KR20210032847A - Self-assembly formed by co-assembling the D-enantiomer and L-enantiomer of a conjugate comprising a peptide molecule capable of self-assembly in an organelle, and a pharmaceutical composition comprising the same - Google Patents

Abstract

The present invention relates to: a self-assembly formed by co-assembly of a D-enantiomer and an L-enantiomer of a conjugate comprising a peptide molecule capable of self-assembly in an organelle; and a pharmaceutical composition for preventing or treating cancer containing the same. According to the self-assembly and the pharmaceutical composition containing the same, it is possible to provide an effect that can be usefully used for preventing or treating cancer by targeting mitochondria of cancer cells.

Description

Self-assembly formed by co-assembly of a conjugate containing a peptide molecule capable of self-assembly in organelles, and a pharmaceutical composition for preventing or treating cancer comprising the same {Self-assembly formed by co- assembling the D-enantiomer and L-enantiomer of a conjugate comprising a peptide molecule capable of self-assembly in an organelle, and a pharmaceutical composition comprising the same}

It relates to a self-assembly formed by co-assembly of a conjugate containing a peptide molecule capable of self-assembly in organelles, and a pharmaceutical composition for preventing or treating cancer comprising the same.

Molecular self-assembly is a field that attracts attention in creating functional nanomaterials that can be applied to various biomedical fields such as drug delivery, tissue engineering, and gene therapy. Self-assembly of most molecules into nanostructures has been studied in solvents, but assembly of synthetic amphiphilic peptides inside cells has not been much studied, making it a promising research topic in the field of biomimetic assembly to regulate cell function. have.

Mitochondria are highly dynamic organelles that produce energy through oxidative phosphorylation, and are involved in cell death processes including fatty acid oxidation, TCA cycle, amino acid metabolism, as well as apoptosis, necrosis and necroptosis. It is known to be related. Mitochondrial dysfunction has been reported in several degenerative diseases associated with the accumulation of protein fibers in the brain, Alzheimer's disease, Parkinson's disease, Huntington's disease, and Amyotrophic lateral sclerosis. have. It is known that abnormally folded proteins rich in beta-sheet conformation, such as Aβ, are observed in the mitochondria of patients with Alzheimer's disease, and that such abnormally folded proteins exhibit cytotoxic effects.

One aspect includes a mitochondrial targeting moiety and a peptide represented by (Xaa)n-Lys, the mitochondrial targeting moiety is linked to the lysine amino acid of the peptide, and n is the amino acid Xaa linked through a peptide bond. As a number, it is an integer of 2 to 200, and Xaa is each independently valine, leucine, isoleucine, methionine, phenylalanine, asparagine, glutamic acid, aspartic acid, glycine, alanine, serine, threonine, cysteine, proline, glutamine, histidine, lysine , Arginine, tyrosine, tryptophan, and C3-C10 cycloalkyl-binding variants thereof, and the amino acid forms a beta-sheet secondary structure between two or more amino acids, and the peptide is It provides a self-assembly formed by co-assembly of the D-enantiomer and the L-enantiomer of a conjugate containing a beta-sheet secondary structure. will be.

Another aspect is to provide a pharmaceutical composition for preventing or treating cancer, comprising the self-assembly as an active ingredient.

One aspect includes a mitochondrial targeting moiety and a peptide represented by (Xaa)n-Lys, the mitochondrial targeting moiety is linked to the lysine amino acid of the peptide, and n is the amino acid Xaa linked through a peptide bond. As a number, it is an integer of 2 to 200, and Xaa is each independently valine, leucine, isoleucine, methionine, phenylalanine, asparagine, glutamic acid, aspartic acid, glycine, alanine, serine, threonine, cysteine, proline, glutamine, histidine, lysine , Arginine, tyrosine, tryptophan, and C3-C10 cycloalkyl-binding variants thereof, and the amino acid forms a beta-sheet secondary structure between two or more amino acids, and the peptide is It provides a self-assembly formed by co-assembly of the D-enantiomer and the L-enantiomer of a conjugate containing a beta-sheet secondary structure. .

The peptide means that 3 to 201 amino acids are linked through a peptide bond, and the peptide may be represented by (Xaa)n-Lys. Wherein n is the number of amino acids linked through a peptide bond, 2 to 200, 2 to 150, 2 to 100, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to It may be an integer of 8, 2 to 6, 2 to 4, or 2 to 3, and each of Xaa is independently valine, leucine, isoleucine, methionine, phenylalanine, asparagine, glutamic acid, aspartic acid, glycine, alanine, serine, threonine, Cysteine, proline, glutamine, histidine, lysine, arginine, tyrosine, tryptophan, and C3-C10 cycloalkyl binding variants thereof may be selected from the group consisting of, for example, n is 2 and Xaa is the same phenylalanine, alanine, cyclohexyl Alanine or valine may form a peptide bond, but is not limited thereto.

The peptide (Xaa)n-Lys is a variant in which a C3-C10 cycloalkyl group is bonded to each amino acid or R group of an amino acid in the order of C-terminus from the N-terminus of the amino acid (C3-C10 cycloalkyl-binding variant) is a peptide bond. It may be connected, but is not limited thereto.

The hydroxy group of the carboxyl group at the C-terminus of Lys of the peptide (Xaa)n-Lys may be substituted with one selected from the group consisting of an amine group, an alcohol group, an ether group, and an acetyl group. In addition, other chemical groups may be chemically bonded, for example, an amine group may be bonded.

The C3-C10 cycloalkyl-linked variant may be one in which cycloalkyl having 3 to 10 carbon atoms is bonded to the R group of the amino acid, for example, cyclohexyl alanine in which cyclohexyl is bonded to the carbon of the methyl group, which is the R group of alanine. alanine; CHA), but is not limited thereto.

The peptide may have a beta-sheet secondary structure, and the peptide is a beta-sheet secondary structure between two or more amino acids present inside the peptide or present in other peptides. It may be to form, but is not limited thereto.

