Inhibitor, therapeutic agent, and prophylactic agent comprising ascopylon p as active ingredient

Abstract

To provide a novel use of Ascopylon P based on a new mechanism.SOLUTION: It has been discovered that Ascopylon P can exhibit a PD-L1 expression inhibitory effect, an endoplasmic reticulum stress inhibitory effect, and a melanin secretion inhibitory effect.SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] The present invention relates to a PD-L1 expression inhibitor containing ascopyrone P as an active ingredient. The present invention also relates to a cancer therapeutic agent containing ascopyrone P as an active ingredient. The present invention also relates to an endoplasmic reticulum stress inhibitor containing ascopyrone P as an active ingredient. The present invention also relates to a melanin secretion inhibitor containing ascopyrone P as an active ingredient. [Background technology]

[0002] Ascopyrone P (APP) is a food ingredient obtained by heating 1,5-anhydro-D-fructose (1,5-AF), which is produced by decomposing starch or glycogen with α-1,4-glucan lyase.

[0003] Ascopyrone P is a substance expected to have various physiological activities. It has been reported that Ascopyrone P has an antioxidant effect 500 times that of 1,5-AF (Non-Patent Document 1). Patent Document 1 discloses an antitumor agent containing Ascopyrone P. Resveratrol, which is contained in red wine, is known as a food ingredient with an antitumor effect. Resveratrol inhibits the glycosylation enzymes α-glucosidase (GAA) in the endoplasmic reticulum, which regulates N-linked glycosylation, and α-ManI (α-mannosidase) in the Golgi apparatus. As a result, PD-L1, an immune checkpoint-related protein, cannot take its original structure, the amount of PD-L1 on the cell membrane decreases, and cytotoxic T cells can easily attack cancer cells (Non-Patent Document 2). Patent Document 2 also discloses an anti-inflammatory agent containing Ascopyrone P.

[0004] However, the effects of Ascopyrone P in suppressing PD-L1 expression, endoplasmic reticulum stress, and melanin secretion are unknown. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 4825413 [Patent Document 2] Patent No. 4778723 [Non-patent literature]

[0006] [Non-Patent Document 1] Journal of Applied Glycoscience, 52(3), 287-291, 2005 [Non-Patent Document 2] Aging (Albany NY), 12(1),8-34, 2020 Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to provide a new use of ascopyrone P based on a new mechanism. [Means for solving the problem]

[0008] As a result of extensive research to solve the above problems, the inventors discovered that Ascopyrone P can suppress PD-L1 expression, endoplasmic reticulum stress, and melanin secretion, and thus completed the present invention.

[0009] That is, the present invention comprises the following. 1. A PD-L1 expression inhibitor containing Ascopyrone P as an active ingredient. 2. A cancer treatment agent containing Ascopyrone P as an active ingredient, A therapeutic agent characterized in that the cancer is immune checkpoint inhibitor resistant cancer and Ascopyrone P suppresses PD-L1 expression on the cell membrane surface. 3. The PD-L1 expression inhibitor according to the preceding item 1 or 2, wherein the inhibition of PD-L1 expression is achieved by inhibiting glycosylation of PD-L1 in cells. 4. The PD-L1 expression inhibitor described in the preceding item 3, wherein the inhibition of glycan modification of PD-L1 is achieved by inhibiting α-glucosidase (GAA) activity in the endoplasmic reticulum. 5. An endoplasmic reticulum stress inhibitor containing ascopyrone P as an active ingredient. 6. The endoplasmic reticulum stress inhibitor described in the preceding item 5, wherein the inhibition of endoplasmic reticulum stress is achieved by inhibiting CHOP expression in macrophages. 7. The endoplasmic reticulum stress inhibitor described in the preceding item 5 or 6, wherein the endoplasmic reticulum stress is suppressed by inhibiting protein disulfide isomerase (PDI) activity. 8. A preventive or therapeutic agent for an endoplasmic reticulum stress-associated disease, comprising the endoplasmic reticulum stress-suppressant according to any one of the preceding items 5 to 7. 9. The preventive or therapeutic agent for an endoplasmic reticulum stress-related disease according to the preceding item 8, wherein the endoplasmic reticulum stress-related disease is any disease selected from the group consisting of cardiac diseases, neurodegenerative diseases and metabolic diseases. 10. A preventive or therapeutic agent for a macrophage-associated disease, comprising the endoplasmic reticulum stress-suppressing agent according to any one of the above items 5 to 7. 11. The preventive or therapeutic agent for a macrophage-associated disease according to the preceding item 10, wherein the macrophage-associated disease is any disease selected from the group consisting of fibrosis, a neurodegenerative disease, a metabolic disease, and a bone-related disease. 12. A melanin secretion inhibitor containing ascopyrone P as an active ingredient. 13. A preventive or therapeutic agent for a melanin-related disease, comprising the melanin secretion inhibitor described in the preceding item 12. 14. The preventive or therapeutic agent for a melanin-related disease according to the preceding item 13, wherein the melanin-related disease is pigmentation disorder. 15. A pharmaceutical or food or beverage comprising a PD-L1 expression inhibitor according to any one of items 1, 3, and 4, an endoplasmic reticulum stress inhibitor according to any one of items 5 to 7, or a melanin secretion inhibitor according to item 12. 16. A preventive or therapeutic agent for a PDI-related disease, comprising the endoplasmic reticulum stress inhibitor according to any one of items 5 to 7 above. 17. The preventive or therapeutic agent for a PDI-related disease according to the preceding item 16, wherein the PDI-related disease is any disease selected from the group consisting of thrombosis, platelet aggregation, viral infection, neurodegenerative disease, cardiovascular disease, inflammatory bowel disease, metabolic disease and bone-related disease. Effect of the Invention

