Antitumor agents by metabolic inhibition

Abstract

It provides a new therapeutic tool for cancer treatment. [Solution] The present invention relates to a metabolic inhibitor containing 7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone (compound KIS079) that suppresses both the mitochondrial respiratory chain and the glycolytic pathway in cells, an antitumor agent or pharmaceutical composition containing this inhibitor, a method for treating cancer using such an antitumor agent, etc. TIFF2026020821000004.tif45170

Description

[Technical Field]

[0001] The present invention relates to an inhibitor containing 7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone (hereinafter also referred to as "compound KIS079") that inhibits both the mitochondrial respiratory chain and glycolysis in cells, a pharmaceutical composition (antitumor agent, anticancer agent, or anticancer agent) containing this compound or inhibitor, a method for treating cancer using such a pharmaceutical composition, etc. Compound KIS079 was developed as a pyruvate dehydrogenase kinase 4 (PDK4) inhibitor and is disclosed in Patent Documents 1 to 3 as a substance for treating or preventing viral infections, heart failure, etc. [ka] [Background technology]

[0002] Antitumor drugs such as 5-fluorouracil (5FU) and oxaliplatin, which are the main drugs used in standard chemotherapy for colon cancer, are unable to kill slow-growing cancer cells because they nonspecifically inhibit proliferating cells, such as DNA replication. For this reason, it is important to develop methods that suppress cancer cells through actions other than cell proliferation, and to use these drugs in combination with chemotherapy drugs that suppress cell proliferation to more completely suppress and kill cancer cells.

[0003] A specific example of a promising method for suppressing effects other than cell proliferation is metabolic inhibition. It has been known that mitochondrial metabolism is suppressed and glycolysis is activated in cancer cells compared to normal cells (the Warburg effect). Therefore, inhibition of glycolysis has been considered important for cancer suppression and has been studied. However, it has recently been discovered that mitochondrial metabolism is also activated and the glycolysis is even more activated in cancer cells compared to normal cells (e.g., Non-Patent Documents 1-3). Therefore, it is believed that effective inhibition of intracellular energy production pathways via mitochondrial metabolism and glycolysis is necessary for suppressing cancer metabolism (e.g., Non-Patent Documents 4-6). However, few single compounds have been developed that can effectively suppress energy production in a multifaceted manner by simultaneously inhibiting both mitochondrial metabolism and glycolysis, thereby enabling effective cancer suppression and death while also reducing polypharmacy (multiple drug use). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2015-160831 [Patent Document 2] International Publication No. 2014 / 103321 Brochure [Patent Document 3] International Publication No. 2013 / 153821 Brochure [Non-patent literature]

[0005] [Non-Patent Document 1] Boudreau, A. et al., Nature Chemical Biology 12, 779-786 (2016) [Non-patent document 2] de Padua, MC et al., Oncotarget 8, 87623-87637 (2017) [Non-patent document 3] Shiratori, R. et al., Scientific Reports 9, 1-15 (2019) [Non-patent document 4] Gatenby, RA & Gillies, RJ, International J of Biochemistry and Cell Biololy 39, 1358-1366 (2007) [Non-Patent Document 5] Hay, N., Nature Reviews Cancer 16, 635-649 (2016). [Non-patent document 6] Vatrinet, R. et al., International J of Biochemistry and Cell Biology 63, 41-45 (2015). Summary of the Invention [Problem to be solved by the invention]

[0006] Under these circumstances, there is a need for the development of substances with different effects than conventional ones that have an inhibitory effect on cancer metabolism, and for the development of cancer treatment methods that can more completely kill cancer cells by using such substances in combination with cell proliferation inhibitors. [Means for solving the problem]

[0007] As a result of conducting research into drugs for cancer treatment, the inventors discovered that compound KIS079 inhibits both the mitochondrial respiratory chain and glycolysis in cancer cells, thereby suppressing energy production, and that it induces cell death in various cancer cells derived from cancers such as colon cancer, pancreatic cancer, and neural tumors. Furthermore, the combined use of compound KIS079 with anticancer agents that inhibit proliferation (e.g., 5-fluorouracil (5FU) and / or oxaliplatin) can further enhance the induction of cell death in cancer cells, leading to the completion of the present invention.

[0008] Examples of the present invention include the following: [1] Mitochondrial respiratory chain inhibitors, including 7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone. [2] The mitochondrial respiratory chain inhibitor according to [1] above, wherein the 7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone inhibits the activity of mitochondrial respiratory chain complex I. [3] The mitochondrial respiratory chain inhibitor according to [1] above, wherein the 7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone inhibits glucose metabolism in cells in an NAD(H)-independent manner. [4] Glycolysis inhibitors, including 7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone. [5] The glycolysis inhibitor according to [5] above, wherein the 7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone inhibits glucose metabolism in cells in an NAD(H)-independent manner. [6] The glycolysis inhibitor described in [5] above, wherein the 7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone inhibits the activity of mitochondrial respiratory chain complex I. [7] A pharmaceutical composition comprising the mitochondrial respiratory chain inhibitor according to any one of [1] to [3] above, and / or the glycolysis inhibitor according to any one of [4] to [6] above. [8] The pharmaceutical composition according to [7] above, which is an anticancer agent. [9] The pharmaceutical composition according to [7] above, which is used in combination with other anticancer drugs.

[10] The pharmaceutical composition according to [9] above, wherein the other anticancer agent is a cell growth inhibitor.

[11] The pharmaceutical composition described in

[10] above, wherein the other anticancer drug comprises one or more selected from the group consisting of 5-fluorouracil, tegafur, doxilfuridine, capecitabine, cytarabine, enocitabine, gemcitabine, bleomycin, BCG vaccine, cisplatin, miriplatin hydrate, carboplatin, nedaplatin, and oxaliplatin.

[12] The pharmaceutical composition according to [9] above, which is an anticancer agent for cancer selected from the group consisting of colorectal cancer (colon cancer, rectal cancer), pancreatic cancer, lung cancer, esophageal cancer, gastric cancer, upper gastrointestinal cancer, liver cancer, head and neck cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular tumor, kidney cancer, bladder cancer, urothelial tumor, oral cancer, malignant skin tumor, gastrointestinal stromal tumor (GIST), glioma, brain tumor, osteosarcoma, malignant lymphoma (Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.), blood tumor, chronic myeloid leukemia, multiple myeloma, and malignant melanoma.

[13] 7-Carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone for the treatment of cancer selected from the group consisting of colorectal cancer (colon cancer, rectal cancer), pancreatic cancer, lung cancer, esophageal cancer, gastric cancer, upper gastrointestinal cancer, liver cancer, head and neck cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, kidney cancer, bladder cancer, urothelial tumor, oral cancer, skin malignancies, gastrointestinal stromal tumor (GIST), glioma, brain tumor, osteosarcoma, malignant lymphoma (Hodgkin lymphoma, non-Hodgkin lymphoma, etc.), blood tumor, chronic myeloid leukemia, multiple myeloma, and malignant melanoma.

