Application of small molecule compounds in the preparation of drugs for antagonizing neurotoxicity or neurological diseases caused by impaired autophagic flux

Abstract

The present invention relates to the technical field of drugs related to neurotoxicity or neurological diseases, and specifically to the use of small molecule compounds in the preparation of drugs for antagonizing neurotoxicity or neurological diseases caused by impaired autophagic flow. The present technical solution studies and discovers the autophagic flow regulating molecule RAB7A, which is a key downstream molecule of SIRT5. Among them, the key site is Lys31, and inhibiting the desuccinylation of RAB7A Lys31 will inhibit the activity of RAB7A, thereby leading to the blockage of autophagic flow. Small molecule compounds were screened for the key site Lys31 of SIRT5 acting on RAB7A, which can antagonize the neurotoxicity induced by heavy metal cadmium exposure and the adverse consequences of the aggravation of neurological diseases including Alzheimer's disease. The present technical solution solves the technical problem of the lack of small molecule drugs for antagonizing neurotoxicity or neurological diseases caused by impaired autophagic flow in the prior art, and has an ideal prospect for application and promotion.

Description

Technical Field

[0001] The present invention relates to the technical field of drugs related to neurotoxicity or neurological diseases, and in particular to the use of small molecule compounds in the preparation of drugs for antagonizing neurotoxicity or neurological diseases caused by impaired autophagic flux. Background Art

[0002] Alzheimer's disease (AD) is a devastating neurodegenerative disease. Studies have shown that the blood cadmium (Cd) level of AD patients is significantly higher than that of healthy controls. In the elderly in the United States, AD mortality is significantly correlated with blood Cd levels. Animal studies have found that Cd exposure is associated with β-amyloid (Aβ) deposition, which is one of the main pathological hallmarks of AD. Studying the effects of Cd-related environmental pollutants on the occurrence and development of AD and the potential mechanisms will help us understand the pathogenesis of AD and explore new therapeutic targets and strategies. Cadmium (Cd), as a common environmental pollutant, can pass through the blood-brain barrier and promote the development of Alzheimer's disease. By studying the mechanism of action of cadmium AD, we can screen drugs that can effectively antagonize the neurotoxicity of cadmium for the prevention or treatment of the occurrence and development of Alzheimer's disease.

[0003] In order to screen and study drugs that antagonize neurotoxicity or neurological diseases induced by heavy metal cadmium exposure, it is first necessary to identify the key targets for the treatment of the occurrence and development of neurodegenerative diseases. Among them, autophagy is the process of degradation of abnormal proteins or defective organelles and renewal of basic cellular components in eukaryotic cells. There is increasing evidence that the pathogenesis of AD is related to impaired autophagy, and autophagy may participate in the metabolism of Aβ by regulating the production and elimination of Aβ. Previous studies have shown that administration of rapamycin (a specific autophagy agonist) to AD mice can significantly reduce intracellular Aβ and extracellular amyloid deposition in brain cells and improve cognitive function. However, the key biological targets of the biological process of autophagy that regulates the metabolism of Aβ and the interaction mechanism between biological molecules need to be further studied and clarified, so as to create conditions for the design and preparation of drugs that antagonize neurotoxicity or neurological diseases induced by heavy metal cadmium exposure. Summary of the invention

[0004] The present invention is intended to provide the use of small molecule compounds in the preparation of drugs for antagonizing neurotoxicity or neurological diseases caused by impaired autophagic flux, so as to solve the technical problem of the prior art that there is a lack of small molecule drugs for antagonizing neurotoxicity or neurological diseases caused by impaired autophagic flux.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] A small molecule compound is used in the preparation of a reagent for antagonizing autophagic flow damage, and its structural formula is:

[0007]

[0008] Furthermore, the autophagic flow damage refers to the damage of fusion between autophagosome and lysosome or the damage of lysosome function.

[0009] Furthermore, the autophagic flux damage is autophagic flux damage caused by cadmium exposure.

[0010] Normal autophagic flux blockage can lead to autophagic flux damage, thus affecting normal physiological processes. SR10 of this technical solution can antagonize autophagic flux damage, and then be used to treat diseases related to autophagic flux damage. In addition, SR10 can also be used in basic scientific research. As an antagonist of autophagic flux damage, it can regulate the autophagic flux of the experimental subject after acting on it, and then study the related mechanism of action and signal pathway. As a small molecule compound, compared with general gene knockout, transgenic, gene overexpression and other methods, the use of small molecule compounds is simpler and more advantageous.

[0011] The technical solution also provides an application of a small molecule compound in the preparation of a drug for treating Alzheimer's disease, and its structural formula is:

[0012]

[0013] Furthermore, the small molecule compound is used to inhibit the accumulation of amyloid-β precursor protein caused by cadmium exposure.

[0014] The accumulation of amyloid-β precursor protein will lead to increased accumulation of amyloid-β, thereby accelerating the progression of Alzheimer's disease. The small molecule compound SR10 screened out by this technical solution can effectively inhibit the accumulation of amyloid-β precursor protein caused by cadmium exposure, thereby achieving the treatment of Alzheimer's disease.

[0015] The technical solution also provides an application of a small molecule compound in the preparation of a drug for antagonizing neurotoxicity caused by cadmium exposure, and its structural formula is:

[0016]

[0017] Furthermore, the effective concentration of the small molecule compound is 5-40 μM.

[0018] Experiments have shown that the small molecule compound SR10 in this scheme can antagonize the neurotoxicity caused by cadmium exposure and can be used as a drug for the treatment of heavy metal poisoning.

[0019] The technical solution also provides an application of a small molecule compound in the preparation of a RAB7A desuccinylation promoter, the structural formula of which is:

[0020]

[0021] Furthermore, the desuccinylation site of RAB7A is Lys31.

[0022] Furthermore, the small molecule compound promotes RAB7A desuccinylation by binding to Tyr255 of SIRT5.

[0023] RAB7A controls the formation, transport and fusion of vesicles, and plays a vital role in regulating autophagosome-lysosome fusion and autophagolysosome maturation. Although RAB7A activity is also regulated by many post-translational modifications of proteins, it is the first discovery of this technical solution that the desuccinylation of RAB7A can affect the autophagic flow. This technical solution further screened out the desuccinylation promoter of RAB7A. The promoter SR10 is a small molecule compound that is easy to add and use as a basic scientific research tool. In the specific scientific research process, applying SR10 to the experimental subjects can promote the desuccinylation process of RAB7A, and then study the downstream action pathways and further clarify a series of action mechanisms.

[0024] In summary, the principle and beneficial effects of this technical solution are:

[0025] This technical solution first discovered new drug design and screening targets for antagonizing cadmium neurotoxicity, regulating autophagic flux, and treating Alzheimer's disease, and then screened small molecule compound drugs based on drug design and screening targets, and obtained two small molecule compounds with different modes of action and molecular structures.

[0026] (1) Drug design and discovery of screening targets

[0027] Cadmium exposure can cause neurotoxicity, leading to decreased neuronal viability, accumulation of amyloid-β precursor protein (APP), and blockage of autophagic flux. Further studies showed that cadmium exposure did not interfere with phagocytic vacuoles or autophagosome maturation in Neuro-2a cells, but cadmium caused blockage of autophagic flux by hindering the fusion of autophagosomes with lysosomes in Neuro-2a cells and impairing lysosomal function. Through omics studies and experimental verification, the potential molecular mechanism of cadmium-induced neurotoxicity was explored, and it was found that there was a significant correlation between the upregulation of APP and the decrease in SIRT5 levels, indicating that SIRT5 may play a vital role in APP accumulation, and in Neuro-2a cells treated with cadmium, SIRT5 protein levels decreased in a dose-dependent manner. Further experimental studies found that SIRT5 plays an important role in cadmium-induced APP accumulation and neuronal cell death.