The beta-sheet secondary structure is one of the secondary structures formed by the interaction between amino acid sequences constituting a protein, and is formed through hydrogen bonds between recognized molecules that are horizontally connected. The beta sheet secondary structure is known to be associated with protein aggregation or fiber formation in many human diseases.

The mitochondrial targeting moiety refers to a function of targeting a corresponding substance to the mitochondria inside a cell, and may be, for example, triphenylphosphonium (TPP), but is not limited thereto.

In addition, due to the mitochondrial targeting moiety constituting the conjugate in the present invention, the conjugate may be targeted to the mitochondria inside the cell, and the conjugate may enter the mitochondria by passing through the double membrane of the mitochondria.

In the present invention, the conjugate can enter the mitochondria due to the mitochondrial targeting moiety, and the conjugate that enters the mitochondria forms a self-assembly of peptides synthesized inside the mitochondria of cancer cells specifically for cancer cells. It can induce apoptosis.

Regarding the formation of self-assembly in the mitochondria in the cancer cells, in the case of cancer cells, peptides contained in the pharmaceutical composition are accumulated due to the large negative internal membrane potential of the cancer cell mitochondria, and the self-assembly process is accelerated through this. In the case of normal cells, since the negative membrane potential is smaller than that of cancer cells, the peptides are not sufficiently accumulated and fibers are not formed by self-assembly in the mitochondria, so that the self-assembly is not formed.

The number of mitochondrial targeting moieties linked to the peptide may be an integer selected from the range of 1 to the number of amine groups present in the peptide represented by (Xaa)n-Lys. When an amine group is present at the C-terminus of the peptide, the number of mitochondrial targeting moieties linked to the peptide may be an integer within m+1, which is a number from 1 to the number of lysine amino acids (m) plus 1. In one example, the number of mitochondrial targeting moieties linked to the peptide may be 1 or 2, but is not limited thereto.

In the present invention, the N-terminus of the peptide of the conjugate may be connected by a fluorophore and an amide bond, but is not limited thereto.

The fluorophore refers to an atomic group necessary for an organic compound to emit fluorescence. In the present invention, the fluorophore is a compound consisting of pyrene, NBD (4-nitro-2, 1, 3-benzoxadiazole), perylene, naphtalene, coronene, and an aromatic compound, which is connected to the N-terminus of the peptide by an amide bond. Since it has a structure, it may be one or more selected from the group consisting of fluorophores that are easily stacked by pi bonds, but is not limited thereto.

The peptide and the mitochondrial targeting moiety, the peptide and the fluorophore, or both may be directly connected or may be connected through a linker. The linker may be an alkyl group having 1 to 10, 1 to 5, 1 to 4, or 2 to 3 carbon atoms, an alkene group, or an alkyne group, but is not limited thereto. In addition, the linker may include a functional group capable of forming an appropriate bond such as a peptide bond and an amide bond at one end.

The conjugate, or a pharmaceutically acceptable salt thereof, may be represented by the following formula (1):

[Formula 1]

In Formula 1,

R ¹ refers to hydrogen or a fluorophore, and is a compound consisting of hydrogen, pyrene, NBD (4-nitro-2, 1, 3-benzoxadiazole), perylene, naphtalene, coronene, and aromatic. It may be one or more selected from the group consisting of fluorophores that are easily stacked on each other,

The linker connects the N-terminus of the peptide and the fluorophore, and does not exist or has 1 to 10, 1 to 5, 1 to 4, or 2 to 3 carbon atoms, an alkyl group, an alkene group (alkene) or alkyne may be used, but the present invention is not limited thereto.

In addition, the linker may include a functional group capable of forming an appropriate bond such as a peptide bond and an amide bond at one end, and pyrene, NBD (4-nitro-2, 1, 3-benzoxadiazole), perylene , naphtalene, coronene, and aromatic compounds, which have a flat structure, and thus connect the peptide with at least one selected from the group consisting of fluorophores that are easily stacked with each other through an amide bond.

Dama, when R ¹ is hydrogen, a linker may not be present,

R ² is a C-terminal (i.e., C-terminal of lysine) moiety of a peptide, a hydroxy group (OH), an amine group, an alkyl group, an alcohol group, an ether group, and an acetyl group, (above, alkyl group, alcohol group, ether The group and the acetyl group may be one or more selected from the group consisting of 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 5 carbon atoms.

In one example, the conjugate, or a pharmaceutically acceptable salt thereof, may have a structure of Formula 2 or Formula 3, but is not limited thereto.

[Formula 2]

[Formula 3]

In Formula 2, the R being an amine group is called Mito-FF in the following examples, and can form fibers by self-assembling with the same or different conjugates in the mitochondria in cancer cells, and in Formula 3, R is an amine The reason is that it is called Mito-FF-NBD in the following examples, and can form fibers by self-assembling with conjugates that are the same or different from each other in mitochondria in cancer cells.

In the present invention, the conjugate may exhibit amphiphilicity, and the amphiphilicity refers to a state having an affinity for both polar and non-polar substances, and in the conjugate of the present invention, non-polar amino acids and fluorophores included in the peptide are hydrophobic. As, the polar amino acid exhibits hydrophilicity, and thus the conjugate may exhibit amphiphilicity, but is not limited thereto.

The conjugate may exist in an isomeric form, and the isomer refers to a compound having the same molecular formula but not the same spatial arrangement, and preferably refers to a stereoisomer. Stereoisomers include enantiomers and diastereomers. An enantiomer refers to an isomer that does not overlap with the mirror image like the relationship between the right hand and the left hand, and is also called an optical isomer. Also, depending on the structure, it can be classified into a D-enantiomer or an L-enantiomer. Diastereoisomers refer to stereoisomers that are not in an enantiomorphic relationship, and are 　cis-trans　isomers in which the spatial arrangement of atoms is different due to the unfree rotation of the carbon-carbon bonds in the diastereomers with different spatial arrangements of constituent atoms and in cycloalkane and alkene compounds. Can be divided by 　.

Another aspect provides a pharmaceutical composition for preventing or treating cancer, comprising the self-assembly as an active ingredient.