[14] A method for treating cancer selected from the group consisting of colorectal cancer (colon cancer, rectal cancer), pancreatic cancer, lung cancer, esophageal cancer, gastric cancer, upper digestive tract cancer, liver cancer, head and neck cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular tumor, kidney cancer, bladder cancer, urothelial tumor, oral cancer, skin malignancy, gastrointestinal stromal tumor (GIST), glioma, brain tumor, osteosarcoma, malignant lymphoma (Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.), blood tumor, chronic myeloid leukemia, multiple myeloma, and malignant melanoma, comprising administering the pharmaceutical composition described in [7] above. [Effects of the Invention]

[0009] Compound KIS079 simultaneously inhibits the mitochondrial respiratory chain and glycolysis in cells, resulting in growth inhibition and / or cell death in cells derived from various cancers, such as colon cancer, pancreatic cancer, and glioma, and can therefore be used to treat these cancers. Furthermore, when compound KIS079 is used in combination with known anticancer agents that inhibit growth, such as 5FU or oxaliplatin, it exhibits enhanced therapeutic effects against cancer cell tissue. Therefore, pharmaceutical compositions containing mitochondrial respiratory chain inhibitors and glycolysis inhibitors, including the compounds of the present invention, provide novel cancer therapeutic effects related to metabolic inhibition, and can further enhance therapeutic effects when used in combination with known anticancer agents, for example. [Brief explanation of the drawings]

[0010] [Figure 1-1] Figure 1 shows that KIS079 induces cell death in cancer cell lines. (A) The chemical structure of KIS079 is shown. (B) The results of a viability assay of SW480 cells using a cell counting kit are shown. Cells were treated with 0 to 40 μM KIS079 for 72 hours before measurement. (C) The results of a cell viability assay using trypan blue staining of HT29 cells, HCT116 cells, Colo205 cells, CK81 cells (left), PANC-1 cells, MIA PaCa-2 cells, A172 cells, and YKG1 cells (right) in response to 0 to 60 μM KIS079. (D) The results of a cell viability assay using trypan blue staining of SW480 cells in response to 0, 10, 20, and 30 μM KIS079. [Figure 1-2] (E) Western blotting of PDH and phosphorylated PDH in extracts from SW480 cells treated with KIS079 (0 or 20 μM) for 0, 3, and 6 hours. (F) Western blotting of PDH and phosphorylated PDH in extracts from HCT116 and HT29 cells treated with KIS079 (0 or 20 μM) for 0, 3, and 6 hours. [Figure 2]FIG. 2 shows the effect of the addition of 20 μM KIS079 compared to the addition of 5 μM or 10 μM 5FU and oxaplatin (Ox) on spheroids of human colorectal cancer cells. [Figure 3] Figure 3 shows that KIS079 induces mitochondrial damage in cancer cells. (A) Representative electron microscopy images of SW480 cells treated with KIS079 (20 μM) for 0, 6, and 12 hours are shown. Scale bar: 1 μm. (B) Fluorescence microscopy images of the TMRE assay of SW480 cells treated with KIS079 (20 μM) for 30, 60, and 120 minutes are shown. Cells were also treated with FCCP (20 μM) for 10 minutes as a control. [Figure 4] Figure 4 shows that KIS079 inhibits the mitochondrial respiratory chain by inhibiting the action of complex I. (A) Seahorse XFp Extracellular Flux Analyzer assay of SW480 cells. The y-axis indicates the oxygen consumption rate (OCR, left) and the extracellular acidification rate (ECAR, right), respectively. Prior to measurement, cells were treated with 20 μM KIS079 or DMSO for 1 hour. Oligomycin (ATP synthase inhibitor), FCCP (proton uncoupler), rotenone (complex I inhibitor), and antimycin A (complex III inhibitor) were sequentially injected. (B) and (C) show the results of a respiratory chain assay of mitochondria isolated from mouse liver. The y-axis indicates the rhodamine fluorescence intensity in solution, which corresponds to mitochondrial function. The respiratory substrates used were succinate (B) and glutamate (C), respectively. KIS079 and CCCP (proton uncoupler) were sequentially injected. (D) Activity assay of mitochondrial complex I isolated from mouse liver in the presence / absence of 20 μM KIS079. Complex I activity was measured by the decrease in absorbance at 340 nm due to the oxidation of NADH. [Figure 5]Figure 5 demonstrates that KIS079 directly inhibits glycolysis. (A) Results of a Seahorse XFp Extracellular Flux Analyzer assay of SW480 cells are shown. The y-axis represents the extracellular acidification rate. Prior to measurement, cells were treated with 20 μM KIS079 or DMSO for 1 hour. Rotenone (complex I inhibitor), antimycin A (complex III inhibitor), and 2-DG (glycolysis inhibitor) were continuously infused. (B) Simplified schematic of the glycolytic pathway. Glucose: glucose, G6P: glucose 6-phosphate, F6P: fructose 6-phosphate, FBP: fructose 1,6-bisphosphate, G3P: glyceraldehyde 3-phosphate, DHAP: dihydroxyacetone phosphate, 1,3-BPG: 1,3-bisphosphoglycerate, Pyruvate: pyruvate, Lactate: lactate. (C) Results of a glycolysis assay using the cytosolic fraction of rat brain tissue. Inorganic phosphate was quantified by measuring absorbance at 660 nm after 0, 30, or 60 minutes of incubation with 20 μM KIS079 or DMSO. (D) NAD+ / NADH assay of SW480 and HCT116 cells. Cells were treated with 20 μM KIS079 or 2 μM rotenone (complex I inhibitor) for 1 hour. (E) Glycolysis rate assay of SW480 cells using a Seahorse XFe96 extracellular flux analyzer. SW480 cells were treated with 1 mM NMN (NAD+ precursor) for 24 hours and 0–5 μM KIS079 for 1 hour. (F) KIS079 dose-dependently reduced the total amount of NAD+ and NADH. [Figure 6] FIG. 6 shows a schematic diagram of the dual inhibitory effect of KIS079 on bioenergy production pathways. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be described in detail below. The scope of the present invention is not limited to these descriptions, and other than the following examples, the present invention can be appropriately modified and implemented within the scope that does not impair the gist of the present invention. In addition, all publications cited in this specification, such as prior art documents, published patent applications, patent publications, and other patent documents, are incorporated herein by reference.

[0012] The inhibitors according to the present invention have the ability to inhibit the mitochondrial respiratory chain and the glycolysis pathway in cells. An example of such a substance is the compound KIS079.

[0013] 1. About KIS079 The present invention uses 7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone (KIS079), and procedures for preparing this compound are disclosed in Patent Documents 1 and 3. Hereinafter, unless otherwise specified, the compound of the present invention refers to compound KIS079. [ka]

[0014] The compound KIS079 was initially developed as a PDK4 inhibitor by Professor Sunazuka Toshiaki and others at the Omura Satoshi Institute, Kitasato University. PDK4 inhibitors are also expected to have therapeutic effects against viral infections, heart failure, sepsis, and the like (Patent Document 1). Patent Documents 1 to 3 disclose various compounds, including the compound KIS079, and describe the effects of these compounds, such as improving the symptoms of a heart failure model mouse (Patent Document 1), inhibiting the anchorage-independent growth of cancer cells (Patent Document 2), and prolonging the survival of influenza-infected mice (Patent Document 3).

[0015] Pyruvate is a substance produced as a result of glucose metabolism in living organisms and serves various functions within cells, including as a raw material for the biosynthesis of the amino acid alanine, malate, lactate, and oxaloacetate, and as a raw material for the citric acid cycle (TCA cycle). Furthermore, pyruvate is converted (metabolized) to acetyl-CoA through decarboxylation by the pyruvate dehydrogenase (PDH) complex localized in mitochondria. This generated acetyl-CoA plays a large number of intracellular functions, including the oxidation of fatty acids and various amino acids, the modification of amino acids such as lysine, and as a precursor for the biosynthesis of steroids such as cholesterol.

