KR102425014B1 - Composition for preventing or treating neurological disorders induced by synaptic formation dysfunction comprising binding modulator of Calcyon and Hevin - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of neurological diseases caused by synaptic formation disorders comprising a binding modulator of Calcyon and Hevin as an active ingredient. , Calcyon and Hevin's mutual binding inhibitor or mutual binding promoter can inhibit the formation of abnormal synapses or promote the formation of synapses. It can be usefully used as a method, a composition for regulating synapse formation, and the like.

Description

Composition for preventing or treating neurological disorders induced by synaptic formation dysfunction comprising binding modulator of Calcyon and Hevin

The present invention relates to a composition for the prevention or treatment of neurological diseases caused by synapse formation disorders comprising a binding modulator of Calcyon and Hevin as an active ingredient.

Calcyon is a single transmembrane protein expressed in the central nervous system, and is located in intramembrane compartments in neuronal dendrites and dendritic spines. In non-human primates and rodents, Calcyon is highly expressed in several brain regions, including the prefrontal cortex, which mediate cognitive performance functions (spatial attention, conversion ability, working memory, and decision-making), whereas in rodent striatum, Calcyon expression is relatively is known to be low. Calcyon is involved in clathrin-mediated endocytosis, an important component of synaptic plasticity. In addition, the Calcyon gene is an important gene for the development and function of the nervous system, and is known to be particularly highly related to schizophrenia, bipolar affective disorder, and attention deficit. In mice with increased expression of Calcyon, abnormal behaviors such as excessive behavioral patterns, anxiety disorders, and memory loss were reported. In mice lacking Calcyon, long-term memory formation was reported to be impaired due to excessive AMPA receptor activity.

On the other hand, Hevin (SPARCL1) is a glycoprotein located in the synaptic cleft of excitatory synapses and plays an important role in linking excitatory synapses. Hevin is highly expressed during the development of the nervous system and promotes synapse formation. Downregulation of Hevin is associated with neurological disorders such as depression and schizophrenia, and Hevin gene mutation is known as the 35th highest risk gene among 107 autism-related genes. In addition, Hevin is increased in expression in activated astrocytes after brain injury, and is known to regulate the formation time of excitatory synapses and synapse maturation. Despite Hevin's fundamental role in the potential link between excitatory synapse development and neurological disorders, the method by which Hevin induces synapse generation is not known to date.

Density regulation of excitatory synapses is highly related to neuropsychiatric diseases. In particular, imbalance of excitatory synaptic density after brain injury can lead to behavioral disorders such as seizures, epilepsy, agitation, aggressive behavior, anxiety disorders, depression and mania. Therefore, the utilization of Calcyon and Hevin in the treatment of diseases caused by synapse formation disorders after brain damage can be a new alternative.

Republic of Korea Patent No. 10-0375018 (Registered on February 22, 2003)

It is an object of the present invention to provide a peptide that inhibits the mutual binding of Calcyon and Hevin.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of neurological diseases.

Another object of the present invention is to provide a method for screening a synapse formation regulator.

Another object of the present invention is to provide a composition for controlling synapse formation.

In order to achieve the above object, the present invention provides a peptide consisting of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of neurological diseases comprising a binding inhibitor for inhibiting the mutual binding of Calcyon and Hevin as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of neurological diseases comprising a binding promoter that promotes the mutual binding of Calcyon and Hevin as an active ingredient.

In addition, the present invention provides a first step of treating a candidate drug to a sample isolated from a subject suspected of a neurological disease; a second step of measuring the level of mutual binding of Calcyon and Hevin before and after administration of the candidate drug; and a third step of selecting a candidate drug that increases or decreases the level of mutual binding between Calcyon and Hevin compared to a normal control sample; provides a synaptic formation regulator screening method comprising a.

In addition, the present invention provides a composition for regulating synapse formation comprising the peptide as an active ingredient.

