Application of SIRT5 Acting on RAB7A K31 in Screening Drugs Antagonizing Impaired Autophagic Flux and Small Molecule Compound Drugs

Abstract

The present invention relates to the technical field of small molecule compound drug screening, and specifically relates to the application of SIRT5 acting on RAB7A K31 in screening drugs antagonizing impaired autophagic flux and small molecule compound drugs. This solution first reveals the interaction and regulatory relationship between SIRT5 and lysosomal Ras-related protein 7a (RAB7A). SIRT5 acts on the RAB7A K31 site, affects the level of protein succinylation, thereby reducing the activity of RAB7A, and affecting the occurrence and development of normal autophagic flux and related diseases. Based on the binding of the K31 site of RAB7A to the Tyr255 site of SIRT5, small molecule drugs that may improve autophagic flux can be screened through compound structure analysis, creating a basis for the development of new drugs. This technical solution can solve the technical problem of the lack of drug screening targets for autophagic flux impairment-related diseases in the prior art, and has an ideal application and promotion prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of drugs for antagonizing impaired autophagy flux, and specifically relates to the application of SIRT5 acting on RAB7A K31 in screening drugs for antagonizing impaired autophagy flux and small molecule compound drugs. Background Art

[0002] Autophagy is a process in eukaryotic cells for the degradation of abnormal proteins or defective organelles and the renewal of basic cell components. More and more evidence shows that the pathogenesis of Alzheimer's disease (AD) is related to impaired autophagy. In AD mice, immature autophagic vacuoles (AVs) accumulate in malnourished synapses or begin to accumulate before synapses and neurons degenerate. In addition, more and more studies have shown that autophagy may be involved in the metabolism of Aβ by regulating the production and elimination of Aβ. Autophagy promotes the degradation and elimination of amyloid-β precursor protein (APP) and its various cleavage products, such as Aβ and APP-cleaved C-terminal fragment (APP-CTF). Impaired autophagy may lead to the accumulation of APP and Aβ, and activating autophagy may be an effective treatment for AD. Therefore, studying the mechanism of autophagy and related regulatory molecules is an effective means to further obtain treatment methods for neurological diseases such as AD.

[0003] Sirtuin 5 (SIRT5) is a member of the Sirtuin family of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases, which regulates the physical and chemical properties of proteins through post-translational modifications (PTMs). SIRT5 regulates a variety of biological processes through its weak deacetylase activity and strong desuccinylase, deacryloylase, and deglutarylase activities. In addition, SIRT5 is related to the pathogenesis of a variety of human diseases, including AD, Parkinson's disease (PD), and cancer. Wu et al. observed a significant downregulation of SIRT5 in the brains of AD mice and found that ectopic expression of SIRT5 could counteract brain damage caused by oxidative stress while inhibiting the activation of microglia and astrocytes. In addition, exogenous Aβ1-42 oligomers (AβO) significantly reduced the expression of SIRT5 in neuronal SH-SY5Y cells. More and more evidence shows that SIRT5 plays an important role in autophagy regulation, thereby affecting the progression of various tumors. However, the exact effect and potential mechanism of SIRT5 in autophagy are still unclear, making it difficult to further develop related small molecule compound drugs targeting this molecule. Summary of the Invention

[0004] The present invention aims to provide the application of SIRT5 in screening drugs for antagonizing impaired autophagy flux, or antagonizing the neurotoxicity caused by cadmium exposure, or treating Alzheimer's disease, or promoting the de-succinylation of RAB7A, so as to solve the technical problem in the prior art that there is a lack of drug screening targets for diseases related to impaired autophagy flux.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The application of SIRT5 in screening drugs for antagonizing impaired autophagy flux, or antagonizing the neurotoxicity caused by cadmium exposure, or treating Alzheimer's disease, or promoting the de-succinylation of RAB7A, and the drug screening target is a small molecule drug binding pocket formed by the amino acids at positions 58-293 of the SIRT5 protein.

[0007] Furthermore, drug screening is carried out using the small molecule drug binding pocket formed by the amino acids at positions 58-293 of the SIRT5 protein; SIRT5 is used to induce the de-succinylation of RAB7A.

[0008] Furthermore, the drug is used to promote the Lys31 de-succinylation of RAB7A induced by SIRT5.

[0009] Furthermore, the Tyr255 site in the small molecule drug binding pocket is used to bind to Lys31 of RAB7A; drug screening is carried out using the Tyr255 site of SIRT5.

[0010] Based on the discovery of a new drug screening target, the principle and beneficial effects of this technical solution are as follows:

[0011] Through molecular structure analysis, it was found that the scoring of the small molecule drug binding pocket at Site1 of the SIRT5 molecule is much higher than that of other sites, and the small molecule drug binding pocket at Site1 contains the Tyr255 site. The Tyr255 of SIRT5 can bind to Lys31 of RAB7A, and Lys31 of RAB7A is the site of protein desuccinylation. SIRT5 regulates the desuccinylation of Lys31 of RAB7A, which can affect the ability of nerve cells to antagonize the neurotoxicity of cadmium exposure and the ability to regulate autophagic flux, and the above regulatory process is closely related to the development process of Alzheimer's disease. Therefore, the target for drug screening is the small molecule drug binding pocket formed by amino acids at positions 58-293 of the SIRT5 protein. In particular, it is the Tyr255 site in the small molecule drug binding pocket. Through the above drug screening sites, it can be used to screen potential drugs that antagonize impaired autophagic flux, or antagonize the neurotoxicity caused by cadmium exposure, or treat Alzheimer's disease, or promote the desuccinylation of RAB7A. By routinely simulating the binding ability between the Tyr255 site of the SIRT5 protein and the candidate drugs in the small molecule compound library through existing technologies, and through drug-likeness analysis, etc., potential drugs can be obtained. These potential drugs can then be further screened and studied through experiments to compare and obtain preferred candidate drugs. Through the above drug screening target of this technical solution, two drugs, SR10 and SR19, were screened and obtained, which can be used to antagonize impaired autophagic flux, antagonize the neurotoxicity caused by cadmium exposure, treat Alzheimer's disease, and promote the desuccinylation of RAB7A. Therefore, the key point in realizing drug screening lies in the confirmation of the exact drug screening target.

[0012] The discovery process of the drug screening target is as follows:

[0013] Cadmium exposure can cause neurotoxicity, leading to a decrease in nerve cell viability, the accumulation of amyloid-β precursor protein (APP), and the blockade of autophagic flux. Further studies showed that cadmium exposure does not interfere with the formation of phagocytic vesicles or the maturation of autophagosomes in Neuro-2a cells, but cadmium triggers autophagic flux blockade by hindering the fusion of autophagosomes and lysosomes in Neuro-2a cells and damaging lysosomal function. Through omics research and experimental verification, the potential molecular mechanism of cadmium-induced neurotoxicity was explored, and a significant correlation was found between the upregulation of APP and the decrease in SIRT5 levels, indicating that SIRT5 may play a crucial role in APP accumulation. Moreover, in cadmium-treated Neuro-2a cells, the SIRT5 protein level decreased in a dose-dependent manner. Further experimental studies found that SIRT5 plays an important role in cadmium-induced APP accumulation and nerve cell death.

[0014] Sirtuin 5 (SIRT5) is a member of the Sirtuin family of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases, which regulate the physical and chemical 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 led to a significant dose-dependent increase in the succinylation level of proteins, while there were no significant changes in the malonylation and glutarylation levels, etc. Combining the analysis of the autophagy-related pathway, 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. Lysosome-associated Ras-related protein 7a (RAB7A) belongs to the small GTPase family, and its members are considered to be mainly involved in regulating vesicle membrane trafficking, fusion with lysosomes and regulating lysosome function. Lys31 is highly conserved in RAB7A homologs from humans to amoeba, indicating that it is crucial for the function of RAB7A. Lys31 is the key succinylation site of RAB7A, and its succinylation status can affect the activity of RAB7A molecules, and thus participate in cadmium-induced autophagic flux blockade and neurotoxicity. Desuccinylated RAB7A at the Lys31 site can antagonize cadmium-induced autophagic flux blockade and neurotoxicity. The interaction between SIRT5 and the Lys31 site of RAB7A desuccinylates it and antagonizes the cadmium-inhibited activity of RAB7A. In the process of related drug design, the interaction between SIRT5 and RAB7A at the Lys31 site can be considered for drug design to regulate autophagy, diseases related to APP accumulation, and nerve damage caused by cadmium exposure, etc.

[0015] In summary, cadmium is released into the environment as an environmental pollutant in various natural and anthropogenic processes (i.e., fossil fuel combustion, mining, manufacturing of batteries and pigments, and plant growth, 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, and then induces autophagic flux damage by impairing autophagosome-lysosome fusion and lysosome function, resulting in abnormal accumulation of APP and neuronal cell death. Quantitative succinylome analysis showed that the decrease in SIRT5 expression led to an increase in the succinylation modification of the RAB7A Lys31 (K31) site, reduced the activity of RAB7A, hindered its recruitment of RILP, resulting in blocked autophagic flux, and ultimately aggravated the pathological progression and cognitive decline of Alzheimer's disease. Therefore, we considered whether there was a certain drug that could antagonize cadmium-induced autophagic flux impairment through the above pathway to achieve the purpose of improving the progression of Alzheimer's disease and the harm caused by cadmium-induced neurotoxicity.

[0016] The present technical solution also provides the application of SR19 in the preparation of a reagent for antagonizing autophagic flux damage, or in the preparation of a drug for treating Alzheimer's disease, or in the preparation of a drug for antagonizing the neurotoxicity caused by cadmium exposure, or in the preparation of a promoter for de-succinylation of RAB7A. The structural formula of SR19 is:

[0017]

[0018] Blockage of normal autophagic flux can lead to autophagic flux damage, which in turn affects normal physiological processes. SR19 of the present technical solution can antagonize autophagic flux damage and thus be used to treat diseases related to autophagic flux damage. In addition, SR19 can also be used in basic scientific research. As an antagonist reagent for autophagic flux damage, after acting on experimental subjects, it can regulate their autophagic flux, and then study the related action mechanisms and signaling pathways. As a small molecule compound, compared with general methods such as gene knockout, transgenic, and gene overexpression, the use method of small molecule compounds is more convenient and has more advantages.

