WO2009023125A1 - Neuronostatin and its uses - Google Patents

Abstract

The invention relates to polypeptides referred to herein as neuronostatin. Neuronostatin, analogs and mimetics thereof act in signaling pathways, and are shown to modulate neurological parameters, and gastric emptying.

Description

NEURONOSTATIN AND ITS USES

INTRODUCTION

[01] Polypeptide hormones and their receptors play important roles in the maintenance of homeostasis in multicellular organisms. Recent sequencing of the genomes of human and several animal models provide an unprecedented opportunity to identify novel polypeptide ligands based on sequence homology among paralogous ligand genes. In addition, a large number of putative G protein-coupled receptors without known ligands have been predicted based on their characteristic seven transmembrane domains. Although the ligands for some of these 'orphan' GPCRs have been identified based on biochemical purification and other approaches, the ligands for many of them are still unknown. There is considerable interest for clinical and research purposes in the discovery and development of agents that act on these receptors.

[02] Originally discovered in 1972 based on its suppression of pituitary growth hormone release (Ling, PMID: 4734245; Burgus PMID: 4514982), somatostatin is the most extensively studied peptide hormone. Somatostatin is widely expressed in neuronal, neuroendocrine, inflammatory, gastrointestinal, immune and cancer cells and plays important roles in the regulation of hormonal secretion, neuromodulation, gastrointestinal functions, nutrient absorption, immune responses, cell growth, and exocrine secretion (Low, PMID: 15533778). Two somatostatin isoforms, SST-14 and SST28, activate five related G protein-coupled receptors with different affinity (Yamada, PMID: 1346068; Patel review, PMID: 10433861 ; Bell, PMID: 7679236). In addition to two somatostatin isoforms, somatostatin receptors are also activated by cortistatin secreted mainly from different brain regions (deLecea PMID: 8622767). Due to the existence of at least four ligands showing differential affinities to five phylogenetically related receptors, the precise physiological roles of individual somatostatin family ligands are unclear and likely determined by tissue-specific expression of ligand-receptor pairs.

SUMMARY OF THE INVENTION

[03] Neuronostatin peptides, mammalian homologs of neuronostatin, and variants thereof are provided. In some embodiments of the invention, the neuronostatin peptide is amidated at the C-terminus. Native neuronostatin is derived from the preprosomatostatin protein through alternative protein processing, although synthetic or recombinantly produced neuronostatin may be synthesized in the absence of somatostatin. Like somatostatin, neuronostatin is widely expressed in neuronal, neuroendocrine, inflammatory, gastrointestinal, immune and cancer cells and plays important roles in the regulation of hormonal secretion, neuromodulation, gastrointestinal functions, nutrient absorption, immune responses, cell growth, and exocrine secretion. Also provided are polynucleotides encoding a neuronostatin peptide, which polynucleotides lack sequences encoding somatostatin polypeptides.

[04] Neuronostatin peptides act on cells in the gastrointestinal tract, resulting in suppression of gastric emptying. When provided to the brain and central nervous system, neuronostatin inceases water drinking activity, and induces expression of c-fos in specific cells in the brain, e.g. cerebellar granule cells, cerebral internal pyramidal cells, hippocampus pyramidal cells, and neurons in caudate-putamen. In some embodiments of the invention, administration of neuronostatin is used in the regulation of motor, cortical, learning, memory, and other brain functions. The identification of neuronostatin allows use of this peptide and its analogs to regulate different brain and gastrointestinal functions.

[05] In addition to use as a therapeutic agent, in another embodiment of the invention neuronostatin peptides are utilized in screening and research methods for the determination of specific analogs, agonists, antagonists mimetics and agents that modulate their interaction with specific receptors, production, metabolism, and disposition. Regulatory peptides are ligands for a subgroup of G protein-coupled receptors (GPCRs) and can play important roles in the gastrointestinal, cardiovascular, hypothalamus-pituitary axis, and the central nervous systems.

[06] In one embodiment of the invention, an isolated polypeptide is provided, usually an amidated peptide, and functional fragments, derivatives and homologs thereof. Such polypeptides may be formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In another embodiment, specific antibodies to block the actions of the endogenous neuronostatin peptide are provided.

[07] Other aspects of the invention and their features and advantages will become apparent from the following description and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS [08] Figure 1. Bioinformatics of neuronostatin. [09] Figs. 2. Neuronostatin suppression of gastric emptying activity. [10] Figure 3. Neuronostatin antagonizes obestatin suppression of water drinking behavior. [11] Figures 4A-4B. Neuronostatin induction of c-fos antigens in stomach and small intestine. Figures 4C. Neuronostaitn inductionof c-fos in anterior pituitary. Figure 4D.

Neuronostatin induction of c-jun in pancreatic alpha cells (a). Figures E and F.

Neuronostatin inductionof c-fos in cerebellar granule cells and hippocampal pyramidal cells.

In all sub-figures, panel b represents animals treated with saline. [12] Figures 5A-5D. Neuronostatin stimulation of c-fos expression, proliferation, and SRE- luciferase reporter activity in human gastric tumor cells. A). KATO-III cells were treated with neuronostatin (10 nM) for different periods before measurement of c-fos transcript levels. B). Cells with or without neuronostatin (10 nM) treatment for 1h were stained using c-fos antibodies (panels b and d). Panels a and c represent cell nuclei staining using the Hoechst 33342 dye. C) Neuronostatin peptides stimulated KATO-III cell proliferation based on the MTT assay. D) Neuronostatin stimulation of luciferase activity in KATO-III cells transfected with an SRE-luciferase reporter construct.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS [13] The regulatory peptide neuronostatin is a ligand for the G protein-coupled receptors (GPCRs) that play important roles in gastrointestinal, cardiovascular, hypothalamus-pituitary axis, and the central nervous system. In addition to use as a therapeutic agent, neuronostatin peptides are utilized in screening and research methods for the determination of specific analogs, agonists, antagonists and mimetics and inhibitors of their interaction with specific receptors, production, metabolism and disposition.

NEURONOSTATIN COMPOSITIONS

[14] Neuronostatin peptides, which can be used in the methods of the invention, comprise at least about 6 amino acids, usually at least about 7 amino acids, at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, at least about 11 , at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, and which may include up to 19 amino acids of a neuronostatin peptide, or modifications thereof, and may further include fusion polypeptides as known in the art in addition to the provided sequences. In some embodiments the neuronostatin polypeptide is amidated at the C-terminus.

[15] The neuronostatin sequence may be from any mammalian or avian species, e.g. primate sp., particularly humans; rodents, including mice, rats and hamsters; rabbits; equines, bovines, canines, felines; etc. Of particular interest are the human proteins. Preferably the peptide is modified, where the C terminal glycine residue is replaced with an amide. A combination of one or more forms may be used.

