WO2022050355A1 - Prophylactic/therapeutic agent for mental disorders involving seretonin transporters, said agent including docosahexaenoic acid, docosahexaenoyl group-containing phosphatidic acid or derivative thereof - Google Patents

Abstract

There is a problem that the effect of known seretonin transporter (SERT) inhibitors (SSRI) on diseases involving SERT (e.g., depression, obsessive-compulsive disorder (OCD), schizophrenia, and Alzheimer's dementia) is limited. In the present invention, a compound that works through a pathway that is separate from the pathway of SSRI and that has a function for reducing SERT itself (e.g., docosahexaenoic acid (DHA), 1-stearoyl-2-docosahexaenoil (18:0/22:6)-phosphatidic acid (PA)[18:0/22:6-PA], etc.) can be expected to exhibit a better function effect (e.g., a stronger effect), and thus the compound is promising for prevention/treatment of diseases involving SERT. In particular, it is expected that DHA serves as a functional food product.

Description

Docosahexaenoic acid, docosahexaenoyl group-containing phosphatidic acid, or a derivative thereof to prevent or treat serotonin transporter-related psychiatric disorders

The present invention relates to drugs such as prophylactic and therapeutic agents for the control of serotonin / 5-hydroxytryptamine (5-HT) transporter (SERT), obsessive-compulsive disorder (OCD), major depressive disorder, autism, integration. It relates to drugs for the prevention and treatment of ataxia, anxiety, depression, obsessive-compulsive disorder, and impulsive psychiatric disorders, as well as foods having such effects.

Serotonin / 5-hydroxytryptamine (5-HT) is known to be involved in anxiety, depression, obsessive-compulsiveness, and impulsivity [Non-Patent Document 1]. Serotonin / 5-HT transporter (SERT) reabsorbs 5-HT from synaptic clefts into presynaptic neurons for recycling and metabolic degradation [Non-Patent Document 2; Non-Patent Document 3]. Selective serotonin reabsorption (SERT) inhibitors (SSRIs) are used to treat obsessive-compulsive disorder (OCD) [Non-Patent Document 2; Non-Patent Document 3] and major depressive disorder [Non-Patent Document 4]. Several studies with SERT-deficient rodents have demonstrated that SERT is associated with neurodevelopmental disorders such as depression, anxiety, and autism [Non-Patent Document 5]. Thus, the hyperactivity of SERT, which reduces the amount of 5-HT in the synaptic cleft, causes the various psychiatric disorders described above. However, the control mechanism of the SERT function is still not well understood.

Diacylglycerol (DG) kinase (DGK), which consists of 10 isozymes (α-κ), is a lipid-metabolizing enzyme that phosphorylates DG to produce phosphatidic acid (PA) [non-patents. Patent Document 6; Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9; Non-Patent Document 10]. DG and PA are established lipid second messengers, which control a wide variety of physiological and pathological events. Recently, the present inventors have generated and analyzed brain-specific DGKδ knockout (KO) mice, and have revealed that KO mice exhibit SSRI (fluoxetine) -sensitive OCD-like behavior [Non-Patent Document 11]. Moreover, the present inventors have demonstrated that DGKδ deficiency increases the mass of SERT protein in the mouse cerebral cortex [Non-Patent Document 12]. These results strongly suggested that OCD-like behavior in DGKδ-KO mice was caused by increased SERT levels.

In addition, recently, the present inventors have described DGKδ as SERT [Non-Patent Document 12; Non-Patent Document 13], melanoma antigen gene-D1 (MAGE-D1) [Non-Patent Document 13], and Praja-1 E3 ubiquitin. -Protein ligase [Non-Patent Document 13] (This Praja-1 E3 ubiquitin-Protein ligase ubiquitinizes SERT [Non-Patent Document 14], and Praja-1-ubiquitin-proteasome system in a DGK activity-dependent manner. It was found that it interacts with [Non-Patent Document 13]), which induces SERT degradation mediated by.
However, it remains unclear how Praja-1 activity is controlled by DGKδ.

Serotonin transporter (SERT) controls the serotonergic system, as well as serotonin / SERTs such as obsessive-compulsive disorder (OCD), depression, autism, and schizophrenia. Involved in the pathophysiology / therapeutics of related diseases. However, there is a problem that known SERT inhibitors (SSRIs) have a limited effect on SERT-related diseases (for example, depression, obsessive-compulsive disorder (OCD), schizophrenia, Alzheimer-type dementia, etc.).
So far, we have found that diacylglcerol (DG) kinase (DGK) δ is DGK active via Praja-1 E3 ubiquitin-protein ligase (Praja-1). It was revealed that it induces ubiquitination / degradation of SERT in a dependent manner. However, it remains unclear how Praja-1 activity is regulated by DGKδ.
Under these circumstances, SERT is controlled to make serotonin / SERT-related diseases such as obsessive-compulsive disorder (OCD), major depressive disorder, autism, schizophrenia, anxiety, depression, obsessive-compulsive disorder, and impulsiveness. It is required to find a drug useful for the prevention and treatment of psychiatric disorders, and a drug having a similar effect and activity that can be easily ingested as a food.

In order to solve the above problems, the present inventors have pursued research focusing on the molecular mechanism by which DGKδ regulates Praja-1 activity.
First, the present inventors in the DGKδ-KO mouse cerebral cortex, 1-stearoyl-2-docosahexaenoyl (18: 0/22: 6) -phosphatidic acid (PA) [18: 0/22: 6- PA] and 1-stearoyl-2-docosahexaenoyl (18: 0/22: 6) -diacylglycerol [18: 0/22: 6-DG] (X: Y = carbon atom of fatty acid acyl portion of glycerol backbone Total number: total number of double bonds) are simultaneously reduced and accumulated, respectively, so that DGKδ selectively phosphorylates 18: 0/22: 6-DG at 18: 0. / 22: 6-Revealed that it indicates that PA is generated.

The present inventors have also found that 18: 0/22: 6-PA selectively interacts with Praja-1 to enhance its E3 ubiquitin-protein ligase activity.
These results strongly suggest that DGKδ activates Praja-1 and degrades SERT through 18: 0/22: 6-PA production. Our findings provide new insights into SERT level control and the pathophysiology / therapeutics of various psychiatric disorders such as OCD, depression, autism, and schizophrenia. ..
We have found that 18: 0/22: 6-PA produced by DGKδ binds to Praja-1 and activates it, destabilizing SERT. Since DGKδ has been found to bind to SERT and Praja-1 in the brain [Non-Patent Document 12, Non-Patent Document 13], in the present invention, by 18: 0/22: 6-PA produced by DGKδ. A new mechanism for controlling SERT protein levels by activated Praja-1 can be proposed (Fig. 4). In addition, the present invention provides new insights into SERT level control and pathophysiology and therapeutic strategies for OCD, depression, autism, and schizophrenia.

In the present invention, 1-stearoyl-2-docosahexaenoyl (18: 0/22: 6) -phosphatidic acid (PA) [18: 0/22: 6-PA] is a serotonin transporter in the brain (serotonin). It is based on interacting with and activating Praja-1 [E3 ubiquitin-protein ligase] acting on transporter: SERT).
In the present invention, in the brain of DGKδ knockout mice, 1-stearoyl-2-docosahexaenoyl (18: 0/22: 6) -phosphatidic acid (PA) [18: 0/22: 6-PA] and 18 0/22: 6-DG are simultaneously reduced and accumulated, respectively, and in the brain of the DGKδ knockout mouse, DGKδ selectively phosphorylates 18: 0/22: 6-DG and 18: 0 / It reveals that it is shown to produce 22: 6-PA, and moreover, 18: 0/22: 6-PA selectively binds to Praja-1 and enhances its activity. I found that. These results strongly suggest that 18: 0/22: 6-PA produced by DGKδ activates Praja-1 and degrades SERT in the brain. Thus, using the present invention, the control of serotonin-operated systems, as well as obsessive-compulsive disorder, depression, autism, Alzheimer's type, through control of SERT, eg, control of degradation of SERT and / or promotion of degradation. It will be possible to obtain drugs for the prevention / treatment of serotonin / SERT-related diseases such as dementia and schizophrenia.
That is, the present inventors selectively bind to Praja-1 to enhance its activity, activate Praja-1 to decompose SERT in the brain, and / or destabilize / decompose it. Drugs that promote squeezing include drugs such as preventive and therapeutic agents for the control of SERT, obsessive-compulsive disorder (OCD), major depressive disorder, autism, schizophrenia, and Alzheimer-type cognition. The present invention has been completed by finding that it is excellent as a medicine for the prevention and treatment of illness and anxiety, depression, obsessive-compulsive disorder, and impulsive psychiatric disorder. In particular, the present invention relates to serotonin transporter-related psychiatric disorders (depression, obsessive-compulsive disorder, schizophrenia, Alzheimer-type dementia, etc.) caused by those selected from docosahexaenoic acid and phosphatidic acid containing a docosahexaenoyl group or derivatives thereof. We will provide preventive and therapeutic agents for dementia, as well as foods that can be expected to have similar effects.