[0028] Sirtuin 5 (SIRT5) is a member of the nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase Sirtuin family, which regulates the physicochemical properties of proteins through post-translational modifications (PTMs). Considering that SIRT5 can induce desuccinylation, deacrylation, or deglutarylation of acyl lysine residues, the levels of these post-translational modifications were evaluated in cadmium-treated cells. Cadmium exposure resulted in a significant dose-dependent increase in the succinylation level of the protein, while the levels of malonylation and glutarylation did not change significantly. Combined with the analysis of cell autophagy-related pathways, it was found that there was a lysine site Lys31 (K31) in RAB7A in the autophagy-related pathway, and the succinylation of this site was significantly upregulated after cadmium treatment. Lysosomal Ras-associated protein 7a (RAB7A) belongs to a small family of GTPases, and its members are believed to be mainly involved in regulating vesicle membrane trafficking and fusion with lysosomes and regulating lysosomal function. Lys31 is highly conserved in RAB7A homologs from humans to amoeba, indicating that it is essential for the function of RAB7A. Lys31 is a key succinylation site of RAB7A, and its succinylation state can affect the activity of RAB7A molecules, thereby participating in cadmium-induced autophagic flux blockade and neurotoxicity. RAB7A desuccinylated at Lys31 can antagonize cadmium-induced autophagic flux blockade and neurotoxicity. SIRT5 interacts with the Lys31 site of RAB7A and desuccinylates it, and antagonizes the cadmium-inhibited RAB7A activity. In the process of related drug design, the interaction between SIRT5 and RAB7A at the Lys31 site can be considered to design drugs that regulate autophagy, diseases related to APP accumulation, and neurological damage caused by cadmium exposure.

[0029] In summary, cadmium is released into the environment as an environmental pollutant in various natural and anthropogenic processes (i.e., fossil fuel combustion, mining, battery and pigment manufacturing, etc.). Cadmium can cross the blood-brain barrier and eventually accumulate in the brain, causing neurotoxicity and promoting the progression of neurological diseases including Alzheimer's disease. Cadmium exposure significantly inhibits the expression of SIRT5, which in turn induces autophagic flux damage by impairing autophagosome-lysosome fusion and lysosomal function, leading to abnormal accumulation of APP and neuronal cell death. Quantitative succinylomics analysis showed that decreased SIRT5 expression led to increased succinylation modification at the Lys31 (K31) site of RAB7A, reduced RAB7A activity, hindered its recruitment to RILP, led to blocked autophagic flux, and ultimately aggravated the pathological progression and cognitive decline of Alzheimer's disease. Therefore, we considered whether there is a drug that can antagonize the cadmium-induced autophagic flux impairment by targeting the above pathway to achieve the purpose of improving the progression of Alzheimer's disease and the harm caused by cadmium-induced neurotoxicity.

[0030] (2) Screening and identification of small molecule drugs

[0031] In the process of new drug design, the binding pocket of small molecule drugs was predicted based on the protein structure of SIRT5, and the binding site and succinylation site between SIRT5 and RAB7A were analyzed. 1.6 million compounds with structural diversity were used for drug screening based on the Site1 site of SIRT5 (especially the key site Tyr255 that binds to the Lys31 site of RAB7A). Finally, 114 compounds that bind to the Site1 site were selected, and 30 small molecule compounds with high scores were selected for subsequent experimental verification. Through screening of a large number of compounds, this scheme found that only two small molecule compounds (SR10 and SR19) had ideal antagonistic effects on cadmium toxicity, and showed regulatory effects on autophagic flow and inhibitory effects on APP accumulation. Both small molecule compounds can be used to effectively inhibit the increase of APP caused by cadmium exposure, and then used to prepare drugs for the treatment of Alzheimer's disease. β-Amyloid protein (Aβ) is one of the main pathological signs of Alzheimer's disease and is derived from the hydrolysis of APP. Since cadmium exposure leads to the blockage of autophagic flux, it will affect the normal physiological activities. Both small molecule compounds can also be used to antagonize the autophagic flux damage caused by cadmium exposure. These two small molecule compounds can also be used as reagents to antagonize autophagic flux damage (mainly to hinder the fusion of autophagosomes and lysosomes and damage the function of lysosomes), that is, an autophagic flux blocking antagonist, as a basic scientific research tool for basic scientific research research, to further study the changes in physiological processes caused by changes in autophagic flux. These two small molecule compounds can also be used as drugs to antagonize neurotoxicity caused by cadmium exposure, to resist nerve damage caused by occupational exposure. In addition, these two small molecule compounds can also be used as promoters of RAB7A desuccinylation, as a basic scientific research reagent, to study the physiological processes related to RAB7A desuccinylation. Small molecule drugs have great advantages as basic scientific research reagents. They can regulate the modification level after gene translation into protein by direct application, and then regulate the function of the genes or proteins involved, without complex gene knockout, introduction of exogenous genes or other operations.

[0032] (3) Advantages of SR10 and SR19

[0033] The two small molecule compounds SR10 and SR19 have their own characteristics. Although both can bind to Tyr255 of SIRT5 and affect SIRT5's succinylation of Lys31 site of RAB7A, the molecular structures of the two small molecule compounds are completely different and do not have similarities in molecular structure. In addition, the two small molecule compounds have different modes of action and their own advantages, but both have the potential for drug development. In the range of 5-40μM, the effect of SR10 increases in a concentration-dependent manner, and the cell survival rate of 5-40μM SR10 is better than that of the blank control exposed to cadmium and not administered, indicating that the drug not only has a good ability to antagonize cadmium exposure, but also the relationship between the drug effect and the dosage is easier to predict and control. In clinical practice, SR10 may have higher safety and can play a positive role in a relatively large dosage range. SR19 has a stronger biological effect than SR10, and can antagonize cadmium exposure at a lower concentration, and the effect of SR19 at the optimal concentration (10μM) is better than that of SR10 at the optimal concentration (40μM). However, the effect of SR19 requires a relatively high control of drug concentration, which needs to be controlled at 5-10μM, otherwise it will produce additional toxic effects on cells. In clinical practice, the drug may have strict requirements for dosage control, and due to the large individual differences in the human body, it is difficult to determine the dosage for a single individual. Therefore, the two small molecule compounds have their own advantages and have the potential to be developed as drugs to antagonize neurotoxicity caused by cadmium exposure, drugs for the treatment of Alzheimer's disease, reagents to antagonize autophagic flow damage, and promoters of RAB7A desuccinylation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1The results of the Cd exposure experiment of Neuro-2a cells in Example 1 (A, B: The cell confluence of Neuro-2a cells treated with 0, 1, 2 and 4 μM CdCl2 for 0, 24, 48 and 72 h was detected by IncuCyteZOOM live cell analysis system, scale: 100 μm; C: Representative immunoblot images and quantitative analysis results of APP after Neuro-2a cells were treated with 0, 1, 2 and 4 μM CdCl2; D: Heat map of differentially expressed proteins in Neuro-2a cells treated with or without 4 μM CdCl2 for 72 h; E: The top 30 pathways identified by KEGG enrichment analysis; F: Representative immunoblot images and quantitative analysis results of MAP1LC3B in Neuro-2a cells treated with 0, 1, 2 and 4 μM CdCl2; G: 4 μM, 72 h Representative immunoblot images and quantitative analysis results of MAP1LC3B in Neuro-2a cells treated with or without CdCl2 and 25μM, 72h chloroquine; H, I: Representative images and quantitative analysis of GFP-LC3B spots according to the treatment method in G; *p<0.05, **p<0.01 compared with the control group; ns: not significant).

[0035] Figure 2 This is a study on the effect of Cd exposure of Neuro-2a cells in Example 1 on phagosome formation or autophagosome maturation (A: representative immunoblot images and quantitative analysis results of MAP1LC3B in Neuro-2a cells treated with or without CdCl2 (4 μM) for 72 hours after transfection with NC or Atg5-siRNA (si-Atg5); B: representative immunoblot images and quantitative analysis results of MAP1LC3B in Neuro-2a cells treated with or without Cd (4 μM) for 72 hours in the presence or absence of 3-MA (1 mM); C, D: representative images and colocalization coefficient analysis of GFP-LC3B and SQSTM1 after Neuro-2a cells were treated with or without CdCl2 (4 μM) for 72 hours, scale: 5 μM; compared with the control group, **p<0.01; compared with the Cd exposure group, #p<0.05, ##p<0.01).

[0036] Figure 3The results of the study on the effect of Cd exposure on autophagosome-lysosome fusion and lysosomal function of Neuro-2a cells in Example 1 (A, B: Representative immunoblot images and quantitative analysis results of LAMP1 and LAMP2 after Neuro-2a cells were treated with different concentrations of Cd (0, 1, 2, 4 μM) for 72 hours; C, D: Representative images and colocalization coefficient analysis of GFP-LC3B and LAMP2 after Neuro-2a cells were treated with or without CdCl2 (4 μM) for 72 hours; E: LysoSensor after Neuro-2a cells were treated with different concentrations of Cd DND-189 fluorescence intensity (0, 1, 2, 4 μM); F: Immunofluorescence analysis of Neuro-2a cells double-transfected with RFP-GFP-LC3B for 24 hours and treated with Cd (4 μM) for 72 hours; The RFP-GFP-LC3B system is a tool for fusion detection, in which the fluorescence of GFP is quenched by an acidic environment; Autophagosomes before fusion are represented by yellow dots, and autophagosomes after fusion are represented by red dots; G: The ratio of yellow dots to red dots after Neuro-2a cells were transfected with RFP-GFP-LC3B and treated with Cd (4 μM) for 72 hours; H, I: CTSB and CTSD activities of Neuro-2a cells treated with different concentrations of Cd (0, 1, 2, 4 μM) for 72 hours, scale bar: 5 μM; compared with the control group, *p<0.05, **p<0.01).