[16] Specific neuronostatin peptides of interest include the following native neuronostatin peptides. There is a high degree of sequence similarity between species, and the human, chimpanzee, pig, dog, sheep and chicken peptides are identical. human PSDPRLRQFLQKSLAAAAG (SEQ ID NO:1 ) mouse/rat PSDPRLRQFLQKSLAAATG (SEQ ID NO:2) frog PTDPRLRQFLQKSLAAAG (SEQ ID NO:3) lungfish PSDLRLRQLLQRSLAAAAG (SEQ ID NO:4) zebrafish PSDAKLRQLLQRSLLSPAG (SEQ ID NO:5) goldfish PTDAKLRQLLQRSLLNPAG (SEQ ID NO:6)

The amino terminus may be truncated to give rise to shorter peptides, for example RLRQFLQKSLAAAAG (SEQ ID NO:7), LRQFLQKSLAAAAG (SEQ ID NO:8), QFLQKSLAAAAG (SEQ ID NO:9), and SLAAAAG (SEQ ID NO: 10), any of which may have the C terminal glycine residue replaced with an amide. In some embodiments of the invention, the neuronostatin peptide is a peptide having the sequence consisting of SEQ ID NO:1-SEQ ID NO:11.

[17] In some embodiments of the invention the neuronostatin peptide has a cyclized N- terminal glutamine residue, resulting in an amino acid sequence where pyro-glutamine is the N-terminal amino acid, and where the C-terminus is amidated, e.g. as set forth in SEQ ID NO:11 , pyro-Glu-FLQKSLAAAA-NH₂.

[18] The GenBank accession numbers for individual somatostatin genes from which neuronostatin is derived are 4507243 (human), 55621730 (chimpanzee), 57528038 (pig), 57163953 (sheep), 73697560 (cattle), 50979130 (dog), 6678035 (mouse), 6981582 (rat), 45385811 (chicken), 32454336 (frog), 9978804 (lungfish), 9978923 (goldfish), 34098954 (zebrafish).

[19] The peptides of the invention have a number of important physiological functions, including modulation of body weight and metabolism. Modulators of weight affect the intake of food, gastric motility, weight homeostasis, etc. The peptides also modulate neural function, e.g. by taking part in neuroprotective pathways upregulated after ischemia, and the like. Such modulators include agonists, which enhance, potentiate and/or mimic the activity of a neuronostatin peptide; and antagonists, which inhibit or decrease the activity of a neuronostatin peptide.

[20] The sequence of neuronostatin peptides as described above may be altered in various ways known in the art to generate targeted changes in sequence. The sequence changes may be substitutions, insertions or deletions. Such alterations may be used to alter properties of the protein, by affecting the stability, specificity, etc. Techniques for in vitro mutagenesis of cloned genes are known. Examples of protocols for scanning mutations may be found in Gustin et al., Biotechniques 14:22 (1993); Barany, Gene 37:111-23 (1985); Colicelli et al., MoI Gen Genet 199:537-9 (1985); and Prentki et al., Gene 29:303-13 (1984). Methods for site specific mutagenesis can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp. 15.3-15.108; Weiner et al., Gene 126:35-41 (1993); Sayers et al., Biotechniques 13:592-6 (1992); Jones and Winistorfer, Biotechniques 12:528-30 (1992); Barton et al., Nucleic Acids Res 18:7349-55 (1990); Marotti and Tomich, Gene Anal Tech 6:67-70 (1989); and Zhu Anal Biochem 177:120-4 (1989).

[21] In some embodiments of the invention, the neuronostatin peptide consists of an oligopeptide of least about 6 amino acids, usually at least about 7 amino acids, at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, at least about 11 , at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, and which may include up to 19 amino acids, and which is at least about 80%, at least about 90%, at least about 95% identical in sequence to an oligopeptide of SEQ ID NO:1 - SEQ ID NO:8, where the C terminal glycine residue may be replaced with an amide moiety.

[22] The peptides may be joined to a wide variety of other oligopeptides or proteins for a variety of purposes. By providing for expression of the subject peptides, various post- expression modifications may be achieved. For example, by employing the appropriate coding sequences, one may provide farnesylation or prenylation. The peptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream. The peptides may also be combined with other proteins in a fusion protein, typically where the two proteins are not normally joined, such as the Fc of an IgG isotype, which may be complement binding, with a toxin, such as ricin, abrin, diphtheria toxin, or the like, or with specific binding agents that allow targeting to specific moieties on a target cell.

[23] The neuronostatin for use in the subject methods may be produced from eukaryotic or prokaryotic cells, or may be synthesized in vitro. Where the protein is produced by prokaryotic cells, it may be further processed by unfolding, e.g. heat denaturation, DTT reduction, etc. and may be further refolded, using methods known in the art.

[24] Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

[25] Also included in the subject invention are peptides that have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non- naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.

[26] The subject peptides may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Foster City, CA, Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.

[27] If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

[28] The polypeptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein.

[29] In one embodiment of the invention, the neuronostatin peptide consists essentially of a polypeptide sequence of at least 14 amino acids in length and having a sequence of an neuronostatin peptide as described above. By "consisting essentially of in the context of a polypeptide described herein, it is meant that the polypeptide is composed of the neuronostatin sequence, which sequence is optionally flanked by one or more amino acid or other residues that do not materially affect the basic characteristic(s) of the polypeptide.

[30] Neuronostatin polypeptides include those provided herein, and variants thereof. Variant polypeptides can include amino acid (aa) substitutions, additions or deletions. The amino acid substitutions can be conservative amino acid substitutions or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, a phosphorylation site or an acetylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function. Variants can be designed so as to retain or have enhanced biological activity of a particular region of the protein (e.g., a functional domain where the polypeptide is a member of a protein family, or a region associated with a consensus sequence). Variants also include fragments of the polypeptides disclosed herein, for example, biologically active fragments and/or fragments corresponding to functional domains.

[31] The invention includes nucleic acids encoding the peptides of the invention. The nucleic acid sequences encoding the above neuronostatin polypeptides may be accessed from public databases, as described above. Identification of additional neuronostatins is accomplished by conventional screening methods of DNA libraries or biological samples for DNA sequences having a high degree of similarity to known neuronostatin sequences. Such polynucleotides preferably lack sequences encoding somatostatin polypeptides, i.e. the polynucleotides of the invention are other than mRNA or corresponding cDNA of naturally occurring somatostatin transcripts. Polynucleotides of interest include those that encode a peptide that consists essentially of a polypeptide sequence of at least about 6 amino acids, usually at least about 7 amino acids, at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, at least about 11 , at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, and which may include up to 19 amino acids having a sequence of a neuronostatin peptide as described above. Such polynucleotides may be operably joined to control sequences, e.g. for transcriptional start, stop, translation, promoters, etc. Polynucleotides may also include a neuronostatin coding sequence combined with fusion polypeptide sequences.

[32] Neuronostatin coding sequences can be generated by methods known in the art, e.g. by in vitro synthesis, recombinant methods, etc. to provide a coding sequence to corresponds to an neuronostatin polypeptide that can serve as an intermediate in the production of the neuronostatin peptide. Using the known genetic code, one can produce a suitable coding sequence. Double or single stranded fragments can be obtained from the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc.

[33] Neuronostatin encoding nucleic acids can be provided as a linear molecule or within a circular molecule, and can be provided within autonomously replicating molecules (vectors) or within molecules without replication sequences. Expression of the nucleic acids can be regulated by their own or by other regulatory sequences known in the art. The nucleic acids can be introduced into suitable host cells using a variety of techniques available in the art, such as transferrin polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated DNA transfer, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, gene gun, calcium phosphate-mediated transfection, and the like.

