Tissues were collected including: hippocampus, temporal area (Broadman 21), parietal area (Broadman 7), occipital area (Broadman 17), upper temporal area (Sup Temproal Ctx) and inferior temporal Field (Inf Temproal Ctx).

  Gene expression was analyzed after normalization using a scaling factor calculated by subtracting the well average (CT average for a specific tissue) from the total average (average CT value for all wells in all runs). The post-scaling CT value is the solution of raw CT value + scaling factor.

Panel CNS neurodegeneration V2.0
The CNS neurodegenerative V2.0 panel included 16 cases of Alzheimer's disease (AD) and 29 normal controls (no symptoms of dementia before death). This includes 14 controls (control) with no dementia and no Alzheimer-like pathology and 15 controls (AH3) with no dementia but manifesting severe Alzheimer-like pathology, including severe Alzheimer-like pathologies In particular, it was senile plaque deposition rated as level 3 in the following 0-3 grades; 0 indicates no plaque symptoms and 3 indicates severe AD senile plaque deposition. Tissue from the temporal field (Broadman 21) included the lower and upper temporal fields pooled from a given individual (Inf & Sup Temp Ctx Pool).

Example Q10. PathCalling® technology
The sequence of NOVX was obtained by screening a cDNA library at the laboratory level. This screening was performed by a two-hybrid approach. Groups of cDNA fragments containing the full-length DNA sequence, or part of the sequence, or both were sequenced. In silico prediction was performed based on sequences available in CuraGen Corporation's private sequence database or public human sequence database to obtain the full length DNA sequence, or any portion thereof.

  Screening at the laboratory level was performed using the following method. The cDNA library was derived from a variety of human samples that were representative of a number of tissue types, normal and disease states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue culture primary cells or cell lines. Cells and cell lines may be treated with biological or chemical substances that control gene expression, such as growth factors, chemokines or steroids. Subsequently, the cDNA thus obtained was cloned with orientation into an appropriate two-hybrid vector (Gal4-activation domain (Gal4-AD) fusion). This cDNA library and a cDNA library commercially available from Clontech (Palo Alto, Calif.) Were then transferred from E. coli to a yeast strain originally developed by CuraGen Corporation (this yeast strain is disclosed in US Pat. No. 6,083,693, which is incorporated herein by reference in its entirety.

  A Gal4 binding domain (Gal4-BD) fusion of a CuraGen Corportion private human sequence library was used to screen a number of Gal4-AD fusion cDNA libraries and to select yeast hybrid diploids. In each diploid, the Gal4-AD fusion contains a separate cDNA. Each sample was amplified using polymerase chain reaction (PCR) using non-specific primers at the cDNA insertion boundary. The PCR product was sequenced; the sequence trace was manually evaluated and edited and corrected if appropriate. The cDNA sequences from all samples are assembled together, which may include public human sequences. For this assembly, a bioinformatics program was used to create a consensus sequence for each assembly. Each assembly is added to the CuraGen Corporation database. A sequence was added as a component for assembly if the degree of identity with another component was at least 95% over 50 bp. Each assembly is representative of the gene or portion thereof and includes information regarding variants such as splicing single nucleotide polymorphisms (SNPs), insertions, deletions and other sequence variations.

  Physical clone: A cDNA fragment containing the entire open reading frame obtained by the screening procedure is cloned as recombinant DNA into the pACT2 plasmid (Clontech). This plasmid is used to create a cDNA library. This recombinant plasmid is inserted into the host and selected by the yeast hybrid diploid generated during the screening procedure. This screening is performed by joining both yeast strains N106 ′ and YULH (US Pat. Nos. 6,057,101 and 6,083,693) independently developed by CuraGen Corporation.

Other Embodiments While the invention has been described in conjunction with the detailed description thereof, the above description is intended to be illustrative and not limiting the scope of the invention. The scope of the invention is defined by the claims. Other aspects, advantages, and modifications are within the scope of the claims.

FIG. 1 is a schematic diagram of the non-oxidative phase of the pentose phosphate pathway, showing the function of transketolase in the pathway. FIG. 2 is a schematic diagram showing the role of SREBP-regulated genes during excessive citrate production. FIG. 3 is a schematic diagram showing representative pathways related to the pathogenesis and pathogenicity of obesity and / or diabetes. FIG. 4 is a schematic diagram of the pyruvate synthesis pathway.