In a representative embodiment, the present invention provides the following:
[1] docosahexaenoic acid (DHA) or its derivative, docosahexaenoyl group-containing phosphatidic acid or its derivative, and 1-stearoyl-2-docosahexaenoyl (18: 0/22: 6) -phosphatidic acid (PA) A drug compound that mimics [18: 0/22: 6-PA], or a SERT activity inhibitor whose active ingredient is selected from the group consisting of salts thereof, selectively bound to Praja-1. , A Praja-1 activity enhancer that enhances its activity, or obsessive-compulsive disorder (OCD), obsessive-compulsive disorder, autism, schizophrenia, Alzheimer-type dementia, and anxiety, depression, obsessive-compulsive disorder, and urge. Prophylactic / therapeutic agents for pathological symptoms selected from the group consisting of sexual psychiatric disorders,
[2] The agent of the above [1], which comprises DHA or 18: 0/22: 6-PA as an active ingredient.
[3] The SERT activity inhibitor is selected from the group consisting of DHA or a derivative thereof, docosahexaenoyl group-containing phosphatidic acid or a derivative thereof, or a salt thereof as an active ingredient. The agent of [1] above,
[4] Selected from the group consisting of docosahexaenoic acid (DHA) or its derivative, docosahexaenoyl group-containing phosphatidic acid or its derivative, and a drug compound that mimics 18: 0/22: 6-PA, or a salt thereof. The agent according to the above [1], which is a Praja-1 activity enhancer that selectively binds to Praja-1 and enhances its activity, which comprises the above-mentioned active ingredient.
[5] Effective one selected from the group consisting of DHA or its derivatives, docosahexaenoyl group-containing phosphatidic acid or its derivatives, and chemical compounds mimic 18: 0/22: 6-PA, or salts thereof. From the group consisting of obsessive-compulsive disorder (OCD), major depressive disorder, autism, schizophrenia, Alzheimer-type dementia, and anxiety, depression, obsessive-compulsive, and impulsive psychiatric disorders. The agent of [1] above, which is a preventive / therapeutic agent for selected pathological symptoms.
[6] Docosahexaenoic acid (DHA) or its derivative, docosahexaenoyl group-containing phosphatidic acid or its derivative, and 1-stearoyl-2-docosahexaenoyl (18: 0/22: 6) -phosphatidic acid (PA) Obsessive-compulsive disorder (OCD), major depressive disorder, autism, with the active ingredient selected from the group consisting of drug compounds mimic [18: 0/22: 6-PA] or salts thereof. , A food having a prophylactic / therapeutic effect on pathological symptoms selected from the group consisting of schizophrenia, Alzheimer-type dementia, and anxiety, depression, obsessive-compulsive, and impulsive psychiatric disorders.

Furthermore, in another representative embodiment, the present invention provides the following:
[7] A selection from the group consisting of DHA or its derivatives, docosahexaenoyl group-containing phosphatidic acid or its derivatives, and chemical compounds mimic 18: 0/22: 6-PA, or salts thereof. A method of suppressing the activity of SERT, which is characterized by administration to a subject, a method of enhancing the activity of Praja-1 that selectively binds to Praja-1 and enhances its activity, or obsessive-compulsive disorder (OCD), major depression. Prevention and treatment of pathological symptoms selected from the group consisting of pathological disorders, autism, schizophrenia, Alzheimer-type dementia, and anxiety, depression, obsessive-compulsive, and impulsive psychiatric disorders.
[8] The method of [7] above, which comprises administering 18: 0/22: 6-PA.
[9] DHA or a derivative thereof, a docosahexaenoyl group-containing phosphatidic acid or a derivative thereof, and a chemical compound mimicking 18: 0/22: 6-PA, or a salt thereof selected from the group. The method of [7] above, which comprises administering to a subject and suppressing the activity of SERT.
[10] A product selected from the group consisting of DHA or its derivatives, docosahexaenoyl group-containing phosphatidic acid or its derivatives, and chemical compounds mimic 18: 0/22: 6-PA, or salts thereof. The method of [7] above, which comprises administering to a subject to selectively bind to Praja-1 and enhancing its activity.
[11] A selection from the group consisting of DHA or its derivatives, docosahexaenoyl group-containing phosphatidic acid or its derivatives, and chemical compounds mimic 18: 0/22: 6-PA, or salts thereof. Administered to subjects from obsessive-compulsive disorder (OCD), major depressive disorder, autism, schizophrenia, Alzheimer-type dementia, and anxiety, depression, obsessive-compulsive, and impulsive psychiatric disorders. The method of the above-mentioned [7], which comprises preventing / treating a pathological symptom selected from the above group.

Moreover, in another typical aspect, the present invention provides the following.
[12] By measuring the activity selected from the group consisting of the interaction activity with Praja-1, the binding activity with Praja-1, and the enhancing or promoting activity of Praja-1 with respect to the drug candidate substance. A screening method, which comprises selecting and / or determining an active substance.
[13] The screening method according to the above [12], which comprises analyzing the reaction of a drug candidate substance to the fusion protein AcGFP-Praja-1 or GST-Praja-1.
[14] The above-mentioned [12], wherein the drug candidate substance is selected and / or determined by measuring the activity of enhancing the E3 ubiquitin-protein ligase activity of Praja-1. Screening method,
[15] Select from the group consisting of obsessive-compulsive disorder (OCD), major depressive disorder, autism, schizophrenia, Alzheimer-type dementia, and anxiety, depression, obsessive-compulsive, and impulsive psychiatric disorders. The screening method according to the above [12], which comprises selecting and / or determining a substance for preventing / treating a depressive pathological symptom.
[16] With respect to the drug candidate substance, the activity selected from the group consisting of the interaction activity with Praja-1, the binding activity with Praja-1, and the enhancing or promoting activity of Praja-1 was measured, and the activity thereof was measured. A screening kit comprising a Praja-1 protein or a tagged Praja-1 protein for selecting and / or determining what to have.

Docosahexaenoic acid (DHA) or a derivative thereof according to the present invention, docosahexaenoyl group-containing phosphatidic acid or a derivative thereof, and 1-stearoyl-2-docosahexaenoyl (18: 0/22: 6) -phosphatidic acid (PA). ) A drug compound that mimics [18: 0/22: 6-PA], or one that has an active ingredient selected from the group consisting of salts thereof, is a SERT activity inhibitor, selectively Praja-1. Praja-1 activity enhancer that binds to and enhances its activity, or obsessive-compulsive disorder (OCD), obsessive-compulsive disorder, autism, schizophrenia, Alzheimer-type dementia, and anxiety, depression, obsessive-compulsive disorder. It is a prophylactic / therapeutic agent for pathological symptoms selected from the group consisting of sexual and impulsive psychiatric disorders, and controls serotonin / 5-hydroxytryptamine (5-HT) transporter (SERT), for example, SERT. It is effective in destabilizing SERT, suppressing SERT activity, degrading SERT, etc., and has an improving effect on various diseases caused by SERT, such as obsessive-compulsive disorder, OCD, and schizophrenia (positive symptoms). ), Autism, Alzheimer-type dementia, etc. are expected to be excellent in prevention and treatment. The agent of the present invention can be expected to have the advantage of being highly safe as a pharmaceutical product or food.

In addition, by administering or ingesting the pharmaceuticals / foods of the present invention, various illnesses / diseases or pathological conditions (particularly anxiety, depression, obsessive-compulsive disorder, and impulsive psychiatric disorders, etc.) can be treated as SERT. Prevention and treatment of (related) are also provided. Further, in the present invention, an efficient screening method for active substances and compounds can be used, and useful active substances and active compounds can be analyzed and determined.
Since 18: 0/22: 6-PA according to the present invention works through an action pathway different from SSRI and reduces SERT itself, a stronger action effect can be expected. It may also lead to increased drug choices for SERT-related psychiatric disorders. In addition, the treatment of the above SERT-related psychiatric disorders may become more effective when used in combination with conventional SSRIs. Ingestion of DHA also makes it possible to provide functional foods that can be expected to prevent SERT-related mental illness.
Other objects, features, excellence and viewpoints thereof of the present invention will be apparent to those skilled in the art from the following description. However, it is understood that the description of the present specification including the following description and the description of specific examples and the like shows a preferable aspect of the present invention and is shown only for the purpose of explanation. sea bream. Making various changes and / or modifications (or modifications) within the intent and scope of the invention disclosed herein will be appreciated by those skilled in the art with the following description and knowledge from other parts of the specification. Will be easy to see. All documents cited herein are cited for purposes of illustration only, and their contents are to be included herein as part of this specification.