[0037] Figure 4 The experimental study results of SIRT5-dependent deacylation in Cd-induced APP accumulation and neuronal cell death in Example 2 (A: ten typical pathways identified by IPA; B: key networks predicted by IPA; C: representative immunoblot images and quantitative analysis results of SIRT5 in Neuro-2a cells treated with different concentrations (0, 1, 2 and 4 μM) of Cd; D, E: Neuro-2a cells were transfected with control vector (vector), wild-type SIRT5 overexpression plasmid (SIRT5-WT) or inactive SIRT5 mutant plasmid (SIRT5-H158Y) with or without CdCl2 (4 μM) for 72 h, and representative Western blot images of APP in Neuro-2a cells Blot images and quantitative analysis results, as well as representative images and quantitative analysis results of Neuro-2a cell fusion, scale bar: 100 μm; compared with the control group, *p<0.05, **p<0.01; compared with the Cd-exposed group, #p<0.05, ##p<0.01).

[0038] Figure 5The experimental results of the SIRT antagonism of Cd-induced autophagy flow blockage in Example 2 (in AG, all Neuro-2a cells were transfected with Vector, SIRT5-WT or SIRT5-H158Y plasmids, with or without CdCl2 (4 μM) for 72 hours; A: representative immunoblot images and quantitative analysis results of MAP1LC3B; B: representative immunofluorescence images of Neuro-2a cells transfected with GFP-LC3B plasmid and detected with anti-LAMP2 antibody, scale bar :5μm; C: Statistical analysis of the colocalization coefficient of GFP-LC3B dots and LAMP2 dots; D, E: Representative immunofluorescence and statistical analysis of Neuro-2a cells 24h after double transfection with RFP-GFP-LC3B (yellow spots: red spots), scale bar: 5μm; F, G: LysoSensorDND-189 fluorescence intensity and CTSB activity in Neuro-2a cells; compared with the control group, **p<0.01; compared with the Cd-exposed group, ##p<0.01).

[0039] Figure 6 These are the experimental results for detecting the levels of lysine succinylated protein in Neuro-2a cells treated with different concentrations (0, 1, 2 or 4 μM) of Cd for 72 hours in Example 3 (A: representative immunoblot images of lysine succinylated protein (K-Succ) and TUBULIN obtained using anti-succinyllysine antibody and anti-TUBULIN antibody, respectively; B: representative immunoblot images of lysine malonyl protein (K-Mal) and TUBULIN obtained using anti-malonyl modification antibody and anti-TUBULIN antibody, respectively; C: representative immunoblot images of lysine-glutarylated protein (K-Glu) and ACTB obtained using anti-glutaryl modification antibody and anti-ACTB antibody, respectively).

[0040] Figure 7 Basic statistical analysis of the quantitative global succinylation analysis MS data of Neuro-2a cells treated with or without Cd for 72 hours in Example 3 (A, B), RSD and PCA of succinylation sites (C, D), motif analysis of identified succinylation sites (E), motif enrichment heat map of amino acids upstream and downstream of the identified modification sites (F), volcano plot of differentially expressed succinylation modification sites (G), clustering heat map (H; orange: increase; green: decrease), and statistical graphs of the number of sites and differentially expressed proteins (I; the number of sites with different succinylation modification levels and differentially expressed proteins at succinylation sites in Neuro-2a cells exposed to Cd compared with control cells).

[0041] Figure 8The analysis of highly succinylated sites in Neuro-2a cells exposed to cd in Example 3 (A: KEGG enrichment analysis of differentially expressed succinylated modification site proteins was performed to screen out the top 20 pathways; B: GO enrichment analysis related to autophagy, lysosome and intracellular pH was obtained from the proteins of differentially expressed succinylated modification sites; C: Gene duplications found in GO enrichment analysis; D: Mass spectrometry analysis results of succinylated RAB7A at K31 site; E: K31 (red mark) is evolutionarily conserved in RAB7A; F: Anti-RAB7A-succinylated K31 antibody was identified by dot blotting, in which nitrocellulose membrane was used to identify different numbers of succinylated K31 peptides (1 and 2) and unmodified peptides, and then the membrane was immunoblotted with anti-RAB7A-succinylated K31 antibody; G: RAB7A K31 after Neuro-2a cells were treated with different concentrations of Cd (0, 1, 2 and 4 μM). su Representative immunoblot images and quantitative analysis results; **p<0.01 compared with the control group).

[0042] Fig. 9 The MS detection results of the three peptides in Example 3 (A: RAB7A K31 su Peptide 1; B: RAB7A K31 su Peptide 2; C: unmodified control).

[0043] Fig.10 The WB results of Example 3 were detected in HeLa cells using rabbit serum.

[0044] Fig.11The results of the study on the effect of RAB7A desuccinylation at the K31 site of Example 3 on Cd-induced autophagic flux blockade and neurotoxicity (all Neuro-2a cells were transfected with blank vector or RAB7AK31R (simulated desuccinylated state) or RAB7AK31E (simulated amberized state) plasmid, with or without CdCl2 (4 μM) for 72 h; A, B: representative immunofluorescence images and statistical results of RILP and RAB7A colocalization, scale bar: 5 μm; C, D: representative immunofluorescence images and colocalization coefficient statistical analysis of GFPLC3B dots and LAMP2 dots, scale bar Ruler: 5 μm; E, F: Representative immunofluorescence images of double-stained RFP-GFP-LC3B and the ratio of yellow dots to red dots, scale bar: 5 μm; G, H: LysoSensorDND-189 fluorescence intensity and CTSB activity detection; I, J: Representative immunoblot images and quantitative analysis results of MAP1LC3B and APP in Neuro-2a cells; K, L: Changes in Neuro-2a cell mass and statistical analysis, scale bar: 100 μm; compared with the control group, **p<0.01; compared with the Cd exposure group, #p<0.05, ##p<0.01).

[0045] Fig.12 The results of the study on the interaction mechanism between SIRT5 and RAB7A at Lys31 site in Example 3 (A: molecular docking model of RAB7A binding to SIRT5 domain, yellow dotted line indicates hydrogen bond; B: MST analysis of direct interaction between SIRT5 and RAB7A; C: SPR analysis of SIRT5 binding affinity curve for RAB7A; D, E: immunofluorescence analysis of endogenous SIRT5 and RAB7A after Neuro-2a cells were treated with or without CdCl2 (4 μM) for 72 h; FH: Neuro-2a cells were transfected with blank vector or SIRT5-WT or SIRT5-H158Y plasmids and treated with or without CdCl2 (4 μM) for 72 h; F: RAB7A K31 in Neuro-2a cells su Representative immunoblot images and quantitative analysis results; G, H: Immunofluorescence analysis of endogenous RILP and RAB7A in Neuro-2a cells, scale bar: 5 μm; compared with the control group, **p<0.01; compared with the Cd exposure group, ##p<0.01).

[0046] Fig.13 This is the prediction of the SIRT5 molecular structure of Example 4 (A: prediction diagram of the SIRT5 small molecule binding pocket; B: analysis of the specific amino acid sites of the SIRT5 action pocket).

[0047] Fig.14 This is the affinity score of the compound of Example 4 in the Site 1 region.

[0048] Fig.15 This is the screening result of the small molecule compounds in Example 5 for antagonizing Cd-induced cytotoxicity (compared with the control group, **p<0.01; compared with the Cd-exposed group, ##p<0.01).

[0049] Fig.16 This is the mass spectrum image of SR10 in Example 5.

[0050] Fig.17 This is the mass spectrum image of SR19 in Example 5.

[0051] Fig.18 This is the chromatogram image of SR19 in Example 5.

[0052] Fig.19 Schematic diagram of the SIRT5 and SR10 binding molecule of Example 6.

[0053] Fig. 20 The experimental results of the cytotoxicity and efficacy study of SR10 in Example 6 (A: SR10 has no obvious cytotoxic effect; B: SR10 can antagonize the neurotoxic effect induced by Cd; compared with the control group, **p<0.01; compared with the Cd exposure group, #p<0.05, ##p<0.01).