Claims (36)

  An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2n-1, wherein n is an integer from 1 to 85.   An isolated nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer from 1 to 85.   An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer from 1 to 85.   The isolated polypeptide is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, 4. The isolated polypeptide of claim 3, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, and SEQ ID NO: 110.   The isolated polypeptide is SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, An amino acid sequence selected from the group consisting of SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, and SEQ ID NO: 154 4. The isolated polypeptide of claim 3, comprising.   The isolated polypeptide of claim 3, wherein the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 28. .   The isolated polypeptide is an amino acid selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 156. 4. The isolated polypeptide of claim 3, comprising a sequence.   4. The isolated polypeptide of claim 3, wherein the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, and SEQ ID NO: 54.   The isolated polypeptide of claim 3, wherein the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 58, and SEQ ID NO: 168.   The isolated polypeptides are SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, and SEQ ID NO: 170. 4. The isolated polypeptide of claim 3, comprising an amino acid sequence selected from the group consisting of: A method for identifying a compound that modulates the activity of a target polypeptide, comprising:
(A) combining a test compound with a target polypeptide and a substrate for the target polypeptide; and (b) determining whether the test compound modulates the activity of the target polypeptide;
Wherein the target polypeptide is an amino acid sequence selected from the group consisting of SEQ ID NO: 2n (n is an integer from 1 to 85), an amino acid sequence that is at least 95% identical to SEQ ID NO: 2n, and at least one of SEQ ID NO: 2n A method comprising an amino acid sequence that is a domain, or an amino acid sequence that is at least 95% identical to at least one domain of SEQ ID NO: 2n.   12. The method of claim 11, further comprising identifying a test compound that modulates the activity of the target polypeptide by inhibiting the activity of the target polypeptide as an inhibitor of the activity of the target polypeptide.   12. The method of claim 11, further comprising identifying a test compound that modulates the activity of the target polypeptide by inhibiting the activity of the target polypeptide as an antagonist of the target polypeptide.   12. The method of claim 11, further comprising identifying a test compound that modulates the activity of the target polypeptide by activating the activity of the target polypeptide as an activator of the activity of the target polypeptide.   12. The method of claim 11, further comprising identifying a test compound that modulates the activity of the target polypeptide by activating the activity of the target polypeptide as an agonist of the target polypeptide.   12. The method of claim 11, further comprising identifying a test compound that modulates the activity of the target polypeptide as a promoter of insulin secretion.   12. The method of claim 11, further comprising identifying a test compound that modulates the activity of the target polypeptide as a therapeutic agent for the treatment of insulin resistance.   12. The method of claim 11, further comprising identifying a test compound that modulates the activity of the target polypeptide as a therapeutic agent for the treatment of obesity.   12. The method of claim 11, further comprising identifying a test compound that modulates the activity of the target polypeptide as a therapeutic agent for the treatment of diabetes.   12. The method of claim 11, wherein the target polypeptide is an isolated polypeptide.   12. The method of claim 11, wherein the target polypeptide is produced by a method comprising culturing a recombinant host cell comprising a nucleic acid encoding the target polypeptide under conditions that promote expression of the target polypeptide. The nucleic acid is the following:
(A) SEQ ID NO: 2n-1 where n is an integer of 1 to 85;
(B) a nucleotide encoding an amino acid sequence of at least one domain of SEQ ID NO: 2n; and (c) an amino acid sequence selected from the group consisting of SEQ ID NO: 2n, an amino acid sequence at least 95% identical to SEQ ID NO: 2n, SEQ ID NO: 2n Or a nucleotide sequence encoding an amino acid sequence that is at least 95% identical to at least one domain of SEQ ID NO: 2n;
24. The method of claim 21, comprising a nucleotide sequence selected from the group consisting of:   12. The method of claim 11, wherein the target polypeptide results from expression of a recombinant vector comprising a nucleic acid, wherein the nucleic acid is selected from the group consisting of SEQ ID NO: 2n (n is an integer from 1 to 85), SEQ ID NO: 2n. A method that encodes an amino acid sequence that is at least 95% identical to, an amino acid sequence that is at least one domain of SEQ ID NO: 2n, or an amino acid sequence that is at least 95% identical to at least one domain of SEQ ID NO: 2n.   24. The method of claim 23, wherein the test compound is combined with the target polypeptide in cultured mammalian cells.   24. The method of claim 23, wherein the test compound is combined with the target polypeptide in vitro. The nucleic acid is the following:
(A) SEQ ID NO: 2n-1 where n is an integer of 1 to 85;
(B) a nucleotide encoding an amino acid sequence of at least one domain of SEQ ID NO: 2n; and (c) an amino acid sequence selected from the group consisting of SEQ ID NO: 2n, an amino acid sequence at least 95% identical to SEQ ID NO: 2n, SEQ ID NO: 2n Or a nucleotide sequence encoding an amino acid sequence that is at least 95% identical to at least one domain of SEQ ID NO: 2n;
24. The method of claim 23, comprising a nucleotide sequence selected from the group consisting of:   12. The method of claim 11, wherein the target polypeptide is generated by expression of an endogenous nucleic acid, wherein the endogenous nucleic acid is selected from the group consisting of SEQ ID NO: 2n (n is an integer from 1 to 85), An amino acid sequence that is at least 95% identical to SEQ ID NO: 2n, an amino acid sequence that is at least one domain of SEQ ID NO: 2n, or an amino acid sequence that is at least 95% identical to at least one domain of SEQ ID NO: 2n.   28. The method of claim 27, wherein the test compound is combined with the target polypeptide in cultured mammalian cells.   28. The method of claim 27, wherein the test compound is combined with the target polypeptide in vitro. Endogenous nucleic acids include the following:
(A) SEQ ID NO: 2n-1 where n is an integer of 1 to 85;
(B) a nucleotide encoding an amino acid sequence of at least one domain of SEQ ID NO: 2n; and (c) an amino acid sequence selected from the group consisting of SEQ ID NO: 2n, an amino acid sequence at least 95% identical to SEQ ID NO: 2n, SEQ ID NO: 2n Or a nucleotide sequence encoding an amino acid sequence that is at least 95% identical to at least one domain of SEQ ID NO: 2n;
24. The method of claim 23, comprising a nucleotide sequence selected from the group consisting of:   An antibody that immunospecifically binds to a target polypeptide, wherein the target polypeptide is an amino acid sequence selected from the group consisting of SEQ ID NO: 2n (n is an integer from 1 to 85), and is at least 95% identical to SEQ ID NO: 2n An antibody comprising an amino acid sequence, an amino acid sequence that is at least one domain of SEQ ID NO: 2n, or an amino acid sequence that is at least 95% identical to at least one domain of SEQ ID NO: 2n.   32. The antibody of claim 31, wherein the antibody is a monoclonal antibody.   32. The antibody of claim 31, wherein the antibody is a humanized antibody.   32. The antibody of claim 31, wherein the antibody is a human antibody. A method for identifying potential therapeutic agents for use in the treatment of pathologies associated with aberrant expression of target polypeptides or abnormal physiological interactions, comprising the steps of:
(A) providing a cell that expresses the target polypeptide and has properties or functions attributable to the target polypeptide;
(B) contacting the cell with a composition comprising a candidate test compound; and (c) determining whether the test compound alters a property or function attributable to the target polypeptide;
Including
Thereby, if a change observed in the presence of a test compound is not observed when the cell is contacted with the composition in the absence of the test compound, the test compound is identified as a potential therapeutic agent;
An amino acid sequence selected from the group consisting of SEQ ID NO: 2n (n is an integer of 1 to 85), an amino acid sequence at least 95% identical to SEQ ID NO: 2n, an amino acid sequence that is at least one domain of SEQ ID NO: 2n, Or a method comprising an amino acid sequence that is at least 95% identical to at least one domain of SEQ ID NO: 2n. A method of screening for modulators or predisposition of pathological activity or latency associated with a target polypeptide, comprising:
(A) administering a test compound to a test animal having an increased risk of pathology associated with the target polypeptide, wherein the test animal recombinantly expresses the target polypeptide;
(B) measuring the activity of the target polypeptide in the test animal after administration of the test compound of step (a); and (c) the activity of the target polypeptide in the test animal, the control animal not receiving the test compound Wherein the change in the activity of the target polypeptide in the test animal relative to the control animal is a modulator of pathological activity or latency in which the test compound is associated with the target polypeptide, or Wherein the target polypeptide is an amino acid sequence selected from the group consisting of SEQ ID NO: 2n (n is an integer from 1 to 85), an amino acid at least 95% identical to SEQ ID NO: 2n A sequence, an amino acid sequence that is at least one domain of SEQ ID NO: 2n, or at least one domain of SEQ ID NO: 2n When those comprising an amino acid sequence at least 95% identical, methods.

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