WO2009042729A1

WO2009042729A1 - Modulation of mglur signaling and methods of use in neurological and psychiatric diseases

Info

Publication number: WO2009042729A1
Application number: PCT/US2008/077584
Authority: WO
Inventors: Paul F. Worley; Sungjin Park; Joomin Park; Chester Gunn Moore; Jia-hua HU
Original assignee: The Johns Hopkins University
Priority date: 2007-09-24
Filing date: 2008-09-24
Publication date: 2009-04-02

Abstract

In accordance with the present invention there are provided methods of modulating the activity of metabotropic glutamate receptors (mGluRs) and/or dopamine receptors involving inhibiting the activity of a prolyl isomerase. Further provided are methods of identifying a test compound that inhibits Pin 1 activity on group 1 metabotropic glutamate receptors (mGluR). Also provided are methods of decreasing dopamine signaling in a subject having enhanced dopamine signaling including administering a prolyl isomerase inhibitor to the subject, wherein the subject has a drug addiction, inflammatory pain, schizophrenia, or a movement disorder. Also provided are methods of treating a disease or disorder related to enhanced dopamine signaling or an mGluR-related disorder. Finally, there are provided transgenic animals having variant mGluRs which exhibit altered Pinl or Homer binding properties.

Description

MODULATION OF mGluR SIGNALING AND METHODS OF USE IN NEUROLOGICAL AND PSYCHIATRIC DISEASES

FIELD OF THE INVENTION

[0001] The present invention relates generally to the modulation of metabotropic glutamate receptor signaling and more specifically, to the involvement of prolyl isomerases in the regulation of metabotropic glutamate receptor signaling.

BACKGROUND OF THE INVENTION

[0002] The metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors capable of activating a variety of intracellular second messenger systems following the binding of agonists such as glutamate. The eight identified mGluRs have been subdivided into three groups based on amino acid sequence identities, the second messenger systems they utilize, and pharmacological characteristics. The Group I mGluRs include mGluRl and mGluR5. Studies of the physiological roles of Group I mGluRs suggest that activation of these receptors elicits neuronal excitation. Other studies examining the physiological roles of mGluRs indicate that metabotropic glutamate receptors, particularly Group I mGluRs, are involved in a number of normal processes in the mammalian CNS, such as nociception and analgesia. In addition. mGluR activation has been suggested to play a modulatory role in a variety of other normal processes including synaptic transmission neuronal development apoptotic neuronal death, synaptic plasticity, spatial learning, olfactory memory, central control of cardiac activity, waking, motor control, and control of the vestibulo-ocular reflex. Metabotropic glutamate receptors also have been suggested to play roles in a variety of pathophysiological processes and disease states affecting the CNS, including stroke, head trauma, anoxic and ischemic injuries, hypoglycemia, epilepsy, and neurodegenerative diseases such as Alzheimer's disease. Much of the pathology in these conditions is thought to be due to excessive glutamate- induced excitation of CNS neurons. Because Group I mGluRs appear to increase glutamate-mediated neuronal excitation via postsynaptic mechanisms and enhanced presynaptic glutamate release, their activation probably contributes to the pathology. Accordingly, selective means of reducing signaling via Group I mGluR receptors could be therapeutically beneficial, specifically as analgesia, as neuroprotective agents or as anticonvulsants. SUMMARY OF THE INVENTION

[0003] The present invention is based on the seminal discovery that group 1 metabotropic glutamate receptors are co-functional with dopamine receptors or growth factor receptors. This discovery includes the finding of regulation of mGluR signaling by the balance between Homer (an adaptor protein that binds the C-terminus of group 1 metabotropic glutamate receptors), and prolyl isomerases.

[0004] Accordingly, there are provided methods of modulating the activity of metabotropic glutamate receptors (mGluRs) involving inhibiting the activity of a prolyl isomerase in a cell expressing a mGluR, thereby reducing the activity of mGluR. In one aspect, the prolyl isomerase is prolyl isomerase 1 (Pin 1). In particular embodiments, the cell expresses a dopamine receptor and the activity of mGluR is decreased, thereby reducing dopamine receptor signaling, and thereby modulating the activity of the dopamine receptor.

[0005] In another embodiment of the present invention, there are provided methods of decreasing dopamine signaling in a subject having enhanced dopamine signaling including administering a prolyl isomerase inhibitor to the subject, wherein the inhibition of prolyl isomerase decreases the activity of mGluR, thereby reducing dopamine receptor signaling. In certain embodiments, the subject has a drug addiction, inflammatory pain, schizophrenia, or a movement disorder, causing enhanced dopamine signaling.

[0006] In another embodiment of the present invention, there are provided methods of identifying a test compound that inhibits Pir 1 activity on group 1 metabotropic glutamate receptors (mGluR). The method includes contacting a cell expressing an mGluR and Pin 1 with a test compound, wherein binding of Pin 1 and mGluR is decreased in the presence of test compound as compared to binding in the absence of test compound. In one aspect, the mGluR receptor has a mutation, wherein the mutated mGluR binds Pin 1 but not Homer.

[0007] In still another embodiment of the present invention, there are provided methods of modulating mGluR sensitization to mGluR agonists, in which the method includes modulating the activity of a prolyl isomerase in a cell expressing a mGluR, thereby modulating the sensitization of mGluR. In one aspect, the activity of the prolyl isomerase is inhibited, thereby reducing the sensitization of mGluR. [0008] In yet another embodiment of the present invention, there are provided methods of modulating the activity of metabotropic glutamate receptors (mGluR), in which the method includes modulating a phosphatase that removes phosphate groups from mGluR, thereby modulating the activity of mGluR. In one aspect, the phosphatase is activated, thereby reducing binding of prolyl isomerase to mGluR. In some embodiments, the phosphorylation of threonine 1123 or serine 1126 of mGluR is reduced.