[34] Expression vectors may be used to introduce a neuronostatin coding sequence into a cell. Such vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences. Transcription cassettes may be prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region. The transcription cassettes may be introduced into a variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like, where the vectors are able to transiently or stably be maintained in the cells, usually for a period of at least about one day, more usually for a period of at least about several days to several weeks.

[35] The nucleic acid may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, or fusion of vesicles. Jet injection may also be used for intramuscular administration, as described by Furth et al. (1992) Anal Biochem 205:365- 368. The DNA may be coated onto gold microparticles, and delivered intradermal^ by a particle bombardment device, or "gene gun" as described in the literature (see, for example, Tang et al. (1992) Nature 356:152-154), where gold microprojectiles are coated with the neuronostatin or DNA, then bombarded into skin cells.

USES OF NEURONOSTATIN

[36] In light of the pharmacologic activities of neuronostatin, numerous clinical indications are evident. For example, clinical indications for which a neuronostatin peptide or variants thereof may find use include treatment of obesity, as a neuroprotective agent.

[37] Human obesity is a widespread and serious disorder, affecting a high percentage of the adult population in developed countries. In spite of an association with heart disease, type Il diabetes, cancer, and other conditions, few persons are able to permanently achieve significant weight loss. The subject peptides are administered to obese patients for purposes of appetite suppression. Patients may use various criteria for determining obesity. Conveniently, a body mass index (BMI) is calculated, where a person having a BMI greater than 25 is overweight and may considered for treatment with the subject peptides. Neuronostatins find use in promoting gastric stasis and anorexic behavior without concomitant activation of the ACTH-glucocorticoid axis.

[38] In a related embodiment, neuronostatin finds use in the treatment of diabetes mellitus, including type I and type Il disease. NIDDM is a metabolic disease that affects about 5% to 7% of the population in western countries (and 10% of individuals over age 70). It is characterized by hyperglycemia and often accompanied by a number of other conditions, including hypertension, obesity and lipid disturbances. Patients are generally categorized as diabetic or hyperglycemic by measuring the level of glucose in the blood, either directly or by monitoring the level of glycosylated hemoglobin. Treatment is recommended where fasting glucose levels are greater 140 mg/dl, where bedtime glucose is greater than 160 mg/dl, or where HbAi_c is greater than 8%. The level of reduction that is desirable depends on the condition of the patient, and the blood glucose levels at the start of treatment, but generally about a 10 to 40 % reduction in blood glucose is desirable, usually about a 25 to 35% reduction.

[39] In some embodiments of the invention, neuronostatin is useful in the treatment of neuronal disease, including but not limited to neuronal ischemia. Therapeutic and prophylactic treatment methods for individuals suffering, or at risk of a neurological disorder such as stroke, involve administering either a therapeutic or prophylactic amount of neuronostatin.

[40] "Neurologic disorder" is defined here and in the claims as a disorder in which dysfunction of neurons occurs either in the peripheral nervous system or in the central nervous system. Examples of neurologic disorders include: chronic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, diabetic peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis; aging; psychiaric diseases and acute neurodegenerative disorders including: stroke, traumatic brain injury, peripheral nerve damage, hypoglycemia, spinal cord injury, epilepsy, anoxia and hypoxia.

[41] The term "stroke" broadly refers to the development of neurological deficits associated with impaired blood flow regardless of cause. Potential causes include, but are not limited to, thrombosis, hemorrhage and embolism. Current methods for diagnosing stroke include symptom evaluation, medical history, chest X-ray, ECG (electrical heart activity), EEG (brain nerve cell activity), CAT scan to assess brain damage and MRI to obtain internal body visuals. Thrombus, embolus, and systemic hypotension are among the most common causes of cerebral ischemic episodes. Other injuries may be caused by hypertension, hypertensive cerebral vascular disease, rupture of an aneurysm, an angioma, blood dyscrasias, cardiac failure, cardiac arrest, cardiogenic shock, septic shock, head trauma, spinal cord trauma, seizure, bleeding from a tumor, or other blood loss.

[42] By "ischemic episode" is meant any circumstance that results in a deficient supply of blood to a tissue. Where the ischemia is associated with a stroke, it can be either global or focal ischemia, as defined below. The term "ischemic stroke" refers more specifically to a type of stroke that is of limited extent and caused due to blockage of blood flow. Cerebral ischemic episodes result from a deficiency in the blood supply to the brain. The spinal cord, which is also a part of the central nervous system, is equally susceptible to ischemia resulting from diminished blood flow.

[43] By "focal ischemia," as used herein in reference to the central nervous system, is meant the condition that results from the blockage of a single artery that supplies blood to the brain or spinal cord, resulting in damage to the cells in the territory supplied by that artery.

[44] By "global ischemia," as used herein in reference to the central nervous system, is meant the condition that results from a general diminution of blood flow to the entire brain, forebrain, or spinal cord, which causes the death of neurons in selectively vulnerable regions throughout these tissues. The pathology in each of these cases is quite different, as are the clinical correlates. Models of focal ischemia apply to patients with focal cerebral infarction, while models of global ischemia are analogous to cardiac arrest, and other causes of systemic hypotension.

[45] The compounds of this invention can be normally administered orally or parenterally, in the treatment of patients in need of neuroprotective therapy. The dosage regimen is that which insures maximum therapeutic response until improvement is obtained and thereafter the minimum effective level that gives relief. Thus, in general, the dosages are those that are therapeutically effective in producing a neuroprotective effect, i.e., amelioration of ischemic injury. It is also anticipated that the peptides would be useful as an injectable dosage form, which may be administered in an emergency to a patient suffering from ischemia, etc.

[46] In one aspect, the invention features a method of beneficially regulating gastrointestinal motility in a subject by administering to said subject a therapeutically effective amount of a neuronostatin peptide or modulator thereof. In one embodiment, the methods of the present invention are directed to reducing gastric motility. In another embodiment, the invention is directed to methods of delaying gastric emptying. These methods may be used on a subject undergoing a gastrointestinal diagnostic procedure, for example radiological examination or magnetic resonance imaging. Alternatively, these methods may be used to reduce gastric motility in a subject suffering from a gastrointestinal disorder, for example, spasm (which may be associated with acute diverticulitis, a disorder of the biliary tract or a disorder of the Sphincter of Oddi). In another aspect, the invention is directed to a method of treating post-prandial dumping syndrome in a subject by administering to the subject a therapeutically effective amount of an neuronostatin modulator. In another aspect, the invention is directed to a method of treating post-prandial hyperglycemia by administering to a subject a therapeutically effective amount of a neuronostatin agonist, e.g. post-prandial hyperglycemia as a consequence of Type 2 diabetes mellitus.

[47] In another aspect, the present invention is directed to a method of treating gastric hypomotility in a subject by administering to the subject a therapeutically effective amount of a neuronostatin antagonist. These methods may be employed where hypomotility is a consequence of diabetic neuropathy or where hypomotility is a consequence of anorexia nervosa. Hypomotility may also occur as a consequence of achlorhydria or as a consequence of gastric surgery. In another aspect, the invention is directed to a method of accelerating gastric emptying in a subject by administering to the subject a therapeutically effective amount of a neuronostatin modulator.