The analysis of PA molecular species and DG molecular species in the mouse cerebral cortex is shown. DGKδ-KO (right column) Mouse cerebral cortex and wild-type (left column) The amounts of major PA molecular species (A) and DG molecular species (B) in the mouse cerebral cortex were quantified using LC-MS. (PA: n = 3, DG: n = 4). Values are shown as mean ± SD. * P <0.05. In the figure, for example, "36: 1" means that "36" (= X) indicates the total number of carbon atoms in the fatty acid acyl portion of the glycerol backbone, and "1" (= Y) indicates the total number of carbon atoms in the glycerol backbone. It shows the total number of double bonds in the fatty acid acyl moiety. It shows the binding activity of AcGFP-Praja-1 to various phospholipids. (A) COS-7 cells were transfected with AcGFP-Praja-1. After 48h incubation, cells are collected and cell lysates are placed in PC liposomes, 18: 1/18: 1-PS liposomes, 18: 0/22: 6-PG liposomes, 16: 0/18: 1-PA liposomes, 18: Incubate with 1/18: 1-PA liposomes, 18: 0/18: 0-PA liposomes, 18: 0/20: 4-PA liposomes, and 18: 0/22: 6-PA liposomes and then by ultracentrifugation. separated. PAcGFP, which encodes AcGFP alone, was transfected as a control and cell lysates were incubated with PC liposomes or 18: 0/22: 6-PA liposomes. AcGFP-Praja-1 was detected by Western blotting with anti-GFP antibody. In the figure, Size marker indicates a size marker, mock indicates a mock, and AcGFP alone indicates AcGFP alone. (B) The amount of AcGFP-Praja-1 protein in the supernatant (S) and the precipitate (P) was quantified by densitometry using ImageJ. Binding activity was calculated as a percentage of precipitation band strength compared to total band strength. Values are shown as the mean ± SD of 3 to 6 independent experiments. *** P <0.005 for PC (control) liposomes. In the figure, for example, "18: 0/22: 6" means that "18" (= X) and "22" (= X) indicate the total number of carbon atoms in each fatty acid acyl portion of the glycerol backbone. , "0" (= Y) and "6" (= Y) indicate the total number of double bonds in each fatty acid acyl moiety of the glycerol backbone. In the figure, mock indicates a mock, and AcGFP alone indicates AcGFP alone. An assay of E3 ubiquitin ligase activity of Praja-1 in the presence of various phospholipids is shown. (A) Purified GST-Praja-1 fusion protein in PC liposomes, 18: 0/22: 6-PG liposomes, 18: 1/18: 1-PA liposomes, 18: 0/18: 0-PA liposomes, and It was incubated with 18: 0/22: 6-PA liposomes and then with E1, E2, ATP, and ubiquitin according to the instructions of the reagent kit manufacturer (left panel). The reaction was analyzed by immunoblotting with anti-ubiquitin antibody. GST alone was used as a negative control (right panel). In the figure, mock indicates a mock, Size marker indicates a size marker, conjugates indicates a conjugate, and GST alone indicates GST alone. (B) The degree of ubiquitination (> 180 kDa) of GST-Praja-1 was quantified by densitometry using ImageJ. Ubiquitinated GST-Praja-1 levels were calculated as a percentage of band intensity compared to mock (set to 100). Values are shown as the mean ± SD of three independent experiments. ** P <0.01; *** P <0.005. In the figure, mock indicates a mock, and GST alone indicates GST alone. The schematic diagram of Praja-1 and SERT control by DGKδ is shown. DGKδ ubiquitinizes SERT [Non-Patent Document 12], MAGE-D1 [Non-Patent Document 13], and Praja-1 E3 ubiquitin-protein ligase [Non-Patent Document 13]. ]) And induces SERT degradation via the ubiquitin (Ub) -proteasome system in a DGK activity-dependent manner [Non-Patent Document 13]. In this study, we show that DGKδ selectively phosphorylates 18: 0/22: 6-DG to produce 18: 0/22: 6-PA. Moreover, 18: 0/22: 6-PA selectively bound to Praja-1 and enhanced its E3 ubiquitin-protein ligase activity. In the figure, Synaptic cleft indicates synaptic cleft, N-Terminal indicates N-terminal, C-Terminal indicates C-terminal, Proteasome indicates proteasome, Degradation indicates degradation, and Presynapse indicates presynapse.

The present invention relates to docosahexaenoic acid (DHA) or a derivative thereof, docosahexaenoyl group-containing phosphatidic acid or a derivative thereof, and 1-stearoyl-2-docosahexaenoyl (18: 0/22: 6) -phosphatidic acid (PA). ) A drug containing a drug compound that mimics [18: 0/22: 6-PA] or a drug selected from the group consisting of salts thereof as an active ingredient, and / or administering it to a subject. It provides preventive and therapeutic methods for diseases characterized by.
The present invention was selected from the group consisting of A) DHA or its derivatives, docosahexaenoyl group-containing phosphatidic acid or its derivatives, and chemical compounds mimic 18: 0/22: 6-PA, or salts thereof. Pharmaceuticals characterized by the active ingredient, and B) DHA or its derivatives, docosahexaenoyl group-containing phosphatidic acid or its derivatives, and chemical compounds mimic 18: 0/22: 6-PA, or Also provided are pharmaceutical compositions characterized by containing pharmaceutically effective amounts of those selected from the group consisting of salts thereof and pharmaceutically acceptable additives.

The above docosahexaenoic acid (DHA) is known as a kind of ω-3 fatty acid and is also called all-cis-4,7,10,13,16,19-docosahexaenoic acid. Also written as (n-3) or simply 22: 6. Examples of the DHA derivative include an ester derivative, an amide derivative, and a DHA derivative known to those skilled in the art, which are hydrolyzed in vivo to give DHA, and further used in vivo. Those that donate a possible docosahexaenoyl group may also be included.
Since the present invention has found that docosahexaenoyl group-containing phosphatidic acids, such as 18: 0/22: 6-PA, reduce the amount of serotonin transporter, 18: 0/22: 6-PA. Ingestion of the precursor DHA leads to a decrease in the amount of serotonin transporter, which enables prevention and treatment of serotonin transporter-related diseases and improvement of symptoms. DHA can be ingested as a functional food, and the present invention may include those that can be ingested in the form of food as the above-mentioned preventive / therapeutic agent.

The above-mentioned docosahexaenoyl group-containing phosphatidic acid or its derivative is, for example, the following general formula.

[In the above formula, R ₁ is a hydrogen atom, a saturated aliphatic alkyl group, or an unsaturated aliphatic alkyl group, and typically R _1- (C = O)-is an acyl residue derived from a saturated fatty acid. A group or acyl residue derived from an unsaturated fatty acid, where R ₂ is a hydrogen atom, a saturated aliphatic alkyl group, or an unsaturated aliphatic alkyl group, typically R _2- (C = O). -Is an acyl residue derived from a saturated fatty acid or an acyl residue derived from an unsaturated fatty acid, and R ₃ is a hydrogen atom, a group known to those skilled in the art of phospholipid chemistry, Examples thereof include groups that are hydrolyzed in vivo, such as choline residues, ethanolamine residues, serine residues, inositol residues, and glycerol residues, and groups that can be formed by in vivo metabolism. However, here, either R _1- (C = O)-and R _2- (C = O)-is an acyl residue derived from docosahexaenoic acid, that is, a docosahexaenoyl group].
It may be a compound having.
In the above general formula, the bond between the fatty acid and the glycerol skeleton is shown to be an ester bond [R- (CO) -OC], and the derivative thereof is the saturated aliphatic alkyl group or unsaturated fat described above. The bond between a hydrocarbon residue including a group alkyl group and a glycerol skeleton is not only an ester bond but also an ether bond (ROC). When an ether bond is used instead of the ester bond, one bond may be replaced or a plurality of bonds may be replaced.

Examples of the saturated fatty acids include formic acid, acetic acid, propionic acid, butyric acid (4: 0), valeric acid (5: 0), caproic acid (6: 0), enanthic acid (7: 0), and capric acid (8: 0). ), Perargonic acid (9: 0), Capric acid (10: 0), Undecic acid (11: 0), Lauric acid (12: 0), Tridecic acid (13: 0), Myristic acid (14: 0), Pentadecylic acid (15: 0), palmitic acid (16: 0), margaric acid (17: 0), stearic acid (18: 0), nonadesilic acid (19: 0), arachidic acid (20: 0), henicosyl acid (21: 0), behenic acid (22: 0), tricosyl acid (23: 0), lignoseric acid (24: 0) and the like.

Examples of the unsaturated fatty acid include ω-3 fatty acid, ω-6 fatty acid, ω-7 fatty acid, ω-9 fatty acid, and ω-10 fatty acid. Examples of the ω-3 fatty acid include cis-7,10,13-hexadecatrienoic acid (16: 3), α-linolenic acid (ALA or 18: 3), stearidonic acid (STD or 18: 4), and the like. Eicosatrienoic acid (ETE or 20: 3), Eicosatetraenoic acid (ETA or 20: 4), Eicosapentaenoic acid (EPA or 20: 5), Docosapentaenoic acid (DPA or 22: 5), Docosahexaenoic acid (DHA or 22: 6), tetracosapentaenoic acid (24: 5), tetracosahexaenoic acid (24: 6) and the like. Examples of the ω-6 fatty acid include linoleic acid (18: 2), γ-linolenic acid (18: 3), eikosazienoic acid (20: 2), dihomo-γ-linolenic acid (20: 3), and arachidonic acid. (20: 4), docosadienoic acid (22: 2), docosatetraenoic acid (22: 4), docosapentaenoic acid (22: 5), calendic acid (18: 3) and the like. Examples of the omega-7 fatty acid include palmitoleic acid (16: 1), vaccenic acid (18: 1), paullinic acid (20: 1) and the like. Examples of the ω-9 fatty acid include oleic acid (18: 1), elaidic acid (18: 1), eicosenoic acid (20: 1), mead acid (20: 3), erucic acid (22: 1), and the like. Examples include erucic acid (24: 1). Examples of the ω-10 fatty acid include sapienic acid (16: 1).