[0009] In another embodiment of the present invention, there are provided methods of modulating mGluR signaling in a subject having an mGluR-related disorder, in which the method includes administering an agent that modulates the activity of a prolyl isomerase, thereby modulating the activity of mGluR. In certain embodiments, the mGluR-related disorder is selected from the group consisting of Alzheimer's disease, Huntington's Disease, Parkinson's disease, Tourette's syndrome, stroke, epilepsy, sleep or circadian rhythm disorder (e.g., insomnia), schizophrenia, depression, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), drug abuse, and pain.

[0010] In other embodiments of the present invention, there are provided methods of treating a disease or disorder related to enhanced dopamine signaling in a subject in need thereof. The method involves administering a prolyl isomerase inhibitor, wherein the inhibition of the prolyl isomerase decreases the activity of mGluR, thereby reducing dopamine receptor signaling. In one aspect, the prolyl isomerase is prolyl isomerase 1 (Pinl). In particular embodiments, the subject has a drug addiction, inflammatory pain, schizophrenia, or a movement disorder, causing enhanced dopamine signaling.

[0011] In another embodiment of the present invention, there are provided methods of treating an mGluR-related disorder in a subject in need thereof. The method includes administering an agent that modulates the activity of a prolyl isomerase, thereby modulating the activity of mGluR. In particular embodiments, the mGluR-related disorder is selected from the group consisting of Alzheimer's disease, Huntington's Disease, Parkinson's disease, Tourette's syndrome, stroke, epilepsy, sleep or circadian rhythm disorder (e.g., insomnia), schizophrenia, depression, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), drug abuse, pain, and cancer. [0012] Also provided are transgenic non-human animals having a transgene encoding mGluR, wherein the mGluR comprises a mutation and the mutated mGluR binds prolyl isomerase but not Homer. In certain embodiments, the mGluR is mGluRl; in other embodiments, the mGluR is mGluR5. In particular aspects, the mutation is an amino acid substitution of phenylalanine to arginine at position 1128 of mGluR5.

[0013] Further provided are transgenic non-human animals having a transgene encoding mGluR, wherein the mGluR comprises a mutation and the mutated mGluR binds Homer but not prolyl isomerase. In certain embodiments, the mGluR is mGluRl; in other embodiments, the mGluR is mGluR5. In particular aspects, the mutation is a threonine to alanine substitution at position 1123 and a serine to alanine substitution at position 1 126 of mGluR5.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figures 1A-1D show plots depicting the behavioral effects of ***e administered to Homer 1 knockout mice (Figures IA and 1C) and Homer 2 knockout mice (Figures IB and ID).

[0015] Figure 2 shows a plot depicting of a dose response study of ***e administered to wild type and mGluR5 knockout mice.

[0016] Figure 3A shows an alignment of mGluRl and mGluR5 C-terminal regions (SEQ ID NO'S 3 & 4). Figures 3B and 3C show immunoblots of phosphorylated and unphosphorylated mGluR5.

[0017] Figure 4A shows representative tracings of mGluR-evoked slow inward currents in neurons derived from Homer null mice. Figures 4B and 4C show plots depicting the relative baseline current in neurons from Homer triple KO and wild type mice.

[0018] Figures 5A-C show representative tracings (Figure 5A) and relative baseline currents (Figures 5B and 5C) of mGluR-mediated slow currents with application of BDNF in wild type striatal neurons.

[0019] Figures 6A-B show representative tracings (Figure 6A) and plots of relative baseline currents (Figure 6B) of mGluR5 -mediated slow currents in Homer triple KO striatal neurons in the presence of a Pin 1 inhibitor. Figure 6C shows a plot of relative baseline currents in the presence of a Pinl inhibitor or control compound.

[0020] Figures 7A-B show representative tracings (Figure 7A) and plots of relative baseline currents (Figure 7B) of mGluR5 -mediated slow currents in Pinl KO striatal neurons in the presence BDNF. Figure 7C shows a plot of relative baseline current in Pin KO in the presence and absence of Homer Ia.

[0021] Figure 8 shows representative tracings (Figures 8A and B) and plots of relative baseline currents (Figure 8C and D) of mGkiR5-mediated slow currents in mGluR5(TS) striatal neurons in the presence BDNF.

[0022] Figure 9 shows immunoblots depicting the results of binding studies between mGluR(F/R) mutant and Homer and Pinl.

[0023] Figure 10 shows representative tracings (Figures 10A) and plots of relative baseline currents (Figures 1OB and C) of mGluR5 -mediated slow currents in mGluR5(F/R) striatal neurons in the presence BDNF.

[0024] Figure 11 shows plots comparing locomotor activity of wild type, mGluR5(TS) KI, and heterozygous mice treated with ***e.

[0025] Figure 12 shows immunoblots depicting the results of binding studies between mGluR5 and Pinl in the presence of Homeric and Homer Ia.

[0026] Figure 13 shows plots depicting the effect of mGluR5 antagonists on inflammatory pain.

[0027] Figure 14 shows plots depicting formalin-induced inflammatory pain in wild type and mGluR5(F/R) KI mice.

[0028] Figure 15 shows plots depicting formalin-induced inflammatory pain in wild type and mGluR5(TS) KI mice.

[0029] Figures 16A and B show representative tracings (Figure 16A) and plots of relative baseline (Figure 16B) currents induced in wild type striatal neurons. Figure 16C shows the HIa mRNA level expressed under various conditions. |0030] Figure 17 shows a schematic of a mGluR interacting with Homer and Pin 1.

[0031] Figure 18A shows the amino acid sequence of an exemplary human Pinl (GenBank Accession No. NP_006212) and Figure 18B shows the corresponding nucleotide sequence (GenBank Accession No. NM_006221).