The self-assembly may exist in the form of a pharmaceutically acceptable salt. The pharmaceutically acceptable salt includes both an acid or base addition salt and a stereochemically isomeric form thereof, and may be, for example, an organic acid or an inorganic acid addition salt. The salt includes any salt that maintains the activity of the parent compound in the administration target and does not cause undesirable effects, and is not particularly limited.

These salts include inorganic and organic salts, for example, acetic acid, nitric acid, aspartic acid, sulfonic acid, sulfonic acid, maleic acid, glutamic acid, formic acid, succinic acid, phosphoric acid, phthalic acid, tannic acid, tartaric acid, hydrobromic acid. , Propionic acid, benzenesulfonic acid, benzoic acid, stearic acid, lactic acid, bicarboxylic acid, bisulfuric acid, bitartrate, oxalic acid, butyric acid, calcium idete, carbonyl acid, chlorobenzoic acid, citric acid, idetic acid, toluene Sulfonic acid, fumaric acid, gluceptic acid, esylic acid, pamoic acid, gluconic acid, methylnitric acid, malonic acid, hydrochloric acid, hydroiodoic acid, hydroxynaphthoic acid, isethionic acid, lactobionic acid, mann Delic acid, mucous acid, napsylic acid, muconic acid, p-nitromethane sulfonic acid, hexamic acid, pantothenic acid, monohydrogen phosphate, dihydrogen phosphate, salicylic acid, sulfamic acid, sulfanilic acid, methanesulphonic acid have.

In addition, in the form of the salt, salts of alkali and alkaline earth metals such as ammonium salt, lithium salt, sodium salt, potassium salt, magnesium salt and calcium salt, for example, benzathine, N-methyl-D-glucamine, hydride It includes salts having an organic base such as a labamine salt, and salts having an amino acid such as arginine and lysine. In addition, the salt form can also be converted to the free form by treatment with a suitable base or acid.

The self-assembly, or a pharmaceutical composition for the prevention or treatment of cancer, containing as an active ingredient, a pharmaceutically acceptable salt thereof may be one that induces apoptosis by forming a self-assembly in the mitochondria in cancer cells.

The apoptosis (apoptosis) is a form of cell death controlled by genes. It is a necrosis or pathological death of cells, which is distinguished from necrosis, which is the death of cells caused by stimulation such as burns, bruises, and poisons. As a result, it starts with the shrinking of the cell, and then a gap is created between adjacent cells, and in the cell, 　DNA is regularly cleaved and fragmented, which means that the cell dies.

Regarding the formation of self-assembly in the mitochondria in the cancer cells, in the case of cancer cells, peptides contained in the pharmaceutical composition are accumulated due to the large negative internal membrane potential of the cancer cell mitochondria, and the self-assembly process is accelerated through this. In the case of normal cells, since the negative membrane potential is smaller than that of cancer cells, the peptides are not sufficiently accumulated and fibers are not formed by self-assembly in the mitochondria, so that the self-assembly is not formed.

The cancer targeted for prevention or treatment of the pharmaceutical composition may be solid cancer or hematologic cancer, and may be primary cancer or metastatic cancer. The cancer may be selected from the group consisting of cervical cancer, liver cancer, skin cancer, prostate cancer, mammary cancer, breast cancer, nasopharyngeal adenocarcinoma, lung cancer, or brain tumor, but is not limited thereto.

The pharmaceutical composition of the present invention may be in any form suitable for the intended method of administration. In the pharmaceutical composition of the present invention, "administration" means introducing a predetermined substance to the patient by any suitable method, and the administration route of the pharmaceutical composition is any general route as long as the drug can reach the target tissue. It can be administered through. For example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc. can be mentioned, but is not limited thereto. Does not. In addition, the pharmaceutical composition of the present invention can be administered by any device capable of moving the active ingredient to target cells.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably administered orally. When administered parenterally, the pharmaceutical composition of the present invention may be administered by intravenous injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. It is preferable that the route of administration of the pharmaceutical composition of the present invention is determined according to the type of disease to be applied.

One embodiment provides a pharmaceutical composition for preventing or treating cancer that is formulated in an oral dosage form or a parenteral dosage form.

The pharmaceutical composition provided herein is formulated according to a conventional method, oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, or suspensions, emulsions, freeze-dried preparations, It can be formulated and used in parenteral formulations such as external preparations, suppositories, sterile injection solutions, and preparations for implantation.

The pharmaceutical composition may further include a pharmaceutically acceptable excipient that can be used for formulation in addition to the active ingredient (ie, a conjugate or a pharmaceutically acceptable salt thereof).

Excipients that can be used in the formulation of the pharmaceutical composition of the present invention are carriers, vehicles, diluents, solvents such as monohydric alcohols such as ethanol, isopropanol and polyhydric alcohols such as glycerol and edible oils such as For example soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, oily esters such as ethyl oleate, isopropyl myristate; Binders, adjuvants, solubilizers, thickeners, stabilizers, disintegrants, lubricants, lubricants, buffers, emulsifiers, wetting agents, suspending agents, sweetening agents, coloring agents, flavoring agents, coating agents, preservatives, antioxidants, processing agents, drug delivery modifiers And enhancers such as calcium phosphate, magnesium state, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpi It may include one or more selected from the group consisting of rolidone, low melting point wax, and ion exchange resin, but is not limited thereto.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It does not become. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like 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 may be formulated in the form of various oral dosage forms. For example, it may be a formulation for oral administration such as tablets, pills, hard/soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, and the like. Such formulations for oral administration are, in addition to the active ingredients, depending on the usual composition of each formulation, for example, diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine, silica, and talc. , Stearic acid and its magnesium or calcium salt, and/or a pharmaceutically acceptable carrier such as a lubricant such as polyethylene glycol.

In addition, when the dosage form for oral administration is a tablet, it may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and if Depending on the case, it may contain a disintegrant such as starch, agar, alginic acid or sodium salt thereof, a boiling mixture and/or an absorbent, a colorant, a flavoring agent or a sweetening agent.

