Novel peptide and MAO-a inhibitory composition comprising same

Abstract

Disclosed are a novel peptide and an MAO-A inhibitory composition comprising same. The peptide may have the sequence CSLPMTAGDDV, GLTGPPTTQDF, or GPDETINLGDF. The peptide can reduce the serotonin-degrading activity of MAO-A by selectively reacting with only the MAO-A enzyme among MAO-A and MAO-B which are serotonin-degrading enzymes. The peptide can bind adjacent to the active site of the MAO-A enzyme to prevent serotonin from approaching the active site of MAO-A.

Description

Novel peptide and composition for inhibiting MAO-A comprising the same

[0001] The present invention relates to a novel peptide and a composition for inhibiting MAO-A comprising the same. More specifically, the present invention relates to a novel peptide, a composition for inhibiting MAO-A comprising the same, a nucleic acid encoding the same, a recombinant expression vector comprising the same, a cell transformed thereby, and the like.

[0002] Monoamine oxidase A (MAO-A) and MAO-B are known as flavoenzymes located in the outer membrane of the mitochondria. These two enzymes share over 70% amino acid sequence homology and contain the cofactor flavin adenine dinucleotide (FAD). Although MAO-A and MAO-B share a high degree of amino acid sequence homology, they are known to differ in substrate specificity and inhibitor sensitivity. Inhibitors of MAO-A have been used to treat depression, anxiety, and migraines. In addition, inhibitors of MAO-B have been used as dopamine-sparing agents for the treatment of Parkinson's disease. The neurotransmitter serotonin is known to be released into the synapse and reabsorbed presynaptly. MAO-A is reported to degrade reabsorbed serotonin by oxidation by its coenzyme (FAD). To maintain serotonin concentration, clorgyline has been used as an MAO-A inhibitor. In the present invention, a monoclonal antibody against serotonin was used to screen serotonin-like mimotopes from an Fv-antibody library.

[0003] The structure of Fv antibodies is based on the VH of immunoglobulin G (IgG), which consists of three clustered region (CDR) regions and four framework regions (FR). CDRs are known as hypervariable regions located in the heavy and light chains of IgG that specifically interact with target antigens. As shown in Figure 1, an Fv antibody library was prepared by randomizing the amino acid sequence of CDR3 through site-directed mutagenesis at 11 amino acid residues. The prepared Fv antibody library was expressed on the outer membrane of E. coli using autodisplay technology. As shown in Figure 2, autodisplay of target proteins has been reported to achieve a high expression yield of >105 Fv antibodies / E. coli, and >90% of the E. coli in the entire E. coli population of the Fv antibody library had autodisplayed Fv antibodies. In addition, the Fv antibody library had >10 5 The Fv-antibody library was reported to have a high diversity of clones / libraries. The Fv-antibody library was used to screen Fv-antibodies with binding affinity to biotin, fluorescent dyes (fluorescein and rhodamine B), biological crystals (sodium urate and calcium pyrophosphate dehydrate), viral proteins, etc.

[0004] One object of the present invention is to provide a peptide having a selective binding ability to monoamine oxidase, particularly MAO-A.

[0005] Another object of the present invention is to provide a nucleic acid encoding the peptide.

[0006] Another object of the present invention is to provide a recombinant expression vector comprising the nucleic acid.

[0007] Another object of the present invention is to provide a cell transformed by the recombinant expression vector.

[0008] Another object of the present invention is to provide a composition for inhibiting MAO-A comprising the peptide.

[0009] In one aspect, the present invention provides a peptide having a binding ability to monoamine oxidase and comprising at least one peptide sequence selected from the group comprising a peptide sequence of SEQ ID NO: 1, a peptide sequence of SEQ ID NO: 2, and a peptide sequence of SEQ ID NO: 3.

[0010] [Sequence number 1]

[0011] CSLPMTAGDDV

[0012] [Sequence number 2]

[0013] GLTGPPTTQDF

[0014] [Sequence number 3]

[0015] GPDETINLGDF

[0016] Since the peptide according to the embodiment of the present invention has the above sequence, the peptide can act as a serotonin mimetic that intervenes in the action of an enzyme that decomposes serotonin.

[0017] In one embodiment, the peptide can selectively react only with MAO-A (Monoamine oxidase A) and MAO-B (Monoamine oxidase B), which are serotonin-decomposing enzymes, thereby reducing the serotonin-decomposing activity of MAO-A.

[0018] Since the peptide according to the embodiment of the present invention selectively acts on MAO-A, it may have advantages such as reducing side effects compared to non-selective inhibition.

[0019] In one embodiment, the peptide may bind adjacent to the active site of the MAO-A enzyme to block serotonin from accessing the active site of the MAO-A.

[0020] By inhibiting MAO-A through the above mechanism, the peptide according to the embodiment of the present invention can act as a serotonin mimotope.

[0021] In another aspect, the present invention provides a nucleic acid encoding the peptide.

[0022] The nucleic acid encoding the above peptide can be used for peptide synthesis using a living organism.

[0023] In another aspect, the present invention provides a recombinant expression vector comprising the nucleic acid.

[0024] It is possible to transform cells, etc., to synthesize the peptide using the above recombinant expression vector.

[0025] In another aspect, the present invention provides a cell transformed with the recombinant expression vector.

[0026] Synthesis of the peptide is possible by utilizing the intracellular mechanism of the above cells.

[0027] In one embodiment, the cell may comprise one or more cells selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells. In one embodiment, the cell is selected from the group consisting of monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp, Pseudomonas sp, Proteus mirabilis or Staphylococcus sp, Aspergillus sp, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and may include one or more cells selected from the group comprising Neurospora crassa.

