METHODS, COMPOSITIONS AND COMPOUND ASSAYS FOR INHIBITING AMYLOID-BETA PROTEIN PRODUCTION

Abstract

compounds that inhibit amyloid-beta precursor protein processing in cells, comprising contacting a test compound with a GPCR polypeptide, or fragment thereof, and measuring a compound-GPCR property related to the production of amyloid-beta peptide. Cellular assays of the method measure indicators including second messenger and/or amyloid beta peptide levels. Therapeutic methods, and pharmaceutical compositions including effective amyloid-beta precursor processing-inhibiting amounts of GPCR expression inhibitors, are useful for treating conditions involving cognitive impairment such as Alzheimers Disease.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No. 12/190,888 filed Aug. 13, 2008, which is a divisional of U.S. application Ser. No. 11/110,274 filed Apr. 20, 2005, now U.S. Pat. No. 7,429,459, which claims priority to U.S. Provisional Application No. 60/563,661, filed Apr. 20, 2004, the disclosures of which are incorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the sequence listing, "Seq_Listing.txt" (109,795 bytes), submitted via EFS-WEB and created on Aug. 12, 2008, is herein incorporated by reference.

FIELD OF THE INVENTION

[0003] This invention relates to the field of mammalian neuronal cell disorders, and in particular, to methods for identifying effective compounds, and therapies and compositions using such compounds, useful for the prevention and treatment of diseases associated with progressive loss of intellectual capacities in humans.

[0004] The neurological disorder that is most widely known for its progressive loss of intellectual capacities is Alzheimer's disease (AD). Worldwide, about 20 million people suffer from Alzheimer's disease. AD is clinically characterized by the initial loss of memory, followed by disorientation, impairment of judgment and reasoning, which is commonly referred to as cognitive impairment, and ultimately by full dementia. AD patients finally lapse into a severely debilitated, immobile state between four and twelve years after onset of the disease.

[0005] The key pathological evidence for AD is the presence of extracellular amyloid plaques and intracellular tau tangles in the brain, which are associated with neuronal degeneration (Ritchie and Lovestone (2002)). The extracellular amyloid plaques are believed to result from an increase in the insoluble amyloid beta peptide 1-42 produced by the metabolism of amyloid-beta precursor protein (APP). Following secretion, these amyloid beta 1-42 peptides form amyloid fibrils more readily than the amyloid beta 1-40 peptides, which are predominantly produced in healthy people. It appears that the amyloid beta peptide is on top of the neurotoxic cascade: experiments show that amyloid beta fibrils, when injected into the brains of P301L tau transgenic mice, enhance the formation of neurofibrillary tangles (Gotz et al. (2001)). In fact, a variety of amyloid beta peptides have been identified as amyloid beta peptides 1-42, 1-40, 1-39, 1-38, 1-37, which can be found in plaques and are often seen in cerebral spinal fluid.

[0006] The amyloid beta peptides are generated (or processed) from the membrane anchored APP, after cleavage by beta secretase and gamma secretase at position 1 and 40 or 42, respectively (FIG. 1A)(Annaert and De Strooper (2002)). In addition, high activity of beta secretase results in a shift of the cleavage at position 1 to position 11. Cleavage of amyloid-beta precursor protein by alpha secretase activity at position 17 and gamma secretase activity at 40 or 42 generates the non-pathological p3 peptide. Beta secretase was identified as the membrane anchored aspartyl protease BACE, while gamma secretase is a protein complex comprising presenilin 1 (PS1) or presenilin 2 (PS2), nicastrin, Anterior Pharynx Defective 1 (APH1) and Presenilin Enhancer 2 (PEN2). Of these proteins, the presenilins are widely thought to constitute the catalytic activity of the gamma secretase, while the other components play a role in the maturation and localization of the complex. The identity of the alpha secretase is still illustrious, although some results point towards the proteases ADAM 10 and TACE, which could have redundant functions.

[0007] A small fraction of AD cases (mostly early onset AD) are caused by autosomal dominant mutations in the genes encoding presenilin 1 and 2 (PS1; PS2) and the amyloid-beta precursor protein (APP), and it has been shown that mutations in APP, PS1 and PS2 alter the metabolism of amyloid-beta precursor protein leading to such increased levels of amyloid beta 1-42 produced in the brain. Although no mutations in PS1, PS2 and amyloid-beta precursor protein have been identified in late onset AD patients, the pathological characteristics are highly similar to the early onset AD patients. These increased levels of amyloid beta peptide could originate progressively with age from disturbed amyloid-beta precursor protein processing (e.g. high cholesterol levels enhance amyloid beta peptide production) or from decreased amyloid beta peptide catabolism. Therefore, it is generally accepted that AD in late onset AD patients is also caused by aberrant increased amyloid peptide levels in the brains. The level of these amyloid beta peptides, and more particularly amyloid-beta peptide 1-42, is increased in Alzheimer patients compared to the levels of these peptides in healthy persons. Thus, reducing the levels of these amyloid beta peptides is likely to be beneficial for patients with cognitive impairment.

Reported Developments

[0008] The major current AD therapies are limited to delaying progressive memory loss by inhibiting the acetylcholinesterase enzyme, which increases acetylcholine neurotransmitter levels, which fall because the cholinergic neurons are the first neurons to degenerate during AD. This therapy does not halt the progression of the disease.

[0009] Therapies aimed at decreasing the levels of amyloid beta peptides in the brain, are increasingly being investigated and focus on the perturbed amyloid-beta precursor protein processing involving the beta- or gamma secretase enzymes.

[0010] The present invention is based on the discovery that certain known polypeptides are factors in the up-regulation and/or induction of amyloid beta precursor processing in neuronal cells, and that the inhibition of the function of such polypeptides are effective in reducing levels of amyloid beta peptides.

SUMMARY OF THE INVENTION

[0011] The present invention relates to the relationship between the function of the G-protein coupled receptor(s) ("GPCR(s)") and amyloid-beta precursor protein processing in mammalian cells.

[0012] One aspect of the present invention is a method for identifying a compound that inhibits the processing of amyloid-beta precursor protein in a mammalian cell, comprising [0013] (a) contacting a compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6, 289-333; and [0014] (b) measuring a compound-polypeptide property related to the production of amyloid-beta protein.

[0015] Aspects of the present method include the in vitro assay of compounds using polypeptide domains of a GPCR, and cellular assays wherein GPCR inhibition is followed by observing indicators of efficacy, including second messenger levels and/or amyloid beta peptide levels.

[0016] Another aspect of the invention is a method of treatment or prevention of a condition involving cognitive impairment, or a susceptibility to the condition, in a subject suffering or susceptible thereto, by administering a pharmaceutical composition comprising an effective amyloid-beta precursor processing-inhibiting amount of a GPCR antagonist or inverse agonist.

[0017] A further aspect of the present invention is a pharmaceutical composition for use in said method wherein said inhibitor comprises a polynucleotide selected from the group of an antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a naturally occurring polynucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6.

[0018] Another further aspect of the present invention is a pharmaceutical composition comprising a therapeutically effective amyloid-beta precursor processing-inhibiting amount of a GPCR antagonist or inverse agonist or its pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof in admixture with a pharmaceutically acceptable carrier. The present polynucleotides and GPCR antagonist and inverse agonist compounds are also useful for the manufacturing of a medicament for the treatment of Alzheimer's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1A: APP processing: The membrane anchored amyloid precursor protein (APP) is processed by two pathways: the amyloidogenic and non amyloidogenic pathway. In the latter pathway, APP is cleaved first by alpha secretase and then by gamma secretase, yielding the p3 peptides (17-40 or 17-42). The amyloidogenic pathway generates the pathogenic amyloid beta peptides (A beta) after cleavage by beta- and gamma-secretase respectively. The numbers depicted are the positions of the amino acids comprising the A beta sequences.

[0020] FIG. 1B: Pictorial representation of transmembrane structure of GPCR proteins.

[0021] FIG. 2: Evaluation of the APP processing assay: Positive (PS1G384L; PS1L392V and BACE1) and negative (eGFP, LacZ and empty) control viruses are infected in Hek293APPwt at random MOI, mimicking a screening. A and B: Transduction is performed respectively with 1 and 0.2 l of virus and amyloid beta 1-42 levels are performed. Data are represented as relative light units and correlate to pM of amyloid beta 1-42.

[0022] FIG. 3: Involvement of GPR3 in APP processing: HEK293 APPwt cells are transduced with Ad5/GPR3 and with negative control viruses (Ad5/empty, Ad5/LacZ, Ad5/eGFP and Ad5/luciferase) at different MOIs (2, 10, 50, 250). Resulting amyloid beta 1-42, 1-40, 11-42, x-42 and 1-x peptides were measured with the appropriate ELISAs. Data are represented in pM or as relative light units (du), which correlates to pM of amyloid beta.

[0023] FIG. 4: Transfection with GPR3 siRNA reduces Amyloid beta 1-42: HEK293 APPwt c129 cells are transfected with siRNA of GPR3, eGFP, Luciferase and BACE and amyloid beta 1-42 levels are determined. Cells are transfected and 24 hours after transfection, medium is refreshed and cells are allowed to accumulate amyloid beta for 24 hours (48 hours post transfection (p.t.)). Amyloid beta is determined by means of the amyloid beta 1-42 ELISA as described above. Data are presented in pM of amyloid beta. RNA levels of GPR3 are determined from these samples.

[0024] FIG. 5: ClustalW protein sequence alignment of GPR3, GPR6 and GPR12.

[0025] FIG. 6: Graph of amyloid beta peptide levels in neurons transfected with a variety of protein expression viruses at different MOI. The graph shows that increased levels of GPR3 overexpression in primary neurons result in a corresponding dose dependent increase of amyloid beta 1-42 levels compared to the negative controls.

DETAILED DESCRIPTION

[0026] The following terms are intended to have the meanings presented therewith below and are useful in understanding the description of and intended scope of the present invention.

DEFINITIONS

[0027] The term "agonist" refers to a ligand that activates the intracellular response of the receptor to which the agonist binds.

[0028] The term "amyloid beta peptide" means amyloid beta peptides processed from the amyloid beta precursor protein (APP). The most common peptides include amyloid beta peptides 1-40, 1-42, 11-40 and 11-42. Other species less prevalent amyloid beta peptides are described as y-42, whereby y ranges from 2-17, and 1-x whereby x ranges from 24-39 and 41.

[0029] The term "antagonist" means a moiety that bind competitively to the receptor at the same site as the agonists but which do not activate the intracellular response initiated by the active form of the receptor, and can thereby inhibit the intracellular responses by agonists. Antagonists do not diminish the baseline intracellular response in the absence of an agonist or partial agonist.

[0030] The term "carrier" means a non-toxic material used in the formulation of pharmaceutical compositions to provide a medium, bulk and/or useable form to a pharmaceutical composition. A carrier may comprise one or more of such materials such as an excipient, stabilizer, or an aqueous pH buffered solution. Examples of physiologically acceptable carriers include aqueous or solid buffer ingredients including phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter ions such as sodium; and/or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0031] The term "compound" is used herein in the context of a "test compound" or a "drug candidate compound" described in connection with the assays of the present invention. As such, these compounds comprise organic or inorganic compounds, derived synthetically or from natural sources. The compounds include inorganic or organic compounds such as polynucleotides, lipids or hormone analogs that are characterized by relatively low molecular weights. Other biopolymeric organic test compounds include peptides comprising from about 2 to about 40 amino acids and larger polypeptides comprising from about 40 to about 500 amino acids, such as antibodies or antibody conjugates.

[0032] The term "constitutive receptor activation" means stabilization of a receptor in the active state by means other than binding of the receptor with its endogenous ligand or a chemical equivalent thereof.

[0033] The term "contact" or "contacting" means bringing at least two moieties together, whether in an in vitro system or an in vivo system.

[0034] The term "condition" or "disease" means the overt presentation of symptoms (i.e., illness) or the manifestation of abnormal clinical indicators (e.g., biochemical indicators), resulting from defects in one amyloid beta protein precursor processing. Alternatively, the term "disease" refers to a genetic or environmental risk of or propensity for developing such symptoms or abnormal clinical indicators.

[0035] The term "endogenous" shall mean a material that a mammal naturally produces. Endogenous in reference to, for example and not limitation, the term "receptor" shall mean that which is naturally produced by a mammal (for example, and not limitation, a human) or a virus. In contrast, the term non-endogenous in this context shall mean that which is not naturally produced by a mammal (for example, and not limitation, a human) or a virus. For example, and not limitation, a receptor which is not constitutively active in its endogenous form, but when manipulated becomes constitutively active, is most preferably referred to herein as a "non-endogenous, constitutively activated receptor." Both terms can be utilized to describe both "in vivo" and "in vitro" systems. For example, and not a limitation, in a screening approach, the endogenous or non-endogenous receptor may be in reference to an in vitro screening system. As a further example and not limitation, where the genome of a mammal has been manipulated to include a non-endogenous constitutively activated receptor, screening of a candidate compound by means of an in vivo system is viable.

[0036] The term "expression" comprises both endogenous expression and overexpression by transduction.

[0037] The term "expressible nucleic acid" means a nucleic acid coding for a proteinaceous molecule, an RNA molecule, or a DNA molecule.

[0038] The term "hybridization" means any process by which a strand of nucleic acid binds with a complementary strand through base pairing. The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., C₀t or R₀t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed). The term "stringent conditions" refers to conditions that permit hybridization between polynucleotides and the claimed polynucleotides. Stringent conditions can be defined by salt concentration, the concentration of organic solvent, e.g., formamide, temperature, and other conditions well known in the art. In particular, reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature can increase stringency.

[0039] The term "inhibit" or "inhibiting", in relationship to the term "response" means that a response is decreased or prevented in the presence of a compound as opposed to in the absence of the compound.

[0040] The term "inverse agonist" mean a moiety that binds the endogenous form of the receptor, and which inhibits the baseline intracellular response initiated by the active endogenous form of the receptor below the normal base level of activity that is observed in the absence of the endogenous ligand, or agonists, or decrease GTP binding to membranes. Preferably, the baseline intracellular response is decreased in the presence of the inverse agonist by at least 30%, more preferably by at least 50%, and most preferably by at least 75%, as compared with the baseline response in the absence of the inverse agonist.

[0041] The term "ligand" means an endogenous, naturally occurring molecule specific for an endogenous, naturally occurring receptor.

[0042] The term "pharmaceutically acceptable prodrugs" as used herein means the prodrugs of the compounds useful in the present invention, which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients with undue toxicity, irritation, allergic response commensurate with a reasonable benefit/risk ratio, and effective for their intended use of the compounds of the invention. The term "prodrug" means a compound that is transformed in vivo to yield an effective compound useful in the present invention or a pharmaceutically acceptable salt, hydrate or solvate thereof. The transformation may occur by various mechanisms, such as through hydrolysis in blood. The compounds bearing metabolically cleavable groups have the advantage that they may exhibit improved bioavailability as a result of enhanced solubility and/or rate of absorption conferred upon the parent compound by virtue of the presence of the metabolically cleavable group, thus, such compounds act as pro-drugs. A thorough discussion is provided in Design of Prodrugs, H. Bundgaard, ed., Elsevier (1985); Methods in Enzymology; K. Widder et al, Ed., Academic Press, 42, 309-396 (1985); A Textbook of Drug Design and Development, Krogsgaard-Larsen and H. Bandaged, ed., Chapter 5; "Design and Applications of Prodrugs" 113-191 (1991); Advanced Drug Delivery Reviews, H. Bundgard, 8 , 1-38, (1992); J. Pharm. Sci., 77, 285 (1988); Chem. Pharm. Bull., N. Nakeya et al, 32, 692 (1984); Pro-drugs as Novel Delivery Systems, T. Higuchi and V. Stella, 14 A.C.S. Symposium Series, and Bioreversible Carriers in Drug Design, E. B. Roche, ed., American Pharmaceutical Association and Pergamon Press, 1987, which are incorporated herein by reference. An example of the prodrugs is an ester prodrug. "Ester prodrug" means a compound that is convertible in vivo by metabolic means (e.g., by hydrolysis) to an inhibitor compound according to the present invention. For example an ester prodrug of a compound containing a carboxy group may be convertible by hydrolysis in vivo to the corresponding carboxy group.

[0043] The term "pharmaceutically acceptable salts" refers to the non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of compounds useful in the present invention.

[0044] The term "polynucleotide" means a polynucleic acid, in single or double stranded form, and in the sense or antisense orientation, complementary polynucleic acids that hybridize to a particular polynucleic acid under stringent conditions, and polynucleotides that are homologous in at least about 60 percent of its base pairs, and more preferably 70 percent of its base pairs are in common, most preferably 90 percent, and in a special embodiment 100 percent of its base pairs. The polynucleotides include polyribonucleic acids, polydeoxyribonucleic acids, and synthetic analogues thereof. The polynucleotides are described by sequences that vary in length, that range from about 10 to about 5000 bases, preferably about 100 to about 4000 bases, more preferably about 250 to about 2500 bases. A preferred polynucleotide embodiment comprises from about 10 to about 30 bases in length. A special embodiment of polynucleotide is the polyribonucleotide of from about 10 to about 22 nucleotides, more commonly described as small interfering RNAs (siRNAs). Another special embodiment are nucleic acids with modified backbones such as peptide nucleic acid (PNA), polysiloxane, and 2'-O-(2-methoxy)ethylphosphorothioate, or including non-naturally occurring nucleic acid residues, or one or more nucleic acid substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-, amino-, propyl-, chloro-, and methanocarbanucleosides, or a reporter molecule to facilitate its detection.

[0045] The term "polypeptide" relates to proteins, proteinaceous molecules, fractions of proteins (such as kinases, proteases, GPCRs), peptides and oligopeptides.

[0046] The term "solvate" means a physical association of a compound useful in this invention with one or more solvent molecules. This physical association includes hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. "Solvate" encompasses both solution-phase and isolable solvates. Representative solvates include hydrates, ethanolates and methanolates.

[0047] The term "subject" includes humans and other mammals.

[0048] The term "effective amount" or "therapeutically effective amount" means that amount of a compound or agent that will elicit the biological or medical response of a subject that is being sought by a medical doctor or other clinician. In particular, with regard to treating an neuronal disorder, the term "effective amount" is intended to mean that effective amyloid-beta precursor processing inhibiting amount of an compound or agent that will bring about a biologically meaningful decrease in the levels of amyloid beta peptide in the subject's brain tissue.

[0049] The term "treating" means an intervention performed with the intention of preventing the development or altering the pathology of, and thereby alleviating a disorder, disease or condition, including one or more symptoms of such disorder or condition. Accordingly, "treating" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treating include those already with the disorder as well as those in which the disorder is to be prevented. The related term "treatment," as used herein, refers to the act of treating a disorder, symptom, disease or condition, as the term "treating" is defined above.

[0050] The background of the present inventors' discovery is described briefly below.

Background of the G-Protein Couple Receptors:

[0051] In 1994, Marchese and co-workers cloned the GPR3 gene (Marchese et al., 1994) and one year later, it was found that a single exon encoded this receptor protein of 330 amino acids, also called adenylate cyclase constitutive activator (ACCA). Based on the amino acid sequence, GPR3 can be classified in the same sub-family as GPR6 and GPR12: GPR3 and GPR6 exhibit 58% identity, and GPR3 and GPR12 57% (FIG. 5).

[0052] G protein-coupled receptors (GPCR) share a common structural motif. All these receptors have seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane forming 7 transmembrane domains, an extracellular N-terminus and an intracellular C-terminus. The transmembrane helices are joined by strands of amino acids having a larger loop between the fourth and fifth transmembrane helix on the extracellular side of the membrane. Another larger loop, composed primarily of hydrophilic amino acids, joins transmembrane helices five and six on the intracellular side of the membrane. See FIG. 1B.

[0053] Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different states or conformations: an "inactive" state and an "active" state. A receptor in an inactive state is unable to link to the intracellular transduction pathway to produce a biological response. Changing the receptor conformation to the active state allows linkage to the transduction pathway and produces a biological response. A receptor may be stabilized in an active state by an endogenous ligand or an exogenous agonist ligand. Recent discoveries, including but not exclusively limited to, modifications to the amino acid sequence of the receptor, provide alternative mechanisms other than ligands to stabilize the active state conformation. These approaches effectively stabilize the receptor in an active state by simulating the effect of a ligand binding to the receptor. Stabilization by such ligand-independent approaches is termed "constitutive receptor activation."

[0054] The major signal transduction cascades activated by GPCRs are initiated by the activation of heterotrimeric G-proteins, built from three different proteins; the G, G and G subunits. It is believed that the loop joining helices five and six, as well as the carboxy terminus, interact with the G protein. Uhlenbrock and colleagues (2002) showed that GPR3, GPR6 and GPR12 all confer constitutive activation of G(a)(s) and G(a)(i/o), and, recently, sphingosine-1-phosphate (S1P) and dihydrosphingosine 1-phosphate (DHS1P) have been identified as bioactive lipid ligands for GPR3, GPR6 and GPR12 (Uhlenbrock et al., 2002). The GPR3, GPR6 and GPR12 expression profile is also similar: they are all primary expressed in brain tissue.

[0055] The signal transduction cascade starts with the activation of the receptor by an agonist. Transformational changes in the receptor are then translated down to the G-protein. The G-protein dissociates into the G subunit and the G subunit. Both subunits dissociate from the receptor and are both capable of initiating different cellular responses. Best known are the cellular effects that are initiated by the G subunit. It is for this reason that G-proteins are categorized by their G subunit. The G-proteins are divided into four groups: G_s, G_i/o, G_q and G₁₂/13. Each of these G-proteins is capable of activating an effector protein, which results in changes in second messenger levels in the cell. The changes in second messenger level are the triggers that make the cell respond to the extracellular signal in a specific manner. The activity of a GPCR can be measured by measuring the activity level of the second messenger.

[0056] The two most important second messengers in the cell are cAMP and Ca²+. The -subunit of the G_s class of G-proteins is able to activate adenylyl cyclase, resulting in an increased turnover from ATP to cAMP. The -subunit of G_i/o G-proteins does exactly the opposite and inhibits adenylyl cyclase activity resulting in a decrease of cellular cAMP levels. Together, these two classes of G-proteins regulate the second messenger cAMP. Ca²+ is regulated by the -subunit of the G_q class of G-proteins. Through the activation of phospholipase C phosphatidylinositol 4,5-bisphosphate (PIP2) from the cell membrane are hydrolyzed to inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, both these molecules act as second messengers. Inositol 1,4,5-trisphosphate binds specific receptors in the endoplasmatic reticulum, resulting in the opening of Ca²+ channels and release of Ca²+ in the cytoplasm.

[0057] No clear functions have been assigned to the GPCRs. The expression level of GPR3 and of GPR12 is increased in human umbilical vein endothelial cells after shear stress (Uhlenbrock et al., 2003). Since sphingosine-1-phosphate is a ligand for GPR3 and GPR12, the above data suggest a role for both GPCRs in sphingosine-1-phosphate-mediated intracellular signaling in human endothelial cells. As the expression of GPR3 and GPR6 is also differentially regulated in rodent obesity models, both GPCRs (+GPR12) are considered as putative drug targets in appetite, hunger and satiety control. GPR12, on the other hand, seems to be involved in the differentiation and maturation of post mitotic neurons (Ignatov et al., 2003).