In addition, the pharmaceutical composition may be formulated in the form of a parenteral dosage form, in which case it is administered by a parenteral administration method such as subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. At this time, in order to formulate the parenteral dosage form, the pharmaceutical composition is prepared as a solution or suspension in which the active ingredient is mixed in water with a stabilizer or buffer, and such a solution or suspension is prepared in a unit dosage form of an ampoule or vial. Can be.

In addition, the pharmaceutical composition may be sterilized or further include adjuvants such as preservatives, stabilizers, hydrating agents or emulsification accelerators, salts and/or buffers for controlling osmotic pressure, or other therapeutically useful substances. And, it may be formulated according to a conventional method of mixing, granulating or coating.

The content of the self-assembly or a pharmaceutically acceptable salt thereof in the pharmaceutical composition can be appropriately adjusted according to the purpose of use of the pharmaceutical composition, the form of the formulation, etc., for example, 0.001 to 99% by weight, 0.001 by weight based on the total weight of the pharmaceutical composition. It may be to 90% by weight, 0.001 to 50% by weight, 0.01 to 50% by weight, 0.1 to 50% by weight, or 1 to 50% by weight, but is not limited thereto.

In addition, the therapeutically effective amount of the self-assembly or a pharmaceutically acceptable salt thereof contained in the pharmaceutical composition of the present invention means an amount required for administration in order to expect a disease treatment effect. Therefore, it can be adjusted according to the patient's disease type, the severity of the disease, the type of the active ingredient to be administered, the type of the dosage form, the patient's age, sex, weight, health status, diet, administration time and administration method of the drug. For example, for mammals, including humans, it may be administered in a pharmaceutically effective amount of 0.01 to 500 mg/kg (body weight) per day. The pharmaceutically effective amount may depend on an amount capable of obtaining a desired effect, for example, a therapeutic and/or prophylactic effect for cancer. The pharmaceutically effective amount may be divided once or twice a day and administered through an oral or parenteral route (eg, intravenous injection, intramuscular injection, etc.).

According to the self-assembly according to an aspect and a pharmaceutical composition comprising the same, there is an effect that can be usefully used in the prevention or treatment of cancer by targeting the mitochondria of cancer cells.

Figure 1 is a schematic diagram of the co-assembly of peptides within the mitochondria of cells:
1A is a picture of Mito-rac co-assembled within the mitochondria; 1B is a picture in which Mito-FF is co-assembled in the mitochondria.
2 is a diagram showing the structures of Mito-FF and Mito-ff.
3 is a graph showing the results of analyzing the CD spectrum.
4 is a graph showing the results of Fourier-transform infrared (FR-IR) analysis.
5A is a graph showing a result of analyzing a critical aggregation concentration (CAC) value; 5B is a photograph and graph showing the results of turbidity analysis and light scattering experiments; 5C is a graph showing the result of analyzing the emission spectrum.
6A is a transmission electron microscopy (TEM) image of Mito-FF; 6B is a TEM image of Mito-ff; 6C is a TEM image of Mito-rac; 6D is a Cryo-TEM image of Mito-FF; 6E is a Cryo-TEM image of Mito-ff; 6f is a Cryo-TEM image of Mito-rac; 6G is a confocal microscope image of Mito-FF; 6H is a confocal microscope image of Mito-ff; 6I is a confocal microscope image of Mito-rac.
7 is a diagram showing a coarse-grained (CG) model.
Figure 8a is a diagram showing the pyrene stacking structure and binding energy in Mito-FF fibers; 8B is a diagram showing a structure in which a pair of Mito-FF backbones are closely facing each other; 8C is a graph showing the probability distribution of the dihedral angle formed by the backbone of Mito-FF and Mito-ff in Mito-rac; 8D and 8E are diagrams showing the pyrene stacking structure and binding energy of Mito-FF and Mito-ff.
9 is a graph showing the results of analyzing radial distribution functions (RDFs).
Figure 10a is a picture showing the formation of a fiber bundle of Mito-rac; 10B is a graph showing the results of RDF analysis; Figure 10c is a picture showing the fiber made up of Mito-FF.
11A is a diagram showing the structure of L-Mito-FF-NBD; 11B is a diagram showing that L-Mito-FF-NBD is co-assembled with Mito-FF or Mito-ff over time; 11C is an image showing the results of time-dependent confocal microscopy analysis; 11D is a graph showing the results of analysis using a flow cytometer.
12 is an image showing the results of confocal microscopy analysis:
12A is an image observing fragmentation of mitochondria; 12B is an image observing the depolarization of the mitochondrial membrane.
13 is a graph showing the results of cytotoxicity analysis:
13A is a graph showing the results of cytotoxicity in HT-29 cancer cells; 13B is a graph showing the results of cytotoxicity in IMR-90 normal cells.
14A is an optical image taken to see the efficacy of tumor treatment in mice; 14B is a graph showing tumor volume over time; 14C is a graph showing the weight of mice over time; 14D and 14E are TEM images of mitochondria of tumor sections treated with Mito-rac.
15 is a diagram showing the CGMD simulation results:
15A is a diagram showing a defect region of cancer cell membranes caused by Mito-rac; 15B is a diagram showing a defect region of a cancer cell membrane caused by Mito-FF.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Experimental materials and equipment

All peptides were manually synthesized at 0.25 μM scale. The product was purified by High-performance liquid chromatography (HPLC, Agilent Technologies, USA) with a C18 reverse column in an ACN/water mixture. Amino acids were purchased from Bead Tech (Korea) and APEX Bio (APEX Bio, Houston, USA). The tetra methyl rhodamine methyl ester (TMRM) was purchased from Santa Cruz Biotechnologies (Korea). MitoSox and Mito Tracker Red FM, products capable of fluorescently staining the mitochondria of living cells, were purchased from Life Technologies. The synthesis of the peptide was confirmed by matrix-assisted laser desorption/ionization (MALDI-TOF / TOF, Bruker, Ultraflex III). Confocal laser scanning microscopy (CLSM) images were taken with LSM 780 (Zeiss). Transmission electron microscopy (TEM) images were obtained using a BioTEM system (JEOL, JEM 1400). Spectroscopic fluorescence analysis was performed using a fluorescence analyzer Cary Eclipse (Agilent). The CD spectrum was confirmed using a CD spectroscopy (JASCO). Count rate analysis was performed using zetasizer (Malvern). Flow cytometry was performed using BD LSRFortessa (BD Biosciences). Cytotoxicity analysis was performed using a plate reader SpectraMax M5 (Molecular Devices).