DETAILED DESCRIPTION OF THE INVENTION

[0032] As provided herein, the present invention is based on the seminal discovery that group 1 metabotropic glutamate receptors are co-functional with dopamine receptors or growth factor receptors. This convergent signaling is central to understanding several diseases including drug addiction, inflammatory pain, schizophrenia and movement disorders. Several lines of evidence provided herein indicate that mGluR signaling is regulated by the balance between Homer (an adaptor protein that binds the C-terminus of group 1 metabotropic glutamate receptors), and prolyl isomerases. The reported discoveries target a specific enzyme and provide technologies to validate screens of compounds that will have therapeutic actions in neurological and psychiatric diseases. Examples of screens that are enabled include small molecules that inhibit the enzyme or screens that maintain Homer binding in the presence of elevated concentrations of enzyme. The discovery has been validated in animal models that express mutant mGluR5s that can differentially bind Homer or the enzyme, and that show differential sensitivity to addictive properties of ***e, inflammatory pain and schizophrenia phenotypes.

[0033] Accordingly, there are provided methods of modulating the activity of metabotropic glutamate receptors (mGluRs) involving inhibiting the activity of a prolyl isomerase in a cell expressing a mGluR, thereby reducing the activity of mGluR. In particular embodiments, the cell expresses a dopamine receptor and the activity of mGluR is decreased, thereby reducing dopamine receptor signaling, and thereby modulating the activity of the dopamine receptor.

[0034] Thus far, eight distinct mGluR subtypes have been isolated via molecular cloning, and named mGluRl to mGluR8 according to the order in which they were discovered. Further diversity occurs through the expression of alternatively spliced forms of certain mGluR subtypes. All of the mGluRs are structurally similar as membrane proteins possessing a large amino-terminal extracellular domain (ECD), followed by seven putative membrane- spanning helices connected by three intracellular and three extracellular loops, and an intracellular carboxy-terminal domain of variable length.

[0035] The eight mGluRs have been subdivided into three groups (groups I, II, and III) based on amino acid sequence identities, the second messenger systems they utilize, and pharmacological characteristics. The amino acid identity between mGluRs within a given group is approximately 70% but drops to about 40% between mGluRs in different groups. For mGluRs in the same group, this relatedness is roughly paralleled by similarities in signal transduction mechanisms and pharmacological characteristics.

[0036] The Group I rnGluRs comprise mGluRl, mGluR5 and their alternatively spliced variants. The binding of agonists to these receptors results in the activation of phospholipase C and the subsequent mobilization of intracellular calcium. Group 1 mGluRs are recognized to be drug targets for treatment of drug addiction, pain, Alzheimer's disease, Parkinson's disease, and schizophrenia, for example. Because of the availability of potent agonists, antagonists and allosteric modulators, relatively more is known about their therapeutic potential than their mechanism of action. This information gap limits the development of next-generation agents.

[0037] One striking property of group 1 mGluRs is that they display prominent physiological interactions with dopamine receptors. For example, mice that lack mGluR5 fail to show behavioral responses to ***e, even at very high doses, and mGluR 5 antagonists (inverse agonists) block ***e-evoked locomotor activity and self-administration (related to addiction) in rodents and monkeys. From disease models, it has been shown that mGluR5 antagonists block development of L-dopa induced dystonia in a rat model of Parkinson's disease.

[0038] As used herein, the term "mGluR-related disorder" includes, but is not limited to, Alzheimer's disease, Huntington's Disease, Parkinson's disease, Tourette's syndrome, stroke, epilepsy, sleep or circadian rhythm disorder (e.g., insomnia), schizophrenia, depression, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), drug abuse, and pain. An mGluR-related disorder also includes, but is not be limited to, a disease (e.g., Parkinson's disease) or a condition (e.g., addiction to ***e) that involves an aberration or dysregulation of a signal transmission pathway, including, but not limited to, neurotransmission mediated by metabotropic glutamate receptors in excitable cells, tissues or organs (e.g., neurons, brain, central nervous system, etc.). A mGluR-related disorder also includes, but is not limited to, a symptom of a mGluR-related disorder.

[0039| Described herein are regulation pathways that mediate convergent signaling with the dopamine receptor. Animal models that target these mGluR modulatory pathways display phenotypes of altered responses to ***e, as well as neuropsychiatric phenotypes that closely mimic schizophrenia. Importantly, as shown herein, dopamine-dependent adaptations of mGluR signaling are required for behavioral changes linked to elevated dopamine signaling, and that crosstalk between the mGluR and dopamine receptor requires the activity of a prolyl isomerase. The present work suggests that this enzyme is a relevant target for development of therapies to prevent cognitive (schizophrenia) and motor (Parkinson's disease and dystonia) effects of enhanced dopamine signaling. Ongoing studies have validated this target in biochemical, electrophysiological, pharmacological (peptidergic antagonists) and genetic mouse models. In one striking example, a mouse in which mGluR5 was replaced with a mGluR5 point mutant (knockin) that is not a substrate for Pin 1 displays acute locomotor responses to ***e, but not the chronic rewarding effects (i.e., absent ***e sensitization). Animal models also support a role for this transduction pathway in inflammatory pain.

[0040] Dopamine is a hormone and neurotransmitter occurring in a wide variety of animals, including both vertebrates and invertebrates. In the brain, dopamine functions as a neurotransmitter, activating the five types of dopamine receptors, Dl, D2, D3, D4 and D5, and their variants. Dopamine is produced in several areas of the brain, including the substantia nigra and the ventral tegmental area. Dopamine is also a neurohormone released by the hypothalamus. Its main function as a hormone is to inhibit the release of prolactin from the anterior lobe of the pituitary. Dopamine has many functions in the brain, including important roles in behavior and cognition, motor activity, motivation and reward, inhibition of prolactin production (involved in lactation), sleep, mood, attention, and learning. Dopaminergic neurons (i.e., neurons whose primary neurotransmitter is dopamine) are present chiefly in the ventral tegmental area (VTA) of the midbrain, substantia nigra pars compacta, and arcuate nucleus of the hypothalamus.

