MXPA06006567A - Interaction of nmda receptor with the protein tyrosine phosphatase step in psychotic disorders - Google Patents

Abstract

The present invention relates to the identification of STEP being as involved in signaling pathways relating to psychotic diseases, including schizophrenia, and other disorders in which NMDA receptor dysfunction is implicated. The present invention provides methods for screening STEP inhibitors that modulate NMDA-R signaling. The present invention also provides methods and compositions for treatment of disorders mediated by abnormal NMDA-R signaling.

Description

INTERACTION OF THE RECEPTOR OF N-METHYL-D-ASPARTATE WITH THE PROTEIN T1ROSINA STEP PHOSPHATASE IN S1COT1COS DISORDERS BACKGROUND OF THE INVENTION In most mammalian excitatory synapses, glutamate (Glu) mediates rapid chemical neurotransmission by binding to four different types of glutamate receptors on the surfaces of brain neurons. Although cellular responses mediated by glutamate receptors are normally activated by exactly the same excitatory amino acid neurotransmitters (EAA) in the brain (eg, glutamate or aspartate), the different subtypes of glutamate receptors have different distribution patterns in the brain. , and mediate different transduction events of the cellular signal. A major class of glutamate receptors is referred to as the N-methyl-D-aspartate (NMDA-Rs) receptors, since these preferentially bind to N-methyl-D-aspartate (NMDA). NMDA is a chemical analog of aspartic acid; it does not normally occur in nature, and NMDA does not occur in the brain. When NMDA molecules are contacted with neurons that have NMDA-Rs, they activate NMDA-R strongly (for example, they act as powerful receptor agonists), causing the same type of neuronal excitation as glutamate. It is known that excessive activation of NMDA-R plays a major role in numerous important disorders of the central nervous system (CNS), while the activity of NMDA-R has been implicated in various psychiatric diseases. The NMDA-Rs contain the NR1 or NR3 subunits and at least one of four different NR2 subunits (designated as NR2A, NR2B, NR2C, and NR2D). NMDA-Rs are "ionotropic" receptors since they make the ions flow, such as Ca2 +. These ion channels allow the ions to flow to a neuron after the depolarization of the postsynaptic membrane, when the receptor is activated by glutamate, aspartate, or an agonist drug. Tyrosine protein phosphorylation plays an important role in the regulation of various cellular processes. The regulation of tyrosine protein phosphorylation is mediated by the reciprocal actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). NMDA-Rs are regulated by protein tyrosine kinases and phosphatases. The phosphorylation of NMDA-R by protein tyrosine kinases results in an improved response to NMDA-R in neurons (Wang et al., Nature 369: 233-235, 1994). It has been shown that NR2B and NR2A are the major sites of phosphorylation by protein tyrosine kinases. The protein tyrosine phosphatases, on the other hand, exert opposite effects on the NMDA-R response in neurons (Wang et al, Proc. Nati, Acad.Sci.U.S.A. 93: 1721-1725, 1996). It is believed that members of the Src family of protein tyrosine kinases mediate the phosphorylation of NMDA-R tyrosine. On the other hand, the identity of the enzyme responsible for the anti-dephosphorylation of NMDA-R has been elusive. The majority of psychiatric disorders are classified as of complex origin, generated from interactions between genetic and environmental causes. One of the most debilitating disorders of these is schizophrenia, which affects approximately 1% of the population. Once the symptoms are present, usually in the young adult stage, they persist throughout the patient's life and are almost totally incapacitating. The diagnosis is based on the simultaneous presentation of two types of symptoms that reflect a psychotic disturbance: "positive" symptoms that include delusions, hallucinations, and bizarre thoughts, and negative symptoms that include social withdrawal with absence of affection, low motivation, and apathy . Although the clinical efficiency of dopamine D2 receptor blockers suggests that the imbalance of dopamine is important in schizophreniaIt has become evident that many other neurotransmitter systems, including the glutamatorgic system, are involved in the pathophysiology of the schizophrenic brain. Positive modulators of cortical glutamaturgic systems can be useful adjuvants in the treatment of schizophrenia. It is known that glutamatorgic transmission plays a fundamental role in cognitive processes. Evidence has been accumulated suggesting that reduced excitatory (glutamatorgic) activity, especially one that participates in select neocortical areas, may underlie some, if not all, of the symptoms of schizophrenia. For example, see Coyle (1996) Harv Rev Psychiatry 3: 241-253; and Tamminga (1998) Crit Rev Neurobiol 12: 21-36. Studies of image formation and postmortem morphometry of schizophrenic brains have found abnormalities in numerous brain regions, such as the prefrontal, temporal and cingulate anterior cortex, hippocampus, amygdala, and stratum, which are connected by glutamatorgic circuits. Phencyclidine, ketamine, and other non-competitive antagonists at the N-methyl-D-aspartate (NMDA) glutamate receptors exacerbate symptoms in patients (Lahti et al. (1995) Neuropsychopharmacology 13: 9-19) and produce an interval of psychotic symptoms in volunteers that are similar to those of schizophrenic patients. Drugs that improve glutamatorgic transmission may compensate for the postulated imbalance between the ascending monoaminergic systems of the midbrain and the cortical descending glutamatorgic systems in the schizophrenic brain (Carlsson and Carlsson (1990) Trends Neurosci., 13: 272-276). One method has focused on the improvement of NMDA receptor activity with glycine or related agonists (D-cycloserine) of the glycine coagonist site insensitive to estriquinin. Some beneficial effects of D-cycloserine have been reported on negative symptoms in patients coadministered with a typical antipsychotic. The methods for the selection of active compounds, and the use of said compounds in the treatment of schizophrenia have a substantial medical interest.

BRIEF DESCRIPTION OF THE INVENTION Methods for the identification of therapeutic agents in the treatment of psychotic disorders, including schizophrenia and related conditions, are provided by the selection of inhibitors of signaling by the N-methyl-D-aspartate (NMDA-R) receptor acting through of one or more isoforms of the protein tyrosine phosphatase STEP. In one embodiment, the modulator is identified by detecting its ability to modulate the STEP phosphatases activity. In another embodiment, the modulator is identified by detecting its ability to modulate the binding of STEP and the NMDA-R. In another embodiment, methods are provided for the identification of a nucleic acid molecule encoding polypeptides that modulate signaling by NMDA-R. Active STEP has been found to down-regulate NMDA-R activity, and STEP inhibitors can increase NMDA-R activity when STEP is present. Methods for the treatment of schizophrenia and related disorders are provided by the administration of an inhibitor of STEP activity, which directly or indirectly modulates the level of tyrosine phosphorylation of NMDA-R. The modulator can affect the ability of STEP to dephosphorylate NMDA-R, to dephosphorylate kinases, for example ERK, in a signaling pathway associated with NMDA-R, and / or the ability of STEP to bind to NMDA-R. In certain embodiments, the modulator is a STEP antagonist and the disease to be treated is mediated by the hypofunction of NMDA-R.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1 B. STEP is selectively expressed in the brain since it has been detected by quantitative PCR in multiple rat tissues. Quantitative PCR using probes that recognize both isoforms STEP46 as STEP61 (figure 1A) and STEP61 alone (figure 1 B) shows that the STEP mRNA is located specifically in the brain. Figure 2. STEP is selectively expressed in the brain as detected by Northern blot in multiple rat tissues. Figure 3. STEP is selectively expressed in the brain as detected by Northern blot in multiple human tissues. Figures 4A-4B. STEP is expressed in rat brain as shown by high levels of in situ hybridization in the stratum and in the hippocampus. In situ hybridization of rat brain sections with probes to STEP shows a strong explosion in the stratum, CA2 and subiculum and an expression detectable in other regions of the hippocampus and cortex. Figure 5. STEP over-expression causes a decreased function of the NMDA receptor. HEK293 cells stably expressing the NR1 and NR2B subunits were transfected with the constructs of STEP61 (61 (WT)), STEP46 (46 (WT)) or the forms of either of the two that contained a CS mutation in their domains catalytic which makes them inactive (61 (CS) and 46 (CS)). The cells were loaded with a calcium indicator dye and the flow of Ca into the cells induced by the application of 1 μM glutamate was measured by evaluating the fluorescence change. The average response to glutamate was normalized to the number of total cells by evaluating the fluorescence change induced by the permeabilization of the cells with 1% NP40. Figure 6. Removal of STEP levels causes an increase in NMDA receptor function. Cultured cortical neurons were transfected with RNA inhibitory molecules designed to specifically inhibit STEP expression using the Amaxa Nucleofection technique. (Top) Four days after transfection, Western blot analysis shows that neurons transfected with STEP inhibitor RNA show lower levels of STEP61 protein than those transfected with the mixed RNA molecule. (Bottom) Measurement of the influx of Ca induced by the application of 1 μM NMDA to neurons four days after transfection shows a much greater response to NMDA in cells whose STEP61 expression levels have been reduced compared to those in which the mixed RNA molecule was introduced.

Figure 7. STEP causes the decrease in ERK phosphorylation in transfected HEK-293 cells. STEP46 causes a decrease in EGF-stimulated ERK phosphorylation in transfected HEK293 cells. HEK293 cells were transfected with several constructions, 2 days after transfection the cells were treated with 50 ng / ml of EGF for 15 minutes. The cells were used and the proteins were separated by SDS-polyacrylamide gel electrophoresis. The proteins were transferred to the nitrocellulose membranes and these were assayed with antibodies that specifically recognize phosphorylated ERK. In the presence of an active form of STEP46 (46WT) the phosphorylation of ERK is reduced compared to untransfected cells. A catalytically inactive form of STEP46 (46CS) shows a greatly increased phosphorylation. The expression of PTP-MEG either in active form (MEG WT) or in inactive form (MEG CS) has no effect on ERK phosphorylation. Figures 8A-8B. STEP modulates NMDAR-mediated ERK phosphorylation in neurons. Cultured cortical neurons (10-13 divisions) show low levels of basal phosphorylation of ERK. After the addition of 100 μM NMDA for 5 minutes the ERK phosphorylation levels increase significantly. The application of the NMDA receptor antagonist D-APV (200 μM) inhibits the phosphorylation of NMDA stimulated by NMDA (left panel). Infection of cells with sindbis virus containing RNA encoding GFP, STEP61, or STEP61cs shows that STEP affects NMDAR-mediated ERK phosphorylation. One day after infection of the cortical neurons cultured with the sindbis virus the cells were treated with 100 μM glutamate for 5 minutes and harvested. SDS-PAGE was run out and the western blot was used to detect the levels of ERK phosphorylation. Neurons infected with active STEP showed less phosphorylation of ERK than cells infected with GFP (control). Neurons infected with dominant negative STEPcs show a higher phosphorylation of ERK than cells infected with GFP (right panel). Figures 9A-9B. HEK293 cells transfected with STEP61 and Fyn (Figure 9A) or Src (Figure 9B) show a decrease in phosphorylation status dependent on the concentration of the kinase. The cells were transfected with constitutively active forms of any kinase and varying amounts of STEP61. Two days after transfection the cells were used and the proteins were separated by SDS-polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose membranes and these were assayed with antibodies that specifically recognize the phosphorylated forms of the kinase (Src-PY-418 or Fyn-PY-420). With increasing amounts of STEP61, phosphorylation levels in these sites decrease. Figures 10A-10B. HEK293 cells stably transfected with NR1, NR2B and STEP61 were harvested. The immunoprecipitation was carried out with anti-NR1 antibody (FIG. 10A) or anti-STEP (FIG. 10B). The used ones were incubated overnight with the antibodies, then the G protein sepharose was added to each lysate for 1 hour and then the immunoprecipitated proteins were isolated by SDS-PAGE. The Western blot shows that NMDAR containing STEP and NR1 co-immunoprecipitates in stably expressed cell lines. Figures 11A-11 F. STEP61 interacts with subunits NR1 and NR2 of NMDAR. HEK293 cells were transfected with NR1, NR2A or NR2B and STEP61. Immunoprecipitation was carried out with appropriate antibodies selective to the subunit. Figures 11A, 11C, 11E show that co-immunoprecipitation of STEP61 with NMDAR subunits occurs when complexes are pulled with antibodies to specific subunits. Figures 11 B, 11 D, 11 F show that the individual NMDAR subunits are co-immunoprecipitated with STEP61 when the complexes are pulled with the anti-STEP antibody. Figures 12A-12F. STEP46 interacts with subunits NR1 and NR2 of NMDAR. HEK293 cells were transfected with NR1, NR2A or NR2B and STEP61. Immunoprecipitation was carried out with appropriate antibodies selective to the subunit. Figures 12A, 12C, 12E show that the co-immunoprecipitation of STEP46 with the NMDAR subunits occurs when the complexes are pulled with antibodies to the specific subunits. Figures 12B, 12D, 12F show that the individual NMDAR subunits are co-immunoprecipitated with STEP46 when the complexes are pulled with anti-STEP antibody.

