# Patent

## Title
Methods for modulating D1-D2 dopamine receptor coupling and function

## Abstract

The present invention provides for prevention and/or treatment of neurological or neuropsychiatric disorders involving abnormal D1-D2 dopamine receptor coupling and/or activation. Methods and agents are provided for modulating dopamine receptor function arising from D1-D2 coupling and/or activation. Agents of the present invention include fragments of D2 receptor or D1 receptor that can disrupt D1-D2 coupling.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. Ser. No. 12/997,813 filed Dec. 13, 2010, now U.S. Pat. No. 8,420,775 issued Apr. 16, 2013, which is the U.S. National Phase of PCT Appln. No. PCT/CA2009/000829 filed Jun. 12, 2009, now expired, which claims the benefit of U.S. provisional application Ser. No. 61/060,948 filed Jun. 12, 2008, the disclosures of which are incorporated in their entirety by reference herein.


SEQUENCE LISTING
The text file dated Jan. 16, 2015 and titled “SequenceListing.txt” of size 15 KB, filed herewith, is hereby incorporated by reference.
FIELD OF INVENTION
The present invention relates to psychiatric diseases or disorders, and more particularly, compositions and methods for modulating the interaction and function of D1-D2 dopamine receptors. Such compositions and methods are useful for prevention and/or treatment of psychiatric diseases or disorders, particularly depression, including major depressive illness and depression in bipolar disorder, and psychiatric conditions that require treatment with medication, including schizophrenia, psychosis in bipolar disorder, and stimulant drug intoxication.
BACKGROUND OF THE INVENTION
Depression:
Depression is a mood disorder characterized by depressed mood; feelings of worthlessness, helplessness or hopelessness; a loss of interest or pleasure; changes in appetite; change in sleeping pattern; fatigue, thoughts of death, inability to concentrate or make decisions. According to Statistics Canada's 2002 Mental Health and Well-being Survey4, 12.2% of all Canadians will experience depression within their lifetime, while 4.8% of Canadians had reported symptoms for major depression. Furthermore, the economical impact of depression is tremendous due to costs in both productivity and health care. In Canada, between 62% and 76% of short-term disability episodes due to mental disorders were attributed to depression5. Work-related productivity losses due to depression have been estimated to be $4.5 billion6. Thus, the prevalence of depression makes this disorder a very important health issue in Canada and abroad.
Once diagnosed, depression can be treated by different therapies including medication, psychotherapy and in more severe cases, with electroconvulsive therapy. The first line of treatment is often through antidepressant medication, sometimes in conjunction with psychotherapy. Antidepressants consist of the classical tricyclic antidepressants (TCA), selective serotonin reuptake inhibitors (SSRI), noradrenaline and serotonin reuptake inhibitor (NSRI), as well as monoamine oxidase inhibitors (MAOI). All antidepressants have acute effects on synaptic levels of neurotransmitters in the brain7-9. The classical TCAs are predominantly noradrenaline and serotonin reuptake inhibitors, similar to NSRIs. The SSRI drugs are more selective serotonin transporter inhibitors, while MAOI block enzymes that are involved in the breakdown of these neurotransmitters.
Dopamine (DA), acting through DA receptors, exerts a major role in regulating neuronal motor control, cognition, event prediction, emotion and pleasure/reward10-15, all of which are affected in depression. The contribution of DA in depression becomes evident when taking into account the major dopaminergic pathways in the mammalian brain: (a) the mesostriatal system consisting of dopaminergic neurons from the substantia nigra (SNc) innervating the striatum; (b) the mesolimbic system in which dopaminergic neurons from the ventral tegmental area (VTA) project into the hippocampus, nucleus accumbens (NAc) and amygdala; (c) the mesocortical system where DA neurons mostly from the VTA project into the cortical regions of the brain including the prefrontal cortex (PFx). Most of these regions have been implicated in depression. Furthermore, numerous studies support the hypothesis of decreased dopaminergic signalling in depression including reports that: (1) the severity of major depression correlates highly with patient response to amphetamine, a drug that facilitates increased synaptic DA levels through multiple mechanisms16, while another study has shown decreased levels of homovanillic acid, a major DA metabolite, in the CSF of depression patients17; (2) animals experiencing learned helplessness, a behavioural paradigm that recapitulates some of the symptoms of depression, have been shown to exhibit DA depletion in the striatum, which can be mitigated by pretreatments with DA agonists18-19; (3) motor effects induced by DA receptor agonists are increased after chronic treatment with antidepressants or electroconvulsive therapy20 suggesting reduced dopaminergic neurotransmission in depression; (4) in forced-swim tests, DA agonists have been shown to inhibit immobility, an indication of antidepressant activity, while DA receptor antagonists have been shown to inhibit the effects of antidepressants21-32; (5) DAT inhibitors nomifensine and bupropion have been shown to be effective antidepressants21, 33-34 and (6) clinical studies have also documented cases where DA receptor agonists have been effective in treating depression35-39. Furthermore, there is some evidence from neuroimaging studies that dopamine D2 receptor (D2R) are elevated in the striatum of depressed patients40-44.
Another brain pathway implicated in depression is the hypothalamic-pituatary-adrenal (HPA) axis. The HPA axis is involved in stress reaction and ultimately leads to increased secretion of glucocorticoids from the adrenal cortex. Although glucocorticoids have effects on the hippocampus it has also been shown to facilitate DA transmission in NAc45. In addition, frequent bouts of stress with intermittent exposure to glucocorticoids sensitize the mesolimbic DA system46. While the hippocampus and frontal cortex are undoubtedly involved in certain aspects of depression, symptoms of anhedonia, lack of motivation and motor deficits implicate other regions of the brain including the dorsal and ventral striatum, which are rich in dopaminergic neurons. Moreover, serotonergic activity has an impact on DA neurotransmission. Studies have shown that stimulation of 5HT1A receptors can stimulate DA release in PFx and NAc but inhibit DA release in the dorsal striatum47. Other studies have shown that activation of serotonergic raphe neurons reduces activity of dopaminergic neurons in VTA (not SNc) and inhibit locomotion, exploratory behaviour48-49.
Schizophrenia:
Schizophrenia is a severe chronic and debilitating mental disorder that strikes in youth and affects not only patients but their families and care-givers50-51. Approximately 2.2 million American adults have schizophrenia in a given year. Clinical symptoms of schizophrenia include delusions, hallucinations, disorganized thinking, and cognitive dysfunction that are divided into two major groups: positive and negative symptoms52.
Accumulated evidence suggests that the positive symptoms result from hyperdopaminergia involving dopamine D2 receptors in the limbic striatum, while the negative/cognitive symptoms arise from a hypodopaminergic function mediated by dopamine D1 receptors in the prefrontal cortex52. Despite decades of intensive research, current antipsychotic medications are still limited to the blockade of D2 receptor function that generally alleviate positive symptoms with only limited impact on cognitive and negative symptoms and can induce serious side effects including extrapyramidal side effects (EPS). Patients continue to experience significant disability and functional impairment that limits their integration in society. The unavailability of effective medications with both D1 agonism and D2 antagonism is mainly due to the unknown therapeutic target, a pathway through which both inhibition of D2 receptor and activation of D1 receptor function can be achieved.
The dopamine hypothesis of schizophrenia, in its original formulation addressed mainly positive symptoms53-54. Early pharmacotherapy for schizophrenia involved the use of reserpine, which blocks dopamine release from presynaptic terminals, and/or the use of antipsychotics55. Moreover, the most compelling evidence for the involvement of dopamine receptors in schizophrenia comes from the fact that most antipsychotics, including atypical antipsychotics, show a dose-dependent threshold of D2 receptor occupancy for their therapeutic effects55. The efficacy of both reserpine and antipsychotics in treating schizophrenia strongly implicate the involvement of dopamine in this neuropsychiatric disorder. More recent versions of this theory suggests that while the positive symptoms result from hyperdopaminergia in the limbic striatum, the negative/cognitive symptoms arise from a hypodopaminergic function in the prefrontal cortex (PFC)56-57. A significant body of literature lends support to this theory. PET & SPECT studies have shown evidence of increased dopamine synthesis, release, and levels in the subcortical/limbic regions58 while functional imaging studies have demonstrated hypofunction in the prefrontal cortex at baseline and while performing cognitive tasks59. More recent studies have focused on the dopamine D1 system in the PFC, as it is the predominant dopamine receptor sub-type in the PFC60-61, and show a decrease in receptor number which correlates with executive dysfunction62 and a compensatory up-regulation which correlates with working-memory dysfunction63. These clinical observations are well supported by preclinical evidence—PFC D1 receptor modulation changes the ‘memory fields’ of prefrontal neurons subserving working memory64-65 and D1 agonist administration improves working memory performance in both aging and dopamine deficient monkeys66 (as it does in aging human subjects)67-68.