Example 1. Synthesis of Mito-FF and Mito-ff

Mito-FF is composed of three peptides of L-form Phe-Phe-Lys (FFK) as basic units, and based on 9-fluorenylmethoxycarbonyl chemistry (Fmoc), standard solid phase synthesis (SSPS) )) was synthesized as follows.

Mito-FF was synthesized using standard 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis at 0.25 nmol scale. For the synthesized peptide, 1-pyrene carboxylic acid (500 umol) and O-(Benzotriazol-1-yl)-N,N,N',N''-tetramethyluronium hexafluorophosphate in the presence of diisopropyl ethyl amine (DIPEA, 500 umol) (HBTU) (500 umol) was treated and mixed with stirring for 24 hours at room temperature in DMF (dimethylformamide). Resin was collected through filtration and washed with DMF to remove unreacted compounds. The product cut from the resin was precipitated in cold ether using a cleavage cock tail TFA/Water/Tri isopropyl amine mixture (9.5:0.5:0.5). The product was purified by HPLC, and mass spectrometry was performed using MALDI-TOF/TOF. For the conjugation of triphenyl phosphonium (TPP), the synthesized peptide (0.02 mmol) was mixed with 1-hexyl triphenylphosphonium bromide salt (0.04 mmol) and tri ethyl amine (0.02 mmol) in DMF, stirring at room temperature for 12 hours. . The pure product is purified using HPLC, dried and then frozen and stored. Mito-ff synthesis was performed in the same manner as above using three peptides of Phe-Phe-Lys (FFK) in D-form form.

The N-terminus of the peptides of Mito-FF and Mito-ff synthesized by the above method is linked to pyrene (butyric acid) to enable fluorescence detection, and the amine group of the side chain of lysine is linked to TPP for mitochondrial targeting. have. In Mito-FF, the pyrene moiety not only acts as a fluorescent source, but also improves the hydrophobic bonding force and pi-pi interconnection to improve self-assembly.

2 is a diagram showing the molecular structures of Mito-FF and Mito-ff according to an embodiment of the present invention.

Experimental Example 1. Characterization of Mito-rac

An experiment was conducted to analyze the properties of Mito-rac. Mito-rac means that Mito-FF prepared in Example 1 and Mito-ff, which is a D-enantiomer thereof, were co-assembled in equimolarity.

First, circular dichroism (CD) spectrum was analyzed. The peptide was dissolved in saline phosphate buffer (250 μM, PBS) to analyze the CD spectrum. As a result, Mito-FF exhibited a negative Cotton effect, Mito-ff exhibited a positive cotton effect, and Mito-rac did not show any signal (FIG. 3). This means that Mito-rac is an equimolar mixture of Mito-FF and Mito-ff, and shows no signal due to destructive interference.

Fourier-transform infrared (FR-IR) analysis was performed to confirm that the structure was not deformed due to the formation of Mito-rac. Specifically, Mito-FF, Mito-ff, and Mito-rac were each dissolved in water to prepare a 100 μM solution, and freeze-dried for one day. The collected powder was analyzed using FT-IR spectroscopy (VARIAN) in ATR mode. As a result, since there was no change in the enantiomer having a peak at 1630 and 1670 cm ^-1 , it was confirmed that the beta-sheet structure was preserved in Mito-rac (FIG. 4). These results indicate that Mito-rac has the same secondary structure as the enantiomer.

3 is a graph showing the results of analyzing the CD spectrum.

4 is a graph showing the results of Fourier-transform infrared (FR-IR) analysis.

Experimental Example 2. Analysis of Aggregation of Mito-rac

Experiments were performed to compare the aggregation phenomenon of Mito-rac with Mito-FF and Mito-ff.

First, in order for molecules to self-assemble inside cells, the concentration of molecules in cells must be much higher than the CAC (Critical Aggregation Concentration) value. In order to check whether Mito-rac of the present invention can be self-assembled inside cells, Mito The CAC value of -rac was analyzed. Specifically, when pyrene exists as a non-aggregated molecular entity and when it is present as agglomerated, the intensity of light of 344 nm and 339 nm in the excitation spectrum is different, and the excitation spectrum at 344 nm and 339 nm is measured. The ratio of the 344 nm and 339 nm excitation spectrum was calculated, and the CAC value of Mito-rac was determined through the standard curve of FIG. 5. As a result, it was confirmed that the CAC value of Mito-rac was 30 μM (FIG. 5A). This is half of the CAC value of Mito-FF or Mito-ff, which is 60 μM, which means that Mito-rac is more easily self-assembled than Mito-FF or Mito-ff.

In addition, when the concentration of Mito-FF, Mito-ff, and Mito-rac was 100 μM and the turbidity was analyzed in PBS, Mito-rac formed large aggregates at a low concentration and showed turbidity. When viewed with the naked eye, it was confirmed that the self-assembled solution of Mito-rac was more turbid than the Mito-FF and Mito-ff solutions of the same concentration (FIG. 5B). These results show that as a result of performing a light scattering experiment on PBS with 200 μM concentration of Mito-FF, Mito-ff, and Mito-rac, Mito-rac at the same concentration showed a counting rate of 251.96 ± 22.3 kcps, Mito-FF and Mito This is a result consistent with that -ff showed a higher counting rate compared to the counting rates of 70.2 ± 14 kcps and 63.8 ± 9.1 kcps, respectively (FIG. 5B).

Since pyrene molecules exhibit different emission characteristics when aggregated, emission spectra were analyzed in PBS with the concentration of Mito-FF, Mito-ff, and Mito-rac as 200 μM. As a result, Mito-rac significantly decreases the emission intensity of Mito-rac with the expansion of the peak in the excimer region, which exhibits less stable or unstable pi-pi interactions between pyrene molecules. (Fig. 5c).