DETAILED DESCRIPTION OF THE MODALITIES OF THE INVENTION The present invention relates to the modulation of the binding interaction between the NR2A or NR2B subunits of the NMDA-R and the tyrosine phosphatase protein STEP. In accordance with the discovery, the present invention provides methods for the identification of STEP agonists and antagonists that modulate NMDA-R signaling, and for the treatment of conditions mediated by abnormal NMDA-R signaling. Of particular interest is the treatment of schizophrenia. The following description provides a guide for the preparation and use of the compositions of the invention, and for carrying out the methods of the invention. In culture models, downstream signaling events in the NMDA-R signaling pathway are affected by STEP expression, while STEP over-expression causes a decrease in either ERK phosphorylation stimulated by EGF or glutamate. Phosphorylated ERK is a key signaling molecule between NMDA receptor activation and nuclear events, which in turn affect the phosphorylation of CREB and genes whose transcription is under the regulation of CREB. Therefore, downstream signaling mediated by NMDA-Rs is affected by STEP, and STEP exacerbates the effects of reduced NMDA-R function in schizophrenia.

STEP causes decreased phosphorylation of tyrosine kinases fyn and src, when it is overexpressed in HEK293 cells. It is known that both src and fyn phosphorylate NMDA receptors when they are in active, phosphorylated forms, so that STEP acts to decrease the level of phosphorylation of NMDA-R. The less phosphorylated NMDA-Rs have lower conductance states and will therefore allow less current and fewer ions to pass through and therefore be less functionally active. This can lead to schizophrenic symptoms.

Definitions Unless otherwise mentioned, all technical and scientific terms used in the present invention have the same meanings that are commonly understood by those skilled in the art to which this invention pertains. The following references provide a person skilled in the art with a general definition of many of the terms used in this invention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2nd ed., 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walkered., 1988); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991). Although any methods and materials similar or equivalent to those described in the present invention may be used, preferred methods and materials are described in the practice or evaluation of the present invention. The following definitions are provided to assist the reader in the practice of the invention. As used in the present invention, the term "psychotic disorder" has the meaning that is commonly known in the art, and as set forth in the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Symptoms of psychotic disorders include delusions, hallucinations, disorganized speech (eg, frequent delirium or incoherence), and strongly disorganized or catatonic behavior. Schizophrenia is a common and severe mental disorder. In the USA, patients with schizophrenia occupy approximately 1/4 of all hospital beds and are considered to currently comprise approximately 20% of all social security disabilities. Schizophrenia is more prevalent than Alzheimer's disease, diabetes, or multiple sclerosis. The symptoms of schizophrenia vary in type and severity. They are generally classified as positive or negative symptoms (deficit). Positive symptoms can be further classified as delusions and hallucinations; or disorder of thinking and bizarre behavior. Delusions and hallucinations are sometimes referred to as the psychotic dimension of schizophrenia. Thought disorder and bizarre behavior are demonized as disorganized symptom clustering. Negative symptoms (deficit) include absence of affection, poverty of speech, anhedonia, and asociality. In some patients with schizophrenia, cognitive functioning decreases, with attention, abstract thinking, and resolution of altered problems. The severity of cognitive impairment is a major determinant of general disability in these patients. Although its specific causes are unknown, schizophrenia has a biological basis. A stress vulnerability model, in which schizophrenia is observed to occur in people with vulnerabilities based on neurological problems, is the most widely accepted explanation. The onset, remission, and recurrence of symptoms are observed as products of the interaction between these vulnerabilities and environmental elements that provide stress. Although many clinical and experimental vulnerability markers have been proposed, none is ubiquitous. Psychophysiologically, deficits in information processing, attention, and inhibition of sensitivity can be markers of vulnerability. Although most people with schizophrenia do not have a family history of schizophrenia, genetic factors have been implicated. People who have a first degree relative with schizophrenia have an approximately 15% risk of developing the disorder, compared to a 1% risk among the general population. A monozygotic twin whose co-twin has schizophrenia has >50% chance of developing it. Conventional antipsychotic (neuroleptic) drugs include chlorpromazine, fluphenazine, haloperidol, loxapine, mesoridazine, molindone, perphenazine, pimozide, thioridazine, thiothixene, and trifluoperazine. These drugs are characterized by their affinity for the dopamine 2 receptor and can be classified as high, intermediate, or low potency. Atypical antipsychotic drugs may have an affinity of selection for brain regions involved in the symptoms of schizophrenia and a reduced affinity for areas associated with motor symptoms and elevated prolactin. These affect other neurotransmitter systems, including serotonin, or have a selective affinity for the dopamine-specific receptor subtypes. Aberrant behaviors induced in rats by metamfetamine (Larson et al., (1996) Brain Res 738: 353-356), is a common and frequently predictive test of the activity of the antipsychotic drug. Implicit in the hypothesis that schizophrenia is generated from an imbalance between opposing neurotransmitter systems is the prediction that the antagonists of one of the systems and positive modulators of the other must be at least additive and probably synergistic. This is of considerable clinical importance because it suggests a novel therapeutic strategy that includes low levels of two completely different classes of drugs. Reducing the dose of commonly used antipsychotics should reduce their side effects that often limit treatment. Psychotic disorders other than schizophrenia include schizophreniform disorder, which is diagnosed when the symptom criteria for schizophrenia are reached, but the duration is too short and social and occupational functioning may not be altered. In schizoaffective disorder, the symptom criteria for schizophrenia are reached, and during the same continuous period there is a major depression, manic episode or mixed. With delusional disorder, prominent non-bizarre delusions are present for at least one month and the symptom criteria for schizophrenia have never been reached. Brief psychotic disorder is diagnosed when psychotic symptoms such as delusions, hallucinations, or speech or disorganized catatonic behavior are present for at least a month and are completely resolved. Shared psychotic disorder is diagnosed when delusions develop in an individual involved in a close relationship with another individual who suffers from delusions that are generated from a different psychosis. Psychotic conditions can also be generated from other diseases, or from substance abuse. Associated with these disorders are: alcohol, drugs similar to amphetamines, cannabis, ***e, hallucinogens, inhalants, opioids, phencyclidine, sedatives, and hypnotics. The term "agent" includes any substance, molecule, element, compound, entity, or a combination thereof. This includes, but is not limited to, a protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, and the like. This can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms "agent", "substance", and "compound" may be used interchangeably. As used in the present invention, an "agonist" is a molecule which, when interacting with (e.g., being bound to) a target protein (e.g., STEP, NMDA-R), increases or prolongs the amount or duration of the effect of the biological activity of the white protein. In contrast, the term "antagonist", as used in the present invention, refers to a molecule which, when interacting with (e.g., binds to) a target protein, decreases the amount or duration of the effect of the biological activity of the target protein (for example, STEP or NMDA-R). Agonists and antagonists can include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules that decrease the effect of a protein. Unless otherwise specified, the term "agonist" can be used interchangeably with "activator", and the term "antagonist" can be used interchangeably with "inhibitor". The term "analog" is used in the present invention to refer to a molecule that structurally resembles a molecule of interest but which has been modified in a controlled and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the initial molecule, an analog may exhibit the same utility, a similar utility, or an improved utility. The synthesis and selection of analogs, to identify variants of the known compounds that have improved traits (such as increased potency in a specific type receptor, or greater selectivity in a targeted receptor type and lower levels of activity in other type receptors) is a method which is well known in pharmaceutical chemistry. The term "biological preparation" refers to biological samples taken in vivo and in vitro (either with or without subsequent manipulation), as well as those prepared synthetically. Representative examples of biological preparations include cells, tissues, solutions and body fluids, a lysate of natural or recombinant cells. As used in the present invention, the term "functional derivative" of a native protein or of a polypeptide is used to define variants of biologically active amino acid sequences that possess biological activities (either functional or structural) that are substantially similar to those of the reference protein or polypeptide. Therefore, a functional derivative of a PTP can retain, among other activities, the ability to bind, and dephosphorylate NMDA-R. Similarly, a functional derivative of NMDA-R may be capable of binding to a PTP, and of being dephosphorylated by a PTP. NMDA receptors are a subclass of excitatory, ionotropic receptors of the neurotransmitter of L-glutamate. These are heteromeric, integral membrane proteins that are formed by the assembly of the obligatory NR1 subunit together with the modulating NR2 subunits. The subunit NR1 is the subunit for binding to glycine and exists as 8 variants of processing of a particular gene. The subunit for glutamate binding is the NR2 subunit, which is generated as the product of four different genes, and provides most of the structural basis for heterogeneity at NMDA receptors. In the hippocampus and cerebral cortex, the active NMDAR1 subunit is associated with 1 of 2 epsilon regulatory subunits: NMDAR2A or NMDAR2B and NR3. Unless otherwise specified, the term "NMDA-R" or "NMDA receptor" as used in the present invention refers to an NMDA receptor molecule having an NR1 subunit and at least one NR2A or NR2B subunit. An exemplary NR1 subunit is the human NMDA-R1 polypeptide. The sequence of the polypeptide and the corresponding nucleic acid can be obtained from Genbank, accession number L05666, and published in Pianells-Cases et al. (1993) P. N. A. S. 90 (11): 5057-5061. An exemplary NR2 subunit is the human NMDAR2A polypeptide. The sequence of the polypeptide and the corresponding nucleic acid can be obtained from Genbank, accession number U09002, and published in Foldes et al. (1994) Biochim. Biophys. Acta 1223 (1): 155-159. Another NR2 subunit is the human NMDAR2B polypeptide. The sequence of the polypeptide and the corresponding nucleic acid can be obtained from Genbank, accession number U11287, and is published in Adams et al. (1995) Biochim. Biophys. Acta 1260 (1): 105-108.