[0016] Pyruvate dehydrogenase kinase (PDK) specifically phosphorylates and inactivates pyruvate dehydrogenase (lipoamide), a component of the pyruvate dehydrogenase complex, thereby inhibiting pyruvate metabolism. Four PDK isotypes (PDK1 to PDK4) have been reported in humans (J Biol Chem. 2001, 276, 37223-37229). Inactivation of the PDH complex by PDK inhibitors is thought to affect various intracellular functions, as described above. PDK inhibitors have been shown to be useful in the treatment of a variety of diseases, including diabetes, heart disease, and cancer (Metabolism. 2012, 61, 175-185; Int J Cancer. 2019, 144, 674-686; Cancer Cell. 2007, 11, 37-51).

[0017] 2. Mitochondrial respiratory chain (electron transport chain) inhibitors The present invention provides a mitochondrial respiratory chain inhibitor (or electron transport chain inhibitor) that inhibits the mitochondrial respiratory chain (also called the electron transport chain) in cells. A specific example of the mitochondrial respiratory chain inhibitor of the present invention is the compound KIS079.

[0018] The mitochondrial respiratory chain inhibitor of the present invention preferably acts on complexes I to IV involved in the mitochondrial respiratory chain, and preferably directly inhibits complex I (which functions as NADH dehydrogenase or NADH-ubiquinone reductase) among complexes I to IV. That is, the mitochondrial respiratory chain inhibitor of the present invention functions as a mitochondrial complex I inhibitor, an NADH-ubiquinone reductase inhibitor, or an NADH dehydrogenase inhibitor. Specifically, the mitochondrial respiratory chain inhibitor of the present invention inhibits ATP production and NAD metabolism in mitochondria by inhibiting the NADH metabolism, glutamine metabolism, etc. of complex I. + Based on these actions, the compounds of the present invention are also substances that suppress or inhibit metabolism in mitochondria.

[0019] Mitochondria are present in the cells of higher animals and are responsible for energy production, such as ATP production. Therefore, in cancer treatment, inhibiting mitochondrial ATP production in cancer cells may suppress cancer cell growth and / or induce cell death.

[0020] ATP production in mitochondria is mediated by the respiratory chain, which is thought to involve a series of membrane proteins in the inner mitochondrial membrane: Complex I or II, Complex III, and Complex IV.

[0021] In more detail regarding the above-mentioned ATP production, Complex I converts NADH to NAD + , H + and two electrons (e - As a result, the inner mitochondrial membrane is divided into two parts: the matrix and the intermembrane space. + A concentration gradient is formed. The resulting H + is transported from the inner to the outer side of the inner membrane by complex I, + The concentration gradient decreases. During this transport, ATP is generated from ADP by the action of the F0F1 complex protein, which is responsible for proton transport.

[0022] Therefore, the mechanism by which ATP synthesis by the mitochondrial respiratory chain is inhibited is by inhibiting the metabolism of NADH in complex I or the metabolism of FADH2 in complex II. + and inhibiting the function of proteins involved in proton transport in complex I, complex III, and / or complex IV (F0F1 complex). The mitochondrial respiratory chain inhibitor of the present invention preferably inhibits the function of complex I, and more preferably, the inhibitor of the present invention inhibits NADH metabolism in complex I, which results in the inhibition of intracellular ATP synthesis.

[0023] The mitochondrial respiratory chain inhibitors of the present invention are used at a dose that reduces mitochondrial oxygen consumption by 60%, 55%, 50%, 45%, 40%, 35%, 30% or less compared to normal oxygen consumption. Alternatively, the mitochondrial respiratory chain inhibitors of the present invention are used at a dose that reduces mitochondrial oxygen consumption by 60%, 55%, 50%, 45%, 40%, 35%, 30% or less compared to normal oxygen consumption. Such a dose may result in a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100 μM or more. Measurement of mitochondrial oxygen consumption is well known to those skilled in the art, and includes, for example, the method used in the Examples below. Measurement can also be performed by measuring the oxygen consumption rate (OCR) associated with cellular respiration, the extracellular acidification rate (ECAR), or the like, as used in the Examples below.

[0024] 3. Glycolysis inhibitors The present invention provides a glycolysis inhibitor that inhibits the glycolysis pathway involved in glucose metabolism in cells. The glycolysis inhibitor used in the present invention is preferably the compound KIS079.

[0025] In the cytoplasm or mitochondria of animal cells, glucose is metabolized to produce pyruvate, and ATP is produced from ADP during this process. Therefore, the inhibitors of the present invention can inhibit intracellular glycolysis, thereby inhibiting intracellular ATP production, pyruvate production, and the TCA cycle driven by pyruvate. Examples of inhibitors of ATP production in intracellular mitochondria include azocyclotine, cyhexatin, diafenthiuron, fenbutatin oxide, provargite, and tetradifon, which are also used in various pesticides. Furthermore, known compounds that inhibit ATP production by suppressing oxygen consumption in cells, particularly in mitochondria, include oligomycin, which is used in the examples below.

[0026] The inhibitors of the present invention preferably inhibit ATP production in the glycolytic pathway, independently of NAD and NADH (also referred to as NAD(H)). Whether or not glycolysis is dependent on NAD(H) can be confirmed by using quantitative detection means for NAD(H) known to those skilled in the art. Examples of such detection means include, but are not limited to, commercially available kits for NAH / NADH assays (e.g., "NAD / NADH Assay Kit-WST" commercially available from Dojindo Laboratories, Inc.) as described in the Examples of the present application.

[0027] Therefore, the inhibitors of the present invention inhibit NAD(H)-independent steps in glucose metabolism (schematically shown in FIG. 5B). Specifically, they inhibit steps upstream of the NAD(H)-dependent step of glucose metabolism where glyceraldehyde 3-phosphate (G3P) is converted to 1,3-bisphosphoglycerate (1,3-BPG), thereby inhibiting ATP production in the glycolytic pathway and, as a result, inhibiting the production of end products such as pyruvate. The glycolysis inhibitors of the present invention are preferably used at a dose that reduces the total amount of NAD(H) in target cells by 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, or 20% or less, and may be a dose that results in a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100 μM or more.

[0028] 4.Ability to induce cell death The inhibitors of the present invention inhibit the mitochondrial respiratory chain and / or intracellular glycolysis, resulting in cell death in cancer cells and are therefore useful for treating cancer. As used herein, the term "inhibitors of the present invention" refers to those that inhibit both the mitochondrial respiratory chain and intracellular glycolysis, and a specific example of such an inhibitor is the compound KIS079. The inhibitors of the present invention are used at doses that reduce cell viability to 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30% or less compared to a control. Such doses can be those that result in concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100 μM or more. Inhibitors of the present invention are expressed as inhibitors of 50% cell viability inhibition (IC 50 The concentration range of ) can be, for example, 1 to 100 μM, 2 to 90 μM, 3 to 80 μM, 4 to 70 μM, or 5 to 60 μM. Means for measuring cell viability include those known to those skilled in the art, such as visual cell counting using a hemocytometer and trypan blue staining, particle measurement using instruments such as flow cytometry or a Coulter counter, and measurement using an automated cell counter (such as ThermoFisher).