REFERENCES

[0058] Annaert, W. and B. De Strooper (2002). "A cell biological perspective on Alzheimer's disease." Annu Rev Cell Dev Biol 18: 25-51. [0059] Gotz, J. F. Chen, et al. (2001). "Formation of neurofibrillary tangles in P3011 tau transgenic mice induced by Abeta 42 fibrils." Science 293(5534): 1491-5. [0060] Ignatov, A.; Lintzel, J.; Hermans-Borgmeyer, I.; Kreienkamp, H-J., Joost, P.; Thomsen, S.; Methner, A. And Schaller, H. C. (2003). Role of the G-protein-coupled receptor GPR12 as high-affinity receptor for sphingosylphosphorylcholine and its expression and function in brain development. J. Neurosci. 23, 3: 907-914. [0061] Lipinski, C. A., Lombardo, F., Dominy, B. W., and Feeney, P. J. Adv. Drug. Deliv. Rev., 23, 3-25, 1997 [0062] Marchese, A.; Docherty, J M.; Nguyen, T.; Heiber, M.; Cheng, R.; Heng, H H.; Tsui, L C.; Shi, X.; George S R. and O'Dowd, B F. (1994). Cloning of human genes encoding novel G protein-coupled receptors. Genomics, 23, 3: 609-618. Marinissen, M. J. and J. S. Gutkind (2001). "G-protein-coupled receptors and signaling networks: emerging paradigms." Trends Pharmacol Sci 22(7): 368-76. [0063] Ritchie, K. and S. Lovestone (2002). "The dementias." Lancet 360(9347): 1759-66. [0064] Uhlenbrock, K.; Gassenhuber, H. And Kostenis, E. (2002). Sphingosine-1-phosphate is a ligand of the human GPR3, GPR6 and GPR12 family of constitutively active G protein-coupled receptors. Cell Signal, 14, 11: 941-953. [0065] Uhlenbrock, K.; Huber, J.; Ardati, A.; Bush, A E. And Kostenis, E. (2003). Fluid shear stress differentially regulates GPR3, GPR6 and GPR12 expression in human umbilical vein endothelial cells. Cell Physiol. Biochem. 13, 2: 75-84. [0066] Wess, J. (1998). "Molecular basis of receptor/G-protein-coupling selectivity." Pharmacol Ther 80(3): 231-64.

Applicants' Invention Based on GPCR Relationship to Amyloid Beta Peptides

[0067] As noted above, the present invention is based on the present inventors' discovery that the G-protein coupled receptor(s) ("GPCR(s)") are factors in the up-regulation and/or induction of amyloid beta precursor processing in mammalian, and principally, neuronal cells, and that the inhibition of the function of such polypeptides is effective in reducing levels of amyloid beta protein peptides.

[0068] The present inventors are unaware of any prior knowledge linking GPCRs, and more particularly GPR3, and amyloid beta peptide formation and secretion. As discussed in more detail in the Experimental section below, the present inventors demonstrate that the overexpression of GPR3 increases, and the knockdown of GPR3 reduces, amyloid beta 1-42 in the conditioned medium of transduced cells. The present invention is based on these findings and the recognition that the GPCRs are putative drug targets for Alzheimer's disease, since the predominant expression of GPR3, GPR6 and GPR12 is in the tissue of the central nervous system.

[0069] One aspect of the present invention is a method based on the aforesaid discovery for identifying a compound that inhibits the processing of amyloid-beta precursor protein in a mammalian cell, and may therefore be useful in reducing amyloid beta peptide levels in a subject. The present method comprises contacting a drug candidate compound with a GPCR polypeptide, or a fragment of said polypeptide, and measuring a compound-polypeptide property related to the production of amyloid-beta protein. The "compound-polypeptide property" is a measurable phenomenon chosen by the person of ordinary skill in the art, and based on the recognition that GPCR activation and deactivation is a causative factor in the activation and deactivation, respectively, of amyloid beta protein precursor processing, and an increase and decrease, respectively, of amyloid beta peptide levels. The measurable property may range from the binding affinity for a peptide domain of the GPCR polypeptide, to the level of any one of a number of "second messenger" levels resulting from the activation or deactivation of the GPCR, to a reporter molecule property directly linked to the aforesaid second messenger, and finally to the level of amyloid beta peptide secreted by the mammalian cell contacted with the compound.

[0070] Depending on the choice of the skilled artisan, the present assay method may be designed to function as a series of measurements, each of which is designed to determine whether the drug candidate compound is indeed acting on the GPCR to amyloid beta peptide pathway. For example, an assay designed to determine the binding affinity of a compound to the GPCR, or fragment thereof, may be necessary, but not sufficient, to ascertain whether the test compound would be useful for reducing amyloid beta peptide levels when administered to a subject. Nonetheless, such binding information would be useful in identifying a set of test compounds for use in an assay that would measure a different property, further down the biochemical pathway. Such second assay may be designed to confirm that the test compound, having binding affinity for a GPCR peptide, actually down-regulates or inhibits, as an agonist or inverse agonist, GPCR function in a mammalian cell. This further assay may measure a second messenger that is a direct consequence of the activation or deactivation of the GPCR, or a synthetic reporter system responding to the messenger. Measuring a different second messenger, and/or confirming that the assay system itself is not being affected directly and not the GPCR pathway may further validate the assay. In this latter regard, suitable controls should always be in place to insure against false positive readings.

[0071] The order of taking these measurements is not believed to be critical to the practice of the present invention, which may be practiced in any order. For example, one may first perform a screening assay of a set of compounds for which no information is known respecting the compounds' binding affinity for GPCR. Alternatively, one may screen a set of compounds identified as having binding affinity for a GPCR peptide domain, or a class of compounds identified as being agonist or inverse agonists of a GPCR. It is not essential to know the binding affinity for GPCR due to the possible compound interaction in the intra-membrane domain of the GPCR polypeptide, which domain conformation may not be possible to reproduce in an affinity experiment. However, for the present assay to be meaningful to the ultimate use of the drug candidate compounds, a measurement of the second messenger(s), or the ultimate amyloid beta peptide levels, is necessary. Validation studies including controls, and measurements of binding affinity to GPCR are nonetheless useful in identifying a compound useful in any therapeutic or diagnostic application.

[0072] The present assay method may be practiced in vitro, using one or more of the GPCR proteins, or fragments thereof, or membrane preparations made from cells transduced with vectors over-expressing the GPCR polypeptides. The amino acid sequences of the GPCRs, and useful fragments thereof are found in SEQ ID NO: 4-6, 289-333. The binding affinity of the compound with the polypeptide can be measured by methods known in the art, such as using surface plasmon resonance biosensors (Biacore), by saturation binding analysis with a labeled compound (e.g. Scatchard and Lindmo analysis), by differential UV spectrophotometer, fluorescence polarization assay, Fluorometric Imaging Plate Reader (FLIPR®) system, Fluorescence resonance energy transfer, and Bioluminescence resonance energy transfer. The binding affinity of compounds can also be expressed in dissociation constant (Kd) or as IC50 or EC50. The IC₅₀ represents the concentration of a compound that is required for 50% inhibition of binding of another ligand to the polypeptide. The EC50 represents the concentration required for obtaining 50% of the maximum effect in any assay that measures receptor function. The dissociation constant, Kd, is a measure of how well a ligand binds to the polypeptide, it is equivalent to the ligand concentration required to saturate exactly half of the binding-sites on the polypeptide. Compounds with a high affinity binding have low Kd, IC50 and EC50 values, i.e. in the range of 100 nM to 1 pM; a moderate to low affinity binding relates to a high Kd, IC50 and EC50 values, i.e. in the micromolar range.

[0073] The present assay method may also be practiced in a cellular assay, A host cell expressing a GPCR polypeptide can be a cell with endogenous expression of the polypeptide or a cell over-expressing the polypeptide e.g. by transduction. When the endogenous expression of the polypeptide is not sufficient to determine a baseline that can easily be measured, one may use using host cells that over express GPCR. Overexpression has the advantage that the level of the second messenger is higher than the activity level by endogenous expression. Accordingly, measuring such levels using presently available techniques is easier. In such cellular assay, the biological activity of the GPCR may be measured using a second messenger, such as cyclic AMP or Ca2+, cyclic GMP, inositol triphosphate (IP₃) and/or diacylglycerol (DAG). Cyclic AMP or Ca2+ are preferred second messengers to measure. Second messenger activation may be measured by several different techniques, either directly by ELISA or radioactive technologies or indirectly by reporter gene analysis, discussed below. Preferably the method further comprises contacting the host cell with an agonist for GPCR before determining the baseline level. The addition of an agonist further stimulates GPCR, thereby further increasing the activity level of the second messenger. Several such agonists (ligands) are known in the art; preferentially the agonist is spingosine-1-phosphate or dihydrosphingosine-1-phosphate. The GPCR polypeptides, when over expressed or activated the level of secreted amyloid beta peptides.

[0074] The present invention further relates to a method for identifying a compound that inhibits amyloid-beta precursor protein processing in a mammalian cell comprising: [0075] (a) contacting a compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6, [0076] (b) determining the binding affinity of the compound to the polypeptide, [0077] (c) contacting a population of mammalian cells expressing said polypeptide with the compound that exhibits a binding affinity of at least 10 micromolar, and [0078] (d) identifying the compound that inhibits the amyloid-beta precursor protein processing in the cells.

[0079] A further embodiment of the present invention relates a method to identify a compound that inhibits the amyloid-beta precursor protein processing in a cell, wherein the activity level of the GPCR polypeptide is measured by determining the level of one or more second messengers, wherein the level of the one or second messenger is determined with a reporter controlled by a promoter, which is responsive to the second messenger. The reporter is a reporter gene under the regulation of a promoter that responds to the cellular level of second messengers. Such preferred second messengers are Cyclic AMP or Ca2+. The reporter gene should have a gene product that is easily detected, and that may be stably infected in the host cell. Such methods are well known by any person with ordinary skill in the art.

[0080] The reporter gene may be selected from alkaline phosphatase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), destabilized green fluorescent protein (dGFP), luciferase, beta-galactosidase among others. The reporter is preferably luciferase or beta-galactosidase, which are readily available and easy to measure over a large range The promoter in the reporter construct is preferably a cyclic AMP-responsive promoter, an NF-KB responsive promoter, or a NF-AT responsive promoter. The cyclic-AMP responsive promoter is responsive to the cyclic-AMP levels in the cell. The NF-AT responsive promoter is sensitive to cytoplasmic Ca²+-levels in the cell. The NF-KB responsive promoter is sensitive for activated NF-B levels in the cell.

[0081] A further embodiment of the present invention relates a method to identify a compound that inhibits the amyloid-beta precursor protein processing in a cell, wherein the activity level of the GPCR polypeptide is measured by determining the level of amyloid beta peptides. The levels of these peptides may be measured with specific ELISAs using antibodies specifically recognizing the different amyloid beta peptide species (see e.g. Example 1). Secretion of the various amyloid beta peptides may also be measured using antibodies that bind all peptides. Levels of amyloid beta peptides can also be measured by Mass spectrometry analysis.

[0082] For high-throughput purposes, libraries of compounds may be used such as antibody fragment libraries, peptide phage display libraries, peptide libraries (e.g. LOPAP®, Sigma Aldrich), lipid libraries (BioMol), synthetic compound libraries (e.g. LOPAC®, Sigma Aldrich) or natural compound libraries (Specs, TimTec).

[0083] Preferred drug candidate compounds are low molecular weight compounds. Low molecular weight compounds, i.e. with a molecular weight of 500 Dalton or less, are likely to have good absorption and permeation in biological systems and are consequently more likely to be successful drug candidates than compounds with a molecular weight above 500 Dalton (Lipinski et al. (1997)). Peptides comprise another preferred class of drug candidate compounds, since peptides are known GPCRs antagonists. Peptides may be excellent drug candidates and there are multiple examples of commercially valuable peptides such as fertility hormones and platelet aggregation inhibitors. Natural compounds are another preferred class of drug candidate compound. Such compounds are found in and extracted from natural sources, and which may thereafter be synthesized. The lipids are another preferred class of drug candidate compound. Lipids may be antagonists of the GPCRs listed in Table 2 (SEQ ID NO: 1-3, 4-6).

[0084] Another preferred class of drug candidate compounds is an antibody. The present invention also provides antibodies directed against the extracellular domains of the GPCR. These antibodies should specifically bind to one or more of the extra-cellular domains of the GPCRs, or as described further below, engineered to be endogenously produced to bind to the intra-cellular GPCR domain. These antibodies may be monoclonal antibodies or polyclonal antibodies. The present invention includes chimeric, single chain, and humanized antibodies, as well as FAb fragments and the products of a FAb expression library, and Fv fragments and the products of an Fv expression library.

[0085] In certain embodiments, polyclonal antibodies may be used in the practice of the invention. The skilled artisan knows methods of preparing polyclonal antibodies. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. Antibodies may also be generated against the intact GPCR protein or polypeptide, or against a fragment such as its extracellular domain peptides, derivatives including conjugates, or other epitope of the GPCR protein or polypeptide, such as the GPCR embedded in a cellular membrane, or a library of antibody variable regions, such as a phage display library.

[0086] It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). One skilled in the art without undue experimentation may select the immunization protocol.

[0087] In some embodiments, the antibodies may be monoclonal antibodies. Monoclonal antibodies may be prepared using methods known in the art. The monoclonal antibodies of the present invention may be "humanized" to prevent the host from mounting an immune response to the antibodies. A "humanized antibody" is one in which the complementarity determining regions (CDRs) and/or other portions of the light and/or heavy variable domain framework are derived from a non-human immunoglobulin, but the remaining portions of the molecule are derived from one or more human immunoglobulins. Humanized antibodies also include antibodies characterized by a humanized heavy chain associated with a donor or acceptor unmodified light chain or a chimeric light chain, or vice versa. The humanization of antibodies may be accomplished by methods known in the art (see, e.g. Mark and Padlan, (1994) "Chapter 4. Humanization of Monoclonal Antibodies", The Handbook of Experimental Pharmacology Vol. 113, Springer-Verlag, New York). Transgenic animals may be used to express humanized antibodies.

[0088] Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, (1991) J. Mol. Biol. 227:381-8; Marks et al. (1991). J. Mol. Biol. 222:581-97). The techniques of Cole, et al. and Boerner, et al. are also available for the preparation of human monoclonal antibodies (Cole, et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77; Boerner, et al (1991). J. Immunol., 147(1):86-95).

[0089] Techniques known in the art for the production of single chain antibodies can be adapted to produce single chain antibodies to the GPCR polypeptides and proteins of the present invention. The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain cross-linking Alternatively; the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent cross-linking.

[0090] Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens and preferably for a cell-surface protein or receptor or receptor subunit. In the present case, one of the binding specificities is for one extracellular domain of the GPCR, the other one is for another extracellular domain of the same or different GPCR.

[0091] Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, (1983) Nature 305:537-9). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Affinity chromatography steps usually accomplish the purification of the correct molecule. Similar procedures are disclosed in Trauneeker, et al. (1991) EMBO J. 10:3655-9.

[0092] According to another preferred embodiment, the assay method comprise using a drug candidate compound identified as having a binding affinity for GPCRs, and/or has already been identified as having down-regulating activity such as antagonist or inverse agonist activity vis-a-vis one or more GPCR. Examples of such compounds are the aryloxydithiourea compounds disclosed in U.S. Pat. No. 6,420,563 (WO 01/62765), hereby incorporated by reference with respect to the active inverse agonists disclosed therein.

[0093] Another aspect of the present invention relates to a method for reducing amyloid-beta precursor protein processing in a mammalian cell, comprising by contacting said cell with an expression-inhibiting agent that inhibits the translation in the cell of a polyribonucleotide encoding a GPCR polypeptide. A particular embodiment relates to a composition comprising an polynucleotide including at least one antisense strand that functions to pair the agent with the target GPCR mRNA, and thereby down-regulate or block the expression of GPCR polypeptide. The inhibitory agent preferably comprises antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a naturally occurring polynucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6.

[0094] A special embodiment of the present invention relates to a method wherein the expression-inhibiting agent is selected from the group consisting of antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ID NO: 4-6, a small interfering RNA (siRNA) that is sufficiently homologous to a portion of the polyribonucleotide corresponding to SEQ ID NO: 4-6 such that the siRNA interferes with the translation of the GPCR polyribonucleotide to the GPCR polypeptide.

[0095] Another embodiment of the present invention relates to a method wherein the expression-inhibiting agent is a nucleic acid expressing the antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ID NO: 4-6, a small interfering RNA (siRNA) that is sufficiently homologous to a portion of the polyribonucleotide corresponding to SEQ ID NO: 4-6 such that the siRNA interferes with the translation of the GPCR polyribonucleotide to the GPCR polypeptide. Preferably the expression-inhibiting agent is an antisense RNA, ribozyme, antisense oligodeoxynucleotide, or siRNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-287 and 340-620.

[0096] The down regulation of gene expression using antisense nucleic acids can be achieved at the translational or transcriptional level. Antisense nucleic acids of the invention are preferably nucleic acid fragments capable of specifically hybridizing with all or part of a nucleic acid encoding a GPCR polypeptide or the corresponding messenger RNA. In addition, antisense nucleic acids may be designed which decrease expression of the nucleic acid sequence capable of encoding a GPCR polypeptide by inhibiting splicing of its primary transcript. Any length of antisense sequence is suitable for practice of the invention so long as it is capable of down-regulating or blocking expression of a nucleic acid coding for a GPCR. Preferably, the antisense sequence is at least about 17 nucleotides in length. The preparation and use of antisense nucleic acids, DNA encoding antisense RNAs and the use of oligo and genetic antisense is known in the art.

[0097] One embodiment of expression-inhibitory agent is a nucleic acid that is antisense to a nucleic acid comprising SEQ ID NO: 1-3. For example, an antisense nucleic acid (e.g. DNA) may be introduced into cells in vitro, or administered to a subject in vivo, as gene therapy to inhibit cellular expression of nucleic acids comprising SEQ ID NO: 1-3. Antisense oligonucleotides preferably comprise a sequence containing from about 17 to about 100 nucleotides and more preferably the antisense oligonucleotides comprise from about 18 to about 30 nucleotides. Antisense nucleic acids may be prepared from about 10 to about 30 contiguous nucleotides selected from the sequences of SEQ ID NO: 1-3, expressed in the opposite orientation.

[0098] The antisense nucleic acids are preferably oligonucleotides and may consist entirely of deoxyribo-nucleotides, modified deoxyribonucleotides, or some combination of both. The antisense nucleic acids can be synthetic oligonucleotides. The oligonucleotides may be chemically modified, if desired, to improve stability and/or selectivity. Since oligonucleotides are susceptible to degradation by intracellular nucleases, the modifications can include, for example, the use of a sulfur group to replace the free oxygen of the phosphodiester bond. This modification is called a phosphorothioate linkage. Phosphorothioate antisense oligonucleotides are water soluble, polyanionic, and resistant to endogenous nucleases. In addition, when a phosphorothioate antisense oligonucleotide hybridizes to its target site, the RNA-DNA duplex activates the endogenous enzyme ribonuclease (RNase) H, which cleaves the mRNA component of the hybrid molecule.

[0099] In addition, antisense oligonucleotides with phosphoramidite and polyamide (peptide) linkages can be synthesized. These molecules should be very resistant to nuclease degradation. Furthermore, chemical groups can be added to the 2' carbon of the sugar moiety and the 5 carbon (C-5) of pyrimidines to enhance stability and facilitate the binding of the antisense oligonucleotide to its target site. Modifications may include 2'-deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxy phosphorothioates, modified bases, as well as other modifications known to those of skill in the art.

[0100] Another type of expression-inhibitory agent that reduces the levels of GPCRs are ribozymes. Ribozymes are catalytic RNA molecules (RNA enzymes) that have separate catalytic and substrate binding domains. The substrate binding sequence combines by nucleotide complementarity and, possibly, non-hydrogen bond interactions with its target sequence. The catalytic portion cleaves the target RNA at a specific site. The substrate domain of a ribozyme can be engineered to direct it to a specified mRNA sequence. The ribozyme recognizes and then binds a target mRNA through complementary base-pairing. Once it is bound to the correct target site, the ribozyme acts enzymatically to cut the target mRNA. Cleavage of the mRNA by a ribozyme destroys its ability to direct synthesis of the corresponding polypeptide. Once the ribozyme has cleaved its target sequence, it is released and can repeatedly bind and cleave at other mRNAs.

[0101] Ribozyme forms include a hammerhead motif, a hairpin motif, a hepatitis delta virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) motif or Neurospora VS RNA motif. Ribozymes possessing a hammerhead or hairpin structure are readily prepared since these catalytic RNA molecules can be expressed within cells from eukaryotic promoters (Chen, et al. (1992) Nucleic Acids Res. 20:4581-9). A ribozyme of the present invention can be expressed in eukaryotic cells from the appropriate DNA vector. If desired, the activity of the ribozyme may be augmented by its release from the primary transcript by a second ribozyme (Ventura, et al. (1993) Nucleic Acids Res. 21:3249-55).

[0102] Ribozymes may be chemically synthesized by combining an oligodeoxyribonucleotide with a ribozyme catalytic domain (20 nucleotides) flanked by sequences that hybridize to the target mRNA after transcription. The oligodeoxyribonucleotide is amplified by using the substrate binding sequences as primers. The amplification product is cloned into a eukaryotic expression vector.

[0103] Ribozymes are expressed from transcription units inserted into DNA, RNA, or viral vectors. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol (I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on nearby gene regulatory sequences. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Gao and Huang, (1993) Nucleic Acids Res. 21:2867-72). It has been demonstrated that ribozymes expressed from these promoters can function in mammalian cells (Kashani-Sabet, et al. (1992) Antisense Res. Dev. 2:3-15).

[0104] A particularly preferred inhibitory agent is a small interfering RNA (siRNA). siRNAs mediate the post-transcriptional process of gene silencing by double stranded RNA (dsRNA) that is homologous in sequence to the silenced RNA. siRNA according to the present invention comprises a sense strand of 17-25 nucleotides complementary or homologous to a contiguous 17-25 nucleotide sequence selected from the group of sequences described in SEQ ID NO: 1-3 and an antisense strand of 17-23 nucleotides complementary to the sense strand. The most preferred siRNA comprises sense and anti-sense strands that are 100 percent complementary to each other and the target polynucleotide sequence. Preferably the siRNA further comprises a loop region linking the sense and the antisense strand.

[0105] A self-complementing single stranded siRNA molecule polynucleotide according to the present invention comprises a sense portion and an antisense portion connected by a loop region linker. Preferably, the loop region sequence is 4-30 nucleotides long, more preferably 5-15 nucleotides long and most preferably 8 nucleotides long. In a most preferred embodiment the linker sequence is UUGCUAUA (SEQ ID NO: 288). Self-complementary single stranded siRNAs form hairpin loops and are more stable than ordinary dsRNA. In addition, they are more easily produced from vectors.

[0106] Analogous to antisense RNA, the siRNA can be modified to confirm resistance to nucleolytic degradation, or to enhance activity, or to enhance cellular distribution, or to enhance cellular uptake, such modifications may consist of modified internucleoside linkages, modified nucleic acid bases, modified sugars and/or chemical linkage the SiRNA to one or more moieties or conjugates. The nucleotide sequences are selected according to siRNA designing rules that give an improved reduction of the target sequences compared to nucleotide sequences that do not comply with these siRNA designing rules (For a discussion of these rules and examples of the preparation of siRNA, WO2004094636, published Nov. 4, 2004, and UA20030198627, are hereby incorporated by reference.

[0107] The present invention also relates to compositions, and methods using said compositions, comprising a DNA expression vector capable of expressing a polynucleotide capable of inhibiting amyloid beta protein precursor processing and described hereinabove as an expression inhibition agent.

[0108] A special aspect of these compositions and methods relates to the down-regulation or blocking of the expression of a GPCR polypeptide by the induced expression of a polynucleotide encoding an intracellular binding protein that is capable of selectively interacting with the GPCR polypeptide. An intracellular binding protein includes any protein capable of selectively interacting, or binding, with the polypeptide in the cell in which it is expressed and neutralizing the function of the polypeptide. Preferably, the intracellular binding protein is a neutralizing antibody or a fragment of a neutralizing antibody having binding affinity to an intra-cellular domain of the GPCR polypeptide of SEQ ID NO: 4-6. More preferably, the intracellular binding protein is a single chain antibody.

