KR101814751B1 - Composition for preventing or treating of cigarette-induced dependence comprising metabotropic glutamate receptor carboxyl-terminus motif - Google Patents
Composition for preventing or treating of cigarette-induced dependence comprising metabotropic glutamate receptor carboxyl-terminus motif Download PDFInfo
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- KR101814751B1 KR101814751B1 KR1020160002133A KR20160002133A KR101814751B1 KR 101814751 B1 KR101814751 B1 KR 101814751B1 KR 1020160002133 A KR1020160002133 A KR 1020160002133A KR 20160002133 A KR20160002133 A KR 20160002133A KR 101814751 B1 KR101814751 B1 KR 101814751B1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
Abstract
The present invention relates to a composition for inhibiting the binding between mGluR1a and JNK1 (c-Jun N-terminal kinase 1) comprising a motif derived from the carboxyl terminal of mGluR1a (metabotropic glutamate receptor 1a), a composition for preventing or treating tobacco poisoning, And a screening method for preventing or treating an addiction. The composition according to the present invention contains a motif derived from the carboxyl terminal of mGluR1a and is characterized by being capable of inhibiting the binding of JNK1 to the carboxyl terminal of mGluR1a and thus is useful for the prevention or treatment of tobacco poisoning or for the screening of a preventive or therapeutic agent for tobacco poisoning Lt; / RTI >
Description
The present invention relates to a composition for inhibiting the binding between mGluR1a and JNK1 (c-Jun N-terminal kinase 1) comprising a motif derived from the carboxyl terminal of mGluR1a (metabotropic glutamate receptor 1a), a composition for preventing or treating tobacco poisoning, And a screening method for preventing or treating an addiction.
Tobacco is the most commonly abused addictive product with alcohol. The number of deaths due to tobacco is around 5 million worldwide, and the World Health Organization (WHO) expects the number to double nearly by 2025-2030. Smoking also has a life-threatening impact on the world, equivalent to 10% of adult deaths. For this reason, WHO prescribes tobacco as a drug. In particular, nicotine, a component of tobacco, has a strong dependency, such as heroin or cocaine, so that people who are addicted to tobacco are classified as medically addicted drug addicts.
As a result of studies on the risk of smoking, many people are attempting to quit smoking, and treatment methods for smoking cessation have increased, but it is known that smoking cessation in the long term is less than 10%. The main reason for the failure to quit smoking, despite the fact that smoking is harmful to health, is due to the strong dependence of nicotine-containing tobacco components, which is primarily due to tolerance and withdrawal symptoms.
The smoke contained in the cigarette smoke and smoke contained toxic, mutagenic, and carcinogenic substances. As mentioned above, tobacco poisoning is a threat to the public health so that it is classified as a disease even in the international disease classification. According to the WHO International Agency for Research on Cancer (IARC), tobacco contains 62 carcinogens, among which 15 carcinogens have been identified for carcinogenicity in humans. It is known that 30% of the causes of cancer, the number one cause of death in Korea, are due to smoking. Therefore, the cost of treatment for diseases caused by smoking and the loss of productivity are caused.
To assess nicotine dependence among tobacco components, smoking habits, Fagerstrom test for nicotine dependence, blood nicotine measurement, reason for smoking, and assessment of associated mental disorders are used. In particular, the Nicotine Dependency Assessment in the quit smoking clinic uses the Fagerstrom Test. However, considering the neurochemical changes occurring at the cerebral reward center, there are limitations in evaluating the dependence of nicotine by self-diagnosis through 6 questionnaires. Therefore, development of a neurochemical evaluation method that can evaluate the dependence of nicotine-containing tobacco product components is required.
The dependence on tobacco components is generally similar to the pattern of drug addiction. The normal person's emotions are controlled by maintaining a 'hedonic state' (negative hedonic state: euphoria) such as pleasure and a negative hedonic state (dysphoria) such as anxiety. Such homeostasis of emotion is closely related to changes in dopamine and glutamate concentrations. Craving for a drug starts with a '-' state in which dopamine concentration decreases over time, which is known to increase glutamate. Thus, dependence can be defined as a neurochemical phenomenon formed by changes in dopamine and glutamate secretion in the cerebral compensation center.