[0028] In another aspect, the present invention provides a composition for inhibiting MAO-A, comprising an amino acid fragment having a binding ability to monoamine oxidase and having at least one peptide sequence selected from the group comprising a peptide sequence of SEQ ID NO: 1, a peptide sequence of SEQ ID NO: 2, and a peptide sequence of SEQ ID NO: 3.

[0029] [Sequence number 1]

[0030] CSLPMTAGDDV

[0031] [Sequence number 2]

[0032] GLTGPPTTQDF

[0033] [Sequence number 3]

[0034] GPDETINLGDF

[0035] Since the peptide included in the composition according to an embodiment of the present invention has the above sequence, the composition can intervene in the action of an enzyme that decomposes serotonin.

[0036] In one embodiment, the MAO-A inhibitory composition selectively reacts only with MAO-A enzyme among serotonin-decomposing enzymes MAO-A (Monoamine oxidase A) and MAO-B (Monoamine oxidase B), thereby reducing the serotonin-decomposing activity of MAO-A.

[0037] Since the peptide included in the composition according to an embodiment of the present invention selectively acts on MAO-A, it may have advantages such as reducing side effects compared to non-selective inhibition.

[0038] In one embodiment, the MAO-A inhibitory composition can bind adjacent to the active site of the MAO-A enzyme to block serotonin from accessing the active site of the MAO-A.

[0039] By inhibiting MAO-A through the above mechanism by the peptide included in the composition according to an embodiment of the present invention, the composition provides a peptide that acts as a serotonin mimotope.

[0040] More specifically, in the present invention, target clones having binding affinity for a monoclonal antibody against serotonin were screened from an Fv-antibody library. The binding affinity of the screened clones to serotonin was analyzed by FACS. The screened CDR3 region (11 residues) of the screened Fv-antibody was synthesized into a peptide (12 residues) by inserting a connecting amino acid residue. The inhibitory activity of the serotonin-like analogues against MAO-A and MAO-B was analyzed in comparison with existing inhibitors. Finally, the screened serotonin-like mimotopes were treated with a serotonin receptor-expressing cell line (SH-SY5Y, ATCC code: CRL-2266) to confirm binding to the serotonin receptor on the cell surface.

[0041] The peptide according to an embodiment of the present invention has a selective binding ability to monoamine oxidase, particularly MAO-A, thereby inhibiting the decomposition of serotonin.

[0042] The nucleic acid according to an embodiment of the present invention can be used for synthesis by encoding the peptide.

[0043] A recombinant expression vector according to an embodiment of the present invention can be used for synthesis by including the nucleic acid.

[0044] A cell according to an embodiment of the present invention can be transformed with the recombinant expression vector and used for synthesis.

[0045] The composition for inhibiting MAO-A according to an embodiment of the present invention can inhibit serotonin decomposition by including the peptide.

[0046] Figure 1 illustrates the preparation of an Fv-antibody library.

[0047] Figure 2 illustrates that autodisplay of target proteins results in high expression yields.

[0048] Figures 3 to 23 are drawings according to embodiments of the present invention.

[0049] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. The present invention may be modified in various ways and may take various forms. Therefore, specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, but should be understood to include all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing each drawing, similar reference numerals are used to indicate similar components. In the attached drawings, the dimensions of structures are shown larger than actual size to ensure clarity of the present invention.

[0050] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, the terms "comprise" or "have" should be understood to indicate the presence of a feature, number, step, operation, component, or combination thereof described in the specification, but do not preclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, or combinations thereof.

[0051] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0052] A peptide according to an embodiment of the present invention has a binding ability to monoamine oxidase and may include one or more peptide sequences selected from the group including the peptide sequence of SEQ ID NO: 1, the peptide sequence of SEQ ID NO: 2, and the peptide sequence of SEQ ID NO: 3.

[0053] [Sequence number 1]

[0054] CSLPMTAGDDV

[0055] [Sequence number 2]

[0056] GLTGPPTTQDF

[0057] [Sequence number 3]

[0058] GPDETINLGDF

[0059] The peptide sequences of the above sequence numbers 1 to 3 are made clear by other parts of this specification, but the above sequence number 1 is 1 CSLPM 6 TAGDD 11 V, the above sequence number 2 1 GLTGP 6 PTTQD 11 F, the above sequence number 3 1 GPDET 6 INLGD 11 F may mean. In the context of this specification, all sequences should be interpreted as including sequences that would be obviously identical or similar to a sequence of ordinary skill in the art. For example, unless otherwise specified herein, all sequences should be considered identical or similar to their reverse sequences and sequences with minor modifications. Furthermore, unless otherwise specified herein, sequences that perform the same function, such as nucleic acid sequences that encode the same amino acid, should be considered identical or similar sequences.

[0060] By having the above sequence, the peptide according to an embodiment of the present invention can act as a serotonin mimetic that interferes with the action of enzymes that decompose serotonin. This effect will be made clear by the following examples.

[0061] In the context of this specification, the term "mimotope" may collectively refer to a molecule, such as a peptide, that mimics the structure and function of an antigen or substrate and induces an immune response or response similar to the original antigen or substrate.

[0062] The above peptide has the function described above, but the peptide of the present invention is not necessarily limited by the function. In one embodiment, the peptide selectively reacts only with MAO-A enzyme among serotonin decomposition enzymes MAO-A (Monoamine oxidase A) and MAO-B (Monoamine oxidase B) to reduce the serotonin decomposition activity of MAO-A. Since the peptide according to the embodiment of the present invention selectively acts on MAO-A, it may have advantages such as reducing side effects compared to non-selective inhibition. Meanwhile, in the context of the present specification, "selective reaction" means that one reaction occurs with a significantly higher level of activity than other reactions, and does not completely exclude the activity of other reactions.