[0109] A special embodiment of this composition comprises the expression-inhibiting agent selected from the group consisting of antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ID NO: 4-6, and a small interfering RNA (siRNA) that is sufficiently homologous to a portion of the polyribonucleotide corresponding to SEQ ID NO: 4-6 such that the siRNA interferes with the translation of the GPCR polyribonucleotide to the GPCR polypeptide,

[0110] The polynucleotide expressing the expression-inhibiting agent or the encoding an intracellular binding protein is preferably included within a vector. The polynucleic acid is operably linked to signals enabling expression of the nucleic acid sequence and is introduced into a cell utilizing, preferably, recombinant vector constructs, which will express the antisense nucleic acid once the vector is introduced into the cell. A variety of viral-based systems are available, including adenoviral, retroviral, adeno-associated viral, lentiviral, herpes simplex viral or a sendaviral vector systems, and all may be used to introduce and express polynucleotide sequence for the expression-inhibiting agents in target cells.

[0111] Preferably, the viral vectors used in the methods of the present invention are replication defective. Such replication defective vectors will usually lack at least one region that is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), or be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution, partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents. Preferably, the replication defective virus retains the sequences of its genome, which are necessary for encapsidating, the viral particles.

[0112] In a preferred embodiment, the viral element is derived from an adenovirus. Preferably, the vehicle includes an adenoviral vector packaged into an adenoviral capsid, or a functional part, derivative, and/or analogue thereof. Adenovirus biology is also comparatively well known on the molecular level. Many tools for adenoviral vectors have been and continue to be developed, thus making an adenoviral capsid a preferred vehicle for incorporating in a library of the invention. An adenovirus is capable of infecting a wide variety of cells. However, different adenoviral serotypes have different preferences for cells. To combine and widen the target cell population that an adenoviral capsid of the invention can enter in a preferred embodiment, the vehicle includes adenoviral fiber proteins from at least two adenoviruses.

[0113] In a preferred embodiment, the nucleic acid derived from an adenovirus includes the nucleic acid encoding an adenoviral late protein or a functional part, derivative, and/or analogue thereof. An adenoviral late protein, for instance an adenoviral fiber protein, may be favorably used to target the vehicle to a certain cell or to induce enhanced delivery of the vehicle to the cell. Preferably, the nucleic acid derived from an adenovirus encodes for essentially all adenoviral late proteins, enabling the formation of entire adenoviral capsids or functional parts, analogues, and/or derivatives thereof. Preferably, the nucleic acid derived from an adenovirus includes the nucleic acid encoding adenovirus E2A or a functional part, derivative, and/or analogue thereof. Preferably, the nucleic acid derived from an adenovirus includes the nucleic acid encoding at least one E4-region protein or a functional part, derivative, and/or analogue thereof, which facilitates, at least in part, replication of an adenoviral derived nucleic acid in a cell. The adenoviral vectors used in the examples of this application are exemplary of the vectors useful in the present method of treatment invention.

[0114] Certain embodiments of the present invention use retroviral vector systems. Retroviruses are integrating viruses that infect dividing cells, and their construction is known in the art. Retroviral vectors can be constructed from different types of retrovirus, such as, MoMuLV ("murine Moloney leukemia virus" MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus. Lentiviral vector systems may also be used in the practice of the present invention. Retroviral systems and herpes virus system may be preferred vehicles for transfection of neuronal cells.

[0115] In other embodiments of the present invention, adeno-associated viruses ("AAV") are utilized. The AAV viruses are DNA viruses of relatively small size that integrate, in a stable and site-specific manner, into the genome of the infected cells. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies.

[0116] In the vector construction, the polynucleotide agents of the present invention may be linked to one or more regulatory regions. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art. Regulatory regions include promoters, and may include enhancers, suppressors, etc.

[0117] Promoters that may be used in the expression vectors of the present invention include both constitutive promoters and regulated (inducible) promoters. The promoters may be prokaryotic or eukaryotic depending on the host. Among the prokaryotic (including bacteriophage) promoters useful for practice of this invention are lac, lacZ, T3, T7, lambda P_r, P₁, and trp promoters. Among the eukaryotic (including viral) promoters useful for practice of this invention are ubiquitous promoters (e.g. HPRT, vimentin, actin, tubulin), intermediate filament promoters (e.g. desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters (e.g. MDR type, CFTR, factor VIII), tissue-specific promoters (e.g. actin promoter in smooth muscle cells, or Flt and Flk promoters active in endothelial cells), including animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift, et al. (1984) Cell 38:639-46; Ornitz, et al. (1986) Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, (1987) Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, (1985) Nature 315:115-22), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl, et al. (1984) Cell 38:647-58; Adames, et al. (1985) Nature 318:533-8; Alexander, et al. (1987) Mol. Cell. Biol. 7:1436-44), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder, et al. (1986) Cell 45:485-95), albumin gene control region which is active in liver (Pinkert, et al. (1987) Genes and Devel. 1:268-76), alpha-fetoprotein gene control region which is active in liver (Krumlauf, et al. (1985) Mol. Cell. Biol., 5:1639-48; Hammer, et al. (1987) Science 235:53-8), alpha 1-antitrypsin gene control region which is active in the liver (Kelsey, et al. (1987) Genes and Devel., 1: 161-71), beta-globin gene control region which is active in myeloid cells (Mogram, et al. (1985) Nature 315:338-40; Kollias, et al. (1986) Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead, et al. (1987) Cell 48:703-12), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, (1985) Nature 314.283-6), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason, et al. (1986) Science 234:1372-8).

[0118] Other promoters which may be used in the practice of the invention include promoters which are preferentially activated in dividing cells, promoters which respond to a stimulus (e.g. steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, E1a, and MLP promoters.

[0119] The vectors may also include other elements, such as enhancers, repressor systems, and localization signals. A membrane localization signal is a preferred element when expressing a sequence encoding an intracellular binding protein, which functions by contacting the intracellular domain of the GPCR and is most effective when the vector product is directed to the inner surface of the cellular membrane, where its target resides. Membrane localization signals are well known to persons skilled in the art. For example, a membrane localization domain suitable for localizing a polypeptide to the plasma membrane is the C-terminal sequence CaaX for farnesylation (where "a" is an aliphatic amino acid residue, and "X" is any amino acid residue, generally leucine), for example, Cysteine-Alanine-Alanine-Leucine, or Cysteine-Isoleucine-Valine-Methionine. Other membrane localization signals include the putative membrane localization sequence from the C-terminus of Bcl-2 or the C-terminus of other members of the Bcl-2 family of proteins.

[0120] Additional vector systems include the non-viral systems that facilitate introduction of polynucleotide agents into a patient. For example, a DNA vector encoding a desired sequence can be introduced in vivo by lipofection. Synthetic cationic lipids designed to limit the difficulties encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner, et. al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7); see Mackey, et al. (1988) Proc. Natl. Acad. Sci. USA 85:8027-31; Ulmer, et al. (1993) Science 259:1745-8). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Felgner and Ringold, (1989) Nature 337:387-8). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO 95/18863 and WO 96/17823, and in U.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages and directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, for example, pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. Targeted peptides, e.g., hormones or neurotransmitters, and proteins for example, antibodies, or non-peptide molecules could be coupled to liposomes chemically. Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, for example, a cationic oligopeptide (e.g., International Patent Publication WO 95/21931), peptides derived from DNA binding proteins (e.g., International Patent Publication WO 96/25508), or a cationic polymer (e.g., International Patent Publication WO 95/21931).

[0121] It is also possible to introduce a DNA vector in vivo as a naked DNA plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859). Naked DNA vectors for therapeutic purposes can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wilson, et al. (1992) J. Biol. Chem. 267:963-7; Wu and Wu, (1988) J. Biol. Chem.

[0122] 263:14621-4; Hartmut, et al. Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990; Williams, et al (1991). Proc. Natl. Acad. Sci. USA 88:2726-30). Receptor-mediated DNA delivery approaches can also be used (Curiel, et al. (1992) Hum. Gene Ther. 3:147-54; Wu and Wu, (1987) J. Biol. Chem. 262:4429-32).

[0123] The present invention also provides biologically compatible compositions comprising the compounds identified as antagonists and/or inverse agonists of GPCR, and the expression-inhibiting agents as described hereinabove.

[0124] A biologically compatible composition is a composition, that may be solid, liquid, gel, or other form, in which the compound, polynucleotide, vector, and antibody of the invention is maintained in an active form, e.g., in a form able to effect a biological activity.

[0125] For example, a compound of the invention would have inverse agonist or antagonist activity on the GPCR; a nucleic acid would be able to replicate, translate a message, or hybridize to a complementary mRNA of a GPCR; a vector would be able to transfect a target cell and expression the antisense, antibody, ribozyme or siRNA as described hereinabove; an antibody would bind a GPCR polypeptide domain.

[0126] A preferred biologically compatible composition is an aqueous solution that is buffered using, e.g., Tris, phosphate, or HEPES buffer, containing salt ions. Usually the concentration of salt ions will be similar to physiological levels. Biologically compatible solutions may include stabilizing agents and preservatives. In a more preferred embodiment, the biocompatible composition is a pharmaceutically acceptable composition. Such compositions can be formulated for administration by topical, oral, parenteral, intranasal, subcutaneous, and intraocular, routes. Parenteral administration is meant to include intravenous injection, intramuscular injection, intraarterial injection or infusion techniques. The composition may be administered parenterally in dosage unit formulations containing standard, well known non-toxic physiologically acceptable carriers, adjuvants and vehicles as desired.

[0127] A particularly preferred embodiment of the present composition invention is a cognitive-enhancing pharmaceutical composition comprising a therapeutically effective amount of an expression-inhibiting agent as described hereinabove, in admixture with a pharmaceutically acceptable carrier. Another preferred embodiment is a pharmaceutical composition for the treatment or prevention of a condition involving cognitive impairment or a susceptibility to the condition, comprising an effective amyloid beta peptide inhibiting amount of a GPCR antagonist or inverse agonist its pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof in admixture with a pharmaceutically acceptable carrier. A particularly preferred class of such compositions comprise an aryloxydithiourea compound.

[0128] Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. Pharmaceutical compositions for oral use can be prepared by combining active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethyl-cellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl-pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

[0129] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

[0130] Preferred sterile injectable preparations can be a solution or suspension in a non-toxic parenterally acceptable solvent or diluent. Examples of pharmaceutically acceptable carriers are saline, buffered saline, isotonic saline (e.g. monosodium or disodium phosphate, sodium, potassium; calcium or magnesium chloride, or mixtures of such salts), Ringer's solution, dextrose, water, sterile water, glycerol, ethanol, and combinations thereof 1,3-butanediol and sterile fixed oils are conveniently employed as solvents or suspending media. Any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid also find use in the preparation of injectables.

[0131] The composition medium can also be a hydrogel, which is prepared from any biocompatible or non-cytotoxic homo- or hetero-polymer, such as a hydrophilic polyacrylic acid polymer that can act as a drug absorbing sponge. Certain of them, such as, in particular, those obtained from ethylene and/or propylene oxide are commercially available. A hydrogel can be deposited directly onto the surface of the tissue to be treated, for example during surgical intervention.

[0132] Embodiments of pharmaceutical compositions of the present invention comprise a replication defective recombinant viral vector encoding the polynucleotide inhibitory agent of the present invention and a transfection enhancer, such as poloxamer. An example of a poloxamer is Poloxamer 407, which is commercially available (BASF, Parsippany, N.J.) and is a non-toxic, biocompatible polyol. A poloxamer impregnated with recombinant viruses may be deposited directly on the surface of the tissue to be treated, for example during a surgical intervention. Poloxamer possesses essentially the same advantages as hydrogel while having a lower viscosity.

[0133] The active expression-inhibiting agents may also be entrapped in microcapsules prepared, for example, by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.

[0134] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT®. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 degree. C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S--S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0135] The present invention also provides methods of inhibiting the processing of amyloid-beta precursor protein in a subject suffering or susceptible to the abnormal processing of said protein, which comprise the administration to said subject a therapeutically effective amount of an expression-inhibiting agent of the invention. Another aspect of the present method invention is the treatment or prevention of a condition involving cognitive impairment or a susceptibility to the condition. A special embodiment of this invention is a method wherein the condition is Alzheimer's disease.

[0136] As defined above, therapeutically effective dose means that amount of protein, polynucleotide, peptide, or its antibodies, agonists or antagonists, which ameliorate the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[0137] For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, age, weight and gender of the patient; diet, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.

[0138] The pharmaceutical compositions according to this invention may be administered to a subject by a variety of methods. They may be added directly to target tissues, complexed with cationic lipids, packaged within liposomes, or delivered to target cells by other methods known in the art. Localized administration to the desired tissues may be done by catheter, infusion pump or stent. The DNA, DNA/vehicle complexes, or the recombinant virus particles are locally administered to the site of treatment. Alternative routes of delivery include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. Examples of ribozyme delivery and administration are provided in Sullivan et al. WO 94/02595.

[0139] Antibodies according to the invention may be delivered as a bolus only, infused over time or both administered as a bolus and infused over time. Those skilled in the art may employ different formulations for polynucleotides than for proteins. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

[0140] As discussed hereinabove, recombinant viruses may be used to introduce DNA encoding polynucleotide agents useful in the present invention. Recombinant viruses according to the invention are generally formulated and administered in the form of doses of between about 10⁴ and about 10¹⁴ pfu. In the case of AAVs and adenoviruses, doses of from about 10⁶ to about 10¹¹ pfu are preferably used. The term pfu ("plaque-forming unit") corresponds to the infective power of a suspension of virions and is determined by infecting an appropriate cell culture and measuring the number of plaques formed. The techniques for determining the pfu titre of a viral solution are well documented in the prior art.

[0141] Still another aspect or the invention relates to a method for diagnosing a pathological condition involving cognitive impairment or a susceptibility to the condition in a subject, comprising determining the amount of polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6 in a biological sample, and comparing the amount with the amount of the polypeptide in a healthy subject, wherein an increase of the amount of polypeptide compared to the healthy subject is indicative of the presence of the pathological condition.

Experimental Section

Example 1

GPR3 Increases Amyloid Beta 1-42 Levels

[0142] To identify novel drug targets that change the APP processing, a stable cell line over expressing APP is generated. This stable cell line is made by transfecting HEK293 cells with APP770 wt cDNA cloned into pcDNA3.1, followed by selection with G418 for 3 weeks. At this time point colonies are picked and stable clones are expanded and tested for their secreted amyloid-beta peptide levels. One clone that secretes amyloid-beta at a high level, HEK293 APPwt, is selected for experiments to identify drug targets. This is accomplished by transducing HEK293 APPwt with adenoviral cDNA libraries and measuring changes to the resulting amyloid beta 1-42 levels via ELISA.

[0143] Cells seeded in collagen-coated plates at a cell density of 15000 cells/well (384 well plate) in DMEM (10% FBS), are infected 24 h later with 1 μl or 0.2 μl of adenovirus (corresponding to an average multiplicity of infection (MOI) of 120 and 24 respectively). The following day, the virus is washed away and DMEM (25 mM Hepes; 10% FBS) is added to the cells. Amyloid-beta peptides are allowed to accumulate during 24 h. The ELISA plate is prepared by coating with a capture antibody (JRF/cAbeta42/26) (obtained from M Mercken, Johnson and Johnson Pharmaceutical Research and Development, B-2340 Beerse, Belgium) overnight in buffer 42 (Table 1) at a concentration of 2.5 g/ml. The excess capture antibody is washed away the next morning with PBS and the ELISA plate is then blocked overnight with casein buffer (see Table 1) at 4° C. Upon removal of the blocking buffer, 30 l of the sample is transferred to the ELISA plate and incubated overnight at 4° C. After extensive washing with PBS-Tween20 and PBS, 30 l of the horseradish peroxidase (HRP) labeled detection antibody (Peroxidase Labeling Kit, Roche), JRF/AbetaN/25-HRP (obtained from M Mercken, Johnson and Johnson Pharmaceutical Research and Development, B-2340 Beerse, Belgium) is diluted 1/5000 in buffer C (see Table 1) and added to the wells for another 2 h. Following the removal of excess detection antibody by a wash with PBS-Tween20 and PBS, HRP activity is detected via addition of luminol substrate (Roche), which is converted into a chemiluminescent signal by the HRP enzyme.

TABLE-US-00001 TABLE 1 buffers and solutions used for ELISA Buffer 42 30 mM NaHCO₃, 70 mM Na₂CO₃, 0.05% NaN₃, pH 9.6 Casein 0.1% casein in PBS 1x buffer EC Buffer 20 mM sodium phosphate, 2 mM EDTA, 400 mM NaCl, 0.2% BSA, 0.05% CHAPS, 0.4% casein, 0.05% NaN₃, pH 7 Buffer C 20 mM sodium phosphate, 2 mM EDTA, 400 mM NaCl, 1% BSA, pH 7 PBS 10x 80 g NaCl + 2 g KCl + 11.5 g Na₂HPO₄•7H₂O + 2 g KH₂PO₄in 1 l milli Q, pH 7.4 PBST PBS 1x with 0.05% Tween 20

[0144] In order to validate the assay, the effect of adenoviral over expression with random titer of two clinical PS1 mutants and BACE on amyloid beta 1-42 production is evaluated in the HEK293 APPwt cells. As is shown in FIG. 2, all PS1 and BACE constructs induce amyloid beta 1-42 levels as expected.

[0145] An adenoviral GPCR cDNA library was constructed as follows. DNA fragments covering the full coding region of the GPCRs, are amplified by PCR from a pooled placental and fetal liver cDNA library (InvitroGen). All fragments are cloned into an adenoviral vector as described in U.S. Pat. No. 6,340,595, the contents of which are herein incorporated by reference, and subsequently adenoviruses are made harboring the corresponding cDNAs. During the screening of the adenoviral GPCR library in the HEK293 APPwt cells, over expression of GPR3 lead to increased levels of amyloid beta 1-42 peptides in the conditioned medium of HEK293 APPwt cells. These results indicate that GPR3 was identified as a modulator of APP processing.

[0146] The stimulatory effect of GPR3 is confirmed upon re-screening of the viruses with a known titer (viral particles/ml), as determined by quantitative real time PCR. GPR3 virus is infected at MOIs ranging from 2 to 250 and the experiment is performed as described above. Amyloid beta 1-42 levels are 2 fold higher compared to the negative controls for Ad5/GPR3 (FIG. 3A). In addition, the effect of GPR3 on amyloid beta 1-40, 11-42, 1-x and y-42 levels are checked under similar conditions as above (FIG. 3B-3E). The respective ELISAs are performed as described above, except that the following antibodies were used: for the amyloid beta 1-40 ELISA, the capture and detection antibody are respectively JRF/cAbeta40/10 and JRF/AbetaN/25-HRP (obtained from M Mercken, Johnson and Johnson Pharmaceutical Research and Development, B-2340 Beerse, Belgium), for the amyloid beta 11-42 ELISA, the capture and detection antibody are respectively JRF/cAbeta42/26 and JRF/hAb11/1 (obtained from M Mercken, Johnson and Johnson Pharmaceutical Research and Development, B-2340 Beerse, Belgium), for the amyloid beta y-42 ELISA (y ranges from 1-17), the capture and detection antibody are respectively JRF/cAbeta42/26 and 4G8-HRP (obtained respectively from M Mercken, Johnson and Johnson Pharmaceutical Research and Development, B-2340 Beerse, Belgium and from Signet, USA) while for the amyloid beta 1-x ELISA (x ranges from 24-42) the capture and detection antibodies are JRF/AbetaN/25 and 4G8-HRP, respectively (obtained respectively from M Mercken, Johnson and Johnson Pharmaceutical Research and Development, B-2340 Beerse, Belgium and from Signet, USA). The amyloid beta 1-x ELISA is used for the detection of amyloid peptides with a variable C-terminus (amyloid beta 1-37; 1-38; 1-39; 1-40; 1-42). The results of these experiments clearly show an increase of amyloid beta 1-40, 11-42, y-42 and 1-x species upon transduction of GPR3 (FIG. 3B-3E). The same procedure is used for the analysis of APP processing by GPR6 and GPR12.

Example 2

Identification of Homologues of GPR3

[0147] The amino acid sequence of the human GPR3 receptor was used as query in a BLAST search against all the human GPCRs in order to find its closest homologues. Table 2 (SEQ ID NO: 5-6) shows the 2 closest homologues of the GPR3 receptor. Using ClustalW an alignment was constructed showing the degree of homology between the GPR3 and its closest homologues, the GPR6 and GPR12 (FIG. 5).