[0010] The PD-L1 expression inhibitor containing the ascopyrone P of the present invention as an active ingredient can inhibit PD-L1 expression on the cell membrane surface. The endoplasmic reticulum stress inhibitor containing the ascopyrone P of the present invention as an active ingredient can inhibit endoplasmic reticulum stress. The melanin secretion inhibitor containing the ascopyrone P of the present invention as an active ingredient can inhibit melanin secretion. [Brief description of the drawings]

[0011] [Figure 1] Example 1 shows the results of confirming the effect of APP stimulation on cell survival in human melanoma cell line A375 cells. [Diagram 2] The expression of PD-L1 in human melanoma cell line A375 cells after stimulation with APP was confirmed by Western blotting, and a graph was created by quantifying the detection intensity of PD-L1 relative to β-actin (Example 1). [Diagram 3] FIG. 1 shows the results of Western blotting to confirm the expression of PD-L1 in A375 cells and HeLa cells after stimulation with 1.5AF, metformin, or APP (Example 1). [Figure 4] The figure shows the results of confirming the amount of PD-L1 in A375 cells after APP stimulation using an immunostaining method. The images in the figure are fluorescent micrographs (magnification 20x). "Merge" in the figure shows a composite image of a PD-L1 stained image and a Hoechst nuclear stained image. The graph shows the graphed results of PD-L1 counts relative to the nucleus (PD-L1 / nucleus (%)) (*P<0.05). (Example 1) [Diagram 5] The change in PD-L1 localization after APP stimulation of A375 cells was confirmed using immunostaining (a composite image of a PD-L1 stained image of a single nucleus and its surroundings photographed with a high-magnification lens (magnification 100x) and a nuclear stained image using Hoechst). The arrowheads in the figure indicate the fluorescence indicating the localization of PD-L1. (Example 1) [Figure 6]The effect of APP stimulation on PD-L1 glycosylation in A375 cells was examined using Western blotting, and a graph was created by quantifying the detection intensity of PD-L1 against β-actin (*P<0.05, ****P<0.0001) (Example 1). [Figure 7] Figure 7A shows the results of Western blot of cell surface proteins (PD-L1 and Na,K-ATPase) and cytoplasmic proteins (β-actin) in A375 cells stimulated with or without APP. Figure 7B shows a graph prepared by quantifying the detection intensity of PD-L1 relative to β-actin (**P<0.01). Figure 7C shows a graph prepared by quantifying the detection intensity of Na,K-ATPase relative to β-actin. Figure 7D shows the average fluorescence intensity of PD-L1 expression on the cell membrane surface by flow cytometry of A375 cells stimulated with or without APP (**P<0.01). (Example 1) [Figure 8] The results of confirming the inhibitory effects of 1,5-AF, APP, or NAC on α-glucosidase (GAA) activity (left panel) and glucose oxidase activity (right panel) using maltose as a substrate are shown (*P<0.05, **P<0.01) (Example 1). [Figure 9] A conceptual diagram of the principle of glucose concentration measurement using an α-glucosidase (GAA) activity inhibition measurement kit is shown. The glucose concentration in a sample can be determined by measuring the red pigment. (Example 1) [Figure 10]FIG. 10A shows the results of confirming the effect of APP and DTT stimulation on CHOP expression in macrophages (THP-1 cell-derived macrophages) after addition of PMA to human monocytic leukemia cell line THP-1 cells using Western blotting (upper row of FIG. 10A) and a graph (lower row of FIG. 10A) showing statistical processing of the detection intensity (*P<0.05, **P<0.01). FIG. 10B shows the results of confirming whether DTT and APP stimulation affect the survival of THP-1 cell-derived macrophages. FIG. 10C shows the results of observing the effect of APP and DTT stimulation on CHOP expression in THP-1 cell-derived macrophages using a fluorescent microscope. FIG. 10D shows a graph showing statistical processing of the detection intensity of Western blotting for the effect of APP and DTT stimulation on CHOP expression in THP-1 cell-derived macrophages (***P<0.0006, ****P<0.0001). (Example 2) [Figure 11] The effect of DTT stimulation on CHOP expression in THP-1 cell-derived macrophages after stimulation with APP or NAC was examined using a Western blot method, and a graph showing statistical analysis of the detection intensity is shown (**P<0.01) (Example 2). [Figure 12] 12 shows the results of measuring the inhibition of the enzyme activity of PDI of APP using the PDI Assay Kit (****P<0.0001). The vertical axis shows the excitation wavelength of 500 nm / fluorescence filter of 603 nm. (Example 2) [Figure 13] FIG. 13A shows a conceptual diagram of the measurement of the melanin secretion inhibitory effect of APP or arbutin (Arb) using B16 melanoma cells. FIG. 13B shows the results of photographing B16 melanoma cells after stimulation with APP or arbutin (Arb). FIG. 13C shows a graph of statistically processed results of absorbance measurement at 405 nm after lysing B16 melanoma cells stimulated with APP or arbutin (Arb) (*P<0.05, **P<0.01, ****P<0.0001). FIG. 13D shows the results of confirming the effect of APP or arbutin (Arb) stimulation of B16 melanoma cells on cell survival using the live cell measurement reagent SF (WST-8). (Example 3) [Figure 14] The effect of APP or arbutin (Arb) stimulation on the expression of tyrosinase in B16 melanoma cells was examined using a Western blot method, and a graph showing statistical analysis of the detection intensity is shown (Example 3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention relates to a PD-L1 expression inhibitor containing ascopyrone P as an active ingredient. The present invention also relates to a cancer therapeutic agent containing ascopyrone P as an active ingredient, characterized in that the cancer is immune checkpoint inhibitor-resistant cancer and ascopyrone P inhibits PD-L1 expression on the cell membrane surface.

