KR20240069480A - Amphipathic conjugates comprising self-assembly peptide molecules and uses thereof - Google Patents

Abstract

An amphipathic conjugate containing a peptide capable of self-assembly and a use thereof. According to one aspect, the conjugate and a pharmaceutical composition containing the same are useful for the prevention or treatment of cancer by destroying the membrane integrity of cancer cell lysosomes. There is a possible effect.

Description

Amphipathic conjugates comprising self-assembly peptide molecules and uses thereof}

It relates to an amphipathic conjugate containing a peptide capable of self-assembly and its use.

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

Lysosomal organelles contain a dynamic system of acidic vesicles that receive material through endocytosis at the plasma membrane or autophagy in the cytoplasm, ultimately degrading and/or recycling the material. This lysosomal-mediated degradation pathway in cancer cells causes various changes in the structure and function of the lysosomal membrane, and lysosomal membrane permeabilization is caused by various endogenous (p53 activation, oxidative stress) and exogenous (cationic amphiphilic drugs, CAD) factors. It is characterized by being more sensitive to permeabilization (LMP). Lysosomal cell death (LCD) is a novel strategy to target cancers resistant to apoptosis therapies and drugs by bypassing the classical caspase-dependent cell death pathway. Accordingly, the development of a drug that can specifically induce lysosomal cell death in cancer cells has strong potential as a variety of anticancer therapy, cancer cell death therapy, and a new treatment for drug-resistant cancer.

The present inventors developed an amphipathic peptide conjugate that induces cancer cell lysosomal apoptosis through carbonic anhydrase IX (CAIX)-mediated endocytosis.

One aspect includes a carbonic anhydrase IX (CAIX) targeting moiety; A binding moiety that interacts with a cell membrane and a peptide represented by (Xaa)n-Lys-Lys, wherein the carbonic anhydrase IX (CAIX) targeting moiety and the binding moiety are each linked to a lysine amino acid of the peptide. where n is the number of amino acids Xaa linked through a peptide bond, and is an integer of 2 to 200, and each , serine, threonine, cysteine, proline, glutamine, histidine, lysine, arginine, tyrosine, tryptophan and their C3-C10 cycloalkyl bond variants, and the peptide is a beta-sheet secondary It provides a conjugate, which contains a structure.

Another aspect is to provide a pharmaceutical composition for preventing or treating cancer containing the conjugate.

One aspect includes a carbonic anhydrase IX (CAIX) targeting moiety; A binding moiety that interacts with a cell membrane and a peptide represented by (Xaa)n-Lys-Lys, wherein the carbonic anhydrase IX (CAIX) targeting moiety and the binding moiety are each linked to a lysine amino acid of the peptide. where n is the number of amino acids Xaa linked through a peptide bond, and is an integer of 2 to 200, and each , serine, threonine, cysteine, proline, glutamine, histidine, lysine, arginine, tyrosine, tryptophan and their C3-C10 cycloalkyl bond variants, and the peptide is a beta-sheet secondary It provides a conjugate, which contains a structure.

In one embodiment, the peptide means 3 to 201 amino acids linked through a peptide bond, and the peptide may be expressed as (Xaa)n-Lys-Lys. The n is the number of amino acids linked through peptide bonds, and is 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 Xaa is each independently valine, leucine, isoleucine, methionine, phenylalanine, asparagine, glutamic acid, aspartic acid, glycine, alanine, serine, threonine, It may be selected from the group consisting of cysteine, proline, glutamine, histidine, lysine, arginine, tyrosine, tryptophan, and C3-C10 cycloalkyl bond variants thereof, for example, where n is 2 and Xaa is the same phenylalanine, alanine, and cyclohexyl. Alanine or valine may form a peptide bond, but are not limited thereto.

In one embodiment, the peptide (Xaa)n-Lys-Lys is a variant in which a C3-C10 cycloalkyl group is bonded to each amino acid or the R group of the amino acid in the order from the N-terminus to the C-terminus of the amino acid (C3-C10 cycloalkyl group). Alkyl bond variants) may be linked with a peptide bond, but are not limited thereto.

In one embodiment, the hydroxy group of the carboxyl group at the C-terminus of Lys of the peptide (Xaa)n-Lys-Lys may be substituted with an amine group, an alcohol group, an ether group, and an acetyl group. , a specific chemical group may not be bonded, or other chemical groups may be chemically bonded, for example, an amine group may be bonded.

In one embodiment, the C3-C10 cycloalkyl bond variant may be one in which a cycloalkyl having 3 to 10 carbon atoms is bonded to the R group of the amino acid, for example, cyclohexyl is bonded to the carbon of the methyl group, which is the R group of alanine. It may be combined cyclohexyl alanine (CHA), but is not limited thereto.

In one embodiment, the peptide may include a beta-sheet secondary structure, and the peptide may have a beta-sheet (beta) between two or more amino acids present inside the peptide or present in another peptide. -sheet) It may form a secondary structure, but is not limited to this.

In one embodiment, the beta-sheet secondary structure is one of the secondary structures formed by interactions between amino acid sequences constituting proteins, and is formed through hydrogen bonds between horizontally connected molecules. . The beta sheet secondary structure is known to be involved in the formation of protein aggregates or fibers in many human diseases.

In one embodiment, the carbonic anhydrase IX (CAIX) targeting moiety functions to recognize, bind, or target carbonic anhydrase IX (CAIX) expressed on the surface of cells cultured under hypoxic conditions or cancer cells. This means that it may be, for example, acetazolamide, but is not limited thereto.