TABLE-US-00002 TABLE 2 GPCRs involved in APP processing (SEQ ID NO: 1-3; 4-6), Sequences for expression-inhibiting agent (SEQ ID NO: 7-287), the hairpin loop sequence of the RNAi (SEQ ID NO: 288), and the domains of GPR3, GPR6, and GPR 12 (SEQ ID NO: 289-333): SEQID NO Galapagos ID Accession Sequence Type 1 1772 NM_005281 GPR3 DNA 2 1780 NM_005284 GPR6 DNA 3 1763 NM_005288 GPR12 DNA 4 1772 NP_005272 GPR3 Protein 5 1780 NP_005275 GPR6 Protein 6 1763 NP_005279 GPR12 Protein 7 NM_005281_idx127 NM_005281 TGGGATGTGGTGCTCTGCATC GPR3 DNA 8 NM_005281_idx129 NM_005281 GGATGTGGTGCTCTGCATCTC GPR3 DNA 9 NM_005281_idx172 NM_005281 AATGCGCTAGTGGTGGCCATC GPR3 DNA 10 NM_005281_idx280 NM_005281 GTCCTGCACTTTGCTGCTGTC GPR3 DNA 11 NM_005281_idx283 NM_005281 CTGCACTTTGCTGCTGTCTTC GPR3 DNA 12 NM_005281_idx286 NM_005281 CACTTTGCTGCTGTCTTCTGC GPR3 DNA 13 NM_005281_idx289 NM_005281 TTTGCTGCTGTCTTCTGCATC GPR3 DNA 14 NM_005281_idx294 NM_005281 TGCTGTCTTCTGCATCGGCTC GPR3 DNA 15 NM_005281_idx297 NM_005281 TGTCTTCTGCATCGGCTCAGC GPR3 DNA 16 NM_005281_idx342 NM_005281 CGTGCTGGCAATGGCCTTTAC GPR3 DNA 17 NM_005281_idx343 NM_005281 GTGCTGGCAATGGCCTTTACC GPR3 DNA 18 NM_005281_idx352 NM_005281 ATGGCCTTTACCGCCAGCATC GPR3 DNA 19 NM_005281_idx370 NM_005281 ATCGGCAGTCTACTGGCCATC GPR3 DNA 20 NM_005281_idx376 NM_005281 AGTCTACTGGCCATCACTGTC GPR3 DNA 21 NM_005281_idx379 NM_005281 CTACTGGCCATCACTGTCGAC GPR3 DNA 22 NM_005281_idx380 NM_005281 TACTGGCCATCACTGTCGACC GPR3 DNA 23 NM_005281_idx390 NM_005281 CACTGTCGACCGCTACCTTTC GPR3 DNA 24 NM_005281_idx392 NM_005281 CTGTCGACCGCTACCTTTCTC GPR3 DNA 25 NM_005281_idx397 NM_005281 GACCGCTACCTTTCTCTGTAC GPR3 DNA 26 NM_005281_idx402 NM_005281 CTACCTTTCTCTGTACAATGC GPR3 DNA 27 NM_005281_idx403 NM_005281 TACCTTTCTCTGTACAATGCC GPR3 DNA 28 NM_005281_idx404 NM_005281 ACCTTTCTCTGTACAATGCCC GPR3 DNA 29 NM_005281_idx406 NM_005281 CTTTCTCTGTACAATGCCCTC GPR3 DNA 30 NM_005281_idx408 NM_005281 TTCTCTGTACAATGCCCTCAC GPR3 DNA 31 NM_005281_idx409 NM_005281 TCTCTGTACAATGCCCTCACC GPR3 DNA 32 NM_005281_idx412 NM_005281 CTGTACAATGCCCTCACCTAC GPR3 DNA 33 NM_005281_idx417 NM_005281 CAATGCCCTCACCTACTATTC GPR3 DNA 34 NM_005281_idx423 NM_005281 CCTCACCTACTATTCAGAGAC GPR3 DNA 35 NM_005281_idx426 NM_005281 CACCTACTATTCAGAGACAAC GPR3 DNA 36 NM_005281_idx432 NM_005281 CTATTCAGAGACAACAGTGAC GPR3 DNA 37 NM_005281_idx434 NM_005281 ATTCAGAGACAACAGTGACAC GPR3 DNA 38 NM_005281_idx438 NM_005281 AGAGACAACAGTGACACGGAC GPR3 DNA 39 NM_005281_idx439 NM_005281 GAGACAACAGTGACACGGACC GPR3 DNA 40 NM_005281_idx449 NM_005281 TGACACGGACCTATGTGATGC GPR3 DNA 41 NM_005281_idx453 NM_005281 ACGGACCTATGTGATGCTGGC GPR3 DNA 42 NM_005281_idx545 NM_005281 CCACATGTGGCGTGGTTTATC GPR3 DNA 43 NM_005281_idx546 NM_005281 CACATGTGGCGTGGTTTATCC GPR3 DNA 44 NM_005281_idx548 NM_005281 CATGTGGCGTGGTTTATCCAC GPR3 DNA 45 NM_005281_idx550 NM_005281 TGTGGCGTGGTTTATCCACTC GPR3 DNA 46 NM_005281_idx552 NM_005281 TGGCGTGGTTTATCCACTCTC GPR3 DNA 47 NM_005281_idx553 NM_005281 GGCGTGGTTTATCCACTCTCC GPR3 DNA 48 NM_005281_idx559 NM_005281 GTTTATCCACTCTCCAAGAAC GPR3 DNA 49 NM_005281_idx560 NM_005281 TTTATCCACTCTCCAAGAACC GPR3 DNA 50 NM_005281_idx563 NM_005281 ATCCACTCTCCAAGAACCATC GPR3 DNA 51 NM_005281_idx572 NM_005281 CCAAGAACCATCTGGTAGTTC GPR3 DNA 52 NM_005281_idx576 NM_005281 GAACCATCTGGTAGTTCTGGC GPR3 DNA 53 NM_005281_idx577 NM_005281 AACCATCTGGTAGTTCTGGCC GPR3 DNA 54 NM_005281_idx582 NM_005281 TCTGGTAGTTCTGGCCATTGC GPR3 DNA 55 NM_005281_idx583 NM_005281 CTGGTAGTTCTGGCCATTGCC GPR3 DNA 56 NM_005281_idx586 NM_005281 GTAGTTCTGGCCATTGCCTTC GPR3 DNA 57 NM_005281_idx589 NM_005281 GTTCTGGCCATTGCCTTCTTC GPR3 DNA 58 NM_008154_idx1099 NM_005281 GCCTTCTTCATGGTGTTTGGC GPR3 DNA 59 NM_005281_idx604 NM_005281 TTCTTCATGGTGTTTGGCATC GPR3 DNA 60 NM_005281_idx608 NM_005281 TCATGGTGTTTGGCATCATGC GPR3 DNA 61 NM_005281_idx611 NM_005281 TGGTGTTTGGCATCATGCTGC GPR3 DNA 62 NM_005281_idx614 NM_005281 TGTTTGGCATCATGCTGCAGC GPR3 DNA 63 NM_005281_idx616 NM_005281 TTTGGCATCATGCTGCAGCTC GPR3 DNA 64 NM_005281_idx619 NM_005281 GGCATCATGCTGCAGCTCTAC GPR3 DNA 65 NM_005281_idx621 NM_005281 CATCATGCTGCAGCTCTACGC GPR3 DNA 66 NM_005281_idx622 NM_005281 ATCATGCTGCAGCTCTACGCC GPR3 DNA 67 NM_005281_idx628 NM_005281 CTGCAGCTCTACGCCCAAATC GPR3 DNA 68 NM_005281_idx631 NM_005281 CAGCTCTACGCCCAAATCTGC GPR3 DNA 69 NM_005281_idx632 NM_005281 AGCTCTACGCCCAAATCTGCC GPR3 DNA 70 NM_005281_idx637 NM_005281 TACGCCCAAATCTGCCGCATC GPR3 DNA 71 NM_005281_idx643 NM_005281 CAAATCTGCCGCATCGTCTGC GPR3 DNA 72 NM_005281_idx644 NM_005281 AAATCTGCCGCATCGTCTGCC GPR3 DNA 73 NM_005281_idx668 NM_005281 ATGCCCAGCAGATTGCCCTTC GPR3 DNA 74 NM_005281_idx775 NM_005281 TGCTGGTTGCCCTTCACTGTC GPR3 DNA 75 NM_005281_idx778 NM_005281 TGGTTGCCCTTCACTGTCTAC GPR3 DNA 76 NM_005281_idx781 NM_005281 TTGCCCTTCACTGTCTACTGC GPR3 DNA 77 NM_005281_idx782 NM_005281 TGCCCTTCACTGTCTACTGCC GPR3 DNA 78 NM_005281_idx785 NM_005281 CCTTCACTGTCTACTGCCTGC GPR3 DNA 79 NM_005281_idx816 NM_005281 CCACTCTCCACCTCTCTACAC GPR3 DNA 80 NM_005281_idx817 NM_005281 CACTCTCCACCTCTCTACACC GPR3 DNA 81 NM_005281_idx821 NM_005281 CTCCACCTCTCTACACCTATC GPR3 DNA 82 NM_005281_idx825 NM_005281 ACCTCTCTACACCTATCTTAC GPR3 DNA 83 NM_005281_idx826 NM_005281 CCTCTCTACACCTATCTTACC GPR3 DNA 84 NM_005281_idx830 NM_005281 TCTACACCTATCTTACCTTGC GPR3 DNA 85 NM_005281_idx832 NM_005281 TACACCTATCTTACCTTGCTC GPR3 DNA 86 NM_005281_idx833 NM_005281 ACACCTATCTTACCTTGCTCC GPR3 DNA 87 NM_005281_idx834 NM_005281 CACCTATCTTACCTTGCTCCC GPR3 DNA 88 NM_005281_idx837 NM_005281 CTATCTTACCTTGCTCCCTGC GPR3 DNA 89 NM_005281_idx838 NM_005281 TATCTTACCTTGCTCCCTGCC GPR3 DNA 90 NM_005281_idx840 NM_005281 TCTTACCTTGCTCCCTGCCAC GPR3 DNA 91 NM_005281_idx847 NM_005281 TTGCTCCCTGCCACCTACAAC GPR3 DNA 92 NM_008154_idx1354 NM_005281 GCCACCTACAACTCCATGATC GPR3 DNA 93 NM_005281_idx859 NM_005281 ACCTACAACTCCATGATCAAC GPR3 DNA 94 NM_008154_idx1358 NM_005281 CCTACAACTCCATGATCAACC GPR3 DNA 95 NM_005281_idx861 NM_005281 CTACAACTCCATGATCAACCC GPR3 DNA 96 NM_005281_idx865 NM_005281 AACTCCATGATCAACCCTATC GPR3 DNA 97 NM_005281_idx868 NM_005281 TCCATGATCAACCCTATCATC GPR3 DNA 98 NM_005281_idx873 NM_005281 GATCAACCCTATCATCTACGC GPR3 DNA 99 NM_005281_idx874 NM_005281 ATCAACCCTATCATCTACGCC GPR3 DNA 100 NM_005281_idx877 NM_005281 AACCCTATCATCTACGCCTTC GPR3 DNA 101 NM_005281_idx878 NM_005281 ACCCTATCATCTACGCCTTCC GPR3 DNA 102 NM_005281_idx880 NM_005281 CCTATCATCTACGCCTTCCGC GPR3 DNA 103 NM_005281_idx883 NM_005281 ATCATCTACGCCTTCCGCAAC GPR3 DNA 104 NM_005281_idx884 NM_005281 TCATCTACGCCTTCCGCAACC GPR3 DNA 105 NM_005281_idx902 NM_005281 ACCAGGATGTGCAGAAAGTGC GPR3 DNA 106 NM_005281_idx909 NM_005281 TGTGCAGAAAGTGCTGTGGGC GPR3 DNA 107 NM_005281_idx916 NM_005281 AAAGTGCTGTGGGCTGTCTGC GPR3 DNA 108 NM_005281_idx941 NM_005281 GCTGTTCCTCTTCCAAGATCC GPR3 DNA 109 NM_005284_idx146 NM_005284 GAGCTAATGGGTCTCTGGAGC GPR6 DNA 110 NM_005284_idx150 NM_005284 TAATGGGTCTCTGGAGCTGTC GPR6 DNA 111 NM_005284_idx151 NM_005284 AATGGGTCTCTGGAGCTGTCC GPR6 DNA 112 NM_005284_idx319 NM_005284 ATGTTCGTGCTGGTAGGCAGC GPR6 DNA 113 NM_005284_idx373 NM_005284 CTCATCTTGCACTTTGTGTTC GPR6 DNA 114 NM_005284_idx374 NM_005284 TCATCTTGCACTTTGTGTTCC GPR6 DNA 115 NM_005284_idx379 NM_005284 TTGCACTTTGTGTTCCAGTAC GPR6 DNA 116 NM_005284_idx386 NM_005284 TTGTGTTCCAGTACTTGGTGC GPR6 DNA 117 NM_005284_idx387 NM_005284 TGTGTTCCAGTACTTGGTGCC GPR6 DNA 118 NM_005284_idx388 NM_005284 GTGTTCCAGTACTTGGTGCCC GPR6 DNA 119 NM_005284_idx390 NM_005284 GTTCCAGTACTTGGTGCCCTC GPR6 DNA 120 NM_005284_idx409 NM_005284 TCGGAGACTGTGAGTCTGCTC GPR6 DNA 121 NM_005284_idx411 NM_005284 GGAGACTGTGAGTCTGCTCAC GPR6 DNA

122 NM_005284_idx496 NM_005284 CGCTACCTGTCCCTGTATAAC GPR6 DNA 123 NM_005284_idx498 NM_005284 CTACCTGTCCCTGTATAACGC GPR6 DNA 124 NM_005284_idx500 NM_005284 ACCTGTCCCTGTATAACGCGC GPR6 DNA 125 NM_005284_idx502 NM_005284 CTGTCCCTGTATAACGCGCTC GPR6 DNA 126 NM_005284_idx504 NM_005284 GTCCCTGTATAACGCGCTCAC GPR6 DNA 127 NM_005284_idx505 NM_005284 TCCCTGTATAACGCGCTCACC GPR6 DNA 128 NM_005284_idx511 NM_005284 TATAACGCGCTCACCTATTAC GPR6 DNA 129 NM_005284_idx513 NM_005284 TAACGCGCTCACCTATTACTC GPR6 DNA 130 NM_005284_idx515 NM_005284 ACGCGCTCACCTATTACTCGC GPR6 DNA 131 NM_005284_idx694 NM_005284 GCCGCCTTCTTCATGGTCTTC GPR6 DNA 132 NM_005284_idx697 NM_005284 GCCTTCTTCATGGTCTTCGGC GPR6 DNA 133 NM_005284_idx700 NM_005284 TTCTTCATGGTCTTCGGCATC GPR6 DNA 134 NM_005284_idx704 NM_005284 TCATGGTCTTCGGCATCATGC GPR6 DNA 135 NM_005284_idx707 NM_005284 TGGTCTTCGGCATCATGCTGC GPR6 DNA 136 NM_005284_idx709 NM_005284 GTCTTCGGCATCATGCTGCAC GPR6 DNA 137 NM_005284_idx710 NM_005284 TCTTCGGCATCATGCTGCACC GPR6 DNA 138 NM_005284_idx715 NM_005284 GGCATCATGCTGCACCTGTAC GPR6 DNA 139 NM_005284_idx719 NM_005284 TCATGCTGCACCTGTACGTGC GPR6 DNA 140 NM_005284_idx819 NM_005284 CACCAGAAAGGGTGTGGGTAC GPR6 DNA 141 NM_005284_idx821 NM_005284 CCAGAAAGGGTGTGGGTACAC GPR6 DNA 142 NM_005284_idx825 NM_005284 AAAGGGTGTGGGTACACTGGC GPR6 DNA 143 NM_005284_idx877 NM_005284 CTGCCCTTCGCCATCTATTGC GPR6 DNA 144 NM_005284_idx889 NM_005284 ATCTATTGCGTGGTGGGCAGC GPR6 DNA 145 NM_005284_idx926 NM_005284 TCTACACTTACGCCACCCTGC GPR6 DNA 146 NM_005284_idx956 NM_005284 CCTACAACTCCATGATCAATC GPR6 DNA 147 NM_005284_idx957 NM_005284 CTACAACTCCATGATCAATCC GPR6 DNA 148 NM_005284_idx958 NM_005284 TACAACTCCATGATCAATCCC GPR6 DNA 149 NM_005284_idx961 NM_005284 AACTCCATGATCAATCCCATC GPR6 DNA 150 NM_005284_idx964 NM_005284 TCCATGATCAATCCCATCATC GPR6 DNA 151 NM_005284_idx969 NM_005284 GATCAATCCCATCATCTATGC GPR6 DNA 152 NM_005284_idx970 NM_005284 ATCAATCCCATCATCTATGCC GPR6 DNA 153 NM_000647_idx981 NM_005284 AATCCCATCATCTATGCCTTC GPR6 DNA 154 NM_005284_idx974 NM_005284 ATCCCATCATCTATGCCTTCC GPR6 DNA 155 NM_005284_idx976 NM_005284 CCCATCATCTATGCCTTCCGC GPR6 DNA 156 NM_005284_idx979 NM_005284 ATCATCTATGCCTTCCGCAAC GPR6 DNA 157 NM_005284_idx980 NM_005284 TCATCTATGCCTTCCGCAACC GPR6 DNA 158 NM_005284_idx1024 NM_005284 CTCCTGCTCTGTGGCTGTTTC GPR6 DNA 159 NM_005284_idx1025 NM_005284 TCCTGCTCTGTGGCTGTTTCC GPR6 DNA 160 NM_005284_idx1029 NM_005284 GCTCTGTGGCTGTTTCCAGTC GPR6 DNA 161 NM_005284_idx1030 NM_005284 CTCTGTGGCTGTTTCCAGTCC GPR6 DNA 162 NM_005284_idx1037 NM_005284 GCTGTTTCCAGTCCAAAGTGC GPR6 DNA 163 NM_005284_idx1038 NM_005284 CTGTTTCCAGTCCAAAGTGCC GPR6 DNA 164 NM_005284_idx1039 NM_005284 TGTTTCCAGTCCAAAGTGCCC GPR6 DNA 165 NM_005284_idx1043 NM_005284 TCCAGTCCAAAGTGCCCTTTC GPR6 DNA 166 NM_005284_idx1047 NM_005284 GTCCAAAGTGCCCTTTCGTTC GPR6 DNA 167 NM_005284_idx1048 NM_005284 TCCAAAGTGCCCTTTCGTTCC GPR6 DNA 168 NM_005284_idx1053 NM_005284 AGTGCCCTTTCGTTCCAGGTC GPR6 DNA 169 NM_005284_idx1055 NM_005284 TGCCCTTTCGTTCCAGGTCTC GPR6 DNA 170 NM_005284_idx1060 NM_005284 TTTCGTTCCAGGTCTCCCAGC GPR6 DNA 171 NM_005288_idx115 NM_005288 GAGCCTGAGCTCGTAGTCAAC GPR12 DNA 172 NM_005288_idx116 NM_005288 AGCCTGAGCTCGTAGTCAACC GPR12 DNA 173 NM_005288_idx138 NM_005288 CTGGGACATTGTCTTGTGTAC GPR12 DNA 174 NM_005288_idx139 NM_005288 TGGGACATTGTCTTGTGTACC GPR12 DNA 175 NM_005288_idx141 NM_005288 GGACATTGTCTTGTGTACCTC GPR12 DNA 176 NM_005288_idx147 NM_005288 TGTCTTGTGTACCTCGGGAAC GPR12 DNA 177 NM_005288_idx148 NM_005288 GTCTTGTGTACCTCGGGAACC GPR12 DNA 178 NM_005288_idx149 NM_005288 TCTTGTGTACCTCGGGAACCC GPR12 DNA 179 NM_005288_idx151 NM_005288 TTGTGTACCTCGGGAACCCTC GPR12 DNA 180 NM_005288_idx154 NM_005288 TGTACCTCGGGAACCCTCATC GPR12 DNA 181 NM_005288_idx156 NM_005288 TACCTCGGGAACCCTCATCTC GPR12 DNA 182 NM_005288_idx184 NM_005288 AATGCCATTGTGGTCCTTATC GPR12 DNA 183 NM_005288_idx187 NM_005288 GCCATTGTGGTCCTTATCATC GPR12 DNA 184 NM_005288_idx191 NM_005288 TTGTGGTCCTTATCATCTTCC GPR12 DNA 185 NM_005288_idx193 NM_005288 GTGGTCCTTATCATCTTCCAC GPR12 DNA 186 NM_005288_idx196 NM_005288 GTCCTTATCATCTTCCACAAC GPR12 DNA 187 NM_005288_idx197 NM_005288 TCCTTATCATCTTCCACAACC GPR12 DNA 188 NM_005288_idx198 NM_005288 CCTTATCATCTTCCACAACCC GPR12 DNA 189 NM_005288_idx232 NM_005288 CCCATGTTCCTGCTAATAGGC GPR12 DNA 190 NM_005288_idx235 NM_005288 ATGTTCCTGCTAATAGGCAGC GPR12 DNA 191 NM_005288_idx236 NM_005288 TGTTCCTGCTAATAGGCAGCC GPR12 DNA 192 NM_005288_idx242 NM_005288 TGCTAATAGGCAGCCTGGCTC GPR12 DNA 193 NM_005288_idx246 NM_005288 AATAGGCAGCCTGGCTCTTGC GPR12 DNA 194 NM_005288_idx312 NM_005288 CTACCTGCTTCAGTCAGAAGC GPR12 DNA 195 NM_005288_idx313 NM_005288 TACCTGCTTCAGTCAGAAGCC GPR12 DNA 196 NM_005288_idx315 NM_005288 CCTGCTTCAGTCAGAAGCCAC GPR12 DNA 197 NM_005288_idx316 NM_005288 CTGCTTCAGTCAGAAGCCACC GPR12 DNA 198 NM_005288_idx320 NM_005288 TTCAGTCAGAAGCCACCAAGC GPR12 DNA 199 NM_005288_idx325 NM_005288 TCAGAAGCCACCAAGCTGGTC GPR12 DNA 200 NM_005288_idx327 NM_005288 AGAAGCCACCAAGCTGGTCAC GPR12 DNA 201 NM_005288_idx343 NM_005288 GTCACGATCGGCCTCATTGTC GPR12 DNA 202 NM_005288_idx352 NM_005288 GGCCTCATTGTCGCCTCTTTC GPR12 DNA 203 NM_005288_idx354 NM_005288 CCTCATTGTCGCCTCTTTCTC GPR12 DNA 204 NM_005288_idx357 NM_005288 CATTGTCGCCTCTTTCTCTGC GPR12 DNA 205 NM_005288_idx358 NM_005288 ATTGTCGCCTCTTTCTCTGCC GPR12 DNA 206 NM_005288_idx360 NM_005288 TGTCGCCTCTTTCTCTGCCTC GPR12 DNA 207 NM_005288_idx364 NM_005288 GCCTCTTTCTCTGCCTCTGTC GPR12 DNA 208 NM_005288_idx367 NM_005288 TCTTTCTCTGCCTCTGTCTGC GPR12 DNA 209 NM_005288_idx370 NM_005288 TTCTCTGCCTCTGTCTGCAGC GPR12 DNA 210 NM_005288_idx382 NM_005288 GTCTGCAGCTTGCTGGCTATC GPR12 DNA 211 NM_005288_idx384 NM_005288 CTGCAGCTTGCTGGCTATCAC GPR12 DNA 212 NM_005288_idx391 NM_005288 TTGCTGGCTATCACTGTTGAC GPR12 DNA 213 NM_005288_idx392 NM_005288 TGCTGGCTATCACTGTTGACC GPR12 DNA 214 NM_005288_idx394 NM_005288 CTGGCTATCACTGTTGACCGC GPR12 DNA 215 NM_005288_idx397 NM_005288 GCTATCACTGTTGACCGCTAC GPR12 DNA 216 NM_005288_idx398 NM_005288 CTATCACTGTTGACCGCTACC GPR12 DNA 217 NM_005288_idx400 NM_005288 ATCACTGTTGACCGCTACCTC GPR12 DNA 218 NM_005288_idx402 NM_005288 CACTGTTGACCGCTACCTCTC GPR12 DNA 219 NM_005288_idx404 NM_005288 CTGTTGACCGCTACCTCTCAC GPR12 DNA 220 NM_005288_idx409 NM_005288 GACCGCTACCTCTCACTGTAC GPR12 DNA 221 NM_005288_idx412 NM_005288 CGCTACCTCTCACTGTACTAC GPR12 DNA 222 NM_005288_idx414 NM_005288 CTACCTCTCACTGTACTACGC GPR12 DNA 223 NM_005288_idx416 NM_005288 ACCTCTCACTGTACTACGCTC GPR12 DNA 224 NM_005288_idx420 NM_005288 CTCACTGTACTACGCTCTGAC GPR12 DNA 225 NM_005288_idx424 NM_005288 CTGTACTACGCTCTGACGTAC GPR12 DNA 226 NM_005288_idx425 NM_005288 TGTACTACGCTCTGACGTACC GPR12 DNA 227 NM_005288_idx429 NM_005288 CTACGCTCTGACGTACCATTC GPR12 DNA 228 NM_005288_idx438 NM_005288 GACGTACCATTCGGAGAGGAC GPR12 DNA 229 NM_005288_idx442 NM_005288 TACCATTCGGAGAGGACGGTC GPR12 DNA 230 NM_005288_idx450 NM_005288 GGAGAGGACGGTCACGTTTAC GPR12 DNA 231 NM_005288_idx451 NM_005288 GAGAGGACGGTCACGTTTACC GPR12 DNA 232 NM_005288_idx457 NM_005288 ACGGTCACGTTTACCTATGTC GPR12 DNA 233 NM_005288_idx461 NM_005288 TCACGTTTACCTATGTCATGC GPR12 DNA 234 NM_005288_idx463 NM_005288 ACGTTTACCTATGTCATGCTC GPR12 DNA 235 NM_005288_idx466 NM_005288 TTTACCTATGTCATGCTCGTC GPR12 DNA 236 NM_005288_idx470 NM_005288 CCTATGTCATGCTCGTCATGC GPR12 DNA 237 NM_005288_idx472 NM_005288 TATGTCATGCTCGTCATGCTC GPR12 DNA 238 NM_005288_idx571 NM_005288 GTCAGACCGCTCACCAAGAAC GPR12 DNA 239 NM_005288_idx574 NM_005288 AGACCGCTCACCAAGAACAAC GPR12 DNA 240 NM_005288_idx576 NM_005288 ACCGCTCACCAAGAACAACGC GPR12 DNA 241 NM_005288_idx583 NM_005288 ACCAAGAACAACGCGGCCATC GPR12 DNA 242 NM_005288_idx586 NM_005288 AAGAACAACGCGGCCATCCTC GPR12 DNA 243 NM_005288_idx601 NM_005288 ATCCTCTCGGTGTCCTTCCTC GPR12 DNA 244 NM_005288_idx604 NM_005288 CTCTCGGTGTCCTTCCTCTTC GPR12 DNA 245 NM_005288_idx612 NM_005288 GTCCTTCCTCTTCATGTTTGC GPR12 DNA 246 NM_005288_idx614 NM_005288 CCTTCCTCTTCATGTTTGCGC GPR12 DNA