[0013] Ascopyrone P is a substance obtained by heating 1,5-anhydro-D-fructose (1,5-AF), a starch hydrolysate, and is known to have antitumor activity, but the mechanism is unclear. Cancer cells avoid attacks by the immune system by using immune checkpoint molecules that suppress autoimmune responses. Immune checkpoint inhibitors, which are antibody drugs that target immune checkpoint molecules and their ligands, are used clinically in cancer treatment. Programmed cell death ligand-1 (PD-L1) is a ligand for the immune checkpoint molecule PD-1 and is expressed on the surface of cancer cells. Anti-PD-1 antibodies and anti-PD-L1 antibodies are used as immune checkpoint inhibitors. However, treatment resistance caused by cancer cells that have acquired resistance to these immune checkpoint inhibitors has become a problem in recent years.

[0014] In the present invention, it has been confirmed by the examples described later that Ascopyrone P has the effect of increasing the amount of PD-L1 in cells while suppressing the expression of PD-L1 on the cell membrane surface. If the expression of PD-L1 on the surface of cancer cells can be suppressed, the immunosuppressive function of immune checkpoints can be inhibited by a mechanism different from that of immune checkpoint inhibitors such as anti-PD-L1 antibodies, which inhibit the function of PD-L1 expressed on the surface of cancer cells by binding to it, and therefore a therapeutic effect on cancers resistant to immune checkpoint inhibitors can be expected.

[0015] Cancers to which the cancer therapeutic agents of the present invention can be applied include any cancer that has resistance to immune checkpoint inhibitors, and the type of cancer is not particularly limited, but examples include blood cancer, cervical cancer, skin cancer (e.g., melanoma, etc.), glioblastoma, head and neck cancer, esophageal cancer, lung cancer, kidney cancer, colon cancer, pancreatic cancer, liver cancer, gastric cancer, hepatic cancer, neuroblastoma, brain tumor, breast cancer, ovarian cancer, testicular cancer, and prostate cancer.

[0016] In addition, in the Examples described below, it was confirmed that Ascopyrone P has the effect of suppressing the activity of α-glucosidase (GAA) and the effect of inhibiting the glycosylation of PD-L1. Ascopyrone P inhibits the activity of α-glucosidase (GAA), which is involved in glycosylation in cancer cells, thereby inhibiting the glycosylation of PD-L1. The incomplete PD-L1 produced as a result is not transported to the cell membrane surface, which is thought to suppress the expression of PD-L1 on the cell membrane surface. Ascopyrone P inhibits the activity of α-glucosidase (GAA), suppressing the breakdown of carbohydrates and slowing their digestion and absorption, thereby slowing the rise in blood sugar levels after meals.

[0017] The present invention relates to an endoplasmic reticulum stress inhibitor containing ascopyrone P as an active ingredient. The present invention also relates to a preventive or therapeutic agent for endoplasmic reticulum stress-related diseases, which contains the endoplasmic reticulum stress inhibitor. The present invention also relates to a preventive or therapeutic agent for macrophage-related diseases, which contains the endoplasmic reticulum stress inhibitor. The present invention also relates to a preventive or therapeutic agent for PDI-related diseases, which contains the endoplasmic reticulum stress inhibitor.