[0063] In one embodiment, the peptide can bind adjacent to the active site of the MAO-A enzyme and block serotonin from accessing the active site of the MAO-A. By inhibiting MAO-A through the above mechanism, the peptide according to an embodiment of the present invention can act as a serotonin mimotope. In the context of the present specification, terms such as reaction inhibition or access blocking are described from the perspective of a phenomenon that occurs overall in a biochemical reaction, and a single molecule or a small number of molecules are not excluded from the scope of the present invention even if they do not probabilistically perform the function described above.

[0064] Meanwhile, the nucleic acid according to an embodiment of the present invention can encode the peptide. Therefore, the description of the nucleic acid according to an embodiment of the present invention can be applied identically or similarly to the same or similar terms or structures in the description of other aspects of the present invention described above.

[0065] In the context of this specification, unless specifically defined otherwise, "coding" is defined based on the relationship between a nucleic acid sequence and a peptide sequence defined by transcription and translation mechanisms known to those skilled in the art. Therefore, as described above, the nucleic acid or nucleic acid sequence referred to herein may be a term encompassing a nucleic acid or nucleic acid sequence encoding a peptide sequence that is identical or similar to each other, taking into account cases where there is one or more nucleic acids corresponding to the amino acids contained in the peptide sequence. Such nucleic acids may be used to derive the peptides or antibodies or antigen-binding fragments thereof discovered in the present invention. The nucleic acids encoding the peptides may be utilized for peptide synthesis using a living organism.

[0066] Meanwhile, a recombinant expression vector according to an embodiment of the present invention may include the nucleic acid. Therefore, the description of the recombinant expression vector according to an embodiment of the present invention may be applied identically or similarly to the same or similar terms or structures in the description of other aspects of the present invention described above. In the context of the present specification, a "recombinant expression vector" or "recombinant expression vector" may refer to a vector used to promote the expression of a gene in a specific host cell or tissue by bringing in a DNA fragment from an external source using genetic engineering technology, and the vector may insert an external gene into a host cell and include a control region and sequence necessary for gene expression. The recombinant expression vector can be used to transform cells or the like so that the peptide can be synthesized. In addition, since the recombinant expression vector includes a nucleic acid according to an embodiment of the present invention, it can be used to express the peptide or an antibody or antigen-binding fragment thereof containing or similar thereto.

[0067] Meanwhile, the cell according to an embodiment of the present invention may be a cell transformed with the recombinant expression vector. Therefore, the description of the cell according to an embodiment of the present invention may be applied identically or similarly to the same or similar terms or structures in the description of other aspects of the present invention described above. In the context of the present specification, the term "transformation" or "transformation" may refer to a form of genetic engineering technology, a process of introducing a gene into a cell or tissue that receives an external gene from the outside, and may be mainly used to induce gene expression by delivering an externally produced recombinant expression vector into a cell using recombinant DNA technology. Therefore, the cell according to an embodiment of the present invention can express the peptide or an antibody containing or similar thereto or an antigen-binding fragment thereof from the recombinant expression vector using any system related to intracellular gene expression. Synthesis of the peptide is possible by utilizing the intracellular mechanism of the cell.

[0068] As long as the above function is performed, the type of the cell is not particularly limited. In one embodiment, the cell may include one or more cells selected from the group including animal cells, plant cells, yeast, Escherichia coli, and insect cells. In one embodiment, the cell is selected from the group consisting of monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp, Pseudomonas sp, Proteus mirabilis or Staphylococcus sp, Aspergillus sp, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and may include one or more cells selected from the group comprising Neurospora crassa.

[0069] Meanwhile, the composition for inhibiting MAO-A according to an embodiment of the present invention may include an amino acid fragment having a binding ability to monoamine oxidase and having at least one peptide sequence selected from the group including the peptide sequence of SEQ ID NO: 1, the peptide sequence of SEQ ID NO: 2, and the peptide sequence of SEQ ID NO: 3.

[0070] [Sequence number 1]

[0071] CSLPMTAGDDV

[0072] [Sequence number 2]

[0073] GLTGPPTTQDF

[0074] [Sequence number 3]

[0075] GPDETINLGDF

[0076] The description of the MAO-A inhibitory composition according to an embodiment of the present invention can be applied identically or similarly to the same or similar configurations or terms in the description of other aspects described above. Since the peptide included in the composition according to an embodiment of the present invention has the above sequence, the composition can interfere with the action of an enzyme that breaks down serotonin.

[0077] In one embodiment, the MAO-A inhibitory composition selectively reacts only with MAO-A enzyme among serotonin-decomposing enzymes MAO-A (Monoamine oxidase A) and MAO-B (Monoamine oxidase B), thereby reducing the serotonin-decomposing activity of MAO-A. Since the peptide included in the composition according to an embodiment of the present invention selectively acts on MAO-A, it may have advantages such as reducing side effects compared to non-selective inhibition.

[0078] In one embodiment, the MAO-A inhibitory composition can bind adjacent to the active site of the MAO-A enzyme to block serotonin from accessing the active site of MAO-A. By inhibiting MAO-A through the above-described mechanism by the peptide included in the composition according to an embodiment of the present invention, the composition provides a peptide that acts as a serotonin mimetic.

[0079] Hereinafter, embodiments of the present invention will be described. However, the embodiments described below are merely some embodiments of the present invention, and the scope of the present invention is not limited to the embodiments described below.