COMPOUND SCREENING [48] In another aspect, the invention relates to methods for assaying or screening compounds to determine their activities as modulators of the function of the polypeptides described above. Compound screening may be performed using an in vitro model, a cell producing neuronostatin, e.g. a genetically altered cell or animal, or purified protein corresponding to an neuronostatin or derivative thereof. One can identify ligands, substrates and/or modulators that bind to, modulate or mimic the action of the peptides, including the identification of inhibitors and potentiators of neuronostatin activity.

[49] Compound screening identifies agents that modulate function of neuronostatin. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein- protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like.

[50] The term "modulator" includes any molecule, e.g. protein or pharmaceutical, with the capability of altering or mimicking the physiological function of an neuronostatin peptide. Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.

[51] Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate modulators comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate modulators often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[52] Candidate modulators are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Test agents can be obtained from libraries, such as natural product libraries or combinatorial libraries, for example. A number of different types of combinatorial libraries and methods for preparing such libraries have been described, including for example, PCT publications WO 93/06121 , WO 95/12608, WO 95/35503, WO 94/08051 and WO 95/30642, each of which is incorporated herein by reference.

[53] Where the screening assay is a binding assay, one or more of the molecules may be joined to a label, where the label can directly or indirectly provide a detectable signal. Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g. magnetic particles, and the like. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin, etc. For the specific binding members, the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures.

[54] A variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used. The components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 and 40° C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 and 1 hours will be sufficient.

[55] Preliminary screens can be conducted by screening for compounds capable of binding to, or interfering in the binding of neuronostatin to target tissues. The binding assays usually involve contacting neuronostatin with one or more test compounds and allowing sufficient time for the protein and test compounds to form a binding complex. Any binding complexes formed can be detected using any of a number of established analytical techniques. Protein binding assays include, but are not limited to, methods that measure co-precipitation, co-migration on non-denaturing SDS-polyacrylamide gels, and co-migration on Western blots, etc. Binding assays may also be conducted in vivo, e.g. in an animal model for a condition of interest. Neuronostatin interacts with tissue receptors in cerebellum, hippocampus, pituitary, different gastrointestinal tissues, etc. Compositions of interest for binding assays include a modified peptide that comprises a detectable label. Such peptides include, without limitation, neuronostatin analogs where an extra tyrosine residue has been added in the zero position of the peptide to allow iodination of this peptide and its use as a tracer for receptor binding assays. Such binding may be determined in competitive and non-competitive assays.

[56] The level of expression or activity can be compared to a baseline value. The baseline value can be a value for a control sample or a statistical value that is representative of a control population. Expression or activity levels can also be determined for cells that do not respond to neuronostatin as a negative control.

[57] Compounds that are initially identified by any of the foregoing screening methods can be further tested to validate the apparent activity. The basic format of such methods involves administering a lead compound identified during an initial screen to an animal that serves as a model for humans and then determining whether the desired biological function is affected. The animal models utilized in validation studies generally are mammals. Specific examples of suitable animals include, but are not limited to, primates, mice, and rats.

[58] Active test agents identified by the screening methods described herein that modulate neuronostatin activity can serve as lead compounds for the synthesis of analog compounds. Typically, the analog compounds are synthesized to have an electronic configuration and a molecular conformation similar to that of the lead compound. Identification of analog compounds can be performed through use of techniques such as self-consistent field (SCF) analysis, configuration interaction (Cl) analysis, and normal mode dynamics analysis. Computer programs for implementing these techniques are available. See, e.g., Rein et al., (1989) Computer-Assisted Modeling of Receptor-Ligand Interactions (Alan Liss, New York).

[59] Once analogs have been prepared, they can be screened using the methods disclosed herein to identify those analogs that exhibit an increased ability to modulate neuronostatin activity. Such compounds can then be subjected to further analysis to identify those compounds that appear to have the greatest potential as pharmaceutical agents. Alternatively, analogs shown to have activity through the screening methods can serve as lead compounds in the preparation of still further analogs, which can be screened by the methods described herein. The cycle of screening, synthesizing analogs and re-screening can be repeated multiple times.

[60] For receptor binding assays, many of the derived compounds are likely to be antagonists. When compounds capable of binding to the neuronostatin receptors are found not to exhibit neuronostatin-like bioactivity, they can be further screened for their antagonistic activities by injecting the compound of interest together with neuronostatin, and testing their antagonistic properties.

PHARMACEUTICAL COMPOSITIONS

[61] Polypeptides and polynucleotides described here, as well as active compounds identified by the screening methods described above and analogs thereof (e.g., pharmaceutically acceptable salts) can serve as the active ingredient in pharmaceutical compositions formulated for the treatment of various disorders as described above. The active ingredient is present in a therapeutically effective amount, i.e., an amount sufficient when administered to substantially modulate the effect of the targeted protein or polypeptide to treat a disease or medical condition mediated thereby.

[62] The compositions can also include various other agents to enhance delivery and efficacy, e.g. to enhance delivery and stability of the active ingredients.

[63] Thus, for example, the compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation can include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like. The compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents. The composition can also include any of a variety of stabilizing agents, such as an antioxidant.

[64] When the pharmaceutical composition includes a polypeptide as the active ingredient, the polypeptide can be complexed with various well-known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance solubility or uptake). Examples of such modifications or complexing agents include sulfate, gluconate, citrate and phosphate. The polypeptides of a composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[65] Further guidance regarding formulations that are suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990).

[66] The pharmaceutical compositions can be administered for prophylactic and/or therapeutic treatments. Toxicity and therapeutic efficacy of the active ingredient can be determined according to standard pharmaceutical procedures in cell cultures and/or experimental animals, including, for example, determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀ZED₅₀. Compounds that exhibit large therapeutic indices are preferred. [67] The data obtained from cell culture and/or animal studies can be used in formulating a range of dosages for humans. The dosage of the active ingredient typically lies within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.

[68] The pharmaceutical compositions described herein can be administered in a variety of different ways. Examples include administering a composition containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, or intracranial method.

[69] For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. The active component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate. Examples of additional inactive ingredients that may be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

[70] The active ingredient, alone or in combination with other suitable components, can be made into aerosol formulations (Ae., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen.

[71] Suitable formulations for rectal administration include, for example, suppositories, which are composed of the packaged active ingredient with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules, which are composed of a combination of the packaged active ingredient with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.

[72] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[73] The components used to formulate the pharmaceutical compositions are preferably of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Moreover, compositions intended for in vivo use are preferably sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is preferably substantially free of any potentially toxic agents, such as any endotoxins, which may be present during the synthesis or purification process. Compositions for parental administration are also preferably sterile, substantially isotonic and made under GMP conditions.

ANTIBODIES SPECIFIC FOR NEURONOSTATIN POLYPEPTIDES

[74] The present invention further provides antibodies specific for neuronostatin polypeptides, e.g. any one of the variants or polypeptides described above. Such antibodies are useful, for example, in methods of detecting the presence of neuronostatin in a biological sample, and in methods of isolating neuronostatin from a biological sample. Antibodies may also be useful as antagonists of neuronostatin activity.