Overview of DA Receptors:
In mammals, five distinct genes, termed D1/D5 for D1-like receptors and D2/D3/D4 for D2-like receptors, encode DA receptors. These receptors belong to a super-family of single polypeptide seven trans-membrane (TM) domain receptors that exert their biological effects via intracellular G-protein coupled signaling cascades1. D1 and D5 receptors preferentially couple to Gs proteins stimulating the activity of adenylate cyclase and PKA dependent pathways. D2 receptors display a more complex pattern of signal transduction primarily due to their coupling to subtype specific members of the Gi/Go protein family.
D2 receptors are known to stimulate a number of signal transduction pathways including the inhibition of adenylate cyclase activity, PI turnover, potentiation of arachidonic acid release, inwardly rectifying K+ and Ca2+ channels and mitogen activated protein kinases2. Moreover, studies have shown that protein interactions can play a large role in DA receptor function. For instance, the D2R has been shown to physically interact with Par-4. Interestingly, Par-4 mutant mice, which are unable to interact with D2R, exhibit depression-like behaviour3.
Dopamine D1-D2 Receptor Link:
While numerous studies have indicated a synergy between D1 and D2 receptors, an interesting study by Seeman et al (1989)69 provided the first evidence of a pharmacological link between D1 and D2 receptors. Briefly, it was shown that dopamine could lower the density of D2 receptors labeled by [3H] raclopride and that the addition of the specific D1 receptor antagonist, SCH-23390, prevented this reduction, suggesting a functional link between D1 and D2 receptors. Interestingly, this pharmacological D1-D2 link was absent or reduced in post-mortem brain tissues of approximately half of the schizophrenia population tested. However, it remains unclear if the absence of the D1-D2 link is due to an inherent dissociation between the D1 and D2 receptor that occurs in schizophrenia or is a result of antipsychotic drug treatment. Furthermore, several studies using a combination of D1 and D2 specific agonists and/or antagonists have shown that co-activation of D1-D2 receptors are required for long-term depression, anandamide-mediated memory consolidation and potentiation of immediate early gene response, suggesting a potential functional interaction between the D1R and D2R70-76.
Recent evidence indicates that D1 and D2 receptors form a protein complex, and co-activation of D1 and D2 receptors results in an increase of intracellular calcium levels via a signaling pathway not activated by either receptor alone, confirming the functional link observed between D1 and D2 receptors77-78. Furthermore, it has been shown that D1 and D2 receptors are co-expressed in neurons of the rat striatum, providing a basis for a functional interaction79-80.
Despite years of research in the field of mental health, there continues to be a need for new and improved medicines for treating psychiatric diseases and disorders, including depression, schizophrenia and psychotic symptoms thereof. The present inventors have accordingly sought to identify new diagnostic and chemotherapeutic methods in this area by investigating the functional association between D1 and D2 classes of DA receptors.
SUMMARY OF THE INVENTION
The present invention accordingly relates to compositions and methods for prevention and/or treatment of diseases and disorders involving abnormal DA receptor association and functionality. More particularly, the present invention relates to methods of modulating the interaction and functionality of D1-D2 receptors, as well as compounds useful in such methods. The invention also relates to methods of diagnosis of diseases and disorders caused by abnormal D1-D2 receptor association and functionality.
The present invention provides compounds, compositions and methods for modulating the interaction of D1-D2 receptors. Furthermore, the present invention provides methods for preventing and/or treating diseases involving abnormal levels of D1-D2 interaction and/or functionality.
According to the present invention there is provided a method for modulating dopamine (DA) receptor function in a mammal in need of such treatment comprising administering a therapeutically effective amount of an agent that disrupts D1-D2 coupling in the mammal. In a preferred embodiment, which is not meant to be limiting in any manner, the agent is a peptide or polypeptide.
In an embodiment, the agent is an antibody that binds to an amino acid sequence that is at least 80% identical to the sequence of any one of the sequences selected from D2IL3-29 (SEQ ID NO:1), D2LIL3-29-2 (SEQ ID NO:2), D2IL3-L (SEQ ID NO:3), and D1CT (SEQ ID NO:4). In a further embodiment, the amino acid sequence is identical to the sequence of D2IL3-29 (SEQ ID NO:1), D2LIL3-29-2 (SEQ ID NO:2), D2IL3-L (SEQ ID NO:3), or D1CT (SEQ ID NO:4).
In a further embodiment, the agent is a nucleic acid encoding a polypeptide of between about 3 and about 140 amino acids and comprising an amino acid sequence that is at least 80% identical to the sequence of any one of the sequences selected from D2IL3-29 (SEQ ID NO:1), D2LIL3-29-2 (SEQ ID NO:2), D2IL3-L (SEQ ID NO:3), and D1CT (SEQ ID NO:4). The polypeptide, in some embodiments, may be identical to a sequence of D2IL3-29 (SEQ ID NO:1), D2LIL3-29-2 (SEQ ID NO:2), D2IL3-L (SEQ ID NO:3), and D1CT (SEQ ID NO:4).
In further embodiments, the agent may be a polypeptide of between about 3 and about 140 amino acids comprising an amino acid sequence that is between about 80% and about 100% identical to any one of the sequences selected from the group consisting of D2IL3-29 (SEQ ID NO:1), D2LIL3-29-2 (SEQ ID NO:2), D2IL3-L (SEQ ID NO:3), and D1CT (SEQ ID NO:4). The polypeptide, in such embodiments, may be identical to a sequence of D2IL3-29 (SEQ ID NO:1), D2LIL3-29-2 (SEQ ID NO:2), D2IL3-L (SEQ ID NO:3), and D1CT (SEQ ID NO:4).
The above method can be for preventing and/or treating a disease selected from the group consisting of depression, including major depressive illness and depression in bipolar disorder, and psychiatric conditions that require treatment with antipsychotic medication, including schizophrenia, psychosis in bipolar disorder, and stimulant drug intoxication.
As a further aspect of the invention, there is provided a polypeptide of between about 3 and about 140 amino acids comprising an amino acid sequence that is at between about 80% and about 100% identical to the sequence of D2IL3-29 (SEQ ID NO:1), D2LIL3-29-2 (SEQ ID NO:2), (SEQ ID NO:3), and D1CT (SEQ ID NO:4).
The polypeptide may comprise an amino acid sequence that is between about 80% and 100% identical to a sequence selected from the group consisting of D2IL3-29 (SEQ ID NO:1), D2LIL3-29-2 (SEQ ID NO:2), D2IL3-L (SEQ ID NO:3), and D1CT (SEQ ID NO:4). More specifically, the polypeptide may comprise an amino acid sequence that is identical to D2IL3-29 (SEQ ID NO:1), D2LIL3-29-2 (SEQ ID NO:2), D2IL3-L (SEQ ID NO:3), or D1CT (SEQ ID NO:4).
In a yet further aspect of the invention, there is provides a nucleic acid encoding a polypeptide of between 3 and 140 amino acids comprising an amino acid sequence that is between about 80% identical and 100% identical to the sequence of D2IL3-29 (SEQ ID NO:1), D2LIL3-29-2 (SEQ ID NO:2), D2IL3-L (SEQ ID NO:3), and D1CT (SEQ ID NO:4).
The nucleic acid may encode a polypeptide having an amino acid sequence that is between 80% and 100% identical to a sequence selected from the group consisting of D2IL3-29 (SEQ ID NO:1), D2LIL3-29-2 (SEQ ID NO:2), D2IL3-L (SEQ ID NO:3), and D1CT (SEQ ID NO:4). In a further embodiment, the nucleic acid encodes a polypeptide that is identical to a sequence selected from D2IL3-29 (SEQ ID NO:1), D2LIL3-29-2 (SEQ ID NO:2), D2IL3-L(SEQ ID NO:3), and D1CT (SEQ ID NO:4).
A protein transduction domain may be fused or linked to any small molecule chemical compound, polypeptide, nucleic acid, or combination thereof, used in the context of the present invention. In certain non-limiting representative examples, the protein transduction domain is selected from the group consisting of TAT, and SynB1/3Cit. Also, other proteins, non-protein carriers or other macromolecules may be attached or conjugated to the compound, polypeptide, nucleic acid described above.
The present invention also provides a kit comprising any component or combination of components as mentioned or described throughout.