Like other addictive drugs, nicotine also causes tolerance and withdrawal with pleasure-reward behavior. Nicotine dependence and reinforcement of addiction by smoking are closely related to the same mesolimbic dopamine pathway that is activated by the addictive drug. Dopaminergic neurons in the ventral tegmental area (VTA) are located in the ascending and descending order of the caudate-putamen (CP), the nucleus accubens (NAc), the prefrontal cortex (PFC), and the amygdala AMG) secretes dopamine. Nicotine, which enters the body through smoking, passes through the brain-blood barrier and affects dopamine secretion in this pathway.
As receptors involved in nicotine poisoning, homologous α7 nicotinic aceticholine receptors (nAChRs) and heterogeneous α4β2 nicotinic acetylcholine receptors (heteromeric α4β2 nAChRs) are known. The a7 and a4beta2 receptors are predominantly present at the end of the glutamate and GABA neurons of the striatum, respectively. Smoking temporarily releases glutamate and GABA neurotransmitters, but by continuous smoking, the secretion of glutamate neurotransmitters increases and the secretion of GABA is reduced in the ventral scalp. This increases the activity of dopaminergic neurons in the dorsal root ganglia and increases the dopamine secretion at the dopaminergic neuron terminals, resulting in a greater pleasure from smoking. In addition, since the increase of dopamine secretion increases the glutamate secretion through the basal ganglia circuit, the dependence on smoking is formed by the increase of dopamine and glutamate secretion in cerebral compensator.
On the other hand, JNK (c-Jun N- terminal kinase) , is activated as a kinase which is activated by the stress through the stress Ca 2 + signal transmission system by an external stimulus, such as smoking, free radicals, UV light. Activated JNK is known to play an important role in life events such as cell growth, differentiation and death by regulating the phosphorylation of c-Jun, ATF2, and Elk-1, which are the sub-proteins of the signal transduction system. Tobacco dependence and mGluR There is no known association.
As described above, the dependence of tobacco is related to neurochemical changes such as the release of dopamine and glutamate, which are major neurotransmitters, centering on cerebral compensation centers, and various posttranslational modifications (PTM) Is a major cause, so there is a limit to controlling dependence at the same gene level as existing microarrays. Therefore, a new method to control the dependence of tobacco components should be developed to reduce the harmful effects of smoking.
Accordingly, the present inventors have continued research on substances capable of controlling tobacco dependence, and as a result, they have identified the mechanism of phosphorylation of metabotropic glutamate receptors, and confirmed that they can control tobacco dependency through this, thereby completing the present invention.
It is an object of the present invention to provide a composition for inhibiting the binding between mGluR1a and JNK1 (c-Jun N-terminal kinase 1) comprising a carboxyl terminus-derived motif of mGluR1a (metabotropic glutamate receptor 1a).
It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating tobacco poisoning which comprises a carboxyl end-derived motif of mGluR1a (metabotropic glutamate receptor 1a).
It is still another object of the present invention to provide a screening method for preventing or treating tobacco poisoning.
In order to achieve the above object, the present invention provides a composition for inhibiting binding between mGluR1a and JNK1 (c-Jun N-terminal kinase 1) comprising a carboxyl end-derived motif of mGluR1a (metabotropic glutamate receptor 1a).
The present invention also provides a binding inhibitor which inhibits binding of mGluR1a and JNK1 comprising the composition.
The present invention also provides a pharmaceutical composition for preventing or treating tobacco poisoning, comprising a carboxyl-derived motif of mGluR1a (metabotropic glutamate receptor 1a).
The present invention also provides a screening method for tobacco poisoning prevention or treatment agent comprising the step of confirming whether the test drug inhibits the binding of JNK1 with the carboxyl terminal motif of mGluR1a.
The composition according to the present invention contains a motif derived from the carboxyl terminal of mGluR1a and is characterized by being capable of inhibiting the binding of JNK1 to the carboxyl terminal of mGluR1a and thus is useful for the prevention or treatment of tobacco poisoning or for the screening of a preventive or therapeutic agent for tobacco poisoning Lt; / RTI >
Figure 1 shows the GST-fusion protein for the five regions of mouse mGluR1a.
Fig. 2 is a diagram showing mGluR1a-CT2 region phosphorylated by JNK1.
FIG. 3 is a graph showing the phosphorylation of the mGluR1a-CT2 region according to the treatment with the inactive JNK1.