In one embodiment, the binding moiety that interacts with the cell membrane refers to a function of increasing membrane interaction through lipophilicity and/or positive charge, and may be, for example, triphenylphosphonium (TPP). , but is not limited to this. Specifically, the cell membrane may include a lysosomal cell membrane.

In one embodiment, due to the carbonic anhydrase IX (CAIX) targeting moiety constituting the conjugate, the conjugate targets CAIX in CAIX overexpressing cells, thereby increasing the concentration of the conjugate locally, leading to the formation of CAIX-induced self-assembly. can be induced. Specifically, CAIX-induced self-assembly may have a nanofiber structure. The CAIX-induced self-assembly may be able to enter the lysosome through CAIX-mediated endocytosis.

In one embodiment, the conjugate entering the lysosome due to the carbonic anhydrase IX (CAIX) targeting moiety changes the structure of the self-assembly into an ordered positively charged chiral structure under acidic pH conditions, destroying the lysosomal membrane integrity. and can induce cell apoptosis. Specifically, under acidic pH conditions, the lipophilicity and positive charge of TPP may lead to destruction of lysosomal membrane integrity.

In one embodiment, 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-Lys. When an amine group is present at the C-terminus of the peptide, the number of mitochondrial targeting moieties connected to the peptide may be an integer within 1 to m+1, which is 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 one embodiment, the N-terminus of the peptide of the conjugate may be connected to a fluorophore through an amide bond, but is not limited thereto.

In one embodiment, the fluorophore refers to an atomic group required for an organic compound to emit fluorescence. In the present invention, the fluorophore is connected to the N-terminus of the peptide with an amide bond, and is a flat compound consisting of pyrene, NBD (4-nitro-2, 1, 3-benzoxadiazole), perylene, naphthalene, coronene, and aromatics. It may be one or more selected from the group consisting of fluorophores that have a structure and are easily stacked together through pi bonds, but are not necessarily limited thereto.

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

In one embodiment, the conjugate may have the structure of Formula 1 below;

[Formula 1]

In one embodiment, in Formula 1, R1 refers to hydrogen or a fluorophore, and is selected from hydrogen, pyrene, NBD (4-nitro-2, 1, 3-benzoxadiazole), perylene, naphthalene, coronene, and aromatic It may be one or more compounds selected from the group consisting of fluorophores that have a flat structure and are easy to stack with each other through pi bonds.

In one embodiment, the linker connects the N-terminus of the peptide and the fluorophore, and may be absent or an alkyl group having 1 to 10, 1 to 5, 1 to 4, or 2 to 3 carbon atoms. It may be (alkyl), alkene, or alkyne, but is not limited thereto.

In one embodiment, the linker may include a functional group at one end capable of forming an appropriate bond such as a peptide and amide bond, and may include pyrene, NBD (4-nitro-2, 1, 3) -benzoxadiazole), perylene, naphthalene, coronene, and aromatics. It has a flat structure and can be connected to one or more fluorophores selected from the group of fluorophores that are easy to stack with each other through pi bonds through an amide bond.

In one embodiment, when R1 is hydrogen, the linker may not exist, and R2 is the C-terminus of the peptide (i.e., the C-terminus of lysine), and includes a hydroxy group (OH), an amine group, and an alkyl group. , alcohol group, ether group, and acetyl group, (the alkyl group, alcohol group, ether group, and acetyl group may have 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 5 carbon atoms). there is.

In one embodiment, the conjugate may be self-assembled to form a nanofiber structure. The nanofiber structure may have amphipathic properties due to the CAIX targeting moiety and the cell membrane interaction binding moiety.

In one embodiment, the structure of the conjugate may change depending on pH. Specifically, the conjugate may change into an ordered chiral structure under acidic conditions. The acidic conditions may mean a pH range that is the same or similar to the environment inside intracellular lysosomes.

In one embodiment, the charge of the conjugate may change depending on pH. Specifically, the conjugate has amphiphilic properties in which positive and negative charges coexist, but the negative charges may change to positive charges or lose amphipathic properties under acidic conditions. The acidic conditions may mean a pH range that is the same or similar to the environment inside intracellular lysosomes.

In one embodiment, the self-assembly may self-assemble near CAIX overexpression and enter intracellular lysosomes through CAIX-mediated endocytosis. Specifically, the self-assembly can change the charge and structure of the conjugate under the acidic conditions of intracellular lysosomes. The integrity of the lysosomal membrane may be damaged due to the positive charge and lipophilic nature of TPP of the altered self-assembly.

In one embodiment, the conjugate may specifically target cancer cells overexpressing CAIX by changing the charge and structure of the conjugate according to changes in pH.

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

[Formula 2]

In one embodiment, in Formula 2, where R is an amine group, it is called Pep-AT in the examples below, and can be self-assembled into a nanofiber structure, which may have amphipathic properties.

In one embodiment, the nanofiber structure may enter the inside of cancer cell lysosomes through CAIX-mediated endocytosis.

In one embodiment, the inside of the cell of the lysosome may be an acidic environment, which may induce a change in the nanofiber structure. Specifically, acetazolamide in Pep-AT may change from a negative charge to a positive charge. Specifically, the nanofiber structure may change into a chiral structure.

In one embodiment, the lysosomal membrane may be destroyed due to the lipophilic and positive charge properties of TPP in Pep-AT, thereby inducing apoptosis.

In one embodiment, the Pep-AT recognizes cells or cancer cells overexpressing CAIX, forms self-assembly of nanofiber structure, induces endocytosis, and induces apoptosis specifically in cancer cells, so it does not act on normal cells but does not act on cancer cells. It may be to specifically target cells.

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

In one embodiment, the conjugate may exist in the form of a pharmaceutically acceptable salt. The pharmaceutically acceptable salt includes both addition salts of acids or bases and their stereochemical isomeric forms, and may be, for example, addition salts of organic acids or inorganic acids. The salt includes any salt that maintains the activity of the parent compound in the subject of administration and does not cause undesirable effects, and is not particularly limited.

In one embodiment, the salts include inorganic salts and organic salts, such as acetic acid, nitric acid, aspartic acid, sulfonic acid, sulfuric 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, bitartaric acid, oxalic acid, butyric acid, calcium idate, carbonic acid, chlorobenzoic acid, Citric acid, idetic acid, toluenesulfonic acid, fumaric acid, gluceptic acid, esilinic acid, pamoic acid, gluconic acid, methylnitric acid, malonic acid, hydrochloric acid, hydroiodoic acid, hydroxynaphtholic acid, isethionic acid, Lactobionic acid, mandelic acid, mucilic acid, naphsilic acid, muconic acid, p-nitromethanesulfonic acid, hexamic acid, pantothenic acid, monohydrogen phosphate, dihydrogen phosphate, salicylic acid, sulfamic acid, sulfanyl. It may be linic acid or methanesulfonic acid.

In one embodiment, the salt forms include salts of alkali and alkaline earth metals such as ammonium salts, lithium salts, sodium salts, potassium salts, magnesium salts and calcium salts, for example benzathine, N-methyl-D- It includes salts with organic bases such as glucamine and hydrabamine salts, and salts with amino acids such as arginine and lysine. Additionally, the salt form may be converted to the free form by treatment with a suitable base or acid.

In one embodiment, the pharmaceutical composition for preventing or treating cancer comprising the conjugate or a pharmaceutically acceptable salt thereof as an active ingredient may induce apoptosis in lysosomes within cancer cells.

In one embodiment, apoptosis is a form of cell death controlled by genes, and is cell necrosis or pathological death, which is cell death caused by stimuli such as burns, bruises, and poisons. It is distinct from necrosis, and refers to a cell death that begins with the cell shrinking, and then a gap is created between adjacent cells, and the DNA within the cell is regularly cut and fragmented.

In one embodiment, regarding the structure of self-assembly in the lysosome within the cancer cell, the integrity of the lysosomal membrane is destroyed through a change in the charge of the self-assembly due to the acidic environment of the lysosome, specifically a change from negative to positive charge. This causes cancer cell-specific apoptosis.

In one embodiment, the cancer targeted for prevention or treatment by the pharmaceutical composition may be solid cancer or hematological 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 necessarily limited thereto.

In one embodiment, the pharmaceutical composition may be in any form suitable for the intended administration method. Specifically, "administration" in a pharmaceutical composition means introducing a predetermined substance into a patient by any appropriate 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, intrarectal administration, etc. may be mentioned, but are not limited thereto. does not Additionally, the pharmaceutical composition of the present invention can be administered by any device that allows the active ingredient to move to target cells.

In one embodiment, the pharmaceutical composition can be administered orally or parenterally, and is preferably administered orally. When administered parenterally, the pharmaceutical composition of the present invention can be administered by intravenous injection, subcutaneous injection, intramuscular injection, and intraperitoneal injection.

In one embodiment, the route of administration of the pharmaceutical composition is preferably determined depending on the type of disease to which it is applied.

In one embodiment, a pharmaceutical composition for preventing or treating cancer formulated as the oral administration formulation or parenteral administration formulation is provided.

In one embodiment, the pharmaceutical composition is an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, or suspension, emulsion, lyophilization, etc., formulated according to a conventional method. It can be formulated and used in parenteral formulations such as preparations, external preparations, suppositories, sterilized injection solutions, and preparations for implantation.

In one embodiment, the pharmaceutical composition may further include pharmaceutically acceptable excipients that can be used in formulation in addition to the active ingredient (i.e., conjugate, or pharmaceutically acceptable salt thereof).

In one embodiment, excipients that can be used in the formulation of the pharmaceutical composition include carriers, vehicles, diluents, solvents, such as monohydric alcohols such as ethanol, isopropanol and polyhydric alcohols such as glycerol and edible substances. Oils such as soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, oily esters such as ethyl oleate, isopropyl myristate; Binders, adjuvants, solubilizers, thickeners, stabilizers, disintegrants, glidants, lubricants, buffers, emulsifiers, wetting agents, suspending agents, sweeteners, colorants, flavors, 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, polyvinyl methylcellulose. It may include one or more selected from the group consisting of rolidone, low melting point wax, ion exchange resin, etc., but is not limited thereto.

In one embodiment, the pharmaceutically acceptable carrier included in the pharmaceutical composition is one commonly used in preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, algae, etc. Contains nitrate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. However, it is not limited to this. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

In one embodiment, the pharmaceutical composition may be formulated into various oral dosage forms. For example, it can be in any dosage form for oral administration, such as tablets, pills, hard/soft capsules, solutions, suspensions, emulsifiers, syrups, granules, and elixirs. These formulations for oral administration may contain, in addition to the above active ingredients, diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine, silica, and talc, depending on the typical composition of each formulation. , stearic acid and its magnesium or calcium salts, and/or lubricants such as polyethylene glycol.

In one embodiment, when the formulation for oral administration is a tablet, it may include a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidine. In some cases, it may include disintegrants such as starch, agar, alginic acid or its sodium salt, boiling mixtures and/or absorbents, colorants, flavoring agents, or sweeteners.

In one embodiment, the pharmaceutical composition may be formulated in the form of a parenteral administration formulation, in which case it is administered by parenteral administration methods 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 by mixing the active ingredient in water with a stabilizer or buffer, and this solution or suspension is prepared in a unit dosage form of an ampoule or vial. It can be.

In one embodiment, the pharmaceutical composition may be sterilized, or may further contain auxiliaries such as preservatives, stabilizers, wetting agents or emulsification accelerators, salts and/or buffers for adjusting osmotic pressure, and other therapeutically useful substances. It may further include, and may be formulated according to conventional methods of mixing, granulating, or coating.

In one embodiment, the content of the conjugate or a pharmaceutically acceptable salt thereof in the pharmaceutical composition can be appropriately adjusted depending on the purpose of use of the pharmaceutical composition, the form of the formulation, etc., for example, from 0.001 to 0.001 based on the total weight of the pharmaceutical composition. It may be 99% by weight, 0.001 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 one embodiment, the therapeutically effective amount of the conjugate or a pharmaceutically acceptable salt thereof included in the pharmaceutical composition of the present invention refers to the amount required for administration to expect a disease treatment effect. Therefore, it can be adjusted according to the type of patient's disease, the severity of the disease, the type of active ingredient administered, the type of dosage form, the patient's age, gender, weight, health status, diet, drug administration time, and administration method. For example, to mammals, including humans, it can 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 the amount that can achieve the desired effect, for example, the effect of treating and/or preventing cancer. The pharmaceutically effective amount may be divided and administered once or twice a day through oral or parenteral routes (eg, intravenous injection, intramuscular injection, etc.).

According to one aspect, the conjugate and the pharmaceutical composition containing the same have the effect of destroying the membrane integrity of cancer cell lysosomes, which can be useful in the prevention or treatment of cancer.

Figure 1 is a diagram showing the molecular structure of Pep-AT.
Figure 2 is a diagram illustrating the change in self-assembly structure and charge of Pep-AT depending on pH.
Figure 3 is a diagram schematically illustrating the mechanism by which Pep-AT acts as a cancer cell treatment within cancer cells.
Figure 4 is an image analyzing the self-assembly structure of Pep-AT according to pH through TEM.
Figure 5 is a graph showing the charges of Pep-AT and control groups (Pep-T, Pep-A, Pep).
Figure 6 is a graph showing the results of analysis of changes in the structure of Pep-AT according to pH through fluorescence analysis (left), UV-vis analysis (middle), and CD spectrum (right).
Figure 7 shows an image analyzing the expression level of carbonic anhydrase IX (CAIX) in HeLa and NIH 3T3 cells through Western blotting (left) and changes in the surface of HeLa and NIH 3T3 cells over time after treatment with Pep-AT. This is an image (right) analyzed through SEM.
Figure 8 is an image confirming the interaction between acetazolamide of Pep-AT and cancer cells through competition analysis.
Figure 9 is an image confirming the intracellular localization of Pep-AT in HeLa and NIH 3T3 cells through confocal laser scanning microscopy.
Figure 10 is an image confirming the formation of co-assemblies when HeLa cells were treated with NBD-AT alone and in combination with Pep-AT using a confocal laser scanning microscope.
Figure 11 is an image confirming the morphological changes in lysosomes depending on the presence or absence of Pep-AT treatment in HeLa cells through TEM.
Figure 12 is an image (left) and graph (right) confirming the membrane integrity of lysosomes according to Pep-AT treatment in HeLa and NIH 3T3 cells through AO analysis.
Figure 13 shows the cytotoxicity of Pep-AT and controls (Pep-T, Pep-A, Pep) on HeLa cells and the cytotoxicity of Pep-AT on NT1, MDA-MB-468, and HEK293K cells through MTT assay. This is the graph shown.
Figure 14 is a graph showing the cytotoxicity of Pep-AT in HeLa, NT1, MDA-MB-468, NIH/3T3, and HEK293K using IC50.
Figure 15 is an image (left) and graph (right) showing apoptosis induced by Pep-AT treatment in HeLa cells through Annexin V/PI staining.
Figure 16 is a graph showing cytotoxicity according to the presence or absence of Z-VAD-FMK, an apoptosis inhibitor, in HeLa cells through MTT assay.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples. The embodiments may be subject to various changes, and the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

Example 1. Synthesis of Pep-AT

Pep-AT is composed of four peptides of Phe-Phe-Lys-Lys (FFKK) as a basic unit, and has a structure in which acetazolamide and TPP (triphenylphosphonium) are bound to each lysine residue. As a practical example, it was synthesized as follows using standard solid phase synthesis (SSPS) method based on 9-fluorenylmethoxycarbonyl (Fmoc). Link amide MBHA resin (0.106 mol) was used as a bead support. The linked amide MBHA resin was swollen in DMF for 30 minutes, and then a 20% piperidine solution in DMF was used for 40 minutes to deprotect Fmoc. The protected Fmoc amino acid (0.5 mol) was incubated with diisopropyl ethyl amine (DIPEA, 1.00 mol) and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylformamide (DMF). It was treated with methyluronium hexafluoramide (HBTU, 0.45 mol) and reacted for 1 hour and 40 minutes. Deprotection and amino acid coupling steps were repeated until the final amino acid conjugation was obtained. After deprotection of the last amino acid, 1-pyrenebutyric acid (0.5 mol) was treated with HBTU (0.45 mol) and DIPEA (1.00 mol) and reacted in DMF overnight. Next, 4-methyltrityl of the lysine residue was deprotected twice with 3% TFA in dichloromethane for 30 minutes. 1-hexyl triphenylphosphonium bromide salt (0.04 mmol) and triethyl amine (0.02 mmol) were treated in DMF and reacted with the resin overnight. The peptide was then cleaved from the resin by treatment with a cleavage cocktail (TFA/triisopropylamine mixture/water (95:2.5:2.5)) for 3 hours. The cleaved peptide was precipitated in cold ether, purified by HPLC, and confirmed by mass spectrometry using MALDI-TOF/TOF. For acetazolamide conjugation, the purified peptide was incubated with 4-ortho-4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)butyric acid (Az-) in DMF. COOH, 2 eq), HBTU (1.95 eq), and DIPEA (4 eq) and stirred overnight. The product was purified by HPLC and mass spectrometry was performed using MALDI-TOF/TOF.

Figure 1 is a diagram showing the molecular structure of Pep-AT.

Figure 2 is a diagram illustrating the change in self-assembly structure and charge of Pep-AT depending on pH.

Figure 3 is a diagram schematically illustrating the mechanism by which Pep-AT acts as a cancer cell treatment within cancer cells.

Experimental Example 1. Characteristic analysis of Pep-AT

To analyze the self-assembly tendency of Pep-AT according to pH, the morphological change of Pep-AT was observed through transmission electron microscopy (TEM) under conditions of pH 7.4 and pH 4.5.

Specifically, in order for molecules to self-assemble inside a cell, the concentration of molecules within the cell must be much higher than the critical aggregation concentration (CAC) value. To determine whether Pep-AT of the present invention can self-assemble inside cells, the critical aggregation concentration (CAC) of Pep-AT was analyzed by the steady-state fluorescence pyrene emission method. did. Specifically, peptide solutions of different concentrations were prepared in PBS (10mM, pH 7.4) and sodium acetate buffer (10mM, pH 4.5) according to each pH condition. 1 μL of pyrene solution in ethanol (2 mM) was added to 999 μL of peptide solution. A peptide solution containing 2 μM pyrene was analyzed using a fluorescence spectrum. The excitation wavelength was measured at 331.0 nm. The slit width of excitation and emission was set to 2.5 nm, and the scan speed was set to 240 nm/min. The ratio of I1 band and I3 band for pyrene was plotted to obtain CAC. The CAC values of Pep-AT, Pep-A, Pep-T, and Pep were found to be 28, 32, 52, and 77 μM, respectively.

100 μM of Pep-AT was dissolved in PBS and observed using TEM (JEOL, JEM-1400), and the results are shown in Figure 4.

Figure 4 is an image analyzing the self-assembly structure of Pep-AT according to pH through TEM.

As shown in Figure 4, the self-assembly tendency of Pep-AT was confirmed to vary depending on pH (pH 7.4 and pH 4.5).

Experimental Example 2. Analysis of changes in self-assembly structure according to pH of Pep-AT

To analyze the self-assembly pattern of Pep-AT according to pH, the surface charge of Pep-AT was measured using a zeta sizer under conditions of pH 7.4 and pH 4.5, and the fluorescence spectrum, UV-vis absorption spectrum, and Circular dichroism (CD) spectra were used to analyze changes in the structure and molecular packing of Pep-AT.

Specifically, the surface zeta potential of Pep-AT and its controls (Pep-A, Pep-T, and Pep) were measured, and the results are shown in FIG. 5.

_ex = 360 nm). UV-vis 스펙트럼은 300 nm에서 400 nm 파장의 범위를 측정하였고, 스캔 속도는 200 nm/min로 설정하였다. 여기(exciation)와 방출(emission)의 슬릿 너비는 10nm로 설정하였다. 원평광 이색성(CD) 스펙트럼은 190 nm에서 400 nm 파장의 범위를 측정하였고, 스캔 속도는 10 nm/min로 설정하였다. 각각의 결과는 도 6에 나타내었다.Specifically, each peptide solution was prepared in PBS (10 mM, pH 7.4) or sodium acetate buffer (10 mM, pH 4.5). The fluorescence spectrum was measured in the wavelength range from 400 nm to 600 nm, and the scan speed was set at 240 nm/min (

_ex = 360 nm). The UV-vis spectrum was measured in the wavelength range from 300 nm to 400 nm, and the scan speed was set at 200 nm/min. The slit width for excitation and emission was set to 10 nm. Circular dichroism (CD) spectra were measured in the wavelength range from 190 nm to 400 nm, and the scan speed was set at 10 nm/min. Each result is shown in Figure 6.

Figure 5 is a graph showing the charges of Pep-AT and control groups (Pep-T, Pep-A, Pep).

Figure 6 is a graph showing the results of analysis of changes in the structure of Pep-AT according to pH through fluorescence analysis (left), UV-vis analysis (middle), and CD spectrum (right).

As shown in Figure 5, it was confirmed that only Pep-AT and Pep-A changed surface charge from negative to positive depending on pH. Pep-T and Pep appear to have no change in self-assembly structure due to pH due to the absence of acetazolamide, and the greater charge conversion in Pep-AT than in Pep-A seems to be due to the high positive charge of TPP. .

As shown in Figure 6, a strong positive Cotton effect was observed due to TPP and a negative band was observed at pH 4.5 due to pyrin. However, no signal appeared at pH 7.4.

Through the above results, it was confirmed that the self-assembly tendency of Pep-AT changes depending on pH. Specifically, it was found that ultramacromolecule J-aggregates were formed with a chiral nanofiber structure along with a change in surface charge at pH 4.5.

Experimental Example 3. Confirmation of CAIX-mediated endocytosis

HeLa and NIH/3T3 cells were cultured at 37°C in 5% CO ₂ in DMEM (Life Technologies) supplemented with 10% FBS and 1% penicillin/streptomycin.

3.1. CAIX expression level and CAIX-induced self-assembly formation in cancer cells

Acetazolamide of Pep-AT acts as a moiety targeting carbonic anhydrase IX (CAIX), and the expression level of carbonic anhydrase IX (CAIX) in cancer cells compared to normal cells was analyzed through Western blotting. . also, To determine whether Pep-AT specifically formed nanofiber structures in cancer cells, the surface of the cells was analyzed over time using scanning electron microscopy (SEM).

Specifically, HeLa, a cancer cell, and NIH/3T3, a normal cell, were seeded in a 6-well plate at a density of 300,000 per well. After 24 hours of culture, the culture medium was removed and each cell was washed with 1X PBS. Each cell was obtained using Trypsin-EDTA (0.25%). The obtained cells were washed with 1X PBS and then suspended in 100 μL of RIPA buffer treated with protease inhibitor cocktail at 4°C for 45 minutes. The cell lysis supernatant obtained by spinning down at 13,000 rpm for 20 minutes was obtained, and the protein concentration was assayed (Bradford reagents). Proteins were separated through SDS-PAGE, transferred to PVDF (polyvinylidene difluoride) membrane, and blocked in TBS-T buffer containing 5% nonfat dry milk solution for 1 hour. Afterwards, the cells were treated with primary antibodies and incubated at 4°C overnight, washed three times for 15 minutes each in TBS-T buffer, treated with HRP (horseradish peroxidase) as secondary antibodies, and incubated at room temperature for 1 hour. After washing with TBS-T buffer three times for 15 minutes each, the protein was imaged with BCIP/NBT solution and observed using ChemiDoc Imaging Systems.

Specifically, HeLa, a cancer cell, and NIH/3T3, a normal cell, were seeded at a density of 100,000 in a 24-well plate containing coverslits. After 24 hours of culture, the peptide solution was diluted in cell culture medium, and the control group was not treated with culture medium. Afterwards, the culture medium was removed and the cells were washed sequentially with 1X PBS and distilled water. After immersing the cover glass containing cells in liquid nitrogen, the completely dehydrated cover glass was observed using a scanning electron microscope. The results are shown in Figure 7.

Figure 7 shows an image analyzing the expression level of carbonic anhydrase IX (CAIX) in HeLa and NIH 3T3 cells through Western blotting (left) and changes in the surface of HeLa and NIH 3T3 cells over time after treatment with Pep-AT. This is an image (right) analyzed through SEM.

As shown in Figure 7, the expression level of CAIX in HeLa cells was measured to be more than three times that of NIH/3T3 cells. In addition, over time, the nanofiber structure of HeLa cells formed a very large structure, but the nanofiber structure was not observed in HeLa cells and NIH/3T3 cells that were not treated with Pep-AT.

Based on the above results, Pep-AT generally self-assembles only in cancer cells that overexpress CAIX, and few targets for normal cells have been found.

3.2. Confirmation of the mechanism of CAIX-mediated endocytosis

To confirm CAIX-mediated endocytosis of Pep-AT, a competition assay was performed using a small molecule inhibitor (e.g., acetazolamide).

Specifically, cancer cells, HeLa, were seeded in an 8-well Lab Tek II slide chamber at a density of 10,000 per well. After 24 hours of incubation, 20uM acetazolamide was incubated with HeLa for 30 minutes, and the negative control group did not contain this. After washing with PBS, the cell culture medium was changed to contain 20 μM of Pep-AT and observed through confocal laser scanning microscopy (CLSM). The results are shown in Figure 8.

Figure 8 is an image confirming the interaction between acetazolamide of Pep-AT and cancer cells through competition analysis.

As shown in Figure 8, the blue fluorescence signal of Pep-AT was reduced.

Through the above results, it was confirmed that CAIX-mediated endocytosis is related to the cellular entry mechanism of Pep-AT.

3.3. Confirmation of Pep-AT intracellular localization

To confirm the intracellular localization of Pep-AT, co-localization experiments were performed and observed through confocal laser scanning microscopy (CLSM).

Specifically, HeLa, a cancer cell, and NIH/3T3, a normal cell, were seeded in an 8-well Lab Tek II slide chamber at a density of 10,000 per well. After 24 hours of cell culture, 20 μM Pep-AT peptide solution was diluted in cell culture medium and cultured for 30 minutes. Then, the cell culture medium was removed, and the cells were cultured by sequentially treating them with Lysotracker ^TM red DND-99 (Invitrogen ^TM ) and Mitotracker ^TM green FM (Invitrogen ^TM ) according to the manufacturer's protocol. Cells were washed with PBS and analyzed using an LSM780 laser confocal scanning microscope. The results are shown in Figure 9.

Figure 9 is an image confirming the intracellular localization of Pep-AT in HeLa and NIH 3T3 cells through confocal laser scanning microscopy.

As shown in Figure 9, HeLa showed overlap between the blue fluorescence of Pep-AT and the red fluorescence of lysotracker, whereas NIH/3T3 did not show blue fluorescence due to lack of expression of CAIX.

The above results indicate that Pep-AT can be spatially and temporally localized to the lysosomes of cancer cells by targeting CAIX.

3.4 Confirmation of the mechanism of self-assembly formation of Pep-AT

To analyze the formation of fiber self-assemblies inside cells, another peptide called NBD-AT was synthesized. In Pep-AT, pyrin was replaced with another environmentally sensitive dye; 4-nitro-2, 1, 3-benzoxadiazole (NBD). NBD dye is a fluorophore used in imaging studies of biological elements. It is known to exhibit more intense fluorescence in a hydrophobic environment compared to a hydrophilic environment, and this property can be used to identify fiber formation inside cells. At this time, NBD-AT was synthesized by reacting with NBD-NH ₂ instead of 1-pyrenebutyric acid in the Pep-AT synthesis process of Example 1.

Specifically, cancer cells, HeLa, were seeded at a density of 10,000 per well in an 8-well plate Lab Tek II slide chamber. After 24 hours of incubation, HeLa was incubated with 20 μM of Pep-AT and 10 μM of NBD-AT for 30 minutes. As a negative control, HeLa was treated with 10 μM of NBD-AT alone and cultured. Cells were washed with PBS and observed with a laser confocal scanning microscope (LSM780). The results are shown in Figure 10.

Figure 10 is an image confirming the formation of co-assemblies when HeLa cells were treated with NBD-AT alone and in combination with Pep-AT using a confocal laser scanning microscope.

As shown in Figure 10, fluorescence was observed when NBD-AT and Pep-AT were treated together, but was not observed when NBD-AT was treated alone.

From the above results, it can be seen that the nanofiber structure formed extracellularly moves into the lysosome in a spatiotemporal manner by CAIX-mediated endocytosis.

Experimental Example 4. Analysis of the effect of Pep-AT on lysosomes

4.1. Morphological effects of Pep-AT on lysosomes

To confirm the morphological effect of Pep-AT on HeLa and NIH 3T3 lysosomes, the results were observed using a transmission electron microscope.

Specifically, cancer cells, HeLa, were seeded at a density of 300,000 per well in a 6-well plate (Thermo Fisher Scientific Inc.). After 24 hours of incubation, the culture medium was replaced with diluted peptide solution. Afterwards, the culture medium is removed and the cells are washed with 1X PBS, harvested using trypsin-EDTA (0.25%), and washed once with 0.1M sodium phosphate buffer containing 5% sucrose. Afterwards, the cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 30 minutes and washed three times with 0.1M sodium phosphate buffer containing 5% sucrose. One part was immobilized for 1 hour with 0.1 M sodium phosphate buffer containing 5% sucrose and 1% osmium tetroxide, and then washed three times with distilled water. Cells were dehydrated sequentially from water to acetone and fixed with epoxy resin. The epoxy resin was synthesized at 80°C for 12 hours and cut into ultrathin sections using a CR-X ultramicrotome. Ultrathin sections were observed with a TEM (JEM-1400) operating at 120 kV, and the results are shown in Figure 11.

Figure 11 is an image confirming morphological changes in lysosomes depending on the presence or absence of Pep-AT treatment in HeLa cells through TEM.

As shown in Figure 11, control HeLa had a smooth spherical shape, while Pep-AT-treated HeLa was noticeably damaged.

4.2. Effect of Pep-AT on membrane integrity of lysosomes

To analyze the membrane integrity of HeLa and NIH 3T3 lysosomes in Pep-AT, Arcridine Orange (AO) staining was performed. Since acridine orange stain can penetrate the cell membrane and accumulates in lysosomes, strong green fluorescence is detected when the lysosome membrane is broken and released into the cytoplasm.

Specifically, HeLa, a cancer cell, and NIH 3T3, a normal cell, were seeded at a density of 10,000 per well in an 8-well Lab Tek II slide chamber. After 24 hours of incubation, 20uM of the peptide solution was diluted in the cell culture medium and incubated for 20 hours. After the culture medium was removed, cells were incubated using acridine orange dye (abcam, ab270791) according to the manufacturer's protocol. Cells were washed with PBS and analyzed with LSM 780 and 880 (ZIESS). The ratio of fluorescence intensity was analyzed using ImageJ software (NIH, USA), and the results are shown in Figure 12.

Figure 12 is an image (left) and graph (right) confirming the membrane integrity of lysosomes according to Pep-AT treatment in HeLa and NIH 3T3 cells through AO analysis.

As shown in Figure 12, green fluorescence with an intensity more than 3 times greater than that of NIH/3T3 was detected in HeLa treated with Pep-AT. This means that the lysosomal nanofibers destroyed the integrity of the lysosomal membrane.

Experimental Example 5. Cytotoxicity analysis of Pep-AT

To confirm the potential of Pep-AT as a cancer treatment, an experiment was performed to analyze in vitro cytotoxicity.

Specifically, cell survival rate was confirmed using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay for 24 hours. Specifically, cancer cells, HeLa, were seeded in a 96-well plate (Thermo Fisher Scientific Inc.) at a density of 10,000 per well. After culturing for 24 hours, different concentrations of peptide solutions (0, 10, 20, and 30 μM) were incubated with the cells. After 24 hours, 100 μL of the MTT solution was replaced with 1/10 diluted cell culture medium and cultured for 4 hours. Then, the MTT solution was dissolved in 100 μL of SDS-HCL solution and cultured for 12 hours. The MTT absorption wavelength measured by the microplate reader was confirmed to be 595 nm. All experiments were repeated three times. The results of the MTT assay are shown in Figures 13 and 14.

Figure 13 shows the cytotoxicity of Pep-AT and controls (Pep-T, Pep-A, Pep) on HeLa cells and the cytotoxicity of Pep-AT on NT1, MDA-MB-468, and HEK293K cells through MTT assay. This is the graph shown.

Figure 14 is a graph showing the cytotoxicity of Pep-AT in HeLa, NT1, MDA-MB-468, NIH/3T3, and HEK293K using IC50.

As shown in Figure 13, Pep-AT showed cytotoxicity against HeLa in a concentration-dependent manner compared to NIH/3T3. Pep-T showed similar behavior to Pep-AT. In contrast, Pep-A and Pep did not show cytotoxicity. Cytotoxicity was also confirmed in other breast cancer cell lines, 4T1 and MDA-MB-468, and in HEK293T, a normal cell line, but showed the same pattern.

As shown in Figure 14, the CAIX-positive cancer cell line had a lower IC50 value compared to the CAIX-negative normal cell line.

Experimental Example 6. Confirmation of apoptosis induced by Pep-AT

To confirm the potential of Pep-AT as a cancer treatment, an experiment was performed to determine whether cancer cell apoptosis was induced. The pattern of apoptosis was examined by labeling Pep-AT treated cells with the apoptosis/necrosis detection dye FITC annexin/PI.

Specifically, cancer cells, HeLa, were seeded in an 8-well Lab Tek II slide chamber at a density of 10,000 per well in DMEM (Life Technologies) supplemented with 10% FBS and 1% penicillin/streptomycin at 37° _C under 5% CO 2 . After 24 hours of cell culture, 20 μM Pep-AT peptide solution was diluted in cell culture medium and cultured for 24 hours. Then, the cell culture medium was removed, replaced with Alexa Fluor488-conjugated Annexin V and PI (Life Technologies, V13241), and incubated for 20 minutes at room temperature. Cells were analyzed using a laser confocal scanning microscope (LSM780) without additional washing steps. The results are shown in Figure 15.

Additionally, after pretreatment with Z-VAD-FMK, a known inhibitor of apoptosis, cytotoxicity of Pep-AT was confirmed in HeLa cells. Example 5 was performed in the same manner as above, and the results of the MTT assay are shown in Figure 16.

Figure 15 is an image (left) and graph (right) showing apoptosis induced by Pep-AT treatment in HeLa cells through Annexin V/PI staining.

Figure 16 is a graph showing cytotoxicity according to the presence or absence of Z-VAD-FMK, an apoptosis inhibitor, in HeLa cells through MTT assay.

As shown in Figure 15, green fluorescence of FITC-annexin and red fluorescence of PI were detected from Pep-AT-treated HeLa cells, confirming that more than 90% of cancer cells were induced into the apoptosis pathway after destruction of the lysosomal membrane.

As shown in Figure 16, it was confirmed that the apoptosis induced by Pep-AT was recovered in HeLa cells pretreated with Z-VAD-FMK.

The above results suggest that Pep-AT can be usefully used as a cancer treatment by directly controlling cell fate.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

Claims (21)

Carbonic anhydrase IX (CAIX) targeting moiety;
a binding moiety that interacts with a cell membrane, and
It contains a peptide represented by (Xaa)n-Lys-Lys,
The carbonic anhydrase IX (CAIX) targeting moiety and binding moiety are each linked to the lysine amino acid of the peptide,
where n is the number of amino acids Xaa linked through peptide bonds and is an integer from 2 to 200,
The -C10 is selected from the group consisting of cycloalkyl bond variants,
The peptide is a conjugate containing a beta-sheet secondary structure. The conjugate of claim 1, wherein n is 2, and the amino acids are selected from the group consisting of phenylalanine, alanine, cyclohexyl alanine, and valine, and are identical to each other. The conjugate of claim 1, wherein the hydroxy group of the C-terminal carboxyl group of the lysine is substituted with a group selected from the group consisting of an amine, an alkyl group, an alcohol group, a ketone group, an acyl group, an ester group, an acyl halide group, and an aldehyde group. The method of claim 1, wherein the number of carbonic anhydrase IX (CAIX) targeting moieties linked to the peptide and binding moieties that interact with the cell membrane is 1 to 1 present in the peptide represented by (Xaa)n-Lys-Lys. A conjugate that is an integer selected from the range of the number of amine groups. The conjugate of claim 1, wherein the carbonic anhydrase IX (CAIX) targeting moiety is acetazolamide. The conjugate according to claim 1, wherein the binding moiety that interacts with the cell membrane is triphenylphosphonium (TPP). The conjugate of claim 1, wherein the peptide and the moiety are connected directly or through a linker. The conjugate of claims 1 to 7, wherein the conjugate further includes a fluorophore, and the fluorophore is linked to the peptide. The conjugate of claim 8, wherein the fluorophore is linked to the N-terminus of the peptide through an amide bond. The conjugate of claim 8, wherein the fluorophore is at least one selected from the group consisting of pyrene, NBD (4-nitoro-2,1,3-venzoxadiazole), perylene, naphthalene, and coronene. The conjugate according to claim 8, having the structure of Formula 2.
[Formula 2]
The conjugate of claim 1, wherein the conjugate is amphipathic. The conjugate according to claim 1, wherein the conjugate self-assembles to form nanofibers. The conjugate of claim 1, wherein the conjugate induces CAIX-mediated endocytosis in carbonic anhydrase IX (CAIX) cancer cells. The conjugate of claim 1, wherein the structure changes depending on pH. The method according to claim 1, wherein the conjugate changes its structure into a chiral self-assembly under acidic conditions. The conjugate according to claim 1, wherein the surface charge of the conjugate changes to a positive charge under acidic conditions. The conjugate according to claim 1, wherein the conjugate specifically targets cancer cells through changes in structure and charge. A pharmaceutical composition for preventing or treating cancer, comprising the conjugate of any one of claims 1 to 7 as an active ingredient. The pharmaceutical composition for preventing or treating cancer according to claim 19, wherein the conjugate of the pharmaceutical composition forms self-assembly to destroy the membrane integrity of cancer cell lysosomes. The pharmaceutical composition for preventing or treating cancer according to claim 19, wherein the conjugate of the pharmaceutical composition forms self-assembly to induce apoptosis in lysosomes within cancer cells.