[0041] Prolyl isomerases are a class of enzymes that interconvert the cis and trans isomers of peptide bonds involving the amino acid proline. In certain embodiments, the prolyl isomerase is prolyl isomerase 1 (Pin 1). In one aspect, the Pin 1 has the amino acid sequence set forth in GenBank Accession No. NP_006212:

MADEEKLPPGWEKRMSRSSGRVYYFNHITNASQWERPSGNSSSGGKN GQGEPARVRCSHLLVKHSQSRRPSSWRQEKITRTKEEALELINGYIQKIK SGEEDFESLASQFSDCSSAKARGDLGAFSRGQMQKPFEDASFALRTGE MSGPVFTDSGIHIILRTE (SEQ ID NO: 1).

In certain embodiments, the prolyl isomerase may be a functional fragment of prolyl isomerase, which possesses the enzymatic activity of the full length prolyl isomerase protein.

[0042] Prolyl isomerase inhibitors may be competitive or non-competitive inhibitors. In certain embodiments, the inhibitor is specific for Pinl. In particular embodiments, the Pinl inhibitor is specific for Pinl's action on group 1 mGluRs. An exemplary prolyl isomerase inhibitor is Fmoc-pSer-Psi[(Z)CHC]-Pro-(2)-N-(3)-ethylaminoindole (Zhao and Etzkorn, Bioorg Med Chem Lett 17(23):6615-8, 2007) having the following structure:

Other prolyl isomerase inhibitors are known in the art (see e.g., Wang and Etzkorn, Biopolymers 84(2): 125-46, 2006). Additional prolyl isomerase inhibitors may be identified by methods disclosed herein.

[0043] Group 1 metabotropic glutamate receptors (mGluRl and mGluR5) couple to intracellular calcium pools by a family of proteins, termed Homer, that cross-link the receptor to inositol trisphosphate receptors. Cross-linking-capable forms of Homer, termed long forms, include Homer Ib, Ic, 2, and 3 and are. Short forms of Homer, which cannot self- multimerize, include Homer Ia and a Homer 2 C-terminal deletion. [0044] In another embodiment of the present invention, there are provided methods of identifying a test compound that inhibits Pin 1 activity on group 1 metabotropic glutamate receptors (mGluRs). The method includes contacting a cell expressing an mGluR and Pin 1 with a test compound, wherein binding of Pin 1 and mGluR is decreased in the presence of test compound as compared to binding in the absence of test compound. In one aspect, the mGluR receptor has a mutation, wherein the mutated mGluR binds Pin 1 but not Homer.

[0045] The term "test compound" is used herein to mean any agent that is being examined for ability to inhibit or facilitate activity of an enzyme or binding between two or more molecules in a method of the invention. In some embodiments the test compound is examined for its ability to inhibit the activity or binding of a prolyl isomerase. Although the method generally is used as a screening assay to identify previously unknown molecules that can act as a therapeutic agent, a method of the invention also can be used to confirm that an agent known to have such activity, in fact has the activity, for example, in standardizing the activity of the therapeutic agent. A test compound can be any type of molecule. In certain embodiments, the test compound may be a peptide, a protein, an antibody, a peptidomimetic, a polynucleotide, antisense RNA, RNAi, a small molecule, or a small organic molecule. A test compound is generally a molecule that one wishes to examine for the ability to inhibit activity or binding or act as a therapeutic agent, which is an agent that provides a therapeutic advantage to a subject receiving it. It will be recognized that a method of the invention is readily adaptable to a high throughput format and, therefore, the method is convenient for screening a plurality of test compounds either serially or in parallel. The plurality of test compounds can be, for example, a library of test agents produced by a combinatorial method library of test agents. Methods for preparing a combinatorial library of molecules that can be tested for therapeutic activity are well known in the art and include, for example, methods of making a phage display library of peptides, which can be constrained peptides (see, for example, U.S. Pat. Nos. 5,622,699; 5,206,347; Scott and Smith, Science 249:386-390, 1992; Markland et al., Gene 109:1319, 1991 ; each of which is incorporated herein by reference); a peptide library (U.S. Pat. No. 5,264,563, which is incorporated herein by reference); a peptidomimetic library (Blondelle et al., Trends Anal. Chem. 14:8392, 1995; a nucleic acid library (O'Connell et al., supra, 1996; Tuerk and Gold, supra, 1990; Gold et al., slpra, 1995; each of which is incorporated herein by reference); an oligosaccharide library (York et al., Carb. Res., 285:99128, 1996; Liang et al., Science, 274:1520-1522, 1996; Ding et al., Adv. Expt. Med. Biol., 376:261-269, 1995; each of which is incorporated herein by reference); a lipoprotein library (de Kruif et al., FEBS Lett., 399:232-236, 1996, which is incorporated herein by reference); a glycoprotein or glycolipid library (Karaoglu et al., J. Cell Biol., 130:567-577, 1995, which is incorporated herein by reference); or a chemical library containing, for example, drugs or other pharmaceutical agents (Gordon et al., J. Med. Chem., 37: 1385-1401, 1994; Ecker and Crooke, Bio/Technology, 13:351-360, 1995; each of which is incorporated herein by reference). Accordingly, the present invention also provides a therapeutic agent identified by such a method, for example, a therapeutic agent useful in the treatment of a neurological, movement, or psychiatric disorder.

[0046] In other embodiments of the present invention, there are provided methods of treating a disease or disorder related to enhanced dopamine signaling in a subject in need thereof. The method involves administering a prolyl isomerase inhibitor, wherein the inhibition of the prolyl isomerase decreases the activity of mGluR, thereby reducing dopamine receptor signaling. In one aspect, the prolyl isomerase is prolyl isomerase 1 (Pinl). In particular embodiments, the subject has a drug addiction, inflammatory pain, schizophrenia, or a movement disorder, causing enhanced dopamine signaling.

[0047] In another embodiment of the present invention, there are provided methods of treating an mGluR-related disorder in a subject in need thereof. The method includes administering an agent that modulates the activity of a prolyl isomerase, thereby modulating the activity of mGluR. In particular embodiments, the mGluR-related disorder is selected from the group consisting of Alzheimer's disease, Huntington's Disease, Parkinson's disease, Tourette's syndrome, stroke, epilepsy, sleep or circadian rhythm disorder (e.g., insomnia), schizophrenia, depression, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), drug abuse, and pain.

