WO2009100384A2 - Heterooligomeric serotonin/glutamate receptor complex - Google Patents

Heterooligomeric serotonin/glutamate receptor complex Download PDF

Info

Publication number
WO2009100384A2
WO2009100384A2 PCT/US2009/033469 US2009033469W WO2009100384A2 WO 2009100384 A2 WO2009100384 A2 WO 2009100384A2 US 2009033469 W US2009033469 W US 2009033469W WO 2009100384 A2 WO2009100384 A2 WO 2009100384A2
Authority
WO
WIPO (PCT)
Prior art keywords
mglur2
receptor
complex
heteroreceptor
compound
Prior art date
Application number
PCT/US2009/033469
Other languages
French (fr)
Other versions
WO2009100384A3 (en
Inventor
Stuart Sealfon
Javier Gonzalez-Maseo
Jay Gingrich
Original Assignee
Mount Sinai School Of Medicine Of New York University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mount Sinai School Of Medicine Of New York University filed Critical Mount Sinai School Of Medicine Of New York University
Publication of WO2009100384A2 publication Critical patent/WO2009100384A2/en
Publication of WO2009100384A3 publication Critical patent/WO2009100384A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor

Definitions

  • the present invention relates generally to heteroreceptor complexes of G- protein coupled receptors, and more specifically to heteroreceptor complexes comprising serotonin and glutamate receptors, as well as to methods for identifying modulators (including agonists and antagonists) of such a receptor complex.
  • the invention also relates to identification of novel heteroreceptor complex ligands and synergistic compositions of ligands for each receptor, which can provide strategies for relieving psychosis associated with schizophrenia or other brain diseases.
  • the psychosis associated with schizophrenia is characterized by alterations in sensory processing and perception (Freedman et al., 2003, N Engl J Med 349; Sawa et al., 2003, Science 296:692-5).
  • Some antipsychotic drugs such as the atypical antipsychotics, were identified by their high affinity for serotonin 5-HT 2A receptors (2AR) (Lieberman, J. A. et al., 1998, Biol Psychiatry 44:1099-117; Miyamoto, S., et al., 2005, J. MoI Psychiatry 10:79-104).
  • atypical antipsychotics e.g., clozapine, olanzapine
  • traditional agents e.g., haloperidol
  • these agents still elicit significant side-effects, such as CNS depression, weight gain, and sexual dysfunction.
  • side-effects reduce patient compliance, leading to relapses of illness and thus a negative impact on the life-long course of this disease.
  • Clozapine which is a very effective antipsychotic medication, has the additional sometimes fatal side effect of agranulcytosis, which severely limits its use and necessitates frequent blood tests when it is prescribed.
  • atypicals only minimally reverse many aspects of schizophrenia, such as negative symptoms, including mood, affect, and cognitive dysfunction.
  • Hallucinogenic drugs such as psilocybin and lysergic acid diethylamide (LSD)
  • LSD lysergic acid diethylamide
  • the present invention relates to the discovery that metabotropic glutamate and serotonin receptors form functional heteroreceptor complexes. These receptor complexes can be exploited for high-throughput screening of compounds to identify heteroreceptor complex modulators (agonists and antagonists).
  • the invention also relates to identification of novel heteroreceptor complex ligands and synergistic compositions, which can form the basis for therapeutic strategies to reduce psychosis associated with, for example, schizophrenia, mania, Alzheimer's disease, or drug induced psychosis.
  • the present invention provides an isolated heteroreceptor complex, which receptor complex comprises a serotonin receptor protein and a glutamate receptor protein. Both receptor proteins are expressed in the same type of cell.
  • the heteroreceptor complex includes the serotonin receptor 2A protein (5-HT 2A receptor; 2AR) and the metabotropic glutamate receptor 2 protein (mGluR2).
  • the invention further provides a recombinant host cell that expresses a functional heteroreceptor complex, which receptor complex comprises a 2AR receptor protein expressed from an expression vector introduced into the host cell, and an mGluR2 receptor protein expressed from an expression vector introduced into the host cell.
  • the host cell stably expresses both receptors.
  • This method comprises observing a change in a property or function of the heteroreceptor complex contacted with a candidate compound. For example, receptor trafficking (e.g., internalization), ligand binding, ligand specificity, or receptor complex activation can be altered in the presence of a test compound.
  • the heteroreceptor complex will exhibit different affinities for various known and test ligands.
  • the invention provides compounds and methods for identifying specific compounds that bind the complex with high affinity and modulate the activity of the complex, but that show absent or limited or indistinguishable modulatory activity or affinity for a 2AR and/or an mGluR2 receptor when expressed alone.
  • the invention provides compounds and methods for identifying compounds that can bind and modulate the function of a 2AR and/or an mGluR2 receptor when expressed alone, but when the receptors are part of a heteroreceptor complex, the compounds have a different binding affinity and/or modulatory effect on the heteroreceptor complex.
  • the invention provides compounds and methods for identifying compounds that can inhibit or prevent the formation of, or otherwise destabilize the presence of, a heteroreceptor complex.
  • the invention provides compounds and methods for identifying compounds that can promote or induce the formation of, or otherwise stabilize the presence of, a heteroreceptor complex.
  • the invention provides a method of increasing the binding affinity of a compound to a 2AR receptor of a heteroreceptor complex by introducing and binding a second compound to the mGluR2 receptor of the same heteroreceptor complex.
  • One embodiment of the present invention provides a method of increasing internalization of a 2AR, mGluR2 or both components of a heteroreceptor complex by introducing and binding of a compound to the 2AR and/or mGluR2 receptor of the heteroreceptor complex.
  • One embodiment of the present invention provides a method of decreasing internalization of a 2AR, mGluR2 or both components of a heteroreceptor complex by introducing and binding of a compound to the 2AR and/or mGluR2 receptor of the heteroreceptor complex.
  • One embodiment of the present invention provides a method of decreasing the binding affinity of a compound to a 2AR receptor of a heteroreceptor complex by introducing and binding a second compound to an mGluR2 receptor of the same heteroreceptor complex.
  • One embodiment of the present invention provides a method of decreasing the ability of a compound to activate a 2AR signal transduction pathway upon binding a 2AR receoptor of a heteroreceptor complex by introducing and binding a second compound to an mGluR2 receptor of the same heteroreceptor complex.
  • One embodiment of the present invention provides a method of increasing the ability of a compound to activate a 2AR signal transduction pathway upon binding a 2AR receptor of a heteroreceptor complex by introducing and binding a second compound to an mGluR2 receptor of the same heteroreceptor complex.
  • One embodiment of the present invention provides a method of activating a 2AR signal transduction pathway by introducing and binding a compound to an mGluR2 receptor of a heteroreceptor complex, wherein the heteroreceptor complex includes a 2AR receptor and an mGluR2 receptor.
  • One embodiment of the present invention provides a method of inhibiting or reducing a 2AR signal transduction pathway by introducing and binding a compound to an mGluR2 receptor of a heteroreceptor complex, wherein the heteroreceptor complex includes a 2AR receptor and an mGluR2 receptor.
  • One embodiment of the present invention provides a method of increasing the binding affinity of a compound to an mGluR2 receptor of a heteroreceptor complex by introducing and binding a compound to a 2AR receptor of the same heteroreceptor complex.
  • One embodiment of the present invention provides a method of decreasing the binding affinity of a compound to an mGluR2 receptor of a heteroreceptor complex by introducing and binding a second compound to a 2AR receptor of the same heteroreceptor complex.
  • One embodiment of the present invention provides a method of increasing the ability of a compound to activate an mGluR2 signal transduction pathway upon binding an mGluR2 receptor of a heteroreceptor complex by introducing and binding a second compound to a 2AR receptor of the same heteroreceptor complex.
  • One embodiment of the present invention provides a method of decreasing the ability of a compound to activate an mGluR2 signal transduction pathway upon binding an mGluR2 receptor of a heteroreceptor complex by introducing and binding a second compound to a 2AR receptor of the same heteroreceptor complex.
  • One embodiment of the present invention provides a method of activating an mGluR2 signal transduction pathway by introducing and binding a compound to a 2AR receptor of a heteroreceptor complex, wherein the heteroreceptor complex includes a 2AR receptor and an mGluR2 receptor.
  • One embodiment of the present invention provides a method of inhibiting or reducing an mGluR2 signal transduction pathway by introducing and binding a compound to a 2AR receptor of a heteroreceptor complex, wherein the heteroreceptor complex includes a 2AR receptor and an mGluR2 receptor.
  • One embodiment of the present invention provides a method of decreasing a hallucinogen induced head-twitch behavior in an organism by introducing and binding a compound to a heteroreceptor complex.
  • One embodiment of the present invention provides a method of increasing a hallucinogen induced head-twitch behavior in an organism by introducing and binding a compound to a heteroreceptor complex.
  • the compounds and compositions of the invention have 2AR/mGluR2 heteroreceptor complex modulating properties and thus therapeutic potential. These compounds address the need that exists for more precise identification of effective compounds for treating a neurological, neurodegenerative, or psychiatric brain disease or disorder of the central nervous system.
  • a neurological, neurodegenerative, or psychiatric brain disease or disorder that is treatable according to the methods of the invention can be, for example, drug abuse, schizophrenia, psychoses associated with schizophrenia, depression, anxiety, obsessive compulsive disorder, bi-polar disorder, neurological diseases associated with psychosis including Alzheimer's and Parkinson's disease, drug induced psychosis, or a dysfunction of the central reward pathway.
  • a heteroreceptor complex comprising a serotonin 2A receptor and an mGluR2 receptor identifies an assay for developing more effective treatments of psychosis in general, and psychoses associated with schizophrenia in particular.
  • Administering a therapeutically effective dose of a compound or a pharmaceutical composition of the invention is expected to have a therapeutic effect on such diseases and disorders.
  • mGluR2/2AR curve is preferably fitted by a saturation curve, F test (pO.OOl).
  • F test pO.OOl
  • the other co-transfection datasets show linear correlations, f, [ 3 H]Ketanserin displacement curves in mouse SCx membranes (top panels). 2AR agonist affinities were higher in the presence of mGluR2/3 agonist 10 ⁇ M LY379. [ 3 H]LY341495 displacement curves (bottom panels). mGluR2/3 agonist affinities were lower in the presence of 2AR agonist 10 ⁇ M DOI.
  • Figure 2a-e shows the lower expression of mGluR2 in the absence of cortical 2AR.
  • Figure 3a-c shows (a) the [ 3 H]Ketanserin binding displacement curves by DOI, DOM and DOM in mouse SCx membrane (top panels). Note that the affinity of DOI displacing [ 3 H]Ketanserin binding was significantly higher in the presence of lO ⁇ M LY379, (see also b.). [ 3 H]LY341495 binding displacement curves by LY379, DCG-IV and L-CCG-I in mouse SCx membranes (bottom panels). Note that the affinity of LY379, DCG-IV and L-CCG-I displacing [ 3 H]LY341495 binding was significantly lower in the presence of lO ⁇ M DOI (see also c).
  • Figure 4a-m shows that mGluR2 transmembrane domains 4/5 mediate association with 2AR.
  • Cells separately expressing each construct were also mixed, c. 2AR competition binding in cells stably expressing 2AR and transfected with mGluR2/mGluR3 chimeras, d. FRET in cells expressing 2AR- eCFP and either mGluR2, mGluR3 or mGluR3 ⁇ TM4,5 chimera, all tagged with eYFP.
  • Pseudo-color images represent normalized values (FRET N ).
  • FRET N normalized values
  • Figure 5a-g shows 2AR/mGluR2 complex-dependent modulation of cellular and behavioral responses, a, DOI-stimulated [ 35 S]GTPyS binding in primary culture membranes followed by immunoprecipitation with anti-G ⁇ q / ⁇ or anti-G ⁇ ,i,2,3 antibodies.
  • DOI Ga 11 ,2 3 activation potency was significantly decreased by 10 ⁇ M LY379. Data are mean ⁇ s.e.m.
  • Double label FISH was performed in SCx layers V and VI in mice injected (i.p.) with vehicle or 0.24 mg/kg LSD 15 min. after being pre-injected with vehicle or 15 mg/kg LY379. Red, green, and blue colors indicate 2AR, c-fos (d) or egr-2 (e), and nucleus (DAPI), respectively. Note that the induction of the hallucinogen signaling marker egr-2 is selectively attenuated by LY379 in mouse SCx.
  • mGluR2 inhibits that specific cellular response induced by 2AR agonists in mouse SCx.
  • Dose-response curves of LY379 on cellular response induced by 2AR agonists in mouse SCx assayed by qRT-PCR. Mice were injected with vehicle, 2 mg/kg DOI, 4 mg/kg DOM, 1 mg/kg DOB, 0.24 mg/kg LSD, 0.4 mg/kg lisuride, or 0.5 mg/kg ergotamine 15 min after being pre-injected with vehicle or 15 mg/kg LY379 (n 4-12 per group).
  • Figure 7a-g shows that 2AR is increased and mGluR2 is decreased in schizophrenia
  • [ 3 H]ketanserin binding was higher and [ 3 H]LY341495 binding was lower (p ⁇ 0.05; Student's ?-test).
  • In treated schizophrenia [ 3 H]ketanserin binding was unaffected and [ 3 H]LY341495 binding was lower (p ⁇ 0.05).
  • f Demographic characteristics and antemortem diagnoses of cases of nontreated schizophrenic subjects, and their respective control subjects
  • g Demographic characteristics and antemortem diagnoses of cases of antipsychotic-treated schizophrenic subjects, and their respective control subjects.
  • Figure 8a-f shows that chronic clozapine modulates the expression of the components of the 2AR/mGluR2 complex in mouse SCx.
  • Animals were chronically (21 days) injected with vehicle (black) or 25 mg/kg clozapine (red) and sacrificed 1 day after the last clozapine injection, a.
  • [ 3 H]Ketanserin binding in mouse SCx after vehicle or chronic clozapine (n 6 per group)
  • [ 3 H[LY341495 binding in htr2A+/+ (b) or htr2A-/- (c) mouse SCx after vehicle or chronic clozapine (n 6 per group)
  • Figure 10 shows the nucleic acid sequences of the oligonucleotide probes used for fluorescence in situ hybridization (FISH.)
  • Figure 11 shows the nucleic acid sequences of the primers pairs used for qRT- PCR of murine genes.
  • Figure 12 shows the nucleic acid sequences of the primers pairs used for qRT- PCR of human genes.
  • Figure 13 shows a sequence alignment comparison of nucleic acid sequences of the transmembrane regions of mGluR2, mGluR3, 2AR, ⁇ 2 -adrenergic receptor, and rhodopsin from different species.
  • Figure 14 shows the evaluation of the specificity of FISH assay, a, FISH assay for 2AR and ⁇ -actin in htr2A+/+ and htr2A-/- mouse SCx. Red, green, and blue colours indicate 2 ⁇ i?, ⁇ -actin, and nucleus (DAPI), respectively, b, Competition of 2AR, mGluR2 and mGluR3 hybridization by specific, unlabeled oligonucleotide probes.
  • a FISH assay in mouse SCx (2AR and mGluR2) and in mouse thalamus (mGluR3) with the fluorescently labelled oligonucleotides used in Fig. 1 was performed with the inclusion of excess of unlabeled oligonucleotides in the hybridization buffers. The presence of specific unlabeled oligonucleotides completely eliminated the signal obtained with the fluorescently labeled oligonucleotide probes.
  • Red, green, and blue colours indicate 2AR, mGluR2 or mGluR3, and nucleus (DAPI), respectively
  • c Similar anatomical pattern of expression of mGluR2 in mouse SCx was obtained with two different sets of fluorescently labeled oligonucleotide probes, and with the combination of probe set 1 and probe set 2. Green, and blue colours indicate mGluR2 and nucleus (DAPI), respectively
  • d Evaluation of FISH assay specificity using scrambled-sequence oligonucleotide probes. FISH was performed by using a mixture of five fluorescently-labeled scrambled oligonucleotide probes. Scale bar, 500 ⁇ m. See Fig. 11 for oligonucleotide sequences.
  • Figure 15 shows Intact HEK293 cells transiently transfected with (a) increasing amounts of mGluR2-i?luc or mGluR3-/?luc or (b) with increasing amounts of 2AR-GFP 2 , 2CR-GFP 2 or pGFP 2 .
  • the amount of each cDNA is noted.
  • Donor (a) and acceptor (b) conjugate relative expression levels were monitored by measuring luminescence and fluorescence. Note that the signals detected are comparable for different donors and acceptors. Data from triplicates assays in a single experiment are displayed. Two further experiments produced similar results.
  • Figure 16 shows characterization of mGluR2/mGluR3 chimeras, a, N- terminally HA-tagged mGluR2, mGluR3 and mGluR2/mGluR3 chimeras were expressed in HEK293 cells, fixed and stained with anti-HA antibody, b, [ 3 H]LY341495 binding saturation curves in HEK293 cells transfected with mock, mGluR2, mGluR3 and mGluR2/mGluR3 chimeras. Note that the level of expression is comparable for the different constructs (see also Fig. 4).
  • Figure 17 shows activation of G protein pathways monitored through ion channels in Xenopus oocytes
  • G, /0 -signalling can be detected through GIRK4* channels.
  • G, /o -coupled receptors G ⁇ , /0 and G ⁇ , /0 dissociate.
  • ⁇ , /0 can then activate the channel increasing potassium influx under the experimental conditions that can be recorded.
  • GIRK4* currents are blocked by barium.
  • G q /n- signalling can be detected through heterologously expressed IRK3 channels (b) and endogenous calcium-activated chloride currents (c).
  • PLC G q m -activation phospholipase C
  • PIP 2 phosphatidylinositol-4,5-biphosphate
  • DAG diacylglycerol
  • IP3 inositol triphosphate
  • IP 3 mobilizes Ca 2+ to the cytosol from the ER producing a characteristic peak current associated with the calcium-activated chloride currents.
  • PIP 2 hydrolysis results in a decrease in IRK3 inward potassium current.
  • Pertussis toxin (PTX) is known to specifically disrupt the function of G, /o proteins.
  • RGS2 exhibits selectivity shutting down the G q/ ⁇ -dependent signalling.
  • Figure 18 shows calcium-activated chloride currents elicited in oocytes expressing mGluR2 alone, mGluR2 and 2AR together, or 2AR alone,
  • Figure 19 shows the relative inhibition of potassium inward currents in oocytes expressing IRK3 together with mGluR2, 2AR or both receptors
  • LY379268 IM glutamate agonist
  • LY379268 Statistical summary of current inhibition from basal level induced by LY379268 (black) and serotonin (white) (mean ⁇ S.E.M.). Values were normalized to the maximum inhibition obtained by 2AR stimulation with serotonin. Results indicate a larger decrease in IRK3 inward current for the 2AR/mGluR2 group than for the negative control (mGluR2) but smaller than the maximum inhibition obtained through 2AR stimulation (**p ⁇ 0.001).
  • Figure 20 shows calcium-activated chloride currents in oocytes expressing 2AR together with mGluR2, mGluR2 ⁇ TM4,5, mGluR3 and mGluR3 ⁇ TM4,5.
  • RGS2 (b) on calcium-activated chloride currents measured in oocytes expressing mGluR2 alone, mGluR2 and 2AR together, or 2AR alone. Representative traces obtained in response to 1 M glutamate agonist (LY379268) and 1 M serotonin are depicted in each panel. Activation of calcium-activated chloride currents by stimulation of mGluR2 was abolished by RGS2 and was insensitive to the presence of PTX confirming Gi/oGq/11 crossactivation. (c) Data corresponding to measured peak currents (mean ⁇ S.E.M.) following LY379268 stimulation.
  • Peak currents for the 2AR/mGluR2 group treated with PTX were larger than the rest of the groups (**p ⁇ 0.001).
  • Figure 23 shows actions of LY379268 in wild-type (htr2A+/+) and 2AR null- mutant mice (htr2A-/-).
  • Figure 24 shows G, /0 -sensitive inward potassium currents measured in oocytes expressing GIRK4* together with mGluR2, mGluR2, mGluR2 ⁇ TM4,5, mGluR3 and mGluR3 ⁇ TM4,5.
  • (b) Statistical summary of basal current (mean ⁇ S.E.M.). Values indicate no significant difference in basal current between groups. (p 0.2419).
  • Figure 25 shows LY379268 displacement of [ 3 H]LY341495 binding was performed in the absence (vehicle) or in the presence of DOI (10 M).
  • Competition curves were analyzed by nonlinear regression to derive dissociation constants for the high (Ki-hig h ) and the low (K 1- I Ow ) affinity states of the receptor.
  • % High refers to the percentage of high-affinity binding sites as calculated from nonlinear fitting.
  • Competition curves of [ 3 H]ketanserin binding showed that the affinity of DOI for the 2AR was increased by 10 M LY379268 (data not shown). Values are best fit ⁇ S.E. of 3-5 experiments performed in duplicate/triplicate.
  • the present invention provides for a heteroreceptor complex comprising a serotonin receptor and a glutamate receptor, and methods of screening for compounds that can bind to and/or modulate the activity of the receptor complex and/or the serotonin and/or the glutamate receptors which form the receptor complex.
  • the invention is based, in part, on biochemical and pharmacological evidence for the physical and functional association of a fully functional serotonin receptor (5- HT 2 A receptor; 2AR), and a fully functional glutamate receptor (mGluR2 receptor).
  • This discovery represents the first observation of a heteroreceptor made up of a class A and a class C type receptors.
  • activation of the mGluR2 receptor inhibits activation of a 2AR G ⁇ i mediated signal transduction pathway upon the binding of a hallucinogenic compound to the 2AR receptor.
  • the discovery of the present invention also demonstrates that when a 2AR and an mGluR2 receptor are comprised in a heteroreceptor complex, binding of an agonist to the mGluR2 receptor activates a 2AR G ⁇ q/ n mediated signal transduction pathway. Such an effect can occur in the absence of the binding of an agonist to the 2AR receptor.
  • the present invention advantageously provides for the development both of more potent therapeutic agents and of a better understanding of the molecular basis of serotonin and glutamate receptor activity.
  • serotonin receptor refers to a receptor that can bind, and is activated, by serotonin.
  • Serotonin receptors include the members of the serotonin receptor families 5-HTi, 5-HT 2 , 5-HT 3 , 5-HT 4 , 5-HT 5A , and 5-HT 7; which are G- protein coupled receptors.
  • these include the specific serotonin receptors 5-HTI A , 5-HTIB, 5-HT 1 D , 5-HTi E , 5-HTi F, 5-HT 2A , 5-HT 2B , 5-HT 2C , 5-HT 3 , 5-HT 4 , 5- HT 5 A, 5-HT 6 , and 5-HT 7 .
  • the serotonin receptor is the 5-HT 2A serotonin receptor (also referred to as 2AR).
  • the serotonin receptor is a human 5-HT 2A serotonin receptor (also referred to as 2AR).
  • the serotonin receptor is a human 5-HT 2A serotonin receptor (also referred to
  • the human 5-HT 2 A serotonin receptor is preferably encoded by the Homo sapiens 5-hydroxytryptamine (serotonin) receptor 2 A (HTR2A) gene (GenBank accession number NM 000621), or any nucleic acid which encodes a human 5-HT 2 A polypeptide (for example, as defined by GenBank accession No. NP_000612.)
  • the serotonin receptor is a mouse 5-HT 2A serotonin receptor.
  • the mouse 5-HT 2A serotonin receptor is preferably encoded by the Mus musculus 5-hydroxytryptamine (serotonin) receptor 2 A (Htr2a) gene (GenBank accession number NM_172812), or any nucleic acid which encodes a mouse 5-HT 2A polypeptide (for example, as defined by GenBankaccession No. NP_766400.)
  • glutamate receptor refers to a receptor that can bind, and is activated, by glutamate.
  • the glutamate receptor can be a metabotropic glutamate receptor.
  • metabotropic glutamate receptors include receptors from glutamate receptor families I, II, and III. These receptor families include the glutamate receptors mGluRi and mGluRs (family I); InGIuR 2 and mGluR 3 (family II); and HiGIuR 4 , mGluR 6 , mGluR 7 , and mGluRg (family III).
  • the glutamate receptor is the mGluR2 receptor.
  • the glutamate receptor is a human mGluR2 glutamate receptor.
  • the human mGluR2 glutamate receptor is preferably encoded by the Homo sapiens glutamate receptor, metabotropic 2 (GRM2) gene (GenBank accession number NM_000839), or any nucleic acid which encodes a human mGluR2 polypeptide (for example, as defined by GenBank accession NPJ3OO83O.)
  • GEM2 metabotropic 2
  • the glutamate receptor is a mouse mGluR2 glutamate receptor.
  • the mouse mGluR2 glutamate receptor may be encoded by the Mus musculus G protein-coupled receptor, family C, group 1, member B (Grm2) gene (GenBank accession number XM_001475814), or any nucleic acid which encodes a mouse mGluR2 polypeptide (for example, as defined by GenBankaccession XPJ)01475864.)
  • the term "heteroreceptor complex” or "receptor complex” or “complex” refers to a G-protein coupled receptor (GPCR) complex made of at least two GPCR molecules with at least one mGluR2 receptor subunit and at least one 2AR receptor subunit.
  • GPCR G-protein coupled receptor
  • Receptors are "expressed endogenously" in a type of cell that expressed that receptor in healthy normal organisms, for example, a mammal, such as a human, mouse, or rat, in vivo or in an a cell line in vitro. In both cases, the cells have not undergone any genetic modification resulting in expression of the receptor, i.e., receptor expression does not depend on such genetic modification.
  • a cell may endogenously express either the serotonin or the glutamate receptor, or it may express both receptors in the same type of cell.
  • endogenous co-expression of a serotonin receptor with a glutamate receptor invovles the co-expression of the 2AR serotonin receptor and the mGluR2 glutamate receptor in the same cell.
  • Such cells include cortical primary cultures as well as membrane preparations from brain, for example, a mouse or rat brain, or any other organism.
  • function refers to an ability to accomplish at least one of the following: bind ligand; bind a selective ligand; bind a non-selective ligand selectively; bind a bifunctional, bi-specif ⁇ c ligand; activate a G- protein mediated signal transduction pathway upon binding of a ligand; attenuate or inhibit activation of a G-protein mediated signal transduction pathway upon binding of a ligand; induce internalization of a receptor, for example, a 2AR and/or an mGluR2 receptor component of a heteroreceptor complex, or a receptor complex; induce formation or stabilize the presence of a heteroreceptor complex, inhibit the formation or destabilize the presence of a heteroreceptor complex, or any combination thereof.
  • Such ability or abilities can be demonstrated in a cell based or cell-free system, for example, in a cell membrane preparation, or in vivo, including a transgenic animal system.
  • the term also refers to an ability to modulate a behavior, for example a locomotor activity, for example, a hallucinogen induced head-twitch behavior.
  • a "synergistically effective amount" of a ligand is an amount able to modulate a heteroreceptor complex's activity or function wherein the binding of one receptor of the complex increases or decreases the "functionality" of another receptor of the complex.
  • the binding of one receptor of the complex to ligand A reduces the ability of ligand B to bind to and/or activate a second receptor of the complex.
  • the term "antagonist” refers to any molecule that binds to a protein and either partially or completely blocks, inhibits, reduces, or neutralizes the function, activity or activation of the protein, for example, a receptor protein.
  • agonist refers to any molecule that binds to a protein and at least partially increases, enhances, or restores the function or activity of the protein, for example, a receptor protein.
  • mixed agonist or “mixed agonist/antagonist” refers to any molecule that binds to binds to a protein and either partially or completely blocks, inhibits, reduces, or neutralizes some function, activity or activation of the protein, and at least partially increases, enhances, or restores some function or activity of the protein, for example, a receptor protein.
  • the compounds of the invention are specific ligands for the hetroreceptor complex.
  • the compounds of the invention are specific ligands for the serotonin and/or glutamate receptor that comprise the heteroreceptor complex.
  • signal transduction pathway refers to the intracellular mechanism by which a serotonin or glutamate or other ligand of a receptor or the heteroreceptor complex induces an alteration of cell function or activity.
  • a key feature of the signal transduction pathway dissected herein is activation of G-protein coupled signaling, such as, for example, cAMP production, cAMP inhibition, or phospholipase C- ⁇ activation, resulting in further signal transduction, including, for example, MAPK phosphorylation.
  • element of a signal transduction pathway refers to a signal transduction factor that is activated or inhibited as a result of ligand binding to a receptor or the heteroreceptor complex.
  • elements of the signal transduction pathway include G-proteins, for example, G ⁇ q or
  • a "signal" in such a pathway can refer to activation, inhibition, increase the amount of, or decrease the amount of an element (or factor) in the pathway.
  • reduction of cAMP is a signal of an agonist-induced serotonin receptor signal transduction pathway.
  • activation of one of these factors involves phosphorylation of one or more proteins.
  • the introduction and binding of a compound (for example, an agonist or antagonist) to one of the receptor components of the heteroreceptor complex increases or decreases the affinity of a second compound for a second receptor type of the complex.
  • a compound for example, an agonist or antagonist
  • binding of a ligand to 2AR may increase or decrease the affinity of a ligand for mGluR2.
  • binding of a ligand to mGluR2 may increase or decrease the affinity of a ligand for 2AR.
  • binding of one compound to the heteroreceptor complex alters the conformation or otherwise facilitate recognition and binding of the second compound.
  • the introduction and binding of a compound (for example, an agonist or antagonist) to one receptor component of the heteroreceptor complex decreases the ability of a second compound to activate a second receptor type of the complex.
  • a compound for example, an agonist or antagonist
  • binding of a ligand to 2AR may decrease the ability of a ligand for mGluR2 to activate mGluR2.
  • binding of a ligand to mGluR2 may decrease the ability of a ligand for 2AR to activate 2AR.
  • the introduction and binding of a compound (for example, an agonist or antagonist) to one receptor component of the heteroreceptor complex increases the ability of a second compound to activate a second receptor type of the complex.
  • a compound for example, an agonist or antagonist
  • binding of a ligand to 2AR may increase the ability of a ligand for mGluR2 to activate mGluR2.
  • binding of a ligand to mGluR2 may increase the ability of a ligand for 2AR to activate 2AR.
  • the introduction and binding of a hallucinogenic compound to a 2AR receptor activates a G ⁇ j and a G ⁇ q/ i i mediated signal transduction pathway.
  • the 2AR comprises a heteroreceptor complex with niGluR2
  • activation of mGluR2 by an agonist inhibits activation of the G ⁇ j mediated signal transduction pathway by a hallucinogenic compound binding to the 2AR.
  • the introduction and binding of a compound (for example, an agonist or antagonist) to one receptor component of the heteroreceptor complex activates a second receptor type of the complex.
  • a compound for example, an agonist or antagonist
  • binding of a ligand to mGluR2 may activate 2AR.
  • binding of a ligand to 2AR may activate mGluR2.
  • the introduction and binding of a compound (for example, an agonist or antagonist) to one receptor component of the heteroreceptor complex inhibits or reduces the activity of a second receptor type of the complex.
  • a compound for example, an agonist or antagonist
  • binding of a ligand to mGluR2 inhibits or reduces activity of 2AR.
  • binding of a ligand to 2AR inhibits or reduces activity of mGluR2.
  • a compound of the invention binds to and modulates the property of a heteroreceptor complex, but has no affinity for, or a limited affinity for, either a 2AR and/or an mGluR2 receptor when expressed alone.
  • the compound when a compound of the invention is introduced and binds to a 2AR and/or an mGluR2 receptor in a complex, the compound has a modulatory effect on the complex that can not be detected, or does not occur, when the compound binds to the 2AR and/or mGluR2 receptors when expressed alone.
  • the binding of an agonist to the mGluR2 receptor of the complex decreases the activation of a G ⁇ ; -protein signal transduction pathway when a hallucinogenic agonist binds to the 2AR receptor of the complex.
  • the compounds identified according to the methods of the invention increase or decrease the stability of the complex.
  • a compound that increases the stability of the complex can be a chaperone, wherein the chaperone has one or more of the following effects: (i) enhancing the formation of a stable molecular conformation of the complex; (ii) enhances proper trafficking of the complex, or the receptors that comprise the complex, from the ER to another cellular location, preferably a native cellular location, i.e., preventing ER- associated degradation of the complex, or the receptors that comprise the complex; (iii) preventing aggregation of conformational ⁇ unstable, i.e., misfolded proteins; (iv) restoring or enhancing at least partial wild-type function, stability, and/or activity of the complex; and/or (v) improving the phenotype or function of the cell harboring the complex.
  • inhibitor is used herein to refer to a compound that can block or reduce the level of signaling in a signal transduction pathway described herein. Such an inhibitor may block the pathway at any point, from blocking binding of ligand to receptor to blocking function of intracellular signals.
  • an inhibitor discovered in accordance with the invention is specific for signals of heteroreceptor complex-induced signaling.
  • Screening refers to a process of testing one or a plurality of compounds (including a library of compounds) for some activity.
  • a “screen” is a test system for screening. Screens can be primary, i.e., an initial selection process, or secondary, e.g., to confirm that a compound selected in a primary screen (such as a binding assay) functions as desired (such as in a signal transduction assay). Screening permits the more rapid elimination of irrelevant or non-functional compounds, and thus selection of more relevant compounds for further testing and development. "High throughput screening” involves the automation and robotization of screening systems to rapidly screen a large number of compounds for a desired activity. Screens are discussed in greater detail below.
  • an isolated biological material can be free of some or all cellular components, i.e., components of the cells in which the native material occurs naturally (e.g., cytoplasmic or membrane component).
  • a material shall be deemed isolated if it is present in a cell extract or if it is present in a heterologous cell or cell extract.
  • an isolated nucleic acid fragment includes a PCR product, an isolated mRNA, a cDNA, or a restriction fragment.
  • an isolated nucleic acid fragment is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined or proximal to non-coding regions (but may be joined to its native regulatory regions or portions thereof), or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome.
  • the isolated nucleic acid fragment lacks one or more introns. Isolated nucleic acid molecules include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like, i.e., when it forms part of a chimeric recombinant nucleic acid construct.
  • a recombinant nucleic acid is an isolated nucleic acid fragment.
  • An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane- associated protein.
  • An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism.
  • An isolated material may be, but need not be, purified.
  • purified refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated and/or unwanted materials, i.e., contaminants, including native materials from which the material is obtained.
  • a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell.
  • substantially free is used operationally, in the context of analytical testing of the material.
  • purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art. Methods for purification are well-known in the art.
  • the terms “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated. Molecular Biology Definitions
  • nucleic acid encoding the protein may be full-length or truncated, so long as the gene encodes a biologically active protein.
  • the coding sequences of the gene to be delivered are operably linked to expression control sequences, e.g., a promoter that directs expression of the gene.
  • expression control sequences e.g., a promoter that directs expression of the gene.
  • operatively linked refers to the functional relationship of a polynucleotide/gene with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences.
  • operative linkage of a nucleic acid to a promoter refers to the physical and functional relationship between the polynucleotide and the promoter such that transcription of DNA is initiated from the promoter by an RNA polymerase that specifically recognizes and binds to the promoter, and wherein the promoter directs the transcription of RNA from the polynucleotide.
  • Expression of a serotonin and/or glutamate receptor may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression.
  • a vector is used in which the coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for expression of the construct from a nucleic acid molecule that has integrated into the genome (Roller and Smithies, 1989, Proc. Natl. Acad. Sci. USA, 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438; U.S. Patent No. 6,244,113 to Zarling et al.; and U.S. Patent No. 6,200,812 to Pati et al.).
  • Each of the above references are hereby incorporated by reference in their entirety.
  • host cell means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or
  • RNA sequence a protein or an enzyme.
  • Host cells can further be used for screening or other assays, as described infra.
  • expression system means a host cell and compatible vector under suitable conditions, e.g., for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.
  • Expression systems include mammalian host cells and vectors. Suitable cells include PC 12 cells, CHO cells, HeLa cells, 293 and 293T (human kidney cells), COS cells, mouse primary myoblasts, NIH 3T3 cells.
  • Suitable vectors include viruses, such as adenoviruses, adeno-associated virus (AAV), vaccinia, herpes viruses, baculoviruses and retroviruses, parvovirus, lentivirus, bacteriophages, cosmids, plasmids, fungal vectors, naked DNA, DNA lipid complexes, and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • viruses such as adenoviruses, adeno-associated virus (AAV), vaccinia, herpes viruses, baculoviruses and retroviruses, parvovirus, lentivirus, bacteriophages, cosmids, plasmids, fungal vectors, naked DNA, DNA lipid complexes, and other recombination vehicles typically used in the art which have been described for expression in a variety
  • Viral vectors especially adenoviral vectors can be complexed with a cationic amphiphile, such as a cationic lipid, polyL-lysine (PLL), and diethylaminoethyldextran (DELAE-dextran), which provide increased efficiency of viral infection of target cells (See, ⁇ ?.g.,PCT/US97/21496 filed Nov. 20, 1997, incorporated herein by reference).
  • AAV vectors such as those disclosed in U.S. Pat. Nos.
  • heterologous refers to a combination of elements not naturally occurring.
  • heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell.
  • the heterologous DNA includes a gene foreign to the cell.
  • a heterologous expression regulatory element is a such an element operatively associated with a different gene than the one it is operatively associated with in nature.
  • the 2AR and/or mGluR2 receptor genes are heterologous to the vector or vectors in which they are inserted for cloning or expression, and they are heterologous to a host cell containing such a vector, in which it is expressed, e.g., a CHO cell.
  • mutant and “mutation” mean any detectable change in genetic material, e.g., DNA, or any process, mechanism, or result of such a change. This includes gene mutations, in which the structure (e.g., DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g., protein or enzyme) expressed by a modified gene or DNA sequence.
  • variant may also be used to indicate a modified or altered gene, DNA sequence, enzyme, cell, etc., i.e., any kind of mutant.
  • Sequence-conservative variants of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position. Such variants can be used in expression of receptor subunits, e.g., where altered codon usage or insertion of a restriction site is desired.
  • “Function-conservative variants” are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like).
  • Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine.
  • Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm.
  • a “function- conservative variant” also includes a polypeptide or enzyme which has at least 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, most preferably at least 85%, and even more preferably at least 90%, and which has the same or substantially similar properties or functions as the native or parent protein or enzyme to which it is compared. Any of these algorithms can be used with defaults provided by the manufacturer, supplier, or provider or provider.
  • two amino acid sequences are "substantially homologous” or “substantially similar” when greater than 80% of the amino acids are identical, or greater than about 90% are similar (functionally identical).
  • the similar or homologous sequences are identified by alignment using, for example, any of the programs described above (BLAST, FASTA, etc.)
  • the present invention provides various screening assays for compounds that modulate the functionality or a property of a heteroreceptor complex, or the receptors that comprise the complex.
  • the assays of the invention are particularly advantageous by permitting rapid evaluation of cellular responses.
  • Biological assays which depend on testing perception, pain sensitivity, survival, or some other response in vivo require substantial amounts of time and resources to evaluate.
  • the present invention short-circuits the more tedious and time consuming biological assays.
  • the signal transduction assays can often be performed with very small amounts of material.
  • a screening assay of the invention makes use of the cells expressing receptor proteins (described above), various heteroreceptor ligands, and a candidate compound for testing.
  • the present invention contemplates screens for small molecule compounds, including peptides and peptidomimetics, and including receptor ligand analogs and mimics, as well as screens for natural compounds that bind to and agonize or antagonize heteroreceptor complexes in vitro.
  • Such agonists or antagonists may, for example, interfere in the phosphorylation or dephosphorylation of signal transduction proteins.
  • natural products libraries can be screened using assays of the invention for such molecules.
  • the term "compound” refers to any molecule or complex of more than one molecule that modulates heteroreceptor complex function.
  • the present invention contemplates screens for synthetic small molecule agents, chemical compounds, chemical complexes, and salts thereof as well as screens for natural products, such as plant extracts or materials obtained from fermentation broths.
  • Other molecules that can be identified using the screens of the invention include, for example, opioids, opiates, narcotics, proteins and peptide fragments, peptides, nucleic acids and oligonucleotides, carbohydrates, phospholipids and other lipid derivatives, steroids and steroid derivatives, prostaglandins and related arachadonic acid derivatives, etc.
  • Test compounds may be screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available, for example, from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal tracts are available from, e.g., Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or are readily producible.
  • test compounds are peptides or peptidomimetic compounds generated by rational drug design based on the structure of known serotonin and/or glutamate receptor agonists and antagonists, or derived from combinatorial libraries.