The STEP protein tyrosine phosphatase is characterized by an association with live NMDA-R, particularly in neural tissues, more particularly in brain tissue. A fundamental process for the regulation of the function of NMDA receptors and other ion channels in neurons is tyrosine phosphorylation. An enzyme phosphatase can act on NMDA-R directly, to dephosphorylate, one or more of the NMDA-R subunits. Alternatively an enzyme phosphatase can act on NMDA-R indirectly, by dephosphorylating a protein tyrosine kinase (PTK) in a signaling pathway. For example, a phosphatase that acts to decrease the activity of a PTK that phosphorylates NMDA-R, which indirectly results in a decreased phosphorylation of NMDA-R. The protein tyrosine phosphatase STEP is also referred to as PTPN5. In the brain, there are STEP transcripts of 3 kb, which are highly enriched in the stratum in relation to other areas, called phosphatase enriched in the stratum (STEP); and a 4.4 kb mRNA, which is more abundant in the cerebral cortex and strange in the stratum. See Genomics (1995) 28 (3): 442-9; and Proc Nati Acad Sci U S A (1991) 88 (16): 7242-6. Among the STEP transcripts are 6 different transcripts, all encoding 6 different protein isoforms. There are 4 probable alternative promoters and 2 alternative terminal exons that do not overlap. Transcripts appear to differ by N-terminal truncation, C-terminal truncation, presence or absence of 2 exons in the cassette, common exons with different ends. The motif of the tyrosine-specific protein phosphatase is found in 3 isoforms from this gene. Among the STEP isoforms is STEP 46, which is the 46 kD total length protein. STEP 20 lacks the tyrosine phosphatase domain. STEP 61 has a 5-prime extended open reading frame that encodes a protein with a predicted molecular mass of 61 kD and contains a particular tyrosine phosphatase domain. You can access the sequences like Genbank: NM 032781; AL832541; AK055450; and B1668912. It has been shown that glutamate-mediated activation of N-methyl-D-aspartate (NMDA) receptors leads to rapid but transient phosphorylation of the kinase related to the extracellular signal (ERK; MAPK1) (Paul et al., ( 2003) Nature Neurosa 6: 34-42). The influx of calcium mediated by NMDA leads to the activation of calcineurin and the subsequent dephosphorylation and activation of STEP. STEP subsequently inactivates ERK through the dephosphorylation of the tyrosine residue in its activation domain and the blocking of the nuclear translocation of the kinase. Therefore, STEP is important in regulating the duration of ERK activation and in downstream signaling in neurons. The sequences of the exemplary STEP nucleic acids and polypeptides can be found as set forth in Table 1, and in the attached sequence listing.

The protein kinases have been found to enhance the function of recombinant NMDA receptors, including the group of mitogen-activated protein kinases (MAP), or ERKs. MAPK1 is also known as ERK, or p42MAPK. MAP kinase ERK is widely involved in the transduction of the eukaryotic signal. After activation, it translocates to the nucleus of the stimulated cell, where it phosphorylates the nuclear targets. The nuclear accumulation of microinjected ERK depends on its phosphorylation status rather than on its activity or on the components upstream of its signaling pathway. The ERK dimeric forms are phosphorylated with phosphorylated and non-phosphorylated ERK partners. Altering dimerization by ERK mutagenesis reduces its ability to accumulate in the nucleus, suggesting that dimerization is essential for its normal ligand-dependent relocation. Other members of the MAP kinase family also form dimers. For a review, see Bhalla et al., (2002) Science 297: 1018-1023. You can access the ERK sequence in Genbank, access number M84489; and is described by Owaki et al. (1992) Biochem. Biophys. Res. Commun. 182 (3), 1416-142. Other protein kinases associated with NMDA-R signaling include the Src family of kinases, which comprises a total of nine members. It is known that five members of this family: Src, Fyn, Lyn, Lck, and Yes, are expressed in the SNC. All members of the Src family contain highly homologous regions of the C-terminal domains, catalytic Src 2 homology, and Src 3 homology. The kinase activity of the Src protein is normally inactivated by phosphorylation of the tyrosine residue at position 527 , which is found at six residues from the C-terminal end. Hydrolysis of phosphotyrosine 527 by an enzyme phosphatase normally activates c-Src. As used in the present invention, the term "NMDA-R signaling" refers to the signal transduction activities in the central nervous system that participate in various cellular processes such as neurodevelopment, neuroplasticity, and excitement. oxycy NMDA-R signaling affects a variety of processes including, but not limited to, neuron migration, neuron survival, synaptic maturation, learning and memory, and neurodegeneration. The term "NMDA-R hypofunction" is used in the present invention to refer to abnormally low levels of signaling activity of NMDA-Rs on CNS neurons. For example, the hypertension of NMDA-R may be caused by an abnormally low level of phosphotyrosine of NMDA-R. The hypofunction of NMDA-R can be presented as a drug-induced phenomenon. It can also present as an endogenous disease process, and is associated with schizophrenia and psychotic disorders. The term "modulation" as used in the present invention refers to both over-regulation, (eg, activation or stimulation), for example by the agonizing action; and down-regulation (e.g. inhibition or suppression), for example by the antagonizing action, of a bioactivity (e.g., direct or indirect phosphorylation of tyrosine NMDA-R, STEP tyrosine phosphatase activity, STEP binding to NMDA-R). As used in the present invention, the term "NMDA-R signaling modulator" refers to an agent that is capable of altering an NMDA-R activity that participates in NMDA-R signaling pathways. Modulators include, but are not limited to, both "activators" and "inhibitors" of tyrosine phosphorylation NMDA-R. An "activator" is a substance that directly or indirectly improves the level of tyrosine phosphorylation of NMDA-R, and therefore causes the NMDA receptor to become more active. The mode of action of the activator can be direct, for example, through binding to the receptor, or indirect, for example, through binding to another molecule which otherwise interacts with NMDA-R (for example, STEP , Src, Fyn, ERK, etc). Conversely, an "inhibitor" directly or indirectly decreases the tyrosine phosphorylation of NMDA-R, and therefore causes the NMDA receptor to become less active. The reduction can be complete or partial. As used in the present invention, modulators of NMDA-R signaling encompass STEP antagonists and agonists. As used in the present invention, the term "PTP modulator" includes both "activators" and "inhibitors" of PTP phosphatase activity. An "activator" of PTP is a substance that causes a PTP to become more active, and therefore directly or indirectly decreases the phosphotyrosine level of NMDA-R. The mode of action of the activator can be through the binding to the PTP; through binding to another molecule which interacts differently with PTP; etc. Conversely, an "inhibitor" of a PTP is a substance that causes the PTP to become less active, and therefore directly or indirectly increases the phosphotyrosine level of the NMDA-R. The reduction can be complete or partial, and is due to a direct effect or an indirect effect. As used in the present invention, the term "protein complex containing STEP / NMDA-R" refers to protein complexes, formed in vitro or in vivo, containing STEP and NMDA-R. In addition, the complex may also comprise other components, for example, a protein tyrosine kinase such as Fyn, Src, etc. The terms "substantially pure" or "isolated", when referring to proteins and polypeptides, for example, a fragment of a PTP, denotes those polypeptides that are separated from the proteins or other contaminants with which they are naturally associated. A protein or polypeptide is considered substantially pure when that protein is considered to be greater than about 50% of the total protein content of the composition containing that protein, and typically, is greater than about 60% of the total protein content. More typically, a substantially pure or isolated protein or polypeptide will consist of at least 75%, more preferably, at least 90%, of the total protein. Preferably, the protein will be more than about 90%, and more preferably, more than about 95% of the total protein in the composition. A "variant" of a molecule such as STEP or NMDA-R is intended to refer to a molecule substantially similar in structure and biological activity with respect to the entire molecule, or to a fragment thereof. Therefore, with the proviso that two molecules possess a similar activity, these are considered variants since that term is used in the present invention if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the sequence of amino acid residues is not identical. As used in the present invention, "recombinant" has the usual meaning in the art, and refers to a polynucleotide synthesized or otherwise manipulated in vitro (eg, "recombinant polynucleotide"), to the methods of use of the recombinant polynucleotides to produce gene products in cells or other biological systems, or to provide a polypeptide ("recombinant protein") encoded by a recombinant polynucleotide. The term "operatively associated" refers to a functional link between a control sequence of nucleic acid expression (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second polynucleotide, wherein the expression control sequence affects the transcription and / or translation of the second polynucleotide. The term "recombinant" when used with reference to a cell indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid. The recombinant cells may contain genes that are not found within the native (non-recombinant) form of the cell. The recombinant cells can also contain genes that are in the native form of the cell where the genes are modified and re-introduced into the cell by artificial means. The term also encompasses cells that contain an endogenous nucleic acid with respect to the cell that has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, site-specific mutation, and related techniques. A "heterologous sequence" or a "heterologous nucleic acid", as used in the present invention, is one that originates from an external source with respect to the particular host cell, or, if it is from the same source , it is modified from its original form. Therefore, a heterologous gene in a prokaryotic host cell includes a gene that, although it is endogenous to the particular host cell, has been modified. Modification of the heterologous sequence can occur, for example, by treating the DNA with a restriction enzyme to generate a DNA fragment that is capable of being operatively associated with the promoter. Techniques such as site-directed mutagenesis are also useful for modifying a heterologous nucleic acid. A "recombinant expression cassette" or simply an "expression cassette" is a nucleic acid construct, generated recombinantly or synthetically, having control elements that are capable of affecting the expression of a structural gene that is operatively associated with the control elements in hosts compatible with said sequences. Expression cassettes include at least promoters and optionally, transcription termination signals. Typically, the recombinant expression cassette includes at least one nucleic acid to be translated (e.g., a nucleic acid encoding a PTP) and a promoter. Additional factors necessary or auxiliary to affect expression may also be used as described in the present invention. For example, transcription termination signals, enhancers, and other nucleic acid sequences that influence gene expression can also be included in an expression cassette. As used in the present invention, "contacting" has its normal meaning and refers to the combination of two or more agents (e.g., two proteins, a polynucleotide and a cell, etc.). The contact can be presented in vitro (for example, two proteins, a polynucleotide and a cell, etc.). The contact can be presented in vitro (for example, two or more agents [for example, a test compound and a cell lysate] are combined in a test tube or in another container) or in situ (for example, two polypeptides can be contacting in a cell by co-expressing in the cell, recombinant polynucleotides encoding the two polypeptides), in a cell lysate. Various biochemical and molecular biology methods referred to in the present invention are well known in the art, and are described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y. Second (1989) and Third (2000) Editions, and Current Protocols in Molecular Biology, (Ausubel, F.M. et al., Eds.) John Wiley & Sons, Inc., New York (1987-1999).