In the present invention, it was confirmed that Calcyon and Hevin regulate synapse formation by mutual bonding, and Calcyon and Hevin mutual binding inhibitor or mutual binding promoter can inhibit abnormal synapse formation or promote synapse formation, synapse formation It can be usefully used as a pharmaceutical composition for the prevention or treatment of neurological diseases caused by disorders, a method for screening a synaptic formation regulator, a composition for synapse formation regulation, and the like.

1 is a schematic diagram showing a protein binding confirmation method using a yeast protein hybridization method.
Figure 2A is a result confirming the mutual binding of Calcyon and Hevin, Figure 2B is a result confirming that Calcyon acts as a receptor, Figure 2C is a result confirming the decrease in the number of synapses by Calcyon or Hevin knockdown, Figure 2D is Calcyon Or, it is the result of confirming the decrease in behavioral recovery due to Hevin knockdown.
Figure 3A is the result of confirming the mutual binding of Calcyon and Hevin fragment peptides (Hevin-C1, Hevin-C2), Figure 3B is the result of confirming the mutual binding inhibition of Calcyon and Hevin by the Hevin fragment peptide (31CP), Figure 3C is the result of confirming the inhibition of the mutual binding of Calcyon and Hevin by the resynthesized CP1 and CP2, FIG. 3D is a result of comparing the inhibition of the mutual binding of CP1 and CP2, and FIG. 3E is the decrease in the behavioral recovery by CP2 is the result of checking

The inventors of the present invention confirmed that Calcyon and Hevin are mutually bound, and that the mutual binding regulates the number of synapses and behavioral recovery in a local brain injury mouse model. has completed the present invention, confirming that it can be used to treat neurological diseases caused by synapse formation disorders by inhibiting the formation of abnormal synapses or promoting the formation of synapses.

Accordingly, the present invention provides a peptide consisting of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3.

The peptide can regulate synapse formation by inhibiting the mutual binding of Calcyon and Hevin.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of neurological diseases comprising a binding inhibitor for inhibiting the mutual binding of Calcyon and Hevin as an active ingredient.

The binding inhibitor may be a peptide consisting of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3, or antisense nucleotides complementary to mRNA of Calcyon and Hevin genes, small interfering RNA (small interfering RNA) RNA; siRNA) and short hairpin RNA (shRNA), a compound that specifically binds to Calcyon and Hevin proteins, peptides, peptide mimetics, aptamers, antibodies and natural products may be any one selected from the group consisting of However, it is specified that it is not limited thereto.

The neurological disease is induced by a synaptic formation disorder, and the neurological disease may be selected from the group consisting of schizophrenia, epilepsy, and neuropathic pain, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of neurological diseases comprising a binding promoter that promotes the mutual binding of Calcyon and Hevin as an active ingredient.

The binding promoter may be any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, and antibodies that specifically bind to Calcyon and Hevin proteins, but is not limited thereto.

The neurological disease is induced by a synaptic formation disorder, and the neurological disease may be selected from the group consisting of autism and dementia, but is not limited thereto.

When the composition of the present invention is a pharmaceutical composition, for administration, a pharmaceutically acceptable carrier, excipient or diluent may be included in addition to the active ingredients described above. The carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods, respectively. . In detail, when formulating, it can be prepared using a diluent or excipient such as a filler, a weight agent, a binder, a wetting agent, a disintegrant, and a surfactant commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. in addition to the active ingredient. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. It can be prepared by adding various excipients, for example, wetting agents, sweetening agents, fragrances, preservatives, and the like, in addition to liquids for oral use and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and tasks. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, Witepsol, Macrosol, Tween 61, cacao butter, laurin fat, glycerogelatin, etc. may be used.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the degree of disease, drug form, and time, but may be appropriately selected by those skilled in the art, and the daily dosage of the composition is preferably It is 0.001 mg/kg to 50 mg/kg, and may be administered once a day or divided into several times a day as needed.