FIG. 4 is a graph showing the phosphorylation of the mGluR1a-CT2 region according to the treatment of [? 32 P] ATP.
FIG. 5 is a graph showing phosphorylation of the mGluR1a-CT2 region according to the treatment with CIP.
Figure 6 shows a potential phosphorylation region of the mGluR1a-CT2 region and a mutant production design thereof.
FIG. 7 is a graph showing the results of phosphoamino acids analysis for the positions at which phosphorylation occurs in the amino acids present in the mGluR1a-CT2 region.
FIG. 8 is a graph showing the results of in vitro kinase assays for the positions where phosphorylation occurs in amino acids present in the mGluR1a-CT2 region.
FIG. 9 shows the results of confirming the region of mGluR1a binding to JNK1 through an in vtiro binding assay.
10 is a schematic diagram of a GST-fusion protein production design for the mGluR1a-CT region of a mouse.
FIG. 11 shows the results of confirming the details of mGluR1a binding to JNK1 through an in vtiro binding assay.
12 is a diagram showing a binding-inhibiting peptide synthesized to identify a motif binding to JNK1 in the mGluR1a-CT2c region.
FIG. 13 is a graph showing the binding affinity of mGluR1a-JNK1 after in vitro binding assay after treatment with a binding inhibitory peptide.
14 shows the production of a cell membrane permeable peptide.
15 shows an experimental design to measure changes in helper activity by binding-inhibiting peptides in mice.
Fig. 16 is a graph showing the results of measurement of changes in helper activity by binding-inhibiting peptides in mice. Fig.
Hereinafter, the present invention will be described in detail.
The present invention provides a composition for inhibiting binding between mGluR1a and JNK1 (c-Jun N-terminal kinase 1) comprising a carboxyl end-derived motif of mGluR1a (metabotropic glutamate receptor 1a).
The above-mentioned "mGluR1a (metabotropic glutamate receptor 1a)" is a cell membrane receptor that senses glutamate, which is a typical excitatory neurotransmitter, and induces an intracellular signal.
The composition of the present invention may include a peptide containing a carboxyl-terminal-derived motif of mGluR1a (metabotropic glutamate receptor 1a), and a peptide comprising a carboxyl-terminal-derived motif of mGluR1a (metabotropic glutamate receptor 1a) is a peptide of mGluR1a-JNK1 Including amino acid-based molecules that may interfere with binding, including, but not limited to, peptide end-length motifs, regardless of peptide length, modification, electrical properties, and the like. The term " peptide " refers to a linear molecule formed by peptide bonds and amino acid residues joined together.
In the present invention, the carboxyl terminus-derived motif may include an mGluR1a-CT2 sequence, preferably an amino acid sequence of YASVILRDYKQSS represented by SEQ ID NO: 1.
The composition of the present invention contains a peptide containing the amino acid sequence shown in SEQ ID NO: 2 as an active ingredient.
The peptides of the present invention can be produced according to chemical synthesis methods known in the art, and in one embodiment of the present invention, a motif (YASVILRDYKQSS) essential for mGluR1a-JNK1 binding and a transactivator of transcription (TAT) The sequence was fused to produce a peptide having cell membrane permeability. Since the peptide itself can not pass through the cell membrane, the TAT sequence that degrades the cell membrane is fused to prevent the peptide of the present invention from interfering with mGluR1a-JNK1 binding in the neuron.
In a specific embodiment of the present invention, the fact that mGluR1a is phosphorylated by JNK1, the region of mGluR1a phosphorylated by JNK1, the region of mGluR1a to which JNK1 binds, and the motif essential for JNK1-mGluR1a binding have been identified. In addition, it was finally confirmed that the activity of the tobacco-dependent behavioral index changes with the interaction of mGluR1a-JNK1 using the motif-containing peptide.
The present invention also relates to a composition for inhibiting the binding of mGluR1a to JNK1 comprising a composition for inhibiting binding of mGluR1a and JNK1 (c-Jun N-terminal kinase 1) comprising carboxyl end-derived motifs of mGluR1a (metabotropic glutamate receptor 1a) Lt; / RTI >
In the present invention, "JNK1 (c-Jun N- terminal kinase 1)" it is activated by the stress Ca 2 + signal transmission system by an external stimulus, such as a kinase which is activated by a stress such as smoking, free radicals, UV cell It is known to play an important role in life phenomena such as growth, differentiation and death.
