Monomer self-assembly polymerized by thioesterase and reactive oxygen species in cancer cells and used thereof

Abstract

The present invention relates to a monomer self-assembled and polymerized by thioesterase and reactive oxygen species in cancer cells and a use thereof. According to the present invention, the monomer represented by chemical formula 1 is selectively accumulated in mitochondria and undergoes the deprotection of the acetyl protecting group from the thioacetyl group by thioesterase within cancerous cells to expose a thiol group which is then activated by reactive oxygen species (ROS) within cancerous cells to form disulfide bonds, leading to self-assembly polymerization into a macromolecular polymer that selectively disrupts cancerous mitochondria. In normal cells, deprotection of the acetyl group does not occur, so the monomer remains as it is, exerting no effect.

Description

Monomers that self-assemble and polymerize by thioesterase and reactive oxygen species of cancer cells and their uses

[0001] The present invention relates to a monomer that undergoes self-assembly polymerization by thioesterase and reactive oxygen species of cancer cells and its use.

[0002] The present invention is a technology developed with the support of the 2024 support projects of the Ministry of Science and ICT, “Polymer polymerization reaction and cell fate control in cells (Project number: 2710000928, Subproject number: 00208386)”, “Artificial intelligence-based drug delivery platform for tissue-specific cell and molecular recognition-self-regulation (Project number: 2710003506, Subproject number: 00281553)”, and “Development of a practical method for selective treatment of senescent cells for senescent cell fate control (Project number: 2710017221, Subproject number: 2020M3A9D8038192)”.

[0003]

[0004] Self-assembly of small building blocks has attracted attention in medical technology due to its unique properties, including multivalent surfaces and multifunctional capacities. These assembled structures can significantly bind to biological targets such as proteins or cell membranes, enabling biomedical applications. Among these, cationic nanostructures have been widely reported as anticancer agents that can specifically interact with the membranes of cancer cells, which are more negatively charged than those of normal cells. Cationic nanoassemblies have been demonstrated as drug-free therapeutics that multifactorially stimulate membranes through physical electrostatic stress, enabling them to overcome drug resistance and inhibit cancer metastasis without being affected by mutations. For example, cationic nanoparticles assembled from diblock polycarbonate selectively bind and lyse cancer cell membranes, killing various cancer cells and even cancer stem cells. While cationic nanomaterials have been intensively studied for their anticancer activity, the presence of positively charged surfaces can potentially render them unstable upon administration and potentially toxic to normal healthy cells. Their lack of targeting ability may lead to interactions with negatively charged blood components or other biological substances, which may pose significant obstacles to their clinical application.

[0005]

[0006] Considering the limitations of nanostructures as therapeutic agents, intracellular polymerization-induced self-assembly (iPISA) is a novel strategy that promotes the in-situ construction of intracellular nanostructures. In this approach, small building blocks accumulate in target cells and form polymeric structures through polymerization and self-assembly. The iPISA strategy can generate cationic nanostructures in situ, thereby minimizing the negative impact of assembled nanostructures, such as unexpected interactions with other cells. Recently, the iPISA strategy has been reported as a potential anticancer system that forms entropically stable cationic structures in cancer cells. However, its selectivity for cancer cells may still be limited due to its limited targeting ability and chemical reaction dynamics, suggesting that polymerization can also occur in non-target cells. Therefore, the development of improved iPISA systems with high selectivity for cancer cells is essential for effective anticancer therapy.

[0007]

[0008] The introduction of enzyme-directed systems is an excellent strategy for increasing selectivity in targeting cancer cells, where specific enzymes are overexpressed due to abnormal metabolism. Increased fatty acid synthesis is the most prominent metabolic change in tumor cells, and thioesterase plays a central role in fatty acid synthesis. Therefore, thioesterase is one of the most abundant enzymes in cancer cells. Thioesterase exhibits esterase activity, which dissociates a thiol-ester group into a thiol group and an acid group in the presence of water. This activity has high specificity for thiol groups, and thus the catalytic reaction can be utilized to activate thiol groups for disulfide bond formation.

[0009]

[0010] Based on these phenomena, the fusion of iPISA and a thioesterase-mediated system is expected to enhance the selectivity of disulfide polymerization. This selective polymerization, in turn, could lead to the specific configuration of cationic polymer structures, leading to the selective elimination of cancer cells with less negative impact on normal cells.

[0011]

[0012] In the present invention, we developed a thioesterase-mediated polymerization system utilizing disulfide bonds for highly selective anticancer treatment based on the thioesterase enzyme overexpressed in cancer cells. Increasing the alkyl chain length in the triphenylphosphonium (TPP) derivatives of monomers (Mito-C3, Mito-C6, and Mito-C10) (Figure 7a) enhances the polymer structure's interaction with the mitochondrial membrane through multivalent bonds (Figures 7b and 7c). While this increased interaction can potently disrupt the mitochondrial membrane, it also reduces toxicological safety due to unexpected interactions with biological substances (Figure 7b). To overcome this drawback, the inventors modified the thiol group to a thioester group (Mito-C10-TE) to prevent unexpected polymerization, thereby activating polymerization only upon exposure to a cancerous environment (Figure 7d). In normal cells, polymerization does not occur due to low expression levels of both thioesterase enzymes and reactive oxygen species (ROS) (Figure 7e). However, overexpressed thioesterase enzymes can induce thiol group activation through esterase reactions, leading to preferential disulfide polymerization in the mitochondria of cancer cells over normal cells (Figure 7f). Polymerization forms cationic nanostructures through polymerization-induced self-assembly, which interact strongly with the mitochondrial membrane. Therefore, our strategy offers enhanced insight into selective mitochondrial dysfunction and apoptosis activation in various cancer types, including drug-resistant cells.

[0013]

[0014] Object 1 of the present invention is to provide a monomer that is non-toxic to normal cells and induces selective mitochondrial destruction in cancer cells.

[0015] Object 2 of the present invention is to provide a method for producing the above monomer.

[0016] Object 3 of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising the monomer.

[0017] Object 4 of the present invention is to provide a pharmaceutical composition for preventing or treating drug-resistant cancer comprising the monomer.

[0018] Object 5 of the present invention is to provide a drug delivery system comprising the monomer.

[0019] Object 6 of the present invention is to provide a method for preventing or treating cancer, comprising a step of administering the monomer to a subject in need thereof.

[0020] Object 7 of the present invention is to provide a method for preventing or treating drug-resistant cancer, comprising a step of administering the monomer to a subject in need thereof.

[0021] Object 8 of the present invention is to provide a use of the monomer for the manufacture of a drug for preventing or treating cancer.

[0022] Object 9 of the present invention is to provide a use of the monomer for the manufacture of a drug for preventing or treating drug-resistant cancer.

[0023]

[0024] A monomer represented by chemical formula 1

[0025] The present invention provides a monomer represented by the following chemical formula 1, which comprises a mitochondrial targeting moiety, an acetyl protecting group deprotected by a thioesterase enzyme, and a thiol group capable of self-crosslinking by reactive oxygen species (ROS).

[0026] [Chemical Formula 1]

[0027]

[0028] In the above chemical formula 1,

[0029] L is C containing a cationic moiety 2-25 is an alkylenyl chain,

[0030] R 2 is a mitochondrial targeting moiety, or mitochondrial targeting peptide.

[0031]

[0032] The above mitochondrial targeting moiety may be triphenylphosphonium, dequalinium, guanidinium, triethylammonium, pyridinium, 3-phenylsulfonyl furoxan, F16, 2,3-dimethylbenzothiazolium iodide, rhodamine 19, rhodamine 123, etc.