As described herein, a direct interaction between D1 and D2 receptors, has been identified, and the present invention provides agents that specifically disrupt this interaction. Furthermore, the present invention provides methods for identifying agents that disrupt the interaction between the D1 and D2 receptors.
This summary of the invention does not necessarily describe all features of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings, in which:
FIG. 1A shows the results of a Western blot analysis in which CO-IP of D1 receptor by D2 receptor (antibody) in solubilized rat striatal tissue;
FIG. 1B shows the results of a Western blot analysis in which D2 receptor is specifically pulled down by GST-D1CT in detergent extracts of rat striatum, but not GST-D1IL3 (third intracellular loop) or GST alone;
FIG. 1C shows the results of a Western blot analysis in which D1 receptor was specifically pulled down by GST-D2IL3-L in detergent extracts of rat striatum, but not GST-D2IL3-S or GST alone;
FIG. 1D shows the results of a Western blot analysis in which D1 receptor was specifically pulled down by GST-D2IL3-L and GST-D2IL3-29 in detergent extracts of rat striatum, but not GST-D2CT, GST-D2IL3-S or GST alone;
FIG. 1E shows the results of a Western blot analysis in which D1 receptor was pulled down by GST-D2IL3-29-2, but not GST-D2IL3-29-1 or GST alone;
FIG. 1F shows the results of a Western blot analysis of an in vitro binding assay, wherein a S35-D1 tail probe specifically bound with GST-D2IL3-29-2 fragment, but not with GST-D2IL3-29-1 or GST alone;
FIG. 1G shows the results of a Western blot analysis of competitive CO-IP in rat striatum. The ability of D1 receptor to co-immunoprecipitate with D2 receptor is inhibited by the addition of GST-D2IL3-29-2 in a concentration dependent manner;
FIG. 2A shows the results of a time course of 2 μM Fluo-4AM fluorescence, corresponding to increases in intracellular Ca2+ levels in HEK-293T cells co-expressing D1 and D2 receptors, following simultaneous activation by both 10 μM SKF81297 and quinpirole. Fluorescence values were monitored using a PerkinElmer multiwell plated fluorometer, collected at 3-s intervals for 150 s. (A.F.U.: arbitrary fluorescence units). The curve shown is representative of three replicate measurements performed;
FIG. 2B shows the measurement of fluorescence, corresponding to the intracellular Ca2+  levels in HEK-293T cells co-expressing D1 and D2 receptors treated with 10 μM SKF81297, 10 μM quinpirole or both. ** Significant vs. control group (p<0.01). Significant vs. SKF+Quin group (#, p<0.05; ##, p<0.01). Data is representative of three replicate measurements performed. Data was analyzed by one-way ANOVA, followed by Newman-Keuls test;
FIG. 3A shows fluorescence measurements for HEK-293T cells co-expressing D1 and D2 receptors and pretreated with 10 μM raclopride or SCH23390. The cells were stimulated with both 10 μM SKF81297 and 10 μM quinpirole. Significant vs. control group (*, p<0.05; **, p<0.01). ### Significant vs. SKF+Quin group (p<0.001) Data is representative of three replicate measurements performed. Data was analyzed by one-way ANOVA, followed by Newman-Keuls test;
FIG. 3B shows fluorescence measurements for HEK-293T cells co-expressing D1 and D2 receptors and pretreated with 10 μM U73122 (PLC inhibitor). The cells were stimulated with 10 μM SKF81297 and 10 μM quinpirole. Significant vs. control group (**, p<0.01). ### Significant vs. SKF+Quin group (p<0.001). Data is representative of three replicate measurements performed. Data was analyzed by one-way ANOVA, followed by Newman-Keuls test;
FIG. 3C shows fluorescence measurements for HEK-293T cells co-expressing D1 and D2 receptors in the presence of D1CT or D5CT mini-genes and stimulated with a 10 μM concentration of both SKF81297 and quinpirole. *, **Significantly from control group (p<0.05; p<0.01). # Significantly from SKF+Quin group (p<0.05). Data is representative of three replicate measurements performed. Data was analyzed by one-way ANOVA, followed by Newman-Keuls test;
FIG. 4 shows the results of characterization of the D1-D2 interaction in post-mortem brain. Striatal post-mortem brain samples (control, schizophrenia, bipolar and depression; 15 samples in each group), obtained from the Stanley Foundation, were incubated with anti-D2 receptor antibodies for coimmunoprecipitation experiments. Precipitated proteins were subject to SDS-PAGE; immunoblotted with D1 antibody. Co-immunoprecipitation of D1 by the D2 antibody is significantly increased in depression brains compared to controls. Data were analyzed by one-way ANOVA followed by post-hoc SNK tests (* P<0.05, n=15);
FIG. 5 (A) illustrates the results of an analysis of D1-D2 interaction, in which it is shown that chronic antidepressant treatment results in a decrease in D1-D2 interaction. Rats were treated with imipramine (IMI, 10 mg/kg/day) or saline for 14 days. On the 14th day, rats were sacrificed, brains quickly extracted and striata were dissected for biochemical analysis. Striata were solubilized and used in coimmunoprecipitation experiments to examine the coprecipitation of the D1 receptor with the D2 receptor. Rats subjected to chronic imipramine (IMI) exhibited a decrease in the D1-D2 interaction, as quantified from Western blots of co-immunoprecipitation samples (*, p<0.05, t-test, n=5);
FIG. 5 (B) illustrates the results of quantification of Western blots from the control (Con) and IMI samples revealed no significant differences in D1 receptor levels (n=5). Tubulin was used as loading controls;
FIG. 5 (C) illustrates the results of quantification of Western blots from the control (Con) and IMI samples revealed no significant differences in D2 receptor levels (n=5). Tubulin was used as loading controls;
FIG. 6 (A) illustrates the results of a study of rats subjected to learned helplessness (LH), in which an increase in the D1-D2 interaction is shown. Rats that were subjected to LH were compared against control rats. Striata were solubilized and used in co-immunoprecipitation experiments to examine the co-precipitation of the D1 receptor with the D2 receptor. Rats exhibited an increase in the D1-D2 interaction after LH, as quantified from Western blots of co-immunoprecipitation samples (**, p<0.01, n=5, t-test).
FIG. 6(B) illustrates the results where prefrontal cortex (PFC) were solubilized and used in co-immunoprecipitation experiments to examine the co-precipitation of the D1 receptor with the D2 receptor. Rats exhibited an increase in the D1-D2 interaction after LH, as quantified from Western blots of co-immunoprecipitation samples (*, p<0.05, n=5, t-test);
FIG. 7 illustrates the results of a study of rats subjected to chronic mild stress (CMS), in which an increase in the D1-D2 interaction is shown. Rats that were subjected to CMS were compared against control rats. Prefrontal cortex were solubilized and used in co-immunoprecipitation experiments to examine the co-precipitation of the D1 receptor with the D2 receptor. Rats exhibited an increase in the D1-D2 interaction after CMS, as quantified from Western blots of co-immunoprecipitation samples (*, p<0.05, n=5, t-test).
FIG. 8 (A) illustrates the results of a study of rats subjected to forced swim tests (FST), in which an increase in the D1-D2 interaction is shown. Rats that were subjected to forced swim tests were compared against control rats for changes in D1 and D2 receptors. Rat striata were solubilized and used in co-immunoprecipitation experiments to examine the co-precipitation of the D1 receptor with the D2 receptor. Rats exhibited an increase in the D1-D2 interaction 3 hours or 3 days after FST trials, as quantified from Western blots of co-immunoprecipitation samples (*, p<0.05, n=4, ANOVA post hoc SNK);
FIG. 8 (B) illustrates the results of quantification of Western blots from the control (Con) and FST samples revealed no significant differences in D1 receptor levels. Tubulin was used as loading controls;
FIG. 8 (C) illustrates the results of quantification of Western blots from the control (Con) and FST samples revealed no significant differences in D2 receptor levels (n=4). Tubulin was used as loading controls;
FIG. 9 shows Western blots illustrating a significant decrease in D1-D2 receptor complex formation in rat with chronic antipsychotic treatment (bottom panel) (by pump 0.25 mg/kg/day for 2 weeks), while direct immunoprecipitated D2 receptors remain unchanged (top panel);
FIG. 10 illustrates the results of a study in which disruption of D1-D2 interaction leads to behavior changes in rats subjected to forced swim tests. Intra-PFC administration of TAT-D2LIL3-29-2, but not TAT alone, decreased the frequencies of rat immobility and increased the frequencies of rat swimming and climbing behaviors in a 5 min forced swimming test. Each value is the mean.+−.S.E.M. for a group of 6 rats. Data were analyzed by one-way analysis of variance (ANOVA).
FIG. 11 shows the protein sequence of human D2R-L, The I256-V270 region shown to be important for D1-D2 binding is underlined.
FIG. 12 shows the DNA sequence encoding the human D2R-L.
FIG. 13 shows the protein sequence of human D1R. The D1CR (A332-T446) is shown in bold, while the third intracellular loop D1IL3 (R219-K272) is underlined in the D1 sequence.
FIG. 14 shows the DNA sequence encoding human DIR.