[75] The neuronostatin polypeptides of the invention are useful for the production of antibodies, where short fragments provide for antibodies specific for the particular polypeptide, and larger fragments or the entire protein allow for the production of antibodies over the surface of the polypeptide. As used herein, the term "antibodies" includes antibodies of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv₁ scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen- binding portion of an antibody and a non-antibody protein. The antibodies may be detectably labeled, e.g., with a radioisotope, an enzyme that generates a detectable product, a green fluorescent protein, and the like. The antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin- avidin specific binding pair), and the like. The antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like. [76] "Antibody specificity", in the context of antibody-antigen interactions indicates that a given antibody binds to a given antigen, wherein the binding can be inhibited by that antigen or an epitope thereof which is recognized by the antibody, and does not substantially bind to unrelated antigens. Methods of determining specific antibody binding are well known to those skilled in the art, and can be used to determine the specificity of antibodies of the invention for a neuronostatin polypeptide, particularly a human neuronostatin polypeptide. [77] Antibodies are prepared in accordance with conventional ways, where the expressed polypeptide or protein is used as an immunogen, by itself or conjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg, other viral or eukaryotic proteins, or the like. Various adjuvants may be employed, with a series of injections, as appropriate. For monoclonal antibodies, after one or more booster injections, the spleen is isolated, the lymphocytes immortalized by cell fusion, and then screened for high affinity antibody binding. The immortalized cells, i.e. hybridomas, producing the desired antibodies may then be expanded. For a more detailed description, see Monoclonal Antibodies: A Laboratory Manual, Harlow and Lane eds., Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, 1988. If desired, the mRNA encoding the heavy and light chains may be isolated and mutagenized by cloning in E. coli, and the heavy and light chains mixed to further enhance the affinity of the antibody. Alternatives to in vivo immunization as a method of raising antibodies include binding to phage display libraries, usually in conjunction with in vitro affinity maturation.

MODULATION OF NEURONOSTATIN EXPRESSION

[78] Neuronostatin genes, gene fragments, or the encoded protein or protein fragments are useful in gene therapy to treat disorders associated with neuronostatin defects. Expression vectors may be used to introduce a neuronostatin coding sequence into a cell. Such vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences. Transcription cassettes may be prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region. The transcription cassettes may be introduced into a variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like, where the vectors are able to transiently or stably be maintained in the cells, usually for a period of at least about one day, more preferably for a period of at least about several days to several weeks.

[79] The gene may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, or fusion of vesicles. Jet injection may also be used for intramuscular administration, as described by Furth et al. (1992) Anal Biochem 205:365- 368. The DNA may be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or "gene gun" as described in the literature (see, for example, Tang et al. (1992) Nature 356:152-154), where gold microprojectiles are coated with the

DNA, then bombarded into skin cells. DIAGNOSTIC USES

[80] DNA-based reagents derived from the sequence of neuronostatin; e.g. PCR primers, oligonucleotide or cDNA probes, as well as antibodies against neuronostatin, are used to screen patient samples, e.g. biopsy-derived tissues, blood samples, and the like, for altered expression of neuronostatin mRNA or proteins. DNA-based reagents are also designed for evaluation of chromosomal loci implicated in certain diseases e.g. for use in loss-of- heterozygosity (LOH) studies, or design of primers based on neuronostatin coding sequence.

[81] The polynucleotides of the invention can be used to detect differences in expression levels between two samples. A difference between protein levels, or the mRNA in the two tissues that are compared, for example, in molecular weight, amino acid or nucleotide sequence, or relative abundance, indicates a change in the gene, or a gene, which regulates it, in the tissue of the human that was suspected of being diseased.

[82] The subject nucleic acid and/or polypeptide compositions may be used to analyze a patient sample for the presence of polymorphisms associated with a disease state or genetic predisposition to a disease state. Biochemical studies may be performed to determine whether a sequence polymorphism in a neuronostatin coding region or control regions is associated with disease, such as stress related disorders, e.g. anxiety disorders. Disease associated polymorphisms may include deletion or truncation of the gene, mutations that alter expression level, that affect the binding activity of the protein, and the like.

[83] Changes in the promoter or enhancer sequence that may affect expression levels of neuronostatin can be compared to expression levels of the normal allele by various methods known in the art. Methods for determining promoter or enhancer strength include quantitation of the expressed natural protein; insertion of the variant control element into a vector with a reporter gene such as β-galactosidase, luciferase, and chloramphenicol acetyltransferase which provides for convenient quantitation; and the like.

[84] A number of methods are available for analyzing nucleic acids for the presence of a specific sequence, e.g. a disease associated polymorphism. Where large amounts of DNA are available, genomic DNA is used directly. Alternatively, the region of interest is cloned into a suitable vector and grown in sufficient quantity for analysis. Cells that express neuronostatin may be used as a source of mRNA, which may be assayed directly or reverse transcribed into cDNA for analysis. The nucleic acid may be amplified by conventional techniques, such as the polymerase chain reaction (PCR), to provide sufficient amounts for analysis. The use of the polymerase chain reaction is described in Saiki et al. (1985) Science 239:487, and a review of techniques may be found in Sambrook et al.,

Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp. 14.2-14.33. [85] A detectable label may be included in an amplification reaction. Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein(6-FAM),2,7-dimethoxy-4,5-dichloro-6- carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxy-2,4,7,4,7- hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N,N-tetramethyl-6- carboxyrhodamine (TAMRA), radioactive labels, e.g. ³²P₁ ³⁵S, and ³H. The label may be a two stage system, where the amplified DNA is conjugated to biotin, haptens, and the like having a high affinity binding partner, e.g. avidin and specific antibodies., where the binding partner is conjugated to a detectable label. The label may be conjugated to one or both of the primers. Alternatively, the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.

[86] The sample nucleic acid, e.g., amplified or cloned fragment, is analyzed by one of a number of methods known in the art. The nucleic acid may be sequenced by dideoxy or other methods, and the sequence of bases compared to a wild-type neuronostatin sequence. Hybridization with the variant sequence may also be used to determine its presence, by Southern blots, dot blots, and the like. The hybridization pattern of a control and variant sequence to an array of oligonucleotide probes immobilized on an array may also be used as a means of detecting the presence of variant sequences. Single strand conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), and heteroduplex analysis in gel matrices may be used to detect conformational changes created by DNA sequence variation as alterations in electrophoretic mobility. Alternatively, where a polymorphism creates or destroys a recognition site for a restriction endonuclease, the sample is digested with that endonuclease, and the products size fractionated to determine whether the fragment was digested. Fractionation is performed by gel or capillary electrophoresis, preferably acrylamide or agarose gels.

[87] Screening for mutations in neuronostatin may be based on the functional or antigenic characteristics of the protein. Protein truncation assays are useful in detecting deletions that may affect the biological activity of the protein. Various immunoassays designed to detect polymorphisms in neuronostatin proteins may be used in screening. Where many diverse genetic mutations lead to a particular disease phenotype, functional protein assays have proven to be effective screening tools. The activity of the encoded neuronostatin peptide in binding assays may be determined by comparison with the wild-type protein. Proteins may also be screened for the presence of post-translational modification of the neuronostatin peptides, e.g. under pathological conditions, including proteolytic fragments, amidation, and acetylation. Proteins of interest for screening also include convertase enzymes specific for the cleavage of neuronostatin from the large pro-protein containing both somatostatin and neuronostatin. The convertase enzyme needed for the derivation of somatostatin may be different from those for neuronostatin derivation.