[0080] ingredient

[0081] Mercaptoundecanoic acid (MUA), ethanolamine, and other chemicals were purchased from Sigma-Aldrich (Seoul, Korea). A monoclonal antibody against serotonin (NB 100-65037) was purchased from Novus Biologicals (Littleton, CO, USA). For the analysis of MAO-A and MAO-B activities, a monoamine oxidase (MAO) assay kit (ab241031) and a monoamine oxidase A (MAO-A) inhibitor screening kit (Fluorometric) (ab284510) were purchased from Abcam (Cambridge, UK). TM 2 and Klenow DNA polymerase were purchased from New England Biolabs (Ipswich, MA, USA). Isopropyl-D-1-thiogalactopyranoside (IPTG) and Kanamycin were purchased from Kisan Bio (Seoul, Korea). Dynabeads™ protein G, FastDigest DpnI, Phusion® High-Fidelity DNA polymerase, trypsin, penicillin / streptomycin (p / s), and SuperSignal® ELISA Femto Maximum Sensitivity Substrate were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). For PCR product purification and plasmid extraction, the PCR purification mini kit and plasmid extraction kit were purchased from Favorgen Biotech Corp. (Pingtung, Taiwan). The peptides listed in Table 1 were synthesized by Peptron Co. (Daejeon, Korea) with a purity of ≥90%. SH-SY5Y cells (ATCC: CRL-2266) were obtained from the Korean Cell Line Bank (Seoul, Korea). Fetal bovine serum (FBS) and minimum essential medium (MEM) medium were purchased from Biowest (Nuaille, France).

[0082] Fv-antibody library screening

[0083] Screening of target clones with binding affinity for monoclonal antibodies against serotonin was performed by magnetic bead-activated cell sorting (MACS) screening method. MACS screening begins with immobilization of monoclonal antibodies on protein G-coated magnetic beads. Monoclonal antibodies against serotonin (1 mg / mL, 5 μL) and Dynabeads TM Protein G (5 μL) was mixed with 0.1% PBST buffer (0.1% Tween 20 in PBS) for 2 h, and the monoclonal antibody-immobilized magnetic beads were mixed with the Fv-antibody library (OD600 nm = 1.0, 100 μL) for 1 h. The screened E. coli with binding affinity to the monoclonal antibody-immobilized magnetic beads were magnetically filtered and washed 15 times with 0.1% PBST and PBS buffer. The monoclonal antibody-immobilized magnetic beads bound to the selected E. coli were resuspended in LB Broth medium and spread on LB agar plates to obtain screened clones with binding affinity to the monoclonal antibody against serotonin.

[0084] Expression and purification of Fv antibodies

[0085] For the expression of Fv antibodies that can act as serotonin-like mimotopes, three plasmids (pJS004, pJS005, and pJS006) containing the open reading frames (ORFs) of Fv antibodies with high binding affinity to monoclonal antibodies against serotonin were used. They were custom synthesized by Cosmogenetech (Seoul, Korea) as shown in Figure 3. The oligonucleotide sequences and amino acid sequences of the CDR regions and framework regions of the three Fv antibodies are summarized in Table 1. The Fv antibodies were expressed intracellularly in E. coli. For expression, the three custom-synthesized plasmids (pJS004, pJS005, and pJS006) were transformed into competent cells using electroporation, and the transformed E. coli were cultured in 20 mL of LB medium containing 1 mM IPTG and 30 μg / mL carbenicillin at 30°C for 16 h. Cultured E. coli was centrifuged (3,000 × g, 3 min) and then resuspended in 30 mL of binding buffer (5 mM Tris-HCl, 0.5 M NaCl, and 3 M urea). The resuspended E. coli was sonicated (Vibracell VCX-130, Sonics, USA), the lysate was centrifuged (25,000 × g, 10 min), and the supernatant containing the expressed Fv-antibody was collected. Purification of the Fv-antibody was performed using a His-tag purification column from Roche (Basel, Switzerland). The elution buffer used was binding buffer containing 100 mM imidazole. After elution, imidazole and urea were removed from the eluted sample by dialysis at 4°C and 150 rpm for 16 h. Finally, the purified Fv-antibody was obtained.

[0086] <h2 style=";text-align:left;direction:ltr">RegionOligonucleotide sequence (5' → 3')Amino acid sequence (N-term → C-term)Frame-1(FR1)GAA GTG CAG CTC GTG GAA AGC GCT GCC GAA GTT CGG CGT CCT GGG GCT AGC GTG AAG ATC ACC TGC AAA GCG TCC GGC TAT TCA TTC AGCEVQLVESAAEVRRPGASVKITCKASGYSFSCDR1ACC TAT GGG ATT CAGTYGIQFrame-2(FR2)TGG ATG CGC CAA GCG CCA GGC CAG CGT CCG GAA TGG CTT GGGWMRQAPGQRPEWLGCDR2TGG ATA CAT GCA GGC ACA GGT GGG ACT AAG TAC TCG CGC AAA TTT CAG GGTWIHAGTGGTKYSRKFQGFrame-3(FR3)CGC ATT ACT ATC ACC CGT GAT ACC AGC GCG AAT ACC GTC TAT CTG GAT CTG AAC TCT CTG ACA TCG GAG GAT ACG GCC GTC TAT TAT TGC GCT CGTRITITRDTSANTVYLDLNSLTSEDTAVYYCARCDR3(Template)GAC AAA GTT ACA GTC TGG GCT TGT CAG GAT AATDKVTVWACQDNFrame-4(FR4)TGG GGT CAA GGT ACT ACG GTT ACG GTC AGC AGTWGQGTTVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0087] <h2 style=";text-align:left;direction:ltr"> 표면 플라즈몬 공명(SPR) 분석<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0088] To measure the binding affinity (KD) of the synthesized peptide and expressed Fv-antibody for a monoclonal antibody, SPR (surface plasmon resonance) analysis was performed using an SPR biosensor from i-Cluebio (Seongnam, Korea). The binding analysis of the synthesized peptide and expressed Fv-antibody was performed by immobilizing the monoclonal antibody against serotonin on the gold surface of an SPR chip (BK-7 glass coated with a 2 nm Ti film and a 48 nm Au layer) used in the SPR biosensor. The SPR chip was modified to contain a carboxylic acid using mercaptoundecanoic acid (MUA, 1 mM), and the monoclonal antibody against serotonin (10 μg / mL) was covalently immobilized using EDC / NHS reagent. After blocking with ethanolamine (1 M), the synthesized peptides or expressed Fv antibodies were bound to the SPR chip immobilized with monoclonal antibodies at a flow rate of 20 μL / min for 10 min. After the binding step, PBS buffer was additionally injected at a flow rate of 20 μL / min for 10 min to facilitate dissociation. The SPR signal results were fitted to Hill's equation for analysis.

