KR102034276B1 - Composition for preventing or treating cancer comprising IDF-11774 and autolysosome formation inhibitor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises IDF-11774 and an autosolubilization inhibitor as an active ingredient.
The pharmaceutical composition of the present invention is excellent in inhibiting the growth and survival of cancer cells can effectively prevent and treat cancer. Specifically, the pharmaceutical composition of the present invention exhibits an excellent synergistic effect of inducing apoptosis on cancer cells by inhibiting the formation of autolysosomes in cancer cells by using IDF-11774 and inhibitors of autolysosomal formation. Efficacy in that it can be used in various ways for the prevention and treatment of cancer. At this time, Bcl-2 may be used as a biomarker to predict synergy efficacy.

Description

Composition for preventing or treating cancer comprising IDF-11774 and autolysosome formation inhibitor}

The present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises IDF-11774 and an autosolubilization inhibitor as an active ingredient.

Colorectal cancer is one of the most common causes of cancer-related deaths worldwide. According to the Cancer Genome Atlas Project (TCGA) report on human colorectal cancer, the most common mutations occur in APC, TP53, KRAS, PIK3CA, FBXW7, in non-hypermutated tumors. SMAD4, TCF7L2 and NRAS, and in hypermutated tumors are BRAF, ACVR2A, APC, TGFBR2, MSH3, MSH6, SLC9A9, and TCF7L2. Genetic changes in the phosphatidylinositide-3-kinase (PI3K) and RAS-MAPK pathways are common in colorectal cancer, and co-occurrence of these two pathways in about one third of colorectal cancers Is reported to be observed. In addition, coexisting mutations in exons 9 and 20 of PIK3CA encoding the p110 catalytic subunit of PI3K are known to be associated with poor prognosis. However, the prognostic significance of PIK3CA mutations in colorectal cancer is not yet clear.

The multikinase inhibitors regorafenib, trametinib, and cobimetinib, as well as the antibodies bevacizumab and cetuximab, have been tested by the FDA for the treatment of colorectal cancer targets. Approved. However, cytotoxic chemotherapy using 5-FU, oxaliplatin and irinotecan has been widely used to delay the growth of incurable metastatic colorectal cancer. Recently, hypoxia-inducible factor (HIF-1) inhibitor IDF-11774 inhibits HSP70 chaperone activity and stimulates HIF-1α degradation, inhibiting mitochondrial respiration and AMP-activated protein kinase (AMPK) It has been reported to activate signal transduction pathways (Ban HS, et al. Bioconjugate chemistry 2016, 27 (8): 1911-1920; Ban HS, et al. Cell Death & Disease 2017). However, there is no known method for treating cancer using information on genes causing synthetic lethal interactions with IDF-11774. In particular, a combination of inhibitors of the gene and IDF-11774 together with the synergistic effects of cancer treatment are significant. There is no known way to derive

Accordingly, the present inventors discovered that IDF-11774 and synthetic lethal gene ATP6V0C treated IDF-11774 and simultaneously knocked down ATP6V0C gene, apoptosis of cancer cells increased rapidly, and in particular, IDF-11774 and autolysettling inhibitors Synergy efficacy was confirmed to complete the present invention.

Ban HS, et al. Bioconjugate chemistry 2016, 27 (8): 1911-1920 Ban HS, et al. Cell Death & Disease 2017

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the active ingredient of IDF-11774 and autosolubilization inhibitor.

The present inventors confirmed that autolysosome formation was inhibited in cancer cells and cancer cell proliferation was inhibited by apoptosis when the lysosomal inhibitor was simultaneously treated with IDF-11774. Thus, the present inventors used IDF-11774 and an autolysosomal inhibitor to treat cancer. It was confirmed that there is an excellent effect.

Based on the above object and the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising IDF-11774 and autolysosome formation inhibitor as an active ingredient.

As used herein, the term “IDF-11774” refers to 2- (4- (adamantan-1-yl) phenoxy) -1- (piperazin) derived from an aryloxyacetylamino benzoic acid backbone. -1-yl) HIF-1 (hypoxia-inducible factor-1) inhibitor of ethanone, which has a structure represented by the following Chemical Formula 1.

[Formula 1]

The IDF-11774 may be prepared according to a method known in Korean Patent Publication No. 10-2013-0033989, but is not limited thereto, and may be prepared according to a conventional manufacturing method.