[0048] The route of administration of the compound will depend, in part, on the chemical structure of the compound and the target tissue. For example, the delivery of a compound to central nervous system (CNS) can be accomplished by administering the compound directly into the CNS or administering it systemically (e.g., by intravenous injection). Intravenous, intranasal, intracerebroventricular, intratheced, intracranial intrapulmonary, or oral administration are commonly used to deliver compounds to the CNS. [0049] The total amount of a compound to be administered in practicing a method of the invention can be administered to a subject as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which multiple doses are administered over a prolonged period of time. The compound can be formulated for oral formulation, such as a tablet, or a solution or suspension form; or can comprise an admixture with an organic or inorganic carrier or excipient suitable for enteral or parenteral applications, and can be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, or other form suitable for use. The carriers, in addition to those disclosed above, can include glucose, lactose, mannose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, medium chain length triglycerides, dextrans, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form. In addition auxiliary, stabilizing, thickening or coloring agents and perfumes can be used, for example a stabilizing dry agent such as triulose (see, for example, U.S. Pat. No. 5,314,695).

[0050] In still another embodiment of the present invention, there are provided methods of modulating mGluR sensitization to mGluR agonists, in which the method includes modulating the activity of a prolyl isomerase in a cell expressing a mGluR, thereby modulating the sensitization of mGluR. In one aspect, the activity of the prolyl isomerase is inhibited, thereby reducing the sensitization of mGluR. Such methods are useful in treatment of drug addiction in subjects addicted to, for example, ***e.

[0051] Signaling through group I mGluRs is regulated, in part, by Homer proteins. Indeed, Homer binding to mGluR can increase some outputs and reduce others (Kammermeier et al. J Neurosci 20(19):7238-45, 2000; Xiao et al. Curr Opin Neurbiol 10(3):370-4, 2000). Further, Homer proteins are naturally down regulated in response to repeated ***e. In particular, the blunting of group I mGluR-induced glutamate release and motor activity after repeated ***e was associated with a reduction in Homer lb/c protein that was selective for the medial nucleus accumbens. These data suggest that repeated ***e produces an enduring inhibition of the neurochemical and behavioral consequences of stimulating mGluRl that is accompanied by changes in the mGluR scaffolding apparatus. (Swanson et al. J Neurosci 21(22)9043-52, 2001). [0052] Knockout of Homer increases the sensitivity of mutant mice to the addictive action of ***e (Szumlinski et al., Neuron 43(3):401-13, 2004). As shown in Figure \, Homerl and Homer2 deletion sensitizes mice to the behavioral effects of ***e. In particular, Homer 1 deletion (Figure IA) and Homer2 deletion (Figure IB) enhanced place conditioning in KO mice relative to wild type controls (n >6 at each dose; data points represent the mean ± SEM difference in time spent in the ***e-paired chamber before and after four conditioning sessions). Moreover, Homer 1 deletion (Figure 1C) and Homer2 deletion (Figure ID) enhanced the capacity of acute ***e to elicit locomotor activation during the first place conditioning session (n > 6).

[0053] In contrast, as shown in Figure 2, reduced ***e sensitivity is observed in mice that lack mGluR5. In particular, mice lacking the mGluR5 gene do not self-administer ***e, and show no increased locomotor activity following ***e treatment, despite showing ***e-induced increases in nucleus accumbens (NAcc) dopamine (DA) levels similar to wild-type (WT) mice. These results demonstrate a significant contribution of mGluS receptors to the behavioral effects of ***e, and suggest that they may be involved in ***e addiction. (Chiamulera, et al., Nat Neurosci 4(9):873-4, 2001.) The opposing phenotypes of the Homer and mGluRi knockout mice suggest that Homer naturally functions to inhibit mGluR5 output that is important for sensitization to ***e.

[0054] Homer 1 knockout mice show behavioral phenotypes that mimic animal models of schizophrenia and are improved by agents that block dopamine receptors (Szumlinski et al., Genes Brain Behav 4(5):273-88, 2005; and Szumlinski et al., Curr Opin Neurobiol 16(3):251-7, 2006). This further supports the notion that Homer is critical for dopamine signaling.

[0055] In yet another embodiment of the present invention, there are provided methods of modulating the activity of metabotropic glutamate receptors (mGluR), in which the method includes modulating a phosphatase that removes phosphate groups from mGluR, thereby modulating the activity of mGluR. In one aspect, the phosphatase is activated, thereby reducing binding of prolyl isomerase to mGluR. In other embodiments the phosphorylation of mGluR may be increased by phosphatase inhibitors (e.g., inhibitors of PPl and PP2A). In some embodiments, the phosphorylation of threonine 1123 or serine 1126 of mGluR is reduced. [0056] Provided herein is the identification of mutant forms of mGluR that selectively lose the ability to bind Pinl or Homer. For example, mGluR5(TS) mutant described herein binds Homer but not Pin. In this mutant no ampification of mGluR signaling by BDNF in striatal neurons prepared from mGluR5(TS) KI mouse, even in the presence of HIa. While not wishing to be bound by any particular theory, it is thought that since this mutant mGluR5 cannot be phosphorylated at the Homer binding site, and binds Homer but not Pinl , it indicates that the critical role of Pinl is to bind mGluR5. mGluR5(TS) KI mice also show selective loss of rewarding effects of ***e but not the increase of locomotor activity. Since this mutant mGluR5 binds Homer but not Pinl, this phenotype suggests that Pinl is required for the rewarding effects of ***e.