[88] Antibodies specific for neuronostatin may be used in staining or in immunoassays. Samples, as used herein, include biological fluids such as blood, cerebrospinal fluid, dialysis fluid and the like; organ or tissue culture derived fluids; and fluids extracted from physiological tissues. Also included are derivatives and fractions of such fluids. The cells may be dissociated, in the case of solid tissues, or tissue sections may be analyzed. Alternatively a lysate of the cells may be prepared.

[89] Diagnosis may be performed by a number of methods to determine the absence or presence or altered amounts of normal or abnormal neuronostatin in patient cells. For example, detection may utilize staining of cells or histological sections, performed in accordance with conventional methods. Cells are permeabilized to stain cytoplasmic molecules. The antibodies of interest are added to the cell sample, and incubated for a period of time sufficient to allow binding to the epitope. The antibody may be labeled with radioisotopes, enzymes, fluorescers, chemiluminescers, or other labels for direct detection. Alternatively, a second stage antibody or reagent is used to amplify the signal. Such reagents are well known in the art. For example, the primary antibody may be conjugated to biotin, with horseradish peroxidase-conjugated avidin added as a second stage reagent. Alternatively, the secondary antibody conjugated to a fluorescent compound, e.g. fluorescein rhodamine, Texas red, and the like. Final detection uses a substrate that undergoes a color change in the presence of the peroxidase. The absence or presence of antibody binding may be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation and counting.

[90] In some embodiments, the methods are adapted for use in vivo. In these embodiments, a detectably-labeled moiety, e.g., an antibody, which is specific for neuronostatin is administered to an individual (e.g., by injection), and labeled cells are located using standard imaging techniques, including, but not limited to, magnetic resonance imaging, computed tomography scanning, and the like.

[91] Diagnostic screening may also be performed for polymorphisms that are genetically linked to a disease predisposition, preferably through the use of microsatellite markers or single nucleotide polymorphisms. The microsatellite polymorphism itself is in many cases not phenotypically expressed, but is linked to sequences that result in a disease predisposition. However, in some cases the microsatellite sequence itself may affect gene expression. Microsatellite linkage analysis may be performed alone, or in combination with direct detection of polymorphisms, as described above. The use of microsatellite markers for genotyping is well known. For examples, see Mansfield et al. (1994) Genomics 24:225-

233; Ziegle et al. (1992) Genomics 14:1026-1031; Dib et al., supra. [92] The detection methods can be provided as part of a kit. Thus, the invention further provides kits for detecting the presence of an mRNA encoding neuronostatin, and/or a polypeptide encoded thereby, in a biological sample. Procedures using these kits may be performed by clinical laboratories, experimental laboratories, medical practitioners, or private individuals. The kits of the invention for detecting a polypeptide comprise a moiety that specifically binds the polypeptide, which may be a specific antibody. The kits of the invention for detecting a nucleic acid comprise a moiety that specifically hybridizes to such a nucleic acid. The kit may optionally provide additional components that are useful in the procedure, including, but not limited to, buffers, developing reagents, labels, reacting surfaces, means for detection, control samples, standards, instructions, and interpretive information.

GENETICALLY ALTERED CELL OR ANIMAL MODELS FOR NEURONOSTATINFUNCTION

[93] The subject nucleic acids can be used to generate transgenic animals or site specific gene modifications in cell lines. Transgenic animals may be made through homologous recombination, where the normal neuronostatin locus is altered. Alternatively, a nucleic acid construct is randomly integrated into the genome. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like.

[94] The modified cells or animals are useful in the study of neuronostatin function and regulation. For example, a series of small deletions and/or substitutions may be made in the neuronostatin coding sequence to determine the role of different residues in receptor binding or signal transduction. In one embodiment, neuronostatin is used to construct transgenic animal models for disorders where expression of neuronostatin is specifically altered, i.e. reduced, increased, or absent. Constructs of interest include anti-sense neuronostatin which will block neuronostatin expression and expression of dominant negative neuronostatin mutations. A detectable marker, such as lac Z, may be introduced into the neuronostatin locus, where up-regulation of neuronostatin expression will result in an easily detected change in phenotype.

[95] One may also provide for expression of the neuronostatin gene or variants thereof in cells or tissues where it is not normally expressed or at abnormal times of development. By providing expression of neuronostatin protein in cells in which it is not normally produced, one can induce changes in cell behavior, e.g. in the control of cell growth and tumorigenesis.

[96] DNA constructs for homologous recombination will comprise at least a portion of the neuronostatin coding sequence with the desired genetic modification, and will include regions of homology to the target locus. The regions of homology may include coding regions, or may utilize intron and/or genomic sequence. DNA constructs for random integration need not include regions of homology to mediate recombination. Conveniently, markers for positive and negative selection are included. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For various techniques for transfecting mammalian cells, see Keown et al. (1990) Methods in Enzymology 185:527-537.

[97] For embryonic stem (ES) cells, an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g. mouse, rat, or guinea pig. Such cells are grown on an appropriate fibroblast-feeder layer or grown in the presence of leukemia inhibiting factor (LIF). When ES or embryonic cells have been transformed, they may be used to produce transgenic animals. After transformation, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct. Those colonies that are positive may then be used for embryo manipulation and blastocyst injection. Blastocysts are obtained from 4 to 6 week old superovulated females. The ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting offspring screened for the construct. By providing for a different phenotype of the blastocyst and the genetically modified cells, chimeric progeny can be readily detected.

[98] The chimeric animals are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogeneic or congenic grafts or transplants, or in culture. The transgenic animals may be any non-human mammal, such as laboratory animals and domestic animals. The transgenic animals may be used in functional studies, drug screening, and the like to determine the effect of a candidate drug on stress responses.

EXPERIMENTAL

[99] The following examples are put forth for illustrative purposes, and are not intended to limit the scope of what the inventors regard as their invention. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. EXAMPLE 1

Materials and Methods

[100] Peptide hormones. All peptides used were synthesized by Phoenix Pharmaceuticals Inc. (Burlingame, CA) or the PAN facility at Stanford University. Peptide purity was verified by analytical reverse phase HPLC and MALDI-TOF (Matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. Unless indicated otherwise, all functional tests utilized human amidated neuronostatin-13.

[101] Animals. All procedures were approved by the institutional animal committees. Adult male BALB/c mice and 7-days-old Sprague-Dawley rats from the Stanford animal facility were used for immunohistochemical staining and c-fos/c-jun induction tests.

[102] Immunohistochemical staining of neuronostatin. Tissues were obtained from adult male mice and embedded with paraffin before cutting into 25-μm sections. After soaking in xylene and series of ethanol concentrations, sections were treated with 0.01 M sodium citrate at 95C for 15 min. followed by 0.1% trypsin at 37C for 15 min. before immunostaining using affinity-purified, rabbit polyclonal antibodies against rat neuronostatin-13 (1 : 200, Phoenix Pharmaceutical Inc.), human somatostatin (1 : 500 dilution, Abeam, Cambridge, MA) or preimmune IgG. Signals were detected using the Histostain®-SP Kit (Zymed LAB, Carlsbad, CA).