As used herein, the term "autolysosome formation inhibitor" refers to a substance that inhibits autophagosomes and lysosomes (solubilizers) to form autolysosomes. Bafilomycin A1 and concanamycin A as specific inhibitors of autolysosomal formation are V0 complexes of vacuolar-ATPase (v-ATPase) that are involved in the regulation of lysosomal activity during aging of cells. Inhibition of ATP6V0C, the C subunit of, can inhibit the formation of autolysosomes. As another specific lysosomal inhibitor, chloroquine can inhibit autolysosomal formation by inhibiting the fusion of autophagosomes and lysosomes.

In the present invention, the autosoluble formation inhibitor may be any one or more selected from the group consisting of bafilomycin A1, concanamycin A, and chloroquine.

Bafilomycin A1 of the present invention is a kind of bacillomycin which is a toxic macrolide antibiotic derived from Streptomyces griseus , and has a structure of Formula 2 below. .

[Formula 2]

Chloroquine (chloroquine) of the present invention is a 4-aminoquinoline derivative developed as an anti-mararia drug, an anti-abeme drug, and has a structure of Formula 3 below.

[Formula 3]

Concanamycin A of the present invention is a kind of antibiotic extracted from the intestines of the larvae of caterpillar moths, and has a structure of the following Chemical Formula 4.

[Formula 4]

The autolysosome formation inhibitors such as bacillomycin A1 or concanamycin A have an inhibitory effect on ATP6V0C, a C subunit of the V0 complex of V-ATPase, thereby significantly inhibiting autolytic soluble body formation. Has an effect. In addition, chloroquine inhibits the formation of autolysosomes by inhibiting the fusion of autophagosomes and lysosomes.

In the present invention, the bacillomycin A1, chloroquine or concanamycin A may be provided in the form of their pharmaceutically acceptable salts.

In the present invention, pharmaceutically acceptable salts mean salts commonly used in the pharmaceutical industry, for example, inorganic ions, hydrochloric acid, nitric acid, phosphoric acid, bromic acid, prepared with calcium, potassium, sodium and magnesium, Inorganic acid salts made with iodic acid, perchloric acid and sulfuric acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galactu Organic acid salts made of lonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanic acid, hydroiodic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfate Amino acid salts made of sulfonic acid salts, glycine, arginine, lysine, etc., made of phosphonic acid and naphthalenesulfonic acid, and the like; Although there are amine salts made of dean, picoline, and the like, the salts used in the present invention are not limited by these salts.

The compound of any one of Formulas 2 to 4 may contain one or more asymmetric carbons and thus exist as racemates, racemic mixtures, single enantiomers, diastereomeric mixtures and respective diastereomers Can be. Such isomers can be separated in the prior art, for example, compounds of any one of Formulas 2 to 4 by column chromatography or HPLC. Alternatively, stereoisomers of each of the compounds of Formulas 2 to 4 may be stereospecifically synthesized using optically pure starting materials and / or reagents in known arrangements.

As used herein, the term “treatment” refers to clinical intervention to alter the natural process of an individual or cell to be treated, which can be performed during or to prevent a clinical pathology. . The desired therapeutic effect includes preventing the occurrence or recurrence of the disease, alleviating the symptoms, reducing all direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, and reducing the disease state. Or temporarily alleviate, drive off, or improve the prognosis. Preferably, the present invention encompasses all actions of improving the course of cancer by administration of a composition comprising IDF-11774 and an autolytic dissolution inhibitor. In addition, "prevention" refers to any action that inhibits or delays the onset of cancer by administration of a composition comprising IDF-11774 and an autosolubilizer forming inhibitor according to the present invention.

The present inventors have first identified that the combination of the IDF-11774 and the autolysosomal formation inhibitors exerts a synergistic effect and inhibits the growth and survival of cancer cells.

Specifically, in combination with IDF-11774 and bacillomycin A1 in the embodiments of the present invention, autologous pagosomes are accumulated by inhibiting the fusion of autologous pagosome-lysosomes in cancer cells by their excellent synergistic effect, It was confirmed that the formation of autolytic bodies was suppressed (FIG. 2). In addition, it was confirmed that this inhibits the growth of cancer cells and induces apoptosis (FIG. 3), and when the combination of IDF-11774 and batillomycin A1 of the present invention is shown to exhibit excellent efficacy in the prevention and treatment of cancer.

In addition, the combination of chloroquine and IDF-11774, which inhibits the formation of autolysosomes, was found to have excellent efficacy in preventing and treating cancer.

In addition, the combination of conkanamycin A and IDF-11774, which inhibits the formation of autolysosomes, has been shown to show excellent efficacy in preventing and treating cancer.

Therefore, the pharmaceutical composition of the present invention may have the effect of inducing the accumulation of autophagosomes in cancer cells or inhibiting the formation of autolysosomes.