Selection for modulators of NMDA-R signaling The present invention provides methods for the identification of therapeutic compounds for the treatment of psychotic disorders, by inhibiting NMDA-R signaling through STEP phosphatase. NMDA-R modulators are identified by the detection of the ability of an agent to inhibit a STEP activity, which is capable of dephosphorylating directly or indirectly an NMDA-R. The modulated activities of PTP include, but are not limited to, its phosphatase activity, its binding to NMDA-R, and its activity on ERK and PTKs. In some aspects of the invention, a soforma is used STEP in the selection of methods where the isoform comprises the STEP phosphatase domain, for example STEP 61; STEP 46; etc. In other embodiments, the STEP isoforms that lack the phosphatase domain, for example STEP 20, etc. they are of interest, for example as negative controls or for comparison; and for the determination of agents that interact with the non-catalytic portions of the enzyme. In one aspect, the NMDA-R modulators of the present invention are identified by monitoring their ability to affect phosphatase activity. As will be detailed below, STEP, the protein complex containing NMDA-R / STEP, or cell lines expressing STEP or the protein complex containing NMDA-R / STEP, are used for the selection of STEP agonists and antagonists that modulate directly or indirectly tyrosine dephosphorylation of NMDA-R, for example in the presence of a protein tyrosine kinase in a signaling pathway with STEP and NMDA-R. An agent that enhances STEP's ability to dephosphorylate directly or indirectly to NMDA-R will result in a net decrease in the amount of phosphotyrosine, while an agent that inhibits STEP's ability to dephosphorylate directly or indirectly to NMDA-R will result in a net increase in the amount of phosphotyrosine. In some embodiments, the ability of an agent to improve or inhibit STEP phosphatase activity is assayed in an in vitro system. In general, the in vitro assay format involves the addition of an agent to STEP (or a functional derivative of STEP) and a STEP substrate, for example Src, Fyn, ERK, NMDA-R, etc., and measuring the level of tyrosine phosphorylation of the substrate. In one embodiment, as a control, the level of tyrosine phosphorylation of the substrate is also measured under the same conditions except that the test agent is not present. By comparing the tyrosine phosphorylation levels of the substrate, PTP antagonists or agonists can be identified. Specifically, the STEP antagonist is identified if the presence of the test agent results in an increased level of tyrosine phosphorylation of the substrate. Conversely, a decreased level of tyrosine phosphorylation in the substrate indicates that the test agent is a STEP agonist. The invention provides the use of said agents to modulate the activity of NMDA-R. STEP used in the trials is obtained from various sources. In some embodiments, STEP used in the assays is purified from cellular or tissue sources, for example, by immunoprecipitation with specific antibodies. In other embodiments, as described below, STEP is purified by affinity chromatography using STEP-specific interactions with known protein substrates. In even other embodiments, STEP, whether in holoenzyme or enzymatically active portions of it, are produced recombinantly either in bacteria or in eukaryotic expression systems. The recombinantly produced variants of STEP may contain short protein labels, such as immunomarks (brand HA, brand c-myc, brand FLAG), brand of 6 x His, brand GST, etc., which can be used to facilitate the purification of STEP produced recombinantly using immunoaffinity chromatography or metal chelation, respectively. In the tests various substrates are used. Preferably, the substrate is Src, Fyn, ERK, NMDA-R, a functional derivative of NMDA-R, or the subunit NR2A or NR2B. In some embodiments, the substrates used are proteins purified from a tissue (such as NR2A or NR2B immunoprecipitated from rat brain). In other embodiments, the substrates are proteins that are expressed recombinantly. Examples of recombinant substrates include, but are not limited to, proteins that are expressed in E coli, yeast, or mammalian expression systems. In even other embodiments, the substrates used are synthetic peptides that are phosphorylated on tyrosine by a specific kinase activity, for example, Src or Fyn kinases. Methods and conditions for the expression of recombinant proteins are well known in the art. See, for example, Sambrook, previously mentioned, and Ausubel, previously mentioned. Typically, the polynucleotides encoding the phosphatase and / or the substrate used in the invention are expressed using expression vectors. Typically, expression vectors include transcriptional and / or translational control signals (e.g., the promoter, ribosome binding site, and ATG start codon). In addition, the efficiency of expression can be improved by the use of appropriate enhancers to the cell system in use. For example, the SV40 enhancer or the CMV enhancer can be used to increase expression in mammalian host cells. Typically, the DNA encoding a polypeptide of the invention is inserted into DNA constructs capable of being introduced and expressed in an in vitro host cell, such as a bacterium (e.g., E. coli, Bacillus subtilus), yeast (e.g. , Saccharomyces), insect (for example, Spodoptera frugiperda), or mammalian cell culture systems. Mammalian cell systems are preferred for many applications. Examples of mammalian cell culture systems useful for the expression and production of the polypeptides of the present invention include the human embryonic kidney line (293).; Graham et al., 1977, J. Gen. Virol. 36: 59); CHO (ATCC CCL 61 and CRL 9618); human cervical carcinoma cells (HeLa, ATCC CCL 2); and others known in the art. The use of tissue culture or mammalian cells to express polypeptides is generally discussed in Winnacker, FROM GENES TO CLONES (VCH Publishers, N.Y., 1987) and Ausubel, previously mentioned. In some embodiments, promoters from mammalian genes or from mammalian viruses are used, for example, for expression in mammalian cell lines. Suitable promoters can be constitutive, cell-specific, stage-specific, and / or modulable or regulatable (for example, by hormones such as glucocorticoids). Useful promoters include, but are not limited to, the metallothionein promoter, the major late promoter constituting the adenovirus, the MMX promoter inducible by dexamethasone, the SV40 promoter, and promoter-enhancer combinations known in the art. The substrate may or may not be in a state of phosphorylated tyrosine (Lau &Huganir, J. Biol. Chem., 270: 20036-20041, 1995). In the case of a non-phosphorylated raw material, the substrate is typically phosphorylated, for example, indicating an exogenous tyrosine kinase activity such as Src, or Fyn. A variety of standard procedures well known to those skilled in the art are used to measure the phosphorylation of the tyrosine levels of the substrates. In some embodiments, an antibody-based assay for phosphotyrosine recognition, eg, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), as well as fluorescently labeled antibodies whose binding can be assessed from fluorescence levels is used. issued. See, for example, the U.S. Patent. No. 5,883,110; Mendoza et al., Biotechniques. 27: 778-788, 1999. In other embodiments, instead of immunoassays, the substrates are directly labeled with a radioactive phosphate group using kinases that carry out selective tyrosine phosphorylation (Braunwaier et al., Anal. Biochem. : 23-26, 1996). The rate of removal of the radioactive label from the labeled substrate can be quantified in a liquid phase (for example, by chromatographic separation) or in a solid phase (gel or Western blot). Comparison of a level of tyrosine phosphorylation under two different conditions (e.g., in the presence and absence of a test agent) sometimes includes the step of recording the level of phosphorylation in a first sample or condition and comparing the level recorded with that of (or recorded for) a second portion or condition. In some embodiments of the invention, other than the addition of STEP to a substrate (eg, NR2A or NR2B), the in vitro assays are carried out with a protein complex containing NMDA-R / STEP. These protein complexes contain NMDA-R and STEP, or their official derivatives. In addition, the complexes may also contain a PTK and other molecules. Protein complexes containing NMDA-R / STEP can be obtained from neuronal cells using methods well known in the art, for example, immunoprecipitation as described in Grant et al. (WO 97/46877). The tyrosine phosphorylation levels of the substrates were tested with standard SDS-PAGE and immunoblot analysis. In other embodiments, the modulators of NMDA-R signaling of the present invention are identified using live assays. Such in vivo assay formats usually encompass culture of cells co-expressing STEP and a substrate (eg, NR2A or NR2B, eg, recombinant forms of STEP and / or substrate (s) of the NMDA-R subunit), adding an agent to the cell culture, and measuring the level of tyrosine phosphorylation of the substrate in the cells. In one embodiment, as a control, the level of tyrosine phosphorylation of the substrate in cells not exposed to the test agent was also measured or determined. In some modalities, the assay can be carried out with non-neural cells expressing NR2A or NR2B, therefore in the absence of synaptic proteins. In one embodiment, the in vivo assay system is modified from the method described in the U.S. Patent. No. 5,958,719. Using this selection system, intact cells expressing STEP and a STEP substrate (e.g., Src, Fyn, ERK, NMDA-R, NR2A, or NR2B) were initially treated (e.g., by NMDA) to stimulate phosphorylation of the substrate. The cells are then incubated with a substance that can penetrate the intact cells and selectively inhibit additional phosphorylation (for example, by a PTK) of the substrate, for example NMDA-R. The degree of phosphorylation of the substrate is then determined by, for example, altering the cells and measuring the phosphotyrosine level of the substrate according to the methods described above, for example with standard SDS-PAGE and immunoblot analysis. The activity of the PTP was determined from the measurement of the degree of phosphorylation of the substrate. An additional measurement is carried out in the presence of an agent. By comparing the degree of phosphorylation, PTP agonists or antagonists that modulate tyrosine phosphorylation of NMDA-R have been identified. In another embodiment, the present invention provides a method for the identification of a nucleic acid molecule encoding a gene product that is capable of modulating the level of tyrosine phosphorylation of NMDA-R. In one embodiment, a test nucleic acid is introduced into the host cells co-expressing STEP and NMDA-R or their functional derivatives. Methods for the introduction of a recombinant or exogenous nucleic acid into a cell are well known and include, without limitation, transfection, electroporation, naked nucleic acid injection, viral infection, liposome-mediated transport (see, for example, Dzau). et al., 1993, Trends in Biotechnology 11: 205-210; Sambrook, previously mentioned, Ausubel, previously mentioned). The cells are cultured so that the gene product encoded by the nucleic acid molecule is expressed in host cells and interacts with STEP and NMDA-R or its functional derivatives, followed by measurement of the phosphotyrosine level of NMDA-R. The effect of the nucleic acid on NMDA-R signaling is determined by comparing the phospholotyrosine levels of NMDA-R measured in the absence or presence of the nucleic acid molecule. It will be appreciated by one skilled in the art that modulation of the binding of STEP and NMDA-R may also affect the level of tyrosine phosphorylation in the NMDA-R by STEP. Therefore, agents identified from the selection using the in vivo and in vitro assay systems described above may also encompass agents that modulate tyrosine phosphorylation of NMDA-R by modulating the binding of STEP and NMDA- R. In some embodiments of the invention, NMDA-R modulators are identified by direct selection of agents that promote or suppress the binding of STEP and NMDA-R. Therefore, the identified agents can be further examined for their ability to modulate the tyrosine phosphorylation of NMDA-R, using the methods described above or standard assays well known in the art. In one embodiment, the modulators of the interaction between STEP and NR2A or NR2B are identified by detecting their capabilities either to inhibit the binding of STEP and NMDA-R (physical contact) to each other or to alter the binding of STEP and NMDA- R that has already been formed. The inhibition or alteration may be either complete or partial. In another embodiment, the modulators are selected for their activities either to promote the binding of STEP and NMDA-R to each other, or to improve the stability of a binding interaction between STEP and NMDA-R that has already been formed. In any case, some of the in vitro and in vivo assay systems discussed above for the identification of agents that modulate the tyrosine phosphorylation level of NMDA-R can be applied directly or can be easily modified to monitor the effect of a agent on the binding of NMDA-R and STEP. For example, a transfected cell to co-express STEP and NMDA-R or the receptor subunit, in which the two proteins interact to form a complex containing NMDA-R / PTP, is incubated with an agent that is suspected of being capable of inhibit this interaction, and the effect on the interaction is measured. Any of numerous means, such as coinmunoprecipitation, is used to measure the interaction and its alteration. Although the foregoing assays or methods are described with reference to the STEP and NMDA-R isoforms, it will be appreciated by one skilled in the art that functional derivatives or subunits of various STEP and NMDA-R isoforms may also be used. For example, in several embodiments, NR2A or NR2B are used to replace an intact NMDA-R in assays for the selection of agents that modulate the binding of STEP and NMDA-R. In a related embodiment, a functional derivative NMDA-R, ERK, Src, Fyn, is used for the selection of agents that modulate phosphatase activity. In addition, functional derivatives of STEP having deletions and / or insertions and / or amino acid substitutions (e.g., conservative substitutions) are used in various embodiments as long as they maintain their catalytic activity and / or binding capacity for the selection of agents. A functional derivative was prepared from a STEP isoform that occurs naturally or that is expressed recombinantly by proteolytic cleavage followed by conventional purification procedures known to those skilled in the art. Alternatively, the functional derivative is produced by recombinant DNA technology by expressing only fragments or combinations of STEP exons in suitable cells. In one embodiment, a partial NMDA receptor or a phosphatase polypeptide is expressed as a fusion polypeptide. The preparation of NMDA mutants that occurs naturally is within the ability of one skilled in the art.; or STEP isoforms that retain the desired properties, and to select the mutants for binding and / or enzymatic activity. The derivatives NR2A and NR2B and can be typically dephosphorylated comprise the cytoplasmic domain of the polypeptides, for example, the C-terminal 900 amino acids or a fragment thereof. In some embodiments, cells expressing STEP and NMDA-R can be used as a source of STEP and / or NMDA-R, without purification or purification, or in a membrane preparation, for the evaluation of these assays. Alternatively, whole cells that are alive or fixed directly in those assays can be used. Methods for the preparation of fixed cells or membrane preparations are well known in the art, see, for example, U.S. Pat. No. 4,996,194. The cells can be genetically engineered to co-express STEP and NMDA-R. The cells can also be used as host cells for the expression of other recombinant molecules for the purpose of putting these molecules in contact with STEP and / or NMDA-R within the cell.