DETAILED DESCRIPTION
The following description is of a preferred embodiment.
The present invention provides a method for modulating dopamine (DA) receptor function, partially as a result of identifying a direct interaction between D1 and D2 receptors (see Examples). Agents that specifically disrupt the D1-D2 receptor interaction, and methods for identifying agents that disrupt this interaction are accordingly provided. Formation of the D1-D2 complex has been shown herein to occur at elevated levels in individuals suffering from depression. Moreover, disruption of the D1-D2 interaction and complex formation is shown to reduce or alleviate symptoms of depression using animal models of depression. Accordingly, modulating DA receptor function through disruption of the D1-D2 complex can be effective for preventing and/or treating a variety of neurological diseases and disorders, for example, but not limited to, depression, including major depressive illness and depression in bipolar disorder, schizophrenia, psychotic symptoms of schizophrenia, and other psychiatric conditions that require treatment with antipsychotic medication, such as psychosis in bipolar disorder, and stimulant drug intoxication.
By disrupting D1-D2 coupling, an agent may for instance inhibit binding or otherwise prevent association or interaction between the D1 and D2 receptor.
The method for modulating DA receptor function may involve administering the D1-D2 disrupting agent in a therapeutically effective amount. Such amount may vary depending upon the disease or disorder to be treated, as well as other pharmacological factors known to those skilled in the art.
By “agent” or “agents” it is meant any small molecule chemical compound, polypeptide, nucleic acid or any combination thereof that can modulate DA receptor function. By “modulate DA receptor function” is meant an altering of function, for instance by inhibition of DA activation and/or signaling function, by disrupting D1-D2 coupling. A polypeptide may be of any length unless otherwise specified and includes, for example and without limitation, antibodies, enzymes, receptors, transporters, D2 receptor, D1 receptor fragment or derivative. A fragment is any polypeptide or nucleic acid that is shorter than its corresponding naturally occurring polypeptide or nucleic acid, respectively. A derivative is any polypeptide or nucleic acid that is altered with respect to a reference polypeptide or nucleic acid, respectively, and includes, for example fragments or mutants.
Accordingly, in a preferred embodiment, the present invention provides a polypeptide of less than 140 amino acids comprising an amino acid sequence that is at least 80% identical to the sequence of D2IL3-29 (SEQ ID NO:1), D2LIL3-29-2 (SEQ ID NO:2), D2IL3-L (SEQ ID NO:3), and D1CT (SEQ ID NO:4), or a fragment of any one thereof. In a preferred embodiment, the polypeptide is between about 4 and about 140 amino acids, for example, but not limited to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90 or 100 amino acids. In an alternate embodiment, the polypeptide is between about 3 and 100 amino acids, for example, but not limited to 5 and 100 amino acids, 7 and 100 amino acids, 9 and 100 amino acids, 11 and 100 amino acids, 13 and 100 amino acids, or 15 and about 140 amino acids, for example. However, it is to be understood that the size of the peptide may be defined by a range of any two of the values listed above or any values therein between. Also, in an alternate embodiment, which is not meant to be limiting in any manner, the present invention contemplates polypeptides as defined above which comprises more than 140 amino acids.
It is to be understood that the polypeptide as described above does not consist of the full amino acid sequence of any naturally occurring D1 or D2 receptor, or any naturally occurring allelic variant thereof.
The sequences of D2LIL3-29-2 (SEQ ID NO:2), D2IL3-(SEQ ID NO:3), and D1CT (SEQ ID NO:4) are as follows, with specific reference to the numbering of the full length amino acid sequences of D1 and D2 shown in FIGS. 11 and 13:







D2 Peptides:


D2IL3-29 G242-V270


(SEQ ID NO: 1)


GNCTHPEDMKLCTVIMKSNGSFPVNRRRV


D2LIL3-29-2 I256-V270


(SEQ ID NO: 2)


IMKSNGSFPVNRRRV


D2IL3-L K211-Q373


(SEQ ID NO: 3)


KIYIVLRRRRKRVNTKRSSRAFRAHLRAPLKGNCTHPEDMKLCTVIMKS


NGSFPVNRRRVEAARRAQELEMEMLSSTSPPERTRYSPIPPSHHQLTLP


DPSHHGLHSTPDSPAKPEKNGHAKDHPKIAKIFEIQTMPNGKTRTSLKT


MSRRKLSQQKEKKATQ


 


D1 Peptides:


D1CT A332-T446


(SEQ ID NO: 4)


AFNADFRKAFSTLLGCYRLCPATNNAIETVSINNNGAAMFSSHHEPRGS


ISKECNLVYLIPHAVGSSEDLKKEEAAGIARPLEKLSPALSVILDYDTD


VSLEKIQPITQNGQHPT






The present invention also contemplates polypeptides having an amino acid sequence that comprises 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino sequences described above. Further, the polypeptides may be defined as comprising a range of sequence identity defined by any two of the values listed above.
In an embodiment of the present invention, which is not meant to be limiting, there is provided a polypeptide from D2LIL3-29-2 comprising or consisting of the amino acid sequence IMK, MKS, KSN, SNG, NGS, GSF, SFP, FPV, PVN, VNR, NRR, RRR, or RRV. Further, the polypeptide may comprise or consist of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids including one or more sequential amino acids attached at the N terminus, C terminus or both of any of the sequences provided above that are found in a naturally occurring D2 receptor or any allelic variant thereof.
In a further embodiment of the present invention, there is provided a polypeptide comprising or consisting of the amino acid sequence from D1CT comprising or consisting of AFN, FNA, NAD, ADF, DFR, FRK, RKA, KAF, AFS, FST, STL, TLL, LLG, LGC, GCY, CYR, YRL, RLC, LCP, CPA, PAT, ATN, TNN, NNA, NAI, AIE, IET, ETV, TVS, VSI, SIN, INN, NNN, NNG, NGA, GAA, AAM, AMF, MFS, FSS, SSH, SHH, HHE, HEP, EPR, PRG, RGS, GSI, SIS, ISK, SKE, KEC, ECN, CNL, NLV, LVY, VYL, YLI, LIP, IPH, PHA, HAV, AVG, VGS, GSS, SSE, SED, EDL, DLK, LKK, KKE, KEE, EEA, EAA, AAG, AGI, GIA, IAR, ARP, RPL, PLE, LEK, EKL, KLS, LSP, SPA, PAL, ALS, LSV, SVI, VIL, ILD, LDY, DYD, YDT, DTD, TDV, DVS, VSL, SLE, LEK, EKI, KIQ, IQP, QPI, PIT, ITQ, TQN, QNG, NGQ, GQH, QHP, and HPT. Further, the polypeptide may comprise or consist of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids including one or more sequential amino acids attached at the N terminus, C terminus or both of any of the sequences provided above that are found in a naturally occurring D1 receptor or any allelic variant thereof.
The present invention also provides a nucleic acid encoding polypeptides as defined above. For example, but not wishing to be limiting in any manner, the present invention contemplates a nucleic acid encoding a polypeptide of between about 3 and less than 140 amino acids, for example, but not limited to between 10 and 135 amino acids, between 10 and 100 amino acids, between 15 and 109 amino acids or between 15 and 100 amino acids and that encodes an amino acid sequence that is at least 80% identical to the sequence of D2LIL3-29-2 (SEQ ID NO:2), D2IL3-(SEQ ID NO:3), and D1CT (SEQ ID NO:4). In an alternate embodiment, the present invention contemplates nucleic acids or nucleotide sequences as described above but that encode more than 140 amino acids. Such nucleic acids may be derived from the amino acid sequences above, or from the corresponding full length nucleic acid sequences of the D1 and/or D2 receptor coding sequences shown in FIGS. 12 and 14.
By “percent identical” or “percent identity”, it is meant one or more than one nucleic acid or amino acid sequence that is substantially identical to a coding sequence or amino acid sequence of peptides that can disrupt D1-D2 coupling. By “substantially identical” is meant any nucleotide sequence with similarity to the genetic sequence of a nucleic acid of the invention, or a fragment or a derivative thereof. The term “substantially identical” can also be used to describe similarity of polypeptide sequences. For example, nucleotide sequences or polypeptide sequences that are at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 98% or 99% identical to the D1 or D2 receptor coding sequence, or the encoded polypeptide, respectively, or fragments or derivatives thereof, and still retain ability to affect D1-D2 coupling are contemplated.
To determine whether a nucleic acid exhibits identity with the sequences presented herein, oligonucleotide alignment algorithms may be used, for example, but not limited to a BLAST (GenBank URL: www.ncbi.nlm.nih.gov/cgi-bin/BLAST/, using default parameters: Program: blastn; Database: nr; Expect 10; filter: default; Alignment: pairwise; Query genetic Codes: Standard(1)), BLAST2 (EMBL URL: http://www.embl-heidelberg.de/Services/index.html using default parameters: Matrix BLOSUM62; Filter: default, echofilter: on, Expect:10, cutoff: default; Strand: both; Descriptions: 50, Alignments: 50), or FASTA, search, using default parameters. Polypeptide alignment algorithms are also available, for example, without limitation, BLAST 2 Sequences (www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html, using default parameters Program: blastp; Matrix: BLOSUM62; Open gap (11) and extension gap (1) penalties; gap x_dropoff: 50; Expect 10; Word size: 3; filter: default).
An alternative indication that two nucleic acid sequences are substantially identical is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. for at least 1 hour (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. for at least 1 hour (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York). Generally, but not wishing to be limiting, stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
By protein transduction domain it is meant a sequence of nucleic acids that encode a polypeptide, or a sequence of amino acids comprising the polypeptide, wherein the polypeptide facilitates localization to a particular site, for example a cell or the like, or it may facilitate transport across a membrane or lipid bilayer. The polypeptides and nucleic acids of the present invention may be fused to a protein transduction domain to facilitate transit across lipid bilayers or membranes. Also, the present invention contemplates that the polypeptide as described herein may be linked or attached to a support, a macromolecule, a non-protein carrier or a protein carrier optionally via a spacer compound, molecule or the like.
Many polypeptides and nucleic acids do not efficiently cross the lipid bilayer of the plasma membrane, and therefore enter into cells at a low rate. However, there are certain naturally occurring polypeptides that can transit across membranes independent of any specific transporter. Antennapedia (Drosophila), TAT (HIV) and VP22 (Herpes) are examples of such polypeptides. Fragments of these and other polypeptides have been shown to retain the capacity to transit across lipid membranes in a receptor-independent fashion. These fragments, termed protein transduction domains, are generally 10 to 27 amino acids in length, possess multiple positive charges, and in several cases have been predicted to be ampipathic. Polypeptides and nucleic acids that are normally inefficient or incapable of crossing a lipid bilayer, can be made to transit the bilayer by being fused to a protein transduction domain.
U.S. Publication 2002/0142299 (which is incorporated herein by reference) describes a fusion of TAT with human beta-glucuronidase. This fusion protein readily transits into various cell types both in vitro and in vivo. Furthermore, TAT fusion proteins have been observed to cross the blood-brain-barrier. Frankel et al. (U.S. Pat. No. 5,804,604, U.S. Pat. No. 5,747,641, U.S. Pat. No. 5,674,980, U.S. Pat. No. 5,670,617, and U.S. Pat. No. 5,652,122; which are incorporated herein by reference) have also demonstrated transport of a protein (beta-galactosidase or horseradish peroxidase) into a cell by fusing the protein with amino acids 49-57 of TAT.
PCT publication WO01/15511 (which is incorporated herein by reference) discloses a method for developing protein transduction domains using a phage display library. The method comprises incubating a target cell with a peptide display library and isolating internalized peptides from the cytoplasm and nuclei of the cells and identifying the peptides. The method further comprised linking the identified peptides to a protein and incubating the peptide-protein complex with a target cell to determine whether uptake is facilitated. Using this method a protein transduction domain for any cell or tissue type may be developed. US Publication 2004/0209797 (which is incorporated herein by reference) shows that reverse isomers of several of the peptides identified by the above can also function as protein transduction domains.
PCT Publication WO99/07728 (which is incorporated herein by reference) describes linearization of protegrin and tachyplesin, naturally occurring as a hairpin type structure held by disulphide bridges. Irreversible reduction of disulphide bridges generated peptides that could readily transit cell membranes, alone or fused to other biological molecules. US Publication 2003/0186890 (which is incorporated herein by reference) describes derivatives of protegrin and tachyplesin that were termed SynB1, SynB2, SynB3, etc. These SynB peptides were further optimized for mean hydrophobicity per residue, helical hydrophobic moment (amphipathicity), or beta hydrophobic moment. Various optimized amphipathic SynB analog peptides were shown to facilitate transfer of doxorubicin across cell membranes. Further, doxorubicin linked to a SynB analog was observed to penetrate the blood-brain-barrier at 20 times the rate of doxorubicin alone.
The protein transduction domains described in the preceding paragraphs are only a few examples of the protein transduction domains available for facilitating membrane transit of small molecules, polypeptides or nucleic acids. Other examples are transportan, W/R, AlkCWK18, DipaLytic, MGP, or RWR. Still many other examples will be recognized by persons skilled in the art