The binding inhibitor of the present invention is characterized by being able to bind to mGluR1a and inhibit the interaction and binding of mGluR1a and JNK1.
The present invention also provides a pharmaceutical composition for preventing or treating tobacco poisoning, comprising a carboxyl-derived motif of mGluR1a (metabotropic glutamate receptor 1a).
The term " tobacco addiction " in the present invention refers collectively to addiction caused by inhaling substances with strong dependence, and is a kind of tobacco dependence, preferably including "nicotine addiction"
The term " prophylactic " in the present invention means inhibiting the occurrence of a disease or a disease in an individual who has never been diagnosed as having a tobacco addiction or tobacco dependence, but tends to be susceptible to such disease or disease. The term "treatment" as used herein also refers to inhibiting tobacco addiction or tobacco-dependent development, alleviating the disease or disease, and eliminating the disease or disease.
The peptide comprising the carboxyl end-derived motif of mGluR1a of the present invention may be used in combination with a known tobacco addiction or tobacco-dependence preventive or therapeutic agent, but may be used alone or in combination with other agents to effectively prevent or prevent tobacco addiction or tobacco dependence, Can be treated.
The pharmaceutical composition for preventing or treating tobacco poisoning of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers in the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, But are not limited to, cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).
The pharmaceutical composition of the present invention may be administered by intravenous infusion, intramuscular injection, local injection, intracerebral infusion, cerebral infarction, spinal cord injection, subcutaneous infusion, intraperitoneal injection, transdermal administration, May be administered by injection. The striatum is a part of neurons that form part of the cerebral basal ganglia, which is associated with poisoning.
The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, the administration method, the age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can easily determine and prescribe dosages effective for the desired treatment. The daily dose of the pharmaceutical composition of the present invention is preferably 0.001-1000 mg / kg.
The pharmaceutical composition of the present invention may be formulated into a unit dosage form by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.
According to a preferred embodiment of the present invention, the animal to which the composition of the present invention can be applied is not particularly limited and preferably a mammal, more preferably a human, a mouse, a rat, a rabbit, a monkey, a pig, Cattle, sheep, nutrition, dogs or cats, and even more preferably human or mouse.
The present invention also provides a food composition for preventing or ameliorating tobacco poisoning comprising a carboxyl terminal-derived motif of mGluR1a (metabotropic glutamate receptor 1a).
When the composition of the present invention is prepared with a food composition, all forms of the functional food, nutritional supplement, health food or food additives including the composition And may be prepared in various forms according to conventional methods known in the art. For example, as a health food, the peptide of the present invention may be prepared in the form of tea, juice, and drink for drinking, granulated, encapsulated, and powdered. However, It is not.
The health functional food of the present invention may contain additional additives, and the kind thereof is not particularly limited. Preferable examples of the functional food that can be added include drinks (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruits, bottled, jam and marmalade), fish, meat and processed foods (Such as butter and cheese), edible vegetable oil, margarine, vegetable protein (such as corn oil), breads and noodles (such as udon, buckwheat noodles, ramen noodles, spaghetti and macaroni), juice, various drinks, cookies, , Retort food, frozen food, various kinds of seasoning (for example, soybean paste, soy sauce, sauce, etc.) by adding the peptide of the present invention.
In addition, the present invention provides a screening method for a tobacco addiction prevention or therapeutic agent, comprising the step of confirming whether the test drug inhibits the binding of JNK1 with a carboxyl-derived motif of mGluR1a.
Terms not otherwise defined herein have meanings as commonly used in the art to which the present invention belongs.
Hereinafter, the present invention will be described in detail with reference to Examples and Preparation Examples. However, the following examples and preparative examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples and preparative examples.
Example 1: Identification of the region of mGluR1a phosphorylated by JNK1
mGluR1a has three intracellular loops (IL) and one long carboxyl terminal (C-terminus, CT). The carboxyl terminus was divided into CT1 and CT2 and mGluR1a was divided into five regions (mGluR1a-IL1, mGluR1a-IL2, mGluR1a-IL3, mGluR1a-CT1 and mGluR1a-CT2) and phosphorylated by JNK1 for each region .