In addition, the present invention provides a first step of treating a candidate drug to a sample isolated from a subject suspected of a neurological disease; a second step of measuring the level of mutual binding of Calcyon and Hevin before and after administration of the candidate drug; and a third step of selecting a candidate drug that increases or decreases the level of mutual binding between Calcyon and Hevin as compared to a normal control sample; provides a synaptic formation regulator screening method comprising a.

In addition, the present invention provides a composition for regulating synapse formation comprising the peptide as an active ingredient.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Plasmid preparation and shRNA construction

cDNA expressing mouse hevin (GenBank accession no. NM_010607) and Calcyon (NM_008430) was obtained using the gateway cloning method (Invitrogen) using the chain polymerase reaction method. Mutants of Hevin and calcyon were obtained using the EZchange site-directed mutagenesis kit (Enzynomics), and the prepared plasmids were pDEST-GFP-N (#31796, Addgene), pDEST-HA-C (#15506, Addgene) and A clone was prepared using the gateway cloning system in pDEST-mCherry-N (#31907, Addgene). Mutants of Hevin and Calcyon were obtained using the EZchange site-directed mutagenesis kit (Enzynomics). Hevin and Calcyon shRNA is ADDGENE using the target sequence (mouse Hevin: 5'-gcagctttatgaaccaaatcc-3', mouse Calcyon: 5'-ggacacgagagagaaagaaga-3', Calcyon shRNA-insensitive form: 5'-agataccagggaaaaggagga-3') shRNA sequences for scrambled (Sc), Hevin, and Calcyon were inserted into the pSicoRmCherryempty vector (#21907) purchased from. Calcyon shRNA-insensitive form was prepared for rescue use for Calcyon KD. After confirming that the correct sequence was inserted through DNA sequencing, the sequence corresponding to the U6 promoter-loxP-shRNA-CMV-mCherry-loxP was amplified by PCR. The amplified sequence was inserted into pAAV-MCS (Stratagene, USA) using an enzyme that cuts MluI and EcoRI sites. After reconfirming the sequence by DNA sequencing, a high titer recombinant AAV virus was prepared by requesting the KIST virus facility. Briefly, an expression vector and a packaging vector were co-transfected into AAV 293 cells (632273, Clontech) using jetPEI (101-10N, Polyplus). For 72 hours after transfection, the obtained virus was infected with HEK293T cells to confirm fluorescence expression. The obtained virus was purified by CsCl gradient method. shRNA (Scr) having a random sequence was used for control experiments.

Example 2: yeast two hybridization assay

As shown in Figure 1, the N-terminal end of Calcyon is pGBKT7 [tryptophan (tryptophan) expression possible, cat. no. 630443, TaKaRa], and the C-terminus or N-terminus of Heivn is pGADT7 [leucine expression capable, cat. no. 630442, TaKaRa). AH109 yeast was transformed with the two plasmids and then mated, and only yeast expressing the two plasmids survived using a medium lacking tryptophan and leucine (TL-). In order to confirm the protein cross-binding, it was cultured in a histidine (H)-deficient medium (TLH-) to enable survival by allowing histidine to be synthesized only when there is protein cross-binding.

Example 3: Cell Culture

HEK293T cells were purchased from the Cell Line Bank of Korea (Seoul National University), and DMEM medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen) and 100 units/ml penicillin-streptomycin was used at 37°C, 95% humidity. , 5% CO ₂ Incubated in an incubator.

Example 4: Immunocytochemical staining

A plasmid in which hemagglutinin (HA) was bound to the N-terminus or C-terminus of Calcyon was expressed in HEK293T cells. After diluting an antibody against HA (monoclonal mouse anti-HA, sc-7392, Santacruz) in a buffer containing a surfactant (permeable) and a buffer containing no surfactant (non-permeable) for 12 hours, the cells were reacted for 12 hours. It was reacted with a secondary antibody (FITC conjugated anti-mouse). All cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole) and used as background staining.