  • peptide is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other bonds, e.g., ester, ether, etc.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • peptides of the invention may comprise D-amino acids, a combination of D- and L- amino acids, and various "designer" amino acids (e.g., ⁇ -methyl amino acids, Ca- methyl amino acids, and N ⁇ -methyl amino acids, etc.) to convey special properties to peptides in the library. Additionally, by assigning specific amino acids at specific coupling steps, peptide libraries with ⁇ -helices, ⁇ -turns, ⁇ . -sheets, ⁇ -turns, and cyclic peptides can be generated. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
  • the coupling of the amino acids may be accomplished by techniques familiar to those in the art (Stewart and Young, 1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, 111.; Fields and Noble, 1990, Int. J. Pept. Protein Res. 35:161-214), or using automated synthesizers, such as sold by ABS.
  • Stepwart and Young, 1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, 111.; Fields and Noble, 1990, Int. J. Pept. Protein Res. 35:161-214 or using automated synthesizers, such as sold by ABS.
  • Intact cells expressing a heteroreceptor complex can be used in screening methods to identify candidate compounds.
  • a permanent cell line is established wherein cells of the cell line express the heteroreceptor complex, or cell membrane preparations are made from cells expressing the heteroreceptor complex.
  • cells including without limitation mammalian, insect, yeast, or bacterial cells
  • Identification of candidate compounds can be achieved using any suitable assay, including without limitation (i) assays that measure selective binding of test compounds; (ii) assays that measure the ability of a test compound to modify (i.e., inhibit or enhance) a measurable activity or function of one or more of the receptors or the receptor complex; (iii) assays that monitor receptor or receptor complex trafficking, e.g., internalization and re-expression; and (iv) assays that measure the ability of a compound to modify (i.e., inhibit or enhance) transcription of proteins induced by the receptors or receptor complex.
  • assays that measure selective binding of test compounds including without limitation (i) assays that measure selective binding of test compounds; (ii) assays that measure the ability of a test compound to modify (i.e., inhibit or enhance) a measurable activity or function of one or more of the receptors or the receptor complex; (iii) assays that monitor receptor or receptor complex trafficking, e.g., internalization and
  • the screening assay of the present invention may utilize a native cell line established from the cortex of a schizophrenic patient, or cells engineered to express a 2AR receptor protein and an mGluR2 receptor protein.
  • engineered cells include recombinant host cells, for example, PC 12 cells, CHO cells, HeLa cells, 293 and 293T cells (human kidney cells), COS cells, mouse primary myoblasts, NIH 3T3 cells, or Xenopus oocytes that have been transformed to express a 2AR receptor protein and an mGluR2 receptor protein.
  • the cells are engineered to express an epitope-tagged 2AR protein and an epitope-tagged mGluR2 protein.
  • heteroreceptor complex in cells engineered to express a 2AR receptor protein and an mGluR2 receptor protein, or in a native cell line, can be determined through the use of, for example, co- immunoprecipitation of the 2AR and mGluR2 receptors.
  • the expression of a heteroreceptor complex may also be determined through the use of bioluminesence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) methods for the detection of protein-protein interactions (described below).
  • BRET bioluminesence resonance energy transfer
  • FRET fluorescence resonance energy transfer
  • a compound of the invention may be identified through the use of a screening assay wherein a candidate compound is contacted to, for example, a cell expressing a 2AR receptor protein and an mGluR2 receptor protein.
  • a compound of the present invention may be identified when, upon binding to a first receptor protein of the heteroreceptor complex, for example mGluR2, the affinity of a ligand for the second receptor protein, for example 2AR, increases or decreases.
  • Such an increase or decrease in the ligand' s affinity for the second receptor may be measured, for example, by a competition assay between the ligand and a second ligand that has been detectably labeled (for example, radiolabeled, fluorescently labeled, or any other detectable label known in the art), for binding the second receptor.
  • detectably labeled for example, radiolabeled, fluorescently labeled, or any other detectable label known in the art
  • a compound of the invention may be identified through the use of a screening assay wherein a candidate compound is contacted to a cell expressing a 2AR receptor protein and an mGluR2 receptor protein, wherein binding of the compound to a first receptor protein of the heteroreceptor complex, for example mGluR2, increases or decreases the ability of a ligand to activate a signal transduction pathway upon binding to the second receptor of the heteroreceptor complex.
  • a screening assay wherein a candidate compound is contacted to a cell expressing a 2AR receptor protein and an mGluR2 receptor protein, wherein binding of the compound to a first receptor protein of the heteroreceptor complex, for example mGluR2, increases or decreases the ability of a ligand to activate a signal transduction pathway upon binding to the second receptor of the heteroreceptor complex.
  • a compound of the invention may be identified through the use of a screening assay wherein a candidate compound is contacted to a cell expressing a 2AR receptor protein and an mGluR2 receptor protein, wherein binding of the compound to a first receptor protein of the heteroreceptor complex, for example mGluR2, activates or inhibits a signal transduction pathway associated with a second receptor of the heteroreceptor complex.
  • a screening assay wherein a candidate compound is contacted to a cell expressing a 2AR receptor protein and an mGluR2 receptor protein, wherein binding of the compound to a first receptor protein of the heteroreceptor complex, for example mGluR2, activates or inhibits a signal transduction pathway associated with a second receptor of the heteroreceptor complex.
  • Agents according to the invention may be identified by screening in high- throughput assays, including without limitation cell-based or cell-free assays. It will be appreciated by those skilled in the art that different types of assays can be used to detect different types of agents. Several methods of automated assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period of time e.g., using a 96- well format). For literature references see, e.g., Beggs et al., 1999, J. Biol. Screening 4(3); Renate de Wit et al, 1998, J. Biol. Screening, 3(4); Fox et al., 1999, J. Biol.
  • Radioligand binding assays allow further characterization of hits from high throughput screens as well as analogs of heteroreceptor complex agonists and antagonists. Any ligand can be radiolabeled for direct binding assays, or alternatively, used in competitive binding assays.
  • G protein coupled receptors are coupled to a variety of heterotrimeric G proteins, which are comprised of ⁇ , ⁇ , and ⁇ subunits. Upon agonist binding to a GPCR at the cell surface, conformational changes occur within the agonist:GPCR complex, which lead to the dissociation of the G protein ⁇ subunit from the ⁇ subunits. The G ⁇ and G ⁇ ⁇ subunits then stimulate a variety of intracellular effectors, which transduce the extracellular signal to the inside of the cell.
  • Various signal transduction systems known to be coupled to GPCRs include adenylate cyclase, phospholipase C, phospholipase A 2 , sodium/hydrogen exchange, etc. Thus, measurements of intracellular calcium concentrations and adenylate cyclase activity, or any component of a signal transduction pathway, indicate whether a hit or test compound is functionally behaving as an agonist or antagonist at the heteroreceptor of the invention.
  • activation of a 2AR or an mGluR2 receptor signal transduction pathway can be determined by observing an increase in the activation of a G ⁇ q and/or a G ⁇ j protein.
  • activation of a 2AR receptor by a hallucinogenic compound activates both a G ⁇ q and a G ⁇ i proteins, while activation of a 2AR by a non-hallucinogenic compound only activates a G ⁇ q protein.
  • activation of a 2AR receptor and/or an mGluR2 receptor can be determined by observing an inhibition of adenylate cyclase and an inhibition in the synthesis of cAMP.
  • activation of a 2AR receptor and/or an mGluR2 receptor can be determined by observing an increase in the phosphorylation of phospholipase C, and the synthesis of inositol triphosphate (IP 3 ) and diacylglycerol (DAG).
  • IP 3 inositol triphosphate
  • DAG diacylglycerol
  • G-protein signal transduction is coupled to expression of a reporter gene, thus permitting a reporter gene screening assay.
  • activation of a G ⁇ q mediated signal transduction pathway can be determined by measuring an increase in the expression of c-fos.
  • Activation of a G ⁇ , mediated signal transduction pathway can be determined by measuring an increase in the expression an egr-1 and/or an egr-2 gene (Early growth Response 1 and 2 genes, respectively).
  • addition of a serotonin-like and/or a glutamate like compound increases the release of calcium from intracellular stores, increasing the ratio of dye: calcium complexes to free dye.
  • G 5 -coupled GPCRs stimulate adenylate cyclase.
  • Adenylate cyclase catalyzes the production of cyclic AMP (cAMP) from adenosine- 5'-triphosphate which, in turn, activates protein kinases.
  • G 1 -coupled GPCRs are also coupled to adenylate cyclase, however, agonist binding to these receptors results in the inhibition of adenylate cyclase and the subsequent inhibition of cAMP accumulation.
  • cells expressing G 1 - coupled receptors must first be stimulated to elevate cAMP levels.
  • activation of a 2AR and/or an mGluR2 receptor can be determined by observing an inhibition of adenylate cyclase, and an inhibition in the synthesis of cAMP.
  • the compounds of the present invention can also act as modulators of the heteroreceptor complex, wherein the compounds modulate the effect of a second compound's binding to a 2AR, mGluR2, or both components of the heteroreceptor complex.
  • the compound can modulate the effect of a 2AR agonist binding to the 2AR receptor component of the heteroreceptor complex.
  • An assay of a candidate compound's modulatory effect on the binding of a second compound to a 2AR, mGluR2, or both components of a heteroreceptor complex may therefore be used to identify a compounds of the invention.
  • the second compound e.g.
  • a 2AR and/or an mGluR2 agonist is a probe for determining the effect of the candidate compound's binding to the heteroreceptor complex (e.g. a 2AR or an mGluR2 or both receptor components of the complex) on the response normally elicited by the second compound (e.g. a 2AR and/or an mGluR2 agonist) binding to a 2AR or an mGluR2 or both receptor components of the complex.
  • the heteroreceptor complex e.g. a 2AR or an mGluR2 or both receptor components of the complex
  • this assay involves observing a change or modulation in the response of the hetroreceptor complex to a 2AR and/or an mGluR2 binding compound, for example, a 2AR and/or mGluR2 agonist, when a candidate compound of the invention is contacted to the heteroreceptor complex at the same time, before, or after the 2AR and/or mGluR2 binding compound is contacted to the heteroreceptor complex.
  • a 2AR and/or an mGluR2 binding compound for example, a 2AR and/or mGluR2 agonist
  • the second compound can be a 2AR agonist, for example, l-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), l-(2,5-dimethoxy-4- methylphenyl)-2-aminopropane (DOM), 1 -(2,5-dimethoxy-4-bromophenyl)-2- aminopropane (DOB), lysergic acid diethylamide (LSD), ergotamine, r-lisuride, s- lisuride, psilocin, mescalin, serotonin, or any other 2AR agonist known in the art.
  • DOI l-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane
  • DOM l-(2,5-dimethoxy-4- methylphenyl)-2-aminopropane
  • DOB 1 -(2,5-dimethoxy-4
  • the second compound can be an mGluR2 agonist, for example, (lR,4R,5S,6R)-4-Amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid
  • Screening assays can also be used in which the behavioral output of a live organism, for example, but not limited to, a mouse or rat, is measured to determine the effect of a compound on the heteroreceptor complex.
  • the administration of a hallucinogenic compound to mice induces an increase in locomotor activity, for example, an increase in head-twitching.
  • Compounds of the invention can be identified based on an increase or decrease in hallucinogenic induced locomotor activity.
  • a compound that binds to and activates an mGluR2 component of a heteroreceptor complex may reduce the hallucinogen induced head-twitching of a mouse.
  • the present invention provides numerous methods for detecting signals, including but not limited to, directly detecting phosphorylation of proteins using radioactive phosphorous compounds, indirectly detecting phosphorylation with antibodies specific for phosphorylated epitopes, or detecting signals from activated signal transduction proteins, such as gene expression.
  • directly detecting phosphorylation of proteins using radioactive phosphorous compounds include directly detecting phosphorylation with antibodies specific for phosphorylated epitopes, or detecting signals from activated signal transduction proteins, such as gene expression.
  • Gctq / ii or G ⁇ >2j3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and phospholipase C (Santa Cruz Biotechnology; Abeam Inc., Cambridge, MA, USA;
  • Reporter genes for use in the invention encode detectable proteins, include, but are not limited to, chloramphenicol transferase (CAT), ⁇ -galactosidase ( ⁇ -gal), luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), alkaline phosphatase, and other genes that can be detected, e.g., immunologically (by antibody assay).
  • CAT chloramphenicol transferase
  • ⁇ -gal ⁇ -galactosidase
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • alkaline phosphatase e.g., immunologically (by antibody assay).
  • the reporter gene signaling activation of a 2AR and/or an mGluR2 receptor can comprise a promoter and regulatory elements from a c-fos, egr-1, or egr-2 gene c fused to one of the reported genes described herein.
  • Such reporter genes can be made, for example, through homologous recombination, wherein the coding sequence for a reporter gene such as, for example, GFP, is interchanged with the coding sequence of, for example, a c-fos, egr-1, or egr-2 gene.
  • expression of the GFP reporter gene would be under the same temporal and spatial expressional control as that of a c-fos, egr-1 , or egr-2 gene.
  • a 2AR and or an mGluR2 receptor protein can be fused to a reporter gene such that the cellular or sub-cellular location of the receptor proteins can be observed, for example, to determine if the compounds of the invention induce internalization of the 2AR, mGluR2, or both components of a heteroreceptor complex.
  • the instant invention discloses the use of the Bioluminescence Resonance Energy Transfer (BRET) or the fluorescence resonance energy transfer (FRET) methods for the detection of protein-protein interactions, for example, to measure an increase in complex stabilization cause by the binding of a compound to the complex ⁇ see, e.g., Xu et al., Proc. Natl. Acad. Sci. USA 96:151- 156; Angers et al., 2000, Proc. Natl. Acad. Sci. USA, 97:3684-3689).
  • BRET Bioluminescence Resonance Energy Transfer
  • FRET fluorescence resonance energy transfer
  • BRET measures the transfer of energy between a luminescent donor ⁇ e.g., luciferase expressed as a fusion protein with one of the receptors) and a fluorescent acceptor ⁇ e.g., YFP expressed as a fusion protein with one of the receptors).
  • FRET measures the transfer of energy between a first fluorescent donor and a second fluorescent acceptor.
  • BRET or FRET can be used (i) to examine the formation of a complex between various receptors ⁇ i.e., serotonin and glutamate receptors) and (ii) to study the effect of agonist/antagonist binding at the level of the complex.
  • a protein-protein interaction can be determined by co- immunoprecipitation of the proteins.
  • a yeast screening assay useful for testing agonists and antagonists of mammalian G-protein coupled receptors, e.g., as disclosed in U.S. Pat. No. 5,482,832, can be used.
  • the serotonin and glutamate receptors which comprise the heteroreceptor complex of the invention, bind synergistically to combinations of ligands (non-linked together), e.g., agonists or antagonists for each of the serotonin and glutamate receptors.
  • the invention further provides such synergistic compositions, e.g., comprising combinations of the ligands described above with at least one member of such combination interacting with each subunit of the heteroreceptor.
  • the compounds and compositions of the invention can be formulated as pharmaceutical compositions by admixture with a pharmaceutically acceptable carrier or excipient.
  • the compounds or compositions are provided in a therapeutically effective amount to an animal in need of treatment therewith for relief of a neurological, neurodegenerative, or psychiatric brain disease.
  • a neurological, neurodegeenerative, or psychiatric brain disease that is treatable according to the methods of the invention can be drug abuse, schizophrenia, psychoses associated with schizophrenia, depression, anxiety, obsessive compulsive disorder, bi-polar disorder, neurological diseases associated with psychosis including Alzheimer's and Parkinson's disease, drug induced psychosis, a dysfunction of the central reward pathway, or some other condition.
  • the animal subject is preferably a human, but the compounds and compositions of the invention have application in veterinary medicine as well, e.g. , for the treatment of domesticated species such as canine, feline, and various other pets; farm animal species such as bovine, equine, ovine, caprine, porcine, etc.; wild animals, e.g., in the wild or in a zoological garden; and avian species, such as chickens, turkeys, quail, songbirds, etc.
  • domesticated species such as canine, feline, and various other pets
  • farm animal species such as bovine, equine, ovine, caprine, porcine, etc.
  • wild animals e.g., in the wild or in a zoological garden
  • avian species such as chickens, turkeys, quail, songbirds, etc.
  • phrases “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a human.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a human.
  • “pharmaceutically acceptable” means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin, 18th Edition, or other editions, hereby incorporated by reference in its entirety.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to reduce by at least about 15%, preferably by at least 50%, more preferably by at least 90%, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host. These parameters will depend on the severity of the condition being treated, other actions, such as diet modification, that are implemented, the weight, age, and sex of the subject, and other criteria, which can be readily determined according to standard good medical practice by those of skill in the art
  • the component or components of a pharmaceutical composition of the invention may be introduced parenterally; transmucosally, e.g., orally, nasally, or rectally; or transdermally.
  • Parenteral administration includes, but is not limited to, intravenous, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration.
  • the therapeutic compound can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., 1989,in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler eds., Liss: New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally Lopez-Berestein, ibid.). To reduce its systemic side effects, this may be a preferred method for introducing the compound.
  • the therapeutic compound can be delivered in a controlled or sustained release system.
  • a compound or composition may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
  • a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321 :574).
  • polymeric materials can be used (see Langer and Wise eds., 1974, Medical Applications of Controlled Release, CRC Press: Boca Raton, FIa; Smolen and Ball eds., 1984, Controlled Drug Bioavailability, Drug Product Design and Performance, Wiley, N. Y.; Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem., 23:61; Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol., 25:351; Howard et al., 9189, J.Neurosurg. 71 :105).
  • a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer (supra). Each of the above references are hereby incorporated by reference in their entirety.
  • [ 3 H]Ketanserin and [ 35 S]GTPyS were purchased from PerkinElmer Life and Analytical Sciences, Inc.
  • [ 3 H]LY341495 was purchased from American Radiolabeled Chemicals, Inc.
  • the injected doses were DOI, 2 mg/kg; DOM, 4 mg/kg; DOB, 1 mg/kg; LSD, 0.24 mg/kg; lisuride, 0.4 mg/kg; ergotamine, 0.5 mg/kg; LY379268, 15 mg/kg; LY341495, 6 mg/kg; clozapine, 25 mg/kg; and haloperidol, 1 mg/kg, unless otherwise indicated.
  • HEK293 Transient Transfection of HEK293 cells.
  • HEK293 were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) foetal bovine serum at 37°C in a 5% CO 2 humidified atmosphere. Transfection was performed using Lipofectamine 2000 reagent (Invitrogen) according to manufacturer's instructions.
  • HEK293 cells stably expressing human 2AR have been described previously (Ebersole et al., 2003, MoI Pharmacol 63:36-43; Gonzalez-Maeso, et al., 2003, J Neurosci 23:8836-43). Co-immunoprecipitation Studies.
  • Equal amounts of proteins were resolved by SDS- polyacrylamide gel electrophoresis. Detection of proteins by immunoblotting using anti-2AR (Santa Cruz Biotechnology), anti-mGluR2 and anti-mGluR3 (Abeam Inc.) in postmortem human brain, or anti-c-myc and anti-HA antibodies (Santa Cruz Biotechnology) in HEK293 was conducted using ECL system according to the manufacturer's recommendations.
  • Bioluminiscence Resonance Energy Transfer (BRET 2 ) in HEK293 live cells.
  • the human 2AR, serotonin 5-HT 2 c (2CR), mGluR2, and mGluR3 receptors with mutated stop codons were subcloned into the pi?luc and pGFP plasmids (PerkinElmer Life Sciences), such that Renilla luciferase (R ⁇ xc) and Green Fluorescent Protein (GFP 2 ) were present at the C-termini of the receptors. All sequences were confirmed by DNA sequencing.
  • transfected cells were washed with PBS, suspended to 1-2 x 10 6 cells/ml, and were treated with DeepBlueC Coelenterazine Substrate (5 ⁇ M final concentration; PerkinElmer Life Sciences). Equivalents amounts of total DNA comprised of various ratios of the i?luc- or GFP 2 -tagged receptors were transfected (James et al., 2006, Nat Methods 3:1001-6). Light emission was monitored by using a Fusion Universal Microplate Analyzer (PerkinElmer Life Sciences). A BRET 2 signal is defined as the light emitted by GFP 2 at 515 nm in response to the light emitted at 410 nm by R ⁇ nc in upon catalysis of DeepBlueC.
  • [ 35 S]GTPyS binding experiments were initiated by the addition of membranes containing 35 ⁇ g protein to an assay buffer (20 mM HEPES, 3 mM MgCl 2 , 100 mM NaCl, 0.2 mM ascorbic acid, and 0.5 nM [ 35 S]GTPyS) supplemented with 0.1 ⁇ M or 10 ⁇ M GDP for G ⁇ q/ ⁇ and Ga 1 , respectively, and containing the indicated concentration of ligands. Nonspecific binding was determined in the presence of 100 ⁇ M GTPyS.
  • an assay buffer (20 mM HEPES, 3 mM MgCl 2 , 100 mM NaCl, 0.2 mM ascorbic acid, and 0.5 nM [ 35 S]GTPyS) supplemented with 0.1 ⁇ M or 10 ⁇ M GDP for G ⁇ q/ ⁇ and Ga 1 , respectively, and containing the indicated concentration of ligands.
  • Nonspecific binding was determined in the presence of 100
  • Reactions were incubated for 30 min at 30°C, and were terminated by the addition of 0.5 ml of ice-cold buffer, containing 20 mM HEPES, 3 mM MgCl 2 , 100 mM NaCl, and 0.2 mM ascorbic acid.
  • the samples were centrifuged at 16,000 ⁇ g for 15 min at 4 0 C, and the resulting pellets resuspended in solubilization buffer (100 mM Tris, 20O mM NaCl, I mM EDTA, 1.25% Nonidet P-40) plus 0.2% sodium dodecylsulfate.
  • HA-tagged wild type human mGluR2 and mGluR3 constructs The rat mGluR5 signal peptide (SP) (Blahos et al., 1998, J Biol Chem 273:25765-9) along with an HA epitope tag was PCR amplified using primers NheI-HA_SP/S (5'- TTTTgctagcGAATTCCTTTCCTAAAATGG-3') and HA_SP-KpnI/A (5'- TTTTggtaccACGCGTGGCGTAGTCGGGTA-3') with pRK5 as template.
  • SP signal peptide
  • Wild type human mGluR2 and mGluR3 were amplified using primers MluI-hGRM2/S (5'- agctacgcgtAAGAAGGTGCTGACCCTGGA-3') hGRM2-XbaI/A (5'-
  • AAtctagaTCAAAGCGATGACGTTGTCGAG-3' KpnI-hGRM3/S (5'- acgtggtaccTTAGGGGACCATAACTTTCT-3')
  • hGRM3-XhoI/A 5'- acgtctcgagTCACAGAGATGAGGTGGTGG-3'
  • the rat mGluR5 signal peptide/HA epitope fragment was digested with Nhel and MIuI
  • the human mGluR2 fragment was digested with MIuI and Xbal, and were simultaneously subcloned into the Nhel and Xbal sites of pcDNA3.1 (Invitrogen, Carlsbad, CA) to yield the HA-tagged mGluR2 construct.
  • the rat mGluR5 signal peptide/HA fragment was digested with Nhel and Kpnl
  • the human mGluR2 PCR product was digested with Kpnl and Xhol, and were simultaneously subcloned into the Nhel and Xhol sites of pcDNA3.1 to give the HA-tagged mGluR2 construct.
  • TTTTCGGGGCACTTGCGAGTTTTGAAGGCGTACACAGTGC-S' The two fragments were annealed and re-amplified using primers hGRM2-1476/S and hGRM3TM5GRM2i3/A.
  • the third intracellular loop to the carboxyl terminal of the human mGluR2 was amplified using primers hGRM3TM5GRM2i3/S (5'- GCACTGTGTACGCCTTCAAAACTCGCAAGTGCCCCGAAAA-S') and hGRM2- Xbal/A. This fragment was then annealed with the previous PCR product and re- amplified using primers hGRM2-1476/S and hGRM2-XbaI/A.
  • the N terminal domain of the HA-tagged wild type human mGluR2 was released using Nhel and BstBI, the final PCR product was digested using BstBI and Xbal, and the two fragments were simultaneously subcloned into the Nhel and Xbal sites of pcDNA3.1.
  • TTTTCTGGGCACTTCCGCGTCTTGAAGGCATAAAGCGTGC-S' The two fragments were annealed and re-amplified using primers hGRM3-2541/S and hGRM2TM5GRM3i3/A.
  • the third intracellular loop to the carboxyl terminal of the human mGluR3 was amplified using primers hGRM2TM5GRM3i3/S (5'- GCACGCTTTATGCCTTCAAGACGCGGAAGTGCCCAGAAAA-S') and hGRM3- Xhol/A. This fragment was then annealed with the previous PCR product and re- amplified using primers hGRM3-2541/S and hGRM3-XhoI/A.
  • the N terminal domain of the HA-tagged wild type human mGluR3 was released using Nhel and Pstl, the final PCR product was digested using Pstl and Xhol, and the two fragments were simultaneously subcloned into the Nhel and Xhol sites of pcDNA3.1.
  • the N terminal domain of the HA-tagged wild type human mGluR3 was released using Nhel and Pstl, the final PCR product was digested using Pstl and Xhol, and the two fragments were simultaneously subcloned into the Nhel and Xhol sites of pcDNA3.1.
  • TM4,5 configuration deriving from atomic force microscopy of rhodopsin in native disk membranes as a template for the heteromer interface between 2AR and mGluR2 (Liang et al., 2003, J Biol Chem 278, 21655-62). This modeling was obtained with the assistance of the Insight II User Graphical Interface (Accelrys Inc.) on a graphics workstation.
  • Neuronal primary culture Primary cultures of cortical and thalamic neurons were prepared as previously described (Gonzalez-Maeso et alo., 2007, Neuron 53, 439-52).
  • mice Mouse brain samples. Experiments were performed as previously described on adult (8-12 weeks old) male 129S6/Sv mice. For experiments involving genetically modified mice, htr2A+/+ or htr2A+/- littermates were used as controls (Gonzalez- Maeso et alo., 2007, Neuron 53, 439-52; Chan et al., 2005, Nucleic Acids Res 33, el 61). Animals were housed at 12 h light/dark cycle at 23 °C with food and water ad libitum. The Institutional Animal Use and Care Committee approved all experimental procedures at Mount Sinai School of Medicine and Columbia University.
  • Fluorescence in situ hybridization FISH. Synthesis of modified DNA oligonucleotide probes, probe labeling, and fluorescence in situ hybridization was performed as previously described(Gonzalez-Maeso et alo., 2007, Neuron 53, 439-52; Chan et al., 2005, Nucleic Acids Res 33, el ⁇ l). (See Figure 10 for oligonucleotide probe sequences).
  • Quantitative real-time PCR Quantitative real-time PCR experiments were performed as previously described (Gonzalez-Maeso et alo., 2007, Neuron 53, 439-52). (See Figures 11 and 12 for primer pair sequences).
  • Behavioural Studies were performed as previously described (Gonzalez-Maeso et alo., 2007, Neuron 53, 439-52; Weisstaub et al., 2006, Science
  • Motor function was assessed using a computerized three-dimentional activity monitorin system (AccuScan Instruments).
  • the activity monitor has 32 infrared sensor pairs with 16 along each side spaced 2.5 cm apart.
  • the system determines motor activity based on the frequency of interruptions to infrared beams traversing the x, y and z planes. Total distance (cm) travelled and vertical activity were automatically determined from the interruptions of beams in the horizontal and vertical planes, respectively.
  • Brain Samples Human brains were obtained at autopsies performed in the Forensic Anatomical Institute, Bilbao, Spain. The study was developed in compliance with policies of research and ethical review boards for postmortem brain studies (Basque Institute of Legal Medicine, Spain).
  • DSM-IV Diagnostic and Statistical Manual of Mental Disorders
  • the toxicological assays were performed at the National Institute of Toxicology, Madrid, Spain, using a variety of standard procedures including radioimmunoassay, enzymatic immunoassay, high-performance liquid chromatography and gas chromatography-mass spectrometry. Controls for the present study were chosen among the collected brains on the basis, whenever possible, of the following cumulative criteria: (1) negative medical information on the presence of neuropsychiatric disorders or drug abuse; (2) appropriate gender, age and postmortem delay to match each subject in the schizophrenia group; (3) sudden and unexpected death (motor vehicle accidents); and (4) toxicological screening for psychotropic drugs with negative results except for ethanol.
  • Tissue pH is assumed to be an indicator of agonal status (Preece et al., 2003, Brain Res MoI Brain Res 118:60-71) .Thus, prolonged terminal hypoxia results in low tissular pH. It has been demonstrated that gene expression patterns are strongly dependent on tissue pH. Brief deaths, associated with accidents, cardiac events or asphyxia, generally had normal pH with minor influence on gene expression changes (Li et al., 2004, Hum MoI Genet 13:609- 16) . All schizophrenic and control subjects showed a sudden and rapid death without long agonal phase. The tissue storage period before assays did not differ between schizophrenic cases (82 + 9 months) and controls (85 + 10 months).
  • Specimens of prefrontal cortex were dissected at autopsy (0.5-1 g tissue) on an ice-cooled surface and immediately stored at -7O 0 C until membrane preparation.
  • the definitive pairs of antipsychotic-untreated schizophrenics and respective matched controls, and the definitive pairs of antipsychotic-treated schizophrenics and respective matched controls are shown in Figure 7.
  • Fluorescent in situ hybridization was used to determine whether mGluR2 or mGluR3 receptor subtypes are co-expressed by the same neurons as 2AR.
  • layer V mouse somatosensory cortex (SCx)
  • 2AR mRNA positive cells were mostly mGluR2 mRNA positive.
  • the level of expression in SCx was much lower for mGluR3 mRNA, which rarely co-localized with 2AR mRNA (Fig. 1).
  • Control studies validated assay sensitivity and specificity, and similar 2AR/mGluR2 mRNA co- localization was found in cortical primary cultures (Fig. 1). Translation of 2AR protein in cortical pyramidal neurons was found to be necessary for normal mGluR2 expression.
  • mice with globally disrupted 2AR expression showed reduced cortical mGluR2 binding and expression, while mice in which 2AR expression was selectively restored in cortical pyramidal neurons (Gonzalez-Maeso, J. et al., 2007, Neuron 53:439-52; Gonzalez-Maeso, J. et al., 2003, J Neurosci 23, 8836-43; Vollenweider, F. X., et al., 1998, Neuroreport 9:3897-902; Gouzoulis- Mayfrank, E. et al., 2005, Pharmacopsychiatry 38:301-11; Colpaert, F.
  • the agonist affinities for the 2AR and mGluR2/3 were decreased when receptor/G protein complexes were uncoupled by GTP ⁇ S (Fig. 3).
  • the glutamate agonist LY379268 (LY379) increased the affinity of all three hallucinogens studied for the 2AR binding site.
  • the 2AR agonist DOI decreased the affinity of the three mGluR2/3 agonists for the glutamate receptor binding site. The allosteric interactions observed were eliminated by antagonist for each modulator (Fig. 3).
  • niGluR2 modulates the G 1 protein coupling of the 2AR (Figs. 4 and 5) suggested that this complex might be important for hallucinogen signalling.
  • the induction of c-fos by hallucinogenic 2AR agonists or by structurally similar non-hallucinogenic 2AR agonists in vivo in mouse SCx and in cortical primary cultures (Fig. 5) was not affected by the mGluR2/3 agonist LY379.
  • the hallucinogen-specific induction of egr-2 was selectively blocked by LY379 in both mouse cortex in vivo and in primary cortical cultures (Fig. 5).
  • HTR head-twitch response
  • the neuropsychological effects of hallucinogenic drugs present commonalities with the psychosis of schizophrenia, and both conditions are accompanied by disruptions of cortical sensory processing (Vollenweider et al., 1998,
  • Schizophrenia is a brain disease that affects perception, emotion and cognition (GonzalezMaeso, J. et al. 2008, Nature 452, 9397).
  • the serotonin (5-HT) and glutamate (GIu) neurotransmitter systems are suspected in the etiology and pathophysiology of schizophrenia, as well as in the mechanism of action of antipsychotic drugs (Marek, G. J., 2004, Curr. Opin. Pharmacol. 4, 18-22; Carlsson, A. et al., 2001, Annu. Rev. Pharmacol. Toxicol. 41, 237-260; Kristiansen, L. V.,
  • a high affinity for the serotonin 5HT2A receptor (2AR) is a common characteristic of all atypical antipsychotics (Miyamoto, S. et al., 2005, MoI. Psychiatry 10, 79-104.) Hallucinogenic 2AR agonists, such as lysergic acid diethylamide (LSD) or psilocybin, produce a psychosislike syndrome in humans that resembles the first episodes of schizophrenia (Vollenweider, F. X., et al., 1998, Neuroreport 9, 3897-3902 and Geyer, M. A. et al., 2008, Trends Pharmacol. Sci.)
  • G proteincoupled receptors are the largest family of signalling proteins in the mammalian genome and the most common target for therapeutic drugs (Pierce, K. L. et al., 2002, Nat. Rev. MoI. Cell Biol. 3, 639-650.) These heptahelical membrane proteins activate heterotrimeric GTPbinding G proteins to modulate cell signalling. G proteins are grouped in four different families (G s , Gj /0 , G q/ n, and Gi 2 ) based on the degree of homology of the primary structure (Oldham, W. M. et al.,
  • GPCRs have been found to interact to form homo and heterocomplexes in the plasma membrane, but the effect of complex formation on signal transduction is not certain for most GPCRs (Terrillon, S. et al., 2004, Proc. Natl. Acad. Sci. U. S. A. 101, 1548-1553; Lopez-Gimenez, J. F. et al., 2007, MoI. Pharmacol. 71, 1015-1029; Guo, W. et al., 2008, EMBO J. 27, 2293- 2304; and Carriba, P. et al., 2008, Nat. Methods 5, 727-733).
  • the 2AR/mGluR2 complex is involved in the cellular responses induced by LSD-like drugs (Gonzalez-Maeso, J. et al., 2008, Nature 452, 93-97; and Gonzalez- Maeso, J. et al., 2007, Neuron 53, 439-452.)
  • LSD-like drugs Nonzalez-Maeso, J. et al., 2008, Nature 452, 93-97; and Gonzalez- Maeso, J. et al., 2007, Neuron 53, 439-452.
  • 5-hydroxytryptamine (serotonin, 5HT), 1- (2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and (+)-MK801 hydrogen maleate (MK801) were purchased from Sigma- Aldrich.
  • (lR,4R,5S,6R)-4-Amino-2- oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268) was obtained from Tocris Cookson Inc. [ 35 S]GTPyS was purchased form PerkinElmer Life and Analytical Sciences, Inc. [ H]LY341495 was purchased from American Radiolabeled Chemicals, Inc.
  • the injected doses (i.p.) were 5 mg/kg LY379268 and 0.5 mg/kg MK801.
  • the human mGluR3 and the mGluR3 ⁇ TM4,5 chimaera were digested with EcoRI and Notl and subcloned into pXOOM.
  • the human RGS2 (University of Missouri-Rolla, UMR) was PCR-amplified using the primers RGS2/S (TTTTggatccATGCAAAGTGCTATGTTCTT) and RGS2/A
  • RNA marker Invitrogen, Carlsbad, CA. Expression of proteins in oocytes was accomplished by injection of the desired amount of cRNA into Xenopus oocytes.
  • oocytes were injected with 1 ng of mGluR2, 2ng of mGluR2 ⁇ TM4,5, 2 ng of mGluR3, 2 ng of mGluR3 ⁇ TM4,5, 2 ng of 2AR, 2 ng of GIRK4*, 2 ng of IRK3, and 1 ng of PTX or 4 ng of RGS2, when present.
  • Oocytes were isolated and microinjected with equal volumes (50 nl), as previously described 52. All oocytes were maintained at 18°C and recordings were performed 14 days following injection adjusting for differences in translation kinetics of each construct.
  • Stable currents obtained at that voltage prior to perfusion of agonist were determined for each trace. Peak currents were measured and normalized to basal current to compensate for size variability in oocytes. Inwardly rectifying potassium currents through GIRK4* and IRK3 were obtained by clamping the cells at 80 mV. Basal currents were defined as the difference between inward currents obtained at 80 mV in the presence of 3 mM BaCl 2 in HK solution and those in the absence OfBa 2+ and measured for each trace.
  • Radioligand binding assays Mouse frontal cortex membrane preparations and radioligand binding assays were performed as previously described with minor modifications (Gonzalez-Maeso, J. et al., 2008, Nature 452, 93-97.)
  • Behavioural Studies were performed as previously described (Gonzalez-Maeso, J. et al., 2008, Nature 452, 93-97.)
  • G protein-coupled inwardly rectifying potassium channels (GIRK), a subfamily of Kir (inwardly rectifying K + ) channels (or Kir3), are known to be sensitive to the G ⁇ subunits of G, /o proteins (Logothetis, D. E. et al., 1987, Nature 325, 321-326.)
  • GIRK4* mutated version of Kir3.4
  • PLC phospholipase C
  • DAG diacylglycerol
  • IP 3 inositol triphosphate
  • ER endoplasmic reticulum
  • G q/ ⁇ -mediated PIP 2 hydrolysis can be detected by measuring changes in the currents through Kir 2.3 (IRK3), a potassium channel member of the Kir2 subfamily (Fig 17b) (Du, X.
  • 2AR allows mGluR2 activation to elicit ion-channel responses indicative of G q/ ⁇ signalling.
  • IRK3 channel cRNA was also injected with the receptors tested in each group of oocytes since, as opposed to calcium-activated chloride channels (Fig 17c), IRK3 channels are not endogenously expressed in Xenopus oocytes (Liu, B. et al., 2007, Eur. J. Pharmacol. 558, 21-26; and Wang, C. et al., 2008, MoI. Pharmacol.
  • mGluR3 which is closely related to mGluR2, is not capable of forming a heterocomplex with 2AR 13.
  • TM transmembrane
  • mGluR3 ⁇ TM4,5 the mGluR3 chimaera containing only this segment from the mGluR2 was capable of forming a receptor complex with the 2AR.
  • mGluR2 ⁇ TM4,5 did not show evidence of complex formation with the 2AR (Gonzalez-Maeso, J.
  • mGluR2/mGluR3 chimaeras were used to study whether formation of a complex between the mGluR2 and 2AR was required for the emergence of G q/ ⁇ activation by the mGluR2/3 agonist LY379268.
  • the functionality of mGluR2, mGluR3, mGluR2TM4,5 and mGluR3TM4,5 was assessed in Xenopus oocytes. While the various mGluR constructs differed in the magnitude of the currents generated, all were functionally coupled to Gj / o (Fig 24).
  • RGS2 GTPase activating protein
  • RGS2 suppresses G q/ ⁇ protein-mediated processes by driving activated G q/ ⁇ (G ⁇ q /n-GTP) proteins into their inactive form (Goiq/ii-GDP).
  • Pertussis Toxin (PTX) is a G protein signalling blocker whose specificity for the Gj/ 0 -pathway has been well established both in native (Logothetis, D. E. et al., 1987, Nature 325, 321-326; Pfaffmger, P. J.
  • FIG 21a shows representative traces of current flow through GIRK4* potassium channels (Fig 17a) measured in oocytes expressing mGluR2 and the channel.
  • the activity of the GIRK4* channel was increased (downward deflection in the trace from the initial steady state) and sustained in the presence of LY379268.
  • Noncompetitive NMDA receptor antagonists such as phencyclidine (PCP) and ketamine are used as pharmacological models for schizophrenia in animals because of their capacity in humans to evoke positive and negative symptoms as well as cognitive deficits resembling those seen in this disease (Kristiansen, L. V. et al., 2007, Curr. Opin. Pharmacol. 7, 48-55; and Morris, B. J. et al., 2005, Curr. Opin. Pharmacol. 5, 101-106.)
  • the potent and selective noncompetitive NMDA receptor antagonists MK801 can also elicit ketamine-like symptoms in healthy volunteers (Reimherr, F. W. et al., 1986, Psychopharmacol. Bull.
  • Behavioural responses induced in rodent models by ketamine-like drugs include increased activity, head weaving, deficits in paired pulse inhibition (PPI) and social interaction, and increased forced swim immobility.
  • PPI paired pulse inhibition
  • Activation of mGluR2, but not mGluR3, has been found to reduce the behavioural stereotypy and hyperlocomotion produced by NMDA antagonists (Spooren, W. P. et al., 2000, Eur. J. Pharmacol. 397, R12; Fell, M. J. et al., 2008, J. Pharmacol. Exp. Ther. 326, 209-217; and Woolley, M. L.
  • GPCR heterocomplexes can also exist between different classes of surface receptors that are each by themselves coupled to different families of heterotrimeric G proteins, and that these complexes may dramatically alter the signalling responses.
  • metabotropic glutamate agonist bind the 2AR/mGluR2 complex leading to activation of both G q/ ⁇ and Gj /0 proteins.
  • the 2AR/mGluR2 complex represents the first demonstration in living cells in which agonist binding to one receptor component of the heteromer results in activation of the G protein normally coupled only with the other receptor.
  • the serotonin2A receptor, 2AR classically couples to intracellular signaling via the heterotrimeric G q subtype G-protein, and the mGluR2 via the Gj/G 0 subtype G proteins.
  • Examples 1 and 2 described herein demonstrate that hallucinogenic drugs such as LSD act at the 2AR component of the 2AR/mGluR2 complex to activate both G, and G q , a response necessary for the hallucinogenic activity and head-twitch behavioral response in mice.
  • the hallucinogenic pattern of signaling elicited at this complex leads to a characteristic pattern of gene changes, including induction o ⁇ c-fos, egrl and egr2.
  • mGluR2/3 antipsychotic drugs act at the mGluR2 component of the complex to disrupt hallucinogen signaling and suppress the head twitch response to hallucinogens in mice and the induction of egrl and egr2, while allowing the induction of c-fos.
  • the c-fos response is also activated by non-hallucinogens such as lisuride acting at the 2AR site in the complex.
  • ERK A prolonged activation of ERK is the signal for differentiation (Santos, SD et al, 2007, Nat Cell Biol, 9: 324-30.) While EGF and NGF act at different receptor targets, the lessons of their signaling specificity are applicable to the 2AR/mGluR2 complex.
  • the critical element in causing a "hallucinogen" response or in an “antipsychotic” response must be in the precise temporal pattern and relative levels of regulation of G 1 and G q .
  • both atypical antipsychotics such as clozapine
  • mGluR antipsychotics act at the complex to modulate the pattern of G, and G q regulation elicited by hallucinogens in the normal brain as well as by glutamate and serotonin in the schizophrenic brain to reduce the generation of a "hallucinogenic" pattern of G, and G q signaling.
  • This "hallucinogenic" pattern is reflected in the precise timing and amplitude of Gi and Gq activation achieved at this complex by hallucinogens such as LSD.
  • Examples 1 and 2 herein also may provide insight into the neurochemical abnormalities of altered brain function in schizophrenia.
  • schizophrenia In contrast with other chronic brain diseases such as Parkinson's and Alzheimer's, schizophrenia lacks diagnostic neuropathological lesions and genetic animal models.
  • Several neurochemical hypotheses of schizophrenia have been proposed, among them abnormalities of serotonin and glutamate systems (Aghajanian, G. K. et al., 2000, Brain Res. Brain Res. Rev. 31, 302-312.)
  • the serotonin hypothesis which is suggested by the 2AR-dependent effects of hallucinogens such as LSD (Vollenweider, F. X. et al., 1998, Neuroreport 9, 3897-3902; Gonzalez-Maeso, J.
  • the 2AR/mGluR2 complex and its heretofore unique signalling mechanisms may contribute to the neuropsychological effects of LSD like drugs, the antipsychotic effects of two different classes of antischizophrenia drugs, and the disordered thought processes in the schizophrenic brain.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Neurology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