[103] Induction of early response genes. Mice were injected (i.p.) with 1 ,000 nmole/kg body weight of neuronostatin or saline to detect c-fos and c-jun expression in diverse tissues. At 3 h after injection, animals were anesthetized with isoflurane and perifused via the tail vein with 4% paraformaldehyde-PBS. Tissues were fixed in 4% paraformaldehyde followed by paraffin-embedding and sectioning before staining with antibodies against c-fos (1 :300 dilution) or c-jun (1 : 1 ,000 dilution)(Abcam, Cambridge, MA). Signals were detected using the ABC staining System (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Immunostaining for insulin and glucagon were performed using specific antibodies from Abeam. For c-fos induction in brain regions, i.c.v. injections of neuronostatin (3 nmol/kg body weight) were performed for 1.5 h using immature rats followed by immunohistochemical staining.

[104] Expression of c-fos in KATO-III cells: Human gastric tumor KATO-MI cells were obtained from ATCC (Manassas, VA) and maintained in DMEM (Dulbecco's minimal essential medium) containing 10% fetal bovine serum. For quantitative real-time RT-PCR analyses of c-fos transcript levels, cells were pre-incubated under serum-free conditions for 16h before hormonal treatment. Total RNA was extracted from cells using the RNeasy kit (Qiagen Science, Valencia, CA) and genomic DNA was eliminated using DNase digestion before reverse transcription using a Sensiscript RT kit (Qiagen Science). Primers were designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA). Standard curves for c-fos and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) transcripts were generated by serial dilutions of individual cDNAs. The primer pairs used were: c-fos forward: δ'-GGACTCAAGTCCTTACCTCTTCC-S'; reverse: 5'- CCTGGCTCAACATGCTACTAACT-3'; GAPDH forward: 5'-

TCACTGCCACTCAGAAGACTGT-3'; reverse: 5'-CGTTCAGCTCTAGGATGACCTT-S'. Real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) and GAPDH levels used for copy number normalization. The assays were performed on a Smart Cycler TD System (Cepheid, Sunnyvale, CA) with an initial enzyme activation step of 15 min at 95C, followed by 45 cycles of two-step PCR (94C₁ 15s; 6OC, 60s). Data are presented as relative expression, normalized to GAPDH. Results represent mean+/- S. E. of fold changes of normalized expression. For immunofluorescence staining of c-fos antigens, cells were cultured on a cover slip in 6-well plates until 50-70% confluent. Cells were treated with 10 nM neuronostatin for 1h and rinsed twice in PBS before fixing in 4% paraformaldehyde in PBS for 20 min at 23C. Following three washes with PBS, cells were incubated with a pre-warmed antigen retrieval buffer (100 mM sodium citrate, pH 6.0) at 95C for 20 min. After further rising in PBS (x3) and incubation in 0.1% Triton X-100 in PBS for 15 min at 23C, cells were treated with 10% goat serum for 1 h at 23C. This was followed by overnight incubation of cells with the c-fos antibodies (1 :300, Abeam) at 4C. After reaction, cells were rinsed in 1 % goat serum in PBS for three times before treatment with fluorophore-conjugated secondary antibodies for 2 h at 23C under darkness. Cells were then washed three times in 1% goat serum for 10 min before staining of cell nuclei using the Hoechst 33342 dye (Invitrogen, San Diego, CA) for visualization under a fluorescence microscope. KATO-III cell proliferation and SRE-luciferase reporter assay: Cell numbers were measured based on the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay that monitored a mitochondrial dehydrogenase enzyme from viable cells using the CellTiter cell proliferation assay (Promega, Madison, Wl). This enzyme cleaves the tetrazolium rings of the pale yellow MTT to form dark blue formazan crystals (15). Cells were seeded on 96-well plates (5,000 cells/well) and incubated at 37C. After incubation for 24h in serum-free media, media were changed. Cells were then treated with or without neuronostatin for 24 h before adding the substrate for 4h to measure absorbance at 570 nm using a scanning spectrophotometer. For the reporter gene assay in KATO-III cells, cells were transfected with the SRE-luciferase reporter plasmid (Stratagene, La JoIIa, CA) for 4h, followed by incubation in serum-free media for 16h to allow recovery. Cells were then treated with different doses of neuronostatin for 6h before measurement of luciferase activity (Luciferase assay system, Promega) using a luminometer. For studying somatostatin stimulation of the inhibitory Gi protein, Chinese hamster ovary cells were co- transfected with plasmids encoding a chimeric Gqi protein (16), the SRE-luciferase reporter construct, and individual somatostatin receptors for 4h, followed by 16h for recovery. Cells were then treated with no hormone (control), 100 nM of somatostatin-14, 100 nM neuronostatin, or fetal bovine serum (FBS, positive controls) for 16h before the luciferase assay. Results

[106] Based on a computer program previously used to identify unique protein signatures, we searched for potential mono- or di-basic cleavage sites in ~200 known preprohormone sequences. Candidate regions were further checked for evolutionary conservation of putative mature peptide regions in diverse species. As shown in Figure 1 , Amino acid sequences of prepro-somatostatin from different vertebrates are shown with the signal peptide (underlined), mature somatostatin (shaded), and the predicted neuronostatin (bold letters). Consensus basic residues representing putative convertase cleavage sites are shown as white letters on a black background. In the consensus sequence, individual residues with conservation in at least 11 of the 13 species are shown in upper case. Due to the existence of a conserved glycine residue at its C-terminus, mature neuronostatin is predicted to be amidated. In addition, the total length of neuronostatin could be variable (6, 11 , 13, and 19 residues) due to the presence of conserved basic residues as potential proteolytic cleavage sites (arrows). GenBank (gi) numbers for individual somatostatin genes are 4507243 (human), 55621730 (chimpanzee), 57528038 (pig), 50979130 (dog), 57163953 (sheep), 73697560 (cattle), 6678035 (mouse), 6981582 (rat), 45385811 (chicken), 32454336 (frog), 9978804 (lungfish), 34098954 (zebrafish), and 9978923 (goldfish).

[107] Due to the existence of a conserved glycine residue at its C-terminus, mature neuronostatin is predicted to be amidated. In addition, the total length of neuronostatin can be variable (6, 11 , 13, and 19 residues) due to the presence of conserved basic residues as potential proteolytic cleavage sites (arrows).

[108] To estimate gastric emptying responses, mice deprived of food for 16 hours were given food pellets for 90 min. before injection of chemically synthesized neuronostatin (500 nmol/kg body weight) or saline. After treatment, mice were deprived of food again and sacrificed 2 hours later. The stomach was excised at the pylorus and cardia before weighing. Gastric emptying was calculated by subtracting the stomach weight of treated mice from those sacrificed at the time of peptide injection. Results are shown in Figure 2.

[109] C57BL6 male mice at eight weeks of age (two/cage) were deprived of water but given free access to food for 16 h before testing. At 10:30h on test day, animals were treated with an intracerebroventricular injection of saline (3 μl), vehicle containing obestatin (15 nmole/kg body weight) or obestatin plus neuronostatin (15 nmole/kg body weight) into the left ventricle. Water bottles were replaced at 15 min after injection and water intake monitored. The results are shown in Figure 3.