Example 5: Photothrombosis-induced brain injury mouse model

After anesthesia of adult C57BL/6 mice (8 weeks old) with isoflurane (3~5% for induction, 1~1.5% for maintaining in a 70% N ₂ O/30% O ₂ mixture), Rose bengal (Rose bengal) ; 100 mg/kg) by intraperitoneal injection. After 10 minutes, a green laser (540 nm, 20 mW) with a diameter of about 2 mm was irradiated to the sensorimotor cortex (1.5 mm right from the center of the brain) for 20 minutes using a stereotaxic device.

Example 6: Brain injection of AAV-shRNA virus

24 hours after brain injury, the mouse was anesthetized with isoflurane, and a hole of 3 mm was made on the right 1.5 mm from the central reference point using a stereotaxic instrument, and then 3 μl of AAV-shRNA virus was injected with a micro-injector ( Thomas Scientific, USA).

Example 7: Preparation of competitive inhibitory peptides for Hevin-Calcyon binding

A recombinant protein mCherry-31 amino acid (31CP) having a sequence of 540-570 amino acids (RSKVKKIYLDEKRLLAGDHPIELLLRDFKKN) among 1-650 amino acids of Hevin was prepared, and shorter peptides of RSKVKKIYLDEKRL (CP1) and IELLLRDFKK (CP2) were prepared It was commissioned and manufactured by a company specializing in peptide synthesis (Peptron, Korea). As a control peptide, the reverse peptides LRKEDLYIKKVKSR (CP1r) and KKFDRLLLEI (CP2r) were prepared in which the sequence order of the CP1 and CP2 peptides was reversed.

Example 8: Brain injection of competitive inhibitory peptides for Hevin-Calcyon binding

On the 7th day after brain injury, the mouse was anesthetized with isoflurane, and a 3 mm hole was made on the right 1.5 mm from the central reference point of the cerebrum using a stereotaxic instrument, and then ALZET Brain infusion kit 1 (Alzet, #0004760) and a micro-osmotic pump (Alzet, 1007D) was used to inject competitive inhibitory peptides.

Example 9: Neurobehavioral Test

An adhesive removal test (tape test) was performed to confirm the sensorimotor ability of the brain. A small tape (0.35 Х 0.45 cm) was attached to the front sole of the mouse, placed in a cage, and observed for 120 seconds to measure the time to remove the tape.

Example 10: Immunoprecipitation method

HEK293T cells were lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF and 1% NP-40, protease inhibitor cocktail) and immunoconjugate protein A/G PLUSAgarose (Santa Cruz). Biotechnology) and an anti-GFP antibody (1:200, B-2; Santa Cruz Biotechnology) were used to immunize for 24 hours under refrigeration. After washing three times with lysis buffer, the proteomic protein was separated on a 10% or 15% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. After reacting with anti-HA antibody (1:1000) and anti-GFP antibody (1:1000), it was reacted with HRP-conjugated anti-rat antibody and anti-mouse IgG antibody, ECL (enhanced chemo-luminal; Amersham) Biosciences) was used to observe the immune reactivity.

Experimental Example 1: Mutual bonding of Hevin and Calcyon

To determine whether Hevin and Calcyon are mutually bound, Hevin's N-terminal (acidic domain, amino acid sequence positions 1-417) or C-terminal (amino acid sequence positions 418-650) and Calcyon's N-terminal (extracellular domain, Amino acid sequence positions 1-88) were expressed in yeast, and mutual association was confirmed by yeast protein hybridization. As a result, as shown in FIG. 2A, it was observed that yeast survived only when the N-terminal end of Calcyon and the C-terminus of Hevin were expressed in TLH- medium, that is, it was confirmed that the two proteins interact with each other. .

To determine whether Calcyon acts as a receptor, hemagglutinin (HA) was bound to the N-terminus or C-terminus of Calcyon and staining was performed by immunocytochemistry. As a result of staining the HA antigen on the cell surface without adding a surfactant to prevent the antibody from penetrating the cell membrane, as shown in FIG. 2B, it was confirmed that the N-terminus of Calcyon was stained and located outside the cell.