Therapeutic applications and pharmaceutical compositions NMDA-R antagonists can be used to treat psychotic symptoms caused by abnormal NMDA-R signaling. As discussed in detail below, the present invention provides pharmaceutical compositions containing STEP antagonists that modulate the tyrosine phosphorylation of NMDA-R. Such antagonists include, but are not limited to, agents that interfere with the expression of the STEP gene, agents that modulate STEP's ability to bind to NMDA-R or to dephosphorylate NMDA-R. In one embodiment, the STEP antisense oligonucleotide or siRNA is used as a STEP antagonist in the pharmaceutical compositions of the present invention. In addition, STEP inhibitors that inhibit the dephosphorylation of NMDA-R will be useful as modulators of NMDA-R signaling. The hypofunction of NMDA-R is causally associated with schizophrenic symptoms (Tamminga, Crit., Rev. Neurobiol 12: 21-36, 1998, Carisson et al., Br. J. Psychiatry Suppl .: 2-6, 1999). Corbett et al., Psychopharmacology (Berl.) 20: 67-74, 1995; Mohn et al., Cell 98: 427-436, 1999). In addition, the hypofunction of NMDA-R is also associated with psychosis and drug addiction (Javitt &Zukin, Am J Psychiatry, 148: 1301-8, 1991). Using a STEP antagonist (NMDA-R agonists) as described in the present invention, the present invention provides methods for the treatment of schizophrenia, and other psychoses by antagonizing the activity of STEP, by inhibiting the the interaction between STEP and the NR2A or NR2B subunit; by interfering with the interaction between STEP and protein tyrosine kinases, by sub-regulation of STEP expression, and the like. It is well known in the art that agonists and antagonists of NMDA-R can be used to treat neurological disorders caused by abnormal NMDA-R signaling, eg acute damage of the central nervous system (CNS). Treatment methods have been described using pharmaceutical compositions comprising NMDA agonists and / or NMDA antagonists, for example, in the U.S. Patent. No. 5,902,815. As discussed in detail below, the present invention provides pharmaceutical compositions containing STEP antagonists and / or agonists that modulate tyrosine phosphorylation of NMDA-R or downstream signaling of NMDA-R. Such agonists and antagonists include, but are not limited to, agents that interfere with the expression of the STEP gene, agents that modulate STEP's ability to bind to NMDA-R or to dephosphorylate NMDA-R. In one embodiment, the antisense oligonucleotide to STEP is used as a STEP antagonist in the pharmaceutical compositions of the present invention. In addition, STEP inhibitors that inhibit the dephosphorylation of NMDA-R will be useful as modulators of NMDA-R signaling. The abnormal activity of NMDA-R induced by endogenous glutamate is implicated in numerous important CNS disorders. In one aspect, the present invention provides STEP modulators which, by modulating the phosphotyrosine level of NMDA-R, can treat or alleviate symptoms mediated by abnormal NMDA-R signaling. Indications of interest include moderate cognitive impairment (MCI), which may progress to Alzheimer's disease (AD). Treatment with acetylcholinesterase inhibitors may provide moderate improvement in memory. Cognitive enhancers can also find use for memory loss associated with aging, and in the general public. An important use for NMDA antagonist drugs includes the ability to prevent or reduce the exitotoxic damage to neurons. In some embodiments, the STEP agonists of the present invention, which promote the dephosphorylation of NMDA-R, are used to alleviate the toxic effects of excessive signaling by NMDA-R. In certain other modalities, STEP antagonists of the present invention, which function as NMDA-R agonists, are used therapeutically to treat conditions caused by NMDA-R hypo-function, for example, abnormally low levels of NMDA-R signaling. in the neurons of the CNS. The hypofunction of NMDA-R can be presented as an endogenous disease process. This can also be presented as a drug-induced phenomenon, after the administration of an NMDA antagonist drug. In some related embodiments, the present invention provides pharmaceutical compositions containing STEP antagonists that are used in conjunction with NMDA antagonists, for example, to prevent the toxic side effects of NMDA antagonists. Excessive glutamatorgic signaling is causatively associated with exitotoxic cell death during acute central nervous system trauma such as stroke (Choi et al., Annu Rev Neurosci.13: 171-182, 1990; Muir & Lees, Stroke 26: 503-513, 1995). Excessive glutamatorgic signaling via NMDA receptors has been implicated in profound consequences and impaired recovery after head trauma or brain injury (Tecoma et al., Neuron 2: 1541-1545, 1989; Mcintosh et al., J. Neurochem 55: 1170-1179, 1990). NMDA receptor-mediated glutamatorgic hyperactivity has also been associated with the process of slow degeneration of neurons in Parkinson's disease (Loopuijt &Schmidt, Amino Acids, 14: 17-23, 1998) and Huntington's disease (Chen et al., J. Neurochem. 72: 1890-1898, 1999). In addition, high signaling by NMDA-R has been reported in different forms of epilepsy (Reid &Stewart, Seizure 6: 351-359,1997).

Accordingly, the STEP agonists of the present invention are useful for the treatment of these diseases or disorders by stimulating the phosphatase activity associated with the NMDA receptor or by promoting the binding of STEP to the NMDA receptor complex. STEP agonists (NMDA-R antagonists) of the present invention can also be used to treat diseases where a mechanism of slow excitotoxicity has been implicated (Bittigau &Ikonomidou, J. Child, Neurol., 12: 471-485, 1997). These diseases include, but are not limited to, spinocerebellar degeneration (e.g., spinocerebellar ataxia), motor neuron diseases (e.g., amyotrophic lateral sclerosis (ALS)), mitochondrial encephalomyopathies. The STEP agonists of the present invention can also be used to alleviate neuropathic pain, or to treat chronic pain without causing tolerance or addiction (see, for example, Davar et al., Brain Res. 553: 327-330, 1991). The hypofunction of NMDA-R has been causally associated with various forms of cognitive impairment, such as dementias (eg, senile dementia and HIV dementia) and Alzheimer's disease (Lipton, Annu, Rev. Pharmacol., Toxicol. 159-177, 1998; Ingram et al., Ann., NY Acad. Sci. 786: 348-361, 1996; Müller et al., Pharmacopsychiatry., 28: 113-124, 1995). In addition, the hypofunction of NMDA-R is also associated with psychosis and drug addiction (Javitt &Zukin, Am J Psychiatry, 148: 1301-8, 1991). In addition, the hypofunction of NMDA-R is also associated with sensitivity to ethanol (Wirkner et al., Neurochem Int. 35: 153-162, 1999; Yagi, Biochem. Pharmacol. 57: 845-850, 1999). The hypofunction of NMDA-R has also been associated with depression. Using a STEP antagonist (NMDA-R agonists) described above, the present invention provides methods for the treatment of schizophrenia, psychosis, cognitive deficits, drug addiction, and ethanol sensitivity by the antagonizing action of the associated STEP activity with NMDA-R, or by inhibiting the interaction between STEP and the NR2A or NR2B subunit. The STEP antagonists of the present invention directly administer it under sterile conditions to the host to be treated. However, although it is possible for the active ingredient to be administered alone, it is often present preferably as a pharmaceutical formulation. Typically the formulations comprise at least one active ingredient together with one or more acceptable vehicles thereof. Each vehicle must be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not deleting the patient. For example, the bioactive agent can form complexes with carrier proteins such as ovalbumin or serum albumin before its administration for the purpose of improving stability or pharmacological properties such as half-life. In addition, the therapeutic formulations of this invention are combined with or used in combination with other therapeutic agents.

The therapeutic formulations are administered by any effective means that could be used for the treatment. Depending on the specific STEP antagonist / NMDA-R agonist to be used, suitable means include but are not limited to oral, rectal, nasal, pulmonary, or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) infusion into the stream blood Therapeutic formulations are prepared by any methods well known in the pharmacy art. See, for example, Gilman et al (eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics (8th edition) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences (17th edition) Mack Publishing Co., Easton, P.a .; Avis et al (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, N. Y .; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets Dekker, N. Y .; and Lieberman et al (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, N.Y. Therapeutic formulations can be conveniently presented in unit dosage forms and administered in a suitable therapeutic dose. The preferred dose and mode of administration of a STEP antagonist will vary for different patients, depending on the factors that will need to be reviewed individually by the attending physician. As a general rule, the amount of an antagonist STEP administered at the smallest dose that effectively prevents or minimizes or confidently conditions the patients.

An appropriate therapeutic dose is determined by any of the well known methods such as clinical studies on mammalian species to determine the maximum tolerable dose and on normal human subjects to determine the safe dose. In human patients, since direct examination of brain tissue is not possible, hallucinations or other psychomotor symptoms, such as disorientation or severe incoherence, may be seen as signs that potentially damage has been caused. neurotoxicity in the CNS by means of an NMDA-R antagonist. Additionally, various types of imaging techniques (such as positron emission tomography and magnetic resonance spectroscopy, which use labeled substrates to identify areas of maximum activity in the brain) can also be used for the determination of preferred doses. of NMDA-R agonists for use as described in the present invention. It is also desirable to evaluate rodents or primates for cellular manifestations in the brain, such as formation of vacuoles, mitochondrial damage, heat shock protein expression, or other pathomorphological changes in the neurons of the cerebral cingulate and retrosplenial cortices. These cellular changes can also be correlated with abnormal behavior in laboratory animals. The effects under certain circumstances when higher doses may be required, the preferred dose of STEP agonist and / or antagonist will usually lie within the range of from about 0.001 to about 1000 mg, more usually from about 0.01 to about 500 mg per day. It should be understood that the amount of any such agent actually administered will be determined by a physician, in light of the relevant circumstances that apply to an individual patient (including the condition or conditions to be treated, the choice of the composition to be administered , including the particular PTP agonist or the particular PTP antagonist, the age, weight, and response of the particular patient, the severity of the patient's symptoms, and the route of administration chosen). Therefore, it is intended that the aforementioned dose ranges provide general guidance and support the teachings in the present invention, but are not intended to limit the scope of the invention. It should be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructions, and described reagents, since these may vary. It is also understood that the terminology used in the present invention is for the purpose of describing only particular embodiments, and is not intended to limit the scope of the present invention, the scope of which will be determined by the language in the claims. It should be mentioned that as used in the present invention and in the appended claims, the singular forms "a / a", "and", and "the" include plural referents unless the context clearly dictates otherwise. . Thus, for example, the reference to "a mouse" includes a plurality of said mice and the reference to "the cytokine" includes reference to one or more cytokines and equivalents thereof known to those skilled in the art., and so on. Unless otherwise mentioned, all technical and scientific terms used in the present invention have the same meanings that are commonly understood by one skilled in the art to which this invention pertains. Although any methods, devices and materials similar or equivalent to those described in the present invention can be used in the practice or evaluation of the invention, preferred methods, devices and materials are described below. All publications mentioned in the present invention are incorporated herein as references for all relevant purposes, for example, the purpose of describing and discovering, for example, the cell lines, constructions, and methodologies described in the publications. they may be used in connection with the presently described invention. The publications discussed above and throughout the text are provided only for description before the filing date of the present application. Nothing in the present invention should be considered as an admission that the inventors do not authorize such a description by virtue of the prior art. The following examples are set forth so as to provide those skilled in the art with a complete description and mention of how to make and use the present invention, and are not intended to limit the scope of what is considered to be the invention. Efforts have been made to ensure accuracy with respect to the numbers used (eg quantities, temperature, concentrations, etc.) but some errors and experimental deviations must be allowed. Unless indicated otherwise, the parts are parts by weight, the molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure at or near atmospheric pressure.