[0029] 5. Pharmaceutical Composition of the Present Invention The inhibitor of the present invention or a pharmaceutical composition comprising the inhibitor of the present invention can be administered to a subject (such as a patient) as an antitumor agent, for example, in an amount sufficient to reduce tumor size or to cause cell death in cancer cells. The pharmaceutical composition of the present invention can contain the inhibitor of the present invention in its original form or in the form of a pharmaceutically acceptable salt, together with other additives such as a carrier. Furthermore, the pharmaceutical composition of the present invention can be provided in a dosage form commonly used in the art depending on the administration route, such as oral or parenteral.

[0030] The inhibitor of the present invention (e.g., KIS079) contained in the pharmaceutical composition of the present invention may be in the form of a pharmaceutically acceptable salt. Examples of such salts include salts of basic groups, such as mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, and nitrate; sulfonates such as methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate; and acid addition salts such as organic acid salts such as oxalate, tartrate, citrate, maleate, succinate, acetate, benzoate, mandelate, ascorbate (e.g., sodium ascorbate, potassium ascorbate, etc.), lactate, gluconate, and malate, preferably hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, p-toluenesulfonate, oxalate, tartrate, citrate, acetate, and lactate. Alternatively, pharmaceutically acceptable salts of the compounds of the inhibitors of the present invention include, for example, salts with acidic groups such as carboxylic acid groups, for example, alkali metal and alkaline earth metal salts such as lithium, sodium, potassium, magnesium, and calcium; salts with amines such as ammonia, methylamine, dimethylamine, trimethylamine, dicyclohexylamine, tris(hydroxymethyl)aminomethane, N,N-bis(hydroxyethyl)piperazine, 2-amino-2-methyl-1-propanol, ethanolamine, N-methylglucamine, and L-glucamine; or salts with basic amino acids such as lysine, δ-hydroxylysine, and arginine. Furthermore, the inhibitor compounds of the present invention also include hydrates or solvates of the above-mentioned pharmaceutically acceptable salts.

[0031] The pharmaceutical composition of the present invention may contain, in addition to the inhibitor of the present invention, one or more conventional additives known in the art. Examples of such additives include dietary fiber and thickeners derived from sources other than vegetables, such as hemicellulose, lignin, guar gum, konjac mannan, isagol, alginic acid, agar, carrageenan, chitin, carboxymethylcellulose, and polydextrose; edible oils; minerals, such as calcium, iron, sodium, zinc, copper, potassium, phosphorus, magnesium, iodine, manganese, and selenium; fat-soluble or water-soluble vitamins, such as vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, niacin, folic acid, and pantothenic acid; glycerin, fatty acid esters, and the like. These include emulsifiers and dispersants such as cellulose, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, phospholipids, gum arabic, xanthan gum, tragacanth gum, and locust bean gum, bulking agents, excipients, preservatives and antioxidants, flavor adjusters and fragrances, flavorings such as sodium chloride, monosodium glutamate, glycine, succinic acid, and sodium lactate, acidulants such as citric acid, sodium citrate, acetic acid, adipic acid, fumaric acid, and malic acid, low-calorie sweeteners such as maltitol and aspartame, coloring agents, and calcium.

[0032] The pharmaceutical compositions of the present invention may be administered to patients orally. Suitable dosage forms for oral administration include, but are not limited to, tablets, capsules, microcapsules, powders, fine granules, granules, liquids, and syrups. In oral dosage forms, the pharmaceutical compositions of the present invention contain the inhibitors of the present invention as the active ingredient and may further contain various excipients known in the art. Examples of such excipients include, but are not limited to, microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate, and glycine. The pharmaceutical compositions of the present invention may also contain a disintegrant. Examples of such disintegrants include, but are not limited to, starch (e.g., corn, potato, or tapioca starch), alginic acid, and various silicates. The pharmaceutical compositions of the present invention may also contain a binder. Examples of such binders include, but are not limited to, polyvinylpyrrolidone, sucrose, gelatin, and gum arabic. Furthermore, the pharmaceutical composition of the present invention may contain a lubricant in tablet form, including, but not limited to, magnesium stearate, sodium lauryl sulfate, and talc. Furthermore, the pharmaceutical composition of the present invention may be filled into capsules made of cellulose, gelatin, or the like. Such capsules may contain lactose, milk sugar, high-molecular-weight polyethylene glycol, or the like. Furthermore, the pharmaceutical composition of the present invention may contain, in the form of an aqueous suspension and / or elixir, a vehicle such as water, ethanol, propylene glycol, or glycerin as a carrier, and may further contain known substances such as sweeteners, flavorings, colorings, emulsifiers, suspending agents, and diluents.

[0033] The pharmaceutical composition of the present invention can be administered to a patient by parenteral administration. Suitable formulations for parenteral administration include, but are not limited to, injections, liniments, suppositories, and the like. For parenteral administration, the active ingredient of the present invention can be dissolved in an oily medium such as sesame oil or peanut oil, or an aqueous medium such as aqueous propylene glycol. The aqueous solution can be buffered (preferably to a pH of 8 or higher) to make it isotonic, as necessary. Such aqueous solutions are suitable for intravenous injection, while oily solutions can be used for intraarticular, intramuscular, and subcutaneous injections. Furthermore, the pharmaceutical composition of the present invention can be administered topically, for example, by application via the skin. Examples of dosage forms for such application include creams, jellies, pastes, ointments, and the like.

[0034] The pharmaceutical composition of the present invention can be administered to patients with cancer, including colorectal cancer (colon cancer, rectal cancer), pancreatic cancer, lung cancer, esophageal cancer, gastric cancer, upper digestive tract cancer, liver cancer, head and neck cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular tumor, kidney cancer, bladder cancer, urothelial tumor, oral cancer, skin malignant tumor, gastrointestinal stromal tumor (GIST), and glioma. 、 Cancers to be treated by the pharmaceutical composition of the present invention include, but are not limited to, brain tumors, osteosarcoma, malignant lymphomas (such as Hodgkin's lymphoma and non-Hodgkin's lymphoma), hematological tumors, chronic myeloid leukemia, multiple myeloma, and malignant melanoma. More preferably, cancers to be treated by the pharmaceutical composition of the present invention include, but are not limited to, colon cancer, pancreatic cancer, lung cancer, esophageal cancer, gastric cancer, liver cancer, head and neck cancer, breast cancer, cervical cancer, ovarian cancer, uterine cancer, and bladder cancer. Patients to which the pharmaceutical composition of the present invention is administered are preferably mammals, more preferably, but not limited to, animals such as humans, monkeys, dogs, horses, pigs, sheep, goats, mice, and rats. Cultured cells to be used with the compound, inhibitor, or pharmaceutical composition of the present invention are cells derived from mammalian species derived from the above-mentioned cancers, most preferably human-derived cells.

[0035] The dose of the inhibitor or composition of the present invention is not particularly limited, and an appropriate amount can be selected depending on various conditions, such as the condition of the subject (e.g., patient), the subject's age, weight, sex, or the type or severity of symptoms, the route of administration, and the purpose of treatment. For example, in the case of oral administration, a dose in the range of 10 mg to 20 g, 100 mg to 10 g, or preferably 150 mg to 5 g per day for an adult (e.g., body weight 60 kg) can be applied. These daily doses may be administered, for example, in two to four divided doses.