247 NM_005288_idx616 NM_005288 TTCCTCTTCATGTTTGCGCTC GPR12 DNA 248 NM_005288_idx620 NM_005288 TCTTCATGTTTGCGCTCATGC GPR12 DNA 249 NM_005288_idx623 NM_005288 TCATGTTTGCGCTCATGCTTC GPR12 DNA 250 NM_005288_idx626 NM_005288 TGTTTGCGCTCATGCTTCAGC GPR12 DNA 251 NM_005288_idx628 NM_005288 TTTGCGCTCATGCTTCAGCTC GPR12 DNA 252 NM_005288_idx631 NM_005288 GCGCTCATGCTTCAGCTCTAC GPR12 DNA 253 NM_005288_idx634 NM_005288 CTCATGCTTCAGCTCTACATC GPR12 DNA 254 NM_005288_idx635 NM_005288 TCATGCTTCAGCTCTACATCC GPR12 DNA 255 NM_005288_idx640 NM_005288 CTTCAGCTCTACATCCAGATC GPR12 DNA 256 NM_005288_idx659 NM_005288 TCTGTAAGATTGTGATGAGGC GPR12 DNA 257 NM_005288_idx661 NM_005288 TGTAAGATTGTGATGAGGCAC GPR12 DNA 258 NM_005288_idx663 NM_005288 TAAGATTGTGATGAGGCACGC GPR12 DNA 259 NM_005288_idx664 NM_005288 AAGATTGTGATGAGGCACGCC GPR12 DNA 260 NM_005288_idx665 NM_005288 AGATTGTGATGAGGCACGCCC GPR12 DNA 261 NM_005288_idx668 NM_005288 TTGTGATGAGGCACGCCCATC GPR12 DNA 262 NM_005288_idx685 NM_005288 CATCAGATAGCCCTGCAGCAC GPR12 DNA 263 NM_005288_idx686 NM_005288 ATCAGATAGCCCTGCAGCACC GPR12 DNA 264 NM_005288_idx691 NM_005288 ATAGCCCTGCAGCACCACTTC GPR12 DNA 265 NM_005288_idx717 NM_005288 CACGTCGCACTATGTGACCAC GPR12 DNA 266 NM_005288_idx718 NM_005288 ACGTCGCACTATGTGACCACC GPR12 DNA 267 NM_005288_idx748 NM_005288 GTCTCCACCCTGGCTATCATC GPR12 DNA 268 NM_005288_idx749 NM_005288 TCTCCACCCTGGCTATCATCC GPR12 DNA 269 NM_005288_idx776 NM_005288 CGTTTGCTGCTTGCTGGATGC GPR12 DNA 270 NM_005288_idx777 NM_005288 GTTTGCTGCTTGCTGGATGCC GPR12 DNA 271 NM_005288_idx781 NM_005288 GCTGCTTGCTGGATGCCTTTC GPR12 DNA 272 NM_005288_idx784 NM_005288 GCTTGCTGGATGCCTTTCACC GPR12 DNA 273 NM_005288_idx811 NM_005288 TCCTTGATAGCGGATTACACC GPR12 DNA 274 NM_005288_idx835 NM_005288 CCCTCCATCTATACCTACGCC GPR12 DNA 275 NM_005288_idx838 NM_005288 TCCATCTATACCTACGCCACC GPR12 DNA 276 NM_005288_idx839 NM_005288 CCATCTATACCTACGCCACCC GPR12 DNA 277 NM_005288_idx842 NM_005288 TCTATACCTACGCCACCCTCC GPR12 DNA 278 NM_005288_idx865 NM_005288 CCCGCCACCTACAATTCCATC GPR12 DNA 279 NM_005288_idx868 NM_005288 GCCACCTACAATTCCATCATC GPR12 DNA 280 NM_005288_idx872 NM_005288 CCTACAATTCCATCATCAACC GPR12 DNA 281 NM_005288_idx877 NM_005288 AATTCCATCATCAACCCTGTC GPR12 DNA 282 NM_005288_idx904 NM_005288 GCTTTCAGAAACCAAGAGATC GPR12 DNA 283 NM_005288_idx912 NM_005288 AAACCAAGAGATCCAGAAAGC GPR12 DNA 284 NM_005288_idx914 NM_005288 ACCAAGAGATCCAGAAAGCGC GPR12 DNA 285 NM_005288_idx928 NM_005288 AAAGCGCTCTGTCTCATTTGC GPR12 DNA 286 NM_005288_idx931 NM_005288 GCGCTCTGTCTCATTTGCTGC GPR12 DNA 287 NM_005288_idx941 NM_005288 TCATTTGCTGCGGCTGCATCC GPR12 DNA 288 Hairpin loop TTGCTATA DNA 289 N-term MMWGAGSPLAWLSAGSGNVNVSSVGPAEGPTGPAAPLPSPKA GPR3 Protein 290 TM1 WDVVLCISGTLVSCENALVVAII GPR3 Protein 291 IL1 VGTPAFRAPMFL GPR3 Protein 292 TM2 LVCSLAVADLLACLCLVLHFAAV GPR3 Protein 293 EL1 FCIGSAEMS GPR3 Protein 294 TM3 LVLVGVLAMAFTASIGSLLAITV GPR3 Protein 295 IL2 DRYLSLYNALTYYSETTVTR GPR3 Protein 296 TM4 TYVMLALVWGGALGLGLLPVLAW GPR3 Protein 297 EL2 NCLDGLTTCGVVYPLSKNH GPR3 Protein 298 TM5 LVVLAIAFFMVFGIMLQLYAQIC GPR3 Protein 299 IL3 RIVCRHAQQIALQRHLLPASHYVATRK GPR3 Protein 300 TM6 GIATLAVVLGAFAACWLPFTVYC GPR3 Protein 301 EL3 LLGDAHSPP GPR3 Protein 302 TM7 LYTYLTLLPATYNSMINPIIYAF GPR3 Protein 303 C-term RNQDVQKVLWAVCCCCSSSKIPFRSRSPSDV GPR3 Protein 304 N-term MNASAASLNDSQVVVVAAEGAAAAATAAGGPDTGEWGPPAAAALG GPR6 Protein AGGGANGSLELSSQLSAGPPGLLLPAVNP 305 TM1 WDVLLCVSGTVIAGENALVVALI GPR6 Protein 306 IL1 ASTPALRTPMFV GPR6 Protein 307 TM2 LVGSLATADLLAGCGLILHFVFQ GPR6 Protein 308 EL1 YLVPSETVS GPR6 Protein 309 TM3 LLTVGFLVASFAASVSSLLAITV GPR6 Protein 310 IL2 DRYLSLYNALTYYSRRTLLG GPR6 Protein 311 TM4 VHLLLAATWTVSLGLGLLPVLGW GPR6 Protein 312 EL2 NCLAERAACSVVRPLARSH GPR6 Protein 313 TM5 VALLSAAFFMVFGIMLHLYV GPR6 Protein 314 IL3 RICQVVWRHAHQIALQQHCLAPPHLAATRK GPR6 Protein 315 TM6 GVGTLAVVLGTFGASWLPFAIYC GPR6 Protein 316 EL3 VVGSHEDPA GPR6 Protein 317 TM7 VVGSHEDPAVYTYATLLPATYNSMINPIIYAF GPR6 Protein 318 C-term RNQEIQRALWLLLCGCFQSKVPFRSRSPSEV GPR6 Protein 319 N-term MNEDLKVNLSGLPRDYLDAAAAENISAAVSSRVPAVEPEPELVVNP GPR12 Protein 320 TM1 WDIVLCTSGTLISCENAIVVLII GPR12 Protein 321 IL1 FHNPSLRAPMFL GPR12 Protein 322 TM2 LIGSLALADLLAGIGLITNFVFA GPR12 Protein 323 EL1 YLLQSEATK GPR12 Protein 324 TM3 LVTIGLIVASFSASVCSLLAITV GPR12 Protein 325 IL2 DRYLSLYYALTYHSERTVTF GPR12 Protein 326 TM4 TYVMLVMLWGTSICLGLLPVMGW GPR12 Protein 327 EL2 NCLRDESTCSVVRPLTKNN GPR12 Protein 328 TMS AAILSVSFLFMFALMLQLYIQIC GPR12 Protein 329 IL3 KIVMRHAHQIALQHHFLATSHYVTTRK GPR12 Protein 330 TM6 GVSTLAIILGTFAACWMPFTLYS GPR12 Protein 331 EL3 LIADYTYPS GPR12 Protein 332 TM7 IYTYATLLPATYNSIINPVIYAF GPR12 Protein 333 C-term RNQEIQKALCLICCGCIPSSLAQRARSPSDV GPR12 Protein

Example 3

Amyloid Beta Peptide Reduction Via Knock Down Of GPCR Expression

[0148] The effect of an antagonist can be mimicked through the use of siRNA-based strategies, which result in decreased expression levels of the targeted protein. For example, transfection with GPR3 siRNA reduces amyloid beta 1-42.

[0149] HEK293 APPwt cells are transfected with a smart pool of siRNAs of GPR3 (Dharmacon, USA: Table 3), eGFP, Luciferase and BACE with Oligofectamine. 24 hours after transfection, the medium is refreshed and the cells are allowed to accumulate amyloid beta peptides in the conditioned medium for 24 hours prior to ELISA analysis as described above. The data clearly show that siRNA targeted against GPR3RNA levels reduce amyloid beta 1-42 levels compared to the control conditions (FIG. 4). In conclusion, these data show that GPR3 modulates the levels of secreted amyloid beta. The same procedure is used for the analysis of APP processing by GPR6 and GPR12.

TABLE-US-00003 TABLE 3 Specific siRNAs for GPR3 (Dharmacon, USA; SEQ ID NO: 334-337) SEQ Gene Dharmacon Full sequence ID symbol NM number Cat. number siRNA NO: GPR3 NM_005281 D-003951-01 GTTTATCCACTCTCCAAGA 334 GPR3 NM_005281 D-003951-02 TTTATCCACTCTCCAAGAA 335 GPR3 NM_005281 D-003951-03 CCACCTCTCTACACCTATC 336 GPR3 NM_005281 D-003951-04 ACCGCTACCTTTCTCTGTA 337

Example 4

Expression Of GPR3 in the Human Brain

[0150] Upon identification of a modulator of APP processing, it is important to evaluate whether the modulator is expressed in the tissue and the cells of interest. This can be achieved by measuring the RNA and/or protein levels in the tissue and cells. In recent years, RNA levels are being quantified through real time PCR technologies, whereby the RNA is first transcribed to cDNA and then the amplification of the cDNA of interest is monitored during a PCR reaction. The amplification plot and the resulting Ct value are indicators for the amount of RNA present in the sample. Determination of the levels of household keeping genes allows the normalization of RNA levels of the target gene between different RNA samples, represented as Ct values.

[0151] To assess whether the GPCRs of the invention are expressed in the human brain, real time PCR with GAPDH specific primers and specific primers for each GPCR of the invention is performed on human total brain, human cerebral cortex, and human hippocampal total RNA (BD Biosciences)(see Table 4).

TABLE-US-00004 TABLE 4 Primers used in quantitative real time PCR-analysis of GPR3 expression levels (SEQ ID NO: 338-339) SEQ ID Gene Primer name NO: Primer sequence GPR3 GPR3_Hs_For 338 GGCCTTTACCGCCAGCAT TCTGAATAGTAGGTGAG GPR3_Hs_Rev 339 GGCATTG

[0152] GAPDH is detected with a Taqman probe, while for the other GPCRs SybrGreen was used. In short, 40 ng of RNA is reverse-transcribed to DNA using the MultiScribe Reverse Transcriptase (50 U/μl) enzyme (Applied BioSystems). The resulting cDNA is amplified with AmpliTaq Gold DNA polymerase (Applied BioSystems) during 40 cycles using an ABI PRISM® 7000 Sequence Detection System.

[0153] Total brain, cerebral cortex and hippocampal total RNA are analyzed for the presence of the GPCR transcripts via quantitative real time PCR. For GPR3, the obtained Ct values indicate that it is detected in all RNA samples (Table 5).

[0154] To gain more insight into the specific cellular expression, immunohistochemistry (protein level) and/or in situ hybridization (RNA level) are carried out on sections from human normal and Alzheimer's brain hippocampal, cortical and subcortical structures. These results indicate whether expression occurs in neurons, microglia cells, or astrocytes. The comparison of diseased tissue with healthy tissue indicates whether GPR3 is expressed in the diseased tissue and whether its expression level is changed compared to the non-pathological situation. The same procedure is used for expression profiling of GPR6 and GPR12.

TABLE-US-00005 TABLE 5 Quantitative real time PCR Ct values: Total human brain, cerebral cortex or hippocampus RNA tested for the presence of GPR3 RNA via quantitative real time PCR. GAPDH RNA is used to normalize all samples (Ct). GAPDH Ct-values GPR3 Ct-values Human Tissue +RT -RT +RT -RT Ct (+RT) Total brain 21.29 NA 24.93 33.07 3.64 Hippocampus 21.65 NA 25.77 36.14 4.12 Cerebral cortex 20.97 NA 25.19 35.73 4.22

Example 5

Amyloid Beta Production In Rat Primary Neuronal Cells

[0155] In order to investigate whether GPR3 affects amyloid beta production in a real neuron, human or rat primary hippocampal or cortical neurons are transduced with adenovirus containing the GPR3 cDNA. Amyloid beta levels are determined by ELISA (see EXAMPLE 1). Since rodent APP genes carry a number of mutations in APP compared to the human sequence, they produce less amyloid beta 1-40 and 1-42. In order to achieve higher amyloid beta levels, co-transduction of GPR3 with human wild type APP or human Swedish mutant APP (which enhances amyloid beta production) cDNA is performed.

[0156] Rat primary neuron cultures are prepared from brain of E18-E19-day-old fetal Sprague Dawley rats according to Goslin and Banker (Culturing Nerve cells, second edition, 1998 ISBN 0-262-02438-1). Briefly, single cell suspensions obtained from the hippocampus or cortices are prepared. The number of viable cells is determined and plated on poly-L-lysine-coated plastic 96-well plates in minimal essential medium (MEM) supplemented with 10% horse serum. The cells are seeded at a density of 50,000 cells per well (i.e. about 166,000 cells/cm²). After 3-4 h, culture medium is replaced by 160 μl serum-free neurobasal medium with B27 supplement (GIBCO BRL). Cytosine arabinoside (5 μM) is added 24 h after plating to prevent normeuronal (glial) cell proliferation.

[0157] Neurons are used at day 5 after plating. Before adenoviral transduction, 150 μl conditioned medium of these cultures is transferred to the corresponding wells in an empty 96-well plate and 50 μl of the conditioned medium is returned to the cells. The remaining 100 μl/well is stored at 37° C. and 5% CO₂. Hippocampal primary neuron cultures are infected with the crude lysate of Ad5C09Att00/A011200-GPR3 v3, Ad5C09Att00/A010801-LacZ_v1, Ad5C09Att00/A010800-eGFP_v1 and Ad5C09Att00/A010800-luc v17 viruses containing the cDNA of GPR3, LacZ, eGFP and luciferase respectively at different MOIs, ranging from 250 to 2000. In addition the cells are also infected with the purified adenovirus Ad5C01Att01/A010800 APP_v6 expressing human wild type APP695 at an MOI of 2000. Sixteen to twenty-four hours after transduction, virus is removed and cultures are washed with 100 μl pre-warmed fresh neurobasal medium. After removal of the wash solution, new medium, containing 50 l of the stored conditioned medium and 50 l of fresh neurobasal medium, is transferred to the corresponding cells. Medium was harvested after 48 and 72 hours. The cell number in the wells was determined by assessing the ATP levels. Amyloid beta concentration was determined by amyloid beta 1-42 specific ELISA (see EXAMPLE 1). Amyloid beta 1-42 levels are normalized for cell number.

[0158] The data (FIG. 6) clearly indicate that increased levels of over expression of GPR3 in the primary neurons result in a corresponding dose dependent increase of amyloid beta 1-42 levels compared to the negative control viruses.

Example 6

Ligand Screens For GPCRs

Reporter Gene Screen.

[0159] Mammalian cells such as HEK293 or CHO-K1 cells are either stably transfected with a plasmid harboring the luciferase gene under the control of a cAMP dependent promoter (CRE elements) or transduced with an adenovirus harboring a luciferase gene under the control of a cAMP dependent promoter. In addition reporter constructs can be used with the luciferase gene under the control of a Ca²+ dependent promoter (NF-AT elements) or a promoter that is controlled by activated NF-B. These cells, expressing the reporter construct, are then transduced with an adenovirus harboring the cDNA of the GPCR of the present invention. Forty (40) hours after transduction the cells are treated with the following:

[0160] a) an agonist for the receptor (e.g. sphingosine 1 phosphate) and screened against a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids (Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6, 289-333, including compounds comprising aryloxydithiourea (see U.S. Pat. No. 6,420,563), its salts, hydrates, or solvates, or

[0161] b) a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids (Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6, 289-333, including compounds comprising aryloxydithiourea (see U.S. Pat. No. 6,420,563), its salts, hydrates, or solvates, only, as GPR3 is considered to be a constitutively active GPCR.

[0162] Compounds, which decrease the agonist induced increase in luciferase activity or the constitutive activity, are considered to be antagonists or inverse agonists for the GPR3. These compounds are screened again for verification and screened against their effect on secreted amyloid beta peptide levels. The compounds are also screened to verify binding to the GPCR. The binding, amyloid-beta peptide and reporter activity assays can be performed in essentially any order to screen compounds.

[0163] In addition, cells expressing the NF-AT reporter gene can be transduced with an adenovirus harboring the cDNA encoding the -subunit of G₁₅ or chimerical G subunits. G₁₅ is a promiscuous G protein of the G_q class that couples to many different GPCRs and as such re-directs their signaling towards the release of intracellular Ca²+ stores. The chimerical G alpha subunits are members of the G_s and G_i/o family by which the last 5 C-terminal residues are replaced by those of G q, these chimerical G-proteins also redirect cAMP signaling to Ca²+ signaling.

FLIPR Screen.

[0164] Mammalian cells such as HEK293 or CHO-K1 cells are stably transfected with an expression plasmid construct harboring the cDNA of a GPCR of the present invention. Cells are seeded, grown, and selected until sufficient stable cells can be obtained. Cells are loaded with a Ca²+ dependent fluorophore such as Fura3 or Fura4. After washing away the excess of fluorophore the cells are screened against a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids (Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6, 289-333, including compounds comprising aryloxydithiourea (see U.S. Pat. No. 6,420,563), its salts, hydrates, or solvates, by simultaneously adding an agonist (alternatively no agonist need be added if the constitutive activity of the receptor is used) and a compound to the cells. Activation of the receptor is measured as an almost instantaneously increase in fluorescence due to the interaction of the fluorophore and the Ca²+ that is released. Compounds that reduce or inhibit the agonist induced increase in fluorescence (or constitutive fluorescence) are considered to be antagonists or inverse agonists for the receptor they are screened against. These compounds will be screened again to measure the amount of secreted amyloid beta peptide as well as binding to the GPCR.

AequoScreen.

[0165] CHO cells, stably expressing Apoaequorin are stably transfected with a plasmid construct harboring the cDNA of a GPCR. Cells are seeded, grown, and selected until sufficient stable cells can be obtained. The cells are loaded with coelenterazine, a cofactor for apoaequorin. Upon receptor activation intracellular Ca.sup.+ stores will be emptied and the aequorin will react with the coelenterazine in a light emitting process. The emitted light is a measure for receptor activation. The CHO, stable expressing both the apoaequorin and the receptor are screened against a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids (Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6, 289-333, including compounds comprising aryloxydithiourea (see U.S. Pat. No. 6,420,563), its salts, hydrates, or solvates, by simultaneously adding an agonist (alternatively no agonist need be added if the constitutive activity of the receptor is used) and a compound to the cells. Activation of the receptor is measured as an almost instantaneously light flash due to the interaction of the apoaequorin, coelenterazine, and the Ca²+ that is released. Compounds that reduce or inhibit the agonist induced increase in light or the constitutive activity are considered to be antagonists or inverse agonists for the receptor they are screened against. These compounds will be screened again to measure the amount of secreted amyloid beta peptide as well as binding to the GPCR.

[0166] In addition, CHO cells stable expressing the apoaequorin gene are stably transfected with a plasmid construct harboring the cDNA encoding the -subunit of G₁₅ or chimerical G subunits. G₁₅ is a promiscuous G protein of the G_q class that couples to many different GPCRs and as such redirects their signaling towards the release of intracellular Ca²+ stores. The chimerical G alpha subunits are members of the G_s and G_i/o family by which the last 5 C-terminal residues are replaced by those of G_q, these chimerical G-proteins also redirect cAMP signaling to Ca²+ signaling.

Screening for compounds that bind to the GPCR polypeptides (displacement experiment)

[0167] Compounds are screened for binding to the GPCR polypeptides. The affinity of the compounds to the polypeptides is determined in a displacement experiment. In brief, the GPCR polypeptides are incubated with a labeled (radiolabeled, fluorescent labeled) ligand that is known to bind to the polypeptide (e.g., spingosine-1-phosphate or dihydrosphingosine-1-phosphate) and with an unlabeled compound. The displacement of the labeled ligand from the polypeptide is determined by measuring the amount of labeled ligand that is still associated with the polypeptide. The amount associated with the polypeptide is plotted against the concentration of the compound to calculate IC₅₀ values. This value reflects the binding affinity of the compound to its target, i.e. the GPCR polypeptides. Strong binders have an IC₅₀ in the nanomolar and even picomolar range. Compounds that have an IC₅₀ of at least 10 micromol or better (nmol to pmol) are applied in beta amyloid secretion assay to check for their effect on the beta amyloid secretion and processing. The GPCR polypeptides can be prepared in a number of ways depending on whether the assay will be run on cells, cell fractions or biochemically, on purified proteins.

Screening for Compounds that Bind to the GPCR Polypeptide (Generic GPCR Screening Assay)

[0168] When a G protein receptor becomes constitutively active, it binds to a G protein (Gq, Gs, Gi, Go) and stimulates the binding of GTP to the G protein. The G protein then acts as a GTPase and slowly hydrolyses the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. However, constitutively activated receptors continue to exchange GDP to GTP. A non-hydrolyzable analog of GTP, [³⁵S]GTPγS, can be used to monitor enhanced binding to membranes which express constitutively activated receptors. It is reported that [³⁵S]GTPγS can be used to monitor G protein coupling to membranes in the absence and presence of ligand. Moreover, a preferred approach is the use of a GPCR-G protein fusion protein. The strategy to generate a GPR3-G protein fusion protein is well known for those known in the art. Membranes expressing GPR3-G protein fusion protein are prepared for use in the direct identification of candidate compounds such as inverse agonist. Homogenized membranes with GPR3-G protein fusion protein are transferred in a 96-well plate. A pin-tool is used to transfer a candidate compound in each well plus [³⁵S]GTPγS, followed by incubation on a shaker for 60 minutes at room temperature. The assay is stopped by spinning of the plates at 4000 RPM for 15 minutes at 22° C. The plates are then aspirated and radioactivity is then read. The same procedure is used for analysis of GPR6 and GPR12.

Receptor Ligand Binding Study On Cell Surface

[0169] The receptor is expressed in mammalian cells (HEK293, CHO, COS7) by adenoviral transducing the cells (see U.S. Pat. No. 6,340,595). The cells are incubated with both labeled ligand (iodinated, tritiated, or fluorescent) and the unlabeled compound at various concentrations, ranging from 10 μM to 10 M (3 hours at 4° C.: 25 mM HEPES, 140 mM NaCl, 1 mM CaCl₂, 5 mM MgCl₂ and 0.2% BSA, adjusted to pH 7.4). Reactions mixtures are aspirated onto PEI-treated GF/B glass filters using a cell harvester (Packard). The filters are washed twice with ice cold wash buffer (25 mM HEPES, 500 mM NaCl, 1 mM CaCl₂, 5 mM MgCl₂, adjusted to pH 7.4). Scintillant (MicroScint-10; 35 μl) is added to dried filters and the filters counted in a (Packard Topcount) scintillation counter. Data are analyzed and plotted using Prism software (GraphPad Software, San Diego, Calif.). Competition curves are analyzed and IC₅₀ values calculated. If one or more data points do not fall within the sigmoidal range of the competition curve or close to the sigmoidal range the assay is repeated and concentrations of labeled ligand and unlabeled compound adapted to have more data points close to or in the sigmoidal range of the curve.