[0089] y = (ad) / [1+(x / c)^b]+d

[0090] Here, a and d represent the maximum and minimum signals, respectively. c represents the concentration of soluble protein or peptide. b represents the Hill's slope of the curve.

[0091] MAO-A and MAO-B activity inhibition assay

[0092] SH-SY5Y cells (ATCC: CRL-2266) from the Korean Cell Line Bank (Seoul, Korea) were cultured in MEM supplemented with 10% heat-inactivated FBS Biowest (Nuaille, France) and 1% penicillin / streptomycin (P / S). For the inhibition assay of MAO-A and MAO-B activity, three synthetic serotonin-mimetic mimotopes (peptides-7, -33, and -40) and Fv antibodies (Fv-7, -33, and 40), a monoamine oxidase (MAO) assay kit (ab241031), and a monoamine oxidase A inhibitor screening kit (fluorometric) (ab284510) from Abcam (Cambridge, UK) were used according to the manufacturers' instructions. The three synthesized peptides were mixed with MAO-A or MAO-B enzymes in the assay kit at concentrations ranging from 100 pM to 10 μM, and fluorescence (λex=535 nm and λem=587 nm) was measured with a fluorescence spectrometer (Victor5) from PerkinElmer Co. (Waltham, MA, USA). For the MAO-A and MAO-B activity inhibition assay using the three expressed Fv-antibodies, the luminol reaction was used, and the luminescence was measured with a Glomax Discover microplate reader from Promega (Sunnyvale, CA, USA).

[0093] For fluorescence imaging, SH-SY5Y cells (1 × 10 5(10 cells / mL) were cultured until 70% - 80% confluency was reached. Subsequently, the cells were further incubated with 20 μM serotonin-mimetic mimotopes (peptides-7, -33, and -40) in phosphate-buffered saline (PBS) buffer for 1 h. After peptide incubation, the cells were washed three times with PBS buffer and fixed with 4% paraformaldehyde for 40 min for immunocytochemistry (ICC) as described above. After washing the cells in PBS, the nuclei and plasma membrane were stained with DAPI and PlasMem Red. Images were acquired using an Olympus BX53 microscope with Olympus Cell Sens software (Carl Zeiss, Jena, Germany). For FACS analysis, SH-SY5Y cells (1 × 10 5 (cells / mL) were cultured to 70% - 80% confluency and further incubated with three synthetic serotonin-like mimotopes in a concentration range of 0.1 - 10 μM in PBS buffer containing 2% FBS for 1 h. Cells were washed three times with 2% FBS in PBS buffer and FACS analysis was performed in the fluorescence channel of FL1 (λex = 488 nm and λem = 525 nm) using a FACSCalibur flow cytometer to measure fluorescein-labeled serotonin-like mimotopes.

[0094] A cell-based MAO-A activity inhibition assay was performed by incubating SH-SY5Y cells (ATCC: CRL-2266) with three synthetic peptides at concentrations ranging from 0.1 to 10 μM for 24 h. After incubation, peptide-treated cells were trypsinized and homogenized with MAO assay buffer (0.1 mg / μL) from an MAO assay kit. MAO-A activity in cell lysates was assayed using an MAO-A inhibitor screening kit according to the manufacturer's instructions. Fluorescence (λex = 535 nm and λem = 587 nm) was measured with a fluorescence spectrometer (Victor5) from PerkinElmer Co. (Waltham, MA, USA).

[0095] Screening for serotonin-like mimotope

[0096] Serotonin-like mimotopes were screened against a Fv-antibody library using a monoclonal antibody against serotonin. To screen for E. coli isolates with binding affinity for the monoclonal antibody, the monoclonal antibody was immobilized on magnetic beads and mixed with the Fv-antibody library, as shown in Figure 4 . The magnetic beads were then separated using an external magnet and cultured on agar plates. Several randomly selected colonies from the cultured colonies were subjected to genetic analysis. Based on the oligonucleotide sequences, three clones (clones Nos. 7, 33, and 40) were selected as valid candidates, possessing different oligonucleotide sequences in the CDR3 region, as shown in Figure 5 . The other clones were determined to have the same oligonucleotide sequence as the template (clone No. 14), as well as deleted and added sequences (summarized in the Supplementary Information).

[0097] The selected clones were analyzed for binding affinity to a fluorescently labeled monoclonal antibody against serotonin. As shown in Figure 6, the selected clones (clones Nos. 7, 33, and 40) exhibited significantly high fluorescence signals in the gated region upon binding of the monoclonal antibody. These high fluorescence signals indicate that the Fv antibody automatically displayed on the outer membrane of the E. coli strain interacted specifically with the fluorescently labeled monoclonal antibody. In the case of the control strain with only the CDR1 and CDR2 regions (without the randomized CDR3 region), no high fluorescence signals were observed in the same gated region. The absence of fluorescence signals in the control strains indicated that the CDR3 region of these E. coli strains contributed to the interaction with the monoclonal antibody.