[0036] The pharmaceutical composition of the present invention can be used in combination with known anticancer drugs for chemotherapy. Examples of known anticancer drugs include, but are not limited to, alkylating agents (e.g., cyclophosphamide, ifosfamide, melphalan, busulfan, dacarbazine, nimustine hydrochloride, ranimustine, bendamustine hydrochloride, temozolomide, carmustine), antimetabolites (e.g., azathioprine, methotrexate, pemetrexed sodium hydrate, mercaptopurine hydrate, fluorouracil, tegafur, tegafur-uracil combination, doxifluridine, capecitabine, cytara). vinocphosphate hydrate, hydroxycarbamide, cytarabine, gemcitabine hydrochloride, fludarabine phosphate, clofarabine, enocitabine, nelarabine), alkaloids (e.g., vincristine sulfate, vinblastine sulfate, vindesine sulfate, docetaxel hydrate, paclitaxel, vinorelbine tartrate, eribulin mesylate), antibiotic anticancer agents (e.g., doxorubicin hydrochloride (adriamycin), epirubicin hydrochloride, pirarubicin hydrochloride, daunorubicin hydrochloride, idarubicin hydrochloride, aclarubicin cin hydrochloride, amrubicin hydrochloride, mitomycin C, actinomycin D, bleomycin hydrochloride, peplomycin sulfate), topoisomerase inhibitors (e.g., etoposide, irinotecan hydrochloride hydrate, nogitecan hydrochloride), hormonal preparations (e.g., estalamustine phosphate sodium, flutamide, bicalutamide, goserelin acetate, leuprorelin acetate, degarelix acetate, tamoxifen citrate, toremifene citrate, anastrozole, letrozolol, exemestane, fulvestrant, mepitiostane, medroxyprogesterone acetate), platinum agents (e.g., cisplatin, miriplatin hydrate, carboplatin, nedaplatin, oxaliplatin), interferon agents (e.g., interferon gamma 1a, interferon alpha, interferon alpha 2a, interferon beta), interleukin agents (e.g., cermoleukin, teseloquin), kinase inhibitors (e.g., gefitinib, imalinib mesylate, erlonib hydrochloride, sorafenib tosylate, sunitinib malate, dasatinib hydrate,Nilotinib hydrochloride hydrate, lapatinib tosylate, crizotinib, axitinib, pazopanib hydrochloride, regorafenib), proteasome inhibitors (e.g., bortezomib), mTOR inhibitors (e.g., everolimus, temsirolimus), other anticancer drugs (mitoxantrone hydrochloride, procarbazine hydrochloride, pentostatin, cladribine, sobuzoxane, tretinoin, tacrolimus), Mybarotene, L-asparaginase, mitotane, porfimer sodium, talaporfin sodium, arsenic trioxide, thalidomide, lenalidomide hydrate, texamethasone, azacitidine, bilinostat, talc), nonspecific antineoplastic agents (e.g., lentinan, ubenimex), topical anticancer agents (5-fluorouracil (5FU), tegafur, doxylofuridine, cape Cytabine, cytarabine, enocitabine, gemcitabine, bleomycin sulfate, BCG vaccine), letrozole, finasunate, rapamycin, leucovorin, lapatinib, lonafarnib, camptothecin, bryostatin, adozelesin, anthracyclines, carzelesin, bizelesin, dolastatins, auristatins, duocarmycins, eluterobin, taxol, prednisone or prednisolone, other alkylating agents (e.g., mechlorethamine, chlorambucil), microtubule inhibitors (e.g., vinca alkaloids such as taxanes), podophyllotoxins (etoposide phosphate and epipodophyllotoxin), other cytotoxins (e.g., actinomycin, plicamycin), and pharmaceutically acceptable salts of the above agents, and Monoclonal antibodies (e.g., abagovomab, adecatumumab, afutuzumab, alacizumab, alemtuzumab, anatumomab, apolizumab, bavituximab, belimumab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, catumaxomab, cetuximab, sitatuzumab bogatox, cixitumumab, clivatuzumab tetraxetan, conatumumab, dacetuzumab , daclizumab, detumomab, ecloneximab, edrecolomab, elotuzumab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab, fresolimumab, galiximab, glenbatumumab vedotin, gemtuzumab, ibritumomab, inotuzumab ozogamicin, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, rituximab, lintuzumab, lucatumumab, lumi Riximab, mapatumumab, matuzumab, milatuzumab, mitumomab, mogamulizumab, nacolomab butafenatox, naptumomab estafenatox, necitumumab, nimotuzumab, ofatumumab, olaratumumab, oportuzumab monatox, oregovomab, panitumumab, pemtumomab, pertuzumab, pintumomab, pritumumab, ramucirumab, rilotumumab, lobatumumab, sibrotuzumab, tacatuzumab tetraxetan , taplitumomab paptox, tenatumomab, ticilimumab, tigatuzumab, tositumomab, trastuzumab, tremelimumab, tuocuzumab celmoleukin, veltuzumab, visilizumab, volociximab, votumumab, zalutumumab, zanolimumab, etc.), and conjugates of these antibodies with the above drugs (e.g., auristatins MMAE and MMAF, maytansinoids such as DM-1, calicheamicin, or various cytotoxins). The pharmaceutical compositions of the present invention can be used in combination with other anticancer agents, preferably, for example, the above-mentioned alkylating agents, antibiotic anticancer agents, microtubule inhibitors, hormone analogs, platinum preparations, topical anticancer agents, topoisomerase inhibitors, biological agents, etc. More preferably, the pharmaceutical compositions of the present invention can be used in combination with one or more anticancer agents, such as cisplatin, miriplatin hydrate, carboplatin, nedaplatin, oxaliplatin, 5-fluorouracil (5FU), tegafur, doxilfuridine, capecitabine, cytarabine, enocitabine, gemcitabine, bleomycin sulfate, BCG vaccine, etc. Even more preferably, the pharmaceutical compositions of the present invention can be used in combination with 5-fluorouracil (5FU) and oxaliplatin. The pharmaceutical composition of the present invention may be administered to a patient simultaneously with other anticancer drugs (or anticancer drugs) containing the above-mentioned components, or may be administered to a patient in a regimen following a treatment regimen using other anticancer drugs containing the above-mentioned components, or may be administered to a patient alternately with other anticancer drugs containing the above-mentioned components. Such administration regimens can be appropriately determined by the attending physician, taking into consideration the condition of the patient to be treated (severity of the target disease, sex, age, drug use history, concomitant drugs, etc.). Furthermore, the pharmaceutical composition of the present invention can be used in combination with various cancer treatment methods other than chemotherapy, such as surgery, radiation therapy, immunotherapy, and hyperthermia.

[0037] In the present invention, when an anticancer composition is prepared, the above-mentioned other anticancer agents can be further blended. In this case, the blending ratio of the inhibitor of the present invention to the other anticancer agent component is appropriately selected depending on the type of cancer to be treated, the age and symptoms of the patient, the administration route, the purpose of treatment, etc., and can be selected, for example, within the range of 1:99 to 99:1. The pharmaceutical composition of the present invention can also be blended with two or more other anticancer agents. In this case, the blending ratio is also appropriately selected depending on the type of cancer, the age and sex of the patient, the severity of symptoms, the administration route, the purpose of treatment, etc.