As used herein, the term "autophagosome" refers to vesicles formed in the process of autophagy, and is also referred to as "autophagy", "autophagy", and the like.

As used herein, the term "autolysosome" means that the autologous pagosome formed in the process of autodigestion is combined with lysosomes, and is also referred to as "autolysosome", "autophagy soluble".

In addition, the pharmaceutical composition of the present invention may have an effect of inducing apoptosis on cancer cells.

Furthermore, the pharmaceutical composition of the present invention may have ATP6V0C inhibitory efficacy in cancer cells.

The disease to be prevented or treated according to the present invention is interpreted to be included as long as it falls within the category of cancer, and the specific disease name is not particularly limited.

As used herein, the term "cancer" medically causes a problem in the regulation function of normal division, differentiation or death of cells and abnormally proliferates to infiltrate surrounding tissues or organs to form lumps and It means any state that destroys or deforms a structure. Cancer is largely divided into primary cancers present at the site of development and metastatic cancers that have spread from one site to another in the body. The cancer may be, for example, any one selected from lung cancer, breast cancer, colon cancer, gastric cancer, brain cancer, pancreatic cancer, thyroid cancer, skin cancer, bone marrow cancer, lymphoma, uterine cancer, kidney cancer and melanoma.

In order to exhibit better therapeutic efficacy, the pharmaceutical composition of the present invention may preferably be a cancer having a low expression level of Bcl-2 in cancer cells, and more preferably colon cancer. Even more preferably, colorectal cancer having a low expression level of Bcl-2 may be used.

As used herein, the term "Bcl-2" is an abbreviation for "B-cell lymphoma 2," and is encoded by the BCL2 gene, which is a type of cellular oncogene activated by chromosomal translocation in human follicular lymphomas. Means protein. Bcl-2 is known as an anti-apoptotic protein. The composition according to the invention shows an excellent therapeutic effect, especially for cancers with low expression levels of Bcl-2.

In the present invention, the measurement of "expression level of Bcl-2" may be performed in a manner of measuring the expression level of a protein of Bcl-2 or the expression level of mRNA of a gene encoding Bcl-2.

Specifically measuring the protein expression level of Bcl-2, for example, by using an antibody, aptamer, antagonist or a combination thereof that specifically binds to Bcl-2 protein, Western blotting, ELISA, radioimmunoassay , Radioimmunoassay, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation analysis, complement fixation analysis, FACS, protein chip analysis, etc., but is not limited thereto. .

In addition, measuring the expression level of mRNA of Bcl-2 can be achieved by, for example, using antisense oligonucleotides, primer pairs, probes, or a combination thereof that specifically binds to mRNA of Bcl-2. Chain reaction, competitive reverse transcriptase chain reaction, real-time reverse transcriptase chain reaction, RNasa protection assay, Northern blotting, DNA microarray chip, etc. can be performed through, but not limited to.

The pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier in addition to the IDF-11774 and autolysosome formation inhibitors, for example, bacillomycin A1, chloroquine or conkanamycin A as active ingredients. It may further comprise.

The kind of carrier that can be used in the present invention is not particularly limited and any carrier can be used as long as it is commonly used in the art. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, and the like. Can be. These may be used alone or in combination of two or more thereof.

In addition, the pharmaceutical composition of the present invention may be used, if necessary, by adding other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostatic agents, fillers, extenders, wetting agents, disintegrants, dispersants, surfactants , Binders or lubricants may be additionally added and used.

In the pharmaceutical composition of the present invention, IDF-11774 and an autosososome formation inhibitor such as batillomycin A1, chloroquine or concanamycin A are 0.01 weight based on the total weight of the pharmaceutical composition. % To 90.00% by weight, preferably 0.01% to 90.00% by weight, more preferably 0.1% to 70% by weight, and more preferably 0.1% to 50% by weight, but The present invention is not limited thereto, and may be variously changed depending on the condition of the administration subject, the type of the specific condition, and the degree of progress. If necessary, it may also be included in the total content of the pharmaceutical composition.

The pharmaceutical compositions of the present invention can be formulated and used in a variety of suitable formulations for oral or parenteral administration.

Non-limiting examples of oral formulations using the pharmaceutical compositions of the present invention include troches, lozenges, tablets, aqueous suspensions, oily suspensions, preparation powders, granules, emulsions, hard capsules, soft Capsules, syrups, or elixirs.

In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin and the like; Excipients such as dicalcium phosphate and the like; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax and the like can be used, and sweeteners, fragrances, syrups and the like can also be used. Furthermore, in the case of a capsule, a liquid carrier such as fatty oil may be additionally used in addition to the above-mentioned materials.