Receptor Ligand Binding Studies On Membrane Preparations

[0170] Membranes preparations are isolated from mammalian cells (HEK293, CHO, COS7) cells over expressing the receptor is done as follows: Medium is aspirated from the transduced cells and cells are harvested in 1×PBS by gentle scraping. Cells are pelleted (2500 rpm 5 min) and resuspended in 50 mM Tris pH 7.4 (10×10⁶ cells/ml). The cell pellet is homogenized by sonicating 3×5 sec (UP50H; sonotrode MS1; max amplitude: 140 μm; max Sonic Power Density: 125 W/cm²). Membrane fractions are prepared by centrifuging 20 min at maximal speed (13000 rpm ˜15 000 to 20 000 g or rcf). The resulting pellet is resuspended in 500 μl 50 mM Tris pH 7.4 and sonicated again for 3×5 sec. The membrane fraction is isolated by centrifugation and finally resuspended in PBS. Binding competition and derivation of IC₅₀ values are determined as described above.

Internalization Screen (1)

[0171] Activation of a GPCR-associated signal transduction pathway commonly leads to translocation of specific signal transduction molecules from the cytoplasm to the plasma membrane or from the cytoplasm to the nucleus. Norak has developed their transfluor assay based on agonist-induced translocation of receptor-β-arrestin-GFP complex from the cytosol to the plasma membrane and subsequent internalization of this complex, which occurs during receptor desensitization. A similar assay uses GFP tagged receptor instead of β-arrestin. HEK293 cells are transduced with a GPR3-eGFP vector that translates for a GPR3-eGFP fusion protein. 48 hours after transduction, the cells are set to fresh serum-free medium for 60 minutes and treated with a ligand (e.g. 100 nM sphingosine 1 phosphate) for 15, 30, 60 or 120 minutes at 37° C. and 5% CO₂. After indicated exposure times, cells are washed with PBS and fixed with 5% paraformaldehyde for 20 minutes at RT. GFP fluorescence is visualized with a Zeiss microscope with a digital camera. This method aims for the identification of compounds that inhibit a ligand-mediated (constitutive activity-mediated) translocation of the fusion protein to intracellular compartments. The same procedure is used for analysis of GPR6 and GPR12.

Internalization Screen (2)

[0172] Various variations on translocation assays exists using β-arrestin and β-galactosidase enzyme complementation and BRET based assays with receptor as energy donor and β-arrestin as energy acceptor. Also the use of specific receptor antibodies labeled with pH sensitive dyes are used to detect agonist induced receptor translocation to acidic lysosomes. All of the translocation assays are used for screening for both agonistic and antagonistic acting ligands.

Melanophore Assay (Arena Pharmaceutical)

[0173] The melanophore assay is based on the ability of GPCRs to alter the distribution of melanin containing melanosomes in Xenopus melanophores. The distribution of the melanosomes depends on the exogenous receptor that is either Gi/o or Gs/q coupled. The distribution of the melanosomes (dispersed or aggregated) is easily detected by measuring light absorption. This type of assay is used for both agonist as well as antagonist compound screens.

Sequence CWU 1

6201993DNAHomo sapiens 1atgatgtggg gtgcaggcag ccctctggcc tggctctcag ctggctcagg caacgtgaat 60gtaagcagcg tgggcccagc agaggggccc acaggtccag ccgcaccact gccctcgcct 120aaggcctggg atgtggtgct ctgcatctca ggcaccctgg tgtcctgcga gaatgcgcta 180gtggtggcca tcatcgtggg cactcctgcc ttccgtgccc ccatgttcct gctggtgggc 240agcctggccg tggcagacct gctggcaggc ctgggcctgg tcctgcactt tgctgctgtc 300ttctgcatcg gctcagcgga gatgagcctg gtgctggttg gcgtgctggc aatggccttt 360accgccagca tcggcagtct actggccatc actgtcgacc gctacctttc tctgtacaat 420gccctcacct actattcaga gacaacagtg acacggacct atgtgatgct ggccttagtg 480tggggaggtg ccctgggcct ggggctgctg cctgtgctgg cctggaactg cctggatggc 540ctgaccacat gtggcgtggt ttatccactc tccaagaacc atctggtagt tctggccatt 600gccttcttca tggtgtttgg catcatgctg cagctctacg cccaaatctg ccgcatcgtc 660tgccgccatg cccagcagat tgcccttcag cggcacctgc tgcctgcctc ccactatgtg 720gccacccgca agggcattgc cacactggcc gtggtgcttg gagcctttgc cgcctgctgg 780ttgcccttca ctgtctactg cctgctgggt gatgcccact ctccacctct ctacacctat 840cttaccttgc tccctgccac ctacaactcc atgatcaacc ctatcatcta cgccttccgc 900aaccaggatg tgcagaaagt gctgtgggct gtctgctgct gctgttcctc ttccaagatc 960cccttccgat cccgctcccc cagtgatgtc tag 99321089DNAHomo sapiens 2atgaacgcga gcgccgcctc gctcaacgac tcccaggtgg tggtagtggc ggccgaagga 60gcggcggcgg cggccacagc agcagggggg ccggacacgg gcgaatgggg accccctgct 120gcggcggctc taggagccgg cggcggagct aatgggtctc tggagctgtc ctcgcagctg 180tcggctgggc caccgggact cctgctgcca gcggtgaatc cgtgggacgt gctcctgtgc 240gtgtcgggga cagtgatcgc tggagaaaac gcgctggtgg tggcgctcat cgcgtccact 300ccggcgctgc gcacgcccat gttcgtgctg gtaggcagcc tggccaccgc tgacctgttg 360gcgggctgtg gcctcatctt gcactttgtg ttccagtact tggtgccctc ggagactgtg 420agtctgctca cggtgggctt cctcgtggcc tccttcgccg cctctgtcag cagcctgctg 480gccattacgg tggaccgcta cctgtccctg tataacgcgc tcacctatta ctcgcgccgg 540accctgttgg gcgtgcacct cctgcttgcc gccacttgga ccgtgtccct aggcctgggg 600ctgctgcccg tgctgggctg gaactgcctg gcagagcgcg ccgcctgcag cgtggtgcgc 660ccgctggcgc gcagccacgt ggctctgctc tccgccgcct tcttcatggt cttcggcatc 720atgctgcacc tgtacgtgcg catctgccag gtggtctggc gccacgcgca ccagatcgcg 780ctgcagcagc actgcctggc gccaccccat ctcgctgcca ccagaaaggg tgtgggtaca 840ctggctgtgg tgctgggcac tttcggcgcc agctggctgc ccttcgccat ctattgcgtg 900gtgggcagcc atgaggaccc ggcggtctac acttacgcca ccctgctgcc cgccacctac 960aactccatga tcaatcccat catctatgcc ttccgcaacc aggagatcca gcgcgccctg 1020tggctcctgc tctgtggctg tttccagtcc aaagtgccct ttcgttccag gtctcccagc 1080gaggtctga 108931005DNAHomo sapiens 3atgaatgaag acctgaaggt caatttaagc gggctgcctc gggattattt agatgccgct 60gctgcggaga acatctcggc tgctgtctcc tcccgggttc ctgccgtaga gccagagcct 120gagctcgtag tcaacccctg ggacattgtc ttgtgtacct cgggaaccct catctcctgt 180gaaaatgcca ttgtggtcct tatcatcttc cacaacccca gcctgcgagc acccatgttc 240ctgctaatag gcagcctggc tcttgcagac ctgctggccg gcattggact catcaccaat 300tttgtttttg cctacctgct tcagtcagaa gccaccaagc tggtcacgat cggcctcatt 360gtcgcctctt tctctgcctc tgtctgcagc ttgctggcta tcactgttga ccgctacctc 420tcactgtact acgctctgac gtaccattcg gagaggacgg tcacgtttac ctatgtcatg 480ctcgtcatgc tctgggggac ctccatctgc ctggggctgc tgcccgtcat gggctggaac 540tgcctccgag acgagtccac ctgcagcgtg gtcagaccgc tcaccaagaa caacgcggcc 600atcctctcgg tgtccttcct cttcatgttt gcgctcatgc ttcagctcta catccagatc 660tgtaagattg tgatgaggca cgcccatcag atagccctgc agcaccactt cctggccacg 720tcgcactatg tgaccacccg gaaaggggtc tccaccctgg ctatcatcct ggggacgttt 780gctgcttgct ggatgccttt caccctctat tccttgatag cggattacac ctacccctcc 840atctatacct acgccaccct cctgcccgcc acctacaatt ccatcatcaa ccctgtcata 900tatgctttca gaaaccaaga gatccagaaa gcgctctgtc tcatttgctg cggctgcatc 960ccgtccagtc tcgcccagag agcgcgctcg cccagtgatg tgtag 10054330PRTHomo sapiens 4Met Met Trp Gly Ala Gly Ser Pro Leu Ala Trp Leu Ser Ala Gly Ser1 5 10 15Gly Asn Val Asn Val Ser Ser Val Gly Pro Ala Glu Gly Pro Thr Gly 20 25 30Pro Ala Ala Pro Leu Pro Ser Pro Lys Ala Trp Asp Val Val Leu Cys 35 40 45Ile Ser Gly Thr Leu Val Ser Cys Glu Asn Ala Leu Val Val Ala Ile 50 55 60Ile Val Gly Thr Pro Ala Phe Arg Ala Pro Met Phe Leu Leu Val Gly65 70 75 80Ser Leu Ala Val Ala Asp Leu Leu Ala Gly Leu Gly Leu Val Leu His 85 90 95Phe Ala Ala Val Phe Cys Ile Gly Ser Ala Glu Met Ser Leu Val Leu 100 105 110Val Gly Val Leu Ala Met Ala Phe Thr Ala Ser Ile Gly Ser Leu Leu 115 120 125Ala Ile Thr Val Asp Arg Tyr Leu Ser Leu Tyr Asn Ala Leu Thr Tyr 130 135 140Tyr Ser Glu Thr Thr Val Thr Arg Thr Tyr Val Met Leu Ala Leu Val145 150 155 160Trp Gly Gly Ala Leu Gly Leu Gly Leu Leu Pro Val Leu Ala Trp Asn 165 170 175Cys Leu Asp Gly Leu Thr Thr Cys Gly Val Val Tyr Pro Leu Ser Lys 180 185 190Asn His Leu Val Val Leu Ala Ile Ala Phe Phe Met Val Phe Gly Ile 195 200 205Met Leu Gln Leu Tyr Ala Gln Ile Cys Arg Ile Val Cys Arg His Ala 210 215 220Gln Gln Ile Ala Leu Gln Arg His Leu Leu Pro Ala Ser His Tyr Val225 230 235 240Ala Thr Arg Lys Gly Ile Ala Thr Leu Ala Val Val Leu Gly Ala Phe 245 250 255Ala Ala Cys Trp Leu Pro Phe Thr Val Tyr Cys Leu Leu Gly Asp Ala 260 265 270His Ser Pro Pro Leu Tyr Thr Tyr Leu Thr Leu Leu Pro Ala Thr Tyr 275 280 285Asn Ser Met Ile Asn Pro Ile Ile Tyr Ala Phe Arg Asn Gln Asp Val 290 295 300Gln Lys Val Leu Trp Ala Val Cys Cys Cys Cys Ser Ser Ser Lys Ile305 310 315 320Pro Phe Arg Ser Arg Ser Pro Ser Asp Val 325 3305362PRTHomo sapiens 5Met Asn Ala Ser Ala Ala Ser Leu Asn Asp Ser Gln Val Val Val Val1 5 10 15Ala Ala Glu Gly Ala Ala Ala Ala Ala Thr Ala Ala Gly Gly Pro Asp 20 25 30Thr Gly Glu Trp Gly Pro Pro Ala Ala Ala Ala Leu Gly Ala Gly Gly 35 40 45Gly Ala Asn Gly Ser Leu Glu Leu Ser Ser Gln Leu Ser Ala Gly Pro 50 55 60Pro Gly Leu Leu Leu Pro Ala Val Asn Pro Trp Asp Val Leu Leu Cys65 70 75 80Val Ser Gly Thr Val Ile Ala Gly Glu Asn Ala Leu Val Val Ala Leu 85 90 95Ile Ala Ser Thr Pro Ala Leu Arg Thr Pro Met Phe Val Leu Val Gly 100 105 110Ser Leu Ala Thr Ala Asp Leu Leu Ala Gly Cys Gly Leu Ile Leu His 115 120 125Phe Val Phe Gln Tyr Leu Val Pro Ser Glu Thr Val Ser Leu Leu Thr 130 135 140Val Gly Phe Leu Val Ala Ser Phe Ala Ala Ser Val Ser Ser Leu Leu145 150 155 160Ala Ile Thr Val Asp Arg Tyr Leu Ser Leu Tyr Asn Ala Leu Thr Tyr 165 170 175Tyr Ser Arg Arg Thr Leu Leu Gly Val His Leu Leu Leu Ala Ala Thr 180 185 190Trp Thr Val Ser Leu Gly Leu Gly Leu Leu Pro Val Leu Gly Trp Asn 195 200 205Cys Leu Ala Glu Arg Ala Ala Cys Ser Val Val Arg Pro Leu Ala Arg 210 215 220Ser His Val Ala Leu Leu Ser Ala Ala Phe Phe Met Val Phe Gly Ile225 230 235 240Met Leu His Leu Tyr Val Arg Ile Cys Gln Val Val Trp Arg His Ala 245 250 255His Gln Ile Ala Leu Gln Gln His Cys Leu Ala Pro Pro His Leu Ala 260 265 270Ala Thr Arg Lys Gly Val Gly Thr Leu Ala Val Val Leu Gly Thr Phe 275 280 285Gly Ala Ser Trp Leu Pro Phe Ala Ile Tyr Cys Val Val Gly Ser His 290 295 300Glu Asp Pro Ala Val Tyr Thr Tyr Ala Thr Leu Leu Pro Ala Thr Tyr305 310 315 320Asn Ser Met Ile Asn Pro Ile Ile Tyr Ala Phe Arg Asn Gln Glu Ile 325 330 335Gln Arg Ala Leu Trp Leu Leu Leu Cys Gly Cys Phe Gln Ser Lys Val 340 345 350Pro Phe Arg Ser Arg Ser Pro Ser Glu Val 355 3606334PRTHomo sapiens 6Met Asn Glu Asp Leu Lys Val Asn Leu Ser Gly Leu Pro Arg Asp Tyr1 5 10 15Leu Asp Ala Ala Ala Ala Glu Asn Ile Ser Ala Ala Val Ser Ser Arg 20 25 30Val Pro Ala Val Glu Pro Glu Pro Glu Leu Val Val Asn Pro Trp Asp 35 40 45Ile Val Leu Cys Thr Ser Gly Thr Leu Ile Ser Cys Glu Asn Ala Ile 50 55 60Val Val Leu Ile Ile Phe His Asn Pro Ser Leu Arg Ala Pro Met Phe65 70 75 80Leu Leu Ile Gly Ser Leu Ala Leu Ala Asp Leu Leu Ala Gly Ile Gly 85 90 95Leu Ile Thr Asn Phe Val Phe Ala Tyr Leu Leu Gln Ser Glu Ala Thr 100 105 110Lys Leu Val Thr Ile Gly Leu Ile Val Ala Ser Phe Ser Ala Ser Val 115 120 125Cys Ser Leu Leu Ala Ile Thr Val Asp Arg Tyr Leu Ser Leu Tyr Tyr 130 135 140Ala Leu Thr Tyr His Ser Glu Arg Thr Val Thr Phe Thr Tyr Val Met145 150 155 160Leu Val Met Leu Trp Gly Thr Ser Ile Cys Leu Gly Leu Leu Pro Val 165 170 175Met Gly Trp Asn Cys Leu Arg Asp Glu Ser Thr Cys Ser Val Val Arg 180 185 190Pro Leu Thr Lys Asn Asn Ala Ala Ile Leu Ser Val Ser Phe Leu Phe 195 200 205Met Phe Ala Leu Met Leu Gln Leu Tyr Ile Gln Ile Cys Lys Ile Val 210 215 220Met Arg His Ala His Gln Ile Ala Leu Gln His His Phe Leu Ala Thr225 230 235 240Ser His Tyr Val Thr Thr Arg Lys Gly Val Ser Thr Leu Ala Ile Ile 245 250 255Leu Gly Thr Phe Ala Ala Cys Trp Met Pro Phe Thr Leu Tyr Ser Leu 260 265 270Ile Ala Asp Tyr Thr Tyr Pro Ser Ile Tyr Thr Tyr Ala Thr Leu Leu 275 280 285Pro Ala Thr Tyr Asn Ser Ile Ile Asn Pro Val Ile Tyr Ala Phe Arg 290 295 300Asn Gln Glu Ile Gln Lys Ala Leu Cys Leu Ile Cys Cys Gly Cys Ile305 310 315 320Pro Ser Ser Leu Ala Gln Arg Ala Arg Ser Pro Ser Asp Val 325 330721DNAHomo sapiens 7tgggatgtgg tgctctgcat c 21821DNAHomo sapiens 8ggatgtggtg ctctgcatct c 21921DNAHomo sapiens 9aatgcgctag tggtggccat c 211021DNAHomo sapiens 10gtcctgcact ttgctgctgt c 211121DNAHomo sapiens 11ctgcactttg ctgctgtctt c 211221DNAHomo sapiens 12cactttgctg ctgtcttctg c 211321DNAHomo sapiens 13tttgctgctg tcttctgcat c 211421DNAHomo sapiens 14tgctgtcttc tgcatcggct c 211521DNAHomo sapiens 15tgtcttctgc atcggctcag c 211621DNAHomo sapiens 16cgtgctggca atggccttta c 211721DNAHomo sapiens 17gtgctggcaa tggcctttac c 211821DNAHomo sapiens 18atggccttta ccgccagcat c 211921DNAHomo sapiens 19atcggcagtc tactggccat c 212021DNAHomo sapiens 20agtctactgg ccatcactgt c 212121DNAHomo sapiens 21ctactggcca tcactgtcga c 212221DNAHomo sapiens 22tactggccat cactgtcgac c 212321DNAHomo sapiens 23cactgtcgac cgctaccttt c 212421DNAHomo sapiens 24ctgtcgaccg ctacctttct c 212521DNAHomo sapiens 25gaccgctacc tttctctgta c 212621DNAHomo sapiens 26ctacctttct ctgtacaatg c 212721DNAHomo sapiens 27tacctttctc tgtacaatgc c 212821DNAHomo sapiens 28acctttctct gtacaatgcc c 212921DNAHomo sapiens 29ctttctctgt acaatgccct c 213021DNAHomo sapiens 30ttctctgtac aatgccctca c 213121DNAHomo sapiens 31tctctgtaca atgccctcac c 213221DNAHomo sapiens 32ctgtacaatg ccctcaccta c 213321DNAHomo sapiens 33caatgccctc acctactatt c 213421DNAHomo sapiens 34cctcacctac tattcagaga c 213521DNAHomo sapiens 35cccctactat tcagagacaa c 213621DNAHomo sapiens 36ctattcagag acaacagtga c 213721DNAHomo sapiens 37attcagagac aacagtgaca c 213821DNAHomo sapiens 38agagacaaca gtgacacgga c 213921DNAHomo sapiens 39gagacaacag tgacacggac c 214021DNAHomo sapiens 40tgacacggac ctatgtgatg c 214121DNAHomo sapiens 41acggacctat gtgatgctgg c 214221DNAHomo sapiens 42ccacatgtgg cgtggtttat c 214321DNAHomo sapiens 43cacatgtggc gtggtttatc c 214421DNAHomo sapiens 44catgtggcgt ggtttatcca c 214521DNAHomo sapiens 45tgtggcgtgg tttatccact c 214621DNAHomo sapiens 46tggcgtggtt tatccactct c 214721DNAHomo sapiens 47ggcgtggttt atccactctc c 214821DNAHomo sapiens 48gtttatccac tctccaagaa c 214921DNAHomo sapiens 49tttatccact ctccaagaac c 215021DNAHomo sapiens 50atccactctc caagaaccat c 215121DNAHomo sapiens 51ccaagaacca tctggtagtt c 215221DNAHomo sapiens 52gaaccatctg gtagttctgg c 215321DNAHomo sapiens 53aaccatctgg tagttctggc c 215421DNAHomo sapiens 54tctggtagtt ctggccattg c 215521DNAHomo sapiens 55ctggtagttc tggccattgc c 215621DNAHomo sapiens 56gtagttctgg ccattgcctt c 215721DNAHomo sapiens 57gttctggcca ttgccttctt c 215821DNAHomo sapiens 58gccttcttca tggtgtttgg c 215921DNAHomo sapiens 59ttcttcatgg tgtttggcat c 216021DNAHomo sapiens 60tcatggtgtt tggcatcatg c 216121DNAHomo sapiens 61tggtgtttgg catcatgctg c 216221DNAHomo sapiens 62tgtttggcat catgctgcag c 216321DNAHomo sapiens 63tttggcatca tgctgcagct c 216421DNAHomo sapiens 64ggcatcatgc tgcagctcta c 216521DNAHomo sapiens 65catcatgctg cagctctacg c 216621DNAHomo sapiens 66atcatgctgc agctctacgc c 216721DNAHomo sapiens 67ctgcagctct acgcccaaat c 216821DNAHomo sapiens 68cagctctacg cccaaatctg c 216921DNAHomo sapiens 69agctctacgc ccaaatctgc c 217021DNAHomo sapiens 70tacgcccaaa tctgccgcat c 217121DNAHomo sapiens 71caaatctgcc gcatcgtctg c 217221DNAHomo sapiens 72aaatctgccg catcgtctgc c 217321DNAHomo sapiens 73atgcccagca gattgccctt c 217421DNAHomo sapiens 74tgctggttgc ccttcactgt c 217521DNAHomo sapiens 75tggttgccct tcactgtcta c 217621DNAHomo sapiens