A protein transduction domain and an agent of the present invention may be placed together in sufficient proximity and maintained together for a sufficient time to allow the protein transduction domain to influence pharmaceutical product performance of the agent. Contemplated associations of protein transduction domain and agent include, for example and without limitation: non-covalent associations such as electrostatic interactions, hydrogen bonding, ionic bonds or complexes, Van der Waals bonds; covalent linkages such as conventional methods of cross-linking; linkages that are activated, in vitro and/or in vivo by electromagnetic radiation; any covalent bond such as a peptide bond; any biochemical interaction known to protein biochemists, such as biotin/streptavidin, nickel/Histidine, glutathione/glutathione-S-transferase, or antigen/antibody; physical associations within matrix structures or encapsulating systems; etc.
The present invention provides an agent that may be any small molecule chemical compound, polypeptide, nucleic acid, or any combination thereof that can modulate DA receptor functionality through disruption of D1-D2 coupling. Accordingly, the present invention provides a polypeptide of about 3 to less than about 140 amino acids, preferably 7 to 109 amino acids, more preferably 15 to 100 amino acids and comprising an amino acid sequence that is at least 80% identical, for example, but not limited to 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of D2IL3-29 (SEQ ID NO:1), D2LIL3-29-2 (SEQ ID NO:2), D2IL3-L (SEQ ID NO:3), or D1CT (SEQ ID NO:4). The present invention also provides a nucleic acid encoding a polypeptide of about 3 to less than about 140 amino acids, preferably about 10 to about 109 amino acids, more preferably about 15 to about 100 amino acids and comprising an amino acid sequence that is at least 80% identical, for example, but not limited to 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of D2IL3-29 (SEQ ID NO:1), D2LIL3-29-2 (SEQ ID NO:2), D2IL3-L (SEQ ID NO:3), or D1CT (SEQ ID NO:4). The polypeptide or nucleic acid may optionally be fused to a protein transduction domain.
A polypeptide of the invention can be synthesized in vitro or delivered to a cell in vivo by any conventional method. As a representative example of an in vitro method, the polypeptide may be chemically synthesized in vitro, or may be enzymatically synthesized in vitro in a suitable biological expression system, such as without limitation, wheat germ extract or rabbit reticulocyte lysate. As a representative example of an in vivo method, a DNA, RNA, or DNA/RNA hybrid molecule comprising a nucleotide sequence encoding a polypeptide of the invention is introduced into an animal, and the nucleotide sequence is expressed within a cell of an animal.
The nucleotide sequence may be operably linked to regulatory elements in order to achieve preferential expression at desired times or in desired cell or tissue types. Furthermore, as will be known to one of skill in the art, other nucleotide sequences including, without limitation, 5′ untranslated region, 3′ untranslated regions, cap structure, poly A tail, translational initiators, sequences encoding signalling or targeting peptides, translational enhancers, transcriptional enhancers, translational terminators, transcriptional terminators, transcriptional promoters, may be operably linked with the nucleotide sequence encoding a polypeptide (see as a representative examples “Genes VII”, Lewin, B. Oxford University Press (2000) or “Molecular Cloning: A Laboratory Manual”, Sambrook et al., Cold Spring Harbor Laboratory, 3rd edition (2001)). A nucleotide sequence encoding a polypeptide or a fusion polypeptide comprising a polypeptide agent and a protein transduction domain may be incorporated into a suitable vector. Vectors may be commercialy obtained from companies such as Stratagene or InVitrogen. Vectors can also be individually constructed or modified using standard molecular biology techniques, as outlined, for example, in Sambrook et al. (Cold Spring Harbor Laboratory, 3rd edition (2001)). A vector may contain any number of nucleotide sequences encoding desired elements that may be operably linked to a nucleotide sequence encoding a polypeptide or fusion polypeptide comprising a protein transduction domain. Such nucleotide sequences encoding desired elements, include, but are not limited to, transcriptional promoters, transcriptional enhancers, transcriptional terminators, translational initiators, translational, terminators, ribosome binding sites, 5′ untranslated region, 3′ untranslated regions, cap structure, poly A tail, origin of replication, detectable markers, afffinity tags, signal or target peptide, Persons skilled in the art will recognize that the selection and/or construction of a suitable factor may depend upon several factors, including, without limitation, the size of the nucleic acid to be incorporated into the vector, the type of transcriptional and translational control elements desired, the level of expression desired, copy number desired, whether chromosomal integration is desired, the type of selection process that is desired, or the host cell or the host range that is intended to be transformed.
The DNA, RNA, or DNA/RNA hybrid molecule may be introduced intracellularly, extracellularly into a cavity, interstitial space, into the circulation of an organism, orally, or by any other standard route of introduction for therapeutic molecules and/or pharmaceutical compositions. Standard physical methods of introducing nucleic acids include, but are not limited to, injection of a solution comprising RNA, DNA, or RNA/DNA hybrids, bombardment by particles covered by the nucleic acid, bathing a cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid.
A nucleic acid may be introduced into suitable eukaryotic cells ex vivo and the cells harboring the nucleic acid can then be inserted into a desired location in an animal. A nucleic acid can also be used to transform prokaryotic cells, and the transformed prokaryotic cells can be introduced into an animal, for example, through an oral route. Those skilled in the art will recognize that a nucleic acid may be constructed in such a fashion that the transformed prokaryotic cells can express and secrete a polypeptide of the invention. Preferably, the prokaryotic cell is part of the animal's endogenous intestinal microflora. With regards to human examples of endogenous microflora are, without wishing to be limiting, Lactobacillus acidophillus, Streptococcus thermophilus, and Bifidobacterium bifidum. A nucleic acid may also be inserted into a viral vector and packaged into viral particles for efficient delivery and expression.
Dosage Forms
An agent of the present invention, for example, D1 or D2 polypeptides or nucleic acids encoding these polypeptides or antibodies or small molecules capable of disrupting D1-D2 coupling, may be formulated into any convenient dosage form. The dosage form may comprise, but is not limited to an oral dosage form wherein the agent is dissolved, suspended or the like in a suitable excipient such as but not limited to water. In addition, the agent may be formulated into a dosage form that could be applied topically or could be administered by inhaler, or by injection either subcutaneously, into organs, or into circulation. An injectable dosage form may include other carriers that may function to enhance the activity of the agent. Any suitable carrier known in the art may be used. Also, the agent may be formulated for use in the production of a medicament. Many methods for the productions of dosage forms, medicaments, or pharmaceutical compositions are well known in the art and can be readily applied to the present invention by persons skilled in the art.
Combination therapy with agents of the present invention or other agents useful for preventing and/or treating neurological diseases or disorders is contemplated. With regards to combination therapy suitable dosage forms again include capsules, tablets, and the like, preferably for oral administration, although any dosage form, for any route of administration is contemplated. Combination therapy can be administered as separate entities, e.g. two tablets or other forms, each containing one agent, or may be administered as a single dosage form containing both drugs, or concomitant use.
In case of oral administration of two or more different agents, the single dose can be, but is not limited to a capsule, tablet, or oral solution, and it may also contain inactive component(s) that is necessary to form the single delivery system.
Combination therapy medications of the present invention may be administered by any desired route, for example without limitation, administration can be transdermal (patch), buccal, sublingual, topical, nasal, parenteral (subcutaneous, intramuscular, intravenous, intradermal,), rectal, vaginal, administration. Various combinations of controlled release/rapid release are also contemplated.
Treatment
The methods and compounds of the present invention are useful for preventing and/or treating diseases that are characterized by abnormal levels of D1-D2 interaction or complex formation. The following are some non-limiting examples of such diseases: depression, including major depressive illness and depression in bipolar disorder, and psychiatric conditions that require treatment with antipsychotic medication, including schizophrenia, psychosis in bipolar disorder, stimulant drug intoxication, delirium, Tourette's syndrome, anxiety or a combination thereof.
Neurological, Neuropsychiatric Diseases
Depression is characterized by profound sadness, pronounced changes in sleep, appetite, and energy. Recurrent thoughts of death or suicide, persistent physical symptoms that do not respond to treatment, such as headaches, digestive disorders, and chronic pain are some symptoms of major depression. Major depression is a unipolar depression, while bipolar disorder (manic depression) involves both depression and mania. Early identification and treatment of depression is required to minimize risk of suicide and self-inflicted injury. The method and compounds of the present invention are useful in decreasing D1-D2 coupling and may be used for preventing and/or treating depression.