Non-limiting examples of parenteral preparations using the pharmaceutical composition of the present invention include injection solutions, suppositories, respiratory inhalation powders, spray aerosols, ointments, application powders, oils, creams and the like.

In order to formulate the pharmaceutical composition for parenteral administration, a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, an external preparation, and the like may be used. The non-aqueous solvent and the suspension may be propylene glycol or polyethylene. Glycols, vegetable oils such as olive oil, injectable esters such as ethyloleate and the like can be used.

When formulating the pharmaceutical composition of the present invention into an injectable solution, the pharmaceutical composition of the present invention is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is intended for unit administration of ampoules or vials. It may be formulated.

When formulating the pharmaceutical composition of the present invention with an aerosol, a propellant or the like may be combined with the additive to disperse the dispersed dispersion or the wet powder.

When the pharmaceutical composition of the present invention is formulated into an ointment, a cream, a powder for application, an oil, an external skin preparation, etc., animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite , Silica, talc, zinc oxide and the like can be formulated using a carrier.

Pharmaceutically effective amounts, effective dosages of the pharmaceutical compositions of the present invention may vary depending on the method of formulating the pharmaceutical composition, mode of administration, time of administration, and / or route of administration, and the type of reaction to be achieved by administration of the pharmaceutical composition. And the severity, type, age, weight, general state of health, general state of health, condition or extent of the disease, sex, diet, excretion, and components of the drug or other composition used concurrently or simultaneously with the subject. It can be varied according to factors and analogous factors well known in the medicinal art, and one of ordinary skill in the art can easily determine and prescribe a dosage effective for the desired treatment.

Administration of the pharmaceutical composition of the present invention may be administered once a day, may be divided into several times. The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. All of the above factors can be considered and administered in an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

"Pharmaceutically effective amount" for treatment means an amount sufficient to inhibit or alleviate the disease at a reasonable rate applicable for medical use, with an effective dosage level of the individual being of type and severity, age, sex, activity of the drug, drug Sensitivity to, time of administration, route of administration and rate of administration, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations.

Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art. For example, the pharmaceutically effective amount is from 10 mg / day / kg to 1000 mg / day / kg, preferably 20 mg / day / kg to 500 mg / day / kg, for IDF-11774, Inhibitors of autolysosomal formation, for example batillomycin A1, are 0.05 mg / day / kg to 500 mg / day / kg in weight, preferably 1.0 mg / day / kg to 50 mg / day / kg in weight. Conkanamycin A is 0.001 mg / day / kg to 10 mg / day / kg / kg, preferably 0.1 mg / day / kg to 1.0 mg / day / kg. Chloroquine is from 1 mg / day / kg to 1000 mg / day / kg, preferably 50 mg / day / kg to 500 mg / day / kg.

The route of administration and mode of administration of the pharmaceutical compositions of the present invention may be independent of each other, and may be any route of administration and mode of administration without particular limitation, so long as the pharmaceutical composition can reach the desired site of interest. The pharmaceutical composition may be administered by oral or parenteral administration.

As a parenteral administration method of the pharmaceutical composition of the present invention, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration or subcutaneous administration can be used, and the method of applying, spraying, or inhaling the composition to a diseased site. It may also be used but is not limited thereto.

The pharmaceutical composition of the present invention may exert an excellent effect even when used alone, but may be used in combination with various cancer treatment methods, such as radiation therapy and chemotherapy, in order to increase the treatment efficiency.

The present invention provides a method for treating cancer comprising administering to a subject a pharmaceutical composition comprising IDF-11774 and an autosososome formation inhibitor, for example, bacillomycin A1, chloroquine or conkanamycin A, as an active ingredient. To provide a method of treatment.

In the present invention, an individual means any animal including a human having or having a cancer disease, and may be an individual except a human. By administering the pharmaceutical composition of the present invention to a subject, it shows an excellent effect in the treatment of cancer.

The present invention provides the use of a composition comprising IDF-11774 and an autosolubilization inhibitor in the manufacture of a medicament for the treatment of cancer.

The present invention provides a composition comprising IDF-11774 and an autosolubilization inhibitor for use in treating cancer.

The pharmaceutical composition of the present invention is excellent in inhibiting the growth and survival of cancer cells can effectively prevent and treat cancer.

Specifically, the pharmaceutical composition of the present invention induces the accumulation of autophagosomes in cancer cells and induces autophagosome accumulation in combination with IDF-11774 and autosolubilization inhibitors such as batillomycin A1, chloroquine or concanamycin A1. It is effective in inhibiting the formation of autolysosomes and exerts an excellent synergistic effect of inducing apoptosis on cancer cells. Therefore, the expression level of Bcl-2 is particularly high. Excellent therapeutic effect against low cancer.