[0057] In another example of a mutant form of mGluR, the mGluR(F/R) mutant described herein binds Pin but not Homer. Ampification of mGluR signaling by BDNF in striatal neurons prepared from mGluR5(FR) KI mouse, even in the absence of HIa. Since this mutant mGluR5 can bind Pin 1 but not Homer its amplification without the requirement for HIa supports the conclusion that the natural function of Homer is to prevent amplification. The mGluR5(F/R) knockin mouse shows enhanced sensitivity to ***e. Since this mutant mGluR5 cannot bind Homer, it supports the notion that Homer functions to limit an activity of mGluR that is critical for ***e sensitization. Since ***e sensitization is a manifestation of enhanced dopamine signaling, these observations suggest that Homer binding regulates convergent dopamine and mGluR signaling.

[0058] Homer and Pinl compete for binding to phosphorylated mGluR. The level of phosphorylated mGluR is defined by the history of dopamine or TrkB signaling. When H 1 a is upregulated, Pinl more effectively competes and amplifies mGluR output. In this way the mGluR integrates signals from dopamine/TrkB with IEG expression. In certain conditions, Homer Ia is up regulated and include sleep deprivation, stress, pain and many forms of developmental plasticity.

[0059] Also provided are transgenic non-human animals having a transgene encoding mGluR, wherein the mGluR comprises a mutation and the mutated mGluR binds prolyl isomerase but not Homer. In certain embodiments, the mGluR is mGluRl ; in other embodiments, the mGluR is mGluR5. In particular aspects, the mutation is an amino acid substitution of phenylalanine to arginine at position 1128. [0060] Further provided are transgenic non-human animals having a transgene encoding mGluR, wherein the mGluR comprises a mutation and the mutated mGluR binds Homer but not prolyl isomerase. In certain embodiments, the mGluR is mGluRl; in other embodiments, the mGluR is mGluR5. In particular aspects, the mutation is a threonine to alanine substitution at position 1123 and a serine to alanine substitution at position 1126 of mGluR5.

[0061] In further embodiments, the above transgenic mice can be crossed to mice that possess altered dopamine signaling or altered Homer signaling, etc. These models can be used for further validation of the model and to test compounds. For example, the mGluR(F/R) may be valuable to test the therapeutic action of Pin 1 inhibitors in the prevention of ***e sensitization.

[0062] The invention will now be described in greater detail by reference to the following non-limiting examples.

EXAMPLES

[0063] Phosphorylation of mGluR in the Homer ligand region (i.e., the Homer binding region) by proline directed kinases was examined. In Figure 3 A, an alignment of mGluRl and mGluR5 C-terminal regions is shown depicting Homer ligand (in red), a peptide used for immunization (in box), and potential sites of phosphorylation with corresponding kinases (as predicted by internet-based algorithms Scansite and NetPhos 2.0). A phosphorylation site- specific antibody (anti-mGlul/5-pSl 154/1126) was derived using the indicated peptide. This antibody detected wild-type mGluR5 C-term from transfected HEK293 cells, but not the mGluR5 S 1126 A mutant which cannot be phosphorylated. λ-phosphatase treatment of the immunoblot abolished its ability to recognize wild-type mGluR5 C-term. A phosphorylation- independent anti-mGluR5-C-terminus antibody (α-mGluR5 C-term) was used to show the expression of both wild-type and mutant mGluR5 C-term (Figure 3B). The anti-mGluRl/5- pSl 154/1126 antibody immunoprecipitated mGluR5 from transfected HEK293 cells. Pre- absorption with Serl 126-phosphorylated mGluR5 peptide (mG5 pS), but not the unphosphorylated peptide (mG5), blocked the immunoprecipitation (Figure 3C).

[0064] Other studies showed that several stimuli that increased proline kinase activity increased specific phosphorylation of mGluR at the Homer binding site. Notable agents included Dopamine, BDNF, NMDA and kainic acid. Active ERK2 directly phosphorylated purified GST fusion proteins corresponding to the C-termini of mGluRl/5 in vitro. EGF- induced MAP kinase phosphorylated the Homer ligand of mGluR5. In this experiment, HEK293 cells transfected with mGluR5 were pre-treated with control DMSO solution, 15OnM AG1478 (EGF receptor inhibitor) or 3.6 μM U0126 (MEKl inhibitor) for 15 minutes, then incubated with EGF (5OnM) for 5 minutes. Further, CDK5 and p35 increased phosphorylation of mGluR5 serine- 1126. In this experiment, HEK293 cells were transfected with HA-mGluR5 alone or together with CDK5 and p35 were treated with Purvalanol A (CDK5 inhibitor) for 15 minutes. Finally, HEK293 cells were transfected with mGluR5 and dominant negative CDK5 or dominant negative p38, a dominant negative p38 blocked phosphorylation of mGluR5 serine- 1126.

[0065] In other experiments, it was demonstrated that phosphorylation of group 1 mGluRs was modulated by multiple stimuli in cultured neurons. For example, in one study, rat El 8 cortical neurons or striatal neurons were pre-treated with control DMSO solution or 3.6 μM U0126 (MEK 1/2 inhibitor) for 15 minutes, then incubated with BDNF (50 ng/mL) for 5 minutes. The results of this study suggested that BDNF induces mGluRl/5 Serl 154/1126 phosphorylation, and is dependent on ERK2. In a further study, rat El 8 striatal neurons were treated with various drugs as indicated. These results indicated that mGluRl/5 Ser 1154/1126 phosphorylation is induced by kainate, mGluR and Dl dopamine receptor activation.

[0066] In still another study, phosphorylation of mGluR was increased by inhibitors of phosphatase PPl and PP2A. PPl and/or PP2A phosphatases regulated mGluRl/5 Serl 154/1126 phosphorylation. In this experiment, rat E18 striatal neurons were incubated with for 30min, then stimulated with 10 uM DHPG for 3min. These data suggested that agents that accelerate dephosphorylation of mGluR (e.g., by activating a specific phosphatase), prevent binding of Pin 1 to mGluR and thereby prevent its action.