[0033] The above mitochondrial targeting peptides are Leu-Leu-Arg-Ala-Ala-Leu-Arg-Lys-Ala-Ala-Leu (LLRAALRKAAL: SEQ ID NO: 1), Met-Leu-Arg-Ala-Ala-Leu-Ser-Thr-Ala-Arg-Arg-Gly-Pro-Arg-Leu-Ser-Arg-Leu-Leu (MLRAALSTARRGPRLSRLL: SEQ ID NO: 2), Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys (MLSLRQSIRFFK: SEQ ID NO: 3), Leu-Ser-Arg-Thr-Arg-Ala-Ala-Pro-Asn-Ser-Arg-Ile-Phe-Thr-Arg (LSRTRAAAPNSRIFTR: SEQ ID NO: 4), Met-Ile-Ala-Ser-His-Leu-Leu-Ala-Tyr-Phe-Phe-Thr-Glu-Leu-Asn (MIASHLLAYFFTELN: SEQ ID NO: 5), Met-Ile-Ala-Ser-His-Leu-Leu-Ala-Tyr-Phe-Phe-Thr-Glu-Leu-Asn (MIASHLLAYFFTELN: SEQ ID NO: 6), Lys-Leu-Ala-Lys-Leu-Ala-Lys (KLAKLAK: SEQ ID NO: 7), Lys-Leu-Ala-Lys-Arg-Gly-Asp (KLAKRGD: SEQ ID NO: 8), Lys-Leu-Ala-Lys-Leu-Ala-Lys-Arg-Gly-Asp (KLAKLAKRGD: SEQ ID NO: 9), etc.

[0034] C containing the above cationic moiety 2-20 In the alkylenyl chain of C, the cationic moiety may be an oxonium ion, a quaternary ammonium ion, or a quaternary phosphonium ion. Here, the C 2-20 The alkylenyl chain may contain one or more cationic moieties, and when two or more are contained, they may contain the same or different types of cationic moieties.

[0035]

[0036] Specifically, the monomer represented by the above chemical formula 1 may include a monomer represented by the following chemical formula 1A.

[0037] [Chemical Formula 1A]

[0038]

[0039] In the above chemical formula 1A,

[0040] R 1 Silver C 1-20 is an alkylenyl chain,

[0041] R 2 is a mitochondrial targeting moiety, or mitochondrial targeting peptide.

[0042]

[0043] Specifically, the monomer represented by the above chemical formula 1A may include a monomer represented by the following chemical formula 1B.

[0044] [Chemical Formula 1B]

[0045]

[0046] In the above chemical formula 1B,

[0047] R 1 Silver C 1-20 is the alkylenyl chain.

[0048]

[0049] Preferably the above R 1 Silver C 3-18 An alkylenyl chain, more preferably C 6-15 alkylenyl chain, more preferably C 7-13 alkylenyl chain, especially preferably C 8-12 It may be an alkylenyl chain.

[0050]

[0051] The monomer represented by chemical formula 1 according to the present invention is characterized in that it selectively accumulates in mitochondria, the acetyl protecting group is deprotected from the thioacetyl group by the thioesterase enzyme in cancer cells, thereby activating the thiol group, and the thiol group activated by reactive oxygen species (ROS) in cancer cells self-assembles and polymerizes through disulfide bonds to form a large polymer, thereby selectively destroying cancerous mitochondria. That is, in normal cells, the deprotection of the acetyl protecting group does not occur, so it continues to be maintained in a monomeric state and does not have any effect.

[0052]

[0053] Manufacturing method

[0054] The present invention is as shown in the following reaction scheme 1,

[0055] A method for producing a monomer represented by chemical formula 1 is provided, comprising a step of reacting compound 100 with a compound containing an acetyl group in an organic solvent to produce a monomer represented by chemical formula 1.

[0056] [Reaction Formula 1]

[0057]

[0058] In the above reaction formula 1,

[0059] L and R 2 is as defined in the above chemical formula 1.

[0060]

[0061] The above acetyl group-containing compound may be acetic anhydride, etc.

[0062]

[0063] The organic solvent that can be used is DIPEA (N,N-Diisopropylethylamine), DMSO (dimethyl sulfoxide), DCM (dichloromethane), THF (tetrahydrofuran), acetone, etc.

[0064]

[0065] Pharmaceutical composition for preventing or treating cancer

[0066] The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a monomer represented by the above chemical formula 1.

[0067]

[0068] The above cancers include brain cancer, brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial tumor, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cancer, paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, urethral cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, female urethral cancer, skin cancer, myeloma, It could be leukemia, malignant lymphoma, etc.

[0069]

[0070] Pharmaceutical composition for preventing or treating drug-resistant cancer

[0071] The present invention provides a pharmaceutical composition for preventing or treating drug-resistant cancer, comprising a monomer represented by the above chemical formula 1.

[0072]

[0073] The above drug-resistant cancers include brain cancer, brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial tumor, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cancer, paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, urethral cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, It could be female urethral cancer, skin cancer, myeloma, leukemia, malignant lymphoma, etc.

[0074]

[0075] drug delivery system

[0076] The present invention provides a drug delivery system comprising a monomer represented by the above chemical formula 1.

[0077]

[0078] The above monomers can self-assemble to form a polymer, and specifically, the polymer can have a micelle structure.

[0079] The above drug delivery system can carry a drug inside the polymer.

[0080] The above drugs include compounds, polynucleotides, polypeptides, etc., which have pharmacological activity, and specifically, compounds used as anticancer agents, for example, doxorubicin, docetaxel, Halaven, vincristine, cisplatin, vinblastine, vinorelbine, paclitaxel, etoposide, topotecan, irinotecan, dactinomycin, doxorubicin, daunorubicin, mitomycin, gleevec, carboplatin, valrubicin, flutamide, gemcitabine, These may include bleomycin, temozolomide, procarbazine, lomustine (CCNU; 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea), carmustine (BCNU), tamoxifen, camptothecin, and anasterozole.

[0081]

[0082] Methods for preventing or treating cancer

[0083] The present invention provides a method for preventing or treating cancer, comprising a step of administering a monomer represented by the above chemical formula 1 to a subject in need thereof.

[0084]

[0085] The above cancers include brain cancer, brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial tumor, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cancer, paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, urethral cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, female urethral cancer, skin cancer, myeloma, It could be leukemia, malignant lymphoma, etc.

[0086]

[0087] Methods for preventing or treating drug-resistant cancer

[0088] The present invention provides a method for preventing or treating drug-resistant cancer, comprising a step of administering a monomer represented by the above chemical formula 1 to a subject in need thereof.

[0089]

[0090] The above drug-resistant cancers include brain cancer, brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial tumor, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cancer, paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, urethral cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, It could be female urethral cancer, skin cancer, myeloma, leukemia, malignant lymphoma, etc.

[0091]

[0092] Use of monomers for the manufacture of drugs for the prevention or treatment of cancer

[0093] The present invention provides the use of a monomer represented by the above chemical formula 1 for the manufacture of a drug for preventing or treating cancer.

[0094]

[0095] The above cancers include brain cancer, brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial tumor, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cancer, paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, urethral cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, female urethral cancer, skin cancer, myeloma, It could be leukemia, malignant lymphoma, etc.

[0096]

[0097] Use of monomers for the manufacture of drugs for the prevention or treatment of drug-resistant cancer

[0098] The present invention provides the use of a monomer represented by the above chemical formula 1 for the manufacture of a drug for the prevention or treatment of drug-resistant cancer.

[0099]

[0100] The above drug-resistant cancers include brain cancer, brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial tumor, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cancer, paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, urethral cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, It could be female urethral cancer, skin cancer, myeloma, leukemia, malignant lymphoma, etc.

[0101]

[0102] The monomer represented by chemical formula 1 according to the present invention selectively accumulates in mitochondria, and the acetyl protecting group is deprotected from the thioacetyl group by the thioesterase enzyme in cancer cells, thereby activating the thiol group, and the thiol group activated by reactive oxygen species (ROS) in cancer cells self-assembles and polymerizes through disulfide bonds to form a large polymer, thereby having the effect of selectively destroying cancerous mitochondria. In normal cells, the acetyl protecting group is not deprotected, so it is maintained in a monomeric state and has no effect.

[0103]

[0104] Figure 1a is a schematic diagram of the formation and multivalency of polymer structures by polymerization-induced self-assembly.