The present invention relates generally to heteroreceptor G-protein coupled receptors, and more specifically to a heteroreceptor complex comprising serotonin and glutamate receptors, as well as to methods for identifying modulators (agonists and antagonists) of such receptors. The invention also relates to identification of novel heteroreceptor ligands and synergistic compositions, which can provide strategies for relieving psychosis associated with schizophrenia or hallucinogenic drug use.

Description

HETEROOLIGOMERIC SEROTONIN/GLUTAMATE RECEPTOR
COMPLEX
GRANT INFORMATION
The subject matter of the invention was developed, at least in part, under National Institutes of Health Grant No. NIH POl DA012923. The United States Government has certain rights herein.
CROSS-REFERENCE TO RELATED APPLICATION
The present application claims the benefit of U.S. Provisional Application No. 61/026,721, filed February 6, 2008, and hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates generally to heteroreceptor complexes of G- protein coupled receptors, and more specifically to heteroreceptor complexes comprising serotonin and glutamate receptors, as well as to methods for identifying modulators (including agonists and antagonists) of such a receptor complex. The invention also relates to identification of novel heteroreceptor complex ligands and synergistic compositions of ligands for each receptor, which can provide strategies for relieving psychosis associated with schizophrenia or other brain diseases.
BACKGROUND
The psychosis associated with schizophrenia is characterized by alterations in sensory processing and perception (Freedman et al., 2003, N Engl J Med 349; Sawa et al., 2003, Science 296:692-5). Some antipsychotic drugs, such as the atypical antipsychotics, were identified by their high affinity for serotonin 5-HT2A receptors (2AR) (Lieberman, J. A. et al., 1998, Biol Psychiatry 44:1099-117; Miyamoto, S., et al., 2005, J. MoI Psychiatry 10:79-104). While the overall profile of atypical antipsychotics (e.g., clozapine, olanzapine) is superior to that of traditional agents (e.g., haloperidol), these agents still elicit significant side-effects, such as CNS depression, weight gain, and sexual dysfunction. These side effects reduce patient compliance, leading to relapses of illness and thus a negative impact on the life-long course of this disease. Clozapine, which is a very effective antipsychotic medication, has the additional sometimes fatal side effect of agranulcytosis, which severely limits its use and necessitates frequent blood tests when it is prescribed. Also, atypicals only minimally reverse many aspects of schizophrenia, such as negative symptoms, including mood, affect, and cognitive dysfunction. The discovery of new agents which could be used alone or in combination with other antipsychotic drugs to enhance their effectiveness at lower doses and/or increase their overall effectiveness against negative symptoms would be a considerable advance in the medical treatment of schizophrenia. Drugs that interact with metabotropic glutamate receptors (mGluR) also show potential for the treatment of schizophrenia (Aghajanian et al., 2000, Brain Res Brain Res Rev 31:302-12; Marek, 2004, Curr Opin Pharmacol 4:18-22; Patil et al., 2007, Nat Med 13:1102-1107). Hallucinogenic drugs, such as psilocybin and lysergic acid diethylamide (LSD), require the 2AR receptor and their effects resemble some of the core symptoms of schizophrenia (Gonzalez-Maeso, et al., 2007, Neuron 53:439-52; Gonzalez-Maeso, et al., 2003, J Neurosci 23:8836-43; Vollenweider, F. X., et al.,
1998, Neuroreport 9:3897-902; Gouzoulis-Mayfrank, E. et al., 2005, Pharmacopsychiatry 38:301-11; Colpaert et al., 2003, Nat Rev Drug Discov 2:315- 20). Additionally, the psychotomimetics PCP and PCP-like drugs (e.g., ketamine, MK-801) are non-competitive NMDA glutamate receptor antagonists (Anis et al., 1983, British Journal of Pharmacology 79:565). The glutamate hypothesis of schizophrenia is supported by the clinical observation that these compounds produce schizophrenia-like symptoms in volunteers and can worsen symptoms in people with schizophrenia (Halberstadt, 1995, Clinical Neuropharmacology 18:237; Krystal et al., 1999, Neuropsychopharmacology 22: S 143). Atypical antipsychotics have been shown to be active in the PCP animal model of schizophrenia, but are not fully effective in this model unless higher doses, which produce significant side effects such as CNS depression or motor performance impairment, are used (Cartmell et al.,
1999, Journal of Pharmacology and Experimental Therapeutics 291:161). Thus a need exists for the identification of drugs that are more effective at treating schizophrenia and that can be used without inducing the deleterious side effects of the atypical or the typical antipsychotics.
SUMMARY OF THE INVENTION The present invention relates to the discovery that metabotropic glutamate and serotonin receptors form functional heteroreceptor complexes. These receptor complexes can be exploited for high-throughput screening of compounds to identify heteroreceptor complex modulators (agonists and antagonists). The invention also relates to identification of novel heteroreceptor complex ligands and synergistic compositions, which can form the basis for therapeutic strategies to reduce psychosis associated with, for example, schizophrenia, mania, Alzheimer's disease, or drug induced psychosis.
The present invention provides an isolated heteroreceptor complex, which receptor complex comprises a serotonin receptor protein and a glutamate receptor protein. Both receptor proteins are expressed in the same type of cell. In one particular embodiment, the heteroreceptor complex includes the serotonin receptor 2A protein (5-HT2A receptor; 2AR) and the metabotropic glutamate receptor 2 protein (mGluR2). The invention further provides a recombinant host cell that expresses a functional heteroreceptor complex, which receptor complex comprises a 2AR receptor protein expressed from an expression vector introduced into the host cell, and an mGluR2 receptor protein expressed from an expression vector introduced into the host cell. Preferably, the host cell stably expresses both receptors. Also provided is a method of screening for compounds that modulate a property or function of a heteroreceptor complex as described above. This method comprises observing a change in a property or function of the heteroreceptor complex contacted with a candidate compound. For example, receptor trafficking (e.g., internalization), ligand binding, ligand specificity, or receptor complex activation can be altered in the presence of a test compound. In particular, the heteroreceptor complex will exhibit different affinities for various known and test ligands.
In one embodiment, the invention provides compounds and methods for identifying specific compounds that bind the complex with high affinity and modulate the activity of the complex, but that show absent or limited or indistinguishable modulatory activity or affinity for a 2AR and/or an mGluR2 receptor when expressed alone.
In another embodiment, the invention provides compounds and methods for identifying compounds that can bind and modulate the function of a 2AR and/or an mGluR2 receptor when expressed alone, but when the receptors are part of a heteroreceptor complex, the compounds have a different binding affinity and/or modulatory effect on the heteroreceptor complex.
In another embodiment, the invention provides compounds and methods for identifying compounds that can inhibit or prevent the formation of, or otherwise destabilize the presence of, a heteroreceptor complex.
In another embodiment, the invention provides compounds and methods for identifying compounds that can promote or induce the formation of, or otherwise stabilize the presence of, a heteroreceptor complex.
In one embodiment, the invention provides a method of increasing the binding affinity of a compound to a 2AR receptor of a heteroreceptor complex by introducing and binding a second compound to the mGluR2 receptor of the same heteroreceptor complex.
One embodiment of the present invention provides a method of increasing internalization of a 2AR, mGluR2 or both components of a heteroreceptor complex by introducing and binding of a compound to the 2AR and/or mGluR2 receptor of the heteroreceptor complex.
One embodiment of the present invention provides a method of decreasing internalization of a 2AR, mGluR2 or both components of a heteroreceptor complex by introducing and binding of a compound to the 2AR and/or mGluR2 receptor of the heteroreceptor complex.
One embodiment of the present invention provides a method of decreasing the binding affinity of a compound to a 2AR receptor of a heteroreceptor complex by introducing and binding a second compound to an mGluR2 receptor of the same heteroreceptor complex. One embodiment of the present invention provides a method of decreasing the ability of a compound to activate a 2AR signal transduction pathway upon binding a 2AR receoptor of a heteroreceptor complex by introducing and binding a second compound to an mGluR2 receptor of the same heteroreceptor complex.
One embodiment of the present invention provides a method of increasing the ability of a compound to activate a 2AR signal transduction pathway upon binding a 2AR receptor of a heteroreceptor complex by introducing and binding a second compound to an mGluR2 receptor of the same heteroreceptor complex.
One embodiment of the present invention provides a method of activating a 2AR signal transduction pathway by introducing and binding a compound to an mGluR2 receptor of a heteroreceptor complex, wherein the heteroreceptor complex includes a 2AR receptor and an mGluR2 receptor.
One embodiment of the present invention provides a method of inhibiting or reducing a 2AR signal transduction pathway by introducing and binding a compound to an mGluR2 receptor of a heteroreceptor complex, wherein the heteroreceptor complex includes a 2AR receptor and an mGluR2 receptor.
One embodiment of the present invention provides a method of increasing the binding affinity of a compound to an mGluR2 receptor of a heteroreceptor complex by introducing and binding a compound to a 2AR receptor of the same heteroreceptor complex.
One embodiment of the present invention provides a method of decreasing the binding affinity of a compound to an mGluR2 receptor of a heteroreceptor complex by introducing and binding a second compound to a 2AR receptor of the same heteroreceptor complex. One embodiment of the present invention provides a method of increasing the ability of a compound to activate an mGluR2 signal transduction pathway upon binding an mGluR2 receptor of a heteroreceptor complex by introducing and binding a second compound to a 2AR receptor of the same heteroreceptor complex.
One embodiment of the present invention provides a method of decreasing the ability of a compound to activate an mGluR2 signal transduction pathway upon binding an mGluR2 receptor of a heteroreceptor complex by introducing and binding a second compound to a 2AR receptor of the same heteroreceptor complex.
One embodiment of the present invention provides a method of activating an mGluR2 signal transduction pathway by introducing and binding a compound to a 2AR receptor of a heteroreceptor complex, wherein the heteroreceptor complex includes a 2AR receptor and an mGluR2 receptor.
One embodiment of the present invention provides a method of inhibiting or reducing an mGluR2 signal transduction pathway by introducing and binding a compound to a 2AR receptor of a heteroreceptor complex, wherein the heteroreceptor complex includes a 2AR receptor and an mGluR2 receptor.
One embodiment of the present invention provides a method of decreasing a hallucinogen induced head-twitch behavior in an organism by introducing and binding a compound to a heteroreceptor complex. One embodiment of the present invention provides a method of increasing a hallucinogen induced head-twitch behavior in an organism by introducing and binding a compound to a heteroreceptor complex.
The compounds and compositions of the invention have 2AR/mGluR2 heteroreceptor complex modulating properties and thus therapeutic potential. These compounds address the need that exists for more precise identification of effective compounds for treating a neurological, neurodegenerative, or psychiatric brain disease or disorder of the central nervous system. In particular, a neurological, neurodegenerative, or psychiatric brain disease or disorder that is treatable according to the methods of the invention can be, for example, drug abuse, schizophrenia, psychoses associated with schizophrenia, depression, anxiety, obsessive compulsive disorder, bi-polar disorder, neurological diseases associated with psychosis including Alzheimer's and Parkinson's disease, drug induced psychosis, or a dysfunction of the central reward pathway. The presence of a heteroreceptor complex comprising a serotonin 2A receptor and an mGluR2 receptor identifies an assay for developing more effective treatments of psychosis in general, and psychoses associated with schizophrenia in particular. Administering a therapeutically effective dose of a compound or a pharmaceutical composition of the invention is expected to have a therapeutic effect on such diseases and disorders.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure la-f shows that the 2AR and mGluR2 receptors co-localize and interact, a, 2AR and mGluR2, but not mGluR3, co-express in neurons. Scale, top=50 μm, bottom=10 μm. Nuclei are blue. Inset: co-expressing neuron, b, FISH for mGluR3 in thalamus. Scale, top=25μm, bottom=10μm. c, mRNA levels by real-time PCR (n=6 per group), d, Specific co-immunoprecipitation of 2AR and mGluR2 in duplicate human cortex samples (arrows), e. BRET shows specific 2AR and mGluR2 interaction in HEK293 cells. Data are mean + s.e.m. (n=3). The mGluR2/2AR curve is preferably fitted by a saturation curve, F test (pO.OOl). The other co-transfection datasets show linear correlations, f, [3H]Ketanserin displacement curves in mouse SCx membranes (top panels). 2AR agonist affinities were higher in the presence of mGluR2/3 agonist 10 μM LY379. [3H]LY341495 displacement curves (bottom panels). mGluR2/3 agonist affinities were lower in the presence of 2AR agonist 10 μM DOI.
Figure 2a-e shows the lower expression of mGluR2 in the absence of cortical 2AR. a. Schematic representation of htr2A+/+, htr2A-/-, htr2A-/-Emx-Cre, and htr2A-/-:Htt-Cre mice. Note that in htr2A-/-:Emc-Cre mice (cortical rescue), 2AR is only expressed in cortical pyramidal neurons, and in htr-/-:Htt-Cre mice (thalamic rescue) 2AR is only expressed in thalamic neurons, b, c. [3H]LY341495 binding saturation curves in mouse SCx membranes (n=6 per group). Bmax values were significantly lower in htr2A-/- mice (p < 0.001; student's t-test), and in htr2A-/-:Htt- Cre mice (p<0.001; ANOVA with Bonferroni's post hoc test), d. Expression of mGluR2 and mGluR3 mRNA in mouse SCx in htr2A+/+:Htt-Cre (black), htr2A-/- (white), htr2A+/- (blue), htr2A-/-:Emx-Cre (red), and htr2A-/-:Htt-Cre (green) mice assayed by qRT-PCR (n=6-12 per group). Expression level was significantly lower for mGluR2 in htr2A-f- mice (p<0.001; student's t-test), and in htr2A-/0:Htt-Cre mice (p<0.05; ANOVA with Bonferroni's post hoc test), e. Relative mRNA expression levels of mGluR in mouse SCx estimated by qRT-PCR.
Figure 3a-c shows (a) the [3H]Ketanserin binding displacement curves by DOI, DOM and DOM in mouse SCx membrane (top panels). Note that the affinity of DOI displacing [3H]Ketanserin binding was significantly higher in the presence of lOμM LY379, (see also b.). [3H]LY341495 binding displacement curves by LY379, DCG-IV and L-CCG-I in mouse SCx membranes (bottom panels). Note that the affinity of LY379, DCG-IV and L-CCG-I displacing [3H]LY341495 binding was significantly lower in the presence of lOμM DOI (see also c).
Figure 4a-m shows that mGluR2 transmembrane domains 4/5 mediate association with 2AR. a. mGluR2/mGluR3 chimeras studied, b. c-myc-2AR and HA- mGluR2/mGluR3 chimera co-immunoprecipitations. Cells separately expressing each construct were also mixed, c. 2AR competition binding in cells stably expressing 2AR and transfected with mGluR2/mGluR3 chimeras, d. FRET in cells expressing 2AR- eCFP and either mGluR2, mGluR3 or mGluR3ΔTM4,5 chimera, all tagged with eYFP. Pseudo-color images represent normalized values (FRETN). eCFP + eYFP (n=19); 2AR-eCFP + mGluR2-eYFP (n-43); 2AR-eCFP + mGluR3-eYFP (n=31); 2AR-eCFP + mGluR3ΔTM4,5-eYFP, (n=27). **p < 0.01; ANOVA with Dunnett's post hoc test. e. DOI-stimulated [35S]GTPyS binding in membranes followed by immunoprecipitation with anti-Gαq/n (top panels), or anti-Gα,]^ (bottom panels). Cells stably expressing 2AR were transfected with mGluR2, mGluR3 or mGluR3ΔTM4,5. The potency of DOI activating Gα,i;2,3 was significantly increased when the 2AR was co-expressed with either mGluR2 or mGluR3ΔTM4,5, an effect abolished by 10 μM LY379 (p<0.001 by F test). Data are mean+s.e.m. of three experiments performed in triplicate, f. Ribbon backbone representation of the transmembrane helices of the 2AR/mGluR2 heteromer model from the intracellular face. g-i. [3H] Ketanserin binding and [3H]LY341495 binding in HEK293 cells stably expressing 2AR and transfected with mock, mGluR2 or mGluR3. g. [3H] Ketanserin binding saturation curve in HEK293 cells stably expressing 2AR. h. [3H]LY341495 binding saturation curves in HEK293 cells stably expressing 2AR and transfected with mock (open squares), lμg (filled triangles), 3μg (inverted filled triangles), 6μg (filled diamonds), 12μg (filled circles), or 24μg mGluR3-eYFP (filled squares), or 24μg mGluR3-eYFP (open triangles). Note that [3H] Ketanserin and [3H]LY341495 Bmax values in mouse SCx were 572+/- 50 fmol/mg prot. and 2986+/- 64 fmol/mg prot, respectively, and that [3H] Ketanserin and [3H]LY341495 Bmax values in cortical primary cultures were 404+/- 12 fmol/prot. and 1246+/- 34 fmol/mg prot. respectively. i. [3H] Ketanserin displacement curves in HEK293 cells stably expressing 2AR and transfected with mock, 24 μg of mGluR2-eYFP (left panels), or 24μg mGluR3-eYFP (right panels), j-m. pharmacological parameters for experiments described in Fig. 4.
Figure 5a-g shows 2AR/mGluR2 complex-dependent modulation of cellular and behavioral responses, a, DOI-stimulated [35S]GTPyS binding in primary culture membranes followed by immunoprecipitation with anti-Gαq/π or anti-Gα,i,2,3 antibodies. DOI Ga11 ,23 activation potency was significantly decreased by 10 μM LY379. Data are mean±s.e.m. of three experiments performed in triplicate, b, FISH in mice injected with vehicle or 2 mg/kg DOI 15 min after being pre-injected with vehicle or 15 mg/kg LY379 (left panels), and in primary cultures treated with 10 μM DOI 15 min after being pre-treated with vehicle or 10 μM LY379 (bottom panels). Nuclei are blue. Scale, left=50 μm, right=10μm. c, Distance and vertical activity induced in htr2A+/+ and htr2A-/- mice by mGluR2/3 antagonist 6 mg/kg LY341495. In htr2A-/- mice, LY341495 effect on distance was reduced (p<0.05, Bonferroni's post hoc test of two-factor ANOVA), and on vertical activity was absent (n=30-32). d, e. Double label FISH was performed in SCx layers V and VI in mice injected (i.p.) with vehicle or 0.24 mg/kg LSD 15 min. after being pre-injected with vehicle or 15 mg/kg LY379. Red, green, and blue colors indicate 2AR, c-fos (d) or egr-2 (e), and nucleus (DAPI), respectively. Note that the induction of the hallucinogen signaling marker egr-2 is selectively attenuated by LY379 in mouse SCx. Scale bar, 60μm. f. Activation of mGluR2 inhibits that specific cellular response induced by 2AR agonists in mouse SCx. Dose-response curves of LY379 on cellular response induced by 2AR agonists in mouse SCx assayed by qRT-PCR. Mice were injected with vehicle, 2 mg/kg DOI, 4 mg/kg DOM, 1 mg/kg DOB, 0.24 mg/kg LSD, 0.4 mg/kg lisuride, or 0.5 mg/kg ergotamine 15 min after being pre-injected with vehicle or 15 mg/kg LY379 (n=4-12 per group). Note that the induction of the hallucinogenic genomic marker egr-2 is selectively attenuated by LY379. Data are means+/-s.e.m. Bonferroni's post hoc test of two-factor ANOVA. *p<0.05, **p<0.01, ***p<0.001. g. Activation of mGluR2 inhibits the specific cellular responses induced by 2AR agonists in cortical primary cultures. Cortical primary cultures were treated for 45 min. with vehicle, lOμM DOI, 10 μM LSD or 10 μM lisuride after being pre-treated for 15 min with vehicle or LY379 (n=4-12 per group). Note that the induction of the hallucinogenic genomic marker egr-2 is selectively attenuated by LY379. Data are means +/- s.e.m. Bonferroni's post hoc test of two-factor ANOVA. p<0.05, **p<0.01, ***p<0.001. Figure 6 shows the head twitch response in mice injected with vehicle, 2 mg/kg DOI or 0.24 mg/kg LSD 15 min after being pre-injected with 15 mg/kg LY379 (n=5-12 per group). Data are means +/- s.e.m. ANOVA with Bonferroni's post hoc test. *p<0.05, **p<0.01, ***p<0.001.
Figure 7a-g. shows that 2AR is increased and mGluR2 is decreased in schizophrenia, a, b, Frontal cortex membrane receptor binding assays from untreated schizophrenic (n = 13) and matched control subjects (n = 13). In schizophrenia, [3H]ketanserin binding was higher and [3H]LY341495 binding was lower (p< 0.05; Student's ?-test). c, d, Receptor binding in antipsychotic-treated schizophrenic (n == 12) and matched control subjects (n = 12). In treated schizophrenia, [3H]ketanserin binding was unaffected and [3H]LY341495 binding was lower (p < 0.05). e, mGluR2 mRNA expression is reduced in untreated schizophrenic subjects (n = 7) compared to matched control subjects (n = 7, p < 0.05, mean ± s.e.m). f. Demographic characteristics and antemortem diagnoses of cases of nontreated schizophrenic subjects, and their respective control subjects g. Demographic characteristics and antemortem diagnoses of cases of antipsychotic-treated schizophrenic subjects, and their respective control subjects.
Figure 8a-f shows that chronic clozapine modulates the expression of the components of the 2AR/mGluR2 complex in mouse SCx. Animals were chronically (21 days) injected with vehicle (black) or 25 mg/kg clozapine (red) and sacrificed 1 day after the last clozapine injection, a. [3H]Ketanserin binding in mouse SCx after vehicle or chronic clozapine (n=6 per group), b., c. [3H[LY341495 binding in htr2A+/+ (b) or htr2A-/- (c) mouse SCx after vehicle or chronic clozapine (n=6 per group), d. Expression of 2AR, mGluR2, and mGluR3 mRNA in mouse SCx assayed by qRT-PCR in htr2A+/+ and htr2A-/- mice after vehicle or chronic clozapine (n=6- 12 per group). Data are means+/- s.e.m. *p<0.05, **p<0.05, ***p<0.001; student's litest, e-f. Chronic haloperidol does not affect the expression of the components of the 2AR.mGluR2 in mouse SCx. Animals were chronically (21 days) injected with vehicle (black) or 1 mg/kg haloperidol (red) and sacrificed 1 day after the last haloperidol injection, e, [3H]Ketanserin binding in mouse SCx after vehicle or chronic haloperidol (n-=6 per group), f. [3H]LY341495 binding in mouse SCx after vehicle or chronic haloperidol (n=6 per group).
Figure 9a-d shows the age-related changes in [3H]Ketanserin (a,b) and [3H]LY341495 (c,d) binding to cortical membranes of control subjects, a, c. Representative saturation curves. Data correspond to a 21 year old subject (black) and an 86 year old subject (white), b, d. [3H]Ketanserin (b) and [3H]LY379268 (d) binding Bmax values expressed in linear relation to the age of control subjects. Estimated linear regressions are represented. Statistical values represent Pearson's correlation coefficients between binding Bmax values and age (n=35). Figure 10 shows the nucleic acid sequences of the oligonucleotide probes used for fluorescence in situ hybridization (FISH.)
Figure 11 shows the nucleic acid sequences of the primers pairs used for qRT- PCR of murine genes.
Figure 12 shows the nucleic acid sequences of the primers pairs used for qRT- PCR of human genes.
Figure 13 shows a sequence alignment comparison of nucleic acid sequences of the transmembrane regions of mGluR2, mGluR3, 2AR, β2-adrenergic receptor, and rhodopsin from different species. Figure 14 shows the evaluation of the specificity of FISH assay, a, FISH assay for 2AR and β-actin in htr2A+/+ and htr2A-/- mouse SCx. Red, green, and blue colours indicate 2^i?, β-actin, and nucleus (DAPI), respectively, b, Competition of 2AR, mGluR2 and mGluR3 hybridization by specific, unlabeled oligonucleotide probes. A FISH assay in mouse SCx (2AR and mGluR2) and in mouse thalamus (mGluR3) with the fluorescently labelled oligonucleotides used in Fig. 1 was performed with the inclusion of excess of unlabeled oligonucleotides in the hybridization buffers. The presence of specific unlabeled oligonucleotides completely eliminated the signal obtained with the fluorescently labeled oligonucleotide probes. Red, green, and blue colours indicate 2AR, mGluR2 or mGluR3, and nucleus (DAPI), respectively, c, Similar anatomical pattern of expression of mGluR2 in mouse SCx was obtained with two different sets of fluorescently labeled oligonucleotide probes, and with the combination of probe set 1 and probe set 2. Green, and blue colours indicate mGluR2 and nucleus (DAPI), respectively, d, Evaluation of FISH assay specificity using scrambled-sequence oligonucleotide probes. FISH was performed by using a mixture of five fluorescently-labeled scrambled oligonucleotide probes. Scale bar, 500 μm. See Fig. 11 for oligonucleotide sequences.
Figure 15 shows Intact HEK293 cells transiently transfected with (a) increasing amounts of mGluR2-i?luc or mGluR3-/?luc or (b) with increasing amounts of 2AR-GFP2, 2CR-GFP2 or pGFP2. The amount of each cDNA is noted. Donor (a) and acceptor (b) conjugate relative expression levels were monitored by measuring luminescence and fluorescence. Note that the signals detected are comparable for different donors and acceptors. Data from triplicates assays in a single experiment are displayed. Two further experiments produced similar results. Figure 16 shows characterization of mGluR2/mGluR3 chimeras, a, N- terminally HA-tagged mGluR2, mGluR3 and mGluR2/mGluR3 chimeras were expressed in HEK293 cells, fixed and stained with anti-HA antibody, b, [3H]LY341495 binding saturation curves in HEK293 cells transfected with mock, mGluR2, mGluR3 and mGluR2/mGluR3 chimeras. Note that the level of expression is comparable for the different constructs (see also Fig. 4). c, [3H]Ketanserin binding displacement curves by DOI in HEK293 cells stably expressing 2AR and transfected with mock mGluR2, mGluR3 and mGluR2/mGluR3 chimeras. Note that the 2AR affinity for DOI was decreased by mGluR2, ΔmGluR2, mGluR3ΔTMl-5 and mGluR3ΔTM4,5 co-expression, and was unaffected by mGluR3 and mGluR2ΔTM4,5 co-expression (see also Figs. 2 and 4).
Figure 17 shows activation of G protein pathways monitored through ion channels in Xenopus oocytes, (a) G,/0-signalling can be detected through GIRK4* channels. Upon stimulation of G,/o-coupled receptors Gα,/0 and Gβγ,/0 dissociate. βγ,/0 can then activate the channel increasing potassium influx under the experimental conditions that can be recorded. GIRK4* currents are blocked by barium. Gq/n- signalling can be detected through heterologously expressed IRK3 channels (b) and endogenous calcium-activated chloride currents (c). Upon Gqm -activation phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-biphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3). IP3 in turn mobilizes Ca2+ to the cytosol from the ER producing a characteristic peak current associated with the calcium-activated chloride currents. Additionally, PIP2 hydrolysis results in a decrease in IRK3 inward potassium current. Pertussis toxin (PTX) is known to specifically disrupt the function of G,/o proteins. RGS2 exhibits selectivity shutting down the Gq/π -dependent signalling.
Figure 18 shows calcium-activated chloride currents elicited in oocytes expressing mGluR2 alone, mGluR2 and 2AR together, or 2AR alone, (a) Representative traces obtained in response to 1 M glutamate agonist (LY379268) and I M serotonin. Stimulation with a selective agonist of mGluR2 elicited calcium- activated chloride currents in the presence of 2AR but not when expressed alone, (b) A statistical summary of measured peak currents (mean ± S.E.M.) following stimulation with L Y379268. Peak currents for 2AR/mGluR2 were larger than for the mGluR2 and 2AR groups (**p<0.001). (c) Data (mean ± S.E.M.) corresponding to the response to serotonin. Peak currents for 2AR/mGluR2 and 2AR were larger than for the mGluR2 group (**p<0.001).
Figure 19 shows the relative inhibition of potassium inward currents in oocytes expressing IRK3 together with mGluR2, 2AR or both receptors, (a) Representative barium-sensitive current traces of IRK3 (below the zero dashed line) corresponding to mGluR2 alone, mGluR2 and 2AR together and 2AR alone. The first two were recorded in response to IM glutamate agonist (LY379268) and the third in response to 1 M serotonin, (b) Statistical summary of current inhibition from basal level induced by LY379268 (black) and serotonin (white) (mean ± S.E.M.). Values were normalized to the maximum inhibition obtained by 2AR stimulation with serotonin. Results indicate a larger decrease in IRK3 inward current for the 2AR/mGluR2 group than for the negative control (mGluR2) but smaller than the maximum inhibition obtained through 2AR stimulation (**p<0.001).
Figure 20 shows calcium-activated chloride currents in oocytes expressing 2AR together with mGluR2, mGluR2ΔTM4,5, mGluR3 and mGluR3ΔTM4,5. (a) Representative traces obtained in response to 1 M glutamate agonist (LY379268) and 1 M serotonin. Replacement of TM4,5 from mGluR3 into mGluR2, which interferes with formation of a complex with the 2AR, did not yield calcium-activated chloride currents. Conversely, substituting TM4,5 from mGluR2 in mGluR3, which allows complex formation with the 2AR, enabled mGluR3 to exhibit crossactivation. (b) Peak currents (mean ± S.E.M.) following LY379268 stimulation were larger for mGluR2 and mGluR3ΔTM4,5 than for the mGluR2ΔTM4,5 and mGluR3 groups (**p<0.001). (c) Data (mean ± S.E.M.) corresponding to the response to serotonin. Peak currents were not significantly different between groups (p=0.6710). Figure 21 shows changes in Qi/o-sensitive inward potassium currents measured in oocytes expressing mGluR2, GIRK4* and PTX or RGS2. (a) Representative barium-sensitive traces obtained in response to 1 M glutamate agonist (LY379268). PTX reduced the basal current and abolished agonist-induced effects, whereas RGS2 did not affect current levels, (b) Summary (mean ± S.E.M.) of measured basal currents. PTX significantly decreased currents when compared to the untreated (control) and RGS2-treated groups (**p<0.001) (c) Data (mean ± S.E.M.) corresponding to LY379268 agonist-induced response. PTX treatment completely removed agonist-induced currents when compared to the untreated and RGS2-treated groups (**p<0.001). Figure 22 shows the effects of G,/o-blocking PTX (a) and Gq/π -blocking
RGS2 (b) on calcium-activated chloride currents measured in oocytes expressing mGluR2 alone, mGluR2 and 2AR together, or 2AR alone. Representative traces obtained in response to 1 M glutamate agonist (LY379268) and 1 M serotonin are depicted in each panel. Activation of calcium-activated chloride currents by stimulation of mGluR2 was abolished by RGS2 and was insensitive to the presence of PTX confirming Gi/oGq/11 crossactivation. (c) Data corresponding to measured peak currents (mean ± S.E.M.) following LY379268 stimulation. Peak currents for the 2AR/mGluR2 group treated with PTX were larger than the rest of the groups (**p<0.001). (d) Summary of data (mean ± S.E.M.) corresponding to the response to serotonin. PTXtreated groups expressing 2AR/mGluR2 and 2AR elicited larger peak currents than the other groups (**p<0.001). (e) Representative signalling scheme of the Gi/oGq/11 crosstalk phenomenon through the 2AR/mGluR2 complex.
Figure 23 shows actions of LY379268 in wild-type (htr2A+/+) and 2AR null- mutant mice (htr2A-/-). (a) [3H]LY341495 binding displacement curves by LY379268 in mouse frontal cortex membranes. Note that the high affinity binding site for LY379268 displacing [3H]LY341495 binding was abolished by DOI at 10 M in wild- type mice, and undetected in 2AR null-mutant mice, (b) LY379268stimulated [35S]GTPγS binding in wild-type and 2AR null-mutant mouse frontal cortex membranes followed by immunoprecipitation with anti-Gα,1)23 (left) or antiGαq/n (right) antibodies (white, vehicle; black, LY379268). Activation of Gαq/π by glutamate agonist L Y379268 (IM) was abolished in htr2A -/- mice. Data are mean ± S.E.M. (*p<0.05; **p <0.001). (c) The effect of the mGluR2/3 agonist LY379268 blocking MK801 -dependent locomotion is absent in 2AR null-mutant mice. Upper panels depict the time course of MK801 -induced locomotion measured in 5 min blocks. Time of injections is indicated by arrows. Lower panels summarize the total MK801 -induced locomotion as a summation of horizontal activity from t=30 min to t=120 min. Mice were administered LY379268 (5 mg/kg), or vehicle followed by MK801 (0.5 mg/kg) or vehicle. Data are mean ± S.E.M. (**p<0.001) (N.S.,not significant).
Figure 24 shows G,/0-sensitive inward potassium currents measured in oocytes expressing GIRK4* together with mGluR2, mGluR2, mGluR2ΔTM4,5, mGluR3 and mGluR3ΔTM4,5. (a) Representative barium-sensitive traces obtained in response to IM glutamate agonist (LY379268). mGluR2, mGluR2ΔTM4,5, mGluR3 and mGluR3ΔTM4,5 were functional as G,/0-coupled GPCRs. These currents were blocked by barium, (b) Statistical summary of basal current (mean ± S.E.M.). Values indicate no significant difference in basal current between groups. (p=0.2419). (c) Statistical summary of glutamate agonist-induced current (mean ± S.E.M.). Results indicate significant Gi/oa-ctivation for all constructs compared to GIRK4* alone (Control) (**p<0.001).
Figure 25 shows LY379268 displacement of [3H]LY341495 binding was performed in the absence (vehicle) or in the presence of DOI (10 M). Competition curves were analyzed by nonlinear regression to derive dissociation constants for the high (Ki-high) and the low (K1-IOw) affinity states of the receptor. % High refers to the percentage of high-affinity binding sites as calculated from nonlinear fitting. Competition curves of [3H]ketanserin binding showed that the affinity of DOI for the 2AR was increased by 10 M LY379268 (data not shown). Values are best fit ± S.E. of 3-5 experiments performed in duplicate/triplicate. One-site model or two-site model as a better description of the data was determined by F test. Two-site model, p=0.001. NA, two-site model not applicable (p>0.05).
DETAILED DESCRIPTION
The present invention provides for a heteroreceptor complex comprising a serotonin receptor and a glutamate receptor, and methods of screening for compounds that can bind to and/or modulate the activity of the receptor complex and/or the serotonin and/or the glutamate receptors which form the receptor complex.
The invention is based, in part, on biochemical and pharmacological evidence for the physical and functional association of a fully functional serotonin receptor (5- HT2A receptor; 2AR), and a fully functional glutamate receptor (mGluR2 receptor). This discovery represents the first observation of a heteroreceptor made up of a class A and a class C type receptors. Furthermore, as this discovery demonstrates, when a 2AR and an mGluR2 receptor are comprised in a heteroreceptor complex, activation of the mGluR2 receptor inhibits activation of a 2AR Gαi mediated signal transduction pathway upon the binding of a hallucinogenic compound to the 2AR receptor. Additionally, the discovery of the present invention also demonstrates that when a 2AR and an mGluR2 receptor are comprised in a heteroreceptor complex, binding of an agonist to the mGluR2 receptor activates a 2AR Gαq/n mediated signal transduction pathway. Such an effect can occur in the absence of the binding of an agonist to the 2AR receptor. Thus, the present invention advantageously provides for the development both of more potent therapeutic agents and of a better understanding of the molecular basis of serotonin and glutamate receptor activity.
