Novel molecular chaperone mediated autophagy activator

Abstract

The invention discloses a novel molecular chaperone-mediated autophagy (CMA) activating agent, relates to the technical field of biological cells, and is characterized in that the novel molecular chaperone-mediated autophagy activating agent is a novel anti-tumor drug chidamide, and the novel molecular chaperone-mediated autophagy activating agent is a novel anti-tumor drug chidamide. The chidamide can obviously up-regulate the mRNA and protein level of the key protein LAMP2A in the CMA process. According to the present invention, by constructing a human THP1-LAMP2luciferase reporter gene system, the compound capable of activating the LAMP2 promoter is screened from the bulk drug; through cell level primary screening, secondary screening and animal level verification, an effective CMA activator chidamide is finally determined. A good tool is provided for basic research of CMA, and novel drugs are further researched and developed by taking CMA as a target spot.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological cells, and more particularly to a novel activator of chaperone-mediated autophagy. BACKGROUND

[0002] Autophagy is a catabolic process in which cells degrade organelles, foreign substances and aggregated denatured proteins in lysosomes. Cells can clear damaged organelles, foreign substances and aggregated denatured proteins through autophagy, and can produce new substances to provide energy for cells, so that cells can adapt to various stress environments. According to the different ways of transporting degradation substrates to lysosomes, autophagy can be divided into three forms: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). The three autophagy processes maintain the homeostasis of the cell.

[0003] CMA is a highly selective autophagy type. CMA is quite different from traditional autophagy (macroautophagy). In the CMA process, the degradation substrate is recognized and combined with heat shock protein 70 (HSC70) to form a substrate-HSC70 complex, which is transported to the lysosome and combined with lysosome-associated membrane protein 2A (LAMP2A) on the lysosome membrane. The substrate enters the lysosome through the pore formed by the polymerization of LAMP2A and is degraded. LAMP2A is a rate-limiting component of the CMA process, and its content on the lysosome membrane directly determines the CMA activity. CMA has strict selectivity, and its substrate is a protein with a specific tag (pentapeptide KFERQ) in the cytoplasm. In recent years, research has found that CMA participates in many physiological processes such as cell energy metabolism, cell proliferation, DNA repair, antigen presentation, etc. by precisely regulating the degradation of many key proteins in the life process of cells. Defects in CMA function are closely related to neurodegenerative diseases, aging, tumors, cardiovascular diseases, metabolic diseases and immune diseases.

[0004] However, the CMA regulation mechanism has not been fully elucidated, and there is a lack of effective CMA activators. In the prior art, the expression of LAMP2A is mainly up-regulated by gene means to activate CMA, and this method has defects such as technical complexity and limited clinical application. Therefore, it is of great scientific significance and application value to develop small molecule CMA activators. SUMMARY

[0005] The object of the present application is to provide a new chaperone-mediated autophagy activator to solve the above technical problems. By constructing a human THP1-LAMP2 luciferase reporter gene system, a compound capable of activating the LAMP2 promoter is screened from the raw drug. After cell level primary screening, rescreening and animal level verification, an effective CMA activator is finally determined.

[0006] The above technical object of the present application is achieved by the following technical solution: a new chaperone-mediated autophagy activator, wherein the active ingredient of the chaperone-mediated autophagy activator is the histone deacetylase inhibitor chidamide.

[0007] The present application further provides the application of chidamide as a new chaperone-mediated autophagy activator. Chidamide can significantly up-regulate the mRNA and protein levels of LAMP2A.

[0008] In summary, the present application has the following beneficial effects: by constructing a human THP1-LAMP2 luciferase reporter gene system, a compound capable of activating the LAMP2 promoter is screened from the raw drug. After cell level primary screening, rescreening and animal level verification, an effective CMA activator chidamide is finally determined. The compound can specifically up-regulate the expression of LAMP2A without affecting other subtypes such as LAMP2B, and has a significant CMA activation effect. It provides a good tool for the basic research of CMA, and further develops a new drug targeting CMA. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 is a schematic diagram of vector construction in the embodiment of the present application;

[0010] Figure 2 is a compound screening by means of the above-mentioned THP1 stable strain carrying the LAMP2 luciferase reporter gene in the embodiment of the present application;

[0011] Figure 3 is the up-regulation of mRNA and protein levels of LAMP2A in mouse peritoneal primary macrophages by chidamide in the embodiment of the present application;

[0012] Figure 4 is the up-regulation of mRNA and protein levels of LAMP2A in human monocyte THP1 cells by chidamide in the embodiment of the present application;

[0013] Figure 5 is the up-regulation of mRNA and protein levels of LAMP2A in mouse fibroblast L929 cells by chidamide in the embodiment of the present application;

[0014] Figure 6These are representative immunoblot images and quantitative analysis images of mice after being fed chidamide in primary peritoneal macrophages in this embodiment of the invention.