[0105] Figure 1b shows the GPC analysis results showing the polymerization behavior of Mito-C3, Mito-C6, and Mito-C10 at various concentrations (5, 10, and 30 mM). (Mw: weight-average molecular weight; PDI (poly dispersity index): polydispersity index)

[0106] Figure 1c is a calibration plot of Nile Red from the emission spectra at 655 nm in solutions of Mito-C3, Mito-C6, and Mito-C10 dissolved in PBS (pH 8.0) containing 10 mM GSH and 1 mM H2O2.

[0107] Figure 1d is a TEM image of the polymer structures of Mito-C3, Mito-C6, and Mito-C10.

[0108] Figure 1e shows the dye release results from model liposomes after incubation with Mito-C3, Mito-C6, and Mito-C10, and the fluorescence of the dye can be recovered by destroying the membrane of the liposomes.

[0109] Figure 1f is a fluorescence image of cancer cells (HeLa) and normal cells (NIH3T3) labeled with TMRM after treatment with Mito-C3, Mito-C6, and Mito-C10.

[0110] Figure 1g shows the results of cell viability analysis for HeLa cells (right) and NIH3T3 cells (left) cultured with Mito-C3, Mito-C6, and Mito-C10 at various concentrations.

[0111] Figure 1h shows the selectivity index (IC of normal cells) of Mito-C3, Mito-C6 and Mito-C10 50 IC of cancer cells 50 The value is the result.

[0112] Figure 2a is a reaction scheme showing the chemical reaction of acetylation of the thiol group of the Mito-C10 monomer and its dissociation by thioesterase.

[0113] Figure 2b shows the HPLC analysis results of Mito-C10-TE in PBS containing 0.5 U of ACOT (Acyl-CoA thioesterase 9) (Mito-C10-TE + Ac). After cleavage of the acetyl group, the peak of Mito-C10-TE was rearranged into the peak of Mito-C10.

[0114] Figure 2c is an IR spectrum result showing the reactivation of the thiol group through deprotection of the acetyl group by ACOT.

[0115] Figure 2d shows the GPC analysis results of Mito-C10-TE in a mitochondrial mimic solution for co-culture with 0.5 U of ACOT.

[0116] Figure 2e shows the DLS measurement results confirming that ACOT-mediated acetyl group deprotection of Mito-C10-TE is reverted to Mito-C10.

[0117] Figure 2f is a TEM image showing ACOT-mediated acetyl group deprotection and polymerization-induced self-assembly of Mito-C10-TE in a mitochondrial-mimetic solution in ACOT and oxidizing (H2O2) environment.

[0118] Figure 2g is a Western blot image and quantitative data showing the expression levels of ACOT in cancer cells (HeLa) and normal cells (NIH3T3).

[0119] Figure 2h shows the results of FACS analysis of HeLa and NIH3T3 cells after immunostaining of ACOT.

[0120] Figure 3a is a diagram illustrating that the thioesterase-mediated disulfide formation induced by the ACOT overexpression environment and high ROS levels in cancer cells effectively causes the FRET phenomenon between Mito-G-TE release and Mito-R-TE uptake.

[0121] Figure 3b is a diagram illustrating that in an environment where ACOT is underexpressed in normal cells, the acetyl protecting group is maintained and disulfide bonds are not formed, so that the FRET phenomenon does not occur between Mito-G-TE release and Mito-R-TE uptake.

[0122] Figure 3c is a confocal image showing mitochondrial localization of Mito-G-TE in HeLa cells after 4 h of incubation. Mitotracker Blue was used to confirm mitochondrial localization.

[0123] Figure 3d is a confocal image showing mitochondrial localization of Mito-R-TE in HeLa cells after 4 h of incubation. Mitotracker Blue was used to confirm mitochondrial localization.

[0124] Figure 3e is an in-situ monitoring fluorescence image of HeLa and NIH3T3 cells cultured with Mito-G-TE and Mito-R-TE for 280 minutes.

[0125] Figure 3f shows the absorption spectra of (a) Mito-G-TE and (b) Mito-R-TE, and (c) the emission spectra of Mito-G-TE and Mito-R-TE under excitation at 450 nm and 560 nm, respectively.

[0126] Figure 3g shows the time-dependent emission spectra of 200-200 μM Mito-G-TE and Mito-R-TE mixture dissolved in mitochondrial mimic solution (pH 8.0 PBS, 10 mM GSH, and 1 mM H2O2) in the presence of 0.5 U acyl-CoA thioesterase 9 (a) and in the absence of 0.5 U acyl-CoA thioesterase 9 (b).

[0127] Figure 4a shows the results of GPC analysis tracking intramitochondrial polymerization induced by ACOT activity in HeLa cells. The polymer properties were analyzed in a mitochondrial separation solution.

[0128] Figure 4b shows the results of a GPC analysis tracking polymerization within mitochondria in NIH3T3 cells. The polymer properties were analyzed in a mitochondrial separation solution.

[0129] Figure 4c is a confocal microscopy image observing mitochondrial morphological changes in HeLa cells labeled with Mitotracker Green after Mito-C10-TE incubation.

[0130] Figure 4d is a confocal microscopy image observing mitochondrial morphological changes in NIH3T3 cells labeled with Mitotracker Green after Mito-C10-TE culture.

[0131] Figure 4e is a fluorescence image of HeLa cells labeled with JC-1 dye to confirm depolarization of the mitochondrial membrane.

[0132] Figure 4f shows the calculated quantitative ratio of green fluorescence to red fluorescence in the confocal image.

[0133] Figure 4g is a confocal microscopy image showing the fluorescence of Rho-2, AM, and MitoSOX in HeLa cells treated with Mito-C10-TE for 24 hours.

[0134] Figure 4h is the quantified fluorescence intensity of Rho-2, AM in HeLa cells cultured with various concentrations of Mito-C10-TE.

[0135] Figure 4i is the quantified fluorescence intensity of MitoSOX in HeLa cells cultured with various concentrations of Mito-C10-TE.

[0136] Figure 4j is a CLSM image of (a) JC-1-labeled and (b) TMRM-labeled HeLa and NIH3T3 cells after treatment with 50 μM Mito-C10-TE for 24 h of culture.

[0137] Figure 5a shows the results of HeLa and NIH3T3 cell survival rates according to treatment with various concentrations of Mito-C10-TE.

[0138] Figure 5b shows the results of calculating the selectivity index of Mito-C10-TE compared to the selectivity index of Mito-C10 for cancer cells.

[0139] Figure 5c shows the IC of Mito-C10-TE against various cell lines including cancer and normal cells. 50 This is the result that shows the value.

[0140] Figure 5d shows the results of Annexin V / PI analysis in HeLa and NIH3T3 cells after 36 hours of incubation with Mito-C10-TE. The results indicate that selective polymerization specifically induced apoptosis in HeLa cells.

[0141] Figure 5e shows the cell viability results of MDA-MB-468 and MDA-MB-458-ADR cells treated with various concentrations of doxorubicin.

[0142] Figure 5f shows the cell viability results of MDA-MB-468 and MDA-MB-458-ADR cells treated with various concentrations of Mito-C10-TE.

[0143] Figure 5g shows the MDR analysis results of MDA-MB-468 cells treated with various concentrations of PBS (control), verapamil (drug efflux pump inhibitor), and Mito-C10-TE.

[0144] Figure 5h shows the cell viability results of MDA-MB-468 cells under repeated treatment with doxorubicin to confirm multi-drug resistance.

[0145] Figure 5i shows the cell viability results of MDA-MB-468 cells under repeated treatment with Mito-C10-TE to confirm multi-drug resistance.

[0146] Figure 5j shows the IC of doxorubicin and Mito-C10-TE after repeated treatment with doxorubicin or Mito-C10-TE to confirm multidrug resistance. 50 It's worth it.

[0147] Figure 6a is a schematic diagram showing the structure of Mito-C10-TE-Dox micelles and the release of doxorubicin by ACOT activity in cancer cells.

[0148] Figure 6b shows the DLS analysis results of Mito-C10-TE-Dox and Mito-C10-TE-Dox after 7 days.