[0054] Fig.21 The experimental study results of Example 6 on the improvement of Cd-induced APP accumulation by SR10 (representative immunoblot images and quantitative analysis results of APP in Neuro-2a cells; compared with the control group, *p<0.05; compared with the Cd-exposed group, #p<0.05).

[0055] Fig. 22 These are the research results of Example 6 on the effect of SR10 on blocking the Cd-induced autophagic flux (A: representative immunoblot images and quantitative analysis results of MAP1LC3B-II in Neuro-2a cells; B: representative immunoblot images and quantitative analysis results of CTSB in Neuro-2a cells; C: representative immunoblot images and quantitative analysis results of SQSTM1 in Neuro-2a cells; compared with the control group, *p<0.05, **p<0.01; compared with the Cd-exposed group, #p<0.05).

[0056] Fig.23 Schematic diagram of the SIRT5 and SR19 binding molecules of Example 6.

[0057] Fig.24The experimental results of the cytotoxicity and efficacy study of SR19 in Example 6 (A: SR19 has no obvious cytotoxic effect; B: SR19 can antagonize the neurotoxic effect induced by Cd; compared with the control group, **p<0.01; compared with the Cd exposure group, #p<0.05, ##p<0.01).

[0058] Fig.25 The experimental study results of Example 6 on the improvement of Cd-induced APP accumulation by SR19 (representative immunoblot images and quantitative analysis results of APP in Neuro-2a cells; compared with the control group, *p<0.05; compared with the Cd-exposed group, #p<0.05).

[0059] Fig.26 These are the research results of Example 6 on the effect of SR19 on blocking Cd-induced autophagic flux (A: representative immunoblot images and quantitative analysis results of MAP1LC3B-II in Neuro-2a cells; B: representative immunoblot images and quantitative analysis results of CTSB in Neuro-2a cells; C: representative immunoblot images and quantitative analysis results of SQSTM1 in Neuro-2a cells; compared with the control group, *p<0.05, **p<0.01; compared with the Cd exposure group, #p<0.05). DETAILED DESCRIPTION

[0060] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0061] Example 1: Cd exposure promotes APP accumulation and blocks autophagic flux in Neuro-2a cells

[0062] A: Experimental part

[0063] 1. Cell Culture and CdCl2 Treatment

[0064] Mouse neuroblastoma cells (Neuro-2a cells) were obtained from the Cell Bank of the Institute of Biochemistry and Cell Biology (TCM29, Shanghai, China) and treated with 0, 1, 2, or 4 μM CdCl2 for 72 h. Neuro-2a cells were cultured in DMEM / H (Gibco, C11995500BT) supplemented with 10% fetal bovine serum (AUSGENEX, FBS500-S) and 1% penicillin / streptomycin (Beyotime, C0222) at 37°C in a 5% CO2 environment. CdCl2 stock solution (Sigma-Aldrich, 202908) was prepared with distilled deionized water and diluted with culture medium. In addition, 25 μM chloroquine (CQ, MedChemExpress, HY-17589A) and 1 mM 3-methyladenine (3-MA, MedChemExpress, HY-19312) were also used in this study.

[0065] 2. Cytotoxicity Assay

[0066] To detect cell proliferation, Neuro-2a cells were plated at 3.5 × 10 3 The density of cells was inoculated in 96-well ImageLock plates (Essen BioScience, USA). After 72 h of treatment, the plates were placed in the Induced Cell ZOOM instrument to record cell images. As described above, cell fusion was analyzed using the Induced Cell ZOOM Live Cell Analysis System (Essen BioScience, USA).

[0067] 3. Immunocytochemistry

[0068] 4. Quantitative proteomics analysis based on tandem mass tag (TMT)

[0069] (1) Protein extraction and peptide enzymatic hydrolysis

[0070] The samples (human, mouse and rat serum samples need to remove high-abundance serum proteins first) were extracted by SDT (4% (w / v) SDS, 100mM Tris / HCl pH7.6, 0.1M DTT) lysis method, and then protein quantification was performed by BCA method. An appropriate amount of protein from each sample was digested with trypsin using the Filter aided proteome preparation (FASP) method, and peptides were quantified (OD280).

[0071] (2) TMT labeling: 100 μg of peptide was taken from each sample and labeled according to the instructions of the TMT labeling kit of Thermo Company.

[0072] (3) High pH Reversed-Phase Peptide Fractionation: Equal amounts of labeled peptides from each group were mixed and fractionated using a High pH Reversed-Phase Peptide Fractionation Kit.

[0073] (4) LC-MS / MS Data Acquisition Each fractionated sample was separated using the HPLC liquid phase system Easy nLC at a nanoliter flow rate.

[0074] (5) Protein identification and quantitative analysis The raw data of mass spectrometry analysis were stored in RAW files and were identified and quantitatively analyzed using Mascot 2.2 and Proteome Discoverer 1.4 software.

[0075] (6) Bioinformatics analysis

[0076] 1) GO functional annotation Blast2GO was used to perform GO annotation on the target protein set. The process can be roughly summarized into four steps: sequence alignment (Blast), GO entry extraction (Mapping), GO annotation (Annotation) and InterProScan supplementary annotation (Annotation Augmentation).

[0077] 2) KEGG pathway annotation The KEGG pathway annotation of the target protein set was performed using KAAS (KEGG Automatic Annotation Server) software.

[0078] 3) Fisher's Exact Test was used to compare the distribution of each GO classification or KEGG pathway in the target protein set and the overall protein set, and the enrichment analysis of GO annotation or KEGG pathway annotation was performed on the target protein set.

[0079] 4) Protein clustering analysis The quantitative information of the target protein set was first normalized (normalized to the interval (-1, 1)). Then, the Complexheatmap R package (RVersion3.4) was used to classify the two dimensions of sample and protein expression (distance algorithm: Euclidean, connection method: Average linkage) and generate a hierarchical clustering heat map.

[0080] 5) Protein interaction network analysis The interaction relationships between target proteins were found based on the information in the STRING (http: / / string-db.org / ) database, and the interaction network was generated and analyzed using CytoScape software (version number: 3.2.1).

[0081] 5. Western Blotting: including total protein extraction, protein concentration quantification, SDS-PAGE electrophoresis, and membrane transfer, which is a conventional method in the prior art.

[0082] 6. GFP-LC3B imaging: Under physiological conditions, most LC3B is diffusely distributed in the cytoplasm in a non-lipidated form (LC3B-I), and a few are lipidated and bound to the autophagosome membrane in a dot-like form (LC3B-II). Therefore, LC3B is most commonly used for autophagosome labeling and dynamic observation of autophagic flow, and fluorescence imaging provides a more intuitive way of observation.

[0083] 7. Analysis of co-localization of GFP-LC3B and SQSTM1 / LAMP2: To further analyze the stages of autophagic flow, we used the co-localization of GFP-LC3B and SQSTM1 or LAMP2 (lysosomal associated membrane protein 2) for analysis. By observing the ability of autophagosomes to bind to substrates during formation and the fusion of autophagosomes with lysosomes, we determined whether the maturation of autophagosomes and the fusion process of autophagosomes with lysosomes were affected.

[0084] 8.RFP-GFP-LC3B transfection: To further observe the fusion of autophagosomes and lysosomes, we used RFP-GFP-LC3B transfection experiment. RFP-GFP-LC3B appears as yellow dots on the autophagosomes, but the GFP fluorescence is sensitive to the acidic environment and will be quenched under acidic conditions. If the autophagosomes bind to the lysosomes normally and the acidic environment in the lysosomes is normal, only red dots will appear after binding. On the contrary, the increase in yellow dots represents the fusion barrier between the autophagosomes and lysosomes, or abnormal lysosomal pH.

[0085] 9. LysoSenor Green DND-189 fluorescence intensity measurement: LysoSensor Green DND-189 dye is an acidophilic probe that accumulates in acidic organelles such as lysosomes and shows a pH-dependent increase in fluorescence intensity upon acidification. Therefore, lysosomal pH can be relatively quantified.

[0086] 10. CTSD (cathepsin D) activity detection: Use Cathepsin D Activity Fluorometric Assay Kit to detect CTSD activity in hippocampal tissue.

[0087] 11. CTSB (cathepsin B) activity detection: Cathepsin B Activity Fluorometric Assay Kit (BioVision, K140-100) was used to detect the CTSB activity in hippocampal tissue.