[0015] Figure 7 This invention relates to the effect of feeding chidamide on the LAMP2A protein level in mouse liver. Detailed Implementation

[0016] The following is in conjunction with the appendix Figures 1-7 The present invention will be described in further detail below.

[0017] Example: A novel molecular chaperone-mediated autophagy activator, such as Figures 1-7 As shown. This invention is achieved through the following technical solution steps:

[0018] 1. Constructing a stable THP1 cell line carrying the LAMP2 luciferase reporter gene system.

[0019] The human LAMP2 promoter (NM_002294-promoter, 2026bp) was cloned using the GV715 vector (element sequence: MCS-firefly_Luciferase-SV40-puromycin). The LAMP2 promoter sequence ( Figure 1 B (red) was placed between the restriction enzyme sites BamHI (GGATCC) and AgeI (ACCGGT). 293T cells were co-transfected using a three-plasmid system (GV715 vector + pHelper1.0 + pHelper2.0). After 72 hours, the viral supernatant was collected and concentrated to a titer ≥1E+8TU / mL by ultracentrifugation (25,000 rpm, 2 hours, 4°C). Well-grown THP-1 cells were cultured and subjected to a preliminary infection experiment to determine an MOI of 50. Luciferase activity was detected 72 hours after infection. Puromycin (2.5 μg / mL) was added for selection until no cell death was observed, then the concentration was halved for maintenance culture, with close monitoring of the suspended cell status. Finally, luciferase activity was again confirmed to be good, and the cells were expanded and cryopreserved.

[0020] like Figure 1 Schematic diagram of carrier construction. Figure 1 A shows the cloning of the human LAMP2 promoter sequence into the GV715 vector (element order: MCS-firefly_Luciferase-SV40-puromycin). Figure 1B is a LAMP2 promoter sequence (red) which is placed between the restriction sites BamHI (GGATCC) and Agel (ACCGGT). ori: origin of replication; Poly(A): polyadenylation signal sequence; AmpR: ampicillin resistance gene; 5'LTR: 5' long terminal repeat; 3'LTR: 3' long terminal repeat; MCS: Multiple Cloning Site; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element; PuroR: puromycin resistance gene; p SV40: SV40 promoter; luciferase: luciferase. BamHI: BamHI restriction endonuclease recognition site; Agel: Agel restriction endonuclease recognition site.

[0021] 2. Screening small molecule compounds up-regulating LAMP2 luciferase expression

[0022] The constructed THP1 stable cell strain carrying LAMP2 luciferase reporter gene was inoculated in a 96-well cell culture plate, and the number of cells per well was strictly controlled. After PMA (phorbol ester) was added to induce THP1 cells to differentiate into macrophages, 3084 compounds in the FDA drug library were added to each well, so that the final concentration of each compound was 10 μmol / L, and the stimulation lasted for 24 hours. Through detection of luciferase activity, 38 small molecule compounds which could significantly up-regulate LAMP2 luciferase expression were preliminarily screened out.

[0023] Wild type THP1 cells were seeded in 6-well plates and induced to differentiate into macrophages by PMA. Then 38 compounds obtained from the primary screening were added (final concentration 10 μmol / L, stimulation for 24 hours). The effects of these compounds on the mRNA expression level of LAMP2A were further verified by qRT-PCR re-screening. Finally, 8 candidate compounds that could significantly up-regulate the transcription level of LAMP2A were obtained. At the same time, the mRNA expression level of another transcript of LAMP2 gene, LAMP2B, was detected to evaluate the subtype selectivity of the compounds. Finally, Tucidinostat was determined as a small molecule compound that specifically up-regulated the mRNA expression of LAMP2A and did not affect LAMP2B and other subtypes.