[0149] Figure 6c is a TEM image of Mito-C10-TE-Dox and Mito-C10-TE-Dox after 7 days.

[0150] Figure 6d is a graph showing ACOT-dependent doxorubicin release from Mito-C10-TE-Dox.

[0151] Figure 6e is a flow cytometry analysis result showing the uptake of Mito-C10-TE-Dox in HeLa cells over the culture time.

[0152] Figure 6f is a time-dependent confocal image showing the cellular uptake of Mito-C10-TE-Dox and release of doxorubicin in HeLa cells over 12 h.

[0153] Figure 6g is a diagram showing Pearson's coefficient values ​​between doxorubicin and lysotracker fluorescence.

[0154] Figure 7a is a diagram showing the chemical structure of mitochondrial targeting monomers with increasing alkyl chain length.

[0155] Figure 7b is a schematic diagram showing the relationship between membrane interactions and biosafety of polymer structures resulting from polymerization-induced self-assembly of Mito-C3, Mito-C6, and Mito-C10.

[0156] Figure 7c is a schematic diagram showing the degree of membrane destruction by polymer structure, with Mito-C10, Mito-C6, and Mito-C3 polymers sequentially destroying the mitochondrial membrane.

[0157] Figure 7d is a chemical reaction scheme for acetylation of thiol groups to prevent unexpected disulfide formation.

[0158] Figure 7e is a schematic diagram showing that thioesterase-mediated polymerization-induced self-assembly does not occur at low ACOT (acyl-CoA thioesterase 9) levels and low ROS (reactive oxygen species) levels in normal cells, indicating no effect on mitochondria.

[0159] Figure 7f is a schematic diagram showing that thioesterase-mediated polymerization-induced self-assembly occurs at high ACOT levels and high ROS levels in cancer cells, resulting in mitochondrial destruction.

[0160]

[0161] Hereinafter, the present invention will be described in detail.

[0162]

[0163] pharmaceutical composition

[0164] The active substance of the present invention can be administered in various oral and parenteral dosage forms during clinical administration, and when formulated, it is manufactured using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0165] Solid preparations for oral administration include tablets, tablets, powders, granules, capsules, troches, etc., and these solid preparations are prepared by mixing one or more active substances of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included.

[0166] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0167] In addition, the effective dosage for the human body of the active substance of the present invention may vary depending on the patient's age, body weight, sex, dosage form, health condition, and disease severity, and is generally about 0.001-100 mg / kg / day, and preferably 0.01-35 mg / kg / day. Based on an adult patient weighing 70 kg, it is generally 0.07-7000 mg / day, and preferably 0.7-2500 mg / day, and may be administered once or several times a day at regular intervals depending on the judgment of a doctor or pharmacist.

[0168]

[0169] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.

[0170]

[0171] Materials and General Methods

[0172] Acetyl anhydride, N,N-diisopropylethylamine, triphenylphosphine, 1,6-dibromohexane, and 1,10-dibromodecane were purchased from Alfa aesar (USA). Dichloromethane, acetonitrile, and methanol were purchased from TCI (Japan). All products were purified by HPLC (Aglient Technologies, US) using a C18 reversed-phase column. TMRM, MitoSOX, Mitotraker Blue, Mitotraker Green, JC-1, and Rho-2 AM were purchased from Thermo Fisher Scientific (USA). Anti-Acyl-CoA Thioesterase 9 (ACOT) antibody was purchased from Abcam (USA). Synthetic identification was performed using NMR and IR. GPC analysis was performed using an Aglient 1200 (column L KD-803, solvent: DMF). LSM980 and LSM780 confocal microscopes were utilized to acquire all CLSM images. Bio-TEM (JEM 1400) was used to acquire all TEM images.

[0173]

[0174] Calculation of critical concentration for polymerization-induced self-assembly (PISA)

[0175] The critical concentration was determined using the NileRed assay, which emits red fluorescence in response to a hydrophobic environment. Compounds were dissolved in a mitochondrial-mimetic solution (pH 8.0 PBS, 10 mM GSH, and 1 mM H2O2) and incubated with 300 nM NileRed. After stirring overnight, NileRed fluorescence was observed under excitation at 550 nm. The maximum fluorescence intensity at 625 nm was then plotted to calculate the critical concentration.

[0176]

[0177] TEM analysis for polymer structure determination

[0178] TEM samples of Mito-C3, Mito-C6, Mito-C10, and Mito-C10-TE were prepared by dissolving 10 μM of the desired compounds in mitochondrial-mimetic solution (pH 8.0 PBS, 10 mM GSH, and 1 mM H2O2). After stirring for 24 h, the samples were placed on 300-mesh copper grids coated with carbon film. Subsequently, 2% uranyl acetate staining was performed for 5 min. The samples were analyzed using a Bio-TEM (JEM 1400).

[0179]

[0180] Determination of membrane disruption of model liposomes

[0181] Calcein-loaded liposomes were treated with 10 mM Mito-C3, Mito-C6, and Mito-C10. The dye in the liposomes emitted weak fluorescence due to quenching, but this fluorescence could be recovered by the dye released from the disrupted liposomes. The green fluorescence kinetics of the liposome solution was monitored at 550 nm for 50 min. To assess the maximum fluorescence intensity, the liposomes were further incubated with an excess of a fluorescent whitening agent (detergent). After confirming the maximum intensity, membrane disruption was assessed by dividing the fluorescence intensity obtained by compound treatment by the maximum intensity.

[0182]

[0183] cell culture

[0184] All cell lines, including cancer cells (HeLa, 4T1, MDA-MB-231, MDA-MB-468, HT-29) and normal cells (NIH3T3, HEK293 cells), were purchased from the Korea Cell Bank. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Life Technologies) at 37°C in a humidified atmosphere of 10% CO2. Cytotoxicity was analyzed using the MTT assay by treating with various concentrations of synthetic compounds. All data points were measured in quadruplicate. MTT fluorescence was monitored using a microplate reader (SpectraMAX 384).

[0185]

[0186] Mitochondrial membrane depolarization imaging using TMRM

[0187] To assess mitochondrial membrane depolarization, HeLa and NIH3T3 cells were treated with 20 μM Mito-C3, Mito-C6, Mito-C10, and 50 μM Mito-C10-TE for 24 h. After washing three times with PBS, cells were incubated with TMRM according to the manufacturer's instructions. Intracellular TMRM images were obtained using an LSM 980 instrument.

[0188]

[0189] Mitochondrial membrane depolarization imaging using JC-1

[0190] To assess mitochondrial membrane depolarization, HeLa and NIH3T3 cells were treated with 20 μM Mito-C3, Mito-C6, Mito-C10, and 50 μM Mito-C10-TE for 24 h. After washing three times with PBS, cells were incubated with JC-1 according to the manufacturer's instructions. Images of JC-1-labeled cells were obtained using an LSM 980 instrument.

[0191]

[0192] Mitochondrial ROS imaging using MitoSOX

[0193] To assess mitochondrial ROS, HeLa and NIH3T3 cells were treated with 50 μM Mito-C10-TE for 24 h. After washing three times with PBS, the cells were incubated with MitoSOX according to the manufacturer's instructions. Images of MitoSOX-labeled cells were obtained using an LSM 980 instrument.

[0194]

[0195] Imaging of released calcium ions using Rho-2, AM

[0196] To assess calcium ion release from mitochondria, HeLa and NIH3T3 cells were treated with 50 μM Mito-C10-TE for 24 h. After washing three times with PBS, cells were incubated with Rho-2 and AM according to the manufacturer's instructions. Images of Rho-2 and AM-labeled cells were obtained using an LSM 980 instrument.

[0197]

[0198] Western blot analysis of the expression level of acyl-CoA thioesterase 9 (ACOT)

[0199] Western blot analysis of HeLa and NIH3T3 cells was performed by separating cells on SDS-PAGE gels and immunoblotting with anti-Acyl-CoA Thioesterase 9 antibody (1:1000, Abcam, ab252978) according to the manufacturer's protocol. Depending on the type of primary antibody, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies for 2 hours. Immunoreactive proteins were detected using an enhanced chemiluminescence substrate (Thermo Fisher, 35480) and quantified using ImageJ (NIH, USA). All experiments were performed at least three times.