Among the compounds of the above general formula, as a typical docosahexaenoyl group-containing phosphatidic acid, the 1-position R _1- (C = O) -group is a stearoyl group and the 2-position R _2- (C =). O)-is a docosahexaenoyl group, and R ₃ is a hydrogen atom. Furthermore, for shorter carbon chains such as acetyl groups at the 1-position, it can be expected that the permeability of the membrane will increase and the effect will increase, and the ester bond between fatty acid and glycerol was replaced with an ether bond. It can be expected that the bond is not easily broken, the stability is increased, and a longer-lasting effect can be obtained.
Since docosahexaenoic acid (DHA) is known as an essential fatty acid, the docosahexaenoyl group at the 2-position is mostly derived from DHA ingested as food (ie, DHA is 18: 0 / Since it can be regarded as a precursor of 22: 6-PA), the prescribed medicinal effect can be obtained more effectively by ingesting DHA or administering DHA.
The above-mentioned DHA or a derivative thereof, and docosahexaenoyl group-containing phosphatidic acid or a derivative thereof can be synthesized by applying a chemical synthesis method. In addition, these compounds can also be isolated and purified from in vivo metabolites when administered to mammals using conventional biochemical and chemical methods.

The chemical compounds that mimic 18: 0/22: 6-PA are not only those designed based on the chemical structural formula of 18: 0/22: 6-PA, but also their three-dimensional three-dimensional structural arrangement. It may be designed with reference to it, or it may be designed with reference to dynamic structural changes. In addition, the drug compound that mimics the above 18: 0/22: 6-PA uses AI by analyzing the structural data of the Praja-1 protein and considering dynamic changes as necessary based on it. It may be obtained by the design. For example, the 1-position may be a shorter carbon chain corresponding to a fatty acid such as acetic acid, or the ester bond between the fatty acid and glycerol may be replaced with an ether bond. The compound can be synthesized by applying a chemical synthesis method, or can be obtained by applying a biochemical method.

Among the above-mentioned active ingredient compounds, if salt-forming is possible, suitable salts include pharmaceutically acceptable salts such as salts with inorganic or organic bases, neutral, basic or acidic amino acids. Salt and the like. Preferable examples of salts with inorganic bases include alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, and salts with aluminum and ammonium. Preferable examples of the salt with an organic base include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N, N-dibenzylethylenediamine and the like. Suitable examples of salts with neutral amino acids include, for example, salts with glycine, valine, leucine and the like, and suitable examples of salts with basic amino acids include, for example, salts with arginine, lysine, ornithine and the like. As a preferred example of the salt with an acidic amino acid, for example, a salt with aspartic acid, glutamic acid and the like can be mentioned.

The drug containing the active ingredient of the present invention is an agent for controlling SERT, for example, an agent for controlling serotoninergic system through control of degradation and / or promotion of degradation of SERT, obsessive-compulsive disorder, depression. , Autism, and as a prophylactic / therapeutic agent for serotonin / SERT-related diseases such as schizophrenia. In addition, the drugs containing the active ingredient of the present invention include SERT activity inhibitors, Praja-1 activity enhancers, and / or obsessive-compulsive disorder (OCD), major depressive disorder, autism, and schizophrenia. , Alzheimer-type dementia, and useful as a prophylactic / therapeutic agent for pathological symptoms selected from the group consisting of anxiety, depression, obsessive-compulsive, and impulsive psychiatric disorders. Disorders or disorders associated with the anxiety include neurosis, generalized anxiety disorder, social anxiety disorder, panic disorder, hyperactivity disorder, attention deficit disorder, personality disorder, bipolar disorder and the like. The active ingredient of the present invention has sufficiently low toxicity and can be safely used as a pharmaceutical product.

The drug containing the active ingredient of the present invention is administered alone or in combination with other drugs as described later, preferably in the form of a preparation containing a pharmaceutically acceptable additive. As the route of administration, oral, transdermal and injectable routes are adopted. Furthermore, as the preparations of the above drugs, external preparations (transdermal preparations, ointments, etc.), suppositories (rectal suppositories, vaginal suppositories, etc.), pellets, nasal preparations, inhalants (nebulizers, etc.), eye drops, liposomes. Examples include pharmaceutical products. Further, in the above-mentioned preparation, an ingredient selected from known preparation additives (hereinafter, also referred to as “formulation component”) can be appropriately used regardless of the administration route. Specific known pharmaceutical additives include, for example, (1) Handbook of Pharmaceutical Additives, Maruzen Co., Ltd., (1989), (2) Encyclopedia of Pharmaceutical Additives, 1st Edition, Yakuji Nippo Co., Ltd. (1994). , (3) Supplement to the Encyclopedia of Pharmaceutical Additives, 1st Edition, Yakuji Nippo Co., Ltd. (1995) and (4) Pharmaceutics, 5th Revised Edition, Nanedo Co., Ltd. (1997) Therefore, it can be appropriately selected according to the administration route and the intended use of the drug.

For example, in the case of oral administration, the additive may be any pharmaceutical component that can constitute an oral preparation and that can achieve the object of the present invention, but is usually an excipient or a binder. Known pharmaceutical ingredients such as agents, disintegrants, lubricants, and coating agents (including taste masking) are selected. Specific oral preparations include tablets (including sublingual tablets and orally disintegrating tablets), capsules (including soft capsules and microcapsules), granules, fine granules, powders, troches, syrups, and liposomes. And so on. The oral preparation is a preparation in which the release of active drugs such as DHA and 18: 0/22: 6-PA, which are active ingredients, is controlled in the body by using known preparation ingredients (eg, immediate release). Formulations, sustained release preparations) are also included.

In the case of injection, as the above additive, a pharmaceutical component that can constitute an aqueous injection or a non-aqueous injection is used, and usually, a solubilizing agent, a solubilizing agent, a suspending agent, an isotonicizing agent, and a buffering agent. , Stabilizers, preservatives and other known pharmaceutical ingredients are used, but they may also be known pharmaceutical ingredients constituting powder injections for use by dissolving or suspending them at the time of administration. The pharmaceutical components of the aqueous injection include, for example, distilled water for injection, isotonic sterilized salt solution (1 sodium or 2 sodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc., or such. Examples of the pharmaceutical components of the non-aqueous injection (including a mixture of salts) include vegetable oils such as olive oil, sesame oil, cottonseed oil, and corn oil, propylene glycol, macrogold, and tricapryrin, which are dissolved in these. Manufactured by suspension or emulsification. Specific examples of the injection include a subcutaneous injection, an intravenous injection, an intramuscular injection, an intraperitoneal injection, a drip infusion, and a liposome. Thus, the usage of the active drug or active compound such as DHA, 18: 0/22: 6-PA for preparing a drug (pharmaceutical or pharmaceutical composition) having the above-mentioned activity containing the active ingredient of the present invention is also possible. Provided.

The effective dose of the drug of the present invention varies depending on the age, weight, symptom of illness, presence or absence of complications, etc. of the patient to be administered, and is appropriately adjusted. However, in the case of oral administration, it is usually 0.1 mg to 3,000. Administer about mg / day, or in the case of injection, about 0.1 mg to 1,000 mg / day.
The drug as the active ingredient of the present invention can also be used in combination with one or more other drugs that do not adversely affect the effect for the purpose of enhancing the action, lowering the dose, reducing side effects and the like. The concomitant drug that can be combined may be a small molecule compound, a polypeptide, an antibody, a vaccine or the like.

When the drug containing the active ingredient of the present invention is used in combination with another drug or drug, the administration form in combination with the concomitant drug is not particularly limited, and the drug may be simply used in combination. For example, both are formulated simultaneously and administered as a single formulation, both are formulated separately and administered simultaneously or at different time intervals on the same route of administration, and both are formulated separately and administered simultaneously or on different routes of administration. The administration etc. may be mentioned.
When a drug containing the active ingredient of the present invention is used in combination with another drug, the concomitant drug can be selected from, for example, a SERT inhibitor (SSRI), and the SERT inhibitor can be, for example, Fluvoxamine, fluoxetine, paroxetine, sertraline, citalopram, escitalopram and the like can be mentioned.

The present invention also provides a preventive / therapeutic method for diseases and the like using the active ingredient drug (compound), for example, by administering an effective amount of the 18: 0/22: 6-PA to a subject. , The preventive / therapeutic method can be implemented. This prevention / treatment method can also be performed while monitoring the health condition and progress of symptoms of the subject. The monitor may be performed at regular or irregular time intervals, or may be performed regularly. In a typical case, it is performed while monitoring blood HDL levels. The administration may be performed at regular or irregular time intervals, or may be performed regularly. In the specification, "prevention / treatment" may refer to prevention and / or treatment, and includes cases where it means prevention and treatment, cases where it means prevention, and cases where it means treatment. is doing.

From the group of the present invention consisting of obsessive-compulsive disorder (OCD), major depressive disorder, autism, schizophrenia, Alzheimer-type dementia, and anxiety, depression, obsessive-compulsive, and impulsive psychiatric disorders. Foods that have the preventive / therapeutic effect of the selected pathological symptoms include DHA or its derivatives, docosahexaenoyl group-containing phosphatidic acid or its derivatives, and 18: 0/22: 6-PA as its active ingredients. It contains a chemical compound selected from the group consisting of chemical compounds or salts thereof. In particular, foods containing DHA can be mentioned. DHA is a fish oil obtained from fish such as herring, mackerel, sardines, tuna, bonito, saury, and yellowtail, and its content is known to be high. It is also known to be produced by microorganisms such as Euglena, which can also be obtained from fermentation products of such microorganisms. As the oil product containing DHA, those having various DHA purity and DHA composition are commercially available or known, and those having the intended effect of the present invention can be selected and used from these products.