EXPERIMENTAL EXAMPLE 1 Characterization of the distribution of STEP and NMDA-R The inventors have shown that STEP is specifically expressed in the brain by quantitative PCR (Figures 1A-1B), in rat tissues by Northern blot (Figure 2) and in the human central nervous system (Figure 3). Schizophrenia is associated with abnormalities in CNS function, and STEP is expressed in regions that are involved in schizophrenia. In situ hybridization shows that STEP is expressed in an interesting pattern in the brain (Figures 4A and 4B), which indicates a connection between STEP and schizophrenia. Schizophrenic brains show abnormalities in numerous brain regions including cortical areas, hippocampus, amygdala and stratum which are connected by glutamatorgic circuits (references in Johnson et al, 1999) and therefore from the data of the inventors, STEP is expressed in abnormal areas in schizophrenia. Quantitative PCR was carried out by standard means. Real-time PCR amplifications with Green SYBR were carried out in an icycler real-time detection system (Bio-Rad Laboratories, Hercules, CA). The reactions were carried out in duplicates in 25 μl of reaction volume with the following PCR conditions: 50 ° C for 2 minutes and 95 ° C for 10 minutes, followed by 45 cycles of 95 ° C for 15 seconds, 60 ° C for 30 seconds followed by 72 ° C for 40 seconds. The primers for Q-PCR were designed using the software Primer 3. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference to normalize the white transcripts and the relative differences were calculated using the PCR efficiencies in accordance with Pfaffl (Pfaffl MW (2001) Nucleic Acids Res. 29 (9): e45). The expression pattern of STEP was also determined by Northern blot. Multiple tissues were obtained for Northern blot of rat and human origin from commercial sources. The membranes were prehybridized in 7% SDS, 0.5 M NaHP04, 1 mM EDTA at 65 ° C for 15 minutes. Using freshly prepared prehybridization solution, the membranes were hybridized with the labeled probe for 18 hours. The hybridized membranes were briefly rinsed in 5% SDS, 40 mM NaHP04, 1 mM EDTA and then washed for 45 minutes at 65 ° C with freshly prepared solution. This wash solution was replaced with 1% SDS, 40 mM NaHP04, 1 mM EDTA and washed twice for 45 minutes at 65 ° C with freshly prepared solution. After washing, the membranes were placed interspersed between self-adhesive plastic and exposed overnight to the Kodak X-OMAT AR film with a Dupont Lightening Plus intensifier screen at -70 ° C. The results are shown in Figure 2 for rat tissues and in Figure 3 for human tissues. The size of STEP's predominant mRNA is 3kb. In situ hybridization was carried out by standard methods. The animals included in the in situ hybridization experiment were sacrificed by decapitation. The brains were removed and placed in a plastic form with the embedment material and frozen on a mixture of dry ice and ethanol. The frozen blocks were stored at -80 ° C before being sectioned. Coronal sections of rat brain were sectioned at 14.5 μm thick in a Microm cryostat at -17 ° C and mounted by thawing on positively charged slides and dried at room temperature for 10 minutes before storage in a freezer. -80 ° C. The prehybridization of the slides was initiated by fixation in paraformaldehyde cooled with 4% ice for 10 minutes followed by a 5 minute rinse in saline with pH regulated with 1X phosphate cooled with ice 0.1 mol / L (PBS pH 7.2) . Then the sections were processed as follows: they were washed for 1 minute in TEA 0.1 mole / L and for 10 minutes in 0.25% acetic anhydride \ TEA. They were rinsed 2 times in 1X SSC and dehydrated in 70% (two minutes), 95% (two minutes) and 100% (two minutes) of ethanol. Finally the sections were incubated for 5 minutes in 100% chloroform followed by 2 minutes incubation in 95% ethanol. The slides were finally air dried for 10 minutes before hybridization. Generation and hybridization of the probe: A linear DNA was generated with the SP6 and T7 transcription sites using PCR amplification. 1 μg of the PCR fragment with SP6 and T7 was used as a template for transcription in vitro. UTP [a-33P] (NEN) was used to generate a sense and antisense hot riboprobe by in vitro transcription using the T7 and SP6 polymerases. The sections were then assayed with a probe with 200 μl of cocktail for hybridization with 105 cpm of specific activity, covered with coverslips and placed in a humidification chamber for 18 hours at 55 ° C. The cocktail for hybridization in addition to the pre-labeled probe consisted of 50% formamide, standard saline 0.3 mol / L, 1X Denhardt's solution, DTT 0.01 mol / L, Tris 0.01 mol / L, 10% dextran sulfate and EDTA 0.001 moles / L. For each hot probe a cold probe was also generated to evaluate the riboprobe specificity, competition experiments were carried out by adding an unlabeled probe to 100 times the concentration of the labeled probe which prevents the binding of the probe. STEP probe. Also, when the STEP warm sensing probe was evaluated no specific binding to the tissue was detected. After hybridization overnight, the sections were rinsed in 1X SSC at room temperature. The sections were then treated for 30 minutes with RNAase A (10 g / L) in pH regulator for RNase consisting of 0.01 mol / L Tris (pH 8.0), 0.5 mol / L NaCl and 0.001 mol / L EDTA at 37 ° C. C. Treatment with RNase A was followed by 30 minutes of rinsing in pH buffer for RNase at 37 ° C, 15 minutes 1X SSC at room temperature and finally 0.5X SSC at 65 ° C for 30 minutes. After the last wash in 0.5X SSC, the slides were dehydrated in 70% (2 minutes), 95% (2 minutes) and 100% (2 minutes) of ethanol and finally air dried for 10 minutes and exposed in the phosphoimager screens (Cyclone) for 5-7 days at room temperature. After 7 days of exposure the phosphoimager screens were recorded. The images obtained are presented in Figures 4A and 4B, STEP is highly expressed in the stratum and hippocampus and at appreciable levels in other brain regions including the cortex and the thalamus.

EXAMPLE 2 Characterization of the effects of STEP on the function of NMDA-R in a heterologous expression system The hypoactivity of the NMDA receptor has been associated with schizophrenia (Coyle et al., 2002) and NMDA receptor antagonists can exacerbate schizophrenic symptoms (Lahti et al, 1995). The inventors have found that STEP reduces the function of the NMDAR by its effects on the influx of Ca influx through the NMDARs in the transfected HEK293 cells stably expressing NMDARs (Figure 5). Cell lines stably expressing the NR1 subunit under the control of a tetracycline-inducible element and the constitutive NR2B subunit were used. These cell lines were transiently transfected with one of the following constructs using Fugene: - STEP61 - STEP61 (CS) - a catalytically inactive form of STEP61 in which the critical residue for the phosphatase activity, cysteine-300, was mutated to a serine. STEP46-STEP46 (CS) -a catalytically inactive form of STEP46 in which the critical residue for the phosphatase activity, cistern 172, was mutated to a serine.

One day after transfection the cells were transferred to a dark-walled, dark-walled, 96-well assay plate and induced to expression of the NMDA receptors by the addition of tetracycline. A day later, the function of the receptors in the presence of STEP constructions was evaluated. The cells were washed with assay pH regulator (saline solution with regulated Hepes pH supplemented with 5 mM HEPES, 10 μM glycine and 1 mM calcium chloride) and loaded with a fluo-3 derivative in pH regulator for assay by 1 hour at 37 ° C. The assay plate was transferred to a Molecular Devices FLEXstation, a registration fluorometer coupled with a fluid transfer system that allows the measurement of rapid, real-time changes of presence in response to the application of the compounds. Baseline measurements of fluorescence were obtained by taking the baseline reading every 1.5 seconds for 30 seconds. The glutamate was added to a final concentration of 1 μM and the fluorescence readings were taken every 1.5 seconds for an additional 2 minutes. At this time NP40 was added to a final concentration of 1% and the readings were taken for an additional 30 seconds. The peak response to was measured and divided by the peak response to NP40 to evaluate the normalized influx of glutamate-induced calcium within the cells for each construct. The comparison of the different constructs indicates that the inactive mutants show a lower function of the NMDA receptor by measuring the calcium flux that activates the STEP forms (figure 5).

STEP, and STEP-61 interacts with NMDA receptors even in the absence of other synaptic proteins, as shown in Figures 10A and 10B. Cell lines stably expressing the NR1 subunit under the control of a tetracycline-inducible element and the constitutive NR2B subunit were used. These cell lines were further stably infected with STEP using lentivirus-mediated gene administration. Stably transfected cell lines expressing the NR1, NR2B and STEP constructs were isolated and confirmed by immunostaining and Western blot. The NR1 / NR2B / STEP cell lines were seeded onto dishes for cell culture and expression of the NMDA receptors was induced by the addition of tetracycline. One day after the cells were harvested for immunoprecipitation experiments. Immunoprecipitation: the cells were harvested, the medium was moved after centrifugation, the cells were resuspended in pH buffer for lysis (150 mM NaCl, 50 mM Tris, pH 7.6, 1% triton). 200 μg of lysate (1 μg / μl) were incubated with 5-10 μg of primary antibody, overnight at 4 ° C, with shaking. After incubation of the antibodies, 100 μl of a slurry of A / G agarose protein (Amersham) was added, the incubation was continued for another hour. To determine the immunoprecipitated proteins, the material bound to the AG agarose protein was separated by concentration of the beads with the bound immunocomplex by centrifugation, washed with PBS and resolved by SDS-PAGE. Resolved proteins in the gel were transferred to membrane to verify the presence of co-immunoprecipitated proteins by Western blot using specific antibodies. The anti-NR1 antibody and an anti-STEP monoclonal antibody (Novus Biologicals Clone # 23E5, Cat # NB300-202) were used as probes (Figures 10A and 10B). The data shows that NR1 co-precipitates with STEP. The co-immunoprecipitation experiments were carried out to additionally identify the specificity of the subunit of the physical interaction between NMDA-R and STEP. HEK-293 cells were transfected with constructs for the expression of any STEP-46, STEP-61, NR1, NR2A or NR2B or a combination thereof using Fugene. Two days after transfection the cells were harvested and used for immunoprecipitation. The cells were harvested, the medium was removed after centrifugation and the cells were suspended in pH buffer for lysis (150 mM NaCl, 50 mM Tris pH 7.6, 1% Triton). 2000 μg of the lysate (1 μg / μl) was incubated with 1-3 μg of primary antibody, overnight at 4 ° C, with shaking. Immunoprecipitation was carried out using an appropriate antibody for each transfected NMDA subunit and the interaction with the NMDA subunits. Following the incubation of the antibodies, 100 μl of a slurry of A / G agarose protein (Amersham) was added, and the incubation was continued for another hour. To determine the immunoprecipitated proteins, the material bound to the AG agarose protein was separated by precipitation of the beads with the bound immunocomplex by centrifugation, washed with PBS and resolved with SDS-PAGE. Resolved proteins in the gel were transferred to the membrane to verify the presence of co-immunoprecipitated proteins by Western blot using the anti-STEP antibody. The data shows that NR1, NR2A and NR2B co-precipitate with STEP (Figures 11A-11F and Figures 12A-12F). Both STEP61 (Figures 11A-11F) and STEP 46 (Figures 12A-12F) are capable of interacting with NMDAR. Therefore both main forms of STEP that are expressed in the brain are able to interact with and modulate the function of NMDAR. In addition both STEP46 and STEP61 are able to interact with subunits NR1, NR2A and NR2B. Therefore STEP is able to interact with all forms of NMDAR present in the adult brain. The importance of this is that STEP acts universally in all brain regions and over all NMDA receptors in the brain and can influence the function of all NMDA receptors.