[0019] RAB7A controls the processes of vesicle formation, transport, and fusion, and plays a crucial role in the regulation of autophagosome-lysosome fusion and autolysosome maturation. Although the activity of RAB7A is also regulated by many post-translational modifications of proteins, the fact that de-succinylation of RAB7A can affect autophagic flux is the first discovery of the present technical solution. The present technical solution has further screened out a promoter for de-succinylation of RAB7A. This promoter, SR19, is a small molecule compound, which is convenient to add and use as a basic scientific research tool. In the specific scientific research process, applying SR19 to experimental subjects can promote the de-succinylation process of RAB7A, and then study the downstream action pathways to further clarify a series of action mechanisms.

[0020] Accumulation of amyloid-β precursor protein will lead to an increase in the accumulation of amyloid-β, which in turn accelerates the disease process of Alzheimer's disease. The small molecule compound SR19 screened out by the present technical solution can effectively inhibit the accumulation of amyloid-β precursor protein caused by cadmium exposure, and thus achieve the treatment of Alzheimer's disease. The small molecule compound SR19 of the present solution can antagonize the neurocytotoxicity brought about by cadmium exposure and can be used as a drug for treating heavy metal poisoning.

[0021] Further, the autophagic flux damage is damage to the fusion of autophagosomes and lysosomes or damage to lysosomal function.

[0022] Further, the autophagic flux damage is caused by cadmium exposure.

[0023] Further, SR19 is used to inhibit the accumulation of amyloid-β precursor protein caused by cadmium exposure.

[0024] Furthermore, SR19 promotes the desuccinylation of the Lys31 site of RAB7A by binding to Tyr255 of SIRT5.

[0025] Furthermore, the working concentration of SR19 is 10 μM.

[0026] Regarding the drug screening based on the new target, the principle and beneficial effects of adopting the above technical solution are as follows:

[0027] During the new drug design process, the small molecule drug binding pocket was predicted based on the protein structure of SIRT5, and the binding site and succinylation site between SIRT5 and RAB7A were analyzed. Using 1.6 million structurally diverse compounds, drug screening was carried out 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 of the top-scoring ones were selected for subsequent experimental verification. Among the 30 small molecule compounds used for experimental research, not all of them have a significant promoting effect on the survival rate of nerve cells exposed to cadmium, and some compounds even further inhibit the activity of nerve cells exposed to cadmium. Through the screening of a large number of compounds in this solution, only two small molecule compounds (SR10 and SR19) were found to have an ideal antagonistic effect on cadmium toxicity, and showed a regulatory effect on autophagic flux and an inhibitory effect on APP accumulation. These two small molecule compounds can both be used to effectively inhibit the increase in APP caused by cadmium exposure, and thus be used to prepare drugs for the treatment of Alzheimer's disease. β-amyloid (Aβ) is one of the main pathological markers of Alzheimer's disease and is hydrolyzed from APP. Since cadmium exposure leads to the blockade of autophagic flux, which will affect the normal physiological activities, these two 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 for antagonizing autophagic flux damage (mainly hindering the fusion of autophagosomes and lysosomes and damaging lysosomal function), that is, an autophagic flux blockade antagonist, as a basic scientific research tool for basic scientific research to further study the physiological process changes caused by autophagic flux alteration. These two small molecule compounds can also be used as drugs for antagonizing the 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 for the desuccinylation of RAB7A, as a basic scientific research reagent for studying the physiological processes related to RAB7A desuccinylation. Small molecule drugs have great advantages as basic scientific research reagents, and can regulate the post-translational modification level of genes by direct application, and then regulate the function of the involved genes or proteins without performing complex gene knockout, introducing exogenous genes or other operations.

[0028] The two small molecule compounds, SR10 and SR19, each have their own characteristics. Although both can bind to Tyr255 of SIRT5 and affect the succinylation of the Lys31 site of RAB7A by SIRT5, the molecular structures of the two small molecule compounds are completely different and do not have structural similarities. Moreover, 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 - 40 μM, the effect of SR10 increases in a concentration-dependent manner, and the cell viability of cells treated with 5 - 40 μM SR10 is better than that of the blank control exposed to cadmium without drug administration, indicating that this drug not only has good ability to antagonize cadmium exposure, but also the relationship between the drug effect and the drug 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 play an effect of antagonizing cadmium exposure at a lower concentration. The effect of SR19 at the optimal concentration (10 μM) is better than that of SR10 at the optimal concentration (40 μM). However, the exertion of the effect of SR19 requires relatively high control of the drug concentration and needs to be controlled within 5 - 10 μM, otherwise it will have an additional toxic effect on cells. Applied to clinical practice, this drug may have strict requirements for dosage control, and due to large individual differences in the human body, it is difficult to determine the dosage for a single individual. Therefore, the two small molecule compounds each have their own advantages and both have the potential to be developed into drugs for antagonizing the neurotoxicity caused by cadmium exposure, drugs for treating Alzheimer's disease, reagents for antagonizing autophagy flux damage, promoters for RAB7A desuccinylation, etc. Description of the Drawings

[0029] Figure 1 Results of the Cd exposure experiment on Neuro-2a cells in Example 1 (A, B: Cell confluence after treating Neuro-2a cells with 0, 1, 2, and 4 μM CdCl 2 for 0, 24, 48, and 72 h detected by the IncuCyte ZOOM live cell analysis system, scale bar: 100 μm; C: Representative immunoblot images and quantitative analysis results of APP after treating Neuro-2a cells with 0, 1, 2, and 4 μM CdCl 2 ; D: Heat map of differentially expressed proteins in Neuro-2a cells treated or not treated with 4 μM, 72 h CdCl 2 ; E: 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 CdCl 2 ; G: 4 μM, 72 h CdCl 2Neuro-2a cells were treated with 25 μM chloroquine for 72 h or not, and representative immunoblot images and quantitative analysis results of MAP1LC3B; H, I: Representative images and quantitative analysis of GFP-LC3B puncta according to the treatment method of G; compared with the control group, *p < 0.05, **p < 0.01; ns: not significant).