Specifically, for the production of GST-fusion protein, mouse cDNA corresponding to five regions as shown in Fig. 1 was cloned into pGEX4T-1 (Amersham) plasmid, and each plasmid was inserted into E. coli BL21, , And the GST-fusion protein was extracted and purified.
Phosphorylation was measured by in vitro kinase assay for each purified GST-fusion protein. The fusion protein GST- reaction buffer [25 mM MOPS, 12.5 mM β-glycerol-phosphate, 25
As shown in Fig. 2, it was confirmed that the phosphorylation by JNK1 was specific to the mGluR1a-CT2 region.
Additional experiments were conducted to clarify these results. First, the in vitro kinase assay confirmed the phosphorylation in the mGluR1a-CT2 region when the inactive JNK1 or active JNK1 was treated, and the results are shown in FIG.
As shown in Fig. 3, mGluR1a-CT2 region was not phosphorylated when inactive JNK1 was treated, while mGluR1a-CT2 region was phosphorylated when active JNK1 was treated. Therefore, mGluR1a is phosphorylated by JNK1.
Secondly, phosphorylation of mGluR1a-CT2 region was confirmed by the presence or absence of [? 32 P] ATP in the presence of JNK1, and the results are shown in FIG.
As shown in FIG. 4, it was confirmed that JNK1-induced phosphorylation of mGluR1a-CT2 region specifically occurred by hydrolysis of ATP only when [? 32 P] ATP was present.
Thirdly, in the presence of JNK1, phosphorylation of the mGluR1a-CT2 region was confirmed in the presence or absence of CIP (calf intestinal alkaline phosphatase) or [? 32 P] ATP, which is known as phosphatase releasing phosphate group, Respectively.
As shown in Fig. 5, since the phosphate group fell off during the CIP treatment, it was confirmed that the reaction between JNK1 and mGluR1a was a phosphorylation reaction.
As a result of synthesizing the results shown in Figs. 2 to 5, phosphorylation was induced by JNK1 in the CT2 region of mGluR1a, and this phosphorylation was confirmed to be specific to [? 32 P] ATP.
Example 2: Identification of the detail region of mGluR1a phosphorylated by JNK1
A total of eight potential phosphorylation regions were screened using www.scansite.mit.edu (Obenauer et al, 2003) to pinpoint which regions of the mGluR1a-CT2 region were phosphorylated by JNK1. As shown in Fig. 6, serine / threonine, which is predicted to be phosphorylated based on the cDNA of the corresponding region, was replaced with alanine (Site-directed mutagenesis) for the selected eight potential phosphorylation regions. Then, codon optimization was performed to induce optimal protein expression in E. coli BL21. After confirming that it was substituted with alanine by DNA sequencing, the cDNA fragment was cloned into pGEX4T-1 vector using BamHI and EcoRI restriction enzymes to obtain GST - fusion proteins were prepared (M1 to M5).
FIG. 7 shows the result of phosphoamino acids analysis of the GST-fusion protein. As shown in FIG. 7, it was confirmed that phosphorylation occurred in serine in the amino acid sequence existing in the mGluR1a-CT2 region.
The result of in vitro kinase assay using the GST-fusion protein is shown in FIG. 8. As shown in FIG. 8, the serine 1098 in mGluR1a-CT2 (S in the
Example 3: Identification of mGluR1a binding to JNK1
First, in vitro binding assay was performed to confirm whether JNK1 binds to mGluR1a.
Specifically, JNK1 (active or inactive form) and 5 GST-fusion proteins were added to binding buffer (200 mM NaCl, 0.2% Triton X-100, 0.1 mg / ml BSA, 50 mM Tris , And reacted for 2 to 3 hours at 4 ° C. Then, the GST-fusion protein complex was precipitated using 10% Glutathione-Sepharose ™ beads and Western blotting was carried out using an antibody specifically recognizing JNK1. 9.
As shown in Fig. 9, it was confirmed that the non-active JNK1 did not bind to mGluR1a, and JNK1 was able to bind mGluR1a-CT1 and mGluR1a-CT2 among the five regions of mGluR1a and strongly binds to mGluR1a-CT2 region .