[0088] 12. Statistical Analysis

[0089] Data were analyzed using GraphPad Prism 9.0 software (GraphPad, USA) and expressed as mean ± SEM. Unpaired two-sided t-test was used for comparison between two experimental groups, and one-way analysis of variance (ANOVA) was used for comparison between multiple groups. Each experiment was repeated at least 3 times, and *p < 0.05 was defined as statistically significant.

[0090] B: Experimental results

[0091] The neurotoxicity of Cd was evaluated using the IncuCyte ZOOM live cell analysis system, and the results showed that the viability of Neuro-2a cells was significantly decreased in a dose- and time-dependent manner (0, 1, 2, and 4 μM cadmium chloride (CdCl2) for 24, 48, and 72 hours (h), respectively) ( Figure 1 AB). In addition, in Neuro-2a cells exposed to Cd for 72 h, it was observed that the expression of APP increased with increasing doses ( Figure 1 C). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway proteomics confirmed that the differentially expressed proteins involved in "autophagy" in Neuro-2a cells treated with Cd were significantly enriched compared with control cells ( Figure 1 DE). In addition, the dose-dependent changes in MAP1LC3B-II protein levels demonstrated that Cd activated autophagy in a dose-dependent manner ( Figure 1 F). Chloroquine (CQ) is a potent autophagy inhibitor that blocks the fusion of autophagosomes with lysosomes, thereby inhibiting the late stage of autophagy. Our study showed that the combination of chloroquine (CQ) and Cd had no effect on the level or number of MAP1LC3B-II spots, suggesting that Cd impedes the degradation of autophagic contents ( Figure 1GI). The above experimental results show that Cd exposure increases APP expression in Neuro-2a cells and blocks autophagic flux. Among them, autophagy is an ancient evolutionarily conserved cytoplasmic component degradation pathway in eukaryotes. Autophagy in the usual sense includes several processes: autophagosome formation, transport of autophagic substrates to lysosomes, fusion of autophagosomes and lysosomes, degradation of autophagosome contents by lysosomes, etc. This dynamic process is called autophagic flux. Blocking the above biological process is called autophagic flux blockage.

[0092] Given that autophagy is a complex process involving multiple stages, our study aimed to determine which specific stage would be impaired by Cd, leading to a blockade of autophagic flux. We first transfected small interfering RNA (siRNA) targeting autophagy-related protein 5 (Atg5) into Neuro-2a cells with or without Cd exposure. Knockdown of Atg5 resulted in a reduction in the accumulation of MAP1LC3B-II triggered by Cd, indicating that Cd does not affect the formation of phagophores ( Figure 2 A). In addition, similar results were obtained with the autophagy inhibitor 3-MA ( Figure 2 B). Autophagosome maturation was assessed by colocalization of GFP-LC3B and SQSTM1. Cd exposure also resulted in increased colocalization of GFP-LC3B and SQSTM1 compared to control cells ( Figure 2 CD). The above experimental results indicate that Cd exposure does not interfere with the phagocytic vacuoles formation or autophagosome maturation of Neuro-2a cells.

[0093] Compared with control cells, the level of lysosomal associated membrane protein 1 (LAMP1), a key protein involved in lysosome formation, was increased in Cd-exposed Neuro-2a cells. Figure 3 A). The reduced colocalization of the autophagosome marker GFP-LC3B with the lysosomal marker lysosomal associated membrane protein 2 (LAMP2) indicated that the fusion of autophagosomes and lysosomes was significantly reduced in Cd-treated Neuro-2a cells compared with control cells ( Figure 3 BD). The acidophilic probe Lysosomal Sensor Green DND-189 dye was observed to accumulate in acidic organelles, especially lysosomes, and showed an increase in fluorescence intensity corresponding to acidification. Cd exposure resulted in a dose-dependent decrease in Lysosomal Sensor Green DND-189 fluorescence, indicating that the lysosomal pH of Neuro-2a cells was disturbed compared with control cells ( Figure 3 E). In addition, using the RFP-GFP-LC3B reporter system, we observed that Cd treatment induced an increase in the ratio of yellow dots to red dots in Neuro-2a cells compared with control cells, and this increase indicated that the acidity of autophagolysosomes was significantly reduced ( Figure 3FG). In addition, the evaluation of CTSB and CTSD activities further supported the idea that lysosomal degradation was impaired in Cd-treated Neuro-2a cells ( Figure 3 HI). These findings suggest that Cd triggers blockade of autophagic flux by hindering the fusion of autophagosomes with lysosomes and impairing lysosomal function in Neuro-2a cells. Collectively, these results provide strong evidence that Cd exposure exacerbates APP accumulation and blocks autophagic flux in Neuro-2a cells.

[0094] Example 2: The deacylating activity of SIRT5 plays an important role in Cd exposure-induced APP accumulation, autophagic flow blockage, and decreased cell proliferation activity

[0095] A: Experimental Methods

[0096] 1. Plasmid construction and transfection:

[0097] Wild-type SIRT5 (SIRT5-WT), enzyme-inactive SIRT5 containing a mutation at amino acid position 158 (H to Y) (SIRT5-H158Y), and mutant RAB7A containing a mutation at amino acid position 31 [K to R (RAB7AK31R) or K to E (RAB7AK31E)] were cloned by GenePharma (Shanghai) and inserted into the pcDNA3.1 vector with a 3×Flag tag. The constructed expression plasmids were verified by Sanger sequencing. All plasmids were transfected using X-tremeGENE HP DNA transfection reagent (Roche, 6366236001), and the empty pcDNA3.1 vector was used as a negative control (Vector). Among them, the gene bank ID numbers of SIRT5 are: 23408 (human), 68346 (mouse); the gene bank ID numbers of RAB7A are 7879 (human), 19349 (mouse).

[0098] 2.IPA analysis

[0099] IPA analysis is an application for molecular mechanism mining of omics data developed based on QIAGEN Knowledge Base (https: / / www.qiagen.com / ), which is used to analyze, integrate and interpret data from omics experiments, such as RNAseq, miRNA, SNP microarrays, metabolomics, proteomics, and small-scale experiments that generate lists of genes and chemicals. Powerful analysis and search tools can analyze the classical pathways of differential molecular enrichment, the diseases and functions that may be affected, etc. in the context of the system. And it can reveal the key molecular networks that have a greater impact on diseases and functions, as well as the interaction relationships between molecules. The differentially expressed proteins obtained by TMT quantitative proteomics analysis in the previous part are entered into the IPA analysis program to obtain relevant pathway information.

[0100] Cytotoxicity detection, western blot, GFP-LC3B imaging, RFP-GFP-LC3B transfection, LysoSenorGreenDND-189 fluorescence intensity measurement, GFP-LC3B and LAMP2 co-localization, CTSB (cathepsin B) activity detection, and statistical analysis are detailed in Example 1.

[0101] B: Experimental results

[0102] In order to explore the potential molecular mechanism of Cd-induced neurotoxicity, the differentially expressed proteins obtained by TMT-based quantitative proteomics analysis were subjected to unique pathway analysis (Ingenuity Pathway analysis, IPA). Classical pathway analysis showed that the Sirtuin signaling pathway was among the top 10 pathways ( Figure 4 A). Subsequently, the molecular interaction network found a significant correlation between the upregulation of APP and the reduction of SIRT5 levels, indicating that SIRT5 may play a crucial role in APP accumulation ( Figure 4 B). We found that SIRT5 protein levels decreased in a dose-dependent manner in Neuro-2a cells treated with Cd ( Figure 4C). SIRT5 is an NAD+-dependent protein deacetylase that regulates the properties and functions of proteins mainly by catalyzing lysine modification, desuccinylation, deacrylation, and deglutarylation. To explore whether the activity of deacetylases in autophagic metabolism and Cd-induced neurotoxicity requires SIRT5 activation, we transfected Neuro-2a cells treated or not with Cd with plasmids containing wild-type SIRT5 (SIRT5-WT), the catalytically inactive mutant SIRT5-H158Y, or empty pcDNA3.1 (Vector). Overexpression of SIRT5-WT, but not overexpression of SIRT5-H158Y, counteracted the increase in APP accumulation and decrease in cell viability induced by Cd treatment. Figure 4 DE). The above experimental data show that SIRT5-dependent deacylation plays an important role in Cd-induced APP accumulation and neuronal cell death.