The food of the present invention may be provided as a composition in various forms, and the food itself containing a functional food, an additive used in producing a food containing various processed foods and functional foods, It can be in the form of an animal feed itself, an additive used in producing an animal feed, or the like. These various forms can be produced by a commonly used method.
The food to which the present invention is applied includes all foods including beverages, and in addition to general processed foods including so-called health foods, foods for specified health use and nutrition specified in the Health Function Food System of the Consumer Affairs Agency of Japan. Includes health functional foods such as functional foods, supplements, etc., as well as health functional foods such as specified health foods and nutritionally functional foods, supplements, etc. that are supported in countries other than Japan, and also includes feeds fed to animals. do.

Foods such as the functional foods of the present invention can take a solid, semi-solid or liquid form, for example, oral liquids (including drinks), biscuits, confectionery, candy, tablets (tablet confectionery, etc.). (Including), granules (including granular confectionery, etc.), powders (including powdered beverages, powdered confectionery, etc.), capsules, jelly and the like.
The food product form of the present invention includes, for example, beverages (soft beverages, tea beverages, coffee beverages, dairy beverages, fruit juice beverages, carbonated beverages, nutritional drinks, powdered beverages, alcoholic beverages, non-alcoholic beverages, sports drinks, etc. ), Soybean processed foods, breads, noodles, rice, gel-like foods (jelly drinks, jelly, bavarois, pudding, mousse, gummy candy, etc.), sweets (various snacks, baked goods, cakes, chocolate , Gum, candy, tablets, etc.), soups, dairy products, frozen foods, processed marine products (fish sausage, kamaboko, chikuwa, hampen, etc.), processed livestock products (hamburger, ham, sausage, wiener, cheese, butter, yogurt) , Raw cream, margarine, fermented milk, etc.), instant foods, supplements, capsules, cereals, other processed foods, seasonings and their ingredients. The food can further contain other necessary raw materials or additives as appropriate, as long as the effects of the present invention are not impaired. Examples of other raw materials or additives include fruit juices, sweeteners, acidulants, vitamins, amino acids, minerals, proteins, thickeners, flavors, pigments and the like.

Matters that are essential for a predetermined purpose and should be followed as necessary so that the user can recognize the efficacy and effect when the composition of the present invention is provided as a product such as a food product or a pharmaceutical product in various forms. Instructions can be attached to the product. This description can be provided by attaching the instruction manual prepared separately from the product to the product package, or by printing the instruction manual on the product itself or on the packaging of the product (including the inner bag that wraps the divided product). .. In this description, information regarding the content of the active ingredient such as DHA of the product, the total intake amount of the active ingredient such as DHA within the ingestion time, the period of continuous ingestion, and the like can be described. In addition, the products can be classified according to the amount to be ingested within the ingestion time range, and the required amount of the divided products can be stored in the product package.

In the screening method of the present invention, the activity selected from the group consisting of the activity of interacting with Praja-1, the activity of binding to Praja-1, and the activity of enhancing or promoting the activity of Praja-1 is measured. The Praja-1 protein is preferably used. The Praja-1 used for screening may be in vivo or in vitro. In a typical case, the fusion protein Praja-1 produced by applying genetic engineering technology is used. For example, a fusion protein tagged with a fluorescent protein derived from Owan jellyfish, glutathione-S-transferase (GST), maltose-binding protein (MBP), cellulose-binding protein (CBP), thioredoxin (TRX), His tag, etc. It is convenient to use a fusion protein or the like with a tag that binds to the ligand of. As the tag of the Praja-1 protein, any suitable marker can be appropriately selected and applied as long as it is a marker that can be detected. Typical examples of the fusion protein include AcGFP-Praja-1, GST-Praja-1 and the like. Detection can also be performed using an antibody (including a monoclonal antibody and a fragment thereof) that specifically recognizes the fusion tag. Expression and purification of such fusion polypeptides or fusion proteins can be performed using commercially available kits suitable for them and can also be performed according to the protocol disclosed by the kit manufacturer or kit distributor.

Performing this screening can be performed using standard techniques that are well known and conventional to those skilled in the art. According to the screening method of the book, SERT activity inhibitor, Praja-1 activity enhancer that selectively binds to Praja-1 and enhances its activity, or obsessive-compulsive disorder (OCD), major depressive disorder, self. Efficient active substances and active compounds useful as prophylactic / therapeutic agents for pathological symptoms selected from the group consisting of obsessive-compulsive disorder, anxiety, depression, obsessive-compulsive disorder, and impulsive psychiatric disorders. It is possible to perform analysis with priority and priority and select it.
Examples of the above-mentioned pharmaceutical candidate substances include compounds or compositions obtained by chemical synthesis, naturally derived compounds or compositions thereof, metabolites, fermentation products, phospholipids, lipids, peptides, proteins, sugar chains and other biopolymer compounds. , Or a mixture thereof. The test substance in the screening method of the present invention is not particularly limited. For example, it may be a compound such as a small molecule compound or a high molecular compound.

In the typical case, this screening involves (i) a system containing the test sample and the Praja-1 protein, and (ii) a system containing the Praja-1 protein but not adding the test sample. In the two types of reaction systems between, the above reaction is allowed to proceed, the presence or absence of binding to the Praja-1 protein, the amount of binding, etc. are measured, and either of these is measured in (i) and (ii). By comparing the values, the Praja-1 binding activity of the test substance and the like can be measured. A suitable detection substrate may be present in the screening system so as to be convenient for measurement. The substrate may be any substrate as long as it can be effectively used for measurement. For example, a compound known as a known substrate can be selected and used, and a synthesized compound or the like can be preferably used. The substrate can be used as it is, but preferably a substrate labeled with a fluorescent, enzyme or radioactive substance such as fluoressein can be used.

In this screening, the Praja-1 protein may be one in which the tagged Praja-1 protein is expressed in animal cells, for example, COS cells, CH0 cells, human-derived cell lines, iPS cells, and the like. For example, when expressed in a transformant using an animal cell as a host cell, an appropriate selection marker can be used to obtain a gene in which the target protein is expressed at a higher level, and the target protein is expressed. It can also be cultivated under possible conditions to produce and accumulate the desired product. The transformant cells can be cultured in a medium widely used in the art.

When culturing animal cells, for example, a MEM medium containing about 5 to about 20% fetal bovine serum, PRMI1640 medium, DMEM medium, or the like may be used as the medium. The pH is preferably about 6 to about 8. Culturing is usually carried out at about 30 to about 40 ° C. for about 15 to about 72 hours, and aeration and stirring are added as necessary. Transformed animal cells expressing a predetermined protein can be used as they are, but they can also be used as cell homodunates thereof, or a predetermined gene product protein can be isolated and used. When extracting from the above cultured cells, after culturing, the cells are collected by a known method, suspended in an appropriate buffer solution, destroyed by ultrasonic waves, lysozyme and / or freeze-thaw, and then centrifuged. A method of obtaining a crude extract by filtration or the like can be appropriately used. A protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (trade name) or Twien-20 (trade name) may be added to the buffer solution. The target protein can be purified by appropriately combining the separation / purification methods known per se.

This screening can be carried out according to a usual method for measuring binding activity or enzyme activity, and can be carried out with reference to, for example, a method known in the art. In addition, various labels, buffer systems, other appropriate reagents, etc. can be used, and the operation can be performed according to the operation described therein. The protein to be used can be treated with an activator, or its precursor or latent form can be converted into an active form in advance. The measurement is usually carried out in a buffer solution such as Tris-HCl buffer solution or phosphate buffer solution which does not adversely affect the reaction, for example, at pH of about 4 to about 10 (preferably pH of about 6 to about 8). It can be carried out. In these individual screenings, a measurement system related to the protein or a protein having substantially the same activity as that of the protein is prepared by adding the usual technical arrangements of those skilled in the art to the usual conditions and operating methods in each method. Just build it. For details of these general technical means, review articles, books, etc. can be referred to [for example, Methods in Enzymology, published by Academic Press (USA), etc.].

The screening kit of the present invention contains a Praja-1 protein or a tagged Praja-1 protein. The screening kit of the present invention can screen a substance or compound having an activity selected from the group consisting of an interaction activity with Praja-1, a binding activity with Praja-1, and an enhancing or promoting activity of Praja-1. .. The screening kit also includes obsessive-compulsive disorder (OCD), major depressive disorder, autism, schizophrenia, Alzheimer-type dementia, and anxiety, depression, obsessive-compulsive, and impulsive psychiatric disorders. It is possible to screen substances or compounds having a preventive / therapeutic effect on pathological symptoms selected from the group.
The screening kit may include a buffer solution for a reaction, a reagent, or the like as described above as a set so that it is convenient to perform the required measurement.
Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is provided merely for the purpose of explaining the present invention and for reference in a specific embodiment thereof. These examples are for explaining specific specific embodiments of the present invention, but do not represent limiting or limiting the scope of the invention disclosed in the present application. It should be understood that in the present invention, various embodiments based on the ideas of the present specification are possible.
All embodiments have been or can be carried out using standard techniques, except as described in detail elsewhere, which are well known and conventional to those of skill in the art. ..