[0024] As shown in Figure 2 , the compound screening was performed by using the above-mentioned THP1 stable strain carrying LAMP2 luciferase reporter gene. Figure 2 A is to add 3084 compounds in the FDA drug library to 96-well cell culture plates, and detect luciferase activity after 24 hours to preliminarily screen small molecule compounds that up-regulate LAMP2 luciferase expression; Figure 2 B-2C is to re-screen the obtained candidate compounds by qRT-PCR, and detect the mRNA levels of two key transcripts of LAMP2 gene, LAMP2A and LAMP2B, to evaluate the subtype selectivity of the compounds. The statistical chart represents mean ± standard deviation, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; β-actin is the internal reference protein; the stimulation concentration of all compounds is adjusted to 10.0 μM, and the time is 24 hours.

[0025] 3. Effects of Tucidinostat on mRNA and protein levels of LAMP2A in mouse peritoneal primary macrophages

[0026] This example was to extract and culture peritoneal primary macrophages of C57BL / 6 mice, and give Tucidinostat (1.0 μM) stimulation for 24 hours. The mRNA and protein levels of LAMP2A in macrophages were detected by western blot and qRT-PCR, respectively. The results showed that Tucidinostat significantly up-regulated the mRNA level (3.1 times, p<0.0001) and protein level (1.4 times, p<0.05) of LAMP2A.

[0027] As shown in Figure 3 , Figure 3 A is a quantitative analysis chart of LAMP2A mRNA expression level; Figure 3B-3C is a representative graph of western blot and quantitative analysis of LAMP2A protein expression; the statistical chart represents mean ± standard deviation, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; β-actin is the internal reference protein; the stimulating concentration of chidamide is 1.0 μM, and the time is 24 hours.

[0028] 4. Effect of chidamide on mRNA and protein levels of LAMP2A in human THP1 cells

[0029] By culturing human monocyte THP1 cell line and inducing it to differentiate into macrophages, chidamide (1.0 μM) was given for 24 hours of stimulation, and the mRNA and protein levels of LAMP2A in macrophages were detected by western blot and qRT-PCR respectively. The results showed that chidamide significantly up-regulated the mRNA level (4.2 times, p<0.0001) and protein level (2.6 times, p<0.001) of LAMP2A, indicating that chidamide promoted the expression of LAMP2A in macrophages of mouse and human origin.

[0030] As shown in Figure 4 , Figure 4 A is a quantitative analysis chart of LAMP2A mRNA expression level; Figure 4 B-4C is a representative graph of western blot and quantitative analysis of LAMP2A protein expression; the statistical chart represents mean ± standard deviation, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; β-actin is the internal reference protein. The stimulating concentration of chidamide is 1.0 μM, and the time is 24 hours.

[0031] 5. Effect of chidamide on mRNA and protein levels of LAMP2A in mouse fibroblast L929 cells

[0032] By culturing mouse fibroblast L929 cells, chidamide (1.0 μM) was given for 24 hours of stimulation, and the mRNA and protein levels of LAMP2A in macrophages were detected by western blot and qRT-PCR respectively. The results showed that chidamide significantly up-regulated the mRNA level (3.7 times, p<0.01) and protein level (1.5 times, p<0.01) of LAMP2A, indicating that chidamide also promoted the expression of LAMP2A in fibroblasts.

[0033] As shown in Figure 5 , Figure 5 A is a quantitative analysis chart of LAMP2A mRNA expression level; Figure 5B-5C is a representative graph of western blot and quantitative analysis of LAMP2A protein expression. The statistical chart represents the mean ± standard deviation, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; β-actin is the internal reference protein. The stimulating concentration of tucidinostat is 1.0 μM, and the time is 24 hours.