Discovery of this receptor complex opens the door to the preparation of synergistic compositions of individual receptor ligands, compounds that bind to the heteroreceptor complex and modulate its activity or stability but that do not bind to either of the two individual receptor components (2AR, mGluR2) when expressed alone, and compounds that bind to a 2AR and/or an mGluR2 receptor when expressed alone, but have a different binding affinity and/or modulatory effect on the receptors when they are part of a heteroreceptor complex. Certain experimental results underlie the present invention: identification of the presence of 2AR and mGluR2 receptors in a single heteroreceptor complex, and the discovery that activation of the mGluR2 receptor of the heteroreceptor complex attenuates the activation of the complex's 2AR receptor by hallucinogenic 2AR agonists. Furthermore, it has been discovered that in the postmortem brains of young untreated schizophrenics, 2AR is expressed at higher densities, and mGluR2 is expressed at lower densities, than in the brains of non-schizophrenic individuals.
The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them.
The term "serotonin receptor" refers to a receptor that can bind, and is activated, by serotonin. Serotonin receptors include the members of the serotonin receptor families 5-HTi, 5-HT2, 5-HT3, 5-HT4, 5-HT5A, and 5-HT7; which are G- protein coupled receptors. For example, these include the specific serotonin receptors 5-HTIA, 5-HTIB, 5-HT1 D, 5-HTiE, 5-HTi F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4, 5- HT5A, 5-HT6, and 5-HT7. In a specific embodiment of the invention, the serotonin receptor is the 5-HT2A serotonin receptor (also referred to as 2AR). In preferred non-limiting embodiments, the serotonin receptor is a human 5-
HT2A serotonin receptor. The human 5-HT2A serotonin receptor is preferably encoded by the Homo sapiens 5-hydroxytryptamine (serotonin) receptor 2 A (HTR2A) gene (GenBank accession number NM 000621), or any nucleic acid which encodes a human 5-HT2A polypeptide (for example, as defined by GenBank accession No. NP_000612.) In other non-limiting embodiments, the serotonin receptor is a mouse 5-HT2A serotonin receptor. The mouse 5-HT2A serotonin receptor is preferably encoded by the Mus musculus 5-hydroxytryptamine (serotonin) receptor 2 A (Htr2a) gene (GenBank accession number NM_172812), or any nucleic acid which encodes a mouse 5-HT2A polypeptide (for example, as defined by GenBankaccession No. NP_766400.)
The term "glutamate receptor" refers to a receptor that can bind, and is activated, by glutamate. The glutamate receptor can be a metabotropic glutamate receptor. For example, metabotropic glutamate receptors include receptors from glutamate receptor families I, II, and III. These receptor families include the glutamate receptors mGluRi and mGluRs (family I); InGIuR2 and mGluR3 (family II); and HiGIuR4, mGluR6, mGluR7, and mGluRg (family III). In a specific embodiment of the invention, the glutamate receptor is the mGluR2 receptor.
In preferred non-limiting embodiments, the glutamate receptor is a human mGluR2 glutamate receptor. The human mGluR2 glutamate receptor is preferably encoded by the Homo sapiens glutamate receptor, metabotropic 2 (GRM2) gene (GenBank accession number NM_000839), or any nucleic acid which encodes a human mGluR2 polypeptide (for example, as defined by GenBank accession NPJ3OO83O.) In other non-limiting embodiments, the glutamate receptor is a mouse mGluR2 glutamate receptor. The mouse mGluR2 glutamate receptor may be encoded by the Mus musculus G protein-coupled receptor, family C, group 1, member B (Grm2) gene (GenBank accession number XM_001475814), or any nucleic acid which encodes a mouse mGluR2 polypeptide (for example, as defined by GenBankaccession XPJ)01475864.) The term "heteroreceptor complex" or "receptor complex" or "complex" refers to a G-protein coupled receptor (GPCR) complex made of at least two GPCR molecules with at least one mGluR2 receptor subunit and at least one 2AR receptor subunit.
Receptors are "expressed endogenously" in a type of cell that expressed that receptor in healthy normal organisms, for example, a mammal, such as a human, mouse, or rat, in vivo or in an a cell line in vitro. In both cases, the cells have not undergone any genetic modification resulting in expression of the receptor, i.e., receptor expression does not depend on such genetic modification. In accordance with this invention, a cell may endogenously express either the serotonin or the glutamate receptor, or it may express both receptors in the same type of cell. In one specific non- limiting example, endogenous co-expression of a serotonin receptor with a glutamate receptor invovles the co-expression of the 2AR serotonin receptor and the mGluR2 glutamate receptor in the same cell. Such cells include cortical primary cultures as well as membrane preparations from brain, for example, a mouse or rat brain, or any other organism.
The term "function," or "property," or "functional property" refers to an ability to accomplish at least one of the following: bind ligand; bind a selective ligand; bind a non-selective ligand selectively; bind a bifunctional, bi-specifϊc ligand; activate a G- protein mediated signal transduction pathway upon binding of a ligand; attenuate or inhibit activation of a G-protein mediated signal transduction pathway upon binding of a ligand; induce internalization of a receptor, for example, a 2AR and/or an mGluR2 receptor component of a heteroreceptor complex, or a receptor complex; induce formation or stabilize the presence of a heteroreceptor complex, inhibit the formation or destabilize the presence of a heteroreceptor complex, or any combination thereof. Such ability or abilities can be demonstrated in a cell based or cell-free system, for example, in a cell membrane preparation, or in vivo, including a transgenic animal system. The term also refers to an ability to modulate a behavior, for example a locomotor activity, for example, a hallucinogen induced head-twitch behavior.
A "synergistically effective amount" of a ligand is an amount able to modulate a heteroreceptor complex's activity or function wherein the binding of one receptor of the complex increases or decreases the "functionality" of another receptor of the complex. In one non-limiting example, the binding of one receptor of the complex to ligand A reduces the ability of ligand B to bind to and/or activate a second receptor of the complex.
As used herein, the term "antagonist" refers to any molecule that binds to a protein and either partially or completely blocks, inhibits, reduces, or neutralizes the function, activity or activation of the protein, for example, a receptor protein. The term "agonist" refers to any molecule that binds to a protein and at least partially increases, enhances, or restores the function or activity of the protein, for example, a receptor protein. The term "mixed agonist" or "mixed agonist/antagonist" refers to any molecule that binds to binds to a protein and either partially or completely blocks, inhibits, reduces, or neutralizes some function, activity or activation of the protein, and at least partially increases, enhances, or restores some function or activity of the protein, for example, a receptor protein.
In one non-limiting embodiment, the compounds of the invention are specific ligands for the hetroreceptor complex.
In another non-limiting embodiment, the compounds of the invention are specific ligands for the serotonin and/or glutamate receptor that comprise the heteroreceptor complex.
Furthermore, the term "synergistic" can be used to describe an activity that two ligands have together that neither one has alone or in the absence of the other. The term "signal transduction pathway" as used in this invention refers to the intracellular mechanism by which a serotonin or glutamate or other ligand of a receptor or the heteroreceptor complex induces an alteration of cell function or activity. A key feature of the signal transduction pathway dissected herein is activation of G-protein coupled signaling, such as, for example, cAMP production, cAMP inhibition, or phospholipase C-β activation, resulting in further signal transduction, including, for example, MAPK phosphorylation.
The term "element of a signal transduction pathway" refers to a signal transduction factor that is activated or inhibited as a result of ligand binding to a receptor or the heteroreceptor complex. In accordance with the present invention, elements of the signal transduction pathway include G-proteins, for example, Gαq or
Gαi, cAMP, MAPK, etc. A "signal" in such a pathway can refer to activation, inhibition, increase the amount of, or decrease the amount of an element (or factor) in the pathway. For example, reduction of cAMP is a signal of an agonist-induced serotonin receptor signal transduction pathway. Generally, activation of one of these factors involves phosphorylation of one or more proteins.
In one non-limiting embodiment of the invention, the introduction and binding of a compound (for example, an agonist or antagonist) to one of the receptor components of the heteroreceptor complex increases or decreases the affinity of a second compound for a second receptor type of the complex. For example, binding of a ligand to 2AR may increase or decrease the affinity of a ligand for mGluR2.
Similarly, binding of a ligand to mGluR2 may increase or decrease the affinity of a ligand for 2AR. Although not bound by any particular explanation or theory, it is believed that binding of one compound to the heteroreceptor complex alters the conformation or otherwise facilitate recognition and binding of the second compound.
In another non-limiting embodiment, the introduction and binding of a compound (for example, an agonist or antagonist) to one receptor component of the heteroreceptor complex decreases the ability of a second compound to activate a second receptor type of the complex. For example, binding of a ligand to 2AR may decrease the ability of a ligand for mGluR2 to activate mGluR2. Similarly, binding of a ligand to mGluR2 may decrease the ability of a ligand for 2AR to activate 2AR.
In another non-limiting embodiment, the introduction and binding of a compound (for example, an agonist or antagonist) to one receptor component of the heteroreceptor complex increases the ability of a second compound to activate a second receptor type of the complex. For example, binding of a ligand to 2AR may increase the ability of a ligand for mGluR2 to activate mGluR2. Similarly, binding of a ligand to mGluR2 may increase the ability of a ligand for 2AR to activate 2AR.
In one non-limiting example, the introduction and binding of a hallucinogenic compound to a 2AR receptor activates a Gαj and a Gαq/i i mediated signal transduction pathway. When the 2AR comprises a heteroreceptor complex with niGluR2, activation of mGluR2 by an agonist inhibits activation of the Gαj mediated signal transduction pathway by a hallucinogenic compound binding to the 2AR.
In another non-limiting embodiment, the introduction and binding of a compound (for example, an agonist or antagonist) to one receptor component of the heteroreceptor complex activates a second receptor type of the complex. For example, binding of a ligand to mGluR2 may activate 2AR. Similarly, binding of a ligand to 2AR may activate mGluR2.
In another non-limiting embodiment, the introduction and binding of a compound (for example, an agonist or antagonist) to one receptor component of the heteroreceptor complex inhibits or reduces the activity of a second receptor type of the complex. For example, binding of a ligand to mGluR2 inhibits or reduces activity of 2AR. Similarly, binding of a ligand to 2AR inhibits or reduces activity of mGluR2.
In another non-limiting embodiment, a compound of the invention binds to and modulates the property of a heteroreceptor complex, but has no affinity for, or a limited affinity for, either a 2AR and/or an mGluR2 receptor when expressed alone.
In another non-limiting embodiment, when a compound of the invention is introduced and binds to a 2AR and/or an mGluR2 receptor in a complex, the compound has a modulatory effect on the complex that can not be detected, or does not occur, when the compound binds to the 2AR and/or mGluR2 receptors when expressed alone. For example, the binding of an agonist to the mGluR2 receptor of the complex decreases the activation of a Gα; -protein signal transduction pathway when a hallucinogenic agonist binds to the 2AR receptor of the complex.
In yet another non-limiting embodiment, the compounds identified according to the methods of the invention increase or decrease the stability of the complex. For example, a compound that increases the stability of the complex can be a chaperone, wherein the chaperone has one or more of the following effects: (i) enhancing the formation of a stable molecular conformation of the complex; (ii) enhances proper trafficking of the complex, or the receptors that comprise the complex, from the ER to another cellular location, preferably a native cellular location, i.e., preventing ER- associated degradation of the complex, or the receptors that comprise the complex; (iii) preventing aggregation of conformational^ unstable, i.e., misfolded proteins; (iv) restoring or enhancing at least partial wild-type function, stability, and/or activity of the complex; and/or (v) improving the phenotype or function of the cell harboring the complex.
The term "inhibitor" is used herein to refer to a compound that can block or reduce the level of signaling in a signal transduction pathway described herein. Such an inhibitor may block the pathway at any point, from blocking binding of ligand to receptor to blocking function of intracellular signals. Preferably, an inhibitor discovered in accordance with the invention is specific for signals of heteroreceptor complex-induced signaling.
"Screening" refers to a process of testing one or a plurality of compounds (including a library of compounds) for some activity. A "screen" is a test system for screening. Screens can be primary, i.e., an initial selection process, or secondary, e.g., to confirm that a compound selected in a primary screen (such as a binding assay) functions as desired (such as in a signal transduction assay). Screening permits the more rapid elimination of irrelevant or non-functional compounds, and thus selection of more relevant compounds for further testing and development. "High throughput screening" involves the automation and robotization of screening systems to rapidly screen a large number of compounds for a desired activity. Screens are discussed in greater detail below.
As used herein, the term "isolated" means that the referenced material is removed from its native environment, e.g., a cell. Thus, an isolated biological material can be free of some or all cellular components, i.e., components of the cells in which the native material occurs naturally (e.g., cytoplasmic or membrane component). A material shall be deemed isolated if it is present in a cell extract or if it is present in a heterologous cell or cell extract. In the case of nucleic acid molecules, an isolated nucleic acid fragment includes a PCR product, an isolated mRNA, a cDNA, or a restriction fragment. In another embodiment, an isolated nucleic acid fragment is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined or proximal to non-coding regions (but may be joined to its native regulatory regions or portions thereof), or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome. In yet another embodiment, the isolated nucleic acid fragment lacks one or more introns. Isolated nucleic acid molecules include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like, i.e., when it forms part of a chimeric recombinant nucleic acid construct. Thus, in a specific embodiment, a recombinant nucleic acid is an isolated nucleic acid fragment. An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane- associated protein. An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism. An isolated material may be, but need not be, purified.
The term "purified" as used herein refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated and/or unwanted materials, i.e., contaminants, including native materials from which the material is obtained. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell. As used herein, the term "substantially free" is used operationally, in the context of analytical testing of the material. Preferably, purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art. Methods for purification are well-known in the art.
The terms "about" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated. Molecular Biology Definitions
In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. (See, e.g., Sambrook, Fritsch & Maniatis, 2001, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover, ed., 1985, DNA Cloning: A Practical Approach, Volumes I and II, Second Edition; Gait, M. J., ed., 1984, Oligonucleotide Synthesis: A practical approach; Hames, B.D. & Higgins, S.J. eds., 1985, Nucleic Acid Hybridization; Hames, B.D. & Higgins, S.J., eds., 1984, Transcription And Translation; Freshney, R.I., 2000, Culture of Animal Cells: A Manual of Basic Technique; Woodward, J., 1986, Immobilized Cells And Enzymes: A practical approach, IRL Press; Perbal, B. E., 1984, A Practical Guide To Molecular Cloning). Each of the above references are hereby incorporated by reference in their entirety. The nucleic acid encoding the protein may be full-length or truncated, so long as the gene encodes a biologically active protein.
The coding sequences of the gene to be delivered are operably linked to expression control sequences, e.g., a promoter that directs expression of the gene. As used herein, the phrase "operatively linked" refers to the functional relationship of a polynucleotide/gene with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences. For example, operative linkage of a nucleic acid to a promoter refers to the physical and functional relationship between the polynucleotide and the promoter such that transcription of DNA is initiated from the promoter by an RNA polymerase that specifically recognizes and binds to the promoter, and wherein the promoter directs the transcription of RNA from the polynucleotide. Expression of a serotonin and/or glutamate receptor may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression.
In one specific embodiment, a vector is used in which the coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for expression of the construct from a nucleic acid molecule that has integrated into the genome (Roller and Smithies, 1989, Proc. Natl. Acad. Sci. USA, 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438; U.S. Patent No. 6,244,113 to Zarling et al.; and U.S. Patent No. 6,200,812 to Pati et al.). Each of the above references are hereby incorporated by reference in their entirety.
The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or
RNA sequence, a protein or an enzyme. Host cells can further be used for screening or other assays, as described infra.
The term "expression system" means a host cell and compatible vector under suitable conditions, e.g., for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Expression systems include mammalian host cells and vectors. Suitable cells include PC 12 cells, CHO cells, HeLa cells, 293 and 293T (human kidney cells), COS cells, mouse primary myoblasts, NIH 3T3 cells.
Suitable vectors include viruses, such as adenoviruses, adeno-associated virus (AAV), vaccinia, herpes viruses, baculoviruses and retroviruses, parvovirus, lentivirus, bacteriophages, cosmids, plasmids, fungal vectors, naked DNA, DNA lipid complexes, and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression. Viral vectors, especially adenoviral vectors can be complexed with a cationic amphiphile, such as a cationic lipid, polyL-lysine (PLL), and diethylaminoethyldextran (DELAE-dextran), which provide increased efficiency of viral infection of target cells (See, <?.g.,PCT/US97/21496 filed Nov. 20, 1997, incorporated herein by reference). AAV vectors, such as those disclosed in U.S. Pat. Nos. 5,139,941, 5,252,479 and 5,753,500 and PCT publication WO 97/09441, the disclosures of which are incorporated herein, are also useful since these vectors integrate into host chromosomes, with a minimal need for repeat administration of vector. For a review of viral vectors in gene therapy, see McConnell et al., 2004, Hum Gene Ther. 15(11):1022-33; Mccarty et al., 2004, Annu Rev Genet. 38:819-45; Mah et al., 2002, Clin. Pharmacokinet. 41(12):901-l 1; Scott et al., 2002,
Neuromuscul. Disord. 12(Suppl l):S23-9. In addition, see U.S. Patent No. 5,670,488. Beck et al., 2004, Curr Gene Ther. 4(4): 457-67, specifically describe gene therapy in cardiovascular cells. Each of the above references are hereby incorporated by reference in their entirety. The term "heterologous" refers to a combination of elements not naturally occurring. For example, heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. Preferably, the heterologous DNA includes a gene foreign to the cell. A heterologous expression regulatory element is a such an element operatively associated with a different gene than the one it is operatively associated with in nature. In the context of the present invention, the 2AR and/or mGluR2 receptor genes are heterologous to the vector or vectors in which they are inserted for cloning or expression, and they are heterologous to a host cell containing such a vector, in which it is expressed, e.g., a CHO cell. The terms "mutant" and "mutation" mean any detectable change in genetic material, e.g., DNA, or any process, mechanism, or result of such a change. This includes gene mutations, in which the structure (e.g., DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g., protein or enzyme) expressed by a modified gene or DNA sequence. The term "variant" may also be used to indicate a modified or altered gene, DNA sequence, enzyme, cell, etc., i.e., any kind of mutant.
"Sequence-conservative variants" of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position. Such variants can be used in expression of receptor subunits, e.g., where altered codon usage or insertion of a restriction site is desired.
"Function-conservative variants" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Such changes are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide. Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm. A "function- conservative variant" also includes a polypeptide or enzyme which has at least 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, most preferably at least 85%, and even more preferably at least 90%, and which has the same or substantially similar properties or functions as the native or parent protein or enzyme to which it is compared. Any of these algorithms can be used with defaults provided by the manufacturer, supplier, or provider or provider.
Similarly, in a particular embodiment, two amino acid sequences are "substantially homologous" or "substantially similar" when greater than 80% of the amino acids are identical, or greater than about 90% are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, any of the programs described above (BLAST, FASTA, etc.)
Screening Assays
The present invention provides various screening assays for compounds that modulate the functionality or a property of a heteroreceptor complex, or the receptors that comprise the complex. The assays of the invention are particularly advantageous by permitting rapid evaluation of cellular responses. Biological assays, which depend on testing perception, pain sensitivity, survival, or some other response in vivo require substantial amounts of time and resources to evaluate. By detecting individual signals in the signal transduction pathway, the present invention short-circuits the more tedious and time consuming biological assays. Furthermore, the signal transduction assays can often be performed with very small amounts of material.
In general, a screening assay of the invention makes use of the cells expressing receptor proteins (described above), various heteroreceptor ligands, and a candidate compound for testing.
The present invention contemplates screens for small molecule compounds, including peptides and peptidomimetics, and including receptor ligand analogs and mimics, as well as screens for natural compounds that bind to and agonize or antagonize heteroreceptor complexes in vitro. Such agonists or antagonists may, for example, interfere in the phosphorylation or dephosphorylation of signal transduction proteins. For example, natural products libraries can be screened using assays of the invention for such molecules. As used herein, the term "compound" refers to any molecule or complex of more than one molecule that modulates heteroreceptor complex function. The present invention contemplates screens for synthetic small molecule agents, chemical compounds, chemical complexes, and salts thereof as well as screens for natural products, such as plant extracts or materials obtained from fermentation broths. Other molecules that can be identified using the screens of the invention include, for example, opioids, opiates, narcotics, proteins and peptide fragments, peptides, nucleic acids and oligonucleotides, carbohydrates, phospholipids and other lipid derivatives, steroids and steroid derivatives, prostaglandins and related arachadonic acid derivatives, etc.
One approach to identifying such compounds uses recombinant bacteriophage to produce large libraries. Using the "phage method" (Scott and Smith, Science, 1990, 249:386-390; Cwirla, et al, 1990, Proc. Natl. Acad. Sci.USA, 87:6378-6382; Devlin et al., 1990, Science 49:404-406), very large libraries can be constructed (106 -108 chemical entities). A second approach uses primarily chemical methods, of which the Geysen method (Geysen et al., 1986, Molecular Immunology 23:709-715; Geysen et al.,1987, J. Immunologic Method 102:259-274) and the method of Fodor et al. (1991, Science 251 :767-773) are examples. Furka et al. (1988, 14th International Congress of Biochemistry, Volume 5, Abstract FR:013; Furka, 1991, Int J. Peptide Protein Res. 37:487-493) and U.S. Pat. Nos. 4,631,211 and 5,010,175 describe methods to produce a mixture of peptides that can be tested as agonists or antagonists. Each of the above references are hereby incorporated by reference in their entirety.
In another aspect, synthetic combinatorial libraries (Needels et al., 1993, Proc. Natl. Acad. Sci. USA, 90:10700-4; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; PCT Publication Nos. WO 92/00252 and WO 94/28028) and the like can be used to screen for compounds according to the present invention. Each of the above references are hereby incorporated by reference in their entirety.
Test compounds may be screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available, for example, from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal tracts are available from, e.g., Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., 1996, Tib Tech 14:60). In one embodiment, test compounds are peptides or peptidomimetic compounds generated by rational drug design based on the structure of known serotonin and/or glutamate receptor agonists and antagonists, or derived from combinatorial libraries.
The term "peptide" is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other bonds, e.g., ester, ether, etc. As used herein the term "amino acid" refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. Thus, peptides of the invention may comprise D-amino acids, a combination of D- and L- amino acids, and various "designer" amino acids (e.g., β -methyl amino acids, Ca- methyl amino acids, and Nα-methyl amino acids, etc.) to convey special properties to peptides in the library. Additionally, by assigning specific amino acids at specific coupling steps, peptide libraries with α-helices, β-turns, β. -sheets, γ-turns, and cyclic peptides can be generated. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
The coupling of the amino acids may be accomplished by techniques familiar to those in the art (Stewart and Young, 1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, 111.; Fields and Noble, 1990, Int. J. Pept. Protein Res. 35:161-214), or using automated synthesizers, such as sold by ABS. Each of the above references are hereby incorporated by reference in their entirety.
Intact cells expressing a heteroreceptor complex can be used in screening methods to identify candidate compounds. In one series of embodiments, a permanent cell line is established wherein cells of the cell line express the heteroreceptor complex, or cell membrane preparations are made from cells expressing the heteroreceptor complex. Alternatively, cells (including without limitation mammalian, insect, yeast, or bacterial cells) are programmed to express receptor genes by introduction of appropriate DNA or mRNA. Identification of candidate compounds can be achieved using any suitable assay, including without limitation (i) assays that measure selective binding of test compounds; (ii) assays that measure the ability of a test compound to modify (i.e., inhibit or enhance) a measurable activity or function of one or more of the receptors or the receptor complex; (iii) assays that monitor receptor or receptor complex trafficking, e.g., internalization and re-expression; and (iv) assays that measure the ability of a compound to modify (i.e., inhibit or enhance) transcription of proteins induced by the receptors or receptor complex.
The screening assay of the present invention, by way of example, and not of limitation, may utilize a native cell line established from the cortex of a schizophrenic patient, or cells engineered to express a 2AR receptor protein and an mGluR2 receptor protein. Such engineered cells include recombinant host cells, for example, PC 12 cells, CHO cells, HeLa cells, 293 and 293T cells (human kidney cells), COS cells, mouse primary myoblasts, NIH 3T3 cells, or Xenopus oocytes that have been transformed to express a 2AR receptor protein and an mGluR2 receptor protein. In one embodiment, the cells are engineered to express an epitope-tagged 2AR protein and an epitope-tagged mGluR2 protein. The presence of a heteroreceptor complex in cells engineered to express a 2AR receptor protein and an mGluR2 receptor protein, or in a native cell line, can be determined through the use of, for example, co- immunoprecipitation of the 2AR and mGluR2 receptors. The expression of a heteroreceptor complex may also be determined through the use of bioluminesence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) methods for the detection of protein-protein interactions (described below).
In one non-limiting embodiment, a compound of the invention may be identified through the use of a screening assay wherein a candidate compound is contacted to, for example, a cell expressing a 2AR receptor protein and an mGluR2 receptor protein. A compound of the present invention may be identified when, upon binding to a first receptor protein of the heteroreceptor complex, for example mGluR2, the affinity of a ligand for the second receptor protein, for example 2AR, increases or decreases. Such an increase or decrease in the ligand' s affinity for the second receptor may be measured, for example, by a competition assay between the ligand and a second ligand that has been detectably labeled (for example, radiolabeled, fluorescently labeled, or any other detectable label known in the art), for binding the second receptor. In another non-limiting embodiment, a compound of the invention may be identified through the use of a screening assay wherein a candidate compound is contacted to a cell expressing a 2AR receptor protein and an mGluR2 receptor protein, wherein binding of the compound to a first receptor protein of the heteroreceptor complex, for example mGluR2, increases or decreases the ability of a ligand to activate a signal transduction pathway upon binding to the second receptor of the heteroreceptor complex.
In another non-limiting embodiment, a compound of the invention may be identified through the use of a screening assay wherein a candidate compound is contacted to a cell expressing a 2AR receptor protein and an mGluR2 receptor protein, wherein binding of the compound to a first receptor protein of the heteroreceptor complex, for example mGluR2, activates or inhibits a signal transduction pathway associated with a second receptor of the heteroreceptor complex.
High Throughput Screen
Agents according to the invention may be identified by screening in high- throughput assays, including without limitation cell-based or cell-free assays. It will be appreciated by those skilled in the art that different types of assays can be used to detect different types of agents. Several methods of automated assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period of time e.g., using a 96- well format). For literature references see, e.g., Beggs et al., 1999, J. Biol. Screening 4(3); Renate de Wit et al, 1998, J. Biol. Screening, 3(4); Fox et al., 1999, J. Biol. Screening 4(4); Boyd et al., 1996, Clin.Chem. 42:1901-10; Broach et al., 1996, Nature 384(Supp.):14-16; Cusack et al., 1993, J. Rec. Res. 13:123-134; U.S. Pat. Nos. 4,980,281 and 5,876,951; PCT Publication Nos. WO 97/45730, WO 97/14812, and WO 97/10502. Each of the above references are hereby incorporated by reference in their entirety. Such high- throughput screening methods are particularly preferred.
Specific Screening Methods
There are several screening methods available for the discovery of specific heteroreceptor complex ligands. These screens include radioligand binding, signal transduction, expression, reporter assays, and structure function of existing agonists and antagonists. This technology can be utilized for screening of novel compounds that are identified in high throughput screens.
Radioligand Binding Assays Radioligand binding assays allow further characterization of hits from high throughput screens as well as analogs of heteroreceptor complex agonists and antagonists. Any ligand can be radiolabeled for direct binding assays, or alternatively, used in competitive binding assays.
Signal Transduction Assays
G protein coupled receptors (GPCR) are coupled to a variety of heterotrimeric G proteins, which are comprised of α, β, and γ subunits. Upon agonist binding to a GPCR at the cell surface, conformational changes occur within the agonist:GPCR complex, which lead to the dissociation of the G protein α subunit from the βγ subunits. The Gα and Gβγ subunits then stimulate a variety of intracellular effectors, which transduce the extracellular signal to the inside of the cell. Various signal transduction systems known to be coupled to GPCRs include adenylate cyclase, phospholipase C, phospholipase A2, sodium/hydrogen exchange, etc. Thus, measurements of intracellular calcium concentrations and adenylate cyclase activity, or any component of a signal transduction pathway, indicate whether a hit or test compound is functionally behaving as an agonist or antagonist at the heteroreceptor of the invention.
In one non-limiting embodiment, activation of a 2AR or an mGluR2 receptor signal transduction pathway can be determined by observing an increase in the activation of a Gαq and/or a Gαj protein.
In a further non-limiting embodiment, activation of a 2AR receptor by a hallucinogenic compound activates both a Gαq and a Gαi proteins, while activation of a 2AR by a non-hallucinogenic compound only activates a Gαq protein.
In another non-limiting embodiment, activation of a 2AR receptor and/or an mGluR2 receptor can be determined by observing an inhibition of adenylate cyclase and an inhibition in the synthesis of cAMP.
In yet another non-limiting embodiment, activation of a 2AR receptor and/or an mGluR2 receptor can be determined by observing an increase in the phosphorylation of phospholipase C, and the synthesis of inositol triphosphate (IP3) and diacylglycerol (DAG).
In a specific embodiment, G-protein signal transduction is coupled to expression of a reporter gene, thus permitting a reporter gene screening assay. For example, activation of a Gαq mediated signal transduction pathway can be determined by measuring an increase in the expression of c-fos. Activation of a Gα, mediated signal transduction pathway can be determined by measuring an increase in the expression an egr-1 and/or an egr-2 gene (Early growth Response 1 and 2 genes, respectively).
Calcium Mobilization Assay
Whole cells expressing the heteroreceptor complex are loaded with a fluorescent dye that chelates calcium ions, such as FURA-2. Upon addition of receptor agonist to these cells, calcium is released from the intracellular stores. The dye chelates these calcium ions. The spectrophotometrically determined ratio of the dye: calcium complexes to free dye provides a numerical measurement of the changes in intracellular calcium concentrations upon addition of serotonin-like and/or glutamate-like substrates. Hits from screens and other test compounds can be similarly tested in this assay to functionally characterize them as agonists or antagonists.
In one non-limiting embodiment, addition of a serotonin-like and/or a glutamate like compound increases the release of calcium from intracellular stores, increasing the ratio of dye: calcium complexes to free dye.