[0103] Furthermore, overexpression of SIRT5-WT, but not SIRT5-H158Y, effectively reversed the Cd-treated induced increase in MAP1LC3B-II levels and decrease in colocalization of GFP-LC3B with LAMP2 ( Figure 5 AC). In addition, overexpression of SIRT5-WT, but not SIRT5-H158Y, improved lysosomal acidification and promoted lysosomal proteolytic activity ( Figure 5 DG). These results confirm that overexpression of SIRT5, but not its inactive mutant SIRT5-H158Y, can antagonize Cd-induced blockade of autophagic flux and that SIRT5 enhances autophagy through its deacetylase activity, thereby alleviating the neurotoxic effects of Cd.

[0104] Example 3: Succinylated proteomics screened RAB7A, a key downstream molecule of SIRT5. SIRT5 antagonized cadmium-induced neurotoxicity by regulating autophagy flux through RAB7A

[0105] A: Experimental part

[0106] 1. Quantitative proteomics of succinylation without 4D labeling:

[0107] 1.1 Sample preparation

[0108] The samples were subjected to protein extraction according to conventional means in the prior art, followed by trypsin digestion, TMT labeling, and high performance liquid chromatography determination.

[0109] 1.2 LC-MS / MS analysis

[0110] (1) 3D mass spectrometer

[0111] Tryptophan peptides were dissolved in solvent A (0.1% formic acid, 2% acetonitrile / water) and directly loaded onto a homemade reverse phase analytical column (25 cm long, 75 μm i.d). The peptides were separated on an EASY-nLC1200UPLC system (Thermo Fisher Scientific) at a constant flow rate of 450 nL / min using a gradient from 5% to 25% solvent B (0.1% formic acid in 90% acetonitrile) for 60 min, from 25% to 35% in 22 min, ramped to 80% in 4 min, and then held at 80% for the last 4 min.

[0112] The separated peptides were analyzed in a Q ExactiveTM HF-X (Thermo Fisher Scientific) using a nano-electrospray ion source. The applied electrospray voltage was 2.0 kV. The full MS scan resolution was set to 60,000 and the scan range was 350-1600 m / z. Up to 20 of the most abundant precursors were then selected for MS / MS analysis and subjected to 30 s of dynamic exclusion. HCD fragmentation was performed under the condition of a normalized collision energy (NCE) of 28%. These fragments were detected in orbitrap at a resolution of 30,000. The fixed first mass was set to 100 m / z. The automatic gain control (AGC) target was set to 1E5, the intensity threshold was 3.3E4, and the maximum injection time was 60 ms.

[0113] (2) Database query The obtained MS / MS data were processed using the MaxQuant search engine (v.1.6.15.0). Tandem mass spectra were searched in the human SwissProt database (20422 entries) and linked to the reverse bait database. Trypsin / P was specified as the cleavage enzyme, and up to 2 missed cleavages were allowed. The first search mass tolerance for precursor ions was 20 ppm, the main search mass tolerance was 5 ppm, and the mass tolerance for fragment ions was 0.02 Da. Carbamylation on Cys was specified as a fixed modification, and protein N-terminal acetylation and oxidation on Met were specified as variable modifications. The FDR was adjusted to <1%.

[0114] 1.3 Bioinformatics methods

[0115] (1) Analysis method

[0116] Conventional GO analysis, domain analysis (sequence analysis software InterProScan was used to annotate the functional description of the identified protein domains), KEGG pathway analysis, and subcellular localization (Wolfpsort subcellular localization prediction software was used to predict subcellular localization) were used.

[0117] (2) Functional enrichment

[0118] 1) Gene ontology analysis enrichment

[0119] Proteins were divided into three categories based on GO annotation: biological process, cellular component and molecular function.

[0120] 2) Enrichment-based clustering: further hierarchical clustering based on the functional classification of differentially expressed proteins (such as GO, Domain, Pathway, Complex).

[0121] 3) Protein-protein interaction network All differentially expressed protein databases were added or sequences were searched for protein-protein interactions in the STRING database version 11.0.

[0122] 2. Preparation and identification of rabbit anti-RAB7A-succinyl-K31 antibody:

[0123] According to conventional methods in the prior art, healthy New Zealand rabbits were immunized to obtain rabbit anti-RAB7A-succinyl-K31 antibodies, that is, antibodies against RAB7A succinylated at position K31 were prepared. RAB7A succinylated at position K31 is referred to as: RAB7AK31 su .

[0124] Among them, the antigen design is shown in Table 1. According to the protein information, 2 modified peptides and 1 non-modified peptide were designed and synthesized. The two modified peptides were coupled with KLH and used for rabbit immunization. After immunization, serum was taken for ELISA and Western blotting serum screening. The results were positive (rabbit-r4 / 5 / 6) for purification (Table 2, Table 3, Table 4). ELISA confirmed the purified Ab4 / 5 / 6 antibodies (Table 5). Finally, Ab5 was selected for Western blotting to detect RAB7A K31 su .

[0125] Table 1: Antigen protein information

[0126] Name Sequences Modified type Weight Peptide1 NQYVN-(Succinyl)K-KFSNQYKC succinyl-modified peptide 1863.75 Peptide2 CNQYVN-(Succinyl)K-KFSN succinyl-modified peptide 1444.30 Peptide3 NQYVNKKFSNQYKC Non-modified control peptide 1763.85

[0127] Table 2: ELISA serum screening results (for RAB7A K31 su Peptide1)

[0128]

[0129]

[0130] Table 3: ELISA serum screening results (for RAB7A K31su Peptide2)

[0131]

[0132] Table 4: ELISA serum screening results (for unmodified Peptide3)

[0133] Table 5: ELISA detection of rabbit anti-RAB7A-succinyl-K31 antibody

[0134]

[0135] 3. Molecular docking

[0136] Molecular docking analysis was performed using the docking web server (GRAMM) and AutoDockTools-1.5.7 software. The SIRT5 crystal structure predicted by the AlphaFold protein structure database was docked with the RAB7A crystal structure obtained from the PubChem protein database (PDBID: 5JRH). Regularized protein identification was used to predict the essential amino acids within the binding site.

[0137] 4. Microscale Thermophoresis (MST)

[0138] To determine the solution equilibrium interaction constant between small molecules and proteins, we used microscale thermophoresis (MST) technology. Small molecules were used as ligands and proteins were used as targets. The proteins were labeled using the RED-NHS protein labeling kit. The small molecules were serially diluted in 16 gradients in the reaction buffer (50 mM HEPES buffer [pH = 7.4], containing 0.05% Tween20), and mixed in equal amounts and placed at room temperature for 20 minutes. The mixture sample was loaded into a capillary (Monolith NT.115 Capillary), and the thermophoresis signal was measured using Monolith NT.115 (Nano Temper). The changes in Kd values ​​were analyzed using Nano Temper analysis software.

[0139] 5. Surface plasmon resonance (SPR)

[0140] Surface plasmon resonance (SPR) measurements: SPR detection was performed by Top Science Co., Ltd. (Shanghai, China) as described above. Recombinant mouse proteins (SIRT5 or RAB7A) were purified from 293F cells. RAB7A protein was immobilized on a CM5 sensor chip (Cytiva, BR-1005-30). A two-fold variable concentration dilution series of SIRT5 was injected into the chip at a flow rate of 10 μl / min for 60 s and then dissociated (90 s). The experiments were performed on a BIAcore T200 instrument (v. 2.0 GE Medical, USA), and the K values ​​were determined according to the 1:1 Langmuir binding model using BIAcore T200458 evaluation software (v. 2.0 GE Medical, USA).

[0141] See Example 1 for cytotoxicity detection, western blot, RFP-GFP-LC3B transfection, LysoSenor GreenDND-189 fluorescence intensity measurement, GFP-LC3B and LAMP2 co-localization, CTSB activity detection, and statistical analysis.

[0142] B: Experimental results

[0143] Considering that SIRT5 can induce desuccinylation, deacryloylation, or deglutarylation of acyl-lysine residues, the levels of these post-translational modifications (PTMs) were evaluated in Neuro-2a cells treated or untreated with Cd for 72 h. After Cd treatment, the succinylation level of the protein showed a significant dose-dependent increase compared with the control group, while the malonylation and glutarylation levels did not change significantly ( Figure 6 The experimental results showed that the level of lysine succinylated protein in Neuro-2a cells treated with different concentrations (0, 1, 2 or 4 μM) of Cd for 72 hours was significantly increased.