[0018] Protein folding and glycosylation take place in the endoplasmic reticulum (ER). The accumulation of improperly folded or improperly modified proteins in the ER is called ER stress. When ER stress occurs, an unfolded protein response (UPR) occurs, protecting the cell. However, when ER stress is severe, the transcription factor CCAAT / enhancer-binding protein-homologous protein (CHOP) is expressed. When CHOP is expressed in macrophages due to ER stress, CHOP promotes differentiation of M0 macrophages into M2 macrophages and their activation. Proteins that are not folded correctly even by the stress signaling cascades stimulated by the UPR and aimed at correcting folding are transported to the cytosol by a pathway called endoplasmic reticulum (ER)-associated degradation (ERAD), which involves protein disulfide isomerase (PDI), and degraded by the proteasome. Viruses use the ERAD pathway to invade the cytosol and cause infection by translocating core virus particles generated by degradation reactions in the cytosol into the nucleus (Nature Reviews Microbiology, 2016, 14.7:407-420). It has been reported that coronaviruses generate double-membrane vesicles (DMVs) by protruding and budding flat sacs in the endoplasmic reticulum of host cells (Frontiers in Cell and Developmental Biology, 2021, 9: 630242). PDI is used to generate double-membrane vesicles. PDI has also been found to play a role in the rapid proliferation of cancer cells, which requires high protein synthesis (Cell & Bioscience, 2022, 12.1: 1-19).In the examples described below, the present invention has induced endoplasmic reticulum stress in macrophages by inhibiting protein folding using a reducing agent, dithiothreitol (DTT), and has clarified that ascopyrone P suppresses the expression of CHOP, an endoplasmic reticulum stress marker, in macrophages under endoplasmic reticulum stress in a reducing state different from known antioxidant effects, and has confirmed that ascopyrone P has an endoplasmic reticulum stress inhibitory effect and further inhibits the activity of PDI. PDI is involved in protein folding in the endoplasmic reticulum and functions in an oxidative environment maintained in the endoplasmic reticulum. When the environment in the endoplasmic reticulum changes, PDI promotes the production and accumulation of defective proteins that have not been folded correctly. Ascopyrone P inhibits PDI activity, thereby suppressing the accumulation of defective proteins and suppressing endoplasmic reticulum stress, and as a result, it is believed that the expression of CHOP is suppressed. In addition, ascopyrone P is believed to suppress macrophage activity by suppressing the expression of CHOP, which promotes the differentiation induction and activation of macrophages. Based on these findings, ascopyrone P is expected to have therapeutic and preventive effects against endoplasmic reticulum stress-related diseases, macrophage-related diseases, and PDI-related diseases.

[0019] Examples of endoplasmic reticulum stress-related diseases include heart diseases (e.g., ischemic heart disease, arrhythmia, valvular heart disease, heart failure, thrombosis, etc.), neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, cerebral ischemia, cystic fibrosis, Huntington's disease, spinocerebellar ataxia, etc.), metabolic diseases (e.g., diabetes, hyperglycemia, hyperinsulinemia, fatty liver, metabolic syndrome, lifestyle-related diseases, etc.), cancer (e.g., blood cancer, cervical cancer, skin cancer (e.g., melanoma, etc.), glioblastoma, head and neck cancer, esophageal cancer, lung cancer, kidney cancer, colon cancer, pancreatic cancer, liver cancer, gastric cancer, liver cancer, neuroblastoma, brain tumor, breast cancer, ovarian cancer, testicular cancer, prostate cancer, etc.), etc.

[0020] Examples of macrophage-related diseases include fibrosis (e.g., pulmonary fibrosis, hepatic fibrosis, renal fibrosis, myocardial fibrosis, skin fibrosis, etc.), cancer (e.g., blood cancer, cervical cancer, skin cancer (e.g., melanoma, etc.), glioblastoma, head and neck cancer, esophageal cancer, lung cancer, kidney cancer, colon cancer, pancreatic cancer, liver cancer, gastric cancer, liver cancer, neuroblastoma, brain tumor, breast cancer, ovarian cancer, testicular cancer, prostate cancer, etc.), neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, cerebral ischemia, cystic fibrosis, Huntington's disease, spinocerebellar ataxia, etc.), metabolic diseases (e.g., diabetes, hyperglycemia, hyperinsulinemia, fatty liver, metabolic syndrome, lifestyle-related diseases, etc.), and bone-related diseases (e.g., osteoporosis, osteoarthritis, rheumatoid arthritis, hypercalcemia, achondroplasia, hypochondroplasia, epiphyseal dysplasia, fractures, etc.).

[0021] Examples of PDI-related diseases include thrombosis, cancer (e.g., blood cancer, cervical cancer, skin cancer (e.g., melanoma, etc.), glioblastoma, head and neck cancer, esophageal cancer, lung cancer, kidney cancer, colon cancer, pancreatic cancer, liver cancer, gastric cancer, hepatic cancer, neuroblastoma, brain tumors, breast cancer, ovarian cancer, testicular cancer, prostate cancer, etc.), platelet aggregation, viruses (e.g., retroviruses (e.g., human immunodeficiency virus (HIV) and human T-lymphotropic viruses, etc.), coronaviruses (e.g., SARS coronavirus 2 (COVID-19), MERS coronavirus, etc.), polyomaviruses (e.g., JC virus, BK virus, etc.), flaviviruses, and the like). Examples of the diseases that may be caused by the following include infections with viruses (e.g., hepatitis C virus, Japanese encephalitis virus, dengue virus, etc.), reovirus (e.g., rotavirus, Colorado tick fever virus, etc.), neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, cerebral ischemia, cystic fibrosis, Huntington's disease, spinocerebellar ataxia, etc.), cardiovascular disease, inflammatory bowel disease, metabolic diseases (e.g., diabetes, hyperglycemia, hyperinsulinemia, fatty liver, metabolic syndrome, lifestyle-related diseases, etc.), and bone-related diseases (e.g., osteoporosis, osteoarthritis, rheumatoid arthritis, hypercalcemia, achondroplasia, hypochondroplasia, epiphyseal dysplasia, fractures, etc.).