The above experimental results show that the self-assembly of Mito-rac is quite different from that of Mito-FF or Mito-ff, and Mito-rac is self-assembled into a larger aggregate, and Mito-rac self-assembled This means that the self-assembly tends to be higher than that of Mito-FF or Mito-ff.

5A is a graph showing a result of analyzing a critical aggregation concentration (CAC) value; 5B is a photograph and graph showing the results of turbidity analysis and light scattering experiments; 5C is a graph showing the result of analyzing the emission spectrum.

Experimental Example 3. Mito-rac fiber structure analysis

The morphology of Mito-FF, Mito-ff, and Mito-rac was observed with a transmission electron microscope (TEM).

Specifically, set the concentration of Mito-FF, Mito-ff and Mito-rac to 100 μM in PBS solution, place a drop on a formvar / carbon-coated copper grid and evaporate under atmospheric conditions. Made it. Samples were stained with 2% by weight uranyl acetate solution, evaporated for 1 minute, and excess solution was removed with filter paper. Then, it was observed with a JEM-1400 TEM operating at 120 kV. Mito-FF, Mito-ff, and Mito-rac fiber structure information self-assembled with more than 100 nanofibers were obtained with more than 10 micrographs using the Image J program. As a result, Mito-FF and Mito-ff were self-assembled into narrow fibers with a diameter of about 10 nm (Figs. 6a and 6b), whereas Mito-rac formed thick fiber bundles and hardly any individual fibers were seen ( Fig. 6c). The diameters of the Mito-rac bundles ranged from 30 to 100 nm, and were polydisperse. This is where many fibers are aggregated to form super-fibers.

In order to further confirm the fiber formation of Mito-FF, Mito-ff, and Mito-rac in the solution, it was observed with a low-temperature transmission electron microscope (cryo-TEM). Cryo-TEM images of Mito-FF and Mito-ff showed a fiber structure with a narrow diameter, similar to that observed in dried TEM (FIGS. 6D and 6E ). In the cryo-TEM image of Mito-rac, in a solution containing Mito-FF and Mito-ff at equimolar concentrations, a super-fiber structure was confirmed that was thicker than Mito-FF and Mito-ff fibers and composed of several different diameters (Fig. 6f).

The structure of the Mito-rac super-fiber was further observed using a high-resolution confocal microsopy.

To confirm the Miro-rac structure under a confocal microscope, another peptide called Mito-FF-NBD was synthesized. Mito-FF-NBD is a peptide analog of Mito-FF, which has 4-nitro-2,1,3-benzoxadiazole (NBD) instead of pyrene. NBD dye is a fluorophore used in imaging studies on biological elements, and is known to exhibit more intense fluorescence in a hydrophobic environment than in a hydrophilic environment, and this property can be used to confirm the formation of fibers inside cells.

Mito-FF, Mito-ff, and Mito-rac in PBS were mixed with 10% of Mito-FF-NBD, respectively, and then observed using a confocal microscope (confocal microsopy). As a result, Mito-FF and Mito-ff showed a faint image without visible fibers (Fig. 6g, Fig. 6h), whereas Mito-rac formed a super-fiber with a diameter of 100 nm, and a distinct green fiber network was observed. Became (Fig. 6i). These results mean that Mito-FF and Mito-ff exist as narrow fibers and Mito-rac exists as super-fibers, which means that there are morphological differences.

6A is a transmission electron microscopy (TEM) image of Mito-FF; 6B is a TEM image of Mito-ff; 6C is a TEM image of Mito-rac; 6D is a Cryo-TEM image of Mito-FF; 6E is a Cryo-TEM image of Mito-ff; 6f is a Cryo-TEM image of Mito-rac; 6G is a confocal microscope image of Mito-FF; 6H is a confocal microscope image of Mito-ff; 6I is a confocal microscope image of Mito-rac.

Experimental Example 4. Mito-rac pyrene-pyrene stacking analysis

In order to confirm the mechanism by which the Mito-rac super-fiber is formed at the molecular level, it was analyzed using coarse-grained molecular dynamics (CGMD). First, the structural differences between Mito-rac, Mito-FF, and Mito-ff were confirmed. Except for Mito-rac fibers with a length of 30 nm, Mito-FF, Mito-ff and Mito-rac, which form narrow fibers with a radius and length of about ~2.5 and 32 nm, respectively, include pyrene, TPP, phenyl and backbone ( BB) There are four components of the group (Fig. 7).

In addition, it was confirmed that the pyrene groups showed different stacking patterns in each fiber. Specifically, the binding energy was calculated by molecular dynamics calculation using the COMPASS II force field. As a result, Mito-FF and Mito-ff showed high binding energy of -138 kcal / mol when packed with stable pyrene stacking (Figs. 8A and 8B), whereas in Mito-rac fibers, Mito-FF When and Mito-ff face each other in the fiber, the backbone dihedral angles of Mito-FF and Mito-ff did not overlap with each other (FIG. 8c ). This means that Mito-FF and Mito-ff have different interactions with pure enantiomers because the dihedral angles of each enantiomer prefer the same distribution in a homo enantiomeric assembly. This particular orientation was obtained due to the closely faced backbones of Mito-FF and Mito-ff with higher binding energies than enantiomers, resulting in loose stacking of pyrenes (Figs. 8D, 8E). In addition, pyrene stacking between Mito-FF and Mito-ff occurred less than that of pure enantiomers, as a significant difference in the first peak of radial distribution functions (RDFs) in FIG. 9 was shown. This low peak intensity of Mito-FF / Mito-ff means lower pyrene stacking than that of Mito-FF / Mito-FF and Mito-ff / Mito-ff, and pyrene excimer of Mito-rac in the emission spectrum of Fig. 5c. The result is consistent with the intensity being lower than that of Mito-FF and Mito-ff.

7 is a diagram showing a coarse-grained (CG) model.