Cyclic AMP Accumulation Assay
Upon agonist binding, G5 -coupled GPCRs stimulate adenylate cyclase. Adenylate cyclase catalyzes the production of cyclic AMP (cAMP) from adenosine- 5'-triphosphate which, in turn, activates protein kinases. G1 -coupled GPCRs are also coupled to adenylate cyclase, however, agonist binding to these receptors results in the inhibition of adenylate cyclase and the subsequent inhibition of cAMP accumulation. To measure the inhibition of cAMP accumulation, cells expressing G1 - coupled receptors must first be stimulated to elevate cAMP levels. This is achieved by treating the cells with forskolin, a diterpene that directly stimulates cAMP production. Co-incubation of cells expressing G1 -coupled receptors with forskolin and a functional agonist will result in the inhibition of forskolin-stimulated cAMP accumulation. For a cAMP assay, cells stably expressing a heteroreceptor of the invention can be incubated with a test compound, and with forskolin plus a test compound. The cells are then lysed and cAMP levels are measured using the [125I]cAMP radioimmunoassay (RIA).
In one non-limiting embodiment, activation of a 2AR and/or an mGluR2 receptor can be determined by observing an inhibition of adenylate cyclase, and an inhibition in the synthesis of cAMP.
Assay Using 2AR and mGluR2 Binding Compounds
The compounds of the present invention can also act as modulators of the heteroreceptor complex, wherein the compounds modulate the effect of a second compound's binding to a 2AR, mGluR2, or both components of the heteroreceptor complex. For example, the compound can modulate the effect of a 2AR agonist binding to the 2AR receptor component of the heteroreceptor complex. An assay of a candidate compound's modulatory effect on the binding of a second compound to a 2AR, mGluR2, or both components of a heteroreceptor complex may therefore be used to identify a compounds of the invention. In such an assay, the second compound (e.g. a 2AR and/or an mGluR2 agonist) is a probe for determining the effect of the candidate compound's binding to the heteroreceptor complex (e.g. a 2AR or an mGluR2 or both receptor components of the complex) on the response normally elicited by the second compound (e.g. a 2AR and/or an mGluR2 agonist) binding to a 2AR or an mGluR2 or both receptor components of the complex. In one embodiment, this assay involves observing a change or modulation in the response of the hetroreceptor complex to a 2AR and/or an mGluR2 binding compound, for example, a 2AR and/or mGluR2 agonist, when a candidate compound of the invention is contacted to the heteroreceptor complex at the same time, before, or after the 2AR and/or mGluR2 binding compound is contacted to the heteroreceptor complex.
In one embodiment, the second compound can be a 2AR agonist, for example, l-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), l-(2,5-dimethoxy-4- methylphenyl)-2-aminopropane (DOM), 1 -(2,5-dimethoxy-4-bromophenyl)-2- aminopropane (DOB), lysergic acid diethylamide (LSD), ergotamine, r-lisuride, s- lisuride, psilocin, mescalin, serotonin, or any other 2AR agonist known in the art. In another embodiment, the second compound can be an mGluR2 agonist, for example, (lR,4R,5S,6R)-4-Amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid
(LY379268) (Eli Lilly and Company), (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)- glycine (DCG-IV), (2S,l'S,2'S)-2-(carboxycyclopropyl)-glycine (L-CCG-I), glutamate or any other mGluR2 agonist known in the art.
Behavioral Assays
Screening assays can also be used in which the behavioral output of a live organism, for example, but not limited to, a mouse or rat, is measured to determine the effect of a compound on the heteroreceptor complex. The administration of a hallucinogenic compound to mice induces an increase in locomotor activity, for example, an increase in head-twitching. Compounds of the invention can be identified based on an increase or decrease in hallucinogenic induced locomotor activity. For example, according to the invention, a compound that binds to and activates an mGluR2 component of a heteroreceptor complex may reduce the hallucinogen induced head-twitching of a mouse.
Methods for Detecting Signals
The present invention provides numerous methods for detecting signals, including but not limited to, directly detecting phosphorylation of proteins using radioactive phosphorous compounds, indirectly detecting phosphorylation with antibodies specific for phosphorylated epitopes, or detecting signals from activated signal transduction proteins, such as gene expression. For example, antibodies to
Gctq/ii or Gαπ>2j3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and phospholipase C (Santa Cruz Biotechnology; Abeam Inc., Cambridge, MA, USA;
Sigma-Aldrich, St. Louis, MO, USA; Millipore, Billerica, MA, USA) are commercially available.
Gene expression is conveniently detected using a reporter gene assay. Alternatively, a downstream element of a signal transduction pathway can be modified to have reporter activity, i.e., the reporter gene can be activated by signals generated as a consequence of receptor binding, rather than as a direct result of receptor binding. Reporter genes for use in the invention encode detectable proteins, include, but are not limited to, chloramphenicol transferase (CAT), β-galactosidase (β-gal), luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), alkaline phosphatase, and other genes that can be detected, e.g., immunologically (by antibody assay). For example, the reporter gene signaling activation of a 2AR and/or an mGluR2 receptor can comprise a promoter and regulatory elements from a c-fos, egr-1, or egr-2 gene c fused to one of the reported genes described herein. Such reporter genes can be made, for example, through homologous recombination, wherein the coding sequence for a reporter gene such as, for example, GFP, is interchanged with the coding sequence of, for example, a c-fos, egr-1, or egr-2 gene. Thus, expression of the GFP reporter gene would be under the same temporal and spatial expressional control as that of a c-fos, egr-1 , or egr-2 gene.
In another embodiment, a 2AR and or an mGluR2 receptor protein can be fused to a reporter gene such that the cellular or sub-cellular location of the receptor proteins can be observed, for example, to determine if the compounds of the invention induce internalization of the 2AR, mGluR2, or both components of a heteroreceptor complex.
In one embodiment, the instant invention discloses the use of the Bioluminescence Resonance Energy Transfer (BRET) or the fluorescence resonance energy transfer (FRET) methods for the detection of protein-protein interactions, for example, to measure an increase in complex stabilization cause by the binding of a compound to the complex {see, e.g., Xu et al., Proc. Natl. Acad. Sci. USA 96:151- 156; Angers et al., 2000, Proc. Natl. Acad. Sci. USA, 97:3684-3689). BRET measures the transfer of energy between a luminescent donor {e.g., luciferase expressed as a fusion protein with one of the receptors) and a fluorescent acceptor {e.g., YFP expressed as a fusion protein with one of the receptors). FRET measures the transfer of energy between a first fluorescent donor and a second fluorescent acceptor. According to the present invention, BRET or FRET can be used (i) to examine the formation of a complex between various receptors {i.e., serotonin and glutamate receptors) and (ii) to study the effect of agonist/antagonist binding at the level of the complex. In another embodiment, a protein-protein interaction can be determined by co- immunoprecipitation of the proteins.
In another embodiment, a yeast screening assay, useful for testing agonists and antagonists of mammalian G-protein coupled receptors, e.g., as disclosed in U.S. Pat. No. 5,482,832, can be used. Synergistic Compositions
It has also been found, as exemplified infra, that the serotonin and glutamate receptors which comprise the heteroreceptor complex of the invention, bind synergistically to combinations of ligands (non-linked together), e.g., agonists or antagonists for each of the serotonin and glutamate receptors. Thus, the invention further provides such synergistic compositions, e.g., comprising combinations of the ligands described above with at least one member of such combination interacting with each subunit of the heteroreceptor.
Pharmaceutical Compositions
The compounds and compositions of the invention can be formulated as pharmaceutical compositions by admixture with a pharmaceutically acceptable carrier or excipient. Preferably, the compounds or compositions are provided in a therapeutically effective amount to an animal in need of treatment therewith for relief of a neurological, neurodegenerative, or psychiatric brain disease. A neurological, neurodegeenerative, or psychiatric brain disease that is treatable according to the methods of the invention can be drug abuse, schizophrenia, psychoses associated with schizophrenia, depression, anxiety, obsessive compulsive disorder, bi-polar disorder, neurological diseases associated with psychosis including Alzheimer's and Parkinson's disease, drug induced psychosis, a dysfunction of the central reward pathway, or some other condition. The animal subject is preferably a human, but the compounds and compositions of the invention have application in veterinary medicine as well, e.g. , for the treatment of domesticated species such as canine, feline, and various other pets; farm animal species such as bovine, equine, ovine, caprine, porcine, etc.; wild animals, e.g., in the wild or in a zoological garden; and avian species, such as chickens, turkeys, quail, songbirds, etc.
The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a human. Preferably, as used herein, the term
"pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, 18th Edition, or other editions, hereby incorporated by reference in its entirety.
The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to reduce by at least about 15%, preferably by at least 50%, more preferably by at least 90%, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host. These parameters will depend on the severity of the condition being treated, other actions, such as diet modification, that are implemented, the weight, age, and sex of the subject, and other criteria, which can be readily determined according to standard good medical practice by those of skill in the art
According to the invention, the component or components of a pharmaceutical composition of the invention may be introduced parenterally; transmucosally, e.g., orally, nasally, or rectally; or transdermally. Parenteral administration includes, but is not limited to, intravenous, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration.
In a specific embodiment, the therapeutic compound can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., 1989,in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler eds., Liss: New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally Lopez-Berestein, ibid.). To reduce its systemic side effects, this may be a preferred method for introducing the compound.
In yet another embodiment, the therapeutic compound can be delivered in a controlled or sustained release system. For example, a compound or composition may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321 :574). In another embodiment, polymeric materials can be used (see Langer and Wise eds., 1974, Medical Applications of Controlled Release, CRC Press: Boca Raton, FIa; Smolen and Ball eds., 1984, Controlled Drug Bioavailability, Drug Product Design and Performance, Wiley, N. Y.; Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem., 23:61; Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol., 25:351; Howard et al., 9189, J.Neurosurg. 71 :105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer (supra). Each of the above references are hereby incorporated by reference in their entirety.
EXAMPLES
The present invention is further described by means of the examples, presented below. The use of such examples are illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the invention will be apparent to those skilled in the art upon reading this specification. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which the claims are entitled.
EXAMPLE 1 The mGluR2 and 5-HT2A Receptors Directly Interact With
Each Other in a Heteroreceptor Complex
Methods
All reagents were purchased from commercial vendors except for LY379268 (Eli Lilly and Company). Mouse lines, treatment protocols, behavioural studies, dissections, and primary neuronal cultures, approved by Institutional Use and Care Committees, have been previously described (Gonzalez-Maeso, J. et al. 2007. Behavior. Neuron 53, 439-52; Gonzalez-Maeso, J. et al. 2003, J Neurosci 23, 8836- 43). Protocols used for FISH, binding assays, real-time PCR, FRET and co- immunoprecipitation were performed as previously described or with minor modifications (Gonzalez-Maeso, J. et al. 2007, Behavior. Neuron 53, 439-52; Lopez- Gimenez, J. F. et al., 2007, MoI Pharmacol 71, 1015-29). Epitope tagged, BRET, FRET and chimera receptor constructs were generated using standard cloning techniques and were confirmed by sequencing. BRET using Renilla luciferase and Green Fluorescent Protein (GFP) was performed in HEK293 cells (Sawa et al., 2002, Science 296, 692-5). Matched schizophrenia and control human brains were obtained from autopsies performed in the Basque Institute of Legal Medicine, Bilbao, Spain in compliance with policies of research and ethical review boards for postmortem brain studies.
Materials and Drug Administration. l-(2,5-dimethoxy-4-iodophenyl)-2- aminopropane (DOI), l-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM), 1- (2,5-dimethoxy-4-bromophenyl)-2-aminopropane (DOB), lysergic acid diethylamide (LSD), and lisuride hydrogen maleate (lisuride) were purchased from Sigma-Aldrich. (lR,4R,5S,6R)-4-Amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid
(LY379268) was obtained from Eli Lilly and Company. 2S-2-amino-2-(lS,2S-2- carboxycyclopropan-l-yl)-3-(xanth-9-yl)-propionic acid (LY341495), (2S,2'R,3'R)-2- (2',3'-dicarboxycyclopropyl)-glycine (DCG-IV), (2S,l'S,2'S)-2-(carboxycyclopropyl)- glycine (L-CCG-I), clozapine, and haloperidol were obtained from Tocris Cookson Inc. [3H]Ketanserin and [35S]GTPyS were purchased from PerkinElmer Life and Analytical Sciences, Inc. [3H]LY341495 was purchased from American Radiolabeled Chemicals, Inc. The injected doses (i.p.) were DOI, 2 mg/kg; DOM, 4 mg/kg; DOB, 1 mg/kg; LSD, 0.24 mg/kg; lisuride, 0.4 mg/kg; ergotamine, 0.5 mg/kg; LY379268, 15 mg/kg; LY341495, 6 mg/kg; clozapine, 25 mg/kg; and haloperidol, 1 mg/kg, unless otherwise indicated.
Transient Transfection of HEK293 cells. HEK293 were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) foetal bovine serum at 37°C in a 5% CO2 humidified atmosphere. Transfection was performed using Lipofectamine 2000 reagent (Invitrogen) according to manufacturer's instructions. HEK293 cells stably expressing human 2AR have been described previously (Ebersole et al., 2003, MoI Pharmacol 63:36-43; Gonzalez-Maeso, et al., 2003, J Neurosci 23:8836-43). Co-immunoprecipitation Studies. Co-immunoprecipitation studies in postmortem human brain, and co-immunoprecipitation studies using N-terminally c-myc tagged form of 2AR, and N-terminally haemagglutinin (HA) tagged forms of mGluR2, mGluR3 or mGluR2/mGluR3 chimeras in HEK293 were performed as previously described with minor modifications (Lopez-Gimenez et al., 2007, MoI Pharmacol 71:1015-29). Briefly, the samples were incubated overnight with protein A/G beads and anti-2AR {postmortem human brain) or anti-c-myc antibody (HEK293 cells) at 4°C on a rotating wheel. Equal amounts of proteins were resolved by SDS- polyacrylamide gel electrophoresis. Detection of proteins by immunoblotting using anti-2AR (Santa Cruz Biotechnology), anti-mGluR2 and anti-mGluR3 (Abeam Inc.) in postmortem human brain, or anti-c-myc and anti-HA antibodies (Santa Cruz Biotechnology) in HEK293 was conducted using ECL system according to the manufacturer's recommendations.
Bioluminiscence Resonance Energy Transfer (BRET2) in HEK293 live cells. The human 2AR, serotonin 5-HT2c (2CR), mGluR2, and mGluR3 receptors with mutated stop codons were subcloned into the pi?luc and pGFP plasmids (PerkinElmer Life Sciences), such that Renilla luciferase (R\\xc) and Green Fluorescent Protein (GFP2) were present at the C-termini of the receptors. All sequences were confirmed by DNA sequencing. After 48 h, transfected cells were washed with PBS, suspended to 1-2 x 106 cells/ml, and were treated with DeepBlueC Coelenterazine Substrate (5 μM final concentration; PerkinElmer Life Sciences). Equivalents amounts of total DNA comprised of various ratios of the i?luc- or GFP2-tagged receptors were transfected (James et al., 2006, Nat Methods 3:1001-6). Light emission was monitored by using a Fusion Universal Microplate Analyzer (PerkinElmer Life Sciences). A BRET2 signal is defined as the light emitted by GFP2 at 515 nm in response to the light emitted at 410 nm by R\nc in upon catalysis of DeepBlueC. The values were corrected by subtracting the background BRET2 signal detected when the receptor-i?luc construct was expressed alone {see Fig. 15 for luminescence and fluorescence values). The specificity of mGluR2-i?luc and 2 AR-GFP2 interactions were assessed by comparison with co-expression of mGluR2-i?luc and 2CR-GFP2, mGluR3-i?luc and 2 AR-GFP2 and mGluR2-i?luc and GFP2. Data from a single experiment, which has been replicated three times, are displayed as mean±s.e.m. (Fig. Ie). Fluorescence Resonance Energy Transfer (FRET). Forms of the 2AR and mGluR2 C-terminally fused to eCFP and eYFP were generated, and FRET microscopy in living cells was conducted as previously reported (Lopez-Gimenez et al., 2007, MoI Pharmacol 71 :1015-29). Results from a single experiment, representative of two-three independent studies, are shown in Fig. 4d.
[3H]Ketanserin, [3H]LY341495 and [35S]GTPyS Binding. Membrane preparations and [3H]ketanserin binding assays were performed as previously reported (Gonzalez- Maeso et al., 2007, Neuron 53:439-52). [3H]LY341495 binding was performed as previously described with minor modifications (Wright et al., 2001, J Pharmacol Exp Ther 298:453-60). Briefly, membrane preparations were incubated for 60 min at 4°C. Non-specific binding was determined in the presence of ImM L-glutamate. [35S]GTPyS binding experiments were initiated by the addition of membranes containing 35 μg protein to an assay buffer (20 mM HEPES, 3 mM MgCl2, 100 mM NaCl, 0.2 mM ascorbic acid, and 0.5 nM [35S]GTPyS) supplemented with 0.1 μM or 10 μM GDP for Gαq/π and Ga1, respectively, and containing the indicated concentration of ligands. Nonspecific binding was determined in the presence of 100 μM GTPyS. Reactions were incubated for 30 min at 30°C, and were terminated by the addition of 0.5 ml of ice-cold buffer, containing 20 mM HEPES, 3 mM MgCl2, 100 mM NaCl, and 0.2 mM ascorbic acid. The samples were centrifuged at 16,000χg for 15 min at 40C, and the resulting pellets resuspended in solubilization buffer (100 mM Tris, 20O mM NaCl, I mM EDTA, 1.25% Nonidet P-40) plus 0.2% sodium dodecylsulfate. Samples were precleared with Pansorbin (Calbiochem), followed by immunoprecipitation with antibody to Gαq/π or Gαπ;2,3 (Santa Cruz Biotechnology). Finally, the immunocomplexes were washed twice with solubilization buffer, and bound [35S]GTPyS was measured by liquid-scintillation spectrometry.
Construction of Receptor Chimeras. All PCR reactions were performed using PfuTurbo Hotstart DNA polymerase (Stratagene, La Jolla, CA) in a PTC-100 thermal cycler (MJ Research, Waltham, MA). Cycling conditions were 30 cycles of 94°C/30 sec, 550C /30 sec and 720C /1 min per kilobase of amplicon, with an initial denaturation/activation of 940C /2 min and a final extension of 720C Il min.
HA-tagged wild type human mGluR2 and mGluR3 constructs. The rat mGluR5 signal peptide (SP) (Blahos et al., 1998, J Biol Chem 273:25765-9) along with an HA epitope tag was PCR amplified using primers NheI-HA_SP/S (5'- TTTTgctagcGAATTCCTTTCCTAAAATGG-3') and HA_SP-KpnI/A (5'- TTTTggtaccACGCGTGGCGTAGTCGGGTA-3') with pRK5 as template. Wild type human mGluR2 and mGluR3 were amplified using primers MluI-hGRM2/S (5'- agctacgcgtAAGAAGGTGCTGACCCTGGA-3') hGRM2-XbaI/A (5'-
AAtctagaTCAAAGCGATGACGTTGTCGAG-3') and KpnI-hGRM3/S (5'- acgtggtaccTTAGGGGACCATAACTTTCT-3') hGRM3-XhoI/A (5'- acgtctcgagTCACAGAGATGAGGTGGTGG-3'), respectively. The rat mGluR5 signal peptide/HA epitope fragment was digested with Nhel and MIuI, the human mGluR2 fragment was digested with MIuI and Xbal, and were simultaneously subcloned into the Nhel and Xbal sites of pcDNA3.1 (Invitrogen, Carlsbad, CA) to yield the HA-tagged mGluR2 construct. Similarly, the rat mGluR5 signal peptide/HA fragment was digested with Nhel and Kpnl, the human mGluR2 PCR product was digested with Kpnl and Xhol, and were simultaneously subcloned into the Nhel and Xhol sites of pcDNA3.1 to give the HA-tagged mGluR2 construct.
Chimeric human mGluR2 with transmembrane domain 4 and 5 from human mGluR3. Fragment of the transmembrane domain TMl to the C terminus of the second intracellular loop of the human niGluR2 was amplified using primers hGRM2-1476/S (5'-GGACACCAGCCTCATCCCAT-S') and hGRM2i2GRM3TM4/A (5'- CAGATGAAAACCTGAGAACTAGGACTGATGAAGCGTGGCC-S'). Fragment of the TM4 through TM5 of the human mGluR3 was amplified using primers hGRM2i2GRM3TM4/S (5'-
GGCCACGCTTCATCAGTCCTAGTTCTCAGGTTTTCATCTG-S ') and hGRM3TM5GRM2i3/A (5'-
TTTTCGGGGCACTTGCGAGTTTTGAAGGCGTACACAGTGC-S'). The two fragments were annealed and re-amplified using primers hGRM2-1476/S and hGRM3TM5GRM2i3/A. The third intracellular loop to the carboxyl terminal of the human mGluR2 was amplified using primers hGRM3TM5GRM2i3/S (5'- GCACTGTGTACGCCTTCAAAACTCGCAAGTGCCCCGAAAA-S') and hGRM2- Xbal/A. This fragment was then annealed with the previous PCR product and re- amplified using primers hGRM2-1476/S and hGRM2-XbaI/A. To reconstitute the complete chimeric receptor, the N terminal domain of the HA-tagged wild type human mGluR2 was released using Nhel and BstBI, the final PCR product was digested using BstBI and Xbal, and the two fragments were simultaneously subcloned into the Nhel and Xbal sites of pcDNA3.1.
Chimeric human mGluR3 with transmembrane domain 4 and 5 from human mGluR2. Fragment of the transmembrane domain TMl to the C terminus of the second intracellular loop of the human mGluR3 was amplified using primers hGRM3-2541/S (5'- TGAAAGTTGGTCACTGGGCA-B') and hGRM3i2GRM2TM4/A (5'- CAGATGGCCACCTGTGAGGCGGGGCTGATGAATTTTGGCC-S'). Fragment of the TM4 through TM5 of the human mGluR2 was amplified using primers hGRM3i2GRM2TM4/S (5'-
GGCCAAAATTCATCAGCCCCGCCTCACAGGTGGCCATCTG-S ') and hGRM2TM5GRM3i3/A (5'-
TTTTCTGGGCACTTCCGCGTCTTGAAGGCATAAAGCGTGC-S'). The two fragments were annealed and re-amplified using primers hGRM3-2541/S and hGRM2TM5GRM3i3/A. The third intracellular loop to the carboxyl terminal of the human mGluR3 was amplified using primers hGRM2TM5GRM3i3/S (5'- GCACGCTTTATGCCTTCAAGACGCGGAAGTGCCCAGAAAA-S') and hGRM3- Xhol/A. This fragment was then annealed with the previous PCR product and re- amplified using primers hGRM3-2541/S and hGRM3-XhoI/A. To reconstitute the complete chimeric receptor, the N terminal domain of the HA-tagged wild type human mGluR3 was released using Nhel and Pstl, the final PCR product was digested using Pstl and Xhol, and the two fragments were simultaneously subcloned into the Nhel and Xhol sites of pcDNA3.1.
Chimeric human mGluR3 with transmembrane domain 1 through 5 from human mGluR2. A small fragment of the N terminal domain to the beginning of TMl of the human mGluR3 was amplified using primers hGRM3-2541/S and hGRM3NGRM2TMl/A (5'-
ACAGCCCAGGCATCGCCCCAGCGGATGTAGTCCTCAGGAAGGT-S'). Fragment of the TMl through TM5 of the human mGluR2 was amplified using primers hGRM3NGRM2TMl/S (5'-
ACCTTCCTGAGGACTACATCCGCTGGGGCGATGCCTGGGCTGT-S') and hGRM2TM5GRM3i3/A. The two fragments were annealed and re-amplified using primers hGRM3-2541/S and hGRM2TM5GRM3i3/A. The third intracellular loop to the carboxyl terminal of the human mGluR3 was amplified using primers hGRM2TM5GRM3i3/S and hGRM3-XhoI/A. This fragment was then annealed with the previous PCR product and re-amplified using primers hGRM3-2541/S and hGRM3-XhoI/A. To reconstitute the complete chimeric receptor, the N terminal domain of the HA-tagged wild type human mGluR3 was released using Nhel and Pstl, the final PCR product was digested using Pstl and Xhol, and the two fragments were simultaneously subcloned into the Nhel and Xhol sites of pcDNA3.1.
Molecular modelling. Three-dimensional molecular models of the seven transmembrane (TM) regions of 2AR and mGluR2 were built using the crystal structures of β 2-adrenergic receptor (Cherezov et al., 2007, Science 318:1258-65) and rhodopsin (Li et al., 2004, J MoI Biol 343:1409-38), respectively, as structural templates, and the latest version of the homology-modeling program MODELLER (SaIi et al., 1993, J MoI Biol 234:779-815). The use of the very recent crystal structure of β 2-adrenergic receptor to build a model of 2AR is justified by the higher sequence identity between these two receptors compared to rhodopsin, and the suitability of the rhodopsin template to build models of family C GPCRs, which includes the mGluR2, has recently been discussed in the literature (Binet et al., 2007, J Biol Chem 282:12154-63). The sequence alignment between the transmembrane helices of β 2-adrenergic receptor and 2AR was obtained with BLAST (Altschul et al., 1990, J MoI Biol 215:403-10). For mGluR2, we used the same alignment with rhodopsin as described in Binet et al. (2007). A multiple alignment of available mGluR2 and mGluR3 sequences was performed with the CLUSTALW program version 1.81 (ThompsonThompson, 1994, Nucleic Acids Res 22, 4673-80). To build a reasonable configuration of the 2AR-mGluR2, we used the TM4,5-
TM4,5 configuration deriving from atomic force microscopy of rhodopsin in native disk membranes as a template for the heteromer interface between 2AR and mGluR2 (Liang et al., 2003, J Biol Chem 278, 21655-62). This modeling was obtained with the assistance of the Insight II User Graphical Interface (Accelrys Inc.) on a graphics workstation.
Neuronal primary culture. Primary cultures of cortical and thalamic neurons were prepared as previously described (Gonzalez-Maeso et alo., 2007, Neuron 53, 439-52).
Mouse brain samples. Experiments were performed as previously described on adult (8-12 weeks old) male 129S6/Sv mice. For experiments involving genetically modified mice, htr2A+/+ or htr2A+/- littermates were used as controls (Gonzalez- Maeso et alo., 2007, Neuron 53, 439-52; Chan et al., 2005, Nucleic Acids Res 33, el 61). Animals were housed at 12 h light/dark cycle at 23 °C with food and water ad libitum. The Institutional Animal Use and Care Committee approved all experimental procedures at Mount Sinai School of Medicine and Columbia University.
Fluorescence in situ hybridization (FISH). Synthesis of modified DNA oligonucleotide probes, probe labeling, and fluorescence in situ hybridization was performed as previously described(Gonzalez-Maeso et alo., 2007, Neuron 53, 439-52; Chan et al., 2005, Nucleic Acids Res 33, elόl). (See Figure 10 for oligonucleotide probe sequences).
Quantitative real-time PCR. Quantitative real-time PCR (qRT-PCR) experiments were performed as previously described (Gonzalez-Maeso et alo., 2007, Neuron 53, 439-52). (See Figures 11 and 12 for primer pair sequences).
Behavioural Studies. Behavioural studies were performed as previously described (Gonzalez-Maeso et alo., 2007, Neuron 53, 439-52; Weisstaub et al., 2006, Science
313, 536-40). Motor function was assessed using a computerized three-dimentional activity monitorin system (AccuScan Instruments). The activity monitor has 32 infrared sensor pairs with 16 along each side spaced 2.5 cm apart. The system determines motor activity based on the frequency of interruptions to infrared beams traversing the x, y and z planes. Total distance (cm) travelled and vertical activity were automatically determined from the interruptions of beams in the horizontal and vertical planes, respectively. Brain Samples. Human brains were obtained at autopsies performed in the Forensic Anatomical Institute, Bilbao, Spain. The study was developed in compliance with policies of research and ethical review boards for postmortem brain studies (Basque Institute of Legal Medicine, Spain). Deaths were subjected to retrospective searching for previous medical diagnosis and treatment using examiner's information and records of hospitals and mental health centers. After searching of antemortem information was fulfilled, 25 subjects who had met criteria of schizophrenia according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) were selected (American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders: DSM-IV 4th edition, Washington , DC, 1994). A toxicological screening for antipsychotics, other drugs and ethanol was performed on blood, urine, liver and gastric contents samples. All subjects who were drug-free before death (as revealed by the absence of prescriptions in medical histories) also gave negative results in the toxicological screening. The toxicological assays were performed at the National Institute of Toxicology, Madrid, Spain, using a variety of standard procedures including radioimmunoassay, enzymatic immunoassay, high-performance liquid chromatography and gas chromatography-mass spectrometry. Controls for the present study were chosen among the collected brains on the basis, whenever possible, of the following cumulative criteria: (1) negative medical information on the presence of neuropsychiatric disorders or drug abuse; (2) appropriate gender, age and postmortem delay to match each subject in the schizophrenia group; (3) sudden and unexpected death (motor vehicle accidents); and (4) toxicological screening for psychotropic drugs with negative results except for ethanol. Tissue pH is assumed to be an indicator of agonal status (Preece et al., 2003, Brain Res MoI Brain Res 118:60-71) .Thus, prolonged terminal hypoxia results in low tissular pH. It has been demonstrated that gene expression patterns are strongly dependent on tissue pH. Brief deaths, associated with accidents, cardiac events or asphyxia, generally had normal pH with minor influence on gene expression changes (Li et al., 2004, Hum MoI Genet 13:609- 16) . All schizophrenic and control subjects showed a sudden and rapid death without long agonal phase. The tissue storage period before assays did not differ between schizophrenic cases (82 + 9 months) and controls (85 + 10 months). Specimens of prefrontal cortex (Brodmann's area 9) were dissected at autopsy (0.5-1 g tissue) on an ice-cooled surface and immediately stored at -7O0C until membrane preparation. The definitive pairs of antipsychotic-untreated schizophrenics and respective matched controls, and the definitive pairs of antipsychotic-treated schizophrenics and respective matched controls are shown in Figure 7.
Results
2AR and mGluR2 Co-localize in Brain Cortex
Fluorescent in situ hybridization (FISH) was used to determine whether mGluR2 or mGluR3 receptor subtypes are co-expressed by the same neurons as 2AR. In layer V mouse somatosensory cortex (SCx), 2AR mRNA positive cells were mostly mGluR2 mRNA positive. The level of expression in SCx was much lower for mGluR3 mRNA, which rarely co-localized with 2AR mRNA (Fig. 1). Control studies validated assay sensitivity and specificity, and similar 2AR/mGluR2 mRNA co- localization was found in cortical primary cultures (Fig. 1). Translation of 2AR protein in cortical pyramidal neurons was found to be necessary for normal mGluR2 expression. Mice with globally disrupted 2AR expression (htr2A-/~ mice) showed reduced cortical mGluR2 binding and expression, while mice in which 2AR expression was selectively restored in cortical pyramidal neurons (Gonzalez-Maeso, J. et al., 2007, Neuron 53:439-52; Gonzalez-Maeso, J. et al., 2003, J Neurosci 23, 8836-43; Vollenweider, F. X., et al., 1998, Neuroreport 9:3897-902; Gouzoulis-Mayfrank, E. et al., 2005, Pharmacopsychiatry 38:301-11; Colpaert, F. C, 2003, Nat Rev Drug Discov 2:315-20.; Marek, G. J., et al.2000, J Pharmacol Exp Ther 292:76-87; Weisstaub, N. V. et al., 2006, Science 313:536-40) showed control expression levels (Fig. 4).
mGluR2 and 2AR Exhibit Synergistic Interactions
Although the 2AR and mGluR2 receptors belong to different GPCR classes, the existence of 2AR/mGluR2 heterocomplexes was established by several methods: co-immunoprecipitation of human brain cortex samples (Fig. 1) and of HEK293 cells transfected with epitope-tagged receptors (Fig. 4), bio luminescence resonance energy transfer (BRET) (Fig. 1), and fluorescence resonance energy transfer (FRET) (Fig. 4) studies in transfected cells.
To determine whether the formation of the 2AR/mGluR2 complex has functional consequences, the effects in mouse SCx membranes of an mGluR2/3 agonist on the competition binding of several hallucinogenic 2AR agonists (Fig. 1) and of a 2AR agonist on the competition binding of several mGluR2/3 agonists (Fig. 1) were examined.
The agonist affinities for the 2AR and mGluR2/3 were decreased when receptor/G protein complexes were uncoupled by GTPγS (Fig. 3). Notably, the glutamate agonist LY379268 (LY379) increased the affinity of all three hallucinogens studied for the 2AR binding site. Furthermore, the 2AR agonist DOI decreased the affinity of the three mGluR2/3 agonists for the glutamate receptor binding site. The allosteric interactions observed were eliminated by antagonist for each modulator (Fig. 3). Although the glutamate agonists studied do not distinguish between the mGluR2 and mGluR3 subtypes, the rarity of mGluR3 and 2AR mRNA co-expression in cortex, the absence of evidence for 2AR/mGluR3 complex formation by co- immunoprecipitation, BRET and FRET, and the detection of 2AR/mGluR2 complexes by these same assays, suggest that the crosstalk identified results from 2AR/mGluR2 complexes.
The changes in high affinity binding caused by 2AR/mGluR2 crosstalk suggested that this complex may serve to integrate serotonin and glutamate signalling and modulate G protein coupling (Kenakin, T., 2002, Nat Rev Drug Discov 1 :103-10; Gonzalez-Maeso et al., 2002, Eur J Pharmacol 452:21-33). This hypothesis was tested by measuring 2AR regulation of Gαq/π and Ga; proteins. High-affinity activation of Gαq/n by the 2AR was reduced by co-expression of mGluR2 (Fig. 4). Interestingly, the activation of Ga; by the 2AR was markedly enhanced by mGluR2 co-expression (Fig. 4). The mGluR2 -dependent effects on both Gαq/n and Goij regulation by the 2AR were reversed in the presence of mGluR2 agonist (Fig. 4). Consonant with the co-immunoprecipitation, allosteric modulation and FRET results, the functional assays of G protein activity also show that the TM4-5 segment of the mGluR2, when substituted into the mGluR3, was sufficient for signalling crosstalk to occur (Fig. 4). These data support the presence of functional and physiological 2AR/mGluR2 complexes that integrate serotonin and glutamate neurotransmission to specify the pattern of G protein regulation.
Similar evidence for specification of G protein subtype regulation was also observed by the endogenous brain 2AR/mGluR2 complex with membranes from cortical primary cultures (Fig 5). The pattern of G protein regulation in cortical pyramidal neurons has been shown to predict specific behavioral responses to 2AR agonists. Hallucinogenic drugs and non-hallucinogenic drugs activate the same population of 2ARs in cortical pyramidal neurons, but differ in the 2AR-dependent pattern of G protein regulation and gene induction they elicit (Gonzalez-Maeso, J. et al., 2007, Neuron 53:439-52; Gonzalez-Maeso et al. 2003, J Neurosci 23:8836-43). In brain cortical neurons, the signalling elicited by hallucinogenic and non- hallucinogenic 2AR agonists causes induction of c-fos and requires Gq/π -dependent phospholipase C activation. However, the signalling of hallucinogens such as DOI and LSD acting at the 2AR also induces egr-2, which is G,/0-dependent. Thus c-fos expression results from any 2AR-signalling, and egr-2 induction is a specific marker for hallucinogen signalling via the 2AR (Gonzalez-Maeso et al., 2007, Neuron 53:439-52; Gonzalez-Maeso et al. 2003, J Neurosci 23:8836-43). The finding that niGluR2 modulates the G1 protein coupling of the 2AR (Figs. 4 and 5) suggested that this complex might be important for hallucinogen signalling. The induction of c-fos by hallucinogenic 2AR agonists or by structurally similar non-hallucinogenic 2AR agonists in vivo in mouse SCx and in cortical primary cultures (Fig. 5) was not affected by the mGluR2/3 agonist LY379. In contrast, the hallucinogen-specific induction of egr-2 was selectively blocked by LY379 in both mouse cortex in vivo and in primary cortical cultures (Fig. 5). The effects of LY379 on the head-twitch response (HTR) behavior, which is hallucinogen-specific, was also studied (Gonzalez-Maeso et al., 2007, Neuron 53, 439-52; Gonzalez-Maeso et al., 2003, J Neurosci 23:8836-43). Similar to its effects on G protein activation and gene induction, the glutamate agonist LY379 suppressed the induction of the HTR by either DOI or LSD (Fig. 6). These results suggest that LY379 acts at the 2AR/mGluR2 complex to reduce the hallucinogen-specific G,/o protein signalling and behaviour. To further establish the functional relevance of 2AR/mGluR2 crosstalk, we compared the responses to the mGluR2/3 antagonist LY341494 in htr2A+/+ and htr2A-/- mice. The locomotor and vertical activities elicited by LY341495 were significantly attenuated in the htr2A-/- mice (Fig. 5), supporting the functional relevance of the 2AR/mGluR2 complex in vivo and suggesting that it also influences the endogenous response to glutamate. mGluR2 Directly Interacts with 2AR