Figure 1 shows the synthetic lethal interaction of IDF-11774 and ATP6V0C in colorectal cancer cells, showing the knockdown effect of ATP6V0C on the growth of HCT116 cells treated with IDF-11774.
Figure 2 confirms the importance of autolysosome formation in the lethal interaction of IDF-11774 and ATP6V0C in colorectal cancer cells. (a) Western blot analysis of the knockdown (using siATP6V0C) effect of ATP6V0C on LC3B-II accumulation in cells treated with IDF-11774, and (b) autolysosome through LysoTracker staining. Formation will be indicated by green fluorescence. (c) shows the results of Western blot analysis of the effect of bacillomycin A1 on the accumulation of LC3B-II in cells treated with IDF-11774 after pretreatment of HCT116 cells with batillomycin A1 (BM), ( d) will show autolysosomal formation in green fluorescence via LysoTracker staining.
Figure 3 shows the results of induction of apoptosis in colorectal cancer cells when combined treatment of barfilomycin A1 with IDF-11774, (a) is treated with IDF-11774 and batillomycin A1 (BM) in HCT116 cells, Treatment with IDF-11774 and Bafilomycin A1 (BM) in WiDr cells shows synergistic effects of the combined treatment of Bafilomycin A1 and IDF-11774. (b) and (c) show Western blot analysis (b) and cell cycle analysis (c) of cells treated with IDF-11774 and bacillomycin A1. (d) shows the results of measuring caspase-3 / 7 activity using the CellPlayer kinetic caspase-3 / 7 reagent, which was treated with IDF-11774 and bacillomycin A1, and the caspase-3 / 7-positive cells were fluorescence microscopy. Turned green under (e) shows scatter plots of FITC-annexin V / PI staining of WiDr and HCT116 cells treated concurrently with IDF-11774 and batillomycin A1. Apoptosis was analyzed by Annexin V staining using a FACSCalibur flow cytometer. .
Figure 4 shows the results of the analysis of the importance of Bcl-2 expression level for the synthetic lethality of IDF-11774 and bacillomycin A1. (a) shows the effect of Bcl-2 overexpression on the growth of cells co-treated with IDF-11774 and bacillomycin A1 using pCMV-SPORT6-Bcl-2. (b) shows the effect of Bcl-2 overexpression on autophagosome formation, showing immunofluorescence against LC3B expression in cells co-treated with IDF-11774 and batillomycin A1. (c) shows HCT116 cells transformed with pCMV-SPORT6-Bcl-2 to identify the effect of Bcl-2 on apoptosis induced by combination treatment with IDF-11774 and batillomycin A1 (BM). And caspase-3 / 7 activity and Annexin V staining by live cell imaging. Caspase-3 / 7-positive cells appear green under fluorescence microscopy and Annexin V positive cells show red. (d) shows the results of isobologram analysis of IDF-11774 and batillomycin A1 in WiDr cells transformed with pCMV-SPORT6-Bcl-2.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Synthetic Lethal Interaction of IDF-11774 and ATP6V0C in HCT116 Cells

Whether ATP6V0C knockdown affects the chemosensitization of cancer cells against IDF-11774 was investigated by real-time growth analysis.

Specifically, HCT116 cells were cultured HCT116 human colon adenocarcinoma cells in 5% CO ₂ conditions at 37 ℃ in DMEM medium containing 10% (v / v) FBS. According to the manufacturer's instructions, sipo (SEQ ID NO: 1) and siRNA (SEQ ID NO: 2) of the control siRNA having the sequence of Table 1 and siRNA (SEQ ID NO: 2) were introduced through Lipofectamine 2000 (Invitrogen). Thereafter, 5 μM of IDF-11774 was treated to analyze the real-time growth of cells by viable cell imaging (IncuCyte Zoom system, Essen Bioscience, Ann Arbor, MI, USA).

TABLE 1

As a result, the knockdown of ATP6V0C itself hardly inhibited the growth of HCT116 cancer cells. However, it was confirmed that the combination treatment of ATP6V0C knockdown and IDF-11774 was significantly superior to the growth inhibitory effect compared to the treatment with IDF-11774 alone (FIG. 1).

EXAMPLE  2: ATP6V0C  At restraint IDF Autodigestion induced by -11774 ( autophagy ) Blocked

(1) LC3B-II Accumulation Effect by ATP6V0C Knockdown

IDF-11774 inhibits mitochondrial respiration and activates AMPK to induce activation of dephosphorylation and autophagy formation of mTOR. Accordingly, Western blotting was performed on HCT116 cells treated with IDF-11774, resulting in accumulation of microtubule-associated protein 1 light chain 3 beta (LC3B) -II (FIG. 2A). This means that autophagosomes were formed by IDF-11774.