[0030] Figure 2 Study on the effect of Cd exposure on phagosome formation or autophagosome maturation in Neuro-2a cells of Example 1 (A: Neuro-2a cells transfected with NC or Atg5-siRNA (si-Atg5) were treated with CdCl 2 (4 μM) for 72 h or not, and representative immunoblot images and quantitative analysis results of MAP1LC3B; B: Representative immunoblot images and quantitative analysis results of MAP1LC3B in Neuro-2a cells treated with Cd (4 μM) for 72 h with or without 3-MA (1 mM); C, D: Representative images and co-localization coefficient analysis of GFP-LC3B and SQSTM1 after Neuro-2a cells were treated with CdCl 2 (4 μM) for 72 h or not, scale bar: 5 μM; compared with the control group, **p < 0.01; compared with the Cd exposure group, #p < 0.05, ##p < 0.01).

[0031] Figure 3 Results of the study on the effect of Cd exposure on autophagosome-lysosome fusion and lysosome function in Neuro-2a cells of 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 h; C, D: Neuro-2a cells with or without CdCl 2(4 μM) Treatment for 72 hours, representative images of GFP-LC3B and LAMP2 and analysis of co-localization coefficient; E: LysoSensor DND-189 fluorescence intensity in Neuro-2a cells treated with different concentrations of Cd (0, 1, 2, 4 μM); F: Immunofluorescence analysis of Neuro-2a cells after double transfection of RFP-GFP-LC3B for 24 h and Cd (4 μM) treatment for 72 h; The RFP-GFP-LC3B system is a tool for detecting fusion, 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: Ratio of yellow dots to red dots in Neuro-2a cells transfected with RFP-GFP-LC3B and treated with Cd (4 μM) for 72 h; H, I: Activities of CTSB and CTSD in Neuro-2a cells treated with different concentrations of Cd (0, 1, 2, 4 μM) for 72 h, scale bar: 5 μM; Compared with the control group, *p < 0.05, **p < 0.01).

[0032] Figure 4 Experimental research results of SIRT5-dependent deacylation in Cd-induced APP accumulation and neuronal cell death in Example 2 (A: Top ten typical pathways identified by IPA; B: Key network 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 inactivated SIRT5 mutant plasmid (SIRT5-H158Y) with or without CdCl 2 (4 μM) Treatment for 72 h, representative Western blot images and quantitative analysis results of APP in Neuro-2a cells, and representative images and quantitative analysis results of cell fusion in Neuro-2a cells, 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).

[0033] Figure 5 Experimental results of SIRT antagonizing Cd-induced autophagic flux blockade phenomenon in Example 2 (In A - G, all Neuro-2a cells were transfected with Vector, SIRT5-WT or SIRT5-H158Y plasmids, with or without CdCl 2(4 μM) treatment for 72 h; 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: 5 μm; C: Statistical analysis of the co-localization coefficient of GFP-LC3B dots and LAMP2 dots; D, E: Representative immunofluorescence and statistical analysis of Neuro-2a cells transfected with RFP-GFP-LC3B for 24 h (yellow dots: red dots), scale bar: 5 μm; F, G: Fluorescence intensity of LysoSensor DND-189 and CTSB activity in Neuro-2a cells; compared with the control group, **p < 0.01; compared with the Cd exposure group, ##p < 0.01).

[0034] Figure 6 Results of the experiment for detecting the levels of lysine succinylated proteins in Neuro-2a cells treated with different concentrations (0, 1, 2, or 4 μM) of Cd for 72 h in Example 3 (A: Representative immunoblot images of lysine succinylated protein (K-Succ) and TUBULIN obtained using anti-succinyl lysine antibody and anti-TUBULIN antibody respectively; B: Representative immunoblot images of lysine malonylated protein (K-Mal) and TUBULIN obtained using anti-malonyl modification antibody and anti-TUBULIN antibody respectively; C: Representative immunoblot images of lysine-glutaryl modified protein (K-Glu) and ACTB obtained using anti-glutaryl modification antibody and anti-ACTB antibody respectively).

[0035] Figure 7 Basic statistical analysis (A, B), RSD and PCA of succinylation sites (C, D), motif analysis of the 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), statistical graphs of the number of sites and differentially expressed proteins (I; the number of sites with different succinylation modification levels and the proteins with differentially expressed succinylation sites in Cd-exposed Neuro-2a cells compared with control cells) of the quantitative global succinylation analysis MS data of Neuro-2a cells treated with or without Cd for 72 h in Example 3.