〔material and method〕
Mice The experimental study of this example was approved by the Animal Care and Use Committee of Chiba University (approval number: 29-195). All animal care and treatment procedures comply with the Commission's Animal Care Guidelines. Both the distress and the number of animals used were sought to be minimized as much as possible.
Animals were housed at 24 ± 2 ° C under a 12-hour light-dark cycle (lit from 7:00 to 19:00), and food and water were free food.
Brain-specific DGKδ-deficient mice [Non-Patent Document 11] were prepared as described above.
Briefly, a targeting construct was created by inserting the LoxP site sandwiching exon 9 of DGKδ. Males of calmodulin-dependent kinase IIα-Cre ⁺ were used to obtain brain-specific DGKδ-KO. Cre ^-/- : loxP ^+/+ mice were mated with cre ^+/- : loxP ^-/- to generate brain-specific DGKδ-deficient mice. cre ^+/- : loxP ^+/+ (brain-specific DGKδ deficiency) As a control group for mice, cre ^-/- : loxP ^+/+ littermates were used. Seven male mice per genotype (brain-specific DGKδ-KO mice and sibling controls) were used.

[Plasmid construct]
Mouse Praja-1 (NCBI accession no. XM_011247544) cDNA was amplified from mouse brain cDNA and inserted into the EcoRI / SalI site of the pAcGFP vector [Non-Patent Document 13]. In addition, in order to express glutathione S-transferase (GST) fusion Praja-1 by mammalian cells, the Praja-1 cDNA should be used in pSF-CMV-Puro-NH2-GST-TEV (Oxford Genetics, Oxford, UK) EcoRI /. I also ligated to the XhoI site.

[Cell culture and transfection]
COS-7 cells are Dulbecco's modified Eagle's medium (D-MEM) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 100 U / mL penicillin + 100 μg / mL streptomycin; Wako Pure Chemical Industries, Osaka, Japan) maintained the temperature at 37 ° C in an atmosphere containing 5% CO ₂ . COS-7 cells were seeded in 6 wells with poly-L-lysine coated coverslips at a density of 1 × 10 ⁵ cells / well.
pAcGFP-Praja-1 or pSF-CMV-Puro-NH2-GST-TEV-Praja-1 was transfected as shown in the legend in the figure in the kit manual. The plasmid was transiently transfected with PolyFect (Qiagen, Hilden, Germany) according to the kit manufacturer's instructions.

[Lipid extraction]
Mouse cerebral cortex was homogenized in ice-cold Lysis buffer (50 mM HEPES, pH7.2, 150 mM NaCl, 5 mM MgCl ₂ , cOmplete ^TM EDTA-free protease inhibitor) and then 1000 g at 4 ° C. Centrifuge for 5 minutes. Total lipids were extracted from mouse brain (cerebral cortex) according to Bligh and Dyer's method [Non-Patent Document 15]. 2 ml of methanol and 1 mL of chloroform were added to 700 μL of the sample.
For PA analysis, 100 μL of 3M HCl was added to the sample to improve the recovery ratio of acidic phospholipids [Non-Patent Document 16]. The lipid-containing solvent was dried under N ₂ gas and the extracted lipid was reconstituted in 100 μL chloroform / methanol (2: 1, v / v).

[Liquid Chromatography (LC) -Mass Spectrometry (MS) and LC-Tandem Mass Spectrometry (MS / MS)]
Phosphatidic acid species in the extracted lipid (10 μL) were analyzed by LC-MS and LC-MS / MS as described in previous literature [Non-Patent Documents 16-19]. The diacylglycerol species in the extracted lipid (10 μL) were analyzed by LC-MS and LC-MS / MS as previously described in [Non-Patent Document 20].

[Purification of GST-Praja-1]
GST-Praja-1 was expressed by COS-7 cells. After cytolysis and centrifugation, affinity-chromatography with glutathione sepharose 4B (GE Healthcare, Chicago, IL, USA) was performed to purify GST fusion α-Syn-N. The column was washed with lysis buffer and the bound protein was eluted with buffer containing 50 mM Tris / HCl, 150 mM NaCl, and 10 mM reduced glutathione. For the lipid overlay assay, the purified protein was dialyzed against HEPES buffer (25 mM HEPES, pH 7.4, 100 mM NaCl). Protein concentration was measured by the Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific).

[Liposome co-precipitation assay]
The lipid binding properties of the Praja-1 protein were determined using the following lipid mixture:
Control liposomes [Cholesterol (30 mol% (Wako Pure Chemical Industries, Ltd.)) and phosphatidylcholine (PC, from egg yolk (Avanti Polar Lipids, Alabaster, AL, USA)) (70 mol%)], phosphatidylserine (PS) liposomes [ Chol (30 mol%), PC (from egg yolk) (60 mol%), and 18: 1/18: 1-PS (10 mol% (Avanti Polar Lipids))], phosphatidylglycerol (PG) liposomes [Chol (30 mol%), PC (from egg yolk) (60 mol%), and 18: 0/22: 6-PG (10 mol% (Avanti Polar Lipids))], and PA liposomes [Chol (30 mol%), PC (from egg yolk) (60 mol%) , And each PA species (10 mol%)].
PA species include 16: 0/18: 1-PA (Avanti Polar Lipids), 18: 1/18: 1-PA (Avanti Polar Lipids), 18: 0/18: 0-PA (Avanti Polar Lipids), 18: 0/20: 4-PA (Avanti Polar Lipids) and 18: 0/22: 6-PA (Avanti Polar Lipids) were used.

For the lipid binding assay, the combined dry lipid mixture was hydrated with HEPES buffer (25 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM dithiothreitol) at 95 ° C for 45 min and 15 min during hydration. Vortexed once for 1 min. The liposomes were then thawed for 5 cycles (3 min -196 ° C, 3 min 95 ° C). Liposomal formation [Non-Patent Document 21] was induced by sonication at 95 ° C using Branson Sonifier 450 (Branson Ultrasonics Corporation, Danbury, CT, USA).

COS-7 cells were transfected with pAcGFP-Praja-1. After incubation for 48 hours, cells were collected with HEPES buffer and the disrupted product (0.3 mg) was incubated with PA-containing liposomes or control liposomes at 4 ° C for 30 min. The sample was ultracentrifuged at 200000 g at 4 ° C for 1 h. The precipitate was dissolved in HEPES buffer. Then, using a CS100GXII centrifuge and an S100-AT4 angle rotor (Hitachi Koki, Tokyo, Japan), the sample was centrifuged at 200,000 g at 4 ° C for 1 h. The precipitate was dissolved in HEPES buffer. The supernatants and pellets were carried over to SDS / PAGE and analyzed by Western blotting using an anti-GFP antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Quantitative densitometry was performed using IMAGEJ software (National Institutes of Health, Bethesda, MD, USA). Since lipids form a bilayer, half of the actual concentration was considered [Non-Patent Document 21].

[E3 Ubiquitin-Protein Ligase Activity Assay]
Purified GST-Praja-1 fusion protein was added to PC liposomes, 18: 0/22: 6-PG liposomes, 18: 1/18: 1-PA liposomes, 18: 0/18: 0-PA liposomes, and 18 :. Incubated with 0/22: 6-PA liposomes. The E3 ligase self-ubiquitination assay kit (BML-UW0970; Enzo Life Sciences, NY, NY, USA) was used to analyze E3 ubiquitin-protein ligase activity. The assay was performed according to the manufacturer's protocol. Briefly, each reaction (25 μL final volume) is 1.25 μL 20 × E1, 1.25 μL 20 × E2, 2.5 μL 10 × Ub E3 ligase buffer, 2.5 μL 10 × ubiquitin, 0.25 μL 100 mM. It was made to contain DTT and 1.25 μL of Mg-ATP. The reaction mixture was incubated at 37 ° C for 1 h and SDS / PAGE using anti-ubiquitin antibody (included in the kit) and horseradish peroxyda-zeconjugated goat anti-mouse IgG (Bethyl Laboratories, Montgomery, AL, USA). / Analyzed by Western blotting.

[Western blotting]
Equal amounts of protein were loaded on a polyacrylamide gel. After separation, the protein is transferred onto a polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA) and at 4 ° C the following primary antibodies: GFP (Santa Cruz Biotechnology) and β-actin (Sigma-Aldrich, St. Louis, MO). , USA) overnight incubation. After washing, the membrane was incubated with a secondary antibody solution [Horseradish peroxydase-zeconjugated goat anti-mouse IgG (Bethyl Laboratories)] for 1 h at room temperature and then detected using enhanced chemiluminescence.

[Statistical analysis]
Data are expressed as mean ± SD, GRAPHPAD PRISM 8 (GraphPad Software) by Student's t-test for comparison of two groups, and by Tukey's post hoc test following one-way ANOVA for multiple comparisons. , San Diego, CA, USA) was used for analysis to determine significant differences. P <0.05 was considered significant.