Example 4: Identification of the detailed region of mGluR1a binding to JNK1
According to the contents confirmed in Example 3, detailed regions binding to JNK1 in mGluR1a-CT1 and mGluR1a-CT2 regions were confirmed. Specifically, the mGluR1a-CT1 region is divided into three regions (mGluR1a-CT1a, mGluR1a-CT1c and mGluR1a-CT1c), and mGluR1a-CT2 region is divided into three regions (mGluR1a-CT2a, GST-fusion protein was prepared for each region as shown in Fig.
The in vitro binding assay of the prepared GST fusion protein was carried out as in Example 3 above, and the results are shown in FIG. As shown in Fig. 11, it was confirmed that JNK1 binds to the mGluR1a-CT1a region and the mGluR1a-CT2a and mGluR1a-CT2c regions.
Through the above Examples 1 to 3, it was confirmed that JNK1 binds to mGluR1a and regulates phosphorylation of mGluR1a. At this time, it was confirmed that the region where JNK1 binds to mGluR1a and the region where mGluR1a phosphorylates are different from each other.
Example 5: Identification of motif sequences of mGluR1a essential for JNK1-mGluR1a binding
In order to identify motifs essential for JNK1 binding in the mGluR1a-CT1a and mGluR1a-CT2c domains as detailed in Example 4 above, binding inhibitory peptides including D-motif (Zhou et al., 2006) (P1, P2) were synthesized and shown in FIG.
The result of pretreatment of the synthesized binding inhibition peptide and in vitro binding assay is shown in FIG.
As shown in Fig. 13, it was confirmed that binding of JNK1 to mGluR1a-CT2 was reduced when P2 peptide in the peptide (P1) for JNK1 and mGluR1a-CT1a and the peptide (P2) for mGluR1a-CT2c was treated.
Therefore, it was confirmed that the peptide containing mGluR1a motif essential for JNK1-mGluR1a binding is P2, which is a peptide for mGluR1a-CT2c, and it can be seen that the peptide interferes with binding of mGluR1a and JNK1.
Example 6: Determination of the effect of mGluR1a phosphorylation on helical activity on JNK1-mGluR1a binding in striatum
The effect of the binding-inhibiting peptide of JNK1-mGluR1a identified in Example 5 above was confirmed in the mouse. Specifically, a transactivator of transcription (YGRKKRRQRRR) derived from an HIV virus that facilitates cell permeability was fused to the N-terminus of the binding-inhibiting peptide containing the motif (YASVILRDYKQSS) in mGluR1a-CT2 identified in Example 5 A cell-penetrating peptide was prepared and is shown in Fig.
FIG. 15 shows an experimental design for quantitatively analyzing the repulsive activity, which is a dependency index of the tobacco component using the peptide shown in FIG. 14, and the experiment was conducted accordingly.
Specifically, 2 nmol / 2 μl of the binding-inhibiting peptide shown in FIG. 14 was injected into mouse striatum, and then the experiment was conducted with two international tobacco products (product tobacco A and B) distributed to the international standard tobacco (3R4F) . Each of the tobacco extracts was repeatedly administered to the peptide-injected mice, and tracking with X-ray, Y-axis, and Z-axis beam breaking using the Opto-
As shown in FIG. 16, it was confirmed that the peptide injected with the peptide of the present invention showed a significant decrease in walkability compared to the control group.
That is, the JNK1-mGluR1a binding-inhibiting peptide of the present invention inhibits binding of JNK1 to mGluR1a, thereby regulating phosphorylation of mGluR1a, and reduces repetitive administration of the tobacco extract to increase the mouse's repulsive activity It can be used as a preventive or therapeutic method for inhibiting tobacco dependence.
Although the present invention has been described in terms of the preferred embodiments mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also to be understood that the appended claims are intended to cover such modifications and changes as fall within the scope of the invention.
Formulation example 1. Preparation of pharmaceutical compositions
1-1. Sanje Produce
20 mg of the peptide of the present invention
The above components are mixed and filled in airtight bags to prepare powders.
1-2. Manufacture of tablets
10 mg of the peptide of the present invention
After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.
1-3. Preparation of capsules
10 mg of the peptide of the present invention
Lactose 14.8 mg
Magnesium stearate 0.2 mg
The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.