Figure 8a is a diagram showing the pyrene stacking structure and binding energy in Mito-FF fibers; 8B is a diagram showing a structure in which a pair of Mito-FF backbones are closely facing each other; 8C is a graph showing the probability distribution of the dihedral angle formed by the backbone of Mito-FF and Mito-ff in Mito-rac; 8D and 8E are diagrams showing the pyrene stacking structure and binding energy of Mito-FF and Mito-ff.

9 is a graph showing the results of analyzing radial distribution functions (RDFs).

Experimental Example 5. Analysis of fiber bundle formation by Mito-rac

To analyze the ability of Mito-rac fibers to interact with adjacent molecules with pyrene groups, three Mito-rac fibers were placed in the Mito-FF/Mito-ff solution. As a result, Mito-FF/Mito-ff was immediately attached to the surface of Mito-rac fibers, and fiber bundles were formed by connecting single fibers (FIG. 10A). In the surface binding region, Mito-molecules were bound through pyrene-TPP and pyrene-pyrene interactions without regular structure or specific formation of binding sites, which was confirmed by RDF analysis (FIG. 10B). On the other hand, in a solvent containing Mito-FF molecules, Mito-FF fibers existed as narrow fibers without forming bundles due to the dense pyrene in the fibers (FIG. 10C). These results imply that the formation of Mito-rac super-fibers is a result of interactions with adjacent molecules, that is, due to an increase in the distance between pyrenes.

Figure 10a is a picture showing the formation of a fiber bundle of Mito-rac; 10B is a graph showing the results of RDF analysis; Figure 10c is a picture showing the fiber made up of Mito-FF.

Experimental Example 6. Analysis of Mito-rac Self-Assembly Formation in Mitochondria

In order to study the self-assembly of Mito-rac inside the mitochondria, we performed a time-dependent confocal microscopic analysis, L-Mito-FF-NBD and Mito-FF or Mito The fluorescence at the time of co-assembly of -ff was observed. Specifically, HeLa cells were cultured simultaneously with 40 μM of L-Mito-FF-NBD / Mito-FF or L-Mito-FF-NBD / Mito-ff mixture and observed green fluorescence emission in mitochondria at different time points. did. As a result, green fluorescence was rapidly observed in L-Mito-FF-NBD / Mito-ff within 30 minutes, whereas green fluorescence was faintly observed in L-Mito-FF-NBD / Mito-FF. After 1 hour, the green fluorescence emission from L-Mito-FF-NBD / Mito-ff further increased (Fig. 11c). This fast and bright green fluorescence emission of L-Mito-FF-NBD / Mito-ff means that co-assembly of racemates is possible in the intracellular environment compared to L-Mito-FF-NBD / Mito-FF.

To quantitatively confirm the observation, flow cytometric analysis (FACS) was performed. Specifically, HeLa cells were seeded in a 25-mL T-flask (Thermo Scientific) containing DMEM (Life Technologies) containing 10% FBS and 1% penicillin/streptomycin under conditions of _{37° C. and 5% CO 2.} Thereafter, 20 μM of L-Mito-FF-NBD / Mito-FF and L-Mito-FF-NBD / Mito-ff were incubated with the cells for 0.5 hours and 1 hour, respectively. Then, the cells were collected, washed with cold PBS, and fluorescence emission was measured at 488 nm with a flow cytometer (FACS ealibur, BD Bioscience). As a result, cells cultured with L-Mito-FF-NBD / Mito-ff showed faster and stronger green fluorescence emission than in L-Mito-FF-NBD / Mito-FF, resulting in racemic co-assembly inside the mitochondria of living cancer cells. The occurrence of was confirmed (Fig. 11D).

11A is a diagram showing the structure of L-Mito-FF-NBD; 11B is a diagram showing that L-Mito-FF-NBD is co-assembled with Mito-FF or Mito-ff over time; 11C is an image showing the results of time-dependent confocal microscopy analysis; 11D is a graph showing the results of analysis using a flow cytometer.

Experimental Example 7. Analysis of Influence of Mito-rac Self-Assembly in Mitochondria

Confocal microscopy analysis was performed to observe the influence of the Mito-rac super-structured fibers inside the mitochondria. Specifically, a human Caucasian prostate cancer cell line (human Caucasian prostate adenocarcinoma, PC-3) was labeled with Mito Tracker Red FM, and 2 μM of Mito-FF, Mito-ff and Mito-rac were cultured together. As a result of confirming the confocal image after 24 hours, in cells cultured with Mito-rac, total destruction and fragmentation of mitochondria were observed along with leakage of Mito Tacker from mitochondria to cytoplasm. In contrast, cells cultured with Mito-FF or Mito-ff had no effect on mitochondria (FIG. 12A). In addition, in the mitochondrial membrane of cells cultured with Mito-rac, rapid membrane depolarization was observed compared to Mito-FF or Mito-ff (FIG. 12B). These results are related to the CAC and morphology of Mito-rac observed above, when Mito-rac is treated, mitochondria are destroyed by the formation of super-structures, whereas Mito-FF or Mito-ff prevents mitochondrial destruction. It means that much more molecular accumulation is needed to induce it.

12 is an image showing the results of confocal microscopy analysis:

12A is an image observing fragmentation of mitochondria; 12B is an image observing the depolarization of the mitochondrial membrane.

Experimental Example 8. Mito-rac cytotoxicity analysis

Cytotoxicity analysis by the super-fibrous structure of Mito-rac was performed. Specifically, it was confirmed whether Mito-FF, Mito-ff, and Mito-RAC prepared in Example 1 have apoptotic activity against cancer cells. Specifically, for cytotoxicity analysis, the above three kinds of peptides were respectively used in DMEM containing HT-29 cells (human colon adenocarcinoma), IMR 90 fibroblasts, 10% FBS, and 1% penicillin/streptomycin (Life Technologies) ( Life Technologies) cultured in a 37° C., 5% CO ₂ incubator under medium. Then, Alamar blue assay (Thermo Fisher Scientific) was used to measure cell viability 72 hours after cell culture using the manufacturer's protocol. As a result, after 72 hours of culture, the HT-29 (human colon adenocarcinoma) cell line showed higher cytotoxicity than Mito-FF and Mito-ff (FIG. 13A). However, in IMR-90 fibroblasts, which are not cancer cell lines, but normal cell lines, there was no molecule that caused toxicity due to low accumulation in mitochondria (FIG. 13B). These results mean that Mito-rac is harmless to normal cells.