Next, the effect of ATP6V0C knockdown on the autophagy of IDF-11774 in HCT116 cells was confirmed. Specifically, Western blotting was performed on HCT116 cells treated with IDF-11774 after knocking down ATP6V0C as in Example 1.

Specifically, for Western blotting experiments, proteins were extracted from cell lines using RIPA buffer (millipore), electrophoresis-separated from polyacrylamide gels, and then gels from polyvinylidene fluoride membranes. The protein was moved. The protein transferred to the membrane was reacted with the LC3B antibody at 4 ° C. overnight and reacted with the conjugated secondary antibody at room temperature for 1 hour and then confirmed by reaction with the substrate.

Experimental results confirm that LC3B-II accumulation induced by IDF-11774 is enhanced by ATP6V0C knockdown (FIG. 2A). This means that autophagosomes accumulate.

(2) Inhibitory Effects of ATP6V0C Knockdown on Autolysosome Formation

Next, LysoTracker, a fluorescent dye for acidic vesicles, was used to evaluate the effect of ATP6V0C knockdown on autolysosomal formation in the presence of IDF-11774.

Specifically, in order to stain the acid endoplasmic reticulum of HCT116 cells treated with IDF-11774 after knocking down ATP6V0C as in Example 1, the cells were lysed at 37 ° C. for 30 minutes at LysoTracker (Molecular Probes, Life Technologies, Carlsbad, CA, USA) were incubated with the label. LysoTracker labeled cells were observed with a confocal fluorescence microscope (LSM5 Live DuoScan, Carl Zeiss, Stuttgart, Germany).

As a result, in the cells treated with siSC as a control, autolysosomes were formed by IDF-11774, but it was confirmed that autolysosomes formation was inhibited when siATP6V0C was treated (FIG. 2B). This means that autophagy is inhibited.

(3) Enhancing LC3B-II Accumulation by Baphyllomycin A1

Similarly to (1) of Example 2, it was confirmed whether the combined treatment of bacillomycin A1 having an ATP6V0C inhibitory effect showed the same effect.

Specifically, HCT116 cells were pre-incubated with 1 nM of Bapilomycin A1 (BM) for 3 hours and then treated with IDF-11774 for 18 hours. Thereafter, Western blotting was performed in the same manner as above.

As a result, LC3B-II was accumulated even in the group treated with IDF-11774 only, but LC3B-II accumulation was enhanced in HCT116 cells treated with IDF-11774 in the presence of bacillomycin A1 (FIG. 2C).

(4) inhibitory effect on autolysosomal formation according to bacillomycin A1

Similarly to (2) of Example 2, it was confirmed whether the combined treatment of bacillomycin A1 showed the same effect.

Specifically, in order to stain the acid endoplasmic reticulum of HCT116 cells incubated with the same method as in Example 2 (3) and treated with Baphyllomycin A1 and IDF-11774, the cells with LysoTracker label for 30 minutes at 37 ℃ Incubated. LysoTracker labeled cells were observed by confocal fluorescence microscopy.

As a result, green fluorescence was visibly formed in cells cultured with only IDF-11774, and autolysosomes were formed, whereas green fluorescence was reduced in cells pre-treated with batillomycin A1 (FIG. 2D). This means that the bacillomycin A1 inhibits autolysosomal formation mediated by IDF-11774.

In conclusion, inhibition of ATP6V0C by Baphyllomycin A1 blocked the production of functional autolysosomes induced by IDF-11774, leading to the accumulation of autologous pagosomes in HCT116 cells.

Example 3: Induction of Apoptosis in Cancer Cells in Combination Treatment with IDF-11774 and Baphyllomycin A1

(1) Inhibition of Cancer Cell Growth by Combination Treatment of IDF-11774 and Bapilomycin A1

When combined with the ATP6V0C inhibitor, Bapilomycin A1 and IDF-11774, it was confirmed that it affected the survival of cancer cells.

Specifically, HCT116 cells were treated with 5 μM of IDF-11774 and 1 nM of Bafilomycin A1, and WiDr cells were treated with 2.5 μM of IDF-11774 and 1 nM of Bapilomycin A1 and cultured. Real time growth of cells was analyzed by imaging (IncuCyte Zoom system, Essen Bioscience, Ann Arbor, MI, USA). HCT116 cells were cultured in DMEM medium containing 10% (v / v) FBS, and WiDr cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS).