[0036] Figure 8Analysis 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 to screen the top 20 pathways; B: Enrichment GO analysis related to autophagy, lysosomes, and intracellular pH obtained from proteins with differentially expressed succinylated modification sites; C: Gene duplicates found in GO enrichment analysis; D: Mass spectrometry analysis results of succinylated RAB7A at the K31 site; E: K31 (red marked) is evolutionarily conserved in RAB7A; F: The anti-RAB7A-succinyl K31 antibody was identified by dot blotting. In the experiment, a nitrocellulose membrane was used to identify different amounts of succinyl k31 peptides (1 and 2) and unmodified peptides, and then the membrane was immunoblotted with the anti-RAB7A-succinyl K31 antibody; G: Representative immunoblot images and quantitative analysis results of RAB7A K31 after treating Neuro-2a cells with different concentrations of Cd (0, 1, 2, and 4 μM); compared with the control group, **p < 0.01). su The representative immunoblot picture and quantitative analysis result of

[0037] Figure 9 MS detection results of three polypeptides in Example 3 (A: RAB7A K31 su peptide 1; B: RAB7A K31 su peptide 2; C: Unmodified control).

[0038] Figure 10 WB results detected in HeLa cells using rabbit serum in Example 3.

[0039] Figure 11Study results on the effect of RAB7A desuccinylation at the K31 site in Example 3 on Cd-induced autophagic flux blockade and neurotoxicity (All Neuro-2a cells were transfected with blank vector or RAB7A K31R (simulating the desuccinylated state) or RAB7A K31E (simulating the succinylated state) plasmids, and treated with or without CdCl2 (4 μM) for 72 h; A, B: Representative immunofluorescence images and statistical results of the co-localization of RILP and RAB7A, scale bar: 5 μm; C, D: Representative immunofluorescence images of GFP-LC3B puncta and LAMP2 puncta and statistical analysis of the co-localization coefficient, scale bar: 5 μm; E, F: Representative immunofluorescence images shown by double-staining RFP-GFP-LC3B and the ratio of yellow puncta to red puncta, scale bar: 5 μm; G, H: Detection of LysoSensor DND-189 fluorescence intensity and CTSB activity; I, J: Representative immunoblot images and quantitative analysis results of MAP1LC3B and APP in Neuro-2a cells; K, L: Changes in the number of Neuro-2a cells and statistical analysis, scale bar: 100 μm; Compared with the control group, **p < 0.01; compared with the Cd-exposed group, #p < 0.05, ##p < 0.01).

[0040] Figure 12 Study results on the interaction mechanism between SIRT5 and RAB7A at the Lys31 site in Example 3 (A: Molecular docking model of RAB7A binding to the SIRT5 domain, yellow dotted lines indicate hydrogen bonds; B: MST analysis of the direct interaction between SIRT5 and RAB7A; C: SPR analysis of the SIRT5-induced RAB7A binding affinity curve; D, E: Immunofluorescence analysis of endogenous SIRT5 and RAB7A in Neuro-2a cells after treatment with or without CdCl 2 (4 μM) for 72 h; F-H: After transfection of Neuro-2a cells with blank vector or SIRT5-WT or SIRT5-H158Y plasmids, they were treated with or without CdCl 2 (4 μM) for 72 h; F: Representative immunoblot images and quantitative analysis results of RAB7A K31 su in Neuro-2a cells; 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-exposed group, ##p < 0.01).

[0041] Figure 13 Molecular structure prediction of SIRT5 in Example 4 (A: Prediction map of the small molecule compound binding pocket of SIRT5; B: Analysis of the specific amino acid sites in the SIRT5 binding pocket).

[0042] Figure 14The affinity score value of the compound in Example 4 in the Site1 region.

[0043] Figure 15 The screening results of the small molecule compound in Example 5 antagonizing Cd-induced cytotoxicity (compared with the control group, **p < 0.01; compared with the Cd exposure group, ##p < 0.01).

[0044] Figure 16 The mass spectrometry image of SR10 in Example 5.

[0045] Figure 17 The mass spectrometry image of SR19 in Example 5.

[0046] Figure 18 The chromatogram image of SR19 in Example 5.

[0047] Figure 19 The schematic diagram of the binding molecule of SIRT5 and SR10 in Example 6.

[0048] Figure 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 Cd-induced neurotoxicity; compared with the control group, **p < 0.01; compared with the Cd exposure group, #p < 0.05, ##p < 0.01).

[0049] Figure 21 The experimental study results of SR10 improving Cd-induced APP accumulation in Example 6 (representative immunoblot pictures and quantitative analysis results of APP in Neuro-2a cells; compared with the control group, *p < 0.05; compared with the Cd exposure group, #p < 0.05).

[0050] Figure 22 The research results of the effect of SR10 in Example 6 on Cd-induced autophagy flux blockade (A: Representative immunoblot pictures and quantitative analysis results of MAP1LC3B-II in Neuro-2a cells; B: Representative immunoblot pictures and quantitative analysis results of CTSB in Neuro-2a cells; C: Representative immunoblot pictures 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).

[0051] Figure 23 The schematic diagram of the binding molecule of SIRT5 and SR19 in Example 6.

[0052] Figure 24Experimental results of cytotoxicity and efficacy of SR19 in Example 6 (A: SR19 has no obvious cytotoxic effect; B: SR19 can antagonize Cd-induced neurotoxicity; compared with the control group, **p < 0.01; compared with the Cd exposure group, #p < 0.05, ##p < 0.01).

[0053] Figure 25 Experimental results of SR19 in Example 6 improving Cd-induced APP accumulation (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 exposure group, #p < 0.05).