Next, after inducing local brain damage in mice, the results of comparing the exercise recovery of mice expressing scramble (Scr) with those in which the Calcyon and Hevin genes were knocked down using AAV virus (Caly KD, Hevin KD), respectively. , As shown in Figure 2C, the number of synapses to be restored is reduced compared to the control group (Scr), and when the knockdown of Caly gene by shRNA Caly is restored using the shRNA Caly insensetive form, it was confirmed that the decrease in synapses is inhibited. In addition, as shown in FIG. 2D , the knockdown of each gene confirmed that the behavioral recovery after brain injury was reduced compared to the control group (Scr).

Experimental Example 2: Mutual binding inhibition peptide of Hevin and Calcyon

In order to determine whether Hevin's fragment peptide inhibits the mutual binding of Hevin and Calcyon, the N-terminal end of Calcyon and Hevin's fragment peptide C1 (Hevin C-terminal peptide 1, amino acid sequence positions 350-650) or C2 ( Hevin C-terminal peptide peptide 2, amino acid sequence positions 418-650) was expressed in yeast and mutual association was confirmed by yeast protein hybridization. As a result, as shown in Figure 3A, it was confirmed that the N-terminal end of Calcyon and Hevin-C1 or Hevin-C2 peptide mutually bind to each other. That is, from the above results, it was confirmed that the N-terminus of Calcyon and the C-terminus of Hevin were mutually bound.

In addition, as shown in FIG. 3B, it was confirmed by co-immunoprecipitation that the binding of Hevin and Calcyon was inhibited by the mCherry-31 peptide (31CP), which is a protein corresponding to 31 amino acids at the Hevin C-terminus, FIG. 3C As shown in , it was confirmed that CP1 (Competitive peptide 1) or CP2 (Competitive peptide 2), a recombinant peptide shorter than the mCherry-31 peptide, inhibits the mutual binding of Hevin (GFP-Hevin) and Calcyon (mCalcyon-HA). did. In addition, as shown in FIG. 3D , it was confirmed that the degree of inhibition of mutual binding of CP2 was better than that of CP1.

Next, as a result of local injection of CP2 peptide after inducing local brain damage in mice to damage the brain region responsible for sensory and motor control of the forelimbs, as shown in FIG. 3E, removing the tape on the sole of the foot during recovery It was confirmed that behavioral recovery was inhibited as the time was delayed compared to the control group (CP2r).

As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, these specific descriptions are only preferred embodiments, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

<110> KYUNGPOOK NATIONAL UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY Korea University Research and Business Foundation <120> Composition for preventing or treating neurological disorders induced by synaptic formation dysfunction comprising binding modulator of Calcyon and Hevin <130> ADP-2019-0187 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> peptide 1 <400> 1 Arg Ser Lys Val Lys Lys Ile Tyr Leu Asp Glu Lys Arg Leu Leu Ala 1 5 10 15 Gly Asp His Pro Ile Glu Leu Leu Leu Arg Asp Phe Lys Lys Asn 20 25 30 <210> 2 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> peptide 2 <400> 2 Arg Ser Lys Val Lys Lys Ile Tyr Leu Asp Glu Lys Arg Leu 1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> peptide 3 <400> 3 Ile Glu Leu Leu Leu Arg Asp Phe Lys Lys 1 5 10

Claims (13)

delete delete As a pharmaceutical composition for the prevention or treatment of neurological diseases comprising a binding inhibitor that inhibits the mutual binding of Calcyon and Hevin as an active ingredient,
The binding inhibitor is a pharmaceutical composition for the prevention or treatment of a neurological disease selected from schizophrenia or epilepsy, characterized in that the peptide consisting of the amino acid sequence of SEQ ID NO: 3. delete delete The pharmaceutical composition for preventing or treating a neurological disease according to claim 3, wherein the neurological disease is induced by a synaptic formation disorder. delete delete delete delete delete delete delete

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