As a result of the experiment, it was confirmed that in the case of co-treatment with bacillomycin A1 and IDF-11774, growth of WiDr and HCT116 was significantly inhibited compared to the treatment with IDF-11774 alone (FIG. 3A).

(2) Changes in apoptosis-related factors following the combined treatment of IDF-11774 and batillomycin A1

As described above, Western blot was performed on the cells cultured by co-treatment with IDF-11774 and Baphyllomycin A1, and it was confirmed that a change in apoptosis-related factors appeared. Specifically, the accumulation of LC3B-II and p21 was increased, cyclin D1 was degraded, and PARP-1 was cleaved (FIG. 3B). Such changes mean that the cell cycle is arrested and that apoptosis occurs.

(3) Induction of apoptosis following the combined treatment of IDF-11774 and batillomycin A1

Flow cytometry showed an increase in the G1 cell population of HCT116 and WiDr cells in the presence of IDF-11774 (FIG. 3C). Specifically, when IDF-11774 and Bafilomycin A1 were simultaneously treated, the sub-G1 cell population of HCT116 and WiDr cells increased rapidly, indicating that apoptosis occurred.

Next, caspase-3 / 7 was analyzed using CellPlayer reagent (Essen Bioscience) (Palechor-Ceron N, et al. The American journal of pathology 2013, 183 (6): 1862-1870). Specifically, frames of cells cultured in 96-well plates were captured at 2 hour intervals from 4 separate regions per well using a 10 × objective. Culture of the cells was maintained in a 37 ℃ incubator. As a result, the combination of IDF-11774 and batillomycin A1 significantly increased caspase-3 / 7 activity compared to IDF-11774 alone treatment (FIG. 3D).

Apoptotic cell populations were analyzed by FITC-annexin V-PI (propidium iodine) double staining. Specifically, FITC-annexin V-PI (propidium iodine) double staining analysis was performed according to the manufacturer's protocol (BD Biosciences). In summary, cells were treated with IDF-11774 and Baphyllomycin A1 and then washed twice with pre-cooled phosphate buffered saline (PBS). The treated cells were stained with FITC-annexin V staining buffer and PI solution for 15 minutes at room temperature and analyzed by FACSCalibur Flow Cytometer (BD Biosciences). As a result, it was confirmed that the combination of IDF-11774 and batillomycin A1 increased the pro-apoptotic and apoptotic cell populations in the HCT116 and WiDr cell lines (FIG. 3E).

These results indicate that IDF-11774 and Bafilomycin A1 synergistically inhibit the growth of HCT116 and WiDr cells by inducing apoptosis through the blocking of autolysosomal formation.

Example 4 Association of Genetic Status of PIK3CA with Synthetic Lethal Interaction of IDF-11774 and Functional Autolysosomal Formation

To determine the relationship between IDF-11774 and Baphilomycin A1, a known inhibitory effect on autolysosomal formation (e.g., with ATP6VOC inhibitory effects), with concanamycin A (CCM) or lysosomal inhibitor chloroquine (CQ). Isobologram analysis was performed to identify genetic markers in cell lines, and the results are shown in Table 2. ATP6V0C and lysosomal inhibitors showed a synergistic effect (CI <1) with IDF-11774 in HCT116, WiDr and HT29 cells. This indicates that the formation of functional autolysosomes plays an important role in synergy.

In addition, we investigated the association of genetic mutations to the CI and functional autolysosomal inhibition of IDF-11774 in cancer cell lines. Experimental results confirmed that PIK3CA mutations significantly induce colorectal cancer cells for the combination treatment of IDF-11774 and bacillomycin A1 (Table 2). In particular, for cancer cells with a PIK3CA mutation and low Bcl-2 expression, the CIs of IDF-11774, Baphyllomycin A1, Concanamycin A, and Chloroquine were measured low in HT29 and WiDr cells (CI <1) to produce synergistic effects. It confirmed that it was shown. This result implies that the genetic status of PIK3CA is associated with the synthetic lethal effects of IDF-11774 and bacillomycin A1.

TABLE 2

Through the above results, it was confirmed that conkanamycin A and chloroquine were also combined with IDF-11774 in a similar effect to bacillomycin A1, and thus have an excellent effect on the prevention or treatment of cancer.

Example 5: Synthesis of IDF-11774 and Autolysosomal Inhibitors Role of Bcl-2 in Lethality

PCTV-SPORT6-Bcl-2 (Bcl-2) plasmid and vector pCMV-SPORT6 (EV) plasmid that induce Bcl-2 overexpression in HCT116 cells were transformed and 10 μM of IDF-11774 and 1 nM of bacillomycin After A1 treatment, real-time growth of cells was analyzed by cell imaging (IncuCyte Zoom system, Essen Bioscience, Ann Arbor, MI, USA). As a result, overexpression of Bcl-2 was confirmed that the growth of HCT116 cells was recovered despite treatment with IDF-11774 and Baphyllomycin A1 (FIG. 4A). Therefore, it was found that overexpression of Bcl-2 confers resistance to IDF-11774.