[0054] Figure 26 Research results of SR19 in Example 6 affecting Cd-induced autophagy flux blockade (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 implementation manners

[0055] The present invention will be further described in detail below with reference to examples, but the implementation manners 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 channels.

[0056] Example 1: Cd exposure promotes APP accumulation and blocks autophagy flux in Neuro-2a cells

[0057] A: Experimental part

[0058] 1. Cell culture and CdCl 2 treatment

[0059] Mouse neuroblastoma cells (Neuro-2a cells) were obtained from the cell bank of the Institute of Biochemistry and Cell Biology (Shanghai, China, TCM29) and were treated with 0, 1, 2, or 4 μM CdCl 2 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% CO 2 environment. CdCl2 The stock solution (Sigma-Aldrich, 202908) was prepared with distilled deionized water and diluted with the 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.

[0060] 2. Cytotoxicity detection

[0061] To detect cell proliferation, Neuro-2a cells were seeded at a density of 3.5×10 3 cells per well in a 96-well ImageLock plate (EssenBioScience, USA). After 72 h of treatment, the plate was placed in an IncuCyte ZOOM instrument to record cell images. As described previously, cell confluence was analyzed using the IncuCyte ZOOM live cell analysis system (EssenBioScience, USA).

[0062] 3. Immunocytochemistry detection

[0063] 4. Tandem Mass Tag (TMT)-based quantitative proteomic analysis

[0064] (1) Protein extraction and peptide digestion

[0065] Samples (human, mouse, and rat serum samples need to remove serum high-abundance proteins first) were used to extract proteins by SDT (4% (w / v) SDS, 100 mM Tris / HCl pH 7.6, 0.1 M DTT) lysis method, and then protein quantification was performed by the BCA method. An appropriate amount of protein from each sample was digested with trypsin using the Filter aided proteome preparation (FASP) method, and peptide quantification (OD280) was carried out.

[0066] (2) TMT labeling: 100 μg of peptides were taken from each sample and labeled according to the instructions of the Thermo TMT labeling kit.

[0067] (3) High pH Reversed-Phase peptide fractionation: The labeled peptides in each group were mixed equally and fractionated using the HighpH Reversed-Phase Peptide Fractionation Kit.

[0068] (4) LC-MS / MS data acquisition: Each fractionated sample was separated using a nano-flow HPLC liquid phase system, Easy nLC.

[0069] (5) Protein identification and quantitative analysis: The original data of mass spectrometry analysis is in RAW format. Database search identification and quantitative analysis are performed using software Mascot 2.2 and Proteome Discoverer 1.4.

[0070] (6) Bioinformatics analysis

[0071] 1) GO functional annotation: Blast2GO is used to perform GO annotation on the target protein set. The process can be generally summarized into four steps: sequence alignment (Blast), GO term extraction (Mapping), GO annotation (Annotation), and InterProScan supplementary annotation (Annotation Augmentation).

[0072] 2) KEGG pathway annotation: The KEGG Automatic Annotation Server (KAAS) software is used to perform KEGG pathway annotation on the target protein set.

[0073] 3) Enrichment analysis of GO annotation and KEGG annotation: Fisher's Exact Test is adopted to compare the distribution of each GO category or KEGG pathway in the target protein set and the overall protein set, and enrichment analysis of GO annotation or KEGG pathway annotation is performed on the target protein set.

[0074] 4) Protein clustering analysis: First, the quantitative information of the target protein set is normalized (normalized to the interval (-1, 1)). Then, the Complexheatmap R package (R Version 3.4) is used to classify both the sample and protein expression levels in two dimensions (distance algorithm: Euclidean, linkage method: Average linkage), and a hierarchical clustering heatmap is generated.

[0075] 5) Protein-protein interaction network analysis: Based on the information in the STRING (http: / / string-db.org / ) database, the interaction relationships between target proteins are searched, and the CytoScape software (version number: 3.2.1) is used to generate an interaction network and analyze the network.

[0076] 5. Western Blotting: It includes total protein extraction, protein concentration quantification, SDS-PAGE electrophoresis, and membrane transfer, which are conventional methods of existing technologies.

[0077] 6. GFP-LC3B Imaging: Under physiological conditions, most of LC3B is diffusely distributed in the cytoplasm in a non-lipidated form (LC3B-I), and a small number exists as punctate (LC3B-II) lipidated forms bound to the autophagosome membrane. Therefore, LC3B is most commonly used for autophagosome labeling and dynamic observation of autophagic flux, and fluorescence imaging provides a more intuitive way of observation.

[0078] 7. Co-localization Analysis of GFP-LC3B with SQSTM1 / LAMP2: To further analyze the stages of autophagic flux, we performed co-localization analysis of GFP-LC3B with SQSTM1 or LAMP2 (lysosomal associated membrane protein 2). By observing the binding ability of autophagosomes to substrates during autophagosome formation and the fusion of autophagosomes with lysosomes, we determined whether autophagosome maturation and the fusion process of autophagosomes with lysosomes were affected.

[0079] 8. RFP-GFP-LC3B Transfection: To further observe the fusion of autophagosomes with lysosomes, we performed RFP-GFP-LC3B transfection experiments. RFP-GFP-LC3B appears as yellow puncta on autophagosomes, but the GFP fluorescence is sensitive to acidic environments and will quench under acidic conditions. If autophagosomes normally bind to lysosomes and the acidic environment in lysosomes is normal, only red puncta will be shown after binding. On the contrary, increased yellow puncta represent a fusion defect between autophagosomes and lysosomes or abnormal lysosomal pH values.