1-4. Injection preparation
10 mg of the peptide of the present invention
180 mg mannitol
Sterile sterilized water for injection 2974 mg
Na 2 HPO 4 2H 2 O 26 mg
(2 ml) per 1 ampoule in accordance with the usual injection preparation method.
1-5. Liquid Produce
20 mg of the peptide of the present invention
10 g per isomer
5 g mannitol
Purified water quantity
Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.
Formulation example 2. Preparation of food composition
2-1. Manufacture of health food
20 mg of the peptide of the present invention
Vitamin mixture quantity
Vitamin A acetate 70 μg
Vitamin E 1.0 mg
Vitamin B1 0.13 mg
0.15 mg of vitamin B2
Vitamin B6 0.5 mg
Vitamin B12 0.2 μg
Biotin 10 μg
Nicotinic acid amide 1.7 mg
Calcium pantothenate 0.5 mg
Mineral mixture quantity
1.75 mg of ferrous sulfate
0.82 mg of zinc oxide
Magnesium carbonate 25.3 mg
Potassium monophosphate 15 mg
Secondary calcium phosphate 55 mg
Potassium citrate 90 mg
Magnesium chloride 24.8 mg
Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.
2-2. Manufacture of health drinks
100 mg of the peptide of the present invention
Vitamin C 15 g
Vitamin E (powder) 100 g
19.75 g of ferrous lactate
3.5 g of zinc oxide
Nicotinic acid amide 3.5 g
Vitamin A 0.2 g
Vitamin B1 0.25 g
Vitamin B2 0.3 g
Water quantification
The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 L container, It is used in the production of the health beverage composition of the invention.
Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.
<110> KOREA FOOD & DRUG ADMINISTRATION Pusan National University Industry-University Cooperation Foundation <120> Composition for preventing or treating of cigarette-induced dependence comprising metabotropic glutamate receptor carboxyl-terminus motif <130> 1.256P <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 13 <212> PRT <213> metabotropic glutamate receptor carboxyl-terminus motif <400> 1 Tyr Ala Ser Val Ile Leu Arg Asp Tyr Lys Gln Ser Ser 1 5 10 <210> 2 <211> 199 <212> PRT Metabotropic glutamate receptor carboxyl-terminus sequence <400> 2 Pro Leu Phe Leu Ala Asp Ser Val Ile Pro Lys Gly Leu Pro Pro Pro 1 5 10 15 Leu Pro Gln Gln Gln Pro Gln Gln Pro Pro Gln Gln Pro Pro Gln 20 25 30 Gln Pro Lys Ser Leu Met Asp Gln Leu Gln Gly Val Val Thr Asn Phe 35 40 45 Gly Ser Gly Ile Pro Asp Phe His Ala Val Leu Ala Gly Pro Gly Thr 50 55 60 Pro Gly Asn Ser Leu Arg Ser Leu Tyr Pro Pro Pro Pro Pro Pro Gln 65 70 75 80 His Leu Gln Met Leu Pro Leu His Leu Ser Thr Phe Gln Glu Glu Ser 85 90 95 Ile Ser Pro Pro Gly Glu Asp Ile Asp Asp Asp Ser Glu Arg Phe Lys 100 105 110 Leu Leu Gln Glu Phe Val Tyr Glu Arg Glu Gly Asn Thr Glu Glu Asp 115 120 125 Glu Leu Glu Glu Glu Glu Asp Leu Pro Thr Ala Ser Lys Leu Thr Pro 130 135 140 Glu Asp Ser Pro Ala Leu Thr Pro Pro Ser Pro Phe Arg Asp Ser Val 145 150 155 160 Ala Ser Gly Ser Ser Val Ser Ser Ser Ser Val Ser Le 165 170 175 Cys Thr Pro Pro Asn Val Thr Tyr Ala Ser Val Ile Leu Arg Asp Tyr 180 185 190 Lys Gln Ser Ser Ser Thr Leu 195
Claims (8)
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| US20050019841A1 (en) | 1999-05-14 | 2005-01-27 | Arbor Vita Corporation | Modulation of signaling pathways |
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| US20050019841A1 (en) | 1999-05-14 | 2005-01-27 | Arbor Vita Corporation | Modulation of signaling pathways |
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| Wiley Interdisciplinary Reviews: Membrane Transport and Signaling. Vol. 1, No. 3, pp. 281-295 (2012) |
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