13 is a graph showing the results of cytotoxicity analysis:

13A is a graph showing the results of cytotoxicity in HT-29 cancer cells; 13B is a graph showing the results of cytotoxicity in IMR-90 normal cells.

Experimental Example 9. Analysis of the efficacy of Mito-rac on tumor treatment in vivo

To analyze the efficacy of treatment by Mito-rac in vivo, the degree of tumor suppression was observed. Specifically, the mouse experiment was performed according to the guidelines of the Korea Institute of Science and Technology (KIST). For animal experiments, the Zoletil-Rompun mixture was anesthetized by injection into the abdominal cavity of BALB/c nude mice (male, ~ 20 g body weight, 5 weeks old; Orient Bio Inc., Korea). Then, a tumor was established by subcutaneous injection of 1.0×10 ⁷ HT-29 (human colon adenocarcinoma) cells into mice. After about 3 weeks, after ^{tumors in a volume of 70-100 mm 3} were established, peptides (1 mg/kg) in 60 μL of PBS (pH7.4) containing 10% DMSO were added around the tumor tissue for tumor treatment. The tumor was injected around the tissue and the control group was injected with PBS. The injection was repeated 7 times every 2 days for 2 weeks. The size of the tumor ^{was measured as x b 2} /2, where a and b mean the largest and smallest diameters, respectively. In order to confirm the in vivo toxicity of the peptide, the body weight of the mouse was observed during the treatment period of the peptide. As a result, the peptide-treated group showed clear signs of tumor cell death and tumor growth inhibition, such as redness, swelling, and scab formation in tumor tissue compared to the control group. In particular, Mito-rac was Mito-FF Or it showed more pronounced tumor suppression than Mito-ff (Fig. 14A, Fig. 14B). In addition, during the treatment period, all experimental groups did not show noticeable weight loss compared to the control group, and thus showed minimal toxicity of the peptide in vivo (FIG. 14C ). In addition, when Mito-rac-treated tumor sections with a thickness of 70 nm were viewed as a TEM image, mitochondrial contraction and damage due to the formation of a super-structure of Mito-rac were observed (FIG. 14d,e). These results imply that Mito-rac actually works in vivo for cancer treatment.

In order to further analyze the cause of the high toxicity caused by Mito-rac, defects on the cancer cell membrane were investigated through CGMD simulation. As a result, both Mito-rac super-structure fibers and Mito-FF fibers caused defects in cell membranes, but the defect area formed by Mito-rac super-fibers was ~57.62 nm ² , about 6.4 times that of Mito-FF fibers. It was wider (Figs. 15a,b). The above results indicate that Mito-rac super-fibers are more effective in treating cancer than individual fibers of Mito-FF or Mito-ff.

14A is an optical image taken to see the efficacy of tumor treatment in mice; 14B is a graph showing tumor volume over time; 14C is a graph showing the weight of mice over time; 14D and 14E are TEM images of mitochondria of tumor sections treated with Mito-rac.

15 is a diagram showing the CGMD simulation results:

15A is a diagram showing a defect region of cancer cell membranes caused by Mito-rac; 15B is a diagram showing a defect region of a cancer cell membrane caused by Mito-FF.

Claims (12)

A mitochondrial targeting moiety and a peptide represented by (Xaa)n-Lys, and the mitochondrial targeting moiety is linked to a lysine amino acid of the peptide,
Wherein n is the number of amino acids Xaa linked through a peptide bond, and is an integer of 2 to 200,
Each of Xaa is independently valine, leucine, isoleucine, methionine, phenylalanine, asparagine, glutamic acid, aspartic acid, glycine, alanine, serine, threonine, cysteine, proline, glutamine, histidine, lysine, arginine, tyrosine, tryptophan and C3 thereof. -C10 cycloalkyl bond variant selected from the group consisting of,
The amino acid forms a beta-sheet secondary structure between two or more amino acids,
The peptide is a self-assembly formed by co-assembly of the D-enantiomer and the L-enantiomer of a conjugate containing a beta-sheet secondary structure. . The self-assembly of claim 1, wherein n is 2, and the amino acid is selected from the group consisting of phenylalanine, alanine, cyclohexyl alanine, and valine, and is the same as each other. The method according to claim 1, wherein the hydroxy group of the C-terminal carboxyl group of the lysine is selected from the group consisting of an amine, an alkyl group, an alcohol group, a ketone group, an acyl group, an ester group, an ether group, an acetyl group, an acyl halide group, and an aldehyde group. Substituted, self-assembly. The self-assembly of claim 1, wherein the number of mitochondrial targeting moieties linked to the peptide is an integer selected from the range of 1 to the number of amine groups present in the peptide represented by (Xaa)n-Lys. The self-assembly of claim 1, wherein the mitochondrial targeting moiety is triphenylphosphonium (TPP). The self-assembly of claim 1, wherein the peptide and the mitochondrial targeting moiety are linked directly or through a linker. The self-assembly according to any one of claims 1 to 6, further comprising a fluorophore, wherein the fluorophore is linked to the peptide. The self-assembly of claim 7, wherein the fluorophore is linked to the N-terminus of the peptide by an amide bond. The self-assembly of claim 7, wherein the fluorophore is at least one selected from the group consisting of pyrene, NBD (4-nitro-2, 1, 3-benzoxadiazole), perylene, naphtalene, and coronene. The self-assembly according to claim 7, having a structure of Formula 2 or Formula 3.
[Formula 2]

[Formula 3]

A pharmaceutical composition for preventing or treating cancer, comprising the self-assembly of any one of claims 1 to 6 as an active ingredient. The pharmaceutical composition of claim 11, wherein the self-assembly of the pharmaceutical composition induces apoptosis in mitochondria in cancer cells.

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