[0098] Quantitative binding analysis was performed on the remaining three clones, and three of the four clones showed quantitatively increased fluorescence signals as shown in Figure 7. For native E. coli without autodisplayed Fv-antibody and the control strain with only CDR1 and CDR2 regions (no CDR3 region), the response was at baseline level over the entire concentration range of monoclonal antibodies. The binding constants (K) of these clones for monoclonal antibodies D ) was measured to be 216 nM for clone No. 7, 7.31 nM for clone No. 33, and 19.9 nM for clone No. 40. Information on the selected clones is summarized in Table 1.

[0099] The Fv antibody of the screened clone was expressed as a soluble protein. As shown in Figure 3, the Fv antibody was composed of three CDRs and four FRs, and the screened CDR3 region (11 residues) of the screened Fv antibody was included. The Fv antibody was expressed as a fusion protein with a red fluorescent protein (tdTomato) with a molecular weight of 69.2 kDa. The binding affinity (K) of the expressed Fv antibody D) was measured using an SPR biosensor. As shown in Fig. 8, a monoclonal antibody against serotonin was immobilized on the gold surface of the SPR biosensor, and then the Fv-antibody was immobilized at a concentration ranging from 3.7 to 300 nM. As shown in Fig. 9, the association and dissociation profiles of the SPR biosensor were obtained, and the binding affinity (K) of the expressed Fv-antibody was measured. D ) were measured to be 20.0 nM for Fv-7, 27.0 nM for FV-33, and 12.4 nM for Fv-40. These results showed that the expressed Fv-antibodies had similar binding affinities compared to E. coli cells. And, these results indicated that the expressed Fv-antibodies could be used as serotonin-like mimotopes. The binding constants (K) of the three synthesized peptides D ) is summarized in Table 1.

[0100] The screened CDR3 region (11 residues) of the screened Fv antibody was synthesized into a peptide (12 residues) by inserting connecting amino acid residues. The binding affinity (K) of the synthesized peptide D ) was measured using an SPR biosensor. As shown in Fig. 10, a monoclonal antibody against serotonin was immobilized on the gold surface of the SPR biosensor, and then the immobilized antibody was treated with a synthetic peptide in the concentration range of 0.37 - 3 μM. As shown in Fig. 11, the association and dissociation profiles of the SPR biosensor were obtained, and the binding affinity (K) of the synthesized peptide D) were estimated to be 681 nM for peptide-7, 693 nM for peptide-33, and 733 nM for peptide-40. These results showed that the synthesized peptides had a significantly improved binding affinity compared to E. coli cells due to an increased degree of freedom in binding to monoclonal antibodies. And these results indicate that the synthesized peptides can be used as serotonin-like mimotopes. The binding constants (K) of the three synthesized peptides D ) is summarized in Table 2.

[0101] Screened CDR3SequencesKD, FACS [M]KD, SPR [M](peptide)KD, SPR [M](FV)Peptide-7(Clone no. 7)Oligonucleotide5'-1TGCCC6GACTC11TGCCG16GGGGT21TCTTG26ATGAT31GT33T-3'2.16 x 10-86.81 x 10-72.00 10-8Amino acid1GLTGP 6PTTQD 11FPeptide-40(Clone no. 40)Oligonucleotide5'-1TGTGG6TCCTC11CGGTT16GTTGC21ACTAC26ATGAT31GA33T-3'1.99 x 10-87.33x 10-71.24 x 10-8Amino acid1GPDET 6INLGD 11F

[0102] Serotonin, a neurotransmitter, is known to be released into synapses and reabsorbed presynaptly. MAO-A is reported to degrade the reabsorbed serotonin by oxidation by the coenzyme A of the enzyme (FAD). Chlorgyline was used as an MAO-A inhibitor to maintain the concentration of serotonin. Since serotonin mitogens were selected using a monoclonal antibody against serotonin, it was expected that the selected mitogens would be recognized as serotonin by MAO-A and occupy the active site of MAO-A, thereby inhibiting the oxidative activity of MAO-A. The activity of MAO-A was analyzed using an activity assay kit based on the reaction of purified MAO-A to generate hydrogen peroxide to produce fluorescence (λex=535 nm, λex=587 nm), as shown in Figure 12. Although MAO-A and MAO-B share a high degree of amino acid sequence homology, they are known to differ in substrate specificity and inhibitor sensitivity. MAO-A inhibitors have been used to treat depression, anxiety, and migraine. Furthermore, MAO-B inhibitors have been used as dopamine-sparing agents for the treatment of Parkinson's disease. Therefore, it was necessary to confirm the selectivity of serotonin-mimetic mimotopes for MAO-A and MAO-B.

[0103] The expressed Fv antibodies were used to inhibit MAO-A and MAO-B enzymes. When MAO-A activity was measured after treatment with three serotonin analogues (Fv antibodies) and clorgyline, it was observed that MAO-A activity decreased depending on the concentration of mimotope (Fv antibodies), as shown in Figure 13. In the case of selegiline, an MAO-B inhibitor, no change in MAO-A activity was observed. The inhibitory activity of serotonin analogues (Fv antibodies), known to oxidize dopamine, was compared with another type of monoamine oxidase called MAO-B. When MAO-B activity was measured after treatment with three serotonin analogues (Fv antibodies) and clorgyline, it was observed that MAO-B activity did not change depending on the concentration of the analogue (Fv antibodies), as shown in Figure 14. In the case of selegiline, an MAO-B inhibitor, MAO-B activity was inhibited depending on the selegiline concentration. These results demonstrated that three serotonin-like analogues (Fv-antibodies) can be recognized as serotonin by MAO-A, and that the analogues (Fv-antibodies) can selectively inhibit MAO-A activity compared to MAO-B.