76ttgcccttca ctgtctactg c 217721DNAHomo sapiens 77tgcccttcac tgtctactgc c 217821DNAHomo sapiens 78ccttcactgt ctactgcctg c 217921DNAHomo sapiens 79ccactctcca cctctctaca c 218021DNAHomo sapiens 80cactctccac ctctctacac c 218121DNAHomo sapiens 81ctccacctct ctacacctat c 218221DNAHomo sapiens 82acctctctac acctatctta c 218321DNAHomo sapiens 83cctctctaca cctatcttac c 218421DNAHomo sapiens 84tctacaccta tcttaccttg c 218521DNAHomo sapiens 85tacacctatc ttaccttgct c 218621DNAHomo sapiens 86acacctatct taccttgctc c 218721DNAHomo sapiens 87cacctatctt accttgctcc c 218821DNAHomo sapiens 88ctatcttacc ttgctccctg c 218921DNAHomo sapiens 89tatcttacct tgctccctgc c 219021DNAHomo sapiens 90tcttaccttg ctccctgcca c 219121DNAHomo sapiens 91ttgctccctg ccacctacaa c 219221DNAHomo sapiens 92gccacctaca actccatgat c 219321DNAHomo sapiens 93acctacaact ccatgatcaa c 219421DNAHomo sapiens 94cctacaactc catgatcaac c 219521DNAHomo sapiens 95ctacaactcc atgatcaacc c 219621DNAHomo sapiens 96aactccatga tcaaccctat c 219721DNAHomo sapiens 97tccatgatca accctatcat c 219821DNAHomo sapiens 98gatcaaccct atcatctacg c 219921DNAHomo sapiens 99atcaacccta tcatctacgc c 2110021DNAHomo sapiens 100aaccctatca tctacgcctt c 2110121DNAHomo sapiens 101accctatcat ctacgccttc c 2110221DNAHomo sapiens 102cctatcatct acgccttccg c 2110321DNAHomo sapiens 103atcatctacg ccttccgcaa c 2110421DNAHomo sapiens 104tcatctacgc cttccgcaac c 2110521DNAHomo sapiens 105accaggatgt gcagaaagtg c 2110621DNAHomo sapiens 106tgtgcagaaa gtgctgtggg c 2110721DNAHomo sapiens 107aaagtgctgt gggctgtctg c 2110821DNAHomo sapiens 108gctgttcctc ttccaagatc c 2110921DNAHomo sapiens 109gagctaatgg gtctctggag c 2111021DNAHomo sapiens 110taatgggtct ctggagctgt c 2111121DNAHomo sapiens 111aatgggtctc tggagctgtc c 2111221DNAHomo sapiens 112atgttcgtgc tggtaggcag c 2111321DNAHomo sapiens 113ctcatcttgc actttgtgtt c 2111421DNAHomo sapiens 114tcatcttgca ctttgtgttc c 2111521DNAHomo sapiens 115ttgcactttg tgttccagta c 2111621DNAHomo sapiens 116ttgtgttcca gtacttggtg c 2111721DNAHomo sapiens 117gttgttccag tacttggtgc c 2111821DNAHomo sapiens 118gtgttccagt acttggtgcc c 2111921DNAHomo sapiens 119gttccagtac ttggtgccct c 2112021DNAHomo sapiens 120tcggagactg tgagtctgct c 2112121DNAHomo sapiens 121ggagactgtg agtctgctca c 2112221DNAHomo sapiens 122cgctacctgt ccctgtataa c 2112321DNAHomo sapiens 123ctacctgtcc ctgtataacg c 2112421DNAHomo sapiens 124acctgtccct gtataacgcg c 2112521DNAHomo sapiens 125ctgtccctgt ataacgcgct c 2112621DNAHomo sapiens 126gtccctgtat aacgcgctca c 2112721DNAHomo sapiens 127tccctgtata acgcgctcac c 2112821DNAHomo sapiens 128tataacgcgc tcacctatta c 2112921DNAHomo sapiens 129taacgcgctc acctattact c 2113021DNAHomo sapiens 130acgcgctcac ctattactcg c 2113121DNAHomo sapiens 131gccgccttct tcatggtctt c 2113221DNAHomo sapiens 132gccttcttca tggtcttcgg c 2113321DNAHomo sapiens 133ttcttcatgg tcttcggcat c 2113421DNAHomo sapiens 134tcatggtctt cggcatcatg c 2113521DNAHomo sapiens 135tggtcttcgg catcatgctg c 2113621DNAHomo sapiens 136gtcttcggca tcatgctgca c 2113721DNAHomo sapiens 137tcttcggcat catgctgcac c 2113821DNAHomo sapiens 138ggcatcatgc tgcacctgta c 2113921DNAHomo sapiens 139tcatgctgca cctgtacgtg c 2114021DNAHomo sapiens 140caccagaaag ggtgtgggta c 2114121DNAHomo sapiens 141ccagaaaggg tgtgggtaca c 2114221DNAHomo sapiens 142aaagggtgtg ggtacactgg c 2114321DNAHomo sapiens 143ctgcccttcg ccatctattg c 2114421DNAHomo sapiens 144atctattgcg tggtgggcag c 2114521DNAHomo sapiens 145tctacactta cgccaccctg c 2114621DNAHomo sapiens 146cctacaactc catgatcaat c 2114721DNAHomo sapiens 147ctacaactcc atgatcaatc c 2114821DNAHomo sapiens 148tacaactcca tgatcaatcc c 2114921DNAHomo sapiens 149aactccatga tcaatcccat c 2115021DNAHomo sapiens 150tccatgatca atcccatcat c 2115121DNAHomo sapiens 151gatcaatccc atcatctatg c 2115221DNAHomo sapiens 152atcaatccca tcatctatgc c 2115321DNAHomo sapiens 153aatcccatca tctatgcctt c 2115421DNAHomo sapiens 154atcccatcat ctatgccttc c 2115521DNAHomo sapiens 155cccatcatct atgccttccg c 2115621DNAHomo sapiens 156atcatctatg ccttccgcaa c 2115721DNAHomo sapiens 157tcatctatgc cttccgcaac c 2115821DNAHomo sapiens 158ctcctgctct gtggctgttt c 2115921DNAHomo sapiens 159tcctgctctg tggctgtttc c 2116021DNAHomo sapiens 160gctctgtggc tgtttccagt c 2116121DNAHomo sapiens 161ctctgtggct gtttccagtc c 2116221DNAHomo sapiens 162gctgtttcca gtccaaagtg c 2116321DNAHomo sapiens 163ctgtttccag tccaaagtgc c 2116421DNAHomo sapiens 164tgtttccagt ccaaagtgcc c 2116521DNAHomo sapiens 165tccagtccaa agtgcccttt c 2116621DNAHomo sapiens 166gtccaaagtg ccctttcgtt c 2116721DNAHomo sapiens 167tccaaagtgc cctttcgttc c 2116821DNAHomo sapiens 168agtgcccttt cgttccaggt c 2116921DNAHomo sapiens 169tgccctttcg ttccaggtct c 2117021DNAHomo sapiens 170tttcgttcca ggtctcccag c 2117121DNAHomo sapiens 171gagcctgagc tcgtagtcaa c 2117221DNAHomo sapiens 172agcctgagct cgtagtcaac c 2117321DNAHomo sapiens 173ctgggacatt gtcttgtgta c 2117421DNAHomo sapiens 174tgggacattg tcttgtgtac c 2117521DNAHomo sapiens 175ggacattgtc ttgtgtacct c 2117621DNAHomo sapiens 176tgtcttgtgt acctcgggaa c 2117721DNAHomo sapiens 177gtcttgtgta cctcgggaac c 2117821DNAHomo sapiens 178tcttgtgtac ctcgggaacc c 2117921DNAHomo sapiens 179ttgtgtacct cgggaaccct c 2118021DNAHomo sapiens 180tgtacctcgg gaaccctcat c 2118121DNAHomo sapiens 181tacctcggga accctcatct c 2118221DNAHomo sapiens 182aatgccattg tggtccttat c 2118321DNAHomo sapiens 183gccattgtgg tccttatcat c 2118421DNAHomo sapiens 184ttgtggtcct tatcatcttc c 2118521DNAHomo sapiens 185gtggtcctta tcatcttcca c 2118621DNAHomo sapiens 186gtccttatca tcttccacaa c 2118721DNAHomo sapiens 187tccttatcat cttccacaac c 2118821DNAHomo sapiens 188ccttatcatc ttccacaacc c 2118921DNAHomo sapiens 189cccatgttcc tgctaatagg c 2119021DNAHomo sapiens 190atgttcctgc taataggcag c 2119121DNAHomo sapiens 191tgttcctgct aataggcagc c 2119221DNAHomo sapiens 192tgctaatagg cagcctggct c 2119321DNAHomo sapiens 193aataggcagc ctggctcttg c 2119421DNAHomo sapiens 194ctacctgctt cagtcagaag c 2119521DNAHomo sapiens 195tacctgcttc agtcagaagc c 2119621DNAHomo sapiens 196cctgcttcag tcagaagcca c 2119721DNAHomo sapiens 197ctgcttcagt cagaagccac c 2119821DNAHomo sapiens 198ttcagtcaga agccaccaag c 2119921DNAHomo sapiens 199tcagaagcca ccaagctggt c 2120021DNAHomo sapiens 200agaagccacc aagctggtca c 2120121DNAHomo sapiens 201gtcacgatcg gcctcattgt c 2120221DNAHomo sapiens 202ggcctcattg tcgcctcttt c 2120321DNAHomo sapiens 203cctcattgtc gcctctttct c 2120421DNAHomo sapiens 204cattgtcgcc tctttctctg c 2120521DNAHomo sapiens 205attgtcgcct ctttctctgc c 2120621DNAHomo sapiens 206tgtcgcctct ttctctgcct c 2120721DNAHomo sapiens 207gcctctttct ctgcctctgt c 2120821DNAHomo sapiens 208tctttctctg cctctgtctg c 2120921DNAHomo sapiens 209ttctctgcct ctgtctgcag c 2121021DNAHomo sapiens 210gtctgcagct tgctggctat c 2121121DNAHomo sapiens 211ctgcagcttg ctggctatca c 2121221DNAHomo sapiens 212ttgctggcta tcactgttga c 2121321DNAHomo sapiens 213tgctggctat cactgttgac c 2121421DNAHomo sapiens 214ctggctatca ctgttgaccg c 2121521DNAHomo sapiens 215gctatcactg ttgaccgcta c 2121621DNAHomo sapiens 216ctatcactgt tgaccgctac c 2121721DNAHomo sapiens 217atcactgttg accgctacct c 2121821DNAHomo sapiens 218cactgttgac cgctacctct c 2121921DNAHomo sapiens 219ctgttgaccg ctacctctca c 2122021DNAHomo sapiens 220gaccgctacc tctcactgta c 2122121DNAHomo sapiens 221cgctacctct cactgtacta c 2122221DNAHomo sapiens 222ctacctctca ctgtactacg c 2122321DNAHomo sapiens 223acctctcact gtactacgct c 2122421DNAHomo sapiens 224ctcactgtac tacgctctga c 2122521DNAHomo sapiens 225ctgtactacg ctctgacgta c 2122621DNAHomo sapiens 226tgtactacgc tctgacgtac c 2122721DNAHomo sapiens 227ctacgctctg acgtaccatt c 2122821DNAHomo sapiens 228gacgtaccat tcggagagga c 2122921DNAHomo sapiens 229taccattcgg agaggacggt c 2123021DNAHomo sapiens 230ggagaggacg gtcacgttta c 2123121DNAHomo sapiens 231gagaggacgg tcacgtttac c 2123221DNAHomo sapiens 232acggtcacgt ttacctatgt c 2123321DNAHomo sapiens 233tcacgtttac ctatgtcatg c 2123421DNAHomo sapiens 234acgtttacct atgtcatgct c 2123521DNAHomo sapiens 235tttacctatg tcatgctcgt c 2123621DNAHomo sapiens 236cctatgtcat gctcgtcatg c 2123721DNAHomo sapiens 237tatgtcatgc tcgtcatgct c 2123821DNAHomo sapiens 238gtcagaccgc tcaccaagaa c 2123921DNAHomo sapiens 239agaccgctca ccaagaacaa c 2124021DNAHomo sapiens 240accgctcacc aagaacaacg c 2124121DNAHomo sapiens 241accaagaaca acgcggccat c 2124221DNAHomo sapiens 242aagaacaacg cggccatcct c 2124321DNAHomo sapiens 243atcctctcgg tgtccttcct c 2124421DNAHomo sapiens 244ctctcggtgt ccttcctctt c 2124521DNAHomo sapiens 245gtccttcctc ttcatgtttg c 2124621DNAHomo sapiens 246ccttcctctt catgtttgcg c 2124721DNAHomo sapiens 247ttcctcttca tgtttgcgct c 2124821DNAHomo sapiens 248tcttcatgtt tgcgctcatg c 2124921DNAHomo sapiens 249tcatgtttgc gctcatgctt c 2125021DNAHomo sapiens 250tgtttgcgct catgcttcag c 2125121DNAHomo sapiens 251tttgcgctca tgcttcagct c 2125221DNAHomo sapiens 252gcgctcatgc ttcagctcta c 2125321DNAHomo sapiens 253ctcatgcttc agctctacat c 2125421DNAHomo sapiens 254tcatgcttca gctctacatc c 2125521DNAHomo sapiens 255cttcagctct acatccagat c 2125621DNAHomo sapiens 256tctgtaagat tgtgatgagg c 2125721DNAHomo sapiens 257tgtaagattg tgatgaggca c 2125821DNAHomo sapiens 258taagattgtg atgaggcacg c 2125921DNAHomo sapiens 259aagattgtga tgaggcacgc c 2126021DNAHomo sapiens 260agattgtgat gaggcacgcc c 2126121DNAHomo sapiens 261ttgtgatgag gcacgcccat c 2126221DNAHomo sapiens 262catcagatag ccctgcagca c 2126321DNAHomo sapiens 263atcagatagc cctgcagcac c 2126421DNAHomo sapiens 264atagccctgc agcaccactt c

2126521DNAHomo sapiens 265cacgtcgcac tatgtgacca c 2126621DNAHomo sapiens 266acgtcgcact atgtgaccac c 2126721DNAHomo sapiens 267gtctccaccc tggctatcat c 2126821DNAHomo sapiens 268tctccaccct ggctatcatc c 2126921DNAHomo sapiens 269cgtttgctgc ttgctggatg c 2127021DNAHomo sapiens 270gtttgctgct tgctggatgc c 2127121DNAHomo sapiens 271gctgcttgct ggatgccttt c 2127221DNAHomo sapiens 272gcttgctgga tgcctttcac c 2127321DNAHomo sapiens 273tccttgatag cggattacac c 2127421DNAHomo sapiens 274ccctccatct atacctacgc c 2127521DNAHomo sapiens 275tccatctata cctacgccac c 2127621DNAHomo sapiens 276ccatctatac ctacgccacc c 2127721DNAHomo sapiens 277tctataccta cgccaccctc c 2127821DNAHomo sapiens 278cccgccacct acaattccat c 2127921DNAHomo sapiens 279gccacctaca attccatcat c 2128021DNAHomo sapiens 280cctacaattc catcatcaac c 2128121DNAHomo sapiens 281aattccatca tcaaccctgt c 2128221DNAHomo sapiens 282gctttcagaa accaagagat c 2128321DNAHomo sapiens 283aaaccaagag atccagaaag c 2128421DNAHomo sapiens 284accaagagat ccagaaagcg c 2128521DNAHomo sapiens 285aaagcgctct gtctcatttg c 2128621DNAHomo sapiens 286gcgctctgtc tcatttgctg c 2128721DNAHomo sapiens 287tcatttgctg cggctgcatc c 212888DNAHomo sapiens 288ttgctata 828942PRTHomo sapiens 289Met Met Trp Gly Ala Gly Ser Pro Leu Ala Trp Leu Ser Ala Gly Ser1 5 10 15Gly Asn Val Asn Val Ser Ser Val Gly Pro Ala Glu Gly Pro Thr Gly 20 25 30Pro Ala Ala Pro Leu Pro Ser Pro Lys Ala 35 4029023PRTHomo sapiens 290Trp Asp Val Val Leu Cys Ile Ser Gly Thr Leu Val Ser Cys Glu Asn1 5 10 15Ala Leu Val Val Ala Ile Ile 2029112PRTHomo sapiens 291Val Gly Thr Pro Ala Phe Arg Ala Pro Met Phe Leu1 5 1029223PRTHomo sapiens 292Leu Val Gly Ser Leu Ala Val Ala Asp Leu Leu Ala Gly Leu Gly Leu1 5 10 15Val Leu His Phe Ala Ala Val 202939PRTHomo sapiens 293Phe Cys Ile Gly Ser Ala Glu Met Ser1 529423PRTHomo sapiens 294Leu Val Leu Val Gly Val Leu Ala Met Ala Phe Thr Ala Ser Ile Gly1 5 10 15Ser Leu Leu Ala Ile Thr Val 2029520PRTHomo sapiens 295Asp Arg Tyr Leu Ser Leu Tyr Asn Ala Leu Thr Tyr Tyr Ser Glu Thr1 5 10 15Thr Val Thr Arg 2029623PRTHomo sapiens 296Thr Tyr Val Met Leu Ala Leu Val Trp Gly Gly Ala Leu Gly Leu Gly1 5 10 15Leu Leu Pro Val Leu Ala Trp 2029719PRTHomo sapiens 297Asn Cys Leu Asp Gly Leu Thr Thr Cys Gly Val Val Tyr Pro Leu Ser1 5 10 15Lys Asn His29823PRTHomo sapiens 298Leu Val Val Leu Ala Ile Ala Phe Phe Met Val Phe Gly Ile Met Leu1 5 10 15Gln Leu Tyr Ala Gln Ile Cys 2029927PRTHomo sapiens 299Arg Ile Val Cys Arg His Ala Gln Gln Ile Ala Leu Gln Arg His Leu1 5 10 15Leu Pro Ala Ser His Tyr Val Ala Thr Arg Lys 20 2530023PRTHomo sapiens 300Gly Ile Ala Thr Leu Ala Val Val Leu Gly Ala Phe Ala Ala Cys Trp1 5 10 15Leu Pro Phe Thr Val Tyr Cys 203019PRTHomo sapiens 301Leu Leu Gly Asp Ala His Ser Pro Pro1 530223PRTHomo sapiens 302Leu Tyr Thr Tyr Leu Thr Leu Leu Pro Ala Thr Tyr Asn Ser Met Ile1 5 10 15Asn Pro Ile Ile Tyr Ala Phe 2030331PRTHomo sapiens 303Arg Asn Gln Asp Val Gln Lys Val Leu Trp Ala Val Cys Cys Cys Cys1 5 10 15Ser Ser Ser Lys Ile Pro Phe Arg Ser Arg Ser Pro Ser Asp Val 20 25 3030474PRTHomo sapiens 304Met Asn Ala Ser Ala Ala Ser Leu Asn Asp Ser Gln Val Val Val Val1 5 10 15Ala Ala Glu Gly Ala Ala Ala Ala Ala Thr Ala Ala Gly Gly Pro Asp 20 25 30Thr Gly Glu Trp Gly Pro Pro Ala Ala Ala Ala Leu Gly Ala Gly Gly 35 40 45Gly Ala Asn Gly Ser Leu Glu Leu Ser Ser Gln Leu Ser Ala Gly Pro 50 55 60Pro Gly Leu Leu Leu Pro Ala Val Asn Pro65 7030523PRTHomo sapiens 305Trp Asp Val Leu Leu Cys Val Ser Gly Thr Val Ile Ala Gly Glu Asn1 5 10 15Ala Leu Val Val Ala Leu Ile 2030612PRTHomo sapiens 306Ala Ser Thr Pro Ala Leu Arg Thr Pro Met Phe Val1 5 1030723PRTHomo sapiens 307Leu Val Gly Ser Leu Ala Thr Ala Asp Leu Leu Ala Gly Cys Gly Leu1 5 10 15Ile Leu His Phe Val Phe Gln 203089PRTHomo sapiens 308Tyr Leu Val Pro Ser Glu Thr Val Ser1 530923PRTHomo sapiens 309Leu Leu Thr Val Gly Phe Leu Val Ala Ser Phe Ala Ala Ser Val Ser1 5 10 15Ser Leu Leu Ala Ile Thr Val 2031020PRTHomo sapiens 310Asp Arg Tyr Leu Ser Leu Tyr Asn Ala Leu Thr Tyr Tyr Ser Arg Arg1 5 10 15Thr Leu Leu Gly 2031123PRTHomo sapiens 311Val His Leu Leu Leu Ala Ala Thr Trp Thr Val Ser Leu Gly Leu Gly1 5 10 15Leu Leu Pro Val Leu Gly Trp 2031219PRTHomo sapiens 312Asn Cys Leu Ala Glu Arg Ala Ala Cys Ser Val Val Arg Pro Leu Ala1 5 10 15Arg Ser His31320PRTHomo sapiens 313Val Ala Leu Leu Ser Ala Ala Phe Phe Met Val Phe Gly Ile Met Leu1 5 10 15His Leu Tyr Val 2031430PRTHomo sapiens 314Arg Ile Cys Gln Val Val Trp Arg His Ala His Gln Ile Ala Leu Gln1 5 10 15Gln His Cys Leu Ala Pro Pro His Leu Ala Ala Thr Arg Lys 20 25 3031523PRTHomo sapiens 315Gly Val Gly Thr Leu Ala Val Val Leu Gly Thr Phe Gly Ala Ser Trp1 5 10 15Leu Pro Phe Ala Ile Tyr Cys 203169PRTHomo sapiens 316Val Val Gly Ser His Glu Asp Pro Ala1 531732PRTHomo sapiens 317Val Val Gly Ser His Glu Asp Pro Ala Val Tyr Thr Tyr Ala Thr Leu1 5 10 15Leu Pro Ala Thr Tyr Asn Ser Met Ile Asn Pro Ile Ile Tyr Ala Phe 20 25 3031831PRTHomo sapiens 318Arg Asn Gln Glu Ile Gln Arg Ala Leu Trp Leu Leu Leu Cys Gly Cys1 5 10 15Phe Gln Ser Lys Val Pro Phe Arg Ser Arg Ser Pro Ser Glu Val 20 25 3031946PRTHomo sapiens 319Met Asn Glu Asp Leu Lys Val Asn Leu Ser Gly Leu Pro Arg Asp Tyr1 5 10 15Leu Asp Ala Ala Ala Ala Glu Asn Ile Ser Ala Ala Val Ser Ser Arg 20 25 30Val Pro Ala Val Glu Pro Glu Pro Glu Leu Val Val Asn Pro 35 40 4532023PRTHomo sapiens 320Trp Asp Ile Val Leu Cys Thr Ser Gly Thr Leu Ile Ser Cys Glu Asn1 5 10 15Ala Ile Val Val Leu Ile Ile 2032112PRTHomo sapiens 321Phe His Asn Pro Ser Leu Arg Ala Pro Met Phe Leu1 5 1032223PRTHomo sapiens 322Leu Ile Gly Ser Leu Ala Leu Ala Asp Leu Leu Ala Gly Ile Gly Leu1 5 10 15Ile Thr Asn Phe Val Phe Ala 203239PRTHomo sapiens 323Tyr Leu Leu Gln Ser Glu Ala Thr Lys1 532423PRTHomo sapiens 324Leu Val Thr Ile Gly Leu Ile Val Ala Ser Phe Ser Ala Ser Val Cys1 5 10 15Ser Leu Leu Ala Ile Thr Val 2032520PRTHomo sapiens 325Asp Arg Tyr Leu Ser Leu Tyr Tyr Ala Leu Thr Tyr His Ser Glu Arg1 5 10 15Thr Val Thr Phe 2032623PRTHomo sapiens 326Thr Tyr Val Met Leu Val Met Leu Trp Gly Thr Ser Ile Cys Leu Gly1 5 10 15Leu Leu Pro Val Met Gly Trp 2032719PRTHomo sapiens 327Asn Cys Leu Arg Asp Glu Ser Thr Cys Ser Val Val Arg Pro Leu Thr1 5 10 15Lys Asn Asn32823PRTHomo sapiens 328Ala Ala Ile Leu Ser Val Ser Phe Leu Phe Met Phe Ala Leu Met Leu1 5 10 15Gln Leu Tyr Ile Gln Ile Cys 2032927PRTHomo sapiens 329Lys Ile Val Met Arg His Ala His Gln Ile Ala Leu Gln His His Phe1 5 10 15Leu Ala Thr Ser His Tyr Val Thr Thr Arg Lys 20 2533023PRTHomo sapiens 330Gly Val Ser Thr Leu Ala Ile Ile Leu Gly Thr Phe Ala Ala Cys Trp1 5 10 15Met Pro Phe Thr Leu Tyr Ser 203319PRTHomo sapiens 331Leu Ile Ala Asp Tyr Thr Tyr Pro Ser1 533223PRTHomo sapiens 332Ile Tyr Thr Tyr Ala Thr Leu Leu Pro Ala Thr Tyr Asn Ser Ile Ile1 5 10 15Asn Pro Val Ile Tyr Ala Phe 2033331PRTHomo sapiens 333Arg Asn Gln Glu Ile Gln Lys Ala Leu Cys Leu Ile Cys Cys Gly Cys1 5 10 15Ile Pro Ser Ser Leu Ala Gln Arg Ala Arg Ser Pro Ser Asp Val 20 25 3033419DNAHomo sapiens 334gtttatccac tctccaaga 1933519DNAHomo sapiens 335tttatccact ctccaagaa 1933619DNAHomo sapiens 336ccacctctct acacctatc 1933719DNAHomo sapiens 337accgctacct ttctctgta 1933818DNAHomo sapiens 338ggcctttacc gccagcat 1833924DNAHomo sapiens 339tctgaatagt aggtgagggc attg 2434019DNAHomo sapiens 340ggatgtggtg ctctgcatc 1934119DNAHomo sapiens 341atgtggtgct ctgcatctc 1934219DNAHomo sapiens 342tgcgctagtg gtggccatc 1934319DNAHomo sapiens 343cctgcacttt gctgctgtc 1934419DNAHomo sapiens 344gcactttgct gctgtcttc 1934519DNAHomo sapiens 345ctttgctgct gtcttctgc 1934619DNAHomo sapiens 346tgctgctgtc ttctgcatc 1934719DNAHomo sapiens 347ctgtcttctg catcggctc 1934819DNAHomo sapiens 348tcttctgcat cggctcagc 1934919DNAHomo sapiens 349tgctggcaat ggcctttac 1935019DNAHomo sapiens 350gctggcaatg gcctttacc 1935119DNAHomo sapiens 351ggcctttacc gccagcatc 1935219DNAHomo sapiens 352cggcagtcta ctggccatc 1935319DNAHomo sapiens 353tctactggcc atcactgtc 1935419DNAHomo sapiens 354actggccatc actgtcgac 1935519DNAHomo sapiens 355ctggccatca ctgtcgacc 1935619DNAHomo sapiens 356ctgtcgaccg ctacctttc 1935719DNAHomo sapiens 357gtcgaccgct acctttctc 1935819DNAHomo sapiens 358ccgctacctt tctctgtac 1935919DNAHomo sapiens 359acctttctct gtacaatgc 1936019DNAHomo sapiens 360cctttctctg tacaatgcc 1936119DNAHomo sapiens 361ctttctctgt acaatgccc 1936219DNAHomo sapiens 362ttctctgtac aatgccctc 1936319DNAHomo sapiens 363ctctgtacaa tgccctcac 1936419DNAHomo sapiens 364tctgtacaat gccctcacc 1936519DNAHomo sapiens 365gtacaatgcc ctcacctac 1936619DNAHomo sapiens 366atgccctcac ctactattc 1936719DNAHomo sapiens 367tcacctacta ttcagagac 1936819DNAHomo sapiens 368cctactattc agagacaac 1936919DNAHomo sapiens 369attcagagac aacagtgac 1937019DNAHomo sapiens 370tcagagacaa cagtgacac 1937119DNAHomo sapiens 371agacaacagt gacacggac 1937219DNAHomo sapiens 372gacaacagtg acacggacc 1937319DNAHomo sapiens 373acacggacct atgtgatgc 1937419DNAHomo sapiens 374ggacctatgt gatgctggc 1937519DNAHomo sapiens 375acatgtggcg tggtttatc 1937619DNAHomo sapiens 376catgtggcgt ggtttatcc 1937719DNAHomo sapiens 377tgtggcgtgg tttatccac 1937819DNAHomo sapiens 378tggcgtggtt tatccactc 1937919DNAHomo sapiens 379gcgtggttta tccactctc 1938019DNAHomo sapiens 380cgtggtttat ccactctcc 1938119DNAHomo sapiens 381ttatccactc tccaagaac 1938219DNAHomo sapiens 382tatccactct ccaagaacc 1938319DNAHomo sapiens 383ccactctcca agaaccatc 1938419DNAHomo sapiens 384aagaaccatc tggtagttc 1938519DNAHomo sapiens 385accatctggt agttctggc 1938619DNAHomo sapiens 386ccatctggta gttctggcc 1938719DNAHomo sapiens 387tggtagttct ggccattgc 1938819DNAHomo sapiens 388ggtagttctg gccattgcc 1938919DNAHomo sapiens 389agttctggcc attgccttc 1939019DNAHomo sapiens 390tctggccatt gccttcttc 1939119DNAHomo sapiens 391cttcttcatg gtgtttggc 1939219DNAHomo sapiens 392cttcatggtg tttggcatc 1939319DNAHomo sapiens 393atggtgtttg gcatcatgc 1939419DNAHomo sapiens 394gtgtttggca tcatgctgc 1939519DNAHomo sapiens 395tttggcatca tgctgcagc 1939619DNAHomo sapiens 396tggcatcatg ctgcagctc 1939719DNAHomo sapiens 397catcatgctg cagctctac 1939819DNAHomo sapiens 398tcatgctgca gctctacgc 1939919DNAHomo sapiens 399catgctgcag ctctacgcc 1940019DNAHomo sapiens 400gcagctctac gcccaaatc 1940119DNAHomo sapiens 401gctctacgcc caaatctgc 1940219DNAHomo sapiens 402ctctacgccc aaatctgcc 1940319DNAHomo sapiens 403cgcccaaatc tgccgcatc 1940419DNAHomo sapiens 404aatctgccgc atcgtctgc 1940519DNAHomo sapiens 405atctgccgca tcgtctgcc 1940619DNAHomo sapiens 406gcccagcaga ttgcccttc 1940719DNAHomo sapiens 407ctggttgccc ttcactgtc 1940819DNAHomo sapiens 408gttgcccttc actgtctac 1940919DNAHomo sapiens 409gcccttcact gtctactgc 1941019DNAHomo sapiens 410cccttcactg