The differences in the capacity of the mGluR2 and mGluR3 to interact with the 2AR and their close sequence similarity provided the basis to identify the specific mGluR2 domains responsible for heterocomplex formation.
A study of a series of molecular chimeras of the mGluR2 and mGluR3 (see Fig. 4) demonstrated that the segment containing transmembrane (TM) helices 4 and 5 of the mGluR2 receptor was both necessary and sufficient for complex formation with the 2AR. The mGluR3 receptor chimera containing only this segment from the mGluR2 (mGluR3ΔTM4,5) was capable of co-immunoprecipitating with the 2AR (Fig 4), mediating allosteric crosstalk (Fig. 4) and maintaining close proximity with the 2AR as indicated by FRET (Fig. 4).
In contrast, mGluR2ΔTM4,5 did not show evidence of complex formation with the 2AR (Fig. 4). The absolute and relative levels of expression of heterologous constructs were comparable to the physiological levels found in mouse SCx, and in cortical primary cultures (Fig. 4).
2AR is Upregulated and mGluR2 is Downregulated in Schizophrenic Brains
The findings that Gj/0 protein regulation, which is necessary for the effects of hallucinogens, is enhanced by the formation of the 2AR/mGluR2 complex and that activation of the mGluR2 component suppresses hallucinogen-specific signalling implicate this complex in the effects of hallucinogens (Gonzalez-Maeso et al., 2007,
Neuron 53:439-52). The neuropsychological effects of hallucinogenic drugs present commonalities with the psychosis of schizophrenia, and both conditions are accompanied by disruptions of cortical sensory processing (Vollenweider et al., 1998,
Neuroreport 9:3897-902; Gouzoulis-Mayfrank, E. et al., 2005, Pharmacopsychiatry
38:301-11; Carlsson, A., 2006, Pharmacopsychiatry 39(Suppl l):S10-4; Vollenweider et al., 2001, Brain Res Bull 56:495-507; Vollenweider et al., 1997,
Neuropsychopharmacology 16:357-72; Umbricht, D. et al., 2003, Neuropsychopharmacology 28:170-81; Gouzoulis-Mayfrank et al., 2005,
Neuropsychopharmacology) .
Whether the components of the 2AR/mGluR2 signalling complex are dysregulated in brain cortex of subjects with schizophrenia was eaxamined. The density of 2AR and mGluR2/3 binding sites in cortex from schizophrenic subjects and controls who were matched by gender, age, and postmortem delay was examined (Fig, 7). The receptor densities in cortical membranes from untreated schizophrenic subjects were significantly altered, showing increased 2AR and reduced mGluR2/3 receptor levels (Fig. 7). mRNA assays showed that expression of mGluR2 but not mGluR3 was reduced in schizophrenia cortex (Fig. 7). The studies in mouse show that activation of the mGluR2 component of the 2AR/mGluR2 complex eliminates the hallucinogen-specific component of the signalling responses to LSD-like drugs. Thus the increased 2AR and decreased mGluR2 found in the brain in schizophrenia may predispose to a hallucinogenic pattern of signalling.
Many laboratories have attempted to determine the density of 2AR in postmortem brain from subjects with schizophrenia, and some studies have reported decreased or unchanged 2AR densities (Dean, B. J., 2003, Neurochem 85:1-13). To try to understand the basis for these discrepancies from our results, the effects of chronic antipsychotic treatment on the 2AR and mGluR2 in mouse was examined. The chronic atypical antipsychotic clozapine specifically down-regulated the level of expression of 2AR and of mGluR2 in mouse SCx (Fig. 8). The down-regulation of mGluR2 by clozapine required expression of the 2AR, as it did not occur in htr2A-/- mice (Fig. 8), and was not induced by the chronic typical antipsychotic haloperidol (Fig. 8). In concordance with the effects of clozapine in murine models, the density of 2AR was reduced to control levels in postmortem human brain cortex of schizophrenics treated with atypical antipsychotic drugs (Fig. 7), and the mGluR2/3 binding sites were also down-regulated (Fig. 7).
The onset of psychosis in schizophrenia usually occurs in later adolescence or early adulthood (Freedman, 2003, R. N Engl J Med 349:1738-49). The relationship of receptor densities with aging and both [3H]ketanserin and [3H]LY341495 binding displayed a highly significant negative correlation with age (Fig. 9). Hallucinations and delusions typically attenuate with aging, which correlates with the lower density of the components of the 2AR/mGluR2 complex that was observed in older subjects (Davidson, M. et al., 1995, Am J Psychiatry 152:197-207). Consequently, the marked dysregulation of both 2AR and mGluR2 expression in schizophrenia would be unlikely to be observed in samples from heterogeneous groups including treated patients or in studies including older patients (Dean, B. J., 2003, Neurochem 85:1-13; Gurevich et al., 1997, J. N. Biol Psychiatry 42:529-45). These studies identify the 2 AR/mGluR2 complex as a possible site of action of hallucinogenic drugs. The glutamate and serotonin systems have both been implicated in psychotic disorders, and the components of this complex are found to be differentially regulated in cortex from individuals with schizophrenia. The results are consistent with the hypothesis that the 2AR/mGluR2 complex integrates serotonin and glutamate signalling to regulate the sensory gating functions of the cortex, a process that is disrupted in psychosis.
EXAMPLE 2 Glutamate Receptor Agonists Target the 5-HT2A/mGluR2 Hetero receptor Complex to modulate signalling and behavior
Schizophrenia is a brain disease that affects perception, emotion and cognition (GonzalezMaeso, J. et al. 2008, Nature 452, 9397). The serotonin (5-HT) and glutamate (GIu) neurotransmitter systems are suspected in the etiology and pathophysiology of schizophrenia, as well as in the mechanism of action of antipsychotic drugs (Marek, G. J., 2004, Curr. Opin. Pharmacol. 4, 18-22; Carlsson, A. et al., 2001, Annu. Rev. Pharmacol. Toxicol. 41, 237-260; Kristiansen, L. V.,
2007, Curr. Opin. Pharmacol. 7, 48-55; Tsai, G. wt al., 2002, Annu. Rev. Pharmacol. Toxicol. 42, 165-179; and Aghajanian, G. K. et al., 2000, Brain Res. Brain Res. Rev.
31, 302-312). A high affinity for the serotonin 5HT2A receptor (2AR) is a common characteristic of all atypical antipsychotics (Miyamoto, S. et al., 2005, MoI. Psychiatry 10, 79-104.) Hallucinogenic 2AR agonists, such as lysergic acid diethylamide (LSD) or psilocybin, produce a psychosislike syndrome in humans that resembles the first episodes of schizophrenia (Vollenweider, F. X., et al., 1998, Neuroreport 9, 3897-3902 and Geyer, M. A. et al., 2008, Trends Pharmacol. Sci.)
G proteincoupled receptors (GPCRs) are the largest family of signalling proteins in the mammalian genome and the most common target for therapeutic drugs (Pierce, K. L. et al., 2002, Nat. Rev. MoI. Cell Biol. 3, 639-650.) These heptahelical membrane proteins activate heterotrimeric GTPbinding G proteins to modulate cell signalling. G proteins are grouped in four different families (Gs, Gj/0, Gq/n, and Gi2) based on the degree of homology of the primary structure (Oldham, W. M. et al.,
2008, Nat. Rev. MoI. Cell Biol. 9, 60-71.) Each of these G protein families is associated with distinct cellular signalling effects. The 2AR and the mGluR2 are coupled to Gq/i i and G,/o G proteins, respectively. GPCRs have been found to interact to form homo and heterocomplexes in the plasma membrane, but the effect of complex formation on signal transduction is not certain for most GPCRs (Terrillon, S. et al., 2004, Proc. Natl. Acad. Sci. U. S. A. 101, 1548-1553; Lopez-Gimenez, J. F. et al., 2007, MoI. Pharmacol. 71, 1015-1029; Guo, W. et al., 2008, EMBO J. 27, 2293- 2304; and Carriba, P. et al., 2008, Nat. Methods 5, 727-733).
The 2AR/mGluR2 complex is involved in the cellular responses induced by LSD-like drugs (Gonzalez-Maeso, J. et al., 2008, Nature 452, 93-97; and Gonzalez- Maeso, J. et al., 2007, Neuron 53, 439-452.) The effect of the functional crosstalk between the 2AR and mGluR2 on G protein coupling and behavioural effects of metabotropic glutamate antipsychotics was examined in vitro and with murine in vivo models.
Methods
Materials and Drug Administration. 5-hydroxytryptamine (serotonin, 5HT), 1- (2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and (+)-MK801 hydrogen maleate (MK801) were purchased from Sigma- Aldrich. (lR,4R,5S,6R)-4-Amino-2- oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268) was obtained from Tocris Cookson Inc. [35S]GTPyS was purchased form PerkinElmer Life and Analytical Sciences, Inc. [ H]LY341495 was purchased from American Radiolabeled Chemicals, Inc. The injected doses (i.p.) were 5 mg/kg LY379268 and 0.5 mg/kg MK801.
Molecular constructs. PCR amplification and subcloning of 2AR, mGluR2, mGluR3, mGluR2ΔTM4,5 and mGluR3ΔTM4,5 into pcDNA3.1 was described previously(Gonzalez-Maeso, J. et al., 2008, Nature 452, 93-97.) The human 2AR, the human mGluR2, and the mGluR2ΔTM4,5 were digested with BamHI and Notl and subcloned into the pXOOM vector (Jespersen, T. et al., 2002, BioTechniques 32, 536- 8, 540.) The human mGluR3 and the mGluR3ΔTM4,5 chimaera were digested with EcoRI and Notl and subcloned into pXOOM. The human RGS2 (University of Missouri-Rolla, UMR) was PCR-amplified using the primers RGS2/S (TTTTggatccATGCAAAGTGCTATGTTCTT) and RGS2/A
(TTTTgcggccgcTCATGTAGCATGAGGCTCTG) and subcloned into the BamHI and Notl sites of pXOOM. Constructs subcloned into the pGEMHE vector with the active point mutants of the human Kir3.4, Kir3.4-S143T or Kir3.4*, Kir2.3 (IRK3) and Pertussis Toxin (PTX) subunit B were previously described (Vivaudou, M. et al., 1997, J. Biol. Chem. 272, 31553-31560.)
Expression of Recombinant Proteins in Xenopus oocytes. The cRNA concentration of all constructs was estimated by optical densitometry and electrophoresed in parallel on formaldehyde gels, compared to known concentrations of RNA marker (Invitrogen, Carlsbad, CA). Expression of proteins in oocytes was accomplished by injection of the desired amount of cRNA into Xenopus oocytes. In all two-electrode voltage-clamp experiments (TEVC), oocytes were injected with 1 ng of mGluR2, 2ng of mGluR2ΔTM4,5, 2 ng of mGluR3, 2 ng of mGluR3ΔTM4,5, 2 ng of 2AR, 2 ng of GIRK4*, 2 ng of IRK3, and 1 ng of PTX or 4 ng of RGS2, when present. Oocytes were isolated and microinjected with equal volumes (50 nl), as previously described 52. All oocytes were maintained at 18°C and recordings were performed 14 days following injection adjusting for differences in translation kinetics of each construct.
Two-electrode voltage-clamp recording and analysis. Whole-cell currents were measured by conventional two-microelectrode voltage clamp (TEVC) with a GeneClamp 500 amplifier (Axon Instruments, Union City, CA), as previously reported (Lopes, C. M. et al., 2002, Neuron 34, 933-944.) A high-potassium (HK) solution was used to superfuse oocytes (96 mM KCl, 1 mM NaCl, 1 mM MgCl2, 5 mM KOH/HEPES; pH 7.4) to obtain a reversal potential for potassium (Ek) close to zero. Calcium-activated chloride currents were obtained clamping the cells at + 80 mV. Stable currents obtained at that voltage prior to perfusion of agonist (basal currents) were determined for each trace. Peak currents were measured and normalized to basal current to compensate for size variability in oocytes. Inwardly rectifying potassium currents through GIRK4* and IRK3 were obtained by clamping the cells at 80 mV. Basal currents were defined as the difference between inward currents obtained at 80 mV in the presence of 3 mM BaCl2 in HK solution and those in the absence OfBa2+ and measured for each trace. Radioligand binding assays. Mouse frontal cortex membrane preparations and radioligand binding assays were performed as previously described with minor modifications (Gonzalez-Maeso, J. et al., 2008, Nature 452, 93-97.)
Behavioural Studies. Behavioural studies were performed as previously described (Gonzalez-Maeso, J. et al., 2008, Nature 452, 93-97.)
Experimental Animals. Experiments were performed on adult (8-12 weeks old) male 129S6/SvEv mice. Animals were housed at 12 h light/dark cycle at 230C with food and water ad libitum. The Institutional Animal Use and Care Committee approved all experimental procedures at the Mount Sinai School of Medicine.
Statistical Methods. Each electrophysiological experiment in Xenopus oocytes was performed in two batches. Every group in each experiment was tested in both batches. Data for both batches were compiled (n = 8-16) and oneway ANOVA tests applied followed by a multiple comparison procedure using Tukey's honestly significant difference test. [3H]LY341495 binding experiments were performed 3-5 times in duplicate/triplicate. One-site model versus two-site model as a better description of the data was determined by F test. [35S]GTPyS binding experiments were performed three times in triplicate. Two-way ANOVA tests were applied to the compiled data followed by a Bonferroni's post hoc test. Horizontal locomotion experiments were analyzed with two-way ANOVA followed by a Bonferroni's post hoc test (n = 5-6).
Results
Ion-channel responses to monitor G protein signalling.
Expression of ion channels in Xenopus oocytes provides a useful system to observe activity of different G protein signalling pathways (Chan, K. W. et al., 1997, J. Biol. Chem. 272, 6548-6555; and Kobrinsky, E. et al., 2000, Nat. Cell Biol. 2, 507- 514.) The distinct G protein coupling and electrophysiological properties of different ion channels allow response patterns that are characteristic of the G,/o-opathway and Gq/! i -pathways to be assayed. The main elements in the G,/o and Gq/π -pathways as well as the specific ionchannel responses used as readouts throughout the experiments are shown in Figure 17. G protein-coupled inwardly rectifying potassium channels (GIRK), a subfamily of Kir (inwardly rectifying K+) channels (or Kir3), are known to be sensitive to the Gβγ subunits of G,/o proteins (Logothetis, D. E. et al., 1987, Nature 325, 321-326.) For monitoring the G,/0-signalling a mutated version of Kir3.4 (GIRK4*) was used that produces large currents and expresses well in oocytes (Fig 17a) (Vivaudou, M. et al., 1997, J. Biol. Chem. 272, 31553-31560; and Zhang, H. et al., 1999, Nat. Cell Biol. 1, 183-188.) Upon Gq/1 ] -activation (Figs 17b and 17c), phospholipase C (PLC) is activated to hydrolyze phosphatidylinositol-4,5-biphosphate (PIP2) in the inner leaflet of the membrane into diacylglycerol (DAG) and inositol triphosphate (IP3). IP3 in turn opens calcium channels in the endoplasmic reticulum (ER) and mobilizes Ca2+ to the cytosol. Gq/π-mediated PIP2 hydrolysis can be detected by measuring changes in the currents through Kir 2.3 (IRK3), a potassium channel member of the Kir2 subfamily (Fig 17b) (Du, X. et al., 2004, J. Biol. Chem. 279, 37271-37281; Liu, B. et al., 2007, Eur. J. Pharmacol. 558, 21-26; and Wang, C. et al., 2008, MoI. Pharmacol. 73, 1185-1194.) Outwardly rectifying chloride currents that are activated by intracellular Ca2+ are induced as a readout of Gq/π signalling (Fig 17c) (Miledi, R. A, 1982, Proc. R. Soc. Lond. B. Biol. Sci. 215, 491-497; and Acuna- Castillo, C. et al., 2002, Br. J. Pharmacol. 136, 510-519.)
2AR allows mGluR2 activation to elicit ion-channel responses indicative of Gq/π signalling.
Currents through calcium-activated chloride channels (Fig 17c) were assayed as a readout of Gq/i i activation in oocytes expressing mGluR2 alone, 2AR alone, or mGluR2 and 2AR together. The responses to the selective mGluR2/3 agonist LY379268 and to serotonin are shown in Figure 18a. LY379268 did not induce currents characteristic of Gq/π signalling in oocytes expressing mGluR2 alone or 2AR alone. However, the metabotropic glutamate agonist elicited a current through calcium-activated chloride channels in cells that coexpressed the mGluR2 and 2AR (Fig 18b). This distinct chloride-current response was characterized by a sharp peak followed by a slower decay. As expected, only the groups expressing 2AR or coexpressing mGluR2 and 2AR had characteristic Gq/π -activated currents elicited by serotonin (Fig 18c).
Whether the metabotropic glutamate agonist also induced IRK3 channel currents characteristic of Gq/i i signalling in oocytes expressing either receptor or both receptors was then tested (Fig 17b). IRK3 channel cRNA was also injected with the receptors tested in each group of oocytes since, as opposed to calcium-activated chloride channels (Fig 17c), IRK3 channels are not endogenously expressed in Xenopus oocytes (Liu, B. et al., 2007, Eur. J. Pharmacol. 558, 21-26; and Wang, C. et al., 2008, MoI. Pharmacol. 73, 1185-1194.) In each group the potassium current flowing through IRK3 was studied (Fig 19a). Consonant with the chloride current results, the metabotropic glutamate agonist L Y379268 elicited a response in oocytes coexpressing mGluR2 and 2AR, but not in oocytes expressing mGluR2 alone (Fig 19a). As expected from the previous experiment (Fig 18), a response through the chloride channel was also observed in this group (outward current data not shown). The decrease in the IRK3 channel activity (current inhibition) was greater in response to serotonin for the group expressing 2AR alone (Fig 19a). The responses induced both by L Y379268 and serotonin were consistent with Gq/n -dependent kinetics (Mark, M. D. et al., 2000, Neuropharmacology 39, 2360-2373; and Keselman, I. et al., 2007, Channels (Austin) 1, 113-123.) A summary of the normalized agonistinduced inhibition for each group expressed as a percentage of the maximal response to serotonin is shown in Figure 19b. Concurrently, these data with calcium-activated chloride channels (Fig 18) and with IRK3 channel currents (Fig 19) suggest that in the presence of a coexpressed 2AR, the mGluR2 is capable of activating Gq/n type G proteins.
Disruption of the 2AR/mGluR2 complex abolishes Gq^ i signalling by mGluR2 stimulation.
As desccribed in Example 1, mGluR3, which is closely related to mGluR2, is not capable of forming a heterocomplex with 2AR 13. Using a series of molecular chimaeras it has been shown that the segment containing transmembrane (TM) helices 4 and 5 of the mGluR2 was both necessary and sufficient for the formation of a complex with the 2AR. Thus, the mGluR3 chimaera containing only this segment from the mGluR2 (mGluR3ΔTM4,5) was capable of forming a receptor complex with the 2AR. In contrast, mGluR2ΔTM4,5 did not show evidence of complex formation with the 2AR (Gonzalez-Maeso, J. et al., 2008, Nature 452, 93-97.) Here the mGluR2/mGluR3 chimaeras were used to study whether formation of a complex between the mGluR2 and 2AR was required for the emergence of Gq/π activation by the mGluR2/3 agonist LY379268. First, the functionality of mGluR2, mGluR3, mGluR2TM4,5 and mGluR3TM4,5 was assessed in Xenopus oocytes. While the various mGluR constructs differed in the magnitude of the currents generated, all were functionally coupled to Gj/o (Fig 24). It was then determined if formation of the 2AR/mGluR2 complex was required for the activation of Gq/π -dependent signalling by the metabotropic glutamate agonist. As in previous experiments (Fig 18), LY379268 elicited a response when the mGluR2 was coexpressed with the 2AR (Fig 20a). In contrast, mGluR3 coexpressed with 2AR did not activate the chloride current in response to LY379268. The signalling response to LY379268 was rescued when the complex was formed in cells coexpressing 2AR with mGluR3ΔTM4,5. Furthermore, disruption of the receptor complex by coexpression of 2AR with mGluR2ΔTM4,5 abolished the signal. A summary of the responses to LY379268 is shown in Figure 20b. All the groups coexpressing mGluR2/mGluR3 chimaeras and 2AR responded to serotonin (Figs 20a and 20c).
Activation of Gq/π signalling through the 2AR/mGluR2 complex is RGS2 sensitive and PTX insensitive.
Regulator of G protein signalling (RGS) proteins have been extensively used to probe the complexity of GPCR cellular signalling mechanisms (Neubig, R. R., 2002, J. Pept. Res. 60, 312-316; and Ross, E. M. et al., 2000, Annu. Rev. Biochem. 69, 795-827.) RGS2 is a GTPase activating protein (GAP) that specifically acts on the Gq/ii -pathway, and has been previously studied in oocytes (Mark, M. D. et al., 2000, Neuropharmacology 39, 2360-2373.) RGS2 suppresses Gq/π protein-mediated processes by driving activated Gq/π (Gαq/n-GTP) proteins into their inactive form (Goiq/ii-GDP). Pertussis Toxin (PTX) is a G protein signalling blocker whose specificity for the Gj/0-pathway has been well established both in native (Logothetis, D. E. et al., 1987, Nature 325, 321-326; Pfaffmger, P. J. et al., 1985, Nature 317, 536- 538) and in heterologous systems, such as Xenopus oocytes (Vivaudou, M. et al., 1997, J. Biol. Chem. 272, 31553-31560) (see Fig 17).
First the effects of PTX and RGS2 on the Gj/0-signalling elicited by the mGluR2 alone was tested. Figure 21a shows representative traces of current flow through GIRK4* potassium channels (Fig 17a) measured in oocytes expressing mGluR2 and the channel. The activity of the GIRK4* channel was increased (downward deflection in the trace from the initial steady state) and sustained in the presence of LY379268. Perfusion of barium, an inhibitor of inwardly rectifying K+ currents, blocked the current through GIRK4* channels. Consistent with previous reports (Pfaffmger, P. J. et al., 1985, Nature 317, 536-538), expression of PTX, but not RGS2, decreased the basal currents (Fig 21b). The current induced by LY379268 through GIRK4* channels was abolished by PTX and unaffected by RGS2 (Fig 21c). Calcium-activated chloride currents were not elicited by LY379268 signalling through the mGluR2 receptor in these groups of oocytes (outward current data not shown). These results demonstrate that mGluR2 alone activates G,/0-dependent but not GqZ11 -dependent signalling.
Next, the effects of disruption of G,/o (PTX) or Gq/i i (RGS2) signalling was investigated by monitoring the calcium-activated chloride currents in oocytes expressing mGluR2 alone, 2AR alone, or mGluR2 and 2AR together (Fig 22). The results indicate that the Gq/n -electrophysiological responses to LY379268 that depend on the 2AR/mGluR2 complex, {see Fig 20) were not affected by PTX (Figs 22a and 22c). The Gq/n mediated calcium-activated chloride currents elicited by serotonin were unaffected by PTX (Figs 22a and 22d). Complementary experiments were performed with RGS2 to interfere with Gq/π signalling. Notably, the responses to L Y379268 that depend on the 2AR/mGluR2 complex {see Fig 20) were abolished by RGS2, further substantiating the role of Gq/i i in these metabotropic glutamate agonist induced currents (Figs 22b and 22c). Control experiments showed that the calcium- activated chloride currents in response to serotonin are also blocked by expression of RGS2 (Figs 22b and 22d). Concurrently, these results demonstrate that the metabotropic glutamate agonist activates both G,/o and Gq/n proteins only when mGluR2 is expressed as a receptor complex with the 2AR in Xenopus oocytes (Fig 22e).
Activation of Gq/π by LY379268 is abolished in 2AR null-mutant mice.
The 2AR expressed by cortical pyramidal neurons is necessary for the cellular and behavioural effects induced by LSDlike drugs (Gonzalez-Maeso, J. et al., 2007, Neuron 53, 439-452.) In order to further investigate the mechanism of action of metabotropic glutamate antipsychotics, the ligand binding properties of LY379268 in mouse frontal cortex was examined. As previously reported in wild-type mouse (Gonzalez-Maeso, J. et al., 2008, Nature 452, 93-97), displacement of the mGluR2/3 antagonist [3H]LY341495 by LY379268 showed high- and low-affinity binding sites, and the hallucinogenic 2AR agonist DOI abolished the high-affinity binding site for LY379268 (Fig 23a). Notably, the fraction of high-affinity binding sites for LY379268 was undetectable in 2AR nullmutant mice, both in the presence and in the absence of DOI (Figs 23a and 25). The effects on high-affinity agonist binding (Kenakin, T., 2002, Annu. Rev. Pharmacol. Toxicol. 42, 349-379) suggested that the 2AR may modulate the pattern of G proteins activated by LY379268 in mouse frontal cortex. This possibility was tested by measuring [35S]GTPyS binding followed by immunoprecipitation with anti-Gαq/π or anti-Gα, antibodies in wild-type and 2AR null-mutant mice (Fig 23b). The absence of 2AR did not affect the activation of G,/o proteins. In contrast, the activation of Gq/i i proteins by the metabotropic glutamate agonist was abolished in the 2AR null-mutant mice. These data indicate that the novel cross-activation of Gq/π by metabotropic glutamate agonist acting at the in vitro 2AR/mGluR2 heterocomplex also occurs with the native receptor complex in cortical neurons in vivo.
The effects of mGluR2 on the behavioural responses induced by psychotomimetic NMDA receptor antagonists are abolished in 2AR null-mutant mice.
Noncompetitive NMDA receptor antagonists, such as phencyclidine (PCP) and ketamine are used as pharmacological models for schizophrenia in animals because of their capacity in humans to evoke positive and negative symptoms as well as cognitive deficits resembling those seen in this disease (Kristiansen, L. V. et al., 2007, Curr. Opin. Pharmacol. 7, 48-55; and Morris, B. J. et al., 2005, Curr. Opin. Pharmacol. 5, 101-106.) The potent and selective noncompetitive NMDA receptor antagonists MK801 (dizocilpine) can also elicit ketamine-like symptoms in healthy volunteers (Reimherr, F. W. et al., 1986, Psychopharmacol. Bull. 22, 237-242.) Behavioural responses induced in rodent models by ketamine-like drugs include increased activity, head weaving, deficits in paired pulse inhibition (PPI) and social interaction, and increased forced swim immobility. Activation of mGluR2, but not mGluR3, has been found to reduce the behavioural stereotypy and hyperlocomotion produced by NMDA antagonists (Spooren, W. P. et al., 2000, Eur. J. Pharmacol. 397, R12; Fell, M. J. et al., 2008, J. Pharmacol. Exp. Ther. 326, 209-217; and Woolley, M. L. et al., 2008, Psychopharmacology (Berl) 196, 431-440.) The in vitro and in vivo findings described above indicate that the 2AR/mGluR2 complex mediates a Gq/n signalling induced by the mGluR2/3 agonist LY379268. We hypothesized that if this novel mGluR2/3 agonist signalling via the 2AR/mGluR2 complex contributed to their antipsychotic effects, then the 2AR would be necessary for their suppression of MK801 -induced behaviour. This was tested by comparing the effects of LY379268 on the locomotor behaviour induced by MK801 in wild-type and 2AR null-mutant mice. The locomotor activity elicited by MK801 in wild-type and 2AR null-mutant mice was indistinguishable (Fig 23c). Notably, the MK801 -stimulated locomotor activity was significantly attenuated by LY379268 in wild-type mice, but it was not affected in 2AR null-mutant mice (Fig 23c). These data support the functional relevance of the 2AR in the behavioural responses induced by metabotropic glutamate antipsychotics.
Discussion
This study investigated the signalling of metabotropic glutamate agonist at the 2AR/mGluR2 complex and the mechanisms for its antipsychotic effects. A surprising and novel cross-activation of G proteins by metabotropic glutamate agonist via this receptor heterocomplex was discovered. The principal coupling of the mGluR2 receptor is through Gj/0 proteins and that of the 2AR through Gq/π proteins. Electrophysiological recordings in Xenopus oocytes and biochemical assays in mouse frontal cortex showed that activation of the 2AR/mGluR2 complex by metabotropic glutamate agonist stimulates both Gj/0 and Gq/n -dependent signalling. Notably, it was shown that the antipsychotic-like effects of metabotropic glutamate agonist on MK801 -induced behaviour were absent in 2AR null-mutant mice. The antipsychotic properties of the mGluR2/3 agonists may therefore be due, in part, to the signalling crosstalk resulting from their activity at the 2AR/mGluR2 complex in cortical neurons. A number of GPCR homo- and hetero-complexes have been identified
(Terrillon, S. et al., 2004, Proc. Natl. Acad. Sci. U. S. A. 101, 1548-1553; and Milligan, G. G., 2004, MoI. Pharmacol. 66, 1-7.) Other than the 2AR/mGluR2 complex, all the receptor heterocomplexes have in common that the two individual receptors are coupled to the same family of heterotrimeric G proteins, and that they belong to the same class of GPCRs. Furthermore, while coactivation of both receptors in a heterocomplex has been reported to alter signalling (Kearn, C. S. et al., 2005, MoI. Pharmacol. 67, 1697-1704; Rios, C. et al., 2006, Br. J. Pharmacol. 148, 387-395; Jordan, B. A. et al., 2003, MoI. Pharmacol. 64, 1317-1324; Vilardaga, J. P. et al., 2008, Nat. Chem. Biol. 4, 126-131; George, S. R. et al., 2000, J. Biol. Chem. 275, 26128-26135; and Mellado, M. et al., 2001, EMBO J. 20, 2497-2507), a heterocomplex formation has not previously been found to alter responses at each individual component of the receptor complex. A new level of complexity arises with the realization that GPCR heterocomplexes can also exist between different classes of surface receptors that are each by themselves coupled to different families of heterotrimeric G proteins, and that these complexes may dramatically alter the signalling responses. As demonstrated here, metabotropic glutamate agonist bind the 2AR/mGluR2 complex leading to activation of both Gq/π and Gj/0 proteins. The 2AR/mGluR2 complex represents the first demonstration in living cells in which agonist binding to one receptor component of the heteromer results in activation of the G protein normally coupled only with the other receptor.
Only the mGluR2, but not the mGluR3, is able to form a receptor complex with the 2AR (Gonzalez-Maeso, J. et al., 2008, Nature 452, 93-97). Furthermore the fragment containing the transmembrane domains 4 and 5 of mGluR2 is necessary and sufficient for complex formation with 2AR. The specificity of 2AR/mGluR2 complex formation was used to investigate in live cells the functional cross-talk between the components of the receptor heterocomplex. It was found that the mGluR2/3 agonist L Y379268 elicited calcium-activated chloride currents when mGluR2 and 2AR, but not mGluR3 and 2AR, were coexpressed in Xenopus oocytes. The RGS2 sensitivity and PTX insensitivity of the calcium-activated chloride currents confirmed that activation of the Gj/0-coupled mGluR2 was eliciting Gq/π signalling in the cell. Enabling or disrupting the 2AR/mGluR2 complex with coexpression of 2AR and mGluR2/mGluR3 chimaeras demonstrated that the LY379268-dependent activation of both Gq/] i and Gj/0 signalling was only achieved when the 2AR and the mGluR2 were expressed in live cells as a receptor complex. The functional significance of the 2AR/mGluR2 complex in brain function was supported by biochemical assays and behavioural models of psychosis in wild-type and 2AR null-mutant mice. The data demonstrate that LY379268 requires the expression of 2AR to activate Gj/0 and Gq/π proteins in mouse frontal cortex, and to abolish the locomotor activity induced by ketamin-elike drugs.
The serotonin2A receptor, 2AR, classically couples to intracellular signaling via the heterotrimeric Gq subtype G-protein, and the mGluR2 via the Gj/G0 subtype G proteins. Examples 1 and 2 described herein demonstrate that hallucinogenic drugs such as LSD act at the 2AR component of the 2AR/mGluR2 complex to activate both G, and Gq, a response necessary for the hallucinogenic activity and head-twitch behavioral response in mice. The hallucinogenic pattern of signaling elicited at this complex leads to a characteristic pattern of gene changes, including induction oϊc-fos, egrl and egr2. mGluR2/3 antipsychotic drugs act at the mGluR2 component of the complex to disrupt hallucinogen signaling and suppress the head twitch response to hallucinogens in mice and the induction of egrl and egr2, while allowing the induction of c-fos. The c-fos response is also activated by non-hallucinogens such as lisuride acting at the 2AR site in the complex. When the effects of the glutamate agonist by itself at the complex are studied, the results described herein show that both G, and Gq are activated, whereas only G1 is activated when the mGluR2 receptor is activated in the absence of the 2AR. These data indicate that the coding of "hallucinogens" or of "antipsychotics" in the signaling pathways in the neurons downstream of this complex is not merely whether G1 or Gq are activated. The importance of the specific pattern of activation of signaling components in determining the effects of signaling is emerging as a critical determinant of cellular effect in many different contexts. For example, epidermal growth factor (EGF) and nerve growth factor (NGF) both cause activation of the mitogen-activated kinase ERK in PC 12 cells. However, only NGF causes neuronal differentiation of cells. The difference in the effects of the two factors is due to differences in the pattern of ERK activation. A prolonged activation of ERK is the signal for differentiation (Santos, SD et al, 2007, Nat Cell Biol, 9: 324-30.) While EGF and NGF act at different receptor targets, the lessons of their signaling specificity are applicable to the 2AR/mGluR2 complex. The critical element in causing a "hallucinogen" response or in an "antipsychotic" response must be in the precise temporal pattern and relative levels of regulation of G1 and Gq. Thus in may be proposed that both atypical antipsychotics (such as clozapine) and mGluR antipsychotics act at the complex to modulate the pattern of G, and Gq regulation elicited by hallucinogens in the normal brain as well as by glutamate and serotonin in the schizophrenic brain to reduce the generation of a "hallucinogenic" pattern of G, and Gq signaling. This "hallucinogenic" pattern is reflected in the precise timing and amplitude of Gi and Gq activation achieved at this complex by hallucinogens such as LSD.
These results described by Examples 1 and 2 herein also may provide insight into the neurochemical abnormalities of altered brain function in schizophrenia. In contrast with other chronic brain diseases such as Parkinson's and Alzheimer's, schizophrenia lacks diagnostic neuropathological lesions and genetic animal models. Several neurochemical hypotheses of schizophrenia have been proposed, among them abnormalities of serotonin and glutamate systems (Aghajanian, G. K. et al., 2000, Brain Res. Brain Res. Rev. 31, 302-312.) The serotonin hypothesis, which is suggested by the 2AR-dependent effects of hallucinogens such as LSD (Vollenweider, F. X. et al., 1998, Neuroreport 9, 3897-3902; Gonzalez-Maeso, J. et al., 2008, Nature 452, 93-97; Gonzalez-Maeso, J. et al., 2007, Neuron 53, 439-452), led to the discovery of several atypical antipsychotics including risperidone (Colpaert, F. C, 2003, Nat. Rev. Drug Discov. 2, 315-320.) The role of glutamate transmission was suggested by the psychotic state elicited by some antagonists of the NMDA-type glutamate receptor (Kristiansen, L. V. et al., 2007, Curr. Opin. Pharmacol. 7, 48-55; and Morris, B. J. et al., 2005, Curr. Opin. Pharmacol. 5, 101-106.) The 2AR/mGluR2 complex brings together several threads of both serotonin and glutamate hypotheses of schizophrenia. Evidence suggests that this complex mediates the signalling changes in cortical pyramidal neurons elicited by LSD-like drugs (Gonzalez-Maeso, J. et al., 2008, Nature 452, 93-97; and Gonzalez-Maeso, J. et al., 2007, Neuron 53, 439- 452.) The 2AR/mGluR2 complex and its heretofore unique signalling mechanisms may contribute to the neuropsychological effects of LSD like drugs, the antipsychotic effects of two different classes of antischizophrenia drugs, and the disordered thought processes in the schizophrenic brain.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