[0104] The synthesized peptides were also used to inhibit MAO-A and MAO-B enzymes. After treating with three serotonin-like mimotope (peptides) and clorgyline, MAO-A activity was measured, and it was observed that MAO-A activity decreased depending on the concentration of mimotope (peptide), as shown in Figure 15. In the case of selegiline, an MAO-B inhibitor, no change in MAO-A activity was observed. The inhibitory activity of serotonin-like mimotope (peptides), known to oxidize dopamine, was compared with another type of monoamine oxidase called MAO-B. After treating with three serotonin-like mimotope (peptides) and clorgyline, MAO-B activity was measured, and it was observed that MAO-B activity did not change depending on the concentration of mimotope (peptide), as shown in Figure 16. In the case of selegiline, an MAO-B inhibitor, MAO-B activity was inhibited depending on the concentration of selegiline. These results showed that three serotonin-like mimotopes (peptides) can be recognized as serotonin by MAO-A, and that the mimotopes (peptides) can selectively inhibit MAO-A activity compared to MAO-B.

[0105] A cell line (SH-SY5Y, ATCC code: CRL-2266) with serotonin receptors was treated. Three types of serotonin-mimetic mimotopes (labeled with fluorescein) were treated to the cell line to confirm binding to serotonin receptors on the cell surface. As shown in Figure 17, the fluorescence signal of mimotopes was observed, and a fluorescent dye for plasma membrane staining (PlasMem Red, Dojindo (Kumamoto, Japan)) was also treated for image comparison. When the images were merged, it was observed that mimotopes (labeled with fluorescein) bound to the cell membrane and partially penetrated into the cell. Among the three types of mimotopes with the highest concentration of penetrated mimotopes, serotonin-mimetic mimotope No. 40 showed the highest fluorescence signal in the cytoplasm. The fluorescence signal of the total number of cells after mimotope treatment was analyzed using FACS. As shown in Fig. 18, the fluorescence signal from the cells was significantly higher than that of the control group that was not treated with Mimotope, and the signal was observed to increase in the three types of Mimotope as the concentration increased from 0.1 to 10 μM. In the case of serotonin-like Mimotope No. 40, the highest fluorescence signal was observed among the three types of Mimotope.

[0106] As mentioned above, mimotop (labeled with fluorescein) was observed to bind to the cell membrane and partially penetrate into the cells. In the next step, MAO-A activity was measured using cell homogenates after treatment with serotonin-like analogs at concentrations of 0.1 to 10 μM. As shown in Figure 19, MAO-A activity was reduced by 50.7% with mimotop No. 7 (10 μM), 65.2% with mimotop No. 33 (10 μM), and 55.6% with mimotop No. 40 (10 μM). In the case of the positive control, a conventional MAO-A inhibitor, MAO-A activity was reduced by 69.8% when treated with clorgyline at a concentration of 10 μM. These results indicate that the serotonin-like mimotop penetrates into the cells and inhibits MAO-A at different rates. Mimotop No. In the case of 40, the inhibition rate was measured to be as high as that of existing MAO-A inhibitors.

[0107] To investigate the interaction between the two mimotopes of the screened clones and MAO-A, docking simulations were performed using AutoDock Vina software from Scripps Research. The interactions were visualized and analyzed with Discovery Studio Visualizer version v21.1.0.20298 from Dassault Systemes BIOVIA (San Diego, CA, USA). The structures of MAO-A (PDB IDs: 2BXR and 2Z5X) were obtained from the PDB (www.rcsb.org), and the interaction with clorgyline, a known MAO-A inhibitor, was performed as shown in Figure 20. The docking results showed that the inhibitor blocks the interaction with the cofactor (FAD) of MAO-A for serotonin oxidation in three loops (loop 1: V93-V95, loop 2: Y108-P118, loop 3: F208-N212). Among the three loops, flexible loop 2 is known to be essential for controlling substrate access to the active site of MAO-A. The amino acids of MAO-A that hydrophobicly interact with chlorgyline were L97, F208, C323, I335, L337, and F352, and Y407 of MAO-A formed a hydrogen bond with chlorgyline.

[0108] When serotonin-like mimotop-1 (peptide) interacts with MAO-A, docking results showed that serotonin-like mimotop-1 (peptide) binds in the middle of the entrance gate region of MAO-A (Figure 21). At this position, serotonin-like mimotop-7 (peptide-7) can interact with three surrounding loops (loop-1, flexible loop-2, and loop-3) as described previously, which are essential for controlling substrate access to the active site of MAO-A, and thus affect the access of the substrate (serotonin) to the cofactor (FAD) of MAO-A for serotonin oxidation. Serotonin-like analogue No. The amino acids of MAO-A that hydrophobically interact with V11, G8, M5, and P4 of 7 (peptide) are V93, L97, R109, F112, and P114, whereas G110, S209, N212, T487, and E492 of MAO-A form hydrogen bonds with M5, D10, D9, G8, and M7 of mimotope.

[0109] When serotonin-like mimotop-2 (peptide) interacted with MAO-A, the docking results showed that serotonin-like mimotop-2 (peptide) bound to the bottom of the single cavity site of MAO-A (Figure 22). At this position, serotonin-like mimotop-2 (peptide) can interact with two loops (flexible loop-2 and loop-3) of the substrate cavity, affecting the access of the substrate (serotonin) to the cofactor (FAD) of MAO-A for serotonin oxidation. The amino acids of MAO-A that hydrophobically interacted with P7, A3 of serotonin-like mimotop No. 33 (peptide) were P114, W116, H488, and A111, V115, P118, N125, N212, I486, T487 of MAO-A. It formed hydrogen bonds with R4, P7, F11, R4, C1, and A3 of Mimotope. Moreover, F112 and E492 of MAO-A interacted electrostatically with R4 of Mimotope.