[0022] The present invention relates to a melanin secretion inhibitor containing as an active ingredient ascopyrone P. The present invention also relates to a preventive or therapeutic agent for melanin-related diseases, which contains the melanin secretion inhibitor.

[0023] Ascopyrone P is known to suppress the activity of tyrosinase, but the mechanism is unknown. Melanin plays a role in preventing the development of malignant tumors caused by ultraviolet rays. On the other hand, if the turnover cycle in which cells are regenerated is disturbed, the tyrosine in melanocytes is activated by tyrosinase to become melanin, which causes spots. In order to prevent spots, it is necessary to suppress the production of melanin. In the examples described below, the present invention confirmed that Ascopyrone P does not suppress the expression of tyrosinase, but has the effect of suppressing the secretion of melanin outside the cells. As a result, Ascopyrone P can be expected to have therapeutic and preventive effects on melanin-related diseases.

[0024] Examples of melanin-related diseases include pigmentation (eg, age spots, freckles, sunburn, etc.) and skin cancer (eg, melanoma, etc.).

[0025] The PD-L1 expression inhibitor, cancer treatment agent, endoplasmic reticulum stress inhibitor, preventive or therapeutic agent for endoplasmic reticulum stress-related disease, preventive or therapeutic agent for macrophage-related disease, preventive or therapeutic agent for PDI-related disease, melanin secretion inhibitor, and preventive or therapeutic agent for melanin-related disease of the present invention (hereinafter, these may be collectively referred to as "various agents of the present invention") can be administered by known methods or any methods to be developed in the future, and the dosage can be appropriately set in consideration of the age, sex, weight, symptoms, number of administrations, frequency of administration, administration time, and use in combination with other drugs or treatments. For example, when orally administered to adults, the daily dosage is 1 to 500 mg / kg body weight, preferably 10 to 100 mg / kg body weight, calculated as the active ingredient, ascopyrone P.

[0026] The route of administration of the various agents of the present invention is not particularly limited, and they can be administered orally or parenterally. Examples of parenteral administration include intravenous, subcutaneous, or intraperitoneal injection, drip infusion, and application.

[0027] The form of the various agents of the present invention is not particularly limited, and they can be formulated into, for example, tablets, granules, fine granules, powders, syrups, solutions, capsules, suspensions, sprays, injections, drops, inhalants, suppositories, transdermal agents, transmucosal agents, etc. By making them into such forms, they can be used, for example, as medicines, foods and beverages, quasi-drugs, cosmetics, or raw materials thereof.

[0028] The form of the food or drink is not particularly limited, and examples thereof include beverages, bread, dairy products, meat products, baked goods, candies, jellies, gums, retort foods, instant foods, frozen foods, food additives, seasonings, etc. In addition, the various agents of the present invention can be added to foods to take the form of specified health foods, nutritional functional foods, foods for the elderly, foods for special uses, functional foods, dietary supplements, etc., for the purpose of preventing or treating symptoms in various diseases described in this specification, and examples of the form of such foods include granules, powders, tablets, capsules, chewables, drinks, jellies, pastes, grains, etc.

[0029] The method for producing the various agents of the present invention is not particularly limited, and they can be produced by any known method or any method to be developed in the future. For the production of liquid preparations such as syrups, solutions, liquid preparations, injections, and drips, additives for preparations such as water, sugars such as sucrose, sorbitol, and fructose; glycols such as polyethylene glycol and propylene glycol; oils such as sesame oil, olive oil, and soybean oil; and preservatives such as p-hydroxybenzoic acid esters can be used. For the production of solid preparations such as capsules, tablets, powders, and granules, excipients such as lactose, glucose, sucrose, and mannitol; disintegrants such as starch and sodium alginate; lubricants such as magnesium stearate and talc; binders such as polyvinyl alcohol, hydroxypropyl cellulose, and gelatin; surfactants such as fatty acid esters; and plasticizers such as glycerin can be used. EXAMPLES

[0030] EXAMPLES In the following, the present invention will be specifically described with reference to examples and comparative examples in order to deepen understanding of the present invention. However, it goes without saying that these do not limit the scope of the present invention.

[0031] (Example 1) Inhibitory effect of APP on PD-LI synthesis

[0032] 1. The effect of APP on cell survival and PD-L1 expression was investigated. APP (final concentrations: 0 μM, 125 μM, 250 μM, 500 μM, 1000 μM) was added to human melanoma cell line A375 cells and stimulated for 20 hours. Then, the effect of APP stimulation on cell survival was confirmed using WST-8 (Nacalai Tesque, 07553-44). In addition, after recovering the cells, the expression of PD-L1 was confirmed using Western blotting. β-actin was used as a loading control. As a result, no significant difference was observed in cell survival confirmation at all concentrations compared to the control (Figure 1). In addition, the expression of PD-L1 with different mobility was observed by Western blotting, and it was confirmed that PD-L1 (Lower), which has a high mobility and was hardly detected in the control, increased in an APP stimulation concentration-dependent manner. The results showed that PD-L1 with different mobilities was quantified and graphed, and that a significant difference was observed from APP 125 μM compared to the control, and that the proportion of lower PD-L1 increased in a concentration-dependent manner (Figure 2). Therefore, it was found that APP does not affect cell survival, but increases the expression of PD-L1 with different mobilities in an APP concentration-dependent manner.