[0144] To identify lysine succinylation (K-Succ) sites in proteins that were significantly altered by Cd treatment, quantitative proteomic analysis of succinylation without 4D labeling was performed by LC-MS / MS in Cd-treated Neuro-2a cells and control cells ( Figure 7 AD). Most of the modification sites are located within the KsuxxxxxxxK motif ( Figure 7 EF). Quantitative data with p values ​​< 0.05 and ratios > 1.5 or < 0.667 were considered differentially succinylated, with 559 sites in 233 proteins showing increased succinylation and 25 sites in 19 proteins showing decreased succinylation ( Figure 7 GI).

[0145] We performed KEGG enrichment analysis to further evaluate the pathways involved in these hypersuccinylated protein changes, and the results showed that the AD pathway was among the top 20 enriched entries ( Figure 8 A). GeneOntology (GO) terms involving “autophagy”, “lysosome”, and “intracellular PH” were selected for further study ( Figure 8 B). RAB7A is the only gene that overlaps with all selected GO terms and thus plays an important role in Cd-induced blockade of autophagic flux ( Figure 8 C). Lysine succinate analysis showed that there is a lysine site Lys31 (K31) in RAB7A, and after Cd treatment, the lysine site in Neuro-2a cells was significantly upregulated ( Figure 8 D) Lys31 is highly conserved among RAB7A homologs from humans to amoebas, indicating that it is essential for the function of RAB7A ( Figure 8 E). To verify the occurrence of succinylation at Lys31 in RAB7A, we developed an antibody that specifically recognizes succinylated K31 in RAB7A ( Figure 9-10 , Table 1-5). Dot blot analysis showed that the anti-Succ-K31 specific antibody selectively recognized the K31 succinylated peptides (1 and 2), but did not react with the unsuccinylated control peptide ( Figure 8 F), indicating that the prepared antibody has high specificity. Western blotting using this site-specific antibody showed that RAB7A K31 in Neuro-2a cells treated with Cd su Levels increased in a dose-dependent manner ( Figure 8 G). Collectively, these observations suggest that Lys31 is a critical succinylation site of RAB7A and may be involved in Cd-induced blockade of autophagic flux and neurotoxicity.

[0146] To verify the role of RAB7A K31 desuccinylation in Cd-induced autophagic flux blockade and neurotoxicity, we used two mutant plasmids: RAB7A K31R [Arginine (R) substituted for lysine (K) 31; mimicking the desuccinate state] and RAB7A K31E [Lysine (K) 31 was replaced by glutamic acid (E); mimicking negatively charged succinylation modification], and transfected into Neuro-2a cells treated or not treated with Cd. Activation of RAB7A promoted its binding to the key effector RILP, so that the activation state of Rab GTPase can be assessed by RILP binding. Compared with empty pcDNA3.1 (Vector), RAB7A K31RThe Cd-induced decrease in the colocalization coefficient of RILP with RAB7A was significantly reversed by AK31E, whereas the presence of RAB7AK31E did not produce any noticeable effect, suggesting that desuccinylation of RAB7A K31 promotes its activity ( Fig.11 AB). It is worth noting that RAB7A K31R , mimicking the deamberized state, rather than the amberized state of RAB7A K31E , significantly reversed the Cd-induced decrease in fusion between autophagosomes and lysosomes, as indicated by the increase in the colocalization coefficient between GFP-LC3B and LAMP2 ( Fig.11 CD). Similarly, RAB7A K31R Overexpression significantly promoted more red spots rather than yellow spots, and the increase in fluorescence intensity was associated with the decrease in pH of acidic organelles (such as lysosomes) and the proteolytic activity of CTSB in Cd-treated Neuro-2a cells ( Fig.11 EH). However, compared with the control vehicle, RAB7A K31E There was almost no change in the overexpression of Fig.11 EH) WB showed that RAB7A was transfected in the absence or presence of Cd. K31R 、RAB7A K31E In Neuro-2a cells treated with or without control vector, MAP1LC3B-II levels were consistently altered ( Fig.11 I). These results indicate that RAB7A K31R In Cd exposure, autophagy was restored. Interestingly, these K31R Rather than RAB7A K31E The changes induced by PD-1 abolished the abnormal accumulation and cytotoxicity of APP induced by Cd in Neuro-2a cells. Fig.11 JL). Collectively, these data suggest that desuccinylation of RAB7A at K31 can abrogate Cd-induced blockade of autophagic flux and neurotoxicity.

[0147] To determine whether SIRT5 has the function of desuccinating K31 in RAB7A and regulating RAB7A, we conducted preliminary studies to determine the direct interaction between SIRT5 and RAB7A. First, we used the crystal structure of SIRT5 predicted by the AlphaFold protein structure database and the crystal structure of RAB7A obtained from the PubChem protein database (PDBID: 5JRH) for molecular docking analysis. Multiple groups of residues were identified to be involved in the formation of hydrogen bonds between RAB7A and SIRT5 ( Fig.12A, Table 6). Notably, a favorable hydrogen bond was observed between Lys31 of RAB7A and Tyr255 of SIRT5 ( Fig.12 A). Subsequently, microscale thermophoresis (MST) was performed using purified recombinant proteins, and it was found that SIRT5 had a strong binding affinity for RAB7A, with an equilibrium dissociation constant (KD) of approximately 1.43 nM ( Fig.12 B). Then, surface plasmon resonance (SPR) experiments confirmed that SIRT5 strongly binds to RAB7A ( Fig.12 C). In addition, immunofluorescence analysis revealed that SIRT5 and RAB7A were significantly co-localized in the cells, and the co-localization was significantly reduced in Neuro-2a cells treated with Cd compared with control cells ( Fig.12 To explore the regulatory effect of SIRT5 on succinylation at RAB7A K31, we used anti-RAB7A Succ-K31 specific antibody to evaluate RAB7A K31. su After transfection of SIRT5-WT, SIRT5-H158Y or empty vector in Neuro-2a cells, overexpression of SIRT5-WT abolished Cd-induced RAB7A K31 su This suggests that K31 succinylation is a physiological PTM of RAB7A and is directly regulated by Sirt5-dependent desuccinylation ( Fig.12 F). In addition, overexpression of SIRT5-WT, but not SIRT5-H158Y, induced RAB7A to bind more to RILP, thereby alleviating the Cd-induced disruption of RAB7A colocalization with RILP ( Fig.12 GH). Collectively, these results suggest that SIRT5 desuccinylates RAB7A at K31, thereby regulating the activity of RAB7A in Cd-treated Neuro-2a cells. SIRT5 interacts with and desuccinylates RAB7A at Lys31 and antagonizes Cd-inhibited RAB7A activity.

[0148] Table 6: Hydrogen bonding determined by molecular docking

[0149]

[0150]

[0151] Example 4: Screening for small molecule compounds that may improve autophagic flux and antagonize cadmium-induced neurotoxicity based on SIRT5 and RAB7A binding sites

[0152] The prediction of the small molecule drug binding pocket of SIRT5 showed that its Site 1 position had a significantly higher PLB value than other sites. Fig.13 , and the screening of small molecule drugs based on Site1 will be considered in the future. Table 6 shows that a hydrogen bond can be formed between Lys31 of RAB7A and Tyr255 of SIRT5. The two proteins can interact through this position to regulate the succinylation of Lys31 of RAB7A. The results of the small molecule drug binding pocket analysis of SIRT5 showed that Tyr255 is located at Site1, which is a potential site for binding to small molecule drugs. That is, Tyr255 may be a key site for the binding of RAB7A and SIRT5 and succinylation regulation, as well as a key site for the binding of small molecule drugs to SIRT5. Small molecule drugs bind to Tyr255 of SIRT5, affecting the succinylation effect of SIRT5 on the Lys31 site of RAB7A, thereby eliminating the blockade of autophagic flow and reducing the neurotoxicity of cadmium. Therefore, Tyr255 of SIRT5 can be used as a more specific site for small molecule drug screening.

[0153] 1.6 million compounds with structural diversity were used to screen drugs based on the Site1 site of SIRT5. After molecular docking, the affinity data of the compounds in the screening results were analyzed, and the interactions between the virtually screened compounds and the amino acid residues were statistically analyzed. Then, effective targeted small molecule compounds were determined based on the docking scores and small molecule binding patterns. Each compound was molecularly docked with the Site1 region of SIRT5. The results showed that 9050 compounds in the compound library bound to the Site1 region, and their affinities were distributed between -5.99 kcal / mol and -12.10 kcal / mol (see Fig.14 ). Due to the difference in affinity distribution for binding to the Site1 region, a total of 930 compounds (red circles) with affinity values ​​less than -9 kcal / mol for binding to the Site1 region were selected. Due to the lack of reports on small molecule inhibitors targeting SIRT5 and studies on key residues for the interaction between SIRT5 and ligands, in order to further select small molecules, the interaction sites and force types of 930 compounds with the SIRT5 structure were analyzed, and 929 compounds were screened out. Among the 929 compounds, 212 compounds were able to interact with Tyr255 amino acids, and finally the 212 compounds that interacted with Tyr255 amino acids were retained for the next step of analysis. Through the analysis of drug properties, 114 compounds were finally selected, and 30 of them with high scores were finally selected for verification in subsequent experiments.