[0067] In another experiment, it was shown that phosphorylation of the Homer ligand in mGluRl/5 enhances its binding to Homer. In particular, phosphorylated (mG5-pS) or unphosphorylated (mG5) peptides were conjugated to Affigel-15 sepharose beads, and were then incubated with the lysate from HA-HomerlC transfected HEK293 cells. mG5-pS showed increased binding to HA-HomerlC as analyzed by Western blotting with anti-HA antibody. Treatment of the beads with λ-PPase before binding to Homer 1 C reduced their interaction. In a further study, HEK293 cells were transiently transfected with myc-Homerl c and mGluR.5. After 5 minutes incubation with EGF (50 nM), Homeric was immunoprecipitated with anti-myc antibody. Co-immunoprecipitation of wild-type or S 1126A mutant mGluR.5 was analyzed by Western blotting with various antibodies indicated above. These data suggested that EGF-mediated phosphorylation of mGluR5 serine- 1 126 increased the interaction of mGluR5 with Homer Ic.

[0068] Homer binds mGluR5 and prevents BDNF-evoked amplification of the delayed inward current. Accordingly, in striatal neurons derived from Homer null mice, BDNF potentiated an mGluR-evoked slow inward current (Figure 4). mGluR-dependent current is amplified by BDNF co-stimulation in the Homer knockout (KO). The amplified current is dependent on niGluRl/5 and BDNF activation of a Trk receptor and MAP Kinase. The fact that the current is amplified in Homer triple KO neurons indicates that amplification does not require Homer.

[0069] In striatal neurons from WT mice, the concomitant expression of Homer Ia was permissive for the ability of BDNF to amplify the delayed inward current (Figure 5). mGluR- dependent current was not amplified by BDNF in wild type striatal neurons unless neurons also expressed the immediate early gene form of Homer, Homer Ia (HIa). HIa functioned as a natural dominant-negative that opposes the crosslinking activity of Homer proteins that are present in unstimulated neurons. This suggested a model in which HIa reversed the action of constitutively expressed Homers and enables the action of BDNF-TrkB-MAPK to amplify the mGluR-dependent current. This co-dependence on both phosphorylation of mGluR and presence of HIa defined a conditional switch for the properties of mGluR signaling that rationalized crosstalk with growth factor and dopamine signaling pathways.

[0070] In another study, Pinl binding to phosphorylated mGluR5 was examined. Unphosphorylated (mG5), Thrl 123 -phosphorylated (mG5-pT), or Serl 126-phosphorylated (mG5-pS) mGlu5 peptides were conjugated to Affigel-15 sepharose beads, and were then incubated with the lysate from HA-Pinl transfected HEK293 cells. Bound Pinl was analyzed by Western blotting with anti-HA antibody. These results suggested that Pin 1 selectively bound phosphorylated Homer ligand of mGluR. In another experiment, GST- fusion proteins corresponding to Pinl wild-type, W34A mutant, and R68,69A mutant were purified and immobilized on glutathione agarose beads, and incubated with the lysates from transfected HEK293 cells expressing mGluR5 wild-type or Tl 123 A₅Sl 126A mutant. Bound mGluR5 was analyzed by Western blotting with the anti-mGluRl/5-pSl 154/1 126 antibody or the anti-mGluR5-C-terminus antibody. These results indicated that Pin 1 binds full length mGluR5 and is dependent on both WW and isomerase domains.

[0071] Phosphorylation of mGluRl/5 was rapidly increased in vivo in response to ***e administration. Phosphorylation of mGluRl/5 was increased in striatum samples. Further, Homer binding to mGluRl/5 is enhanced as a consequence of phosphorylation.

[0072] In another study, an inhibitor of prolyl isomerase Pinl, blocked the BDNF potentiation of mGluR5-mediated slow current in Homer triple KO striatal neurons (Figure 6). Moreover, BDNF did not change the mGluR5 -mediated slow inward currents in striatal neurons derived from Pinl knockout mice.

[0073] In another study, a knockin mouse having a mutant mGluR5 transgene resulting in an mGluR that binds Homer but not Pinl was generated (termed "mGluR5(TS)"). Specifically, both the threonine and serine in the homer ligand (at positions 1 123 and 1 126 of mGluR5, respectively) are mutated to alanine. Detergent lysates were prepared from forebrain of WT mice and mGluR5(TS) mice. Lysates were assayed for the ability of mGluR to co-immunoprecipitate with Homeric or for mGluR to bind to GST-Pin 1. It was noted that mGluR5(TS) cannot be phosphorylated by proline directed kinases (absence of band in immunoblot when using the phosphospecific Ab) and does not bind Pinl. In a study of mGluR-mediated currents in mGluR5(TS) mice (also termed "TSAA"), BDNF did not change the mGluR5 -mediated slow inward currents in striatal neurons derived from these mice (Figure 8). In these experiments, even with the expression of HIa, BDNF did not evoke amplification of the delayed inward current. Without wishing to be bound by any particular theory, this is likely because mGluR5(TS) cannot be phosphorylated at the Homer ligand, Pinl cannot bind and by its enzyme activity cause conformational change required for amplification.

[0074] In another study, a knockin mouse having a mutant mGluR5 transgene resulting in an mGluR that binds Pinl but not Homer was generated (termed "mGluR5(F/R)"). Specifically, the phenylalanine at position 1128 of mGluR5 was substituted with arginine. Co-immunoprecipitation studies confirmed mGluR(F/R) binds Pinl but not Homer (Figure 9). Amplification of mGluR signaling by BDNF in striatal neurons prepared from mGluR5(F/R) KI mouse was shown, even in the absence of HIa (Figure 10). Since this mutant mGluR5 can bind Pin 1 but not Homer its amplification without the requirement for HIa supports the conclusion that the natural function of Homer is to prevent amplification.

[0075] mGluR5(TS) knockin (KI) mouse show selective loss of rewarding effects of ***e but not the increase of locomotor activity. In a study of place conditioning between wild type (WT), mGluR5(TS) knockin, and the heterozygote (HET), group differences were not observed for total distance traveled in response to acute ***e (Figure 1 IA) or in the change in locomotor activity with repeated ***e administration (although comparisons revealed sensitization only in heterozygote) (Figures 1 IA and B). While WT mice showed a place-preference following 4 X 10 mg/kg ***e, both HET and KI mice showed a significant place aversion (Figures 11C and D).