[0038] Preferably, when used in combination with a drug capable of inhibiting proliferation or inducing cell death, the inhibitor or pharmaceutical composition of the present invention provides effective growth inhibition of cancer tissues from the early stage after administration (1 to 3 days after administration, preferably 1 to 2 days after administration, more preferably 1 day after administration). Typically, when an anticancer drug is administered to cancer tissues, a certain amount of cell proliferation occurs in the early stage immediately after administration, during which the anticancer drug is taken up, and then the drug's efficacy is exerted, resulting in cell death of the cancer cells (e.g., the control in Figure 2). When used in combination with a drug capable of inhibiting proliferation or inducing cell death, the inhibitor of the present invention preferably reduces the size of cancer tissues to at most 110% or less, 105% or less, 100% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, or 70% or less of their initial size. In such cases, the administered dose may be, for example, a dose that results in a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100 μM or more.

[0039] Cancer tissue size can be measured using methods known to those skilled in the art. Examples of such methods include visual size measurement, spheroid measurement devices (e.g., Beckman Coulter's Multisizer 4e), and measurement devices that combine devices such as the electrochemical detection zone method (also known as the Coulter principle) with images or computer graphics. Furthermore, the cancer tissue may be a colony, spheroid, or aggregate formed by cancer cells in an in vitro or in vivo experimental system using a scaffold such as cellulose. For example, methods known to those skilled in the art can be used to form spheroids containing cultured cells, such as the method described in the Examples below or a method using a commercially available spheroid formation vessel (e.g., "Wellbag" manufactured by Toyo Seikan Group Holdings Co., Ltd.).

[0040] Unless otherwise specified, technical terms used herein have the same meaning as commonly understood by those skilled in the art. [Example]

[0041] 1. Materials and Procedures (1) Preparation of KISS079 The compound KISS079 (7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone) is disclosed in Patent Documents 1 and 3 (JP 2015-160831 A, WO 2014 / 103321 A), and can be prepared by the methods described in these documents.

[0042] (2) Cell culture and reagents SW480, HT29, HCT116, Colo205, CK81, A172, and YKG1 were maintained in D-MEM (high glucose), McCoy's 5A, McCoy's 5A, RPMI-1640, MEM, and RPMI-1640 + D-MEM (high glucose) media containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, respectively. MICa-2 and PANC-1 were provided by the RIKEN BRC through the National BioResource Project of MEXT / AMED (Japan). Each cell line was maintained in D-MEM (low glucose) + RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin. D-MEM (high glucose), D-MEM (low glucose) + RPMI-1640 medium were purchased from Wako Pure Chemical Industries, Ltd., Japan. McCoy's 5A and MEM medium were purchased from Thermo Fisher Scientific.

[0043] (3) Cell proliferation assay Cell proliferation was assessed by trypan blue exclusion assay or Cell Counting Kit-8 (Dojindo, Japan). For the trypan blue exclusion assay, 5 × 10 cells were cultured on the day before KIS079 administration. 5Cells were seeded at 1 × 10 cells / dish into 35 mm dishes containing 1.5 mL of culture medium. Total media and single-cell suspensions prepared by trypsinization were collected at 0, 4, 8, 12, and 24 hours after administration. 50 μL of the cell suspension was mixed 1:1 with trypan blue solution. Live and dead cells were counted using a hemocytometer. For the Cell Counting Kit-8 assay, cells were diluted to 1 × 10 cells / dish on the day before administration of KIS079. 4 Cells were seeded per well in a 96-well plate using 100 μL of culture medium. 72 hours after administration, 10 μL of CCK-8 solution was added to the cells and incubated for 1 hour at 37° C. The absorbance at 450 nm was measured by a Chromate 4300 microplate reader (Awareness Technologies, USA).

[0044] (4) Formation and size measurement of colorectal cancer cell spheroids Colon cancer-derived cell line HCT116 was cultured at 10 cells / 100 μL of medium (DMEM medium containing 10% FBS and 1% penicillin / streptomycin). 3 ~10 4 The cells were suspended to a density of 1000 cells / well and seeded into each well of a low-adhesive 96-well plate (PrimeSurface, Sumitomo Bakelite Co., Ltd.). After 1-2 days, spheroid formation was confirmed. 50 μL of medium containing 5-FU and oxaliplatin (both 5 μM or 10 μM), and in some cases 20 μM KIS079, was added, and the culture was continued for 9 days or longer. The size change over time of spheroids formed in approximately 10 or more wells was calculated based on the results of measurements using a SCREEN CELL3IMAGER DUOS.

[0045] (5) Western blotting Complete the cell count. TMThe samples were dissolved in modified RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM KCl, 0.2% NP-40, 10% glycerol) supplemented with Lysis-M (Sigma-Aldrich, USA). Protein concentrations were determined using the Protein Assay Rapid Kit Wako II (Wako, Japan). Proteins were separated by SDS-PAGE using Bolt™ 4-12% Bis-Tris Plus gels (Thermo Fisher Scientific, USA) and transferred to PVDF membranes using a Mini Gel Tank (Thermo Fisher Scientific, USA). Primary antibodies were as follows: PDHA1 (phopho-S293) (#ab177461; 1:300; Abcam); PDHA1 (#ab168379; 1:300; Abcam); PDK4 (#12949-1-AP; 1:300; Proteintech); β-actin (#3700; 1:1000; Cell Signaling Technology).

[0046] (6) Transmission electron microscope. SW480 cells were seeded in 35 mm dishes containing D-MEM (high glucose) medium and treated with KIS079. Cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) at room temperature, then fixed in 1% osmium tetroxide in the same buffer at 4°C, dehydrated through a dilution series of ethanol, and embedded in Ketol 812 epoxy resin (Nissin EM Co., Ltd.). 70-nm-thick sections were cut using an ultramicrotome (Ultracut UCT, LEICA), stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (HT7700, Hitachi Hi-Tech).

[0047] (6) TMRE (tetramethylrhodamine ethyl ester) assay Mitochondrial membrane potential was measured using a TMRE-mitochondrial membrane potential assay kit (Abcam). Briefly, SW480 cells seeded in a 96-well plate were treated with 30 μM KIS079 for up to 2 hours, incubated with 100 nM TMRE for 20 minutes, rinsed with PBS, and examined under a fluorescence microscope.

[0048] (7) Extracellular Flux Analysis Cell metabolism was analyzed using a Seahorse XFp Extracellular Flux Analyzer (Agilent Technologies). To analyze mitochondrial function, oxygen consumption rate (OCR) was measured using a Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies). SW480 cells were cultured in Seahorse XFp cell culture miniplates at 2 × 10 4 Cells were seeded at 1000 x g / well and cultured overnight. The cells were washed twice with assay medium (Seahorse XF basal medium supplemented with 2 mM glutamine, 1 mM pyruvate, and 10 mM glucose) and incubated with assay medium containing the indicated concentrations of KIS079 for 1 h in a CO2-free incubator at 37°C. During OCR measurements, oligomycin (1.5 μM final concentration), FCCP (1.0 μM final concentration), and rotenone / antimycin A (0.5 μM final concentration) were continuously injected. To analyze glycolytic function, extracellular acidification rate (ECAR) was measured using the Seahorse XF Glycolytic Rate Assay Kit (Agilent Technologies). The cell seeding density and pretreatment conditions were the same as those used in the Mito Stress Test assay. During ECAR measurements, rotenone / antimycin A (0.5 μM final concentration) and 2-DG (50 mM final concentration) were continuously injected.