[0154] The above-mentioned computer-assisted virtual screening process can be entrusted to relevant biotechnology companies, that is, targeting the Tyr255 site of SIRT5, through molecular docking, affinity data analysis, and drugability analysis, the process of screening 114 compounds from 1.6 million compounds is a conventional method of existing technology and can be entrusted to complete. This scheme uses mouse SIRT5 protein for molecular docking and small molecule drug screening. The key point to achieve drug screening lies mainly in the confirmation of drug screening targets, that is, determining the small molecule drug binding pocket of SIRT5 (formed by amino acids 58-293 of SIRT5 protein) and the Tyr255 site as key targets for drug screening. Through the experimental studies of Examples 1-4, this scheme confirmed the key effects of SIRT5 on the succinylation of Lys31 site of RAB7A on neurotoxicity, APP accumulation and autophagy flow blockage caused by cadmium exposure, and discovered the binding relationship between the Tyr255 site of SIRT5 and the Lys31 site of RAB7A through analysis, and that the Tyr255 site of SIRT5 is located in the pocket Site 1 of SIRT5 with a high probability of binding to small molecule drugs. It is precisely because of the discovery of drug screening targets that subsequent drug screening can be implemented, and the potential drugs in Example 5 are obtained.

[0155] Example 5: Experimental screening of small molecule drugs that improve autophagic flux by acting on SIRT5 and RAB7A binding sites

[0156] A: Experimental part

[0157] 1. Cell culture: Mouse neuroblastoma cells (Neuro-2a cells) were treated with 4 μM CdCl2 for 72 hours. Neuro-2a cells were cultured in DMEM / H (Gibco, C11995500BT), supplemented with 10% fetal bovine serum (AUSGENEX, FBS500-S) and 1% penicillin / streptomycin (Beyotime, C0222), and cultured at 37°C in a 5% CO2 environment. CdCl2 stock solution (Sigma-Aldrich, 202908) was prepared with distilled deionized water and diluted with culture medium.

[0158] 2. Cell viability detection and drug screening: CCK-8 (Cell Counting Kit-8) cell proliferation-toxicity detection kit from Tokyo Tongren was used to detect cell viability. This is an orange-yellow formazan dye generated by the oxidation and reduction of WST-8 by intracellular dehydrogenases, which can be dissolved in the culture medium, and the amount of formazan generated is proportional to the number of living cells. The concentration of small molecule compounds was 10μM.

[0159] B: Experimental results: In response to the above results, we experimentally verified the small molecule drugs that can antagonize Cd-induced neurotoxicity. 30 small molecule drugs were added to the Cd exposure cell model established in the early stage. To observe the antagonistic effect of small molecule drugs on the neurotoxicity induced by Cd exposure. The screening results showed that drugs No. 10 and No. 19 had the most significant effects in antagonizing the toxic effects of Cd, and the cell survival rates of the remaining 28 compounds were statistically significantly lower than those of No. 10 or No. 19. The CCK8 experimental research results of No. 7, No. 10, No. 14, No. 18, No. 19, No. 21, No. 26, and No. 28 are listed below, as shown in Tables 7 and Fig.15 shown.

[0160] Table 7: Compound information

[0161]

[0162] We named drug No. 10 SR10 (merchant: Chemdiv; number: 6158-0051; https: / / www.chemdiv.com / ), the structural formula is shown in formula (1), and the mass spectrum is shown in Fig.16 Drug No. 19 is named SR19 (merchant: Chemdiv; number: D576-0016; https: / / www.chemdiv.com / ), the structural formula is shown in formula (2), and the mass spectrum is shown in Fig.17 , chromatogram see Fig.18 .

[0163]

[0164] Example 6: Experimental screening of small molecule compounds (SR10) that improve autophagic flux by acting on the SIRT5 and RAB7A binding sites

[0165] Molecular docking was used to analyze the interaction between SR10 and SIRI5 binding sites. The results showed that Tyr255 was found to be an effective binding site for SR10 and SIRT5. In previous studies, we found that the K31 site (succinylation site) of RAB7A binds to the Tyr255 site of SIRT5. Therefore, SR10 can affect the effect of RAB7A by binding to the Tyr255 site of Site1 ( Fig.19 ). We then treated the cells with different concentrations of SR10 to detect the effective concentration range of SR10 in cells. CCK8 detection found that SR10 did not produce cytotoxicity when the concentration was 10-40μM ( Fig. 20A). In order to clarify the optimal concentration of SR10 to antagonize Cd-induced neurotoxicity, the Cd-exposed cell model was treated with 5, 10, 20, and 40 μM SR10. We found that 10, 20, and 40 μM SR10 could effectively antagonize Cd-induced neurotoxicity, and 40 μM SR10 had the best effect ( Fig. 20 B).

[0166] In previous studies, we found that Cd exposure blocked cell autophagy, induced increased APP expression and inhibited cell activity. In order to study the specific mechanism by which SR10 affects the toxic effects of Cd, we used Western blot to detect the expression of autophagy-related proteins and APP in Neuro-2a cells with or without Cd (4μM, 72h) exposure before and after SR10 (40μM, 72h) treatment. The results showed that Cd exposure can significantly increase APP expression, while SR10 treatment can effectively inhibit the increase of APP caused by Cd exposure ( Fig.21 ); Cd exposure significantly increased the protein expression of MAP1LC3B and SQSTM1 and inhibited the expression of CTSB, indicating that autophagy was impaired, while SR10 treatment effectively inhibited the autophagy inhibition caused by Cd exposure ( Fig. 22 ), therefore, we believe that SR10 can antagonize the neurotoxicity of Cd by inhibiting the autophagy blockade caused by Cd exposure.

[0167] Example 7: Experimental screening of small molecule compounds (SR19) that improve autophagic flux by acting on the SIRT5 and RAB7A binding sites

[0168] Molecular docking was used to analyze the interaction between SR19 and SIRI5 binding sites. The results showed that SR19 can bind to the Tyr255 site of SIRT5 through hydrogen bonds ( Fig.23 ). In previous studies, we found that the K31 site (succinylation site) of RAB7A binds to the Tyr255 site of SIRT5. Therefore, SR19 can affect RAB7A by binding to the Tyr255 site of SIRT5. We then treated cells with different concentrations of SR19 to detect the effective concentration range of SR19 in cells. CCK8 detection found that SR19 did not produce cytotoxicity when the concentration was 10-40μM ( Fig.24 A). In order to clarify the optimal concentration of SR19 to antagonize Cd-induced neurotoxicity, 5, 10, 20, and 40 μM SR19 was used to treat the Cd-exposed cell model. We found that 5 and 10 μM SR19 could effectively antagonize Cd-induced neurotoxicity, and the antagonistic effect decreased at 20 and 40 μM. Among them, 10 μM SR19 had the best effect ( Fig.24 B).

[0169] In previous studies, we found that Cd exposure blocked cell autophagy to inhibit cell activity. In order to study the specific mechanism by which SR19 affects the toxic effects of Cd, we used Western blot to detect the expression of autophagy-related proteins and APP in Neuro-2a cells with or without Cd (4μM, 72h) exposure before and after SR19 (10μM, 72h) treatment. The results showed that Cd exposure significantly increased APP expression, while SR19 treatment effectively inhibited the increase in APP caused by Cd exposure ( Fig.25 ); Cd exposure significantly increased the protein expression of MAP1LC3B and SQSTM1 and inhibited the expression of CTSB, indicating that autophagy was impaired, while SR19 treatment effectively inhibited the autophagy inhibition caused by Cd exposure ( Fig.26 ), therefore, we believe that SR19 can antagonize the neurotoxicity of Cd by inhibiting the autophagy blockade caused by Cd exposure.

[0170] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. Use of a small molecule compound in the preparation of a drug for treating Alzheimer's disease, characterized in that: Its structural formula is:

2. Use of a small molecule compound according to claim 1 in the preparation of a drug for treating Alzheimer's disease, characterized in that: The small molecule compound is used to inhibit the accumulation of amyloid-β precursor protein caused by cadmium exposure.

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