[0080] 9. Measurement of LysoSenor Green DND-189 Fluorescence Intensity: The LysoSensor Green DND-189 dye is an eosinophilic probe that accumulates in acidic organelles (such as lysosomes) and shows a pH-dependent increase in fluorescence intensity after acidification. Therefore, the lysosomal pH can be relatively quantified.

[0081] 10. Detection of CTSD (cathepsin D) Activity: The activity of CTSD in hippocampal tissues was detected using the Cathepsin D Activity Fluorometric Assay Kit.

[0082] 11. Detection of CTSB (cathepsin B) Activity: The activity of CTSB in hippocampal tissues was detected using the Cathepsin B Activity Fluorometric Assay Kit (BioVision, K140-100).

[0083] 12. Statistical Analysis

[0084] Data were analyzed using GraphPad Prism 9.0 software (GraphPad, USA) and expressed as mean ± SEM. Unpaired two-tailed t-tests were used for comparisons between two experimental groups, and one-way analysis of variance (ANOVA) was used for comparisons among multiple groups. Each experiment was repeated at least 3 times, and *p < 0.05 was defined as statistically significant.

[0085] B: Experimental results

[0086] The neurotoxicity of Cd was evaluated using the IncuCyte ZOOM live cell analysis system, and the results showed a significant decrease in Neuro-2a cell viability in a dose- and time-dependent manner (0, 1, 2, and 4 μM cadmium chloride (CdCl 2 ), for 24, 48, and 72 hours (h), respectively) ( Figure 1 A - B). In addition, an increase in APP expression with increasing dose was observed in Neuro-2a cells exposed to Cd for 72 hours ( Figure 1 C). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway proteomics confirmed that differentially expressed proteins involved in "autophagy" were significantly enriched in Cd-treated Neuro-2a cells compared with control cells ( Figure 1 D - E). In addition, dose-dependent changes in the MAP1LC3B-II protein level demonstrated that Cd activates autophagy in a dose-dependent manner ( Figure 1 F). Chloroquine (CQ) is an effective autophagy inhibitor that can hinder the fusion process of autophagosomes with lysosomes, thus inhibiting the late stage of autophagy. Our study showed that the combination of chloroquine (CQ) and Cd had no effect on the MAP1LC3B-II level or the number of puncta, indicating that Cd blocked the degradation of autophagic contents ( Figure 1 G - I). The above experimental results indicate that Cd exposure increases APP expression in Neuro-2a cells and blocks autophagic flux. Among them, autophagy is an ancient and evolutionarily conserved cytoplasmic component degradation pathway in eukaryotes. Generally speaking, autophagy includes several processes: autophagosome formation, transport of autophagic substrates to lysosomes, fusion of autophagosomes with lysosomes, and degradation of autophagosome contents by lysosomes. This dynamic process is called autophagic flux. Blocking the above biological processes is called autophagic flux blockade.

[0087] Given that autophagy is a complex process involving multiple stages, our study aimed to determine which specific stage would be damaged by Cd, leading to the 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 led to a decrease in the accumulation of Cd-triggered MAP1LC3B-II, indicating that Cd does not affect the formation of phagocytes ( Figure 2A). Additionally, similar results were obtained with the autophagy inhibitor 3-MA treatment ( Figure 2 B). The autophagosome maturation process was evaluated by the co-localization of GFP-LC3B and SQSTM1. Cd exposure also led to an increased co-localization of GFP-LC3B with SQSTM1 compared to control cells ( Figure 2 C-D). The above experimental results indicate that Cd exposure does not interfere with phagosome formation or autophagosome maturation in Neuro-2a cells.

[0088] The level of lysosome-associated membrane protein 1 (LAMP1), a key protein involved in lysosome formation, was elevated in Cd-exposed Neuro-2a cells compared to control cells ( Figure 3 A). The decreased co-localization of the autophagosome marker GFP-LC3B with the lysosome marker lysosome-associated membrane protein 2 (LAMP2) indicates that the fusion of autophagosomes and lysosomes was significantly reduced in Cd-treated Neuro-2a cells compared to control cells ( Figure 3 B-D). The eosinophilic probe Lysosensor Green DND-189 dye was observed to accumulate in acidic organelles, particularly lysosomes, and showed an increase in fluorescence intensity corresponding to acidification. Cd exposure led to a dose-dependent decrease in Lysosensor Green DND-189 fluorescence, indicating that the lysosomal pH of Neuro-2a cells was disturbed compared to control cells ( Figure 3 E). Additionally, using the RFP-GFP-LC3B reporter system, it was observed that Cd treatment induced an increase in the ratio of yellow dots to red dots in Neuro-2a cells compared to control cells, and this increase indicates a significant decrease in the acidity of autolysosomes ( Figure 3 F-G). Additionally, the evaluation of CTSB and CTSD activities further supported the view that lysosomal degradation was impaired in Cd-treated Neuro-2a cells ( Figure 3 H-I). These findings suggest that Cd triggers the blockade of autophagic flux by hindering the fusion of autophagosomes and lysosomes and impairing lysosomal function in Neuro-2a cells. Overall, these results provide strong evidence that Cd exposure exacerbates APP accumulation and blocks autophagic flux in Neuro-2a cells.