[0110] For serotonin-like mimotope No. 40 (peptide), docking simulation analysis for MAO-A revealed that serotonin-like mimotope-3 (peptide) binds to the bottom of the entrance gate region of MAO-A (Fig. 23). At this position, serotonin-like mimotope-3 (peptide) may interact with the substrate cavity surrounding loop-3 and affect the access of the substrate (serotonin) to the cofactor (FAD) of MAO-A for serotonin oxidation. The amino acids of MAO-A that hydrophobically interact with F11 and P4 of serotonin-like mimotope No. 40 (peptide) are Y106, R109, and H488, whereas P107, A111, F112, P114, W116, Y121, and T487 of MAO-A interacted hydrophobically. It formed hydrogen bonds with F11, P4, P4, N5, L7, N5, and G1 of Mimotope. Furthermore, R96 of MAO-A interacted electrostatically with D10 of Mimotope. The interactions of MAO-A with chlorgiline and the three Mimotopes are summarized in Table 3.

[0111] Binding site of MAO-AHydrophobic interactionHydrogen bondElectrostatic interactionClorgilineL97, F208, C323, I335, L337, F352Y407-Mimotope No. 7V93, L97, R109, F112, P114G110, S209, N212, T487, E492 - Mimotope No. 33P114, W116, H488A111, V115, P118, N125, N212, I486, T487F112, E492Mimotope No. 40Y106, R109, H488P107, A111, F112, P114, W116, Y121, T487R96

[0112] Conclusion: Serotonin-like mimotopes were screened from a Fv-antibody library for use as inhibitors of monoamine oxidase-A (MAO-A). Target clones with binding affinity for serotonin were screened from the Fv-antibody library. Finally, three clones were screened by measuring their binding affinity for serotonin using FACS. The screened Fv-antibodies were expressed as soluble proteins as fusion proteins with green fluorescent protein, and the screened CDR3 region (11 residues) of the screened Fv-antibodies was also synthesized as peptides (12 residues) by inserting connecting amino acid residues. Three serotonin-like mimotopes (Fv-antibodies and peptides) were synthesized, and their binding constants were measured using a SPR biosensor. The binding affinities (K) of the expressed Fv-antibodies were D ) was measured to be 20.0 nM for Fv-7, 27.0 nM for FV-33, and 12.4 nM for Fv-40. And, the binding affinity (K) of the synthesized peptides D) were measured as 681 nM for peptide-7, 693 nM for peptide-33, and 733 nM for peptide-40 using a SPR biosensor. The inhibitory activity of serotonin analogues on MAO-A and MAO-B was analyzed in comparison with existing inhibitors such as clorgyline for MAO-A and selegiline for MAO-B. The three serotonin analogues can be recognized as serotonin by MAO-A, and the mimotopes can selectively inhibit MAO-A activity compared to MAO-B inhibitors. Finally, the screened serotonin analogues (peptides) were treated with a cell line (SH-SY5Y, ATCC code: CRL-2266) with a serotonin receptor to confirm binding to the serotonin receptor on the cell surface. Analysis of cell lysates after treatment with three serotonin-mimetic mimotope (peptides) showed that MAO-A activity was reduced by 50.7% with mimotope No. 7 (10 μM), 65.2% with mimotope No. 33 (10 μM), and 55.6% with mimotope No. 40 (10 μM). These results indicate that serotonin-mimetic mimotopes penetrate cells and inhibit MAO-A at different rates, with mimotope No. 40 exhibiting an inhibitory effect as high as that of conventional MAO-A inhibitors.

[0113] Although the present invention has been described above with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

Claims

1. It has binding ability to monoamine oxidase, Comprising one or more peptide sequences selected from the group comprising the peptide sequence of SEQ ID NO: 1, the peptide sequence of SEQ ID NO: 2 and the peptide sequence of SEQ ID NO:

3. Peptides: [Sequence number 1] CSLPMTAGDDV [Sequence number 2] GLTGPPTTQDF [Sequence number 3] GPDETINLGDF.

2. In paragraph 1, The above peptide selectively reacts only with MAO-A enzyme among serotonin-decomposing enzymes MAO-A (Monoamine oxidase A) and MAO-B (Monoamine oxidase B), thereby reducing the serotonin-decomposing activity of MAO-A. Peptide.

3. In paragraph 2, The peptide binds adjacent to the active site of the MAO-A enzyme and blocks serotonin from accessing the active site of the MAO-A. Peptide.

4. Coding a peptide according to any one of claims 1 to 3; Nucleic acid.

5. Containing nucleic acid according to Article 4, Recombinant expression vector.

6. Transformed with a recombinant expression vector according to Article 5, cell.

7. In paragraph 6, The above cell comprises one or more cells selected from the group including animal cells, plant cells, yeast, E. coli, and insect cells. cell.

8. In paragraph 7, The above cells are monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell line, HuT 78 cells and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp, Pseudomonas sp, Proteus mirabilis or Staphylococcus sp, Aspergillus sp, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp and Containing one or more cells selected from the group comprising Neurospora crassa, cell.

9. It has binding ability to monoamine oxidase, Comprising an amino acid fragment having at least one peptide sequence selected from the group comprising the peptide sequence of SEQ ID NO: 1, the peptide sequence of SEQ ID NO: 2 and the peptide sequence of SEQ ID NO: 3; Compositions for inhibiting MAO-A: [Sequence number 1] CSLPMTAGDDV [Sequence number 2] GLTGPPTTQDF [Sequence number 3] GPDETINLGDF.

10. In paragraph 9, The above MAO-A inhibitory composition selectively reacts only to the MAO-A enzyme among the serotonin-decomposing enzymes MAO-A (Monoamine oxidase A) and MAO-B (Monoamine oxidase B), thereby reducing the serotonin-decomposing activity of MAO-A. A composition for inhibiting MAO-A.

11. In paragraph 10, The above MAO-A inhibitory composition binds adjacent to the active site of the MAO-A enzyme and blocks serotonin from accessing the active site of the MAO-A. A composition for inhibiting MAO-A.

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