[0034] 6. In vivo experiment verifies the effect of tucidinostat on the level of LAMP2A protein in mouse peritoneal primary macrophages

[0035] 8-week-old male C57BL / 6 mice were divided into control and tucidinostat groups. The control group (NC) was fed with ordinary feed, and the tucidinostat group was fed with different concentrations of feed added with tucidinostat, setting low (Low), middle (Middle), and high (High) three different dose groups (10 mg, 20 mg, and 30 mg of tucidinostat were added per kilogram of feed, respectively). After 14 days of feeding, mouse peritoneal primary macrophages were extracted, and the protein level of LAMP2A in macrophages was detected by western blot. The results showed that the protein level of LAMP2A in the peritoneal macrophages of the tucidinostat group mice was significantly higher than that of the control group (up-regulated by 2.1 times, 2.6 times, and 3.0 times in the low, middle, and high concentration groups, respectively), and showed a dose-dependent effect. It is shown that feeding tucidinostat can promote the expression of LAMP2A in mouse macrophages in vivo.

[0036] As shown in Figure 6 , the western blot and quantitative analysis of the level of LAMP2A protein in mouse peritoneal primary macrophages were detected after feeding tucidinostat to mice. C57BL / 6 mice were divided into control (NC) and tucidinostat (Tucidinostat) groups, and the tucidinostat group was set to low (Low), middle (Middle), and high (High) three dose concentrations (i.e. 10, 20, and 30 mg of tucidinostat were added per kilogram of feed, respectively). The statistical chart represents the mean ± standard deviation, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; β-actin is the internal reference protein; tucidinostat is fed for 14 days.

[0037] 7. In vivo experiments verify the effect of Tucidinostat on LAMP2A protein levels in mouse liver. Eight-week-old male C57BL / 6 mice were divided into control and Tucidinostat groups. The control group (NC) was fed with ordinary feed, and the Tucidinostat group was fed with feed containing different concentrations of Tucidinostat, setting up low (Low), medium (Middle), and high (High) three different dose groups (10 mg, 20 mg, and 30 mg of Tucidinostat were added per kilogram of feed, respectively). After 14 days of feeding, the expression level of LAMP2A in the liver of mice was detected by western blot and immunohistochemistry. Western blot results showed that the protein level of LAMP2A in the liver of mice in the low, medium, and high dose Tucidinostat groups was significantly higher than that in the control group (the protein level was up-regulated by 1.9 times, 2.0 times, and 3.0 times, respectively); immunohistochemical results showed that the staining area of LAMP2A in the liver of mice in the Tucidinostat group was up-regulated by 3.8 times compared with the control group.

[0038] As shown in Figure 7 , the effect of feeding Tucidinostat on the protein level of LAMP2A in the liver of mice. Figure 7 A-7B is an immunoblot representative graph and quantitative analysis graph of LAMP2A expression in the liver. C57BL / 6 mice were divided into a control group (NC) and a Tucidinostat group (Tucidinostat), and the Tucidinostat group was set up in low (Low), medium (Middle), and high (High) three dose concentrations (i.e. 10, 20, and 30 mg of Tucidinostat were added per kilogram of feed, respectively). Figure 7 C-7D is an immunohistochemical representative graph and quantitative analysis graph of LAMP2A expression in the liver. The Tucidinostat group is the medium dose group. The statistical graph represents the mean ± standard deviation, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; β-actin is the internal reference protein; Tucidinostat was fed for 14 days.

[0039] By constructing a human THP1-LAMP2 luciferase luciferase reporter gene system, compounds that can activate the LAMP2 promoter were screened from 3035 FDA-approved and pharmacopoeia-included raw materials. After cell level primary screening, rescreening and animal level verification, it was finally determined that the new anti-tumor drug Tucidinostat (English name Tucidinostat or Chidamide) is an effective CMA activator. Tucidinostat can significantly up-regulate the mRNA and protein levels of LAMP2A in various cells such as mouse peritoneal primary macrophages, THP1 cells and mouse fibroblasts. In animal experiments, after feeding with feed containing different concentrations of Tucidinostat, the protein level of LAMP2A in mouse peritoneal primary macrophages and liver also increased in a concentration-dependent manner.

[0040] The specific embodiments are only illustrative of the present application, and are not intended to limit the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, and the modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A novel activator of chaperone-mediated autophagy, characterized in that, The chaperone-mediated autophagy activator is the histone deacetylase inhibitor chidamide.

2. Use of chidamide as claimed in claim 1 as an activator of molecular chaperone-mediated autophagy, characterized in that, The histone deacetylase inhibitor chidamide can up-regulate the mRNA and protein levels of LAMP2A.

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