[0033] 2. A375 cells and HeLa cells 2x10 5 Each well was cultured in FBS(-)DMEM and stimulated with 1.5AF, metformin or APP (1.25 mM, 2.5 mM, 5.0 mM), and PD-L1 expression was confirmed by Western blotting. Metformin is a known PD-L1 expression inhibitor. In addition, the effects of the protein synthesis inhibitor cycloheximide (CHX) and the antioxidant N-Acety1 Cysteine ​​(NAC) were examined. As a result, only APP changed the mobility of PD-L1 (Figure 3A-C), and no change in mobility was observed with CHX treatment (Figure 3B), and it was not affected by antioxidants (Figure 3C). Therefore, it was found that the difference in the mobility of PD-L1 in APP acts on the glycosylation process, not during translation.

[0034] 3. APP (final concentrations 0 and 500 μM) was added to A375 cells and stimulated for 20 hours. After that, the localization of PD-L1 was examined using immunostaining. IgG was used as an isotype control. As a result, APP stimulation was confirmed to significantly increase the accumulation of PD-L1 in cells compared to the PD-L1 control (Figure 4).

[0035] 4. Using the same experimental method as in 3 above, but with a higher magnification lens, we confirmed changes in the localization of PD-L1. As a result, in the control group, PD-L1 was observed near the cell membrane, but after APP stimulation, PD-L1 accumulation was observed around the nucleus, i.e., in the endoplasmic reticulum (Figure 5).

[0036] 5. The same experiment was performed up to the collection of 1. above. The samples were then heat denatured at 100°C for 10 minutes and deglycosylated using PNGase F and Endo H, respectively. PD-L1 was then detected by Western blotting. As a result, in the case of PNGase F treatment, similar amounts of PD-L1 were detected around 35 kDa in both the unstimulated and APP (final concentration 500 μM) stimulated cases. However, in the case of Endo H treatment, PD-L1 with a high mobility shifted to 35 kDa when stimulated with APP. In other words, incomplete PD-L1 in the glycosylation process was detected (Figure 6). The results of Figures 5 and 6 indicate that APP inhibits the enzymatic activity of glycosylation in the endoplasmic reticulum, which performs post-translational modification, thereby expressing incomplete PD-L1.

[0037] 6. After the stimulation described in 1 above, cell surface proteins were collected using a Cell Surface Isolation kit (Funakoshi, K295-10) and detected by Western blotting. β-actin in the cytoplasm collected in the same experiment was used as a loading control. As a result, no significant difference was observed in Na,K-ATPase when comparing the control and stimulation with APP (final concentration 500 μM). In addition, a significant difference was observed in PD-L1 when stimulated with APP compared to the control (Figure 7A-C). The reason why no significant difference was observed in Na,K-ATPase is due to the difference in molecular weight with PD-L1. PD-L1 is 45 kDa, while Na,K-ATPase is about twice as large, 110 kDa. Protein expression and maturation process require time depending on the size of the molecular weight. This suggests that no significant difference was observed in Na,K-ATPase. Therefore, it was shown that APP significantly suppresses PD-L1 expression on the cell membrane surface.

[0038] 7. After the stimulation described above in 1, the cells were transferred to a test tube using Cell Dissociation Solution (non-enzymatic) (BLG 03-071-1B). Next, the cells were fixed using optiLyseC (Beckman Coulter, A11895). Then, 1% BSA solution was added and the cells were left to stand. After washing, PerCP / Cyanine5.5 anti-human CD274 (B7-H1, PD-L1) Antibody (BioLegend 329738) or PerCP / Cyanine5.5 Mouse IgG2b, κ Isotype Ctrl Antibody (BioLegend 400338) was added. Then, the expression of PD-L1 was confirmed by flow cytometry (RF500, Sysmex). The results showed that APP stimulation significantly suppressed PD-L1 expression (mean fluorescence intensity) on the cell membrane surface (Figure 7D).

[0039] 8. The α-glucosidase (GAA) inhibitory activity of each reagent (1,5-AF, APP, and NAC, each at a final concentration of 500 μM) was measured using the α-glucosidase (GAA) activity inhibition measurement kit (Wako, 297-78601) and Labo Assay (trademark) Glucose (Wako, 638-50971). The disaccharide maltose was used as the substrate. As a result, the antioxidants APP and NAC inhibited α-glucosidase (GAA) activity without inhibiting GOD (glucose oxidase) activity, which reacts when measuring glucose concentration (Figure 8). These results revealed that APP inhibits the enzymatic activity of α-glucosidase (GAA), which is involved in post-translational modification in the endoplasmic reticulum (Figure 9).

[0040] (Example 2) Inhibitory effect of APP on endoplasmic reticulum stress The effect of APP on endoplasmic reticulum stress was examined by inducing endoplasmic reticulum stress using the reducing agent dithiothreitol (DTT).