EXAMPLE 3 Characterization of the effects of STEP on the function of NMDA-R in cultured cortical neurons The use of RNAi to reduce STEP levels in cultured cortical neurons occasioned an increase in the influx of Ca mediated by the NMDA receptor within the neurons (Figure 6). This suggests that STEP actively causes a decrease in NMDAR function in neurons, which could lead to hypoactivity of the NMDAR and thus to schizophrenia. The single-stranded intervening RNA (RNAi) molecules were designed to be complementary to the STEP sequence by standard means. The sequence used was 5 'AAA CAU GCG AAC AGU AUC AGU 3'. A standard mixed RNA molecule of the sequence 5'-CAG TCG CGT TTG CGA CTG G-3 'was used as a control. Dissociated cortical neurons were prepared from E18 rat embryos by standard protocols. Dissociated neurons were mixed with 90 ul of rat Nucleofector solution to produce a final concentration of 4.8 x 10 6 cells / 90 μl. The cells were mixed with 20 μg of RNAi and transferred to a cuvette for electroporation. Using an AMAXA Nucleofector the cells were electroporated using standard measurements. Cells were transferred from the electroporation cuvette to 96-well plates coated with Poly-D-lysine for calcium flux assays or 6-well plates for Western blot procedures and growth in standard neuronal medium at 37 ° C with 5% of C02 for 4 days. The elimination of the endogenous levels of STEP protein was visualized by Western blot. Four days after electroporation the cells were harvested and used with pH regulator for lysis (150 mM NaCl, 50 mM Tris pH 7.6, 1% triton, in the presence of a protease inhibitor cocktail and 1 mM sodium orthovanadate ) on ice. The protein samples were separated by SDS-polyacrylamide gel electrophoresis and the proteins were transferred to nitrocellulose membranes. STEP protein levels were determined by Western blot using anti-STEP antibodies (Figure 6). The functional experiments were carried out using the FLEXstation of Molecular Devices as previously described. To work with neuronal cultures, the pH regulators were supplemented with 1 uM tetrodotoxin and 100 nM nifedipine and the specific activation of the NMDA receptors was achieved by applying 1 μM NMDA instead of glutamate. The analysis of the influence of calcium mediated by NMDA indicates that when STEP protein levels are reduced by interfering RNA, there is an increased calcium influx mediated by NMDA in cultured cortical neurons (figure 6).

EXAMPLE 4 Characterization of STEP effects on ERK phosphorylation The HEK293 cell lines stably expressing the NMDA receptor (as previously described) were transfected with the STEP61, STEP61 CS, STEP46 or STEP46CS constructs by standard means and grown in 6-well plates for two days. The cells were washed with PBS and then treated in the absence or presence of 50 ng / ml of EGF (in PBS) for 15 minutes. The cells were then harvested on ice in pH buffer for lysis (150 mM NaCl, 50 mM Tris pH 7.6, 1% Triton, in the presence of a protease inhibitor cocktail and 1 mM sodium orthovanadate) and used for 1 hour with stirring at 4 ° C. The solubilized proteins were separated by centrifugation and resolved by SDS-polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose membranes and Western blotting was carried out using antibodies that specifically recognize the phosphorylated form or ERK (Biosource). To ensure that the samples were loaded equally, the antibodies were removed from the membranes using pH regulator for elimination (β-mercaptoethanol 100 M, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) at 55 ° C for 30 minutes and the membranes were retested with an antibody that recognizes the total levels of ERK. This shows that there is much less phosphorylation of ERK in the presence of active forms of STEP compared to the forms that have a mutation to alter their catalytic activity (figure 7). The phosphorylation of ERK in cultured cortical neurons ^ (10-13 divisions) can be produced by the activation of NMDARs. Neurons show low levels of basal phosphorylation of ERK. After stimulation for 5 minutes with NMDA (100 μM), significant phosphorylation of ERK was observed. This effect is blocked by incubation with the competitive antagonist of the acid NMDA receptor D-2-amino-5-phosphonopentanoic acid (D-APV). Therefore in the neurons a main route that leads to the phosphorylation of ERK via activation of the NMDARs. Cortical neurons cultured 10 to 13 days in vitro were infected with Sindbis virus containing RNA encoding STEP61, STEP61CS (a catalytically inactive form of STEP61 in which the critical residue for phosphatase activity, cysteine-300, was mutated to a serine) or GFP (control). 150 ul of Sindbis virus viruses expressing GFP, STEP-61 or STEP61 (CS) were used to infect a 10 cm Petri dish with neurons. Sindbis virus infection was allowed to proceed for 18 hours before stimulation and harvest. 18 hours after infection the neurons were washed with PBS and then treated with 100 μM glutamate for 5 minutes. Then the cells were harvested, used and the proteins were separated and the phospho-ERK levels were detected by western blot. This demonstrates that there is much less phosphorylation of ERK in the presence of active forms of STEP compared to the forms that have a mutation to alter their catalytic activity (Figures 8A-8B).

EXAMPLE 5 Characterization of STEP effects on phosphorylation of protein tyrosine kinase The HEK293 cell lines that stably express the NMDA receptor (as previously described) were transfected with a mutated form of src [Src (KP)] or Fyn [Fyn (Y531F) J in the presence or absence of varying concentrations of STEP61 by standard means and growth in 6-well plates for two days. The cells were then harvested on ice in pH buffer for lysis (150 mM NaCl, 50 mM Tris pH 7.6, 1% triton, in the presence of a protease inhibitor cocktail and 1 mM sodium orthovanadate) and used by 1 hour with stirring at 4 ° C. The solubilized proteins were separated by centrifugation and resolved by SDS-polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose membranes and Western blotting was carried out using antibodies that specifically recognize the phosphorylated form or Src at the tyrosine residue 418 (which also recognizes phosphorylated Fyn at residue Y420). To ensure that the samples were similarly loaded, the antibodies were removed from the membranes using pH regulator for elimination (100 M β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) at 55 ° C 30 minutes and the membranes were retested with an antibody that recognizes the total levels of src or fyn. This shows that there is a dephosphorylation of src and fyn dependent on the concentration at this critical site in the presence of STEP61 (Figures 9A-9B).

EXAMPLE 6 Selection for agents that modulate NMDA-R signaling Expression and purification of STEP. A 1.1 Kb DNA fragment encoding hSTEP46 (residues E2 through E369) preceded by the HHHHHH tag was subcloned into the vector pET-17b (Novagen) between the Ndel and Hindlll sites. The resulting pyramid was transformed into both BL21 (DE3) (Invitrogen) and Tuner (DE3) cells (Novagen), which were used for large-scale expression of hSTEP46. Cells were grown in LB medium at 37 ° C and induced an A6oo = 0.6-1.0 with 0.1 mM IPTG for 6 hours before harvest. Cell paste is sonic in pH buffer for lysis composed of 50 mM HEPES, pH 8.0, 0.3 M NaCl, 1 mM PMSF, 1 mM mercaptoethanol, and 0.1% Triton X-100. The cell lysate was centrifuged at 27,000 x g for 20 minutes, and the supernatant was loaded onto a column of Ni2 + -NTA (Qiagen) equilibrated with 10 mM imidazole, 0.3 M NaCI, 50 mM HEPES, pH buffer at pH 8.0. The column was washed with the same pH regulator, and the protein was eluted with 250 mM imidazole, 0.3 M NaCl, 50 mM HEPES, pH regulator at pH 8.0. The eluate from the Ni2 + -NTA column was adjusted to 1 M ammonium sulfate and subjected to chromatography on a Macro-Prep methyl HIC column (BioRad). The protein was eluted with 0.5 M ammonium sulfate and the pH regulator was changed to 50 mM HEPES, pH regulator at pH 7.5. The protein obtained from two chromatographies was at least 95% pure by Coomassie staining.

Test development Numerous in vitro tests are used to evaluate the activity of STEP and subsequently to select compounds that modulate their function. An example is TR-FRET, but for those skilled in the art alternative phosphatase activity assays will be evident.

TR-FRET test Material: pH regulator with HEPES phosphatase 50 mm, pH 8; 1 mM DDT; 2 mM EDTA; 0.01% Brij solution; 10 mM MgCl2 pH regulator for detection: 25 mM Tris, pH 7.5 + 0.2% Tritium 100; 0.5 μl of Eu PY20 Ab; 1.5 μl streptavidin-APC per 5 ml of pH regulator substrate for detection: AGY 1336. enzyme: STEP. Sodium orthovanadate. DMSO (CLAR grade). Plates with the compound: the plates with the compound were thawed overnight at room temperature.

Method: The storage solution of the enzyme is elaborated by the addition of 24.4 μl of STEP storage solution at 100 ml pH regulator with phosphatase. The substrate storage solution is made by the addition of 2 μl of AGY-1336 (at 5 mM) to 100 ml of pH regulator with phosphatase. The storage solution for the control inhibitor is made by the addition of 90 ul of sodium orthovanadate (100 μM) to 30 ml of pH regulator with phosphatase. The solution for storage of the reagent for detection is elaborated by the addition of 15 μL of Eu-anti-phosphotyrosine antibody + 45 μL of APC to 150 ml of the pH regulator for detection. This produces the initial concentrations of: enzyme: 10 μM; Substrate: 100 nM; vanadate: 300 nM. The reagents from the control wells were dispensed by the robots Biomek 2000 (B2K) and Biomek FX. The B2K controls are dispensed in six test plates. 12.5 μl of enzyme, 2.5 μl of DMSO, and 10 μl of pH regulators are placed inside columns 1 and 2, rows A to H. A substrate volume of 12.5 μl, 2.5 μl of DMSO, and 10 μl of regulators of pH are placed inside columns 1 and 2, rows I to P. Column 23, row A to P will contain 5.0 μl of orthovanadate solution. Column 24 is left empty.