〔result〕
[Analysis of PA and DG molecular species in the cerebral cortex of DGKδ-KO mice]
Applying the recently established LC-MS method [Non-Patent Document 16; Non-Patent Document 22], first, the amount of PA molecular species (DGK product) is higher in the brain of DGKδ-KO mice compared to wild-type mice (Non-Patent Document 16; It was examined whether it decreased in the cerebral cortex). The results are shown in Figure 1A. As shown in Figure 1A, the levels of almost all PA species were unaffected. However, 40: 6-PA was significantly reduced in the cerebral cortex of DGKδ-KO mice compared to wild-type mice (Fig. 1A). Moreover, LC-MS / MS analysis shows that 40: 6-PA contains docosahexaenoic acid (DHA, 22: 6, ω-3) at the sn-2 position 18: 0/22: 6-PA (94.6). %) Shown that it is mainly (Table 1 and Table 2).

Next, it was evaluated whether or not the DG molecular species (DGK substrate) was accumulated in the cerebral cortex of DGKδ-KO mice. The results are shown in Figure 1B. As shown in Figure 1B, only 40: 6-DG was significantly increased in the DGKδ-KO mouse cerebral cortex, and the levels of other DG species were not significantly affected (Figure 1B). ). Similar to 40: 6-PA (Tables 1 and 2), 40: 6-DG mainly consists of 18: 0/22: 6-DG (76.1%) (Tables 3 and 4), which is DGKδ. In the defective cerebral cortex, it means that 18: 0/22: 6-PA and 18: 0/22: 6-DG of PA and DG species containing the same fatty acid moiety are simultaneously decreased and increased, respectively. is doing. Therefore, these results strongly suggest that DGKδ selectively uses 18: 0/22: 6-DG as a substrate for making 18: 0/22: 6-PA in the mouse cerebral cortex.

Tables 1 and 2 below show the results of identifying acyl species of each PA molecular species in the mouse cerebral cortex using LC-MS / MS.

Tables 3 and 4 below show the results of identifying acyl species of each DG molecular species in the mouse cerebral cortex using LC-MS / MS.

[Praja-1 selectively interacts with 18: 0/22: 6-PA]
Since it was strongly suggested that DGKδ produces 18: 0/22: 6-PA in the mouse cerebral cortex (Fig. 1), the AcGFP-tagged Praja for 18: 0/22: 6-PA using the liposome sedimentation assay was used. The binding activity of -1 (expressed by COS-7 cells) was determined.

Praja-1 did not precipitate with mock control (without liposomes) (Fig. 2A). Liposomes containing PC (neutral phospholipid) alone as a background control showed moderate precipitation (~ 50%) of Praja-1 (FIGS. 2A, B). PS (acidic phospholipid) liposomes did not precipitate Praja-1 more strongly than PC-controlled liposomes. Moreover, 18: 0/22: 6-PG liposomes as acidic phospholipid controls containing the same fatty acid moieties as 18: 0/22: 6-PA also have almost the same Praja-1 binding activity as background controls. showed.
In contrast, nearly 100% of Praja-1 co-precipitated with 18: 0/22: 6-PA liposomes (Fig. 2A, B). However, AcGFP alone as a negative control was not detectable in precipitated 18: 0/22: 6-PA liposomes (Fig. 2A).

In addition, the Praja-1 binding activity of other PA species was also determined (Fig. 2A, B).
Unlike 18: 0/22: 6-PA liposomes, 16: 0/18: 1-PA liposomes, 18: 0/18: 0-PA liposomes, and 18: 1/18: 1-PA liposomes are PCs. Only the co-precipitation ability, which is almost the same as that of the control liposome, was shown (Fig. 2A, B). Liposomes containing 18: 0/20: 4-PA, also with polyunsaturated fatty acid (PUFA) arachidonic acid (20: 4, ω-6), have stronger Praja-1 binding activity than PC control liposomes. Was not shown (Fig. 2A, B).
Therefore, these results show that the following liposomes containing various phospholipids evaluated in the experiments of this example [PS liposome (acidic phospholipid control), 18: 0/22: 6-PG liposome (same fatty acid moiety). Contains acidic phospholipid controls), other PA species (16: 0/18: 1-PA, 18: 0/18: 0-PA, 18: 1/18: 1-PA, and 18: 0/20: 4-PA) -containing liposomes, as well as PC-only liposomes (neutral lipid / background control)], which means that they interacted most strongly and highly selectively with 18: 0/22: 6-PA liposomes. (Fig. 2).

[Praja-1 is selectively activated by 18: 0/22: 6-PA]
Then PC liposomes, 18: 0/22: 6-PG liposomes, 18: 0/18: 0-PA liposomes, 18: 1/18: 1-PA liposomes, or 18: 0/22: 6-PA liposomes. An E3 ligase self-ubiquitination assay was performed using GST-tagged Praja-1 expressed by COS-7 cells and purified by glutathione Sepharose affinity-chromatography.
As shown in FIG. 3, PC liposomes only conservatively activated the self-E3 ubiquitin-protein ligase activity of Praja-1. Moreover, it contains 18: 0/22: 6-PG, 18: 0/18: 0-PA, or 18: 1/18: 1-PA, which exhibit almost the same Praja-1 binding activity as that of PC liposomes. Liposomes (Fig. 2) were not able to enhance Praja-1 activity compared to PC alone (Fig. 3). Interestingly, as with Praja-1 binding activity (Figure 2), 18: 0/22: 6-PA liposomes are PC-only liposomes, 18: 0/22: 6-PG liposomes, 18: 0/18 :. Compared with 0-PA liposomes and 18: 1/18: 1-PA liposomes, Praja-1 self-ubiquitination was more strongly enhanced (Fig. 3). However, GST alone did not self-ubiquitinate even in the presence of 18: 0/22: 6-PA liposomes (Fig. 3).
Thus, these results mean that 18: 0/22: 6-PA selectively and highly enhances the E3 ubiquitin-protein ligase activity of Praja-1. Taken together, it is strongly suggested that 18: 0/22: 6-PA produced by DGKδ activates Praja-1, which ubiquitinates and destabilizes SERT (Fig. 4).

[Discussion]
The study of this example demonstrated for the first time that the E3 ubiquitin-protein ligase Praja-1, which ubiquitinates SERT, interacts selectively and strongly with 18: 0/22: 6-PA species (Fig. 2). ).
Praja-1 is a liposome containing phospholipids, such as PS liposomes (acidic phospholipid control), 18: 0/22: 6-PG liposomes (acidic phospholipid control containing the same fatty acid moiety), and other PA species. Of the liposomes containing (16: 0/18: 1-PA, 18: 0/18: 0-PA, 18: 1/18: 1-PA, and 18: 0/20: 4-PA), 18: 0 / 22: 6-The strongest interaction with PA liposomes (Fig. 2). Therefore, it can be said with some high possibility that the binding of 18: 0/22: 6-PA to Praja-1 is highly selective. Interestingly, Praja-1 was selectively activated by the 18: 0/22: 6-PA species (Fig. 3).

The results of the present inventors using the newly developed LC-MS method [Non-Patent Document 16, Non-Patent Document 20, Non-Patent Document 22] are obtained in the DGKδ-KO mouse cerebral cortex at 18: 0/22: 6 -It was demonstrated that PA and 18: 0/22: 6-DG were simultaneously decreasing and accumulating (Fig. 1). This result strongly suggests that DGKδ utilizes 18: 0/22: 6-DG to generate 18: 0/22: 6-PA. Interestingly, we found that 18: 0/22: 6-PA selectively binds to Praja-1 and enhances the E3 ubiquitin-protein ligase activity of purified Praja-1. (Figs. 2 and 3).

Moreover, we have previously stated that DGKδ interacts with SERTs in brain and neuronal cells, and with Praja-1 [Non-Patent Document 14], which ubiquitinates SERTs [Non-Patent Document 14]. It was clarified that Document 12, Non-Patent Document 13] and that Praja-1 induces SERT ubiquitination and degradation in a DGK activity-dependent manner [Non-Patent Document 13]. Therefore, the increase in 18: 0/22: 6-PA produced by DGKδ directly enhances Praja-1 E3 ubiquitin-protein ligase activity, resulting in a decrease in SERT protein stability in the brain. Is possible (Fig. 4). As shown in Figure 1, the amount of 18: 0/22: 6-PA is low. However, DGKδ interacts with Praja-1 and co-localizes with Praja-1 [Non-Patent Document 13]. Therefore, it can be said with high possibility that 18: 0/22: 6-PA is concentrated in the local region where DGKδ and Praja-1 are present and may affect the activity of Praja-1 in the proximal region.

In the brain and neurons, phosphatidylinositol (PI) 4,5-bisphosphate (which is made by PI turnover and consists primarily of 18: 0/20: 4-PI 4,5-diphosphate species) , Has been demonstrated to play an essential role in control [Non-Patent Document 23, Non-Patent Document 24]. However, in the study of this example, the results of the present inventors found that in addition to PI 4,5-diphosphate, the structurally simple lipid 18: 0/22: 6-PA was also Praja-1. It suggests that it plays an essential role in the central nervous system through the activation of E3 ubiquitin-protein ligase and the degradation of SERT.