[0076] In another study, Pinl and Homeric were shown to compete for binding to mGluRl/5. HIa did not compete with Pinl (Figure 12).

[0077] In still another study, paraformaldehyde (PFA), which induces two phases of pain response-acute (0-10 min) and inflammatory (10-60 min) was injected into animals. The inverse agonist of mGluRS, MPEP, (30mg/kg, ip injection) 20min prior to formalin injection reduced the intensity of the second phase of pain but did not alter the first phase (Figure 13). In a related study, formalin-induced inflammatory pain was increased in mGluR5(F/R) knockin mice (Figure 14). In contrast formalin-induced inflammatory pain was decreased in mGluR5(TS) knockin mice (Figure 15).

[0078] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

We claim:

1. A method of modulating the activity of metabotropic glutamate receptors (mGluR), comprising: inhibiting the activity of a prolyl isomerase in a cell expressing a mGluR, thereby reducing the activity of mGluR.

2. The method of claim 1, wherein the prolyl isomerase is prolyl isomerase 1 (Pin 1).

3. The method of claim 2, wherein the Pin 1 has the sequence set forth in SEQ ID NO: 1.

4. The method of claim 1, wherein the inhibitor is specific for the action of the prolyl isomerase on group 1 mGluRs.

5. The method of claim 1, wherein the cell expresses a dopamine receptor, and wherein further the activity of mGluR is modulated, thereby modulating dopamine receptor signaling.

6. The method of claim 5, wherein the dopamine receptor signaling is reduced.

7. A method of decreasing dopamine signaling in a subject having enhanced dopamine signaling; comprising: administering a prolyl isomerase inhibitor, wherein the inhibition of prolyl isomerase decreases the activity of mGluR, thereby reducing dopamine receptor signaling.

8. The method according to claim 7, wherein the subject has a drug addiction, inflammatory pain, schizophrenia, or a movement disorder, causing enhanced dopamine signaling.

9. A method of identifying a compound that inhibits Pin 1 activity on group 1 metabotropic glutamate receptors (mGluR) comprising: contacting a cell expressing an mGluR and Pin 1 with a test compound, wherein when binding of Pin 1 and mGluR is decreased in the presence of test compound as compared to binding in the absence of test compound, it is indicative of an inhibitor of Pin 1 activity.

10. The method of claim 9, wherein the test compound is selected from the group consisting of a peptide, a protein, an antibody, a peptidomimetic, a polynucleotide, antisense RNA, RNAi, a small molecule, and a small organic molecule.

11. The method of claim 9, wherein the mGluR receptor comprises a mutation, wherein the mutated mGluR binds Pin 1 but not Homer.

12. A method of modulating mGluR sensitization to mGluR agonists, comprising: modulating the activity of a prolyl isomerase in a cell expressing a mGluR, thereby modulating the sensitization of mGluR.

13. The method of claim 12, wherein the activity of the prolyl isomerase is inhibited, thereby reducing the sensitization of mGluR.

14. A method of modulating the activity of metabotropic glutamate receptors (mGluR), comprising: modulating a phosphatase that removes phosphate groups from mGluR, thereby modulating the activity of mGluR.

15. The method of claim 14, wherein the phosphatase is activated, thereby reducing binding of prolyl isomerase to mGluR.

16. The method of claim 14, wherein phosphorylation of threonine 1123 or serine 1126 of mGluR is reduced.

17. The method of claim 14, wherein the phosphatase is inhibited, thereby increasing binding of prolyl isomerase to mGluR.

18. A method of modulating mGluR signaling in a subject having an mGluR- related disorder; comprising: administering an agent that modulates the activity of a prolyl isomerase, thereby modulating the activity of mGluR.

19. The method of claim 18, wherein the mGluR-related disorder is selected from the group consisting of Alzheimer's disease, Huntington's Disease, Parkinson's disease, Tourette's syndrome, stroke, epilepsy, sleep or circadian rhythm disorder (e.g., insomnia), schizophrenia, depression, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), drug abuse, and pain.

20. The method of claim 18, wherein the activity of the prolyl isomerase is inhibited, thereby decreasing the activity of mGluR.

21. A method of treating a disease or disorder related to enhanced dopamine signaling in a subject in need thereof; comprising: administering a prolyl isomerase inhibitor, wherein the inhibition of prolyl isomease decreases the activity of mGluR, thereby reducing dopamine receptor signaling.

22. The method according to claim 21, wherein the subject has a drug addiction, inflammatory pain, schizophrenia, or a movement disorder, causing enhanced dopamine signaling.

23. A method of treating an mGluR-related disorder in a subject in need thereof; comprising: administering an agent that modulates the activity of a prolyl isomerase, thereby modulating the activity of mGluR.

24. The method of claim 23, wherein the mGluR-related disorder is selected from the group consisting of Alzheimer's disease, Huntington's Disease, Parkinson's disease, Tourette's syndrome, stroke, epilepsy, sleep or circadian rhythm disorder (e.g., insomnia), schizophrenia, depression, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), drug abuse, pain, and cancer.

25. A transgenic non-human animal comprising a transgene encoding mGluR5 wherein the mGluR comprises a mutation, wherein the mutated mGluR binds prolyl isomerase but not Homer.

26. The transgenic animal of claim 25, wherein the mutation is an amino acid substitution of phenylalanine at position 1128 with arginine.

27. A transgenic non-human animal comprising a transgene encoding mGluR5 wherein the mGluR comprises a mutation, wherein the mutated mGluR binds Homer but not prolyl isomerase.

28. The transgenic animal of claim 27, wherein the mutation comprises a threonine to alanine substitution at position 1123 and a serine to alanine substitution at position 1 126.