Accordingly the present invention provides methods for modulating DA functionality by disrupting D1-D2 coupling in a mammal. Any mammal including, without limitation, human, rat, cow, pig, dog, or mouse, may be treated with the agents and methods of the present invention.
The present invention will be further illustrated in the following examples.
EXPERIMENTS
Experiment 1
D1-D2 Receptor Complex is Facilitated by the D1R Carboxyl Tail (CT) and the Third Intracellular Loop (IL3) of D2R
To confirm previous reports of a D1-D2 receptor complex we used rat striata in co-immunoprecipitation (co-IP) experiments. As shown in FIG. 1A, the D1R was able to co-precipitate with the D2R, confirming the presence of a D1-D2 receptor complex. In an attempt to define the structural basis for the observed D1-D2 coupling, we carried out affinity purification using GST (glutathione-S-transferase)-fusion proteins encoding the D1CT and the third intracellular loop (D1IL3), since both D1CT: A332-T446 and D1IL3: R219-K272 contain putative consensus sequences for receptor phosphorylation, desensitization and potential binding sites for various proteins important for signalling (e.g. G proteins, NMDA receptor NR1, NR2A subunits)81-82. As shown in FIG. 1B, GST-D1CT, but not GST-D1IL3 or GST alone, precipitated solubilized striatal D2R as illustrated by the D2 antibody immunolabeled Western blot, indicating that the D1R can interact with D2R through the D1CT region. To locate the interacting site on D2R, GST-fusion proteins encoding IL3 from both the D2 short (D2S) and D2 Long (D2L) (GST-D2IL3-L: K211-Q373, GST-D2IL3-S:K211-Q315) were used in affinity purification assays. As shown in FIG. 1C, GST-D2IL3-L, but not GST-D2IL3-S or GST alone was able to pull-down D1 receptors. Since the D2L and D2S are differentiated by the additional 29 amino-acid within the third intracellular loop, the fact that only D2L but not D2S interacts with D1R made us suspect that this specific 29 amino-acid may contain the D1-D2 interacting site. The affinity “pull down” results revealed that the sequence encoded by the D2IL3-29 facilitates the interaction between D1 and D2 receptors since only the GST-D2IL3-L and GST-D2IL3-29:G242-V270 but not GST-D2IL3-S, GST-D2CT:T399-C414 or GST alone was able to pull-down D1 receptors (FIG. 1D). Further experiments using fragments of D2IL3-29 show that GST-D2LIL3-29-2:I256-V270, but not the GST-D2LIL3-29-1:G242-V255 or GST, can successfully pull-down D1R from solubilized rat striatum (FIG. 1E). Furthermore, in vitro binding assay suggested direct interaction between D1R and D2R (FIG. 1F). These results provide evidence that the D1-D2 direct interaction is dependent on sequences located in the I256-V270 region of D2R. Further, as shown in FIG. 1G, preincubation of GST-D2-IL3-29-2 inhibits the D1-D2 interaction as indexed by the co-immunoprecipitation in a concentration dependent manner.
Experiment 2
Co-Activation of the D1-D2 Receptor Induces an Increase in Intracellular Ca2+ Mediated by Phospholipase C Activation
To investigate the functional implication of the D1-D2 coupling, we examined the ability of this complex to promote Gq/11 signaling as indexed by changes in intracellular Ca2+ levels83-85. In HEK-293T cells co-transfected with D1R and D2R we examined changes in intracellular Ca2+ levels when co-treated with 10 μM SKF81297 and 10 μM quinpirole. Cells that were treated with both agonists exhibited a rapid and significant increase in intracellular Ca2+ levels that peaked 30 seconds after addition of agonists (FIG. 2A). However, cells treated with either SKF81297 or quinpirole alone had no change in Ca2+ levels when compared to nontreated control cells (FIG. 2B). The increase in Ca2+ levels could be blocked by pretreatment with either 10 μM raclopride (D2 antagonist) or 10 μM SCH23390 (D1 antagonist), as shown in FIG. 3A. Furthermore, this unique D1-D2 co-activation dependent signaling is mediated by phospholipase C (PLC) activation, since pretreatment with the PLC inhibitor U73122 (10 μM) inhibited the increase in Ca2+ levels induced by co-activation of D1R and D2R (FIG. 3B). These data are in line with previous studies showing that this signaling likely recruits Gq/11 upon co-activation of DA receptors, leading to downstream activation of PLC83-85.
Experiment 3
Disruption of D1-D2 Coupling Abolished the D1-D2 Co-Activation Induced Increases in Intracellular Ca2+
Our preliminary data has shown that coexpression of the GST-D1CT is able to affinity pull down the D2R (FIG. 1B). Thus, to test whether D1-D2 coupling is necessary for the D1-D2 co-activation induced increases in intracellular Ca2+, we examined changes in intracellular Ca2+ levels when co-treated with 10 μM SKF81297 and 10 μM quinpirole in HEK-293T cells cotransfected with mini-genes encoding D1CT, D5CT along with D1R and D2R. As shown in FIG. 3C, coexpression of the D1CT mini-gene but not the D5CT mini-gene abolished D1-D2 co-activation induced increases in intracellular Ca2+ suggesting that the D1-D2 coupling may be responsible for the observed increases in intracellular Ca2+ induced by D1/D2 co-activation.
Experiment 4
D1-D2 Coupling is Upregulated in Post-Mortem Brain Tissue of Depression Patients
Both D1R and D2R have been implicated in the pathology of psychiatric diseases such as schizophrenia. To test whether D1-D2 coupling is altered in disease, we carried out co-immunoprecipitation experiments in a double-blind manner on 60 post-mortem brain striatum samples from the Stanley Foundation, which includes 15 samples from each of four groups: control, schizophrenia, bipolar and severe depression. The four groups were matched by age, sex, race, postmortem interval, pH, side of brain, and mRNA quality by the Stanley Foundation brain bank. The same amount of protein from each sample was incubated with anti-D2 receptor antibody and protein A/G agarose. The precipitated proteins were divided equally into two groups before being subjected to SDS-PAGE and immunoblotted with either D1 antibody or D2 antibody. Each Western blot included 3 samples from each group and the intensity of each protein band was quantified by densitometry (software: AIS from Imaging Research Inc). Each sample is presented as the percentage of the mean of three control samples on the same blot. As shown in FIG. 4A, the co-immunoprecipitation of D1 by the D2 receptor antibody was significantly enhanced in the depression post-mortem brain samples compared to control brains. The levels of directly immunoprecipitated D2 receptors were not significantly different between the control and depression groups (data not shown). Therefore, the observed D1-D2 coupling upregulation seen in the depression brain samples may be a primary aspect of depression pathophysiology. However, we are aware that two issues may have influenced the results: (i) we do not have the patient history of antidepressant usage, and (ii) that some of the depression patients had history of drug abuse (5 out of 15) and/or alcohol abuse (10 out of 15), which may account for the large standard error in our results. Given the complex nature of data involving human brain tissue, we have confirmed the D1-D2 coupling is enhanced in rats with depressive-like behaviours induced by three different uncontrollable stress paradigms.
Experiment 5
Chronic Antidepressant Treatment Leads to a Decrease in the D1-D2 Coupling
If the observed increases in the D1-D2 coupling in the postmortem brain tissue of depression patients were indeed part of the pathological foundation of depression, one would imagine that chronic antidepressant treatment might correct such a change in the D1-D2 coupling. To test this hypothesis, we injected rats subcutaneously with 10 mg/kg/day of imipramine, a TCA, for a period of 14 days. On the 14th day, rats were sacrificed, striata dissected and were processed for co-IP assays and Western blots. Co-immunoprecipitation experiments revealed that chronic antidepressant treatment led to a decrease in D1-D2 coupling (FIG. 5A). Furthermore, this decrease in the D1-D2 coupling could not be attributed to changes in receptor levels, since Western blots revealed no significant change in D1R and D2R levels when comparing chronically treated rats with control rats (FIG. 5B,C).
Experiment 6
D1-D2 Coupling is Up-Regulated in Animals with Depressive-Like Behaviours
This was tested by co-immunoprecipitation of the D1R by the D2R primary antibody from solubilized proteins extracted from striatum, PFC of rats from the two animal models with depressive-like behaviours compared to the control groups. Briefly, anti-D2 antibody is incubated with solubilized protein for 4 hours followed by the addition of protein A/G beads. Incubation with protein A/G beads continues overnight followed by high stringency washes. The immunoprecipitated proteins are eluted from the beads and subjected to SDS-PAGE for Western blot analysis. Control experiments without D2 antibody are carried out concurrently. The co-immunoprecipitated proteins were immunoblotted with the D1, antibody and the intensity of each protein band was quantified by densitometry. Each co-immunoprecipitation is in parallel with western blot analysis of the initial levels of solubilized protein and directly immunoprecipitated proteins. As shown in FIG. 6, 7, striata (LH model) and PFC from rats (LH model and CMS model) showed a significant increase in the D1-D2 coupling compared to control rats (n=5 p<0.05). Furthermore, there was no significant change in D1R or D2R protein levels (data not shown).
Experiment 7
Animal Model #1: Learned Helplessness Induced by Inescapable Shock