[0041] CHOP expression suppression The cellular expression of CCAAT / enhancer-binding protein-homologous protein (CHOP) was confirmed by Western blot and immunostaining. THP-1 cells were cultured in a 6-well plate at 2 × 10 6 The cells were seeded at 100x the concentration of 100x the number of cells / we11 and differentiated into macrophages by stimulation with PMA (50 nM) for 48 hours. The medium was replaced with Opti-MEM, and the cells were stimulated with APP (0.5 mg / mL) for 1 hour and DTT (0.7 mM) for 3 hours, and then harvested. After sonication, protein quantification was performed, and CHOP expression was confirmed by Western blotting using β-actin as a loading control. Next, 24-well glass bottom plates were coated with ε-poly-L-lysine, and 2x10 human monocytic leukemia cell line THP-1 cells were cultured. 5Cells were seeded per well and differentiated into macrophages by stimulation with PMA (50 nM) for 48 hours. The medium was replaced with Opti-MEM, and the cells were stimulated with APP (100 μg / mL, 300 μg / mL, 500 μg / mL) for 1 hour, and then with DTT (0.7 mM) for 3 hours. The control was treated with solvent alone. The cells were harvested, sonicated, and CHOP was detected by Western blotting. The localization and expression of CHOP were confirmed by cell immunostaining using anti-CHOP antibody and Hoechst 33342 for nuclear staining. CHOP and nuclei were confirmed under a fluorescent microscope at 20x magnification, and the expression rate of CHOP was calculated by cell counting.

[0042] As a result of Western blotting, the expression of CHOP in the DTT-stimulated sample decreased in the amount of CHOP detected depending on the concentration of APP (Figure 10A, upper row, CHOP). β-actin was used as a loading control (Figure 10A, upper row, β-actin). This result was also supported by a graph in which the detection intensity was measured using Image J and statistical processing was performed using one-way Anova (Figure 10A, lower row). At the same time, cell viability was measured and statistical processing was performed for the 500 μg / mL APP-added sample, and there was no significant difference between the APP-free sample and the APP-free sample, and there was no effect on cell viability (Figure 10B). As a result of fluorescent immunostaining, when 500 μg / mL APP was added in addition to DTT, the fluorescence intensity indicating CHOP expression decreased compared to DTT stimulation alone (Figure 10C). Regarding the expression rate of CHOP calculated by cell counting, the addition of 500 μg / mL APP significantly suppressed CHOP expression, which was expressed in about 70% of cells with DTT stimulation alone, to about 30% (Figure 10D). Therefore, it was demonstrated that APP suppresses the expression of CHOP induced by DTT stimulation.

[0043] Antioxidant effects We investigated whether the suppression of CHOP expression by APP was due to the antioxidant effect of APP by using the antioxidant NAC. 6Cells / we11 were seeded and differentiated into macrophages by stimulation with PMA (50 nM) for 48 hours. The medium was replaced with Opti-MEM, and cells were stimulated with APP (0.5 mg / mL) or the antioxidant NAC (3.5 mM) for 1 hour, and then stimulated with DTT (0.7 mM) for 3 hours, and then harvested. After sonication, protein quantification was performed, and CHOP expression was confirmed by Western blotting using β-actin as a loading control.

[0044] As a result, NAC did not suppress the expression of CHOP by DTT stimulation, and this result was supported by a graph in which the detection intensity was measured using Image J and statistical processing was performed using one-way anova (Figure 11). Therefore, it was found that the suppression of CHOP expression by APP is not due to its antioxidant effect.

[0045] PDI enzyme activity inhibition DTT acts on protein disulfide isomerase (PDI) in the endoplasmic reticulum and induces endoplasmic reticulum stress. Therefore, we investigated whether APP inhibits the enzymatic activity of PDI. Using a PDI Assay Kit (Cosmo Bio Co., Ltd., ENZ-51024-KP050), we measured the inhibition of PDI enzymatic activity of APP. 50 μL of 10 mM insulin, 10 μL of PDI, and 10 μL of APP (final concentration 5 mg / mL) were added to a 96-well plate. Next, the volume was adjusted to 90 μL using PBE buffer. Then, the plate was left to stand at room temperature for 30 minutes in the dark. 10 μL each of PROTEOSTAT (trademark) and STOP solution were added, and the plate was left to stand for 15 minutes in the dark at room temperature. Insulin aggregation was quantified using a fluorometer (500 nm / 603 nm).

[0046] As a result, PDI aggregated insulin with a significant difference, and APP was found to suppress the insulin-aggregating activity of PDI with a significant difference (Figure 12). Therefore, it was found that APP suppresses PDI enzyme activity.

[0047] (Example 3) Inhibitory effect of APP on melanin pigment secretion The inhibitory effect of APP and the positive control arbutin (Arb) on melanin secretion was examined (FIG. 13A).

[0048] Detection of melanin in culture supernatant B16 melanoma cells (1.0 × 10 6 Six mice (6 wells / dish, 6 wells plates) were stimulated with APP or Arb at 0 to 100 μM, and the culture supernatant was collected 24 hours later and photographed (FIG. 13B).