[0089] Example 2: The deacylation activity of SIRT5 plays an important role in Cd exposure-induced APP accumulation, autophagic flux blockade, and decreased cell proliferation viability

[0090] A: Experimental methods

[0091] 1. Plasmid construction and transfection:

[0092] Wild-type SIRT5 (SIRT5-WT), enzymatically inactivated 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 (RAB7A K31R) or K to E (RAB7A K31E)] 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 the 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).

[0093] 2. IPA analysis

[0094] IPA analysis is an application for mining the molecular mechanisms of omics data developed based on the 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 gene and chemical substance lists. Powerful analysis and search tools can analyze classical pathways enriched with differential molecules, possible affected diseases and functions, etc. in the context of the system. And it can reveal 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 from the TMT quantitative proteomics analysis in the previous part were input into the IPA analysis program to obtain relevant pathway information.

[0095] Cytotoxicity detection, western blot, GFP-LC3B imaging, RFP-GFP-LC3B transfection, LysoSenorGreen DND-189 fluorescence intensity measurement, co-localization of GFP-LC3B and LAMP2, CTSB (cathepsin B) activity detection, and statistical analysis are shown in detail in Example 1.

[0096] B: Experimental results

[0097] To explore the potential molecular mechanism of Cd-induced neurotoxicity, unique pathway analysis (Ingenuity Pathway analysis, IPA) was performed on the differentially expressed proteins obtained by TMT-based quantitative proteomics analysis. Classical pathway analysis showed that the Sirtuin signaling pathway was among the top 10 pathways (Figure 4 A). Subsequently, a molecular interaction network revealed a significant correlation between the upregulation of APP and the decrease in SIRT5 levels, indicating that SIRT5 may play a crucial role in APP accumulation ( Figure 4 B). We found that the protein level of SIRT5 decreased in a dose-dependent manner in Cd-treated Neuro-2a cells. Figure 4 C). SIRT5 is a NAD+-dependent protein deacetylase that mainly regulates the properties and functions of proteins by catalyzing lysine modifications, desuccinylation, deacrylation, and deglutarylation. To explore whether the activation of deacetylase is required for autophagy metabolism and Cd-induced neurotoxicity, we transfected Cd-treated or untreated Neuro-2a cells with plasmids encoding wild-type SIRT5 (SIRT5-WT), catalytically inactive mutant SIRT5-H158Y, or empty pcDNA3.1 (Vector). Overexpression of SIRT5-WT, but not SIRT5-H158Y, counteracted the increase in APP accumulation and the decrease in cell viability induced by Cd treatment. Figure 4 D-E). From the above experimental data, it can be seen that SIRT5-dependent deacylation plays an important role in Cd-induced APP accumulation and neuronal cell death.

[0098] In addition, overexpression of SIRT5-WT, but not SIRT5-H158Y, effectively reversed the increase in MAP1LC3B-II levels and the decrease in the co-localization of GFP-LC3B and LAMP2 induced by Cd treatment. Figure 5 A-C). In addition, overexpression of SIRT5-WT, but not SIRT5-H158Y, improved lysosomal acidification and promoted lysosomal proteolytic activity. Figure 5 D-G). These results confirmed that overexpression of SIRT5, but not the inactivated mutant SIRT5-H158Y, could antagonize Cd-induced autophagic flux blockade, and SIRT5 enhanced autophagy through its deacetylase activity, thereby reducing the neurotoxic effects of Cd.

[0099] Example 3: Key molecule RAB7A downstream of SIRT5 was screened by succinylome, and SIRT5 regulated autophagic flux through RAB7A to antagonize cadmium-induced neurotoxicity

[0100] A: Experimental section

[0101] 1. Label-free quantitative proteomics of succinylation:

[0102] 1.1 Sample preparation

[0103] Extract proteins from the samples by conventional means according to the prior art, and then perform trypsin digestion; TMT labeling; determination by high performance liquid chromatography.

[0104] 1.2 LC-MS / MS analysis

[0105] (1) 3D mass spectrometer

[0106] Dissolve the tryptophan peptides in solvent A (0.1% formic acid, 2% acetonitrile / water) and directly load them onto a self-made reversed-phase analytical column (25 cm long, 75 μm i.d.). The peptides are separated on an EASY-nLC 1200 UPLC system (Thermo Fisher Scientific) at a constant flow rate of 450 nL / min with a gradient from 5% to 25% of solvent B (0.1% formic acid in 90% acetonitrile) for 60 minutes, from 25% to 35% in 22 minutes, ramping up to 80% in 4 minutes, and then held at 80% for the last 4 minutes.

[0107] The separated polypeptides are analyzed in a Q ExactiveTM HF-X (Thermo Fisher Scientific) using a nanoelectrospray ion source. The applied electrospray voltage is 2.0 kV. The full MS scan resolution is set to 60,000, and the scan range is 350 - 1600 m / z. Then, up to 20 of the most abundant precursors are selected for MS / MS analysis, and 30 s of dynamic exclusion is performed. HCD fragmentation is carried out under the condition of a normalized collision energy (NCE) of 28%. These fragments are detected in the orbitrap at a resolution of 30,000. The fixed first mass is set to 100 m / z. The automatic gain control (AGC) target is set to 1E5, the intensity threshold is 3.3E4, and the maximum injection time is 60 ms.

[0108] (2) Database query Use the MaxQuant search engine (