[110] To investigate potential target cells for neuronostatin, adult male mice were treated i.p. with neuronostatin. lmmunostaining was performed 3 h later to detect the expression of c- fos and c-jun, early response genes known to be induced after diverse hormonal stimulation. Nuclear staining of c-fos was found mainly in the chief cells of gastric mucosa in adult mice treated with neuronostatin but not in saline-treated controls. In the jejunum, neuronostatin stimulated nuclear c-fos expression in cells of the intestinal villi but not crypts. Non-specific staining was found in the lumen of both groups. In the anterior pituitary, neuronostatin-treated animals showed c-fos staining, unlike saline-treated controls. Although no c-fos stimulation was found in the pancreatic islets, neuronostatin treatment induced c-jun expression mainly in the periphery of islets, likely representing alpha cells expressing glucagon and not beta cells expressing insulin. Following i.c.v. (intracerebroventricular) injection of neuronostatin for 1.5 h in immature rats, nuclear c-fos staining was found in granule cells and cells in the Purkinje layer in the cerebellum as well as in pyramidal cells of the hippocampus. In contrast, treatment with saline was ineffective. Figure 4A, 4B, 4C, and 4D.

[111] Adult male C57BL6 mice were treated with an intracerebroventricular injection of saline (3 μl), or vehicle containing neuronostatin (15 nmole/kg body weight) into the left ventricle. Brain tissues were obtained at 1 h after injection and fixed using Bouin's solution before embedding in paraffin for making slides. After treating tissue sections with 0.01 M sodium citrate at 95C for 15 min followed by 0.1% trypsin at 37C for 15 min, immunostaining was performed using affinity-purified, rabbit polyclonal antibodies raised against a synthetic peptide corresponding to the N-terminus of human c-Fos (Santa Cruz Biotech, Santa Cruz, CA). After pretesting the optimal antibody titers, tissue staining was performed with c-Fos antibody (1 :3,000 dilution) and signals were detected using the Histostain®-SP Kits Zymed® LAB-SA Detection System. The staining is shown in Figure 4E and 4F.

[112] To further test neuronostatin actions in vitro, human gastric tumor KATO-III cells were treated with neuronostatin before analyzing c-fos expression. As shown in Fig. 5A, realtime RT-PCR analyses indicated a time-dependent stimulation of c-fos transcript levels. Also, c-fos staining was detected 1 h after treatment with neuronostatin (Fig. 5Bb) but not in untreated cells (Fig. 5Bd). We also tested the ability of neuronostatin to regulate KATO-III cell proliferation based on the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay that monitored a mitochondrial dehydrogenase enzyme from viable cells. As shown in Fig. 5C, treatment with either human or mouse neuronostatin led to a dose- dependent stimulation of the proliferation of KATO-III cells with ED50 values of ~5 nM. Because the promoter of the c-fos gene contains multiple SRE (serum response element) sites, the ability of neuronostatin to stimulate an SRE-driven luciferase reporter activity was also tested. As shown in Fig. 5D, treatment with either human or mouse neuronostatin led to a dose-dependent stimulation of luciferase activity. In contrast, treatment with somatostatin-14 was ineffective.

[113] Based on bioinformatic prediction followed by immuno-affinity purification, we have demonstrated herein the existence of a new peptide hormone encoded by the pro- somatostatin protein. Neuronostatin induced the expression of early response genes in a variety of tissues. We further demonstrated the ability of this hormone to modulate gastric emptying responses, and gastric tumor cell proliferation, as well as to modulate water drinking.

[114] After the synthesis of pro-somatostatin, this precursor is likely to be cleaved by one or more convertase enzymes at basic residues to yield somatostatin and neuronostatin peptides. Unlike somatostatin and cortistatin, neuronostatin is not a cyclic polypeptide and is amidated. Although derived from the same preproprotein, the processing of neuronostatin and somatostatin can be differentially regulated. Due to the need for the amidation of neuronostatin but not somatostatin, expression of amidation enzymes in cells expressing somatostatin transcripts could determine neuronostatin levels. Because the neuronostatin sequence, including the Gly-Lys residues at its C-terminus, is conserved from mammals to fish, this amidated hormone likely plays important roles in diverse vertebrates.

[115] Neuronostatin is a brain/gut peptide due to its site of production and its ability to induce early response genes c-fos or c-jun in neuronal, anterior pituitary, and gastrointestinal tissues. Studies using human stomach tumor KATO-III cells indicated that neuronostatin acts directly on gastric cells and may also serve as a paracrine hormone. Similar to somatostatin, neuronostatin is likely expressed in pancreatic delta cells and neuronostatin could act on alpha cells in a paracrine manner.

[116] Neuronostatin directly regulates human tumor KATO-III cell functions as reflected by the stimulation of c-fos induction, cell proliferation, and SRE-luciferase activity. The ability of both human and mouse neuronostatin to stimulate the proliferation and SRE-luciferase activity in KATO-III cells at nM levels suggests mediation by specific receptors. Neuronostatin, unlike somatostatin, does not stimulate Gi signaling mediated by the somatostatin receptors. Neuronostatin also did not modulate GH release by cultured pituitary cells.

[117] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. Accordingly, it should be understood that the scope of the invention is not limited by this detailed description, but by the appended claims as properly construed under principles of patent law.

Claims

WHAT is CLAIMED IS:

1. An isolated neuronostatin polypeptide.

2. An isolated polypeptide having at least 6 contiguous amino acids of the sequence PSDPRLRQFLQKSLAAAAG (SEQ ID NO:1).

3. The polypeptide of Claim 2, wherein the C-terminal glycine is converted to an amide moiety.

4. The polypeptide of Claim 2, wherein the polypeptide consists of a fragment selected from the group consisting of: PSDPRLRQFLQKSLAAAA-NH₂; RLRQFLQKSLAAAA-NH₂; LRQFLQKSLAAAA-NH₂; QFLQKSLAAAA-NH₂; and SLAAAA- NH₂.

5. The polypeptide of Claim 2, wherein the polypeptide consists of the sequence pyro-Glu-FLQKSLAAAA-NH₂.

6. A polypeptide of from 6 to 19 amino acids, having at least 95% sequence similarity to SEQ ID NO:1.

7. An isolated polypeptide consisting essentially of a sequence set forth in claim 2, 4 or 5.

8. An isolated polynucleotide encoding a polypeptide as set forth in any one of Claims 1-7.

9. A vector comprising an isolated nucleic acid molecule as defined in Claim 8, operably linked with a promoter sequence.

10. A host cell transformed with the vector of Claim 9.

11. A pharmaceutical composition comprising a therapeutically effective amount of a neuronostatin peptide; and a pharmaceutically acceptable carrier.

12. A method of modulating gastrointestinal motility, the method comprising administering to an individual a pharmaceutical composition as set forth in Claim 11.

13. A method of neuroprotection, the method comprising administering to an individual a pharmaceutical composition as set forth in Claim 11.

14. An antibody that specifically recognizes a neuronostatin peptide.

15. A method of screening compounds to identify biologically active agents that modulate neuronostatin function, the method comprising: combining a compound with:

(a) a mammalian neuronostatin peptide;

(b) a cell comprising a nucleic acid encoding a mammalian neuronostatin peptide; or

(c) a non-human transgenic animal model for neuronostatin gene function comprising (i) a knockout of a fragment of somatostatin gene encoding neuronostatin sequence or (ii) an exogenous and stably transmitted mammalian fragment of somatostatin gene encoding neuronostatin sequence; and determining the effect of said compound on neuronostatin function.