[0049] (8) Mitochondrial respiratory chain assay To prepare the mitochondrial solution, fresh mouse liver was cut into small pieces, homogenized in homogenization buffer, and centrifuged at 2,000 rpm for 10 min. The supernatant was centrifuged at 10,000 rpm for 10 min, and the pellet was homogenized in homogenization buffer. To analyze mitochondrial function in the resulting mitochondrial solution, rhodamine fluorescence was measured using an FP-8200 fluorescence spectrometer (JASCO, Japan). Prior to measurement, respiratory substrates (10 mM succinate or 4 mM glutamate) and 1 μL of rhodamine 123 were added to 1 mL of reaction buffer. Then, 10 μL of the above mitochondrial solution, KIS079 (final concentrations of 20–40 μM), and CCCP (proton uncoupler, final concentrations of 4–24 μM) were injected continuously during the measurement.

[0050] (9) Complex I assay Isolated mouse liver mitochondria prepared as described above were resuspended in hypotonic buffer (25 mM K2PO4, 5 mM MgCl2) (pH 7.2) and freeze-thawed three times to disrupt the mitochondrial membrane (Oncotarget 8, 67269-67286 (2017)). 800 μL of HO, 20 μM KIS079, or DMSO (control) was added to the mitochondrial solution and incubated at 37 °C for 1 min. Then, 200 μL of 50 mM Tris-HCl (pH 8.0) buffer supplemented with 5 mg / ml BSA, 0.8 mM NADH, 240 μM KCN, and 4 μM antimycin A was added. To analyze Complex I activity, the decrease in absorbance at 340 nm due to the oxidation of NADH was measured using a GENESYS 10S Vis spectrophotometer (Thermo Fisher Scientific, USA). The redox reaction was initiated by adding 50 μM decylubiquinone (electron acceptor). After a baseline measurement, 4 μM rotenone was added, and the rotenone-sensitive activity of complex I was calculated.

[0051] (10) NAD + / NADH assay NAD +and NADH concentrations were measured using the NAD / NADH Assay Kit-WST (Dojindo Laboratories, Japan: manufacturer code N509) according to the manufacturer's instructions. + For the NAD / NADH degradation inhibition test, SW480 whole cell extracts were incubated with 50 mM K2PO4 buffer (pH 7.2), 1 mM MgCl2, and KIS079 (0 or 20 μM) in the presence or absence of 1 mM nicotinamide at 37°C for 1 hour. After incubation, 100 μL of each cell extract sample was mixed with 200 μL of NAD / NADH extraction buffer provided with the kit. The samples were centrifuged at 12,000 rpm for 5 minutes, and the supernatant was filtered using a 10K MWCO filter tube (molecular weight cutoff 10 kDa). To prepare the NADH sample, total NAD was extracted with 10 μL of K2PO4 buffer (pH 7.2), 1 mM MgCl2, and KIS079 (0 or 20 μM). + The NAD / NADH samples were incubated for 1 hour at 60° C. Samples were diluted with NAD / NADH control buffer and incubated with the working solution for 1 hour at 60° C. The absorbance at 450 nm was measured by a ChroMate 4300 microplate reader.

[0052] 2. Experimental Results and Discussion (1) KIS079 induces cancer cell death The chemical structure of KIS079 (7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone) is shown in Figure 1A. This molecule was originally developed as one of the KIS compounds (natural diterpene quinones) as inhibitors of pyruvate dehydrogenase kinase 4 (PDK4) (Patent Documents 1 to 3). PDK4 inactivates pyruvate dehydrogenase (PDH), the gatekeeper enzyme of the TCA cycle, via phosphorylation. Therefore, PDK4 inhibitors are expected to induce a metabolic shift from glycolysis to mitochondrial respiration. First, we evaluated the effect of KIS079 on the human colon cancer cell line SW480. The viability of SW480 cells was reduced to almost 0% after 72 hours with 20 μM KIS079 (IC 50= 12.7 μM) (Figure 1B). KIS079 also reduced the viability of other human colon cancer cell lines (HT29, HCT116, Colo205, and CK81), human pancreatic cancer cell lines (MIA PaCa-2, PANC-1), and human glioblastoma cell lines (A172, YKG1) (Figure 1C). The IC of these cells was 1.2 μM. 50 was in the range of 5-30 μM. Time course analysis of SW480 cell viability revealed that cell viability and viable cell counts began to decrease within 4 hours at 20–30 μM KIS079 (Figure 1D). Considering that KIS079 was originally developed as a PDK4 inhibitor, we next examined the effect of KIS079 on PDH phosphorylation in SW480 cell extracts by Western blotting. Unexpectedly, PDH phosphorylation was not reduced by treatment with 20 μM KIS079 for 3–6 hours (Figure 1E, F). These results suggested that the antitumor effects of KIS079 are mediated by mechanisms other than PDK4 inhibition.

[0053] (2) KIS079 enhances the antitumor effect of 5FU and oxaliplatin on colorectal cancer cell spheroids. To investigate whether KIS079 inhibits or kills cancer cells through cell proliferation or non-cell proliferation, we investigated the effect of KIS079 in combination with standard chemotherapeutic agents that inhibit proliferating cells. The addition of KIS079 to 5FU and oxaliplatin (Ox) significantly suppressed the spheroid size of human colorectal cancer cells (Figure 2), demonstrating that KIS079 significantly enhances the antitumor effects of 5FU and oxaliplatin. These results indicated that KIS079 has the effect of suppressing cancer cells through a mechanism other than cell proliferation, such as DNA replication.

[0054] (3) KIS079 inhibits mitochondrial respiration To further investigate the effects of KIS079 on cancer cells, we performed transmission electron microscopy of SW480 cells treated with KIS079. Six hours after KIS079 administration, we observed cytoplasmic vacuolization and mitochondrial swelling in cancer cells (Figure 3A). The mitochondrial matrix swelled more severely at 12 hours. This observation confirmed that mitochondrial damage occurred in cancer cells treated with KIS079. A TMRE assay was performed to visualize mitochondrial membrane potential. Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), a proton uncoupler, was used as a control. As expected, KIS079 significantly reduced the membrane potential, as indicated by fluorescence intensity, at 30 min (Figure 3B). Next, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of KIS079-treated SW480 cells were measured using a Seahorse Extracellular Flux Analyzer. OCR and ECAR correspond to the activities of mitochondrial respiration and glycolysis, respectively. During the assay, mitochondrial function was assessed using three reagents. After measuring basal respiration, we injected oligomycin, an ATP synthase inhibitor, and observed a decrease in OCR after oligomycin injection, which corresponds to new ATP production. The second reagent, FCCP, causes proton leak without ATP production, resulting in maximal respiration. Finally, rotenone (complex I inhibitor) and antimycin A (complex III inhibitor) were injected to block total mitochondrial respiration, respectively. After 1 hour of treatment with 20 μM KIS079, basal (Fig. 4A, left) and maximum (Fig. 4A, right) respiration of SW480 cells were significantly suppressed. These results suggest that KIS079 treatment significantly reduces or disables new ATP production, and that cells are largely unresponsive to subsequent respiratory induction. This suggests that KIS079 severely damages mitochondrial structure and respiratory function.