In addition, it was investigated whether the overexpression of Bcl-2 in HCT116 cells affected the autophagy following the combination of IDF-11774 and batillomycin A1. Immunofluorescence staining was performed on the cells by conventionally reported methods (Palechor-Ceron N, et al. The American journal of pathology 2013, 183 (6): 1862-1870). Specifically, the cells were incubated with LC3B antibody at 4 ° C. overnight and incubated with conjugated secondary antibody for 1 hour at room temperature. After washing with PBS, the cells were stained with 4 ', 6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole) for 5 minutes, followed by confocal fluorescence microscopy (LSM5 Live DuoScan; The cells were observed with Carl Zeiss, Stuttgart, Germany). As a result, overexpression of Bcl-2 reduced LC3B-II accumulation induced by IDF-11774 and bacillomycin A1 and inhibited autologous pagosome formation (FIG. 4B).

Next, it was investigated whether overexpression of Bcl-2 in HCT116 cells affected apoptosis following the combined treatment of IDF-11774 and batillomycin A1. Specifically, HCT116 cells transformed with the pCMV-SPORT6-Bcl-2 plasmid were treated with 10 μM of IDF-11774 and 1 nM of bacillomycin A1. Thereafter, FITC-annexin V-PI (propidium iodine) double staining was performed, followed by live cell imaging to analyze real-time growth of cells. As a result, despite the combination treatment of IDF-11774 and batillomycin A1, it was confirmed that induction of apoptosis was suppressed by overexpression of Bcl-2 (FIG. 4C).

To investigate the effect of Bcl-2 expression levels on the synthetic lethal interaction of IDF-11774, isobologram analysis was performed on WiDr cells with low Bcl-2 expression. Specifically, WiDr cells were transformed with pCMV-SPORT6 (EV) or pCMV-SPORT6-Bcl-2, and then treated with IDF-11774 and bacillomycin A1 for 72 hours. As a result, overexpression of Bcl-2 in WiDr increased the CI from 0.85 to 1.05 (FIG. 4D). This means that the expression of Bcl-2 inhibits the synthetic lethality of IDF-11774 and bacillomycin A1.

Statistical analysis

Student's t-test or Chi-square test was used for statistical analysis. Bars indicate S.D. and asterisks indicate significant differences between the two groups (*** p ≦ 0.005, ** p ≦ 0.01, * p ≦ 0.05).

In the present specification, those skilled in the art of the present invention can fully recognize and infer the details that have been omitted, and the technical spirit or essential configuration of the present invention in addition to the specific examples described in this specification are changed. Many more variations are possible without departing. Therefore, the present invention may be implemented in a manner different from that specifically described and illustrated herein, which can be understood by those skilled in the art.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> Composition for preventing or treating cancer configuring          IDF-11774 and autolysosome formation inhibitor <130> P17-100-KRI <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siSC <400> 1 ccuacgccac caauuucgu 19 <210> 2 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> ATP6V0C siRNA <400> 2 cgcccucauc cucuccacaa a 21

Claims (9)

IDF-11774 (2- (4- (adamantan-1-yl) phenoxy) -1- (piperazin-1-yl) ethanone) and autolysosome formation inhibitor as active ingredients As a pharmaceutical composition for preventing or treating cancer, comprising
The autosoluble formation inhibitor is to inhibit the fusion of autopagosomes and lysosomes, the cancer is colorectal cancer, a pharmaceutical composition for preventing or treating cancer. The pharmaceutical composition of claim 1, wherein the IDF-11774 is a compound of Formula 1 below.
[Formula 1]

The method for preventing or treating cancer according to claim 1, wherein the autosoluble formation inhibitor is at least one selected from the group consisting of bafilomycin A1, chloroquine, and concanamycin A. Composition. delete According to claim 1, wherein the pharmaceutical composition is to have the effect of inducing the accumulation of autophagosome (autophagosome) in cancer cells, pharmaceutical composition for preventing or treating cancer. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition has an ATP6V0C inhibitory effect in cancer cells. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition has an effect of inhibiting the formation of autolysosomes in cancer cells. The pharmaceutical composition for preventing or treating cancer according to any one of claims 1 to 3 and 5 to 7, wherein the cancer is a cancer having a low expression level of Bcl-2 in cancer cells. delete

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