tctactgcc 1941119DNAHomo sapiens 411ttcactgtct actgcctgc 1941219DNAHomo sapiens 412actctccacc tctctacac 1941319DNAHomo sapiens 413ctctccacct ctctacacc 1941419DNAHomo sapiens 414ccacctctct acacctatc 1941519DNAHomo sapiens 415ctctctacac ctatcttac 1941619DNAHomo sapiens 416tctctacacc tatcttacc 1941719DNAHomo sapiens 417tacacctatc ttaccttgc 1941819DNAHomo sapiens 418cacctatctt accttgctc 1941919DNAHomo sapiens 419acctatctta ccttgctcc 1942019DNAHomo sapiens 420cctatcttac cttgctccc 1942119DNAHomo sapiens 421atcttacctt gctccctgc 1942219DNAHomo sapiens 422tcttaccttg ctccctgcc 1942319DNAHomo sapiens 423ttaccttgct ccctgccac 1942419DNAHomo sapiens 424gctccctgcc acctacaac 1942519DNAHomo sapiens 425cacctacaac tccatgatc 1942619DNAHomo sapiens 426ctacaactcc atgatcaac 1942719DNAHomo sapiens 427tacaactcca tgatcaacc 1942819DNAHomo sapiens 428acaactccat gatcaaccc 1942919DNAHomo sapiens 429ctccatgatc aaccctatc 1943019DNAHomo sapiens 430catgatcaac cctatcatc 1943119DNAHomo sapiens 431tcaaccctat catctacgc 1943219DNAHomo sapiens 432caaccctatc atctacgcc 1943319DNAHomo sapiens 433ccctatcatc tacgccttc 1943419DNAHomo sapiens 434cctatcatct acgccttcc 1943519DNAHomo sapiens 435tatcatctac gccttccgc 1943619DNAHomo sapiens 436catctacgcc ttccgcaac 1943719DNAHomo sapiens 437atctacgcct tccgcaacc 1943819DNAHomo sapiens 438caggatgtgc agaaagtgc 1943919DNAHomo sapiens 439tgcagaaagt gctgtgggc 1944019DNAHomo sapiens 440agtgctgtgg gctgtctgc 1944119DNAHomo sapiens 441tgttcctctt ccaagatcc 1944219DNAHomo sapiens 442gctaatgggt ctctggagc 1944319DNAHomo sapiens 443atgggtctct ggagctgtc 1944419DNAHomo sapiens 444tgggtctctg gagctgtcc 1944519DNAHomo sapiens 445gttcgtgctg gtaggcagc 1944619DNAHomo sapiens 446catcttgcac tttgtgttc 1944719DNAHomo sapiens 447atcttgcact ttgtgttcc 1944819DNAHomo sapiens 448gcactttgtg ttccagtac 1944919DNAHomo sapiens 449gtgttccagt acttggtgc 1945019DNAHomo sapiens 450tgttccagta cttggtgcc 1945119DNAHomo sapiens 451gttccagtac ttggtgccc 1945219DNAHomo sapiens 452tccagtactt ggtgccctc 1945319DNAHomo sapiens 453ggagactgtg agtctgctc 1945419DNAHomo sapiens 454agactgtgag tctgctcac 1945519DNAHomo sapiens 455ctacctgtcc ctgtataac 1945619DNAHomo sapiens 456acctgtccct gtataacgc 1945719DNAHomo sapiens 457ctgtccctgt ataacgcgc 1945819DNAHomo sapiens 458gtccctgtat aacgcgctc 1945919DNAHomo sapiens 459ccctgtataa cgcgctcac 1946019DNAHomo sapiens 460cctgtataac gcgctcacc 1946119DNAHomo sapiens 461taacgcgctc acctattac 1946219DNAHomo sapiens 462acgcgctcac ctattactc 1946319DNAHomo sapiens 463gcgctcacct attactcgc 1946419DNAHomo sapiens 464cgccttcttc atggtcttc 1946519DNAHomo sapiens 465cttcttcatg gtcttcggc 1946619DNAHomo sapiens 466cttcatggtc ttcggcatc 1946719DNAHomo sapiens 467atggtcttcg gcatcatgc 1946819DNAHomo sapiens 468gtcttcggca tcatgctgc 1946919DNAHomo sapiens 469cttcggcatc atgctgcac 1947019DNAHomo sapiens 470ttcggcatca tgctgcacc 1947119DNAHomo sapiens 471catcatgctg cacctgtac 1947219DNAHomo sapiens 472atgctgcacc tgtacgtgc 1947319DNAHomo sapiens 473ccagaaaggg tgtgggtac 1947419DNAHomo sapiens 474agaaagggtg tgggtacac 1947519DNAHomo sapiens 475agggtgtggg tacactggc 1947619DNAHomo sapiens 476gcccttcgcc atctattgc 1947719DNAHomo sapiens 477ctattgcgtg gtgggcagc 1947819DNAHomo sapiens 478tacacttacg ccaccctgc 1947919DNAHomo sapiens 479tacaactcca tgatcaatc 1948019DNAHomo sapiens 480acaactccat gatcaatcc 1948119DNAHomo sapiens 481caactccatg atcaatccc 1948219DNAHomo sapiens 482ctccatgatc aatcccatc 1948319DNAHomo sapiens 483catgatcaat cccatcatc 1948419DNAHomo sapiens 484tcaatcccat catctatgc 1948519DNAHomo sapiens 485caatcccatc atctatgcc 1948619DNAHomo sapiens 486tcccatcatc tatgccttc 1948719DNAHomo sapiens 487cccatcatct atgccttcc 1948819DNAHomo sapiens 488catcatctat gccttccgc 1948919DNAHomo sapiens 489catctatgcc ttccgcaac 1949019DNAHomo sapiens 490atctatgcct tccgcaacc 1949119DNAHomo sapiens 491cctgctctgt ggctgtttc 1949219DNAHomo sapiens 492ctgctctgtg gctgtttcc 1949319DNAHomo sapiens 493tctgtggctg tttccagtc 1949419DNAHomo sapiens 494ctgtggctgt ttccagtcc 1949519DNAHomo sapiens 495tgtttccagt ccaaagtgc 1949619DNAHomo sapiens 496gtttccagtc caaagtgcc 1949719DNAHomo sapiens 497tttccagtcc aaagtgccc 1949819DNAHomo sapiens 498cagtccaaag tgccctttc 1949919DNAHomo sapiens 499ccaaagtgcc ctttcgttc 1950019DNAHomo sapiens 500caaagtgccc tttcgttcc 1950119DNAHomo sapiens 501tgccctttcg ttccaggtc 1950219DNAHomo sapiens 502ccctttcgtt ccaggtctc 1950319DNAHomo sapiens 503tcgttccagg tctcccagc 1950419DNAHomo sapiens 504gcctgagctc gtagtcaac 1950519DNAHomo sapiens 505cctgagctcg tagtcaacc 1950619DNAHomo sapiens 506gggacattgt cttgtgtac 1950719DNAHomo sapiens 507ggacattgtc ttgtgtacc 1950819DNAHomo sapiens 508acattgtctt gtgtacctc 1950919DNAHomo sapiens 509tcttgtgtac ctcgggaac 1951019DNAHomo sapiens 510cttgtgtacc tcgggaacc 1951119DNAHomo sapiens 511ttgtgtacct cgggaaccc 1951219DNAHomo sapiens 512gtgtacctcg ggaaccctc 1951319DNAHomo sapiens 513tacctcggga accctcatc 1951419DNAHomo sapiens 514cctcgggaac cctcatctc 1951519DNAHomo sapiens 515tgccattgtg gtccttatc 1951619DNAHomo sapiens 516cattgtggtc cttatcatc 1951719DNAHomo sapiens 517gtggtcctta tcatcttcc 1951819DNAHomo sapiens 518ggtccttatc atcttccac 1951919DNAHomo sapiens 519ccttatcatc ttccacaac 1952019DNAHomo sapiens 520cttatcatct tccacaacc 1952119DNAHomo sapiens 521ttatcatctt ccacaaccc 1952219DNAHomo sapiens 522catgttcctg ctaataggc 1952319DNAHomo sapiens 523gttcctgcta ataggcagc 1952419DNAHomo sapiens 524ttcctgctaa taggcagcc 1952519DNAHomo sapiens 525ctaataggca gcctggctc 1952619DNAHomo sapiens 526taggcagcct ggctcttgc 1952719DNAHomo sapiens 527acctgcttca gtcagaagc 1952819DNAHomo sapiens 528cctgcttcag tcagaagcc 1952919DNAHomo sapiens 529tgcttcagtc agaagccac 1953019DNAHomo sapiens 530gcttcagtca gaagccacc 1953119DNAHomo sapiens 531cagtcagaag ccaccaagc 1953219DNAHomo sapiens 532agaagccacc aagctggtc 1953319DNAHomo sapiens 533aagccaccaa gctggtcac 1953419DNAHomo sapiens 534cacgatcggc ctcattgtc 1953519DNAHomo sapiens 535cctcattgtc gcctctttc 1953619DNAHomo sapiens 536tcattgtcgc ctctttctc 1953719DNAHomo sapiens 537ttgtcgcctc tttctctgc 1953819DNAHomo sapiens 538tgtcgcctct ttctctgcc 1953919DNAHomo sapiens 539tcgcctcttt ctctgcctc 1954019DNAHomo sapiens 540ctctttctct gcctctgtc 1954119DNAHomo sapiens 541tttctctgcc tctgtctgc 1954219DNAHomo sapiens 542ctctgcctct gtctgcagc 1954319DNAHomo sapiens 543ctgcagcttg ctggctatc 1954419DNAHomo sapiens 544gcagcttgct ggctatcac 1954519DNAHomo sapiens 545gctggctatc actgttgac 1954619DNAHomo sapiens 546ctggctatca ctgttgacc 1954719DNAHomo sapiens 547ggctatcact gttgaccgc 1954819DNAHomo sapiens 548tatcactgtt gaccgctac 1954919DNAHomo sapiens 549atcactgttg accgctacc 1955019DNAHomo sapiens 550cactgttgac cgctacctc 1955119DNAHomo sapiens 551ctgttgaccg ctacctctc 1955219DNAHomo sapiens 552gttgaccgct acctctcac 1955319DNAHomo sapiens 553ccgctacctc tcactgtac 1955419DNAHomo sapiens 554ctacctctca ctgtactac 1955519DNAHomo sapiens 555acctctcact gtactacgc 1955619DNAHomo sapiens 556ctctcactgt actacgctc 1955719DNAHomo sapiens 557cactgtacta cgctctgac 1955819DNAHomo sapiens 558gtactacgct ctgacgtac 1955919DNAHomo sapiens 559tactacgctc tgacgtacc 1956019DNAHomo sapiens 560acgctctgac gtaccattc 1956119DNAHomo sapiens 561cgtaccattc ggagaggac 1956219DNAHomo sapiens 562ccattcggag aggacggtc 1956319DNAHomo sapiens 563agaggacggt cacgtttac 1956419DNAHomo sapiens 564gaggacggtc acgtttacc 1956519DNAHomo sapiens 565ggtcacgttt acctatgtc 1956619DNAHomo sapiens 566acgtttacct atgtcatgc 1956719DNAHomo sapiens 567gtttacctat gtcatgctc 1956819DNAHomo sapiens 568tacctatgtc atgctcgtc 1956919DNAHomo sapiens 569tatgtcatgc tcgtcatgc 1957019DNAHomo sapiens 570tgtcatgctc gtcatgctc 1957119DNAHomo sapiens 571cagaccgctc accaagaac 1957219DNAHomo sapiens 572accgctcacc aagaacaac 1957319DNAHomo sapiens 573cgctcaccaa gaacaacgc 1957419DNAHomo sapiens 574caagaacaac gcggccatc 1957519DNAHomo sapiens 575gaacaacgcg gccatcctc 1957619DNAHomo sapiens 576cctctcggtg tccttcctc 1957719DNAHomo sapiens 577ctcggtgtcc ttcctcttc 1957819DNAHomo sapiens 578ccttcctctt catgtttgc 1957919DNAHomo sapiens 579ttcctcttca tgtttgcgc 1958019DNAHomo sapiens 580cctcttcatg tttgcgctc 1958119DNAHomo sapiens 581ttcatgtttg cgctcatgc 1958219DNAHomo sapiens 582atgtttgcgc tcatgcttc 1958319DNAHomo sapiens 583tttgcgctca tgcttcagc 1958419DNAHomo sapiens 584tgcgctcatg cttcagctc 1958519DNAHomo sapiens 585gctcatgctt cagctctac 1958619DNAHomo sapiens 586catgcttcag ctctacatc 1958719DNAHomo sapiens 587atgcttcagc tctacatcc 1958819DNAHomo sapiens 588tcagctctac atccagatc 1958919DNAHomo sapiens 589tgtaagattg tgatgaggc 1959019DNAHomo sapiens 590taagattgtg atgaggcac 1959119DNAHomo sapiens 591agattgtgat gaggcacgc 1959219DNAHomo sapiens 592gattgtgatg aggcacgcc 1959319DNAHomo sapiens 593attgtgatga ggcacgccc 1959419DNAHomo sapiens 594gtgatgaggc acgcccatc 1959519DNAHomo sapiens 595tcagatagcc ctgcagcac 1959619DNAHomo sapiens 596cagatagccc tgcagcacc 1959719DNAHomo sapiens 597agccctgcag caccacttc 1959819DNAHomo sapiens 598cgtcgcacta tgtgaccac

1959919DNAHomo sapiens 599gtcgcactat gtgaccacc 1960019DNAHomo sapiens 600ctccaccctg gctatcatc 1960119DNAHomo sapiens 601tccaccctgg ctatcatcc 1960219DNAHomo sapiens 602tttgctgctt gctggatgc 1960319DNAHomo sapiens 603ttgctgcttg ctggatgcc 1960419DNAHomo sapiens 604tgcttgctgg atgcctttc 1960519DNAHomo sapiens 605ttgctggatg cctttcacc 1960619DNAHomo sapiens 606cttgatagcg gattacacc 1960719DNAHomo sapiens 607ctccatctat acctacgcc 1960819DNAHomo sapiens 608catctatacc tacgccacc 1960919DNAHomo sapiens 609atctatacct acgccaccc 1961019DNAHomo sapiens 610tatacctacg ccaccctcc 1961119DNAHomo sapiens 611cgccacctac aattccatc 1961219DNAHomo sapiens 612cacctacaat tccatcatc 1961319DNAHomo sapiens 613tacaattcca tcatcaacc 1961419DNAHomo sapiens 614ttccatcatc aaccctgtc 1961519DNAHomo sapiens 615tttcagaaac caagagatc 1961619DNAHomo sapiens 616accaagagat ccagaaagc 1961719DNAHomo sapiens 617caagagatcc agaaagcgc 1961819DNAHomo sapiens 618agcgctctgt ctcatttgc 1961919DNAHomo sapiens 619gctctgtctc atttgctgc 1962019DNAHomo sapiens 620atttgctgcg gctgcatcc 19

Claims

1. A method for identifying a compound that inhibits the processing of amyloid-beta precursor protein in a mammalian cell, comprising (a) contacting a compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6, 289-333; and (b) measuring a compound-polypeptide property related to the production of amyloid-beta protein.

2. The method according to claim 1, wherein said polypeptide comprises SEQ ID NO: 289-333 in an in vitro cell-free preparation.

3. The method according to claim 1, wherein said polypeptide is membrane-bound.

4. The method according to claim 2, wherein said polypeptide is present as a transmembrane cell receptor in a mammalian cell.

5. The method of claim 1, wherein said property is a binding affinity of said compound to said polypeptide.

6. The method of claim 4, wherein said property is activation of a biological pathway producing an indicator of the processing of amyloid-beta precursor protein.

7. The method of claim 6 wherein said indicator is a second messenger.

8. The method of claim 7 wherein said second messenger is cyclic AMP or Ca²+.

9. The method of claim 6 wherein said indicator is amyloid-beta peptide.

10. The method of claim 9 wherein said amyloid-beta protein is selected from the group consisting of one or more of amyloid-beta peptide 1-42, 1-40, 11-42 and 11-40.

11. The method of claim 10 wherein said amyloid-beta protein is amyloid-beta peptide 1-42.

12. The method according to claim 6 wherein said indicator induces the expression of a reporter in said mammalian cell.

13. The method according to claim 12 wherein the reporter is selected from the group consisting of alkaline phosphatase, GFP, eGFP, dGFP, luciferase and B galactosidase.

14. The method according to claim 1, wherein said compound is selected from the group consisting of compounds of a commercially available screening library and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6, 289-333.

15. The method according to claim 2, wherein said compound is a peptide in a phage display library or an antibody fragment library.

16. The method according to claim 1, wherein said compound is an aryloxydithiourea, its salts, hydrates, or solvates.

17. An agent for the inhibition of amyloid-beta precursor processing selected from the group consisting of an antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a naturally occurring polynucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6.

18. The agent according to claim 17, wherein a vector in a mammalian cell expresses said agent.

19. The agent according to claim 18, wherein said vector is an adenoviral, retroviral, adeno-associated viral, lentiviral, a herpes simplex viral or a sendaiviral vector.

20. The agent according to claim 19, wherein said antisense polynucleotide and said siRNA comprise an antisense strand of 17-25 nucleotides complementary to a sense strand, wherein said sense strand is selected from 17-25 continuous nucleotides of a naturally occurring nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6.

21. The agent according to claim 20, wherein said siRNA further comprises said sense strand.

22. The agent according to claim 20, wherein said sense strand is selected from 17-25 continuous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-3.

23. The agent according to claim 17, wherein said siRNA further comprises a loop region connecting said sense and said antisense strand.

24. The agent according to claim 23 wherein said loop region comprises a nucleic acid sequence defined of SEQ ID NO: 288.

25. The agent according to claim 17, wherein said agent is an antisense polynucleotide, ribozyme, or siRNA comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 7-287.

26. A cognitive enhancing pharmaceutical composition comprising a therapeutically effective amount of an agent of claim 17 in admixture with a pharmaceutically acceptable carrier.

27. The cognitive enhancing pharmaceutical composition according to claim 26 wherein said agent comprises a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 7-287, a polynucleotide complementary to said nucleic acid sequence, and a combination thereof.

28. A method of inhibiting the processing of amyloid-beta precursor protein in a subject suffering or susceptible to the abnormal processing of said protein, comprising administering to said subject a pharmaceutical composition according to claim 26.

29. A method according to claim 28 for treatment or prevention of a condition involving cognitive impairment or a susceptibility to the condition.

30. The method according to claim 29 wherein the condition is Alzheimer's disease.

31. A pharmaceutical composition for the treatment or prevention of a condition involving cognitive impairment or a susceptibility to the condition, comprising an effective amyloid-beta precursor processing-inhibiting amount of a GPCR antagonist or inverse agonist.

32. A composition according to claim 31, wherein said GPCR inverse agonist is an aryloxydithiourea, its pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof in admixture with a pharmaceutically acceptable carrier.

Images