[0055] (4) KIS079 directly inhibits mitochondrial complex I To elucidate the mechanism of inhibition of mitochondrial respiration by KIS079, an electron transport assay was performed using mitochondria isolated from mouse liver. Mitochondrial function was assessed by measuring the fluorescence intensity of rhodamine in the culture medium. Since rhodamine is taken up by active mitochondria, a decrease in rhodamine in the culture medium indicates active mitochondria. When succinate was added as a respiratory substrate, KIS079 did not inhibit mitochondrial function (Figure 4B). Specifically, succinate is oxidized by complex II (succinate dehydrogenase), and the resulting electrons are transferred to complexes III and IV, allowing us to confirm the activity of complexes II, III, and IV. When KIS079 was added after the addition of rhodamine and succinate, the rhodamine fluorescence intensity did not change significantly before and after the addition of KIS079. This indicates that KIS079 does not affect, or inhibit, complexes II, III, or IV. In contrast, when glutamate was added as a respiratory substrate, mitochondrial function was suppressed by KIS079 (Figure 4C). Specifically, glutamate is a substrate for complex I, and because electrons derived from glutamate enter the electron transport chain via complex I, the activity of complexes I–IV can be confirmed. When KIS079 (20 μM) and then KIS079 (40 μM) were added after the addition of rhodamine + glutamate, the rhodamine fluorescence intensity fluctuated over time. This indicates that the addition of KIS079 affected complexes I–IV, resulting in abnormalities in mitochondrial activity. These results indicate that KIS079 inhibits complex I among complexes I to IV of the mitochondrial respiratory chain (electron transport system). To further verify this observation, we next performed a complex I assay using isolated mouse liver mitochondria (Current Protocols in Human Genetics 63, 19-3 (2009)). Complex I activity was determined by the decrease in absorbance at 340 nm due to the oxidation of NADH. Consistently, KIS079 significantly reduced complex I activity (NADH dehydrogenase activity) in this assay (Figure 4D). These results revealed that KIS079 directly inhibits mitochondrial complex I.

[0056] (5) KIS079 also inhibits glycolysis independently of NAD(H). We also observed a significant decrease in ECAR in OCR measurements of SW480 cells treated with KIS079 compared with controls (Figure 4A, right panel). This result prompted us to examine the inhibitory effect of KIS079 on glycolysis using a glycolytic rate assay. After baseline measurements, rotenone and antimycin A were injected into the assay medium to inhibit mitochondrial respiration and thereby promote compensatory glycolysis. We then administered the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) and measured the ECAR. We confirmed that the ECAR of SW480 cells was significantly suppressed by KIS079, indicating the inhibitory effect of KIS079 on glycolysis (Figure 5A). To confirm that KIS079 inhibits glycolysis, we performed an in vitro glycolysis assay using the cytosolic fraction of rat brain tissue. Glycolytic activity was measured as phosphate consumption by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Figure 5B). KIS079 almost completely inhibited phosphate consumption by glycolysis (Figure 5C). The reaction catalyzed by GAPDH is an NAD(H)-dependent process. + To examine the effect of KIS079 on the intracellular NAD / NADH ratio in SW480 cells treated with KIS079, + Unexpectedly, KIS079 increased NAD + The total amount of NAD(H) and NADH was significantly reduced (Fig. 5D). + We added nicotinamide mononucleotide (NMN), a precursor of NAD(H), to SW480 cells and measured glycolytic activity using an extracellular flux analyzer. Despite an increase in the total amount of NAD(H), glycolytic activity suppressed by KIS079 was not restored by NMN (Figure 5E). These results indicated that the inhibition of glycolysis by KIS079 is independent of the amount of NAD(H), i.e., it inhibits a step in glycolysis upstream of the conversion of glyceraldehyde 3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG), which involves NAD(H) (schematic diagram shown in Fig. 5B). This inhibition inhibits ATP production via glycolysis, which in turn inhibits pyruvate production, which in turn inhibits the TCA cycle and lactate production (Fig. 6).

[0057] From the above results, it was confirmed that the compound KIS079 of the present invention has at least the following effects. 1) It has the effect of suppressing and killing cancer cells. 2) When used in combination with 5FU and oxaliplatin, which are anti-proliferative anti-cancer drugs used in standard chemotherapy for colorectal cancer, the anti-tumor effect is significantly enhanced. 3) Acting on mitochondrial complex I and inhibiting mitochondrial metabolism. 4) Simultaneously suppress the glycolytic pathway. [Industrial Applicability]

[0058] The inhibitors and pharmaceutical compositions of the present invention simultaneously suppress the metabolic functions of the mitochondrial respiratory chain and glycolysis in cells, and induce growth inhibition and / or cell death in cells derived from various cancers such as colon cancer, pancreatic cancer, and glial cell carcinoma, and can therefore be used to treat these cancers. Furthermore, when used in combination with known growth-inhibiting anticancer agents such as 5FU and oxaliplatin, the inhibitors and pharmaceutical compositions of the present invention more potently induce cell death in cancer cells and cancer cell aggregates, thereby providing a new therapeutic method for cancer treatment.

Claims

1. A mitochondrial respiratory chain inhibitor containing 7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone.

2. The mitochondrial respiratory chain inhibitor according to claim 1, wherein the 7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone inhibits the action of mitochondrial respiratory chain complex I.

3. The mitochondrial respiratory chain inhibitor according to claim 1, wherein the 7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone inhibits glucose metabolism in cells in an NAD(H)-independent manner.

4. A glycolysis inhibitor containing 7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone.

5. The glycolysis inhibitor according to claim 4, wherein the 7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone inhibits glucose metabolism in cells in an NAD(H)-independent manner.

6. The glycolysis inhibitor according to claim 5, wherein the 7-carbomethoxy-5-hydroxy-2-methyl-1,4-naphthoquinone inhibits the action of mitochondrial respiratory chain complex I.

7. A pharmaceutical composition comprising a mitochondrial respiratory chain inhibitor according to any one of claims 1 to 3, and / or a glycolysis inhibitor according to any one of claims 4 to 6.

8. The pharmaceutical composition according to claim 7, which is an anticancer agent.

9. The pharmaceutical composition according to claim 7, which is used in combination with other anticancer agents.

10. The pharmaceutical composition according to claim 9, wherein the other anticancer agent is a cell proliferation inhibitor.

11. The pharmaceutical composition according to claim 10, wherein the other anticancer agent comprises one or more selected from the group consisting of 5-fluorouracil, tegafur, doxylfridine, capecitabine, cytarabine, enocitabine, gemcitabine, bleomycin, BCG vaccine, cisplatin, miriplatin hydrate, carboplatin, nedaplatin, and oxaliplatin.

12. The pharmaceutical composition according to claim 8, which is an anticancer agent for cancer selected from the group consisting of colorectal cancer, pancreatic cancer, lung cancer, esophageal cancer, gastric cancer, upper gastrointestinal cancer, liver cancer, head and neck cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular tumor, kidney cancer, bladder cancer, urothelial tumor, oral cancer, skin malignancies, gastrointestinal stromal tumors (GIST), glioma, brain tumor, osteosarcoma, malignant lymphoma (Hodgkin lymphoma, non-Hodgkin lymphoma), hematological malignancies, chronic myeloid leukemia, multiple myeloma, and malignant melanoma.

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