Previously, the present inventors reported that DGKδ produces 16: 0-containing PA species and 16: 1-containing PA species in C2C12 myoblasts in response to high glucose stimulation [Non-Patent Document 22]. .. However, the study of this example strongly suggested that DGKδ produces 18: 0/22: 6-PA in the brain (Fig. 1). DGKδ has no obvious DG species selectivity in vitro [Non-Patent Document 22]. Therefore, it can be said with some high possibility that DGKδ utilizes different DG species pools in different cells and tissues. On the other hand, in the brains of DGKη-deficient mice, 36: 3 (18: 1/18: 2) -PA, 38: 3 (18: 0/20: 3) -PA, 40: 5 (18: 0/22:: 5)-PA, 40: 4 (18: 0/22: 4)-PA, and 40: 3 (18: 0/22: 3) -PA were significantly reduced [Non-Patent Document 18]. This profile is distinctly different from that of the brain of DGKδ-KO mice (Fig. 1).

In addition, in neuroblastoma cells, DGKζ is 30: 0 (14: 0/16: 0) -PA, 32: 0 (16: 0/16: 0) -PA, and 34: 0 (16: 0 / 18: 0)-PA was generated [Non-Patent Document 25]. Therefore, it is possible that different PA species are produced by DGK isozymes in different tissues and cells [Non-Patent Document 26]. Like DGKδ, DGKη and DGKζ showed no apparent DG species selectivity in vitro [Non-Patent Document 27, Non-Patent Document 28]. Therefore, they may also access different DG species pools. Since different DGK isozymes produce different PA species in different tissues and cells, it is speculated that these different PA species have their own unique targets. In fact, the present inventors have found that 18: 1/18: 1-PA selectively interacts with α-synuclein [Non-Patent Document 29, Non-Patent Document 30].
Moreover, muscular creatine kinases are selectively 16: 0/16: 0-PA, 16: 0/18: 1-PA, 18: 1/18: 1-PA, and 18: 0/18: 0- Bound to PA [Non-Patent Document 31], L-lactic dehydrogenase A selectively 16: 0/16: 0-PA, 18: 0/18: 0-PA, 18: 0/20: 4-PA, And 18: 0/22: 6-bonded to PA.

It is known that docosahexaenoic acid (DHA), which is mainly derived from fish oil, is essential for the development and function of the brain [Non-Patent Document 32, Non-Patent Document 33]. DHA increases membrane fluidity and has important anti-inflammatory, antioxidant, and anti-Alzheimer's disease roles [Non-Patent Document 32, Non-Patent Document 33]. Moreover, many reports have repeatedly demonstrated that ω-3 PUFAs such as DHA have anxiolytic effects [Non-Patent Document 34]. In the study of this example, DHA-containing PA selectively binds to and activates SERT (which is closely related to anxiety [Non-Patent Document 5]) E3 ubiquitin-protein ligase Praja-1. (Figs. 2 and 3). Therefore, it is possible that DHA incorporated into 18: 0/22: 6-PA, which enhances SERT degradation, could explain the anxiolytic effect of DHA. In addition to its free fatty acid form, it can be said with some high probability that DHA incorporated into PA also plays an important role in brain function.

A female patient with DGKδ gene disruption was recently investigated [Non-Patent Document 35]. Patients showed seizures and experienced self-stimulating behaviors such as pulling hair, tapping their feet, and fluttering their hands [Non-Patent Document 35]. These are observed in OCD and OC spectrum disorders [Non-Patent Document 36, Non-Patent Document 37]. Therefore, abnormalities in DGKδ cause OCD-like psychiatric disorders in humans, meaning that DGKδ is a key enzyme for maintaining mental health. However, the regulatory mechanism of DGKδ activity / expression remains unknown. In the present inventors, the protein kinase C (PKC) α (which regulates SERT activity [Non-Patent Documents 38-40]) interacts with DGKδ [Non-Patent Document 41], and phosphorus is used. It was reported that it oxidizes [Non-Patent Document 42] and controls the localization of its plasma membrane [Non-Patent Document 42]. Thus, PKCα can regulate the intracellular localization and activity of DGKδ in the brain. Moreover, formal esters increased the expression level of DGKδ [Non-Patent Document 43], suggesting that conventional PKC and Nobel PKC, including PKCα, regulate their expression.

SERT inhibitors (SSRIs) have been used to treat depression or major depressive order and OCD [Non-Patent Documents 2-4], with excess SERT protein / activity in these. It means that it is closely related to the onset of the disorder. Moreover, SERT is associated with anxiety and autism [Non-Patent Document 5]. Hyperactivity of 5-HT is associated with negative signs of schizophrenia [Non-Patent Document 44]. In this regard, treatment with a potent 5-HT _2A receptor antagonist in combination with a weak dopamine D2 receptor antagonist is the first-line treatment for patients suffering from schizophrenia (negative symptoms) [Non-Patent Document 44]. ..
Therefore, the new discoveries of the present inventors provide new findings on the molecular mechanism of the onset of these mental disorders and the development of therapeutic strategies.
For example, drug compounds that mimic 18: 0/22: 6-PA can be therapeutic agents for obsessive-compulsive disorder, OCD, schizophrenia (positive symptoms), and autism. In contrast, DGKδ inhibitors can potentially be used to treat the negative sign of schizophrenia.
This application claims benefits under FEBS Letters 2020 Jun; 594 (11): 1787-1796, published March 5, 2020, and the disclosure in this journal is hereby by reference in its entirety. Incorporated into the book.

According to the present invention, active substances and active compounds can be identified by using the interaction activity with Praja-1, the binding activity with Praja-1, the enhancing or promoting activity of Praja-1 as an index, and further. Drugs with identified activity, including DHA or derivatives thereof, docosahexaenoyl group-containing phosphatidic acid or derivatives thereof, drug compounds mimic 18: 0/22: 6-PA, or salts thereof, are SERTs. Activity inhibitor, Praja-1 activity enhancer that selectively binds to and enhances its activity, or obsessive-compulsive disorder (OCD), major depressive disorder, autism, schizophrenia, It can be used as a prophylactic / therapeutic agent for Alzheimer-type dementia and pathological symptoms selected from the group consisting of anxiety, depression, obsessive-compulsive, and impulsive psychiatric disorders, and is highly useful.
It is clear that the present invention can be carried out other than those specifically described in the above description and examples. In view of the above teachings, many modifications and variations of the invention are possible, and thus they are also within the scope of the appended claims.

Claims (8)

1. Docosahexaenoic acid (DHA) or its derivatives, docosahexaenoyl group-containing phosphatidic acid or its derivatives, and 1-stearoyl-2-docosahexaenoyl (18: 0/22: 6)-phosphatidic acid (PA) [18: 0/22: 6-PA] is a drug compound that mimics], or a SERT activity inhibitor whose active ingredient is selected from the group consisting of salts thereof, selectively binds to Praja-1 and its activity. Praja-1 activity enhancer, or obsessive-compulsive disorder (OCD), obsessive-compulsive disorder, autism, schizophrenia, Alzheimer-type dementia, and anxiety, depression, obsessive-compulsive, and impulsive psychiatry. A prophylactic / therapeutic agent for pathological symptoms selected from the group consisting of medical disorders.
2. The agent according to claim 1, which comprises DHA or 18: 0/22: 6-PA as an active ingredient.
3. The active ingredient is selected from the group consisting of DHA or its derivatives, docosahexaenoyl group-containing phosphatidic acid or its derivatives, and chemical compounds that mimic 18: 0/22: 6-PA, or salts thereof. , Obsessive-compulsive disorder (OCD), major depressive disorder, autism, schizophrenia, Alzheimer-type dementia, and anxiety, depression, obsessive-compulsive, and impulsive psychiatric disorders. The agent according to claim 1, wherein the agent is a preventive / therapeutic agent for pathological symptoms.
4. Docosahexaenoic acid (DHA) or its derivatives, docosahexaenoyl group-containing phosphatidic acid or its derivatives, and 1-stearoyl-2-docosahexaenoyl (18: 0/22: 6)-phosphatidic acid (PA) [18: 0/22: 6-PA] as the active ingredient selected from the group consisting of drug compounds mimic or salts thereof, obsessive-compulsive disorder (OCD), major depressive disorder, autism, schizophrenia Foods having prophylactic / therapeutic effects selected from the group consisting of illness, Alzheimer-type dementia, and anxiety, depression, obsessive-compulsive, and impulsive psychiatric disorders.
5. The drug candidate substance has the activity by measuring the activity selected from the group consisting of the activity of interacting with Praja-1, the activity of binding to Praja-1, and the activity of enhancing or promoting the activity of Praja-1. A screening method comprising selecting and / or determining one.
6. The screening method according to claim 5, wherein the reaction of the drug candidate substance to the fusion protein AcGFP-Praja-1 or GST-Praja-1 is analyzed.
7. Diseases selected from the group consisting of obsessive-compulsive disorder (OCD), major depressive disorder, autism, schizophrenia, Alzheimer-type dementia, and anxiety, depression, obsessive-compulsive, and impulsive psychiatric disorders. The screening method according to claim 5, wherein a substance for preventing / treating a depressive symptom is selected and / or determined.
8. For drug candidate substances, the activity selected from the group consisting of the activity of interacting with Praja-1, the activity of binding to Praja-1, and the activity of enhancing or promoting the activity of Praja-1 was measured, and those having the activity were measured. A screening kit comprising a Praja-1 protein or a tagged Praja-1 protein for selection and / or determination.

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