WHAT IS CLAIMED IS:
1. An isolated heteroreceptor complex, wherein the receptor complex comprises a serotonin receptor protein and a glutamate receptor protein.
2. The heteroreceptor complex of claim 1 , wherein the serotonin receptor protein is a 5-HT2A serotonin receptor.
3. The heteroreceptor complex of claim 1, wherein the glutamate receptor protein is a metabotropic glutamate receptor.
4. The heteroreceptor complex of claim 3, wherein the glutamate receptor protein is an mGluR2 glutamate receptor.
5. The heteroreceptor complex of claim 1 , wherein the serotonin receptor protein is a 5-HT2A serotonin receptor and the glutamate receptor protein is an mGluR2 glutamate receptor.
6. A recombinant host cell that expresses a functional heteroreceptor complex, wherein the receptor complex comprises a serotonin receptor protein expressed from an expression vector introduced into the host cell, and a glutamate receptor protein expressed from an expression vector introduced into the same host cell, wherein the host cell does not endogenously express the receptor proteins.
7. The host cell of claim 6, wherein the serotonin receptor protein is a 5- HT2A serotonin receptor.
8. The host cell of claim 6, wherein the glutamate receptor protein is an mGluR2 glutamate receptor.
9. The host cell of claim 6, wherein the serotonin receptor protein is a 5- HT2A serotonin receptor and the glutamate receptor protein is an mGluR2 glutamate receptor.
10. A method of screening for a compound that modulates a property of a heteroreceptor complex, wherein the complex comprises a 5-HT2A serotonin receptor protein and an mGluR2 glutamate receptor protein, which method comprises observing a change in a property of the heteroreceptor complex contacted with a candidate compound.
11. The method of claim 10, wherein the method comprises contacting a heteroreceptor complex with a second compound, wherein the second compound is an agonist of one receptor of the heteroreceptor complex, and wherein the property change is a change in the response elicited by contacting the second compound to the heteroreceptor complex in the absence of the first compound.
12. The method of claim 11, wherein if the first compound binds to 2AR, the second compound is an mGluR2 agonist, and if the first compound binds to mGluR2, the second compound is a 2AR agonist.
13. The method according to claim 10, wherein both receptor proteins are heterologously expressed in a host cell.
14. The method according to claim 10, wherein both receptor proteins are endogenously expressed in a primary brain cortical culture.
15. The method according to claim 14, wherein the primary brain cortical culture is a mouse primary brain cortical culture.
16. The method according to claim 14, wherein the primary brain cortical culture is a rat primary brain cortical culture.
17. The method according to claim 10, wherein both receptor proteins are endogenously expressed in a primary brain cortical culture cell membrane preparation.
18. The method according to claim 10, wherein the heteroreceptor complex property is inhibition of a 5-HT2A signal transduction pathway.
19. The method according to claim 10, wherein the heteroreceptor complex property is activation of a 5-HT2A signal transduction pathway.
20. The method of claim 18 or 19, wherein the 5-HT2Λ signal transduction pathway is a Gαi or Gαq mediated signal transduction pathway.
21. The method of claim 20, wherein the 5-HT2A signal transduction pathway is a Gα, mediated signal transduction pathway.
22. The method according to claim 10, wherein the heteroreceptor complex property is inhibition of an mGluR2 signal transduction pathway.
23. The method according to claim 10, wherein the heteroreceptor complex property is activation of an mGluR2 signal transduction pathway.
24. The method of claim 22 or 23, wherein the mGluR2 signal transduction pathway is a Gα, or Gαq mediated signal transduction pathway.
25. The method of claim 10, wherein the heteroreceptor complex property is expression of a reporter gene.
26. The method of claim 25, wherein the reporter gene is selected from the group consisting of an early growth response 2 gene (egr-2), an early growth response 1 gene (egr-1), and a c-fos gene.
27. The method of claim 25, wherein the reporter gene comprises a promoter selected from the group consisting of an early growth response 2 gene (egr- 2) promoter, an early growth response 1 gene (egr-1) promoter, and a c-fos promoter.
28. The method of claim 10, wherein the heteroreceptor complex property is an increase in a hallucinogen induced head-twitching behavior.
29. The method of claim 10, wherein the heteroreceptor complex property is a decrease in a hallucinogen induced head-twitching behavior.
30. The method of claim 10, wherein the heteroreceptor complex property is a decrease in locomotor activity induced by PCP-like psychotomimetics.
31. The method of claim 10, wherein the heteroreceptor complex property is an increase in locomotor activity incuded by PCP-like psychotomimetics.
32. The method according to claim 10, wherein the heteroreceptor complex property is internalization of either the 2AR, mGluR2 or both components of the heteroreceptor complex.
33. The method according to claim 10, wherein the heteroreceptor complex property is binding affinity for a ligand.
34. The method according to claim 10, wherein the heteroreceptor complex property is binding affinity of a 5-HT2A serotonin receptor protein for a ligand.
35. The method according to claim 10, wherein the heteroreceptor complex property is binding affinity of an mGluR2 glutamate receptor protein for a ligand.
36. The method of claim 10, wherein the heteroreceptor complex property is an increase in stability and formation of the heteroreceptor complex.
37. The method of claim 10, wherein the heteroreceptor complex property is a decrease in stability and formation of the heteroreceptor complex.
38. The method of claim 10, wherein the compound does not bind a 5- HT2A or mGluR2 receptor when the 5-HT2A or mGluR2 receptors are expressed alone and not part of a heteroreceptor complex.
39. A pharmaceutical composition comprising synergistically effective amounts of a ligand of a 5-HT2A receptor and a ligand of an mGluR2 receptor.
40. The pharmacological composition of claim 39, wherein the 5-HT2A ligand is specific for the 5-HT2A receptor and the mGluR2 ligand is specific for the mGluR2 receptor.
41. A method of treating a neurological, neurodegenerative or psychiatric brain disease, which method comprises administering a therapeutically effective dose of a compound according to claim 10.
42. A method of treating a neurological, neurodegenerative or psychiatric brain disease, which method comprises administering a therapeutically effective dose of a compound according to claim 39.
43. The method of claim 41 or 42, wherein the neurological, neurodegenerative, or psychiatric brain disease is schizophrenia.
PCT/US2009/033469 2008-02-06 2009-02-06 Heterooligomeric serotonin/glutamate receptor complex WO2009100384A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US2672108P 2008-02-06 2008-02-06
US61/026,721 2008-02-06