[0049] Melanin quantification The culture supernatant in the above plate was subjected to absorbance measurement at 405 nm and converted into numerical values, and graphs were created and statistical processing was performed using Prism8 (FIG. 13C).

[0050] As a result, there was a significant difference between APP at 50 μM and 100 μM and the negative control without addition of Arb at 25-100 μM, but there was no significant difference between Arb at 25-100 μM and the negative control (Figure 13B, C). When comparing APP and Arb at the same concentration (50 μM and 100 μM), APP significantly suppressed melanin secretion.

[0051] cell survival To confirm whether the melanin secretion inhibitory effect of APP affects cell viability, WST-8 was used. The plate was washed three times with 500 μL, and 400 μL of medium and 40 μL of viable cell measurement reagent were added, and measurements were performed at 450 nm.

[0052] As a result, there was no significant difference between APP and Arb and the control (Figure 13D), confirming that APP does not affect cell survival.

[0053] Detection of intracellular tyrosinase protein To confirm whether APP suppresses the expression of tyrosinase, Western blotting was performed using tyrosinase, β-actin (loading control), and tyrosinase as primary antibodies. RIPA was added to the above plate in 200 μL each, and the plate was collected with a scraper and stored in a 1.5 mL tube. The plate was sonicated and centrifuged at 15,000 rpm, 4°C, for 2 minutes to collect the cells. Protein quantification was then performed, and the sample was diluted 1:1 with 2×SB and stored at -80°C. The collected sample (2 μg) was subjected to 10% SDS-PAGE electrophoresis (25 mA, 70 minutes). After electrophoresis, the gel was transferred (200 mA, 120 minutes). The transferred membrane was stained with Ponceau S to confirm the transfer. After blocking for 1 hour, primary antibodies (anti-mouse tyrosinase antibody, β-actin) were added and rotated at 4°C overnight. After removing and washing the primary antibody, the secondary antibody was added and washed after 1 hour. Tyrosinase and actin were photographed using a luminescence reagent. The amount of the target protein was measured using Image J and statistical processing was performed using one-way Anova.

[0054] As a result, no significant difference was observed between APP and Arb, indicating that APP does not suppress the expression of tyrosinase (Figure 14). [Industrial Applicability]

[0055] As described above in detail, various agents containing the ascopyrone P of the present invention as an active ingredient have the effect of inhibiting PD-L1 expression on the cell membrane surface, the effect of inhibiting endoplasmic reticulum stress, and / or the effect of inhibiting melanin secretion, and can provide new uses for ascopyrone P in the fields of pharmaceuticals, food and beverages, etc.

Claims

1. A PD-L1 expression inhibitor containing ascopirone P as the active ingredient.

2. A cancer treatment agent containing ascopirone P as an active ingredient, A therapeutic agent characterized in that the cancer is an immune checkpoint inhibitor resistant cancer and Ascopyrone P suppresses PD-L1 expression on the cell membrane surface.

3. The PD-L1 expression inhibitor according to claim 1 , wherein the inhibition of PD-L1 expression is achieved by inhibiting glycosylation of PD-L1 in cells.

4. The PD-L1 expression inhibitor according to claim 3, wherein the inhibition of glycosylation of PD-L1 is achieved by inhibiting α-glucosidase (GAA) activity in the endoplasmic reticulum.

5. An endoplasmic reticulum stress inhibitor containing ascopirone P as an active ingredient.

6. The endoplasmic reticulum stress inhibitor according to claim 5, wherein the endoplasmic reticulum stress is inhibited by inhibiting CHOP expression in macrophages.

7. The endoplasmic reticulum stress inhibitor according to claim 5 , wherein the endoplasmic reticulum stress is suppressed by inhibiting protein disulfide isomerase (PDI) activity.

8. A preventive or therapeutic agent for an endoplasmic reticulum stress-related disease, comprising the endoplasmic reticulum stress inhibitor according to any one of claims 5 to 7.

9. The preventive or therapeutic agent for an endoplasmic reticulum stress-related disease according to claim 8, wherein the endoplasmic reticulum stress-related disease is any disease selected from the group consisting of heart disease, neurodegenerative disease and metabolic disease.

10. A preventive or therapeutic agent for a macrophage-related disease, comprising the endoplasmic reticulum stress inhibitor according to any one of claims 5 to 7.

11. The preventive or therapeutic agent for a macrophage-associated disease according to claim 10, wherein the macrophage-associated disease is any disease selected from the group consisting of fibrosis, a neurodegenerative disease, a metabolic disease, and a bone-related disease.

12. A melanin secretion inhibitor containing ascopirone P as an active ingredient.

13. A preventive or therapeutic agent for a melanin-related disease, comprising the melanin secretion inhibitor according to claim 12.

14. The preventive or therapeutic agent for a melanin-related disease according to claim 13, wherein the melanin-related disease is pigmentation disorder.

15. A medicine or a food or drink comprising a PD-L1 expression inhibitor according to any one of claims 1, 3 and 4, an endoplasmic reticulum stress inhibitor according to any one of claims 5 to 7, or a melanin secretion inhibitor according to claim 12.

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