The learned helplessness procedure consists of two separate stress sessions. On day 1, animals are placed in sound-attenuated operant boxes (Med Associates, St. Albans, Vt.) where they receive inescapable shock, with lights off and no levers present. Shock is delivered as a scrambled pulsed 0.8 mA current through metal floor bars. Both shock duration and intertrial intervals are randomly varied (1.5-60 sec and 1-30 sec, respectively), for a total shock exposure of 25 min. On day 2 rats are placed in the operant boxes, and given exactly 15 trials of escapable shock, each lasting for a maximum 60 sec, with a fixed intertrial interval of 24 seconds. Initiation of shock (0.8 mA) is accompanied by the onset of a red cue light placed directly above a lever which when pressed terminates the shock and turns off the cue light. A houselight outside the immediate chamber is kept on during the entire trial. A bar press within the first 20 seconds of shock initiation is recorded as an escape response. A response between 20-60 seconds is classified as failure to escape. After 60 seconds the shock is automatically terminated and the trial counted as a failure. Escape performance and latency to escape are recorded for each animal over the 15 trials. Animals are classified as learned helpless (LH) if they fail to escape in 10 or more of the 15 trials. Rats that fail to escape in 5 or less of the 15 trials are termed resistant or non-learned helpless (nLH)86-87. Animals that fail 5-10 times are considered borderline. In addition to home cage controls, another group may be placed in the boxes in day 1 without receiving any shock. On day 2 this group receives escapable shock. It serves as a behavioral control only, by demonstrating that control rats can learn to escape during the 15 trials. For each treatment condition, 8 LH, 8 nLH and 8 cage controls are sacrificed 24 hr after the escapable shock session via decapitation. Brains are quickly dissected and frozen on dry ice. All samples are kept at −80° C. for biochemistry and pharmacology analysis.
Experiment 8
Animal Model #2: Chronic Mild Stress 
Rats are first trained to drink a 1% sucrose solution, by exposing them to sucrose in place of water for 48 h. They then receive a series of sucrose preference tests, preceded by 23 h food and water deprivation, where each animal is presented simultaneously with 2 bottles, one containing 1% sucrose the other water. The position of the 2 bottles (right/left) is varied randomly from trial to trial and, within each trial, is counterbalanced across the animals in each group. During the test, both bottles are removed after 30 min for weighing, and replaced by a second pair of preweighed bottles (with the positions of the 2 bottles reversed), which are removed and weighed at 60 min. Tests are timetabled at the start of the dark cycle (1800-1900 h) for half of the animals and in the first half of the light cycle (1000-1100 h) for the other half. Following the final baseline test, each group of animals will be divided into 2 subgroups, matched on the basis of their total (60-min) sucrose intake in the final baseline test. One pair of subgroups will be exposed to CMS for 6 weeks; the control subgroups will not be stressed, other than the food/water deprivation that precedes each sucrose preference test. In each of the first four weeks, the CMS schedule (adopted from ref 88) will consist of the following elements: Tues.1900-Wed.1000 h Paired housing (new partner); Wed.1000-1800 h Stroboscopic illumination (in dark); Wed.1800-Thurs.1000 h Food deprivation in soiled cage (water in sawdust); Thurs.1000-1800 h 45° cage tilt; Thurs.1800-Fri.1000 h Mouse cage; Fri.1000-1800 h Paired housing (new partner); Fri.1800-Sat.1000 h Water deprivation; Sat.1000-1800 h Stroboscopic illumination (in dark); Sat.1800-Sun.0600 h Light on; Sun.0600-1800 h Intermittent lighting (off/on every 2 h); Mon.1100 or 1900 h 23-h food and water deprivation; Tue.1000 or 1800 h 1-h sucrose intake test. In the final two weeks, the CMS timetable will be rearranged, with paired housing (Mon. night) and food/water deprivation (Tues.night) immediately before sucrose intake test. Body weight will be monitored daily. After the last sucrose preference test, animals will be left undisturbed until next morning during when they will be decapitated.
Experiment 9
Increase in the D1-D2 Coupling in Rats Subjected to Forced-Swim Tests (FST) 
To investigate the possibility that the D1-D2 coupling plays a role in the pathology of depression, we examined the D1-D2 coupling using D1R antibody to co-IP the D2R from striata of rats subjected to FST. The FST (or behavioural despair) is a good model to test for the efficacy of antidepressant drugs. As shown in FIG. 8A, Striata from rats sacrificed 3 hours or 3 days after the last FST trial exhibited a significant increase in the D1-D2 coupling compared to control rats. Furthermore, there was no change in D1R or D2R protein levels that could account for the increase in the D1-D2 complex formation (FIG. 8B, C). Surprisingly, rats sacrificed 3 days after the last FST trial exhibited a larger increase in the D1-D2 coupling compared to rats sacrificed 3 hours after the last FST trial and suggested long-term changes occur after FST trials.
Experiment 10
Disruption of the D1-D2 Protein Complex in Schizophrenic Brain Tissue
To identify the physiological relevance for the D1-D2 receptor complex formation and specifically the effect of the antipsychotic medication, we carried out co-immunoprecipitation experiments in a double-blind manner with 60 post-mortem brain striatum samples from the Stanley Foundation, which includes 15 samples from each of the four groups: control, schizophrenia, bipolar and severe depression. The four groups were matched by age, sex, race, postmortem interval, pH, side of brain, and mRNA quality by the brain bank. The same amount of protein from each sample was co-immunoprecipitated with D2 receptor antibody. The precipitated proteins were divided equally into two groups and immunoblotted with either D1 antibody or D2 antibody. Consistent with our hypothesis, the co-immunoprecipitated D1 by the D2 antibody was significantly decreased in the post-mortem brain samples from both schizophrenia and bipolar patients compared to control group (FIG. 4). The levels of directly immunoprecipitated D2 were not significantly different among the four groups (data not shown). Interestingly, all the schizophrenia samples and 12 out of 15 of bipolar samples were from patients treated with antipsychotics, indicating that the observed D2-D1 interaction deficit seen in schizophrenia patients may not be a primary aspect of schizophrenia pathophysiology, it may actually reflect the pharmacological effects of antipsychotics/D2 antagonists. Thus, we further tested the D1-D2 protein complex formation in rats chronically treated with haloperidol, a clinical antipsychotic as well as a D2 antagonist (By pump 0.25 mg/kg/day for 2 weeks, to get continuous clinical occupancy89). As shown in FIG. 9, while the direct immunoprecipitated D2 receptors remain unchanged (top panel), the D1-D2 receptor complex formation is significantly decreased in rat with chronic antipsychotic treatment (bottom panel), suggesting that antagonizing D2 function disrupts D1-D2 receptor coupling.
Experiment 11
Interfering Protein Peptide that is Able to Disrupt the D1-D2 Coupling Exerts Antidepressant Effect in the Forced-Swim Test
The FST is a stress model commonly used to test for the efficacy of antidepressant drugs. Thus we will examine the effect of the interfering TAT peptides on rats using the FST, developed by Porsolt and colleagues146. Phase 1 of the FST consists of a preconditioning period in which rats are forced to swim in an enclosed container of water (height of container, 40 cm; diameter of container, 20 cm; depth of water, 13 cm; temperature, 25° C.) for 15 min without a way to escape. In this predicament, rats will respond by becoming immobile. Twenty-four hours after being removed from the container, each rat will be returned to the water for a 5 min test (phase 2), and the behavior will be recorded by a video camera from the side of the cylinder. Rat's behavior will be classified for each 5 seconds and assigned to different categories according to the standard described previously90 (climbing, diving, swimming, immobility and latency to immobility) from videotapes by a trained observer who is blinded to the experimental conditions). To examine the potential antidepressant effect of the interfering peptide, TAT-D2LIL3-29-2 (5 pmol) and TAT alone peptide were given ICV three times similar to antidepressant drugs: 1 hr and 5 hrs after the pre-swim trial (phase 1) respectively and 1 hour before the swim test (phase 2). As shown in FIG. 10, Intra-PFC administration of TAT-D2LIL3-29-2, but not TAT alone, decreased the frequencies of rat immobility and increased the frequencies of rat swimming and climbing behaviors in a 5 mins forced swimming test. Each value is the mean±S.E.M. for a group of 6 rats. Data were analyzed by one-way analysis of variance (ANOVA).
One or more currently preferred embodiments have been described by way of example. It will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.
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## Claims

1. A method for modulating dopamine (DA) receptor function in a mammal in need of such treatment comprising administering an effective amount of a nucleic acid expressing a polypeptide of up to 140 amino acids and comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 and comprising SEQ ID NO:2; wherein the polypeptide disrupts D1-D2 coupling in the mammal.

2. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is identical to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

3. The method of claim 1, wherein the nucleic acid further encodes a protein transduction domain.

4. The method of claim 1, wherein the method is for reducing the likelihood of depression and/or treatment of depression.

5. The method of claim 1, wherein the amino acid sequence comprises SEQ ID NO: 1, SEQ ID NO: 3, or both.