[0200]

[0201] <Manufacturing Example 1> Preparation of Mito-C3

[0202] <Manufacturing Example 2> Preparation of Mito-C6

[0203] <Manufacturing Example 3> Preparation of Mito-C10

[0204] Mito-C3, Mito-C6, and Mito-C10 (see chemical structures below) were prepared according to the inventor's previous research protocol.

[0205]

[0206]

[0207] <Example 1> Preparation of Mito-C10-TE

[0208] According to the reaction scheme shown in Figure 7d, the starting materials Mito-C10 (0.35 g, 0.52 mmol), acetic anhydride (0.53 g, 5.2 mmol), and DIPEA (363 μL, 2.08 mmol) were further dissolved in DCM. The mixture was then stirred for 24 h. The product was purified by HPLC, producing Mito-C10-TE.

[0209] 1 H NMR (400 MHz, MeOD): δ 1.35 (m, 12H), 1.55 (m, 2H), 1.61 (m, 6H), 2.04 (m, 2H), 2.45 (s, 6H), 3.06 (s, 6H), 3.38 (t, 2H), 3.47-3.53 (m, 4H), 3.61 (t, 2H), 7.42 (s, 1H), 7.52(s, 2H), 7.65-7.9 (m, 15H).

[0210]

[0211] <Experimental Example 1> Increased binding affinity of cationic nanostructures and its negative effects

[0212] Nanostructures have multivalency on their surfaces, allowing them to interact strongly with biomacromolecules, whereas individual building blocks do not exhibit surface multivalency. From this perspective, we anticipated that the mitochondrial-targeting monomers would be localized to small organelles, polymerize via disulfide formation, and self-assemble to exhibit highly robust interactions with the negative mitochondrial membrane (Fig. 1a). Furthermore, the increased hydrophobicity of the molecular design was expected to enhance the binding affinity of the assembled structures. To investigate this, we synthesized various mitochondrial-targeting monomers with increasing lengths of the alkyl chain (propyl, hexyl, and decyl) between the quaternary ammonium ion and triphenylphosphonium (TPP), designated Mito-C3, Mito-C6, and Mito-C10, respectively. After synthesis, the products were purified using high-performance liquid chromatography (HPLC). 1 The products were confirmed by analyzing H NMR and mass spectra (Preparation Examples 1 to 3).

[0213]

[0214] Next, the polymerization behavior of these three monomers was investigated using gel permeation chromatography (GPC) after stirring for 24 h at various concentrations (5, 10, and 30 mM) in a mitochondrial-mimetic solution (pH 8.0 PBS, 10 mM GSH, and 1 mM H2O2).

[0215] GPC analysis results showed that all three monomers exhibited similar polymerization tendencies in 5 mM, 10 mM, and 30 mM solutions, with molecular weights of approximately 8.0 kDa, 14.0 kDa, and 20.0 kDa, respectively. Increasing the alkyl chain length did not appear to affect the polymerization characteristics (Fig. 1b).

[0216]

[0217] As disulfide polymerization induces self-assembly behavior, we first investigated the critical aggregation concentration (CAC) of Mito-C3, Mito-C6, and Mito-C10 in mitochondrial-mimetic solutions.

[0218] Mito-C3 exhibited a critical concentration of ~1.22 mM, but due to increased hydrophobicity, the concentration decreased to 0.83 mM and 0.59 mM in Mito-C6 and Mito-C10 solutions, respectively (Fig. 1c). After 24 h of incubation in a mitochondrial-mimetic solution, a 5 mM Mito-C3 solution, which is above the critical concentration, showed fibril structures using transmission electron microscopy (TEM). More robust fibril structures were observed in the 5 mM Mito-C6 and Mito-C10 solutions (Fig. 1d). These results suggest a correlation with enhanced self-assembly behavior.

[0219]

[0220] We then investigated whether a rigid fibrillar structure could better disrupt the mitochondrial membrane. To analyze this, mitochondrial-mimicking liposomes were prepared by loading calcein AM into the liposomes using a known method. While calcein AM fluorescence quenches in the liposomes, green fluorescence can be recovered by dye release from the liposomes upon membrane disruption. Therefore, green fluorescence was monitored after treatment with 5 mM Mito-C3, Mito-C6, and Mito-C10 in mitochondrial-mimicking solutions. Up to 60% dye release was observed within 50 minutes after treatment with Mito-C10, whereas lower releases of up to 50% and 35% were observed in liposomes treated with Mito-C6 and Mito-C3 solutions, respectively (Fig. 1e). These results indicate that nanostructures composed of monomers with longer alkyl chains can better disrupt the membrane.

[0221]

[0222] The effect of the nanostructures on the mitochondrial membrane was further investigated in HeLa cells labeled with tetramethylrhodamine methyl ester (TMRM), which loses red fluorescence in response to mitochondrial membrane depolarization. Bright red fluorescence was observed in untreated HeLa cells, but the fluorescence slightly disappeared after treatment with 20 μM Mito-C3 and Mito-C6. However, fluorescence completely disappeared in cells treated with 20 μM Mito-C10 for 12 h (Fig. 1f). We confirmed that Mito-C10 was more potent than Mito-C3 and Mito-C6 in disrupting the mitochondrial membrane. However, due to its lack of selectivity, a similar membrane disruption trend was observed in normal cells (NIH3T3 cells) (Fig. 1f). Specifically, treatment of NIH3T3 cells with 20 μM Mito-C3 had little negative effect, whereas the fluorescence of normal cells was completely reduced after treatment with 20 μM Mito-C10.

[0223]

[0224] Since mitochondria are involved in programmed cell death, non-targeted disruption of the mitochondrial membrane leads to the activation of apoptosis in both cancer and normal cells. In this regard, the effects of non-targeted cationic nanostructures were investigated by analyzing cell viability in cancer (HeLa) and normal (NIH3T3) cells. As expected, Mito-C3, Mito-C6, and Mito-C10 exhibited increased toxicity toward cancer cells with increasing alkyl chain length, but their toxicity toward normal cells also increased (Fig. 1g).

[0225]

[0226] To specifically estimate the negative effects on normal cells, the selectivity index, which represents the targeting ability of the molecules, was calculated. The selectivity index of Mito-C3 was calculated to be 2.7, while the values ​​for Mito-C6 and Mito-C10 decreased to 2.5 and 2.3, respectively (Fig. 1h). These results suggest that cationic nanostructures with high binding affinity without selectivity significantly disrupt the mitochondrial membrane of cancer cells, but also have negative effects on normal cells.

[0227]

[0228] <Experimental Example 2> Thioesterase-mediated polymerization for selective construction of cationic fibrillar structures

[0229] We hypothesized that incorporating enzyme (thioesterase)-mediated cleavage into a thiol group would enhance the selectivity of disulfide polymerization, allowing for selective disulfide bond formation in the mitochondria of cancer cells. To verify this hypothesis, we first conjugated the acetyl group to the thiol group using acetyl anhydride (Figure 2a). The product was purified by HPLC. 1 It was confirmed by H NMR, and the peak of the acetyl group was confirmed here (Example 1).

[0230]

[0231] We investigated whether thioesterases could induce cleavage of the acetyl group and reactivate the thiol group for disulfide bond formation. Thioesterase-mediated cleavage was analyzed using HPLC after incubation of 0.5 U of Acyl-CoA thioesterase 9 (ACOT), a member of the thioesterase superfamily, in 10 mM Mito-C10-TE dissolved in phosphate-buffered saline (PBS) at pH 7.4 for 12 h. As shown in Fig. 2b, HPLC analysis revealed peaks for Mito-C10 and Mito-C10-TE at retention times of 1.4 and 7.3 min, respectively. However, the intensity of Mito-C10-TE decreased, and a new peak correlated with the peak of Mito-C10 appeared at approximately 1.4 min after 12 h of incubation with ACOT (Fig. 2b). This indicates that ACOT induces the conversion of Mito-C10-TE to Mito-C10 through cleavage of the acetyl group.