For the assay of enzymatic activity, 2.5 ul of the compound, 12.5 μl of enzyme, and 10 μl of the substrate (separated by air spaces) are added to columns 3 to 24 by the Biomek FX for application. After application, the tips were washed with DMSO and water for re-use between each quadrant. Once the test plates have been established, they are incubated at 27 ° C for 45 minutes. Subsequently, 20 μl of pH regulators were added for detection to stop the reaction and to allow the Europium antibody (Eu-Ab) and the streptavidin-APC to bind to the substrate. Subsequently the plates are placed in the plate reader, a Analyst HT. The excitation light at 360 nm is used to excite the Europium antibody with an emission at 620 nm. The fluorescence resonance energy (FRET) transfer from Eu-Ab to APC will only occur when they are in close proximity. Therefore, when an APC emission is observed at 665 nm the enzyme has been inhibited for the removal of the phosphate group from the substrate. The FRET test is resolved in time (TR), where there is a delay between the excitation light and the collection of emission signals. This reduces the amount of straight light created by short-lived fluorescent molecules. The Analyst HT measures the emission signals of APC and Europium and calculates the relationship between the two intensities. Typical intensities for Europiumis -2000 and APC is -600. The specificity of inhibition is evaluated using a broad panel of phosphatase to determine the inhibition of phosphatases other than STEP. Once the records are identified as specific to STEP, the inhibitor is evaluated in secondary assays as described below, for example HEK293 cells expressing the NR1 / NR2A and NR1 / NR2B subunits. The functional characterization of the active compounds is carried out in primary neurons of the hippocampus by electrophysiology. In vivo validation of STEP inhibitors uses behavioral tests in mouse or rat animal models. Design of profiling trials. The development of cell-based secondary assays is used in the profiling of compounds. The key parameters of the increased NMDAR activity were evaluated including increased NR2 phosphorylation; increased NMDAR current; increased permeability Ca2 +. The transient expression of the glutamate subunit of the receptors is used in HEK293 cells. The phosphorylation status of the NR2 subunits was determined by endogenous kinases in HEK293 cells, and evaluated for an effect on NMDA receptor activity. The profiling assays include transient expression of the binary receptor channels NR1 / NR2B and NR1 / NR2A in the presence and absence of the glutamate agonist. Stable cell lines can also be used. Glutamate, by activating the NMDA receptor channels, also leads to increased phosphorylation (only in the presence of activated PTK) of the NR2 subunits and therefore to increase the current and permeability to Ca2 +. The specifically identified compounds will inhibit STEP and lead to increased phosphorylation of NR2 and Ca2 + flux after activation of NMDAR with glutamate. The functionality of NMDA receptors and their modulation is evaluated initially using calcium flow measurements. The different calcium indicator dyes were evaluated. For profiling assays, primary neurons of the hippocampus or cortical neurons are used in a non-infected or infected manner with either the Sindbis or Lentivirus constructs that express STEP, STEPCS and a GFP control. Organotypic cultures are also used. Currents induced by NMDA or L-glutamate are selectively recorded in the presence / absence of identified compounds. In order to measure the NMDA currents, the cells are fixed with the pipette for voltage fixation and the characteristic NMDA-R currents are recorded at different membrane potentials (Kohr & amp;; Seeburg, J. Physiol (London) 492: 445-452, 1996). The function of the NMDA neuronal receptor is measured using either electrophysiology or the FLEX station, for example by measuring the influx of Ca2 +. A calcium imaging experiment is carried out as follows. The measurements are performed in the presence / absence of compounds in a primary neuronal cell expressing NMDA-R subunits as described above by measuring the use of a FLEX FLIPR station or Ca2 + imaging (see Renard, S. et al., Eur. J. Physicology 366: 319-328 (1999)). The FLEX station in combination with the calcium indicator dyes is used to measure the activity of the NMDA receptor.

Similar to the experiments in HEK293, it is expected to observe a decrease in the NMDAR current in neurons infected with the virus STEP wt. The compounds could restore NMDAR function / activity by inhibiting STEP. The STEP mutant (cs) serves as a control. The additional assays utilize the additional role of STEP in the dephosphorylation of ERK and the protein tyrosine kinases as previously described. The assays are carried out using Western blotting or ELISA techniques to evaluate the effects of the compounds on the phosphorylation status of these proteins which are substrates of STEPs either in heterologous expression systems or in neuronal preparations.

EXAMPLE 7 Inhibition by prepulse Schizophrenia is a chronic and debilitating syndrome, which is usually associated with a wide range of cognitive and emotional disturbances. A preclinical study for the disease is the paradigm of prepulse inhibition. Prepulse inhibition (PPI) refers to the inhibition of an alarm reflex that occurs when an intense alarm stimulus (acoustic or tactile) is preceded by a barely detectable prepulse. PPI provides an operational measurement of sensorimotor activation and may reflect the ability to evaluate exteroceptive stimuli for physiological or cognitive relevance. Several clinical studies have shown that schizophrenic patients have poor PPI and habituation to alarm (SH). Habituation is seen as the simple form of non-associative learning and reflects a decrease in the response of the repeated presentation of an initially novel exteroceptive stimulus. Common neuropathological mechanisms have been proposed to underlie the clinical signs and reduced PPI and habituation in schizophrenic patients. As shown by numerous studies, reliable alarm and PPI reflex can be obtained in mice using stimulus parameters almost identical to those used in rats. More importantly, the genetic differences marked in PPI are also reported through the strains of mice, with strain C57BL / 6J showing a low PPI. Therefore, in the present study we evaluate whether different doses of antipsychotics could improve PPI in mice that show poor sensorimotor activation.

Methods Animals. Adult mice of the following strains were used: C57BL / 6J. The animals that weighed between 20 and 24 g were thrown four per cage with water and food ad libitum. They let acclimatize for 1 week before the evaluation.

Instruments. The outstanding evaluation in startle devices (SRLAB, San Diego Instruments, San Diego, Calif., USA) each consisting of a 5.1cm Plexiglas cylinder (external diameter) mounted on a Plexiglas platform in a ventilated cubicle, with attenuated sound with a high speaker frequency (28 cm above the cylinder) producing acoustic stimuli. The background noise for each camera is 70 dB. The movements inside the cylinder that are detected and transduced by a piezoelectric accelerometer attached to the Plexiglas base, digitized and stored in a computer. Beginning with the stimuli, 65 readings of 1 ms of duration are recorded to obtain the amplitude of the animal's alarm reflex. Drugs STEP inhibitors are evaluated in comparison with the vehicle, and with clozapine as a positive control. Inhibition by prepulse. Twelve mice without affection were evaluated. Each session starts with a 5-minute acclimation period followed by five successive 110 dB trials. These trials are not included in the analysis. Subsequently, six different types of tests are presented: alarm pulse (ST110, 110 dB / 40 ms), low prepulse stimulus given only (P74.74 dB / 20 ms), high prepulse stimulus given alone (P90, 90 dB / 20 ms), P74 or P90 given 100 ms before the start of the alarm pulse (PP74 and PP90, respectively), and finally an assay in which only the background noise is present (NST) in order to measure the movement of the baseline in the cylinders. All tests are applied 10 times and are presented in random order (P74 and P90 where only 5 times are provided) and the average inter-assay interval (ITI) was 15 s (10-20 s). Habituation of alarm. Twelve mice without affection were used in this experiment. Following a 5 minute acclimation period, a defined number of 110 dB tests is presented during a 45-minute test session. The interassay interval varied randomly from 10 to 20 s, with an average of 15 s. the data from the first trial were analyzed separately, because the alarm responses from the first presentation stimulus are considered to reflect the initial reactivity to a single event. The remaining trials are grouped into blocks of ten trials each. The amount of habituation (percentage of habituation) is calculated by the following equation: 100 - [(average alarm amplitude for block 1- average alarm amplitude for block 11)] / average alarm altitude for block 1. A High percentage value reflects a high degree of habituation. Effects of antipsychotics on PPI in C57BL / 6J mice. Separate groups of animals received an injection of clozapine (0.3, 1, 3 and 30 mg / kg) or STEP modulating antagonist (0, 1, 0.3 and 1 mg / kg) and were evaluated for 30 minutes, using the procedures mentioned above. Statistic analysis. The data analysis was carried out with one or two entity ANOVA analysis followed by Duncan's test for post-hoc comparisons as long as the ANOVAs will indicate main effects or statistically significant interactions. The alarm reflex and% PPI are analyzed with two-way ANOVA analysis with the strain (or drug dose), as the factor between the subject and the stimuli as the repeated measurement. The analysis of the habituation of alarm on the session is carried out using two-way ANOVA with strains as the factor between subjects and blocks as repeated measurements (11 levels). The percentage of alarm habituation is analyzed with one-way ANOVA with the strain as a factor among the subjects.

Amphetamine-induced hyperactivity Hyperactivity induced by d-Amfetamine: C57BL / 6J mice were used, aged 5-6 weeks. Interactivity was induced by administration s. c. of d-amphetamine sulfate, at a dose of 4 mg / kgy1, 30 minutes before the test. Clozapine or STEP modulator plus vehicle was administered i. p / icv. 30 minutes before the d-amphetamine. For the evaluation, each mouse was placed in an open field cage and locomotion activity and stereotyped behavior was recorded for 10 minutes. The minimum active dose, defined as the lowest dose that significantly inhibits the hyperactivity induced by d-amphetamine, is calculated using the 2-tailed Mann-Whitney U test.

Claims (19)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for the identification of a therapeutic agent for the treatment of schizophrenia, the method comprising: detecting the ability of an agent to inhibit the phosphatase activity of a STEP isoform on a substrate or to inhibit the binding of STEP to NMDA-R , thus identifying an inhibitor that is useful as a therapeutic agent, wherein the inhibition of STEP that increases activity by NMDA-R signaling is therapeutic in the treatment of schizophrenia.

2. The method according to claim 1, further characterized in that said agent modulates the dephosphorylation by STEP of a protein kinase in the NMDA-R signaling pathway.

3. The method according to claim 2, further characterized in that said kinase is Src.

4.- The method of compliance with the. claim 2, further characterized in that said kinase is Fyn.

5. The method according to claim 2, further characterized in that said kinase is ERK.

6. The method according to claim 1, further characterized in that the STEP isoform is human. 7

7. - The method according to claim 1, further characterized in that the indicator is identified by detecting its ability to inhibit the phosphatase activity of the STEP isoform.

8. The method according to claim 1, further characterized in that the inhibitor is identified by detecting its ability to inhibit the binding of the STEP isoform to NMDA-R.

9. The method according to claim 1, further characterized in that the inhibitor is identified by detecting its ability to modulate the dephosphorylation of NMDA-R by STEP.

10. The use of a modulator of a STEP activity, which modulates the level of tyrosine phosphorylation of NMDA-R, to prepare a medicament for the treatment of a disease by neurological disorder associated with abnormal NMDA-R signaling.

11. The use claimed in claim 10, wherein said neurological disorder is a psychotic disorder.

12. The use claimed in claim 11, wherein said psychotic disorder is schizophrenia.

13. The use claimed in claim 10, wherein said inhibitor modulates the ability of STEP to dephosphorylate a protein kinase in the NMDA-R signaling pathway.

14. The use claimed in claim 13, wherein said kinase is Src.

15. - The use claimed in claim 13, wherein said kinase is Fyn.

16. The use claimed in claim 13, wherein said kinase is ERK.

17. The use claimed in claim 10, wherein the inhibitor modulates the ability of STEP to dephosphorylate directly or indirectly to NMDA-R.

18. The use claimed in claim 10, wherein the inhibitor modulates the ability of STEP to bind to NMDA-R.

19. The use claimed in claim 10, wherein the neurological disease is selected from the group consisting of stroke; head trauma or brain injury; Huntington's disease; Parkinson's disease; spinocerebellar degeneration; motor neuron diseases; epilepsy; neuropathic pain; chronic pain; tolerance to alcohol; schizophrenia; Alzheimer disease; dementia; psychosis; drug addiction; sensitivity to ethanol, moderate cognitive impairment; and depression.