Publications (2)

Publication Number Publication Date
WO2009100384A2 true WO2009100384A2 (en) 2009-08-13
WO2009100384A3 WO2009100384A3 (en) 2010-01-07

Family

ID=40952722

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/033469 WO2009100384A2 (en) 2008-02-06 2009-02-06 Heterooligomeric serotonin/glutamate receptor complex

Country Status (1)

Country Link
WO (1) WO2009100384A2 (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020006925A1 (en) * 2000-04-13 2002-01-17 Xue-Feng Pei Sulfur containing dihydrophthalazine antagonists of excitatory amino acid receptors
US6358975B1 (en) * 1997-08-15 2002-03-19 Johns Hopkins University Method of using selective parp inhibitors to prevent or treat neurotoxicity
US20030153576A1 (en) * 2000-12-20 2003-08-14 Robichaud Albert J. Aryl and aminoaryl substituted serotonin receptor agonist and antagonist ligands
US6855807B1 (en) * 1999-06-16 2005-02-15 New York University Heterodimeric opioid G-protein coupled receptors
WO2005038006A2 (en) * 2003-10-17 2005-04-28 Nps Pharmaceuticals, Inc. Chimeric metabotropic glutamate receptors and uses thereof
US20060292694A1 (en) * 2005-06-22 2006-12-28 Roslin Institute Reporter hepatocytes and other cells for drug screening and toxicity testing

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6358975B1 (en) * 1997-08-15 2002-03-19 Johns Hopkins University Method of using selective parp inhibitors to prevent or treat neurotoxicity
US6855807B1 (en) * 1999-06-16 2005-02-15 New York University Heterodimeric opioid G-protein coupled receptors
US20020006925A1 (en) * 2000-04-13 2002-01-17 Xue-Feng Pei Sulfur containing dihydrophthalazine antagonists of excitatory amino acid receptors
US20030153576A1 (en) * 2000-12-20 2003-08-14 Robichaud Albert J. Aryl and aminoaryl substituted serotonin receptor agonist and antagonist ligands
WO2005038006A2 (en) * 2003-10-17 2005-04-28 Nps Pharmaceuticals, Inc. Chimeric metabotropic glutamate receptors and uses thereof
US20060292694A1 (en) * 2005-06-22 2006-12-28 Roslin Institute Reporter hepatocytes and other cells for drug screening and toxicity testing

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MOGHADDAM ET AL.: 'Reversal of Phencyclidine Effects by a Group ll Metabotropic Glutamate Receptor Agonist in Rats.' SCIENCE vol. 281, 1998, pages 1349 - 1352 *
SCHOEPP.: 'Unveiling the Functions of Presynaptic Metabotropic Glutamate Receptors in the Central Nervous System.' J PHARMACOL & EXP THERAPEUT vol. 299, no. 1, 2001, pages 12 - 20 *

Also Published As

Publication number Publication date
WO2009100384A3 (en) 2010-01-07

Similar Documents

Publication Publication Date Title
Tiger et al. The 5-HT1B receptor-a potential target for antidepressant treatment
Callier et al. Evolution and cell biology of dopamine receptors in vertebrates
Kuzhikandathil et al. Human dopamine D3 and D2L receptors couple to inward rectifier potassium channels in mammalian cell lines
Ferré et al. G protein–coupled receptor oligomerization revisited: functional and pharmacological perspectives
Espinoza et al. Functional interaction between trace amine-associated receptor 1 and dopamine D2 receptor
Smith et al. Allostery at G protein-coupled receptor homo-and heteromers: uncharted pharmacological landscapes
Ng et al. Neto1 is a novel CUB-domain NMDA receptor–interacting protein required for synaptic plasticity and learning
Bai Dimerization of G-protein-coupled receptors: roles in signal transduction
Paez-Pereda et al. Corticotropin releasing factor receptor antagonists for major depressive disorder
Koshimizu et al. Complex formation between the vasopressin 1b receptor, β-arrestin-2, and the μ-opioid receptor underlies morphine tolerance
Sager et al. Proteins interacting with monoamine transporters: current state and future challenges
Bohn et al. Serotonin receptor signaling and regulation via β-arrestins
CA2930810C (en) Pharmacological chaperones for treating obesity
Convertino et al. μ-Opioid receptor 6-transmembrane isoform: A potential therapeutic target for new effective opioids
Price et al. Endogenous serotonin 5-HT2A and 5-HT2C receptors associate in the medial prefrontal cortex
Kozek et al. Discovery and characterization of VU0529331, a synthetic small-molecule activator of homomeric G protein-gated, inwardly rectifying, potassium (GIRK) channels
Werkman et al. Dopamine receptor pharmacology: interactions with serotonin receptors and significance for the aetiology and treatment of schizophrenia
Gross et al. Role of RGS12 in the differential regulation of kappa opioid receptor-dependent signaling and behavior
Simen et al. The C terminus of the Ca channel α1B subunit mediates selective inhibition by G-protein-coupled receptors
Kong et al. Agonist-induced cell surface trafficking of an intracellularly sequestered D1 dopamine receptor homo-oligomer
Zhang et al. Correlations between clinical and physiological consequences of the novel mutation R878C in a highly conserved pore residue in the cardiac Na+ channel
Chiu et al. A suite of engineered mice for interrogating psychedelic drug actions
WO2003073107A2 (en) Neuropeptide receptor and uses thereof
WO2009100384A2 (en) Heterooligomeric serotonin/glutamate receptor complex
Bushman et al. Characterization and functional restoration of a potassium channel Kir6. 2 pore mutation identified in congenital hyperinsulinism

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09708162

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09708162

Country of ref document: EP

Kind code of ref document: A2