[0232]

[0233] The reactivation of the thiol group was further monitored using infrared spectroscopy in a 10 mM Mito-C10-TE solution incubated with 0.5 U of ACOT. In the initial state, the peak intensity was 2700–2900 cm -1 No SH binding peaks were detected in the range. However, a peak appeared after 12 h of incubation, indicating that the thiol group was revealed by cleavage of the acetyl group (Fig. 2c).

[0234]

[0235] Because the thiol group can be activated by cleavage of the acetyl group under the activity of a thioesterase, specific polymerization is expected in an environment rich in thioesterase and oxidants. Specific polymerization was monitored by GPC analysis in 10 mM Mito-C10-TE dissolved in a mitochondrial-mimetic solution. In the absence of ACOT, no polymer was observed in the solution even after stirring for 24 h. However, after incubation for 24 h with 0.5 U of ACOT, a polymer peak was observed at a retention time of 7–8 min, which was consistent with the results obtained for 10 mM Mito-C10. Self-assembly behavior was observed using dynamic light scattering (DLS) and transmission electron microscopy (TEM) after stirring for 24 h. DLS analysis revealed nanostructures of approximately 20 nm in size in the Mito-C10-TE solution, which increased to ~400 nm after incubation with 0.5 U of ACOT, consistent with the nanostructures of Mito-C10 (Fig. 2e). Furthermore, TEM analysis revealed that spherical structures were observed in the 10 mM Mito-C10-TE solution even after 1 mM H2O2 treatment, whereas robust fibrous structures were observed in the Mito-C10-TE solution incubated in 0.5 U of ACOT for 24 h (Fig. 2f). These results suggest that the construction of fibrillar structures through disulfide polymerization can only be generated under the activity of ACOT, even in an oxidizing environment.

[0236]

[0237] Because ACOT is overexpressed in cancer cells due to abnormal metabolism, we anticipated that this thioesterase-mediated fibril structure formation might occur preferentially in cancer cells rather than in normal cells. To demonstrate this, we first analyzed the expression levels of ACOT in both cancer and normal cells. Western blot analysis showed that ACOT expression levels were significantly increased in cancer cells (HeLa) compared to normal cells (NIH3T3) (Figure 2g). As shown in Figure 2h, quantitative analysis revealed that the amount of ACOT in HeLa cells was approximately 18-fold higher than that in NIH3T3 cells. This suggests that the thioesterase-mediated polymerization system can be selectively activated in a cancer environment.

[0238]

[0239] <Experimental Example 3> In situ monitoring of disulfide bond formation in response to ACOT within cancer cells.

[0240] To realize the selective formation of disulfide bonds in cancer cells, we utilized the fluorescence resonance energy transfer (FRET) characteristic to monitor disulfide bond formation within cells. The designed Mito-G-TE (Fig. 3a) has maximal absorption and emission at approximately 450 nm and 555 nm, respectively, under 450 nm excitation (Fig. 3f a and c). Furthermore, the synthesized Mito-R-TE (Fig. 3a) exhibits maximal absorption and emission at approximately 560 nm and 630 nm under 560 nm excitation, indicating that the emission of Mito-G-TE overlaps well with that of Mito-R-TE (Fig. 3f b and c). Therefore, when Mito-G-TE is close to Mito-R-TE, red fluorescence can be observed even under 450 nm excitation due to the FRET phenomenon. By leveraging this characteristic, we expected to be able to monitor the bright red fluorescence through disulfide bond formation between Mito-G-TE and Mito-R-TE. To verify this hypothesis, we first analyzed the fluorescence changes in 200 μM Mito-G-TE and Mito-R-TE solutions. After treatment with 0.5 U of ACOT and 1 mM H2O2, a decrease in the green fluorescence of Mito-G-TE and an increase in the red fluorescence of Mito-R-TE were observed over time (Fig. 3g, a). In contrast, there was little fluorescence change in the solution even after treatment with 1 mM H2O2 in the absence of ACOT, likely due to the inability to form disulfide bonds (Fig. 3g, b). These results demonstrated that this FRET strategy can reflect the formation of disulfide bonds in real time.

[0241]

[0242] Leveraging the FRET characteristic, the formation of disulfide bonds within cells can be monitored in situ through changes in fluorescence. In the monomeric state, disulfide bonds are not formed, so bright red fluorescence may not be observed. However, when the thiol group activated by ACOT forms a disulfide bond between Mito-G and Mito-R, green fluorescence decreases and red fluorescence increases. Therefore, this FRET phenomenon is observed only in the mitochondria of cancer cells, not in normal cells, because ACOT is selectively and highly overexpressed in cancer cells (Figures 3a and 3b).

[0243]

[0244] To demonstrate this, the mitochondrial localization of compounds was analyzed in cancer cells (HeLa) and normal cells (NIH3T3) treated with 20 μM of each compound, Mito-G-TE and Mito-R-TE. The fluorescence of Mito-G-TE and Mito-R-TE overlapped well with the blue fluorescence of Mitotracker, with Pearson coefficients of 0.862 and 0.876, respectively (Fig. 3c, 3d). Next, the fluorescence changes were observed in both HeLa and NIH3T3 cells treated with 20 μM of Mito-G-TE and Mito-R-TE for 2 h. As the incubation time increased, a decrease in green fluorescence and an increase in red fluorescence were monitored in HeLa cells (Fig. 3e), whereas only a minimal change was observed in NIH3T3 cells (Fig. 3f). These results indicate that disulfide bonds between acetyl-modified monomers can be selectively formed in the mitochondria of cancer cells.

[0245]

[0246] <Experimental Example 4> Selective mitochondrial dysfunction through cancer-specific disulfide bond formation

[0247] We attempted to utilize polymer production through selective formation of disulfide bonds in cancer cells. Cancer cells (HeLa) and normal cells (NIH3T3) were cultured with 50 μM Mito-C10-TE for 24 hours, and mitochondria were isolated. Polymer production was then analyzed using GPC in the mitochondrial isolation solution. Polymer formation was observed in the mitochondria of HeLa cells, but not in NIH3T3 cells (Fig. 4a, b).

[0248]

[0249] It was expected that the polymer structure, which selectively forms disulfide bonds in cancer cells, would favor mitochondrial dysfunction in cancer cells and have less negative effects on normal cells. As expected, mitochondrial morphology disruption was observed in HeLa cells after 24 h of treatment with 50 μM Mito-C10-TE (Fig. 4c). In contrast, mitochondrial morphology was maintained in NIH3T3 cells after 24 h of treatment with 50 μM Mito-C10-TE (Fig. 4d).

[0250]

[0251] Mitochondrial dysfunction was further analyzed using the JC-1 assay, which demonstrates that dimerization of the JC-1 probe elicits bright red fluorescence in healthy mitochondria, whereas membrane depolarization results in the emission of green fluorescence. The JC-1 assay revealed that bright red fluorescence was observed in HeLa cells treated with 50 μM Mito-C10-TE for 6 h, whereas green fluorescence appeared within 18 h of incubation (Fig. 4e). Furthermore, the ratio of green to red fluorescence increased with increasing incubation time (Fig. 4f). In contrast, bright red fluorescence remained at its initial level in NIH3T3 cells even after 24 h of treatment with 50 μM Mito-C10-TE (Fig. 4j, a). Mitochondrial membrane depolarization was further observed in cells labeled with TMRM. After 24 h of incubation with 50 μM Mito-C10-TE, the bright red fluorescence disappeared in HeLa cells, whereas fluorescence remained in NIH3T3 cells (Fig. 4j, b). This suggests that Mito-C10-TE can selectively destroy the mitochondrial membrane of cancer cells.

[0252]

[0253] Membrane depolarization contributes to mitochondrial stress, inducing the production of mitochondrial ROS and the release of calcium ions from mitochondria. Therefore, we assessed mitochondrial ROS production using MitoSOX, which emits red fluorescence in response to mitochondrial ROS. As shown in Figure 4g, bright red fluorescence was observed in MitoSOX-labeled HeLa cells after 24 h of treatment with 50 μM Mito-C10-TE. Furthermore, calcium ion release was monitored using Rho-2, AM, whose fluorescence is enhanced upon interaction with calcium ions. Confocal images showed an increase in red fluorescence in HeLa cells incubated with 50 μM Mito-C10-TE within 24 h (Figure 4g). Additionally, increases in MitoSOX, Rho-2, and AM intensities were assessed at various concentrations of Mito-C10-TE, verifying that Mito-C10-TE induces mitochondrial dysfunction in cancer cells (Fig. 4h, Fig. 4i).

[0254]

[0255] <Experimental Example 5> Anticancer effect of thioesterase-mediated polymerization without drug resistance development

[0256] Considering the results on selective mitochondrial dysfunction in cancer cells, we envisioned that Mito-C10-TE would specifically inhibit cancer cell viability without reducing toxicity to normal cells. To verify the selective inhibition of cancer cells, the toxicity of Mito-C10-TE was assayed against both HeLa and NIH3T3 cells using 2,5-diphenyl-2H-tetrazolium bromide (MTT).

[0257]

[0258] Mito-C10-TE was cultured for 48 hours before IC 50 While it showed toxicity against HeLa cells with an IC value of ~43.7 μM (Fig. 5a, Fig. 5c), 50It was found to be less toxic to NIH3T3 cells at a concentration of ~356.5 μM (Fig. 5c). As expected, Mito-C10-TE showed higher selectivity for targeting cancer cells with a selectivity index of ~8.2 than Mito-C10 with a selectivity index of ~2.3, as shown in Fig. 5b. In addition, it was shown to be able to inhibit various cancer cell lines, including 4T1, MDA-MB-231, HeLa, and HT-29 cells, while showing lower toxicity to normal cells (NIH3T3 and HEK293) (Fig. 5c).

[0259]

[0260] This selective anticancer effect was further investigated using fluorescence-activated single cell sorting (FACS) in both Annexin V-FITC and propidium iodide (PI)-labeled HeLa and NIH3T3 cells. HeLa cells were found to progress to necrosis within 36 h after treatment with 50 μM Mito-C10-TE. In contrast, Mito-C10-TE did not induce necrosis in NIH3T3 cells even after 36 h of incubation. These results demonstrate that Mito-C10-TE can selectively induce apoptosis in cancer cells (Fig. 5d).

[0261]

[0262] Beyond metabolism, mitochondria contribute to the development of drug resistance through the synthesis of specific mitochondrial proteins, enabling cancer cell survival during drug treatment. From this perspective, mitochondrial targeting systems are expected to be a promising therapeutic approach for drug-resistant cells.

[0263]

[0264] To investigate whether the present system can overcome drug resistance, the viability of doxorubicin-resistant human breast cancer cells (MDA-MB-468-ADR) and drug-sensitive cells (MDA-MB-468) was evaluated upon treatment with various concentrations of doxorubicin and Mito-C10-TE. Doxorubicin at 20 μM exhibited clear anticancer activity in MDA-MB-468 cells, but no activity was observed in MDA-MB-468-ADR cells (Fig. 5e). In contrast, Mito-C10-TE exhibited similar anticancer activity against MDA-MB-468 and MDA-MB-468-ADR cells, with a nearly identical trend in cell viability (Fig. 5f).

[0265]

[0266] To investigate the ability to overcome drug resistance, a multidrug resistance (MDR) assay was additionally performed to analyze the activity of drug efflux pumps in MDA-MB-468-ADR. The results showed that Mito-C10-TE effectively reduced drug efflux activity in MDA-MB-468-ADR cells, similar to the group treated with verapamil, a known drug efflux pump inhibitor (Fig. 5g).

[0267]

[0268] Given the therapeutic efficacy of the present invention against drug-resistant cancer cells, the development of drug resistance to Mito-C10-TE was investigated. Following a previously reported protocol, the development of resistance was assessed in MDA-MB-468 cells treated with 20 μM Mito-C10-TE or 75 nM doxorubicin for 1, 2, 4, 12, 24, and 48 h. As shown in Figures 5h and 5j, the IC of doxorubicin 50 The value increased by more than 22 times. In contrast, the IC of Mito-C10-TE 50Little change was observed in the values ​​(Figures 5i and 5j). These data demonstrate that multiple treatments with doxorubicin induce the development of drug resistance, whereas repeated treatments with Mito-C10-TE do not lead to the development of resistance due to its mitochondrial targeting ability.

[0269]

[0270] <Experimental Example 6> Enzyme-responsive drug release capacity of drug-loaded thioesterase-mediated polymerization

[0271] Additionally, considering that thioesterase-mediated polymerization self-assembles into a spherical shape and is enzymatically reactive, its potential as a drug delivery system was evaluated.

[0272] As illustrated in Fig. 6a, doxorubicin (Dox) can be delivered into cancer cells in the form of Mito-C10-TE polymer micelles by encapsulating it within the Mito-C10-TE polymer, and the Mito-C10-TE polymer containing the doxorubicin internalized into the cancer cells releases the doxorubicin loaded therein into the cancer cells by converting Mito-C10-TE into Mito-C10 by ACOT activity.

[0273] Specifically, to confirm the formation of the micelle, Mito-C10-TE and doxorubicin (Dox) were dissolved in a mitochondrial-mimetic solution and stirred to form Mito-C10-TE polymerized micelles loaded with doxorubicin (hereinafter, Mito-C10-TE-Dox). The self-assembly behavior of the formed micelles was observed using dynamic light scattering (DLS) and TEM.

[0274] Mito-C10-TE-Dox showed a size of ~40 nm, which was larger than the size of ~20 nm of micelles formed by self-assembling only Mito-C10-TE by stirring due to drug loading (Fig. 6b). In addition, when the size change of the Mito-C10-TE-Dox was measured by stirring for 7 days, the size of the Mito-C10-TE-Dox loaded with doxorubicin did not change for up to 7 days, confirming the formation of stable micelles (Figs. 6b and 6c).

[0275] Next, the release of doxorubicin was evaluated by treating a solution containing Mito-C10-TE-Dox dispersed in PBS with ACOT. As shown in Fig. 3d, doxorubicin release immediately increased after ACOT treatment, reaching approximately 68% of the cumulative release after 20 hours of treatment with 0.5 U ACOT. In contrast, no doxorubicin release was observed in the absence of ACOT.

[0276] To confirm the mechanism of doxorubicin release in cancer cells by Mito-C10-TE-Dox, the uptake of Mito-C10-TE-Dox in HeLa cells cultured with 200 μg / mL of Mito-C10-TE-Dox was measured using a confocal microscope. As shown in Fig. 6e, the fluorescence of Mito-C10-TE-Dox increased over time (0, 2, and 4 h), confirming that Mito-C10-TE-Dox was effectively internalized into cells.

[0277] Additionally, flow cytometry analysis was used to measure the uptake of doxorubicin into HeLa cells, which increased with incubation time (Fig. 6f). After 12 h of incubation, doxorubicin release was confirmed. After 12 h of incubation, doxorubicin fluorescence was observed exclusively in the cytoplasm, and the overlap between lysotracker and doxorubicin fluorescence decreased with increasing incubation time (Fig. 6g). This suggests that doxorubicin can be released from Mito-C10-TE-Dox into the cytoplasm in response to the activity of thioesterase in cancer cells.

[0278]

[0279] Example of manufacturing a drug

[0280] The active substance according to the present invention can be formulated in various forms depending on the intended purpose. The following are examples of formulation methods containing the active substance according to the present invention as an active ingredient, but the present invention is not limited thereto.

[0281]

[0282] <Pharmaceutical Manufacturing Example 1> Manufacturing of powder

[0283] 2 g of active ingredient

[0284] 1 g lactose

[0285] After mixing the above ingredients, the powder was prepared by filling it into a sealed bag.

[0286]

[0287] <Pharmaceutical Manufacturing Example 2> Manufacturing of tablets

[0288] 100 mg of active ingredient

[0289] 100 mg of corn starch

[0290] 100 mg of lactose

[0291] Magnesium stearate 2 mg

[0292] After mixing the above ingredients, tablets were manufactured by pressing them according to a conventional tablet manufacturing method.

[0293]

[0294] <Pharmaceutical Manufacturing Example 3> Manufacturing of capsules

[0295] 100 mg of active ingredient

[0296] 100 mg of corn starch

[0297] 100 mg of lactose

[0298] Magnesium stearate 2 mg

[0299] After mixing the above ingredients, the mixture was filled into a gelatin capsule according to a conventional capsule manufacturing method to produce a capsule.

[0300]

[0301] <Pharmaceutical Manufacturing Example 4> Manufacturing of Injectables

[0302] 10 μg / ml of active substance

[0303] Dilute hydrochloric acid BP until pH 3.5

[0304] Sodium chloride for injection BP up to 1 ml

[0305] The active ingredient according to the present invention was dissolved in an appropriate volume of sodium chloride BP for injection, the pH of the resulting solution was adjusted to pH 3.5 using diluted hydrochloric acid BP, the volume was adjusted using sodium chloride BP for injection, and the mixture was thoroughly mixed. The solution was filled into a 5 ml Type I ampoule made of transparent glass, sealed under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120°C for more than 15 minutes to prepare an injection solution.

[0306]

[0307] <Pharmaceutical Manufacturing Example 5> Manufacturing of nasal spray

[0308] Active ingredient 1.0 g

[0309] 0.3 g of sodium acetate

[0310] 0.1 g of methylparaben

[0311] Propylparaben 0.02 g

[0312] Sodium chloride appropriate amount

[0313] Appropriate amount of HCl or NaOH for pH adjustment

[0314] Appropriate amount of purified water

[0315] According to the manufacturing method of a conventional absorbent, 3 mg of the active substance was prepared per 1 mL of saline solution (0.9% NaCl, w / v, solvent: purified water), filled into an opaque spray container, and sterilized to prepare a absorbent.

[0316]

[0317] <Pharmaceutical Manufacturing Example 6> Preparation of liquid preparation

[0318] 100 mg of active ingredient

[0319] 10 g of isoflavonoids

[0320] 5 g of mannitol

[0321] Appropriate amount of purified water

[0322] According to the usual method of manufacturing a liquid, each ingredient was dissolved in purified water, lemon scent was added, the above ingredients were mixed, purified water was added, the total volume was adjusted to 100 mL, and the liquid was filled into a brown bottle and sterilized to manufacture a liquid.

[0323]

[0324] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A monomer represented by the following chemical formula 1, which comprises a mitochondrial targeting moiety, an acetyl protecting group that is deprotected by a thioesterase enzyme, and a thiol group that is self-crosslinkable by reactive oxygen species (ROS). [Chemical Formula 1] (In the above chemical formula 1, L is C containing a cationic moiety 2-25 is an alkylenyl chain, R 2 is a mitochondrial targeting moiety, or mitochondrial targeting peptide.) 2. In paragraph 1, The above mitochondrial targeting moiety is one of triphenylphosphonium, dequalinium, guanidinium, triethylammonium, pyridinium, 3-phenylsulfonyl furoxan, F16, 2,3-dimethylbenzothiazolium iodide, rhodamine 19, and rhodamine 123. The above mitochondrial targeting peptides are Leu-Leu-Arg-Ala-Ala-Leu-Arg-Lys-Ala-Ala-Leu (LLRAALRKAAL: SEQ ID NO: 1), Met-Leu-Arg-Ala-Ala-Leu-Ser-Thr-Ala-Arg-Arg-Gly-Pro-Arg-Leu-Ser-Arg-Leu-Leu (MLRAALSTARRGPRLSRLL: SEQ ID NO: 2), Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys (MLSLRQSIRFFK: SEQ ID NO: 3), Leu-Ser-Arg-Thr-Arg-Ala-Ala-Pro-Asn-Ser-Arg-Ile-Phe-Thr-Arg (LSRTRAAAPNSRIFTR: SEQ ID NO: 4), One of Met-Ile-Ala-Ser-His-Leu-Leu-Ala-Tyr-Phe-Phe-Thr-Glu-Leu-Asn (MIASHLLAYFFTELN: SEQ ID NO: 5), Met-Ile-Ala-Ser-His-Leu-Leu-Ala-Tyr-Phe-Phe-Thr-Glu-Leu-Asn (MIASHLLAYFFTELN: SEQ ID NO: 6), Lys-Leu-Ala-Lys-Leu-Ala-Lys (KLAKLAK: SEQ ID NO: 7), Lys-Leu-Ala-Lys-Arg-Gly-Asp (KLAKRGD: SEQ ID NO: 8) and Lys-Leu-Ala-Lys-Leu-Ala-Lys-Arg-Gly-Asp (KLAKLAKRGD: SEQ ID NO: 9), C containing the above cationic moiety 2-20 A monomer wherein the cationic moiety in the alkylenyl chain is an oxonium ion, a quaternary ammonium ion or a quaternary phosphonium ion.

3. In paragraph 1, A monomer represented by the above chemical formula 1, which includes a monomer represented by the following chemical formula 1A. [Chemical Formula 1A] (In the above chemical formula 1A, R 1 Silver C 1-20 is an alkylenyl chain, R 2 is a mitochondrial targeting moiety, or mitochondrial targeting peptide.) 4. In paragraph 3, A monomer represented by the above chemical formula 1A, which includes a monomer represented by the following chemical formula 1B. [Chemical Formula 1B] (In the above chemical formula 1B, R 1 Silver C 1-20 ) is an alkylenyl chain.

5. In paragraph 4, The above R 1 Silver C 3-18 A monomer having an alkylenyl chain.

6. In paragraph 5, The above R 1 Silver C 6-15 A monomer having an alkylenyl chain.

7. In paragraph 6, The above R 1 Silver C 7-13 A monomer having an alkylenyl chain.

8. In paragraph 1, The monomer represented by the above chemical formula 1 is It selectively accumulates in mitochondria, The acetyl protecting group is deprotected from the thioacetyl group by the thioesterase enzyme in cancer cells, thereby activating the thiol group. A monomer in which thiol groups activated by reactive oxygen species (ROS) within cancer cells self-assemble into a large polymer through disulfide bonds, thereby selectively destroying cancerous mitochondria.

9. As shown in the following reaction formula 1, A method for producing a monomer represented by chemical formula 1, comprising the step of reacting compound 100 with a compound containing an acetyl group in an organic solvent to produce a monomer represented by chemical formula 1. [Reaction Formula 1] (In the above reaction formula 1, L and R 2 is as defined in Chemical Formula 1 of Article 1.) 10. In paragraph 9, A manufacturing method wherein the above acetyl group-containing compound is acetic anhydride.

11. In paragraph 9, A manufacturing method, wherein the organic solvent is at least one selected from the group consisting of DIPEA (N,N-Diisopropylethylamine), DMSO (dimethyl sulfoxide), DCM (dichloromethane), THF (tetrahydrofuran), and acetone.

12. A pharmaceutical composition for preventing or treating cancer, comprising a monomer represented by the chemical formula 1 of paragraph 1.

13. In paragraph 12, The above cancers include brain cancer, brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial tumor, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cancer, paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, urethral cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, female urethral cancer, skin cancer, myeloma, A pharmaceutical composition comprising at least one selected from the group consisting of leukemia and malignant lymphoma.

14. A pharmaceutical composition for preventing or treating drug-resistant cancer, comprising a monomer represented by the chemical formula 1 of paragraph 1.

15. In paragraph 14, The above drug-resistant cancers include brain cancer, brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial tumor, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cancer, paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, urinary bladder cancer, kidney cancer, male genital tumor, penile cancer, urethral cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, A pharmaceutical composition comprising at least one selected from the group consisting of female urethral cancer, skin cancer, myeloma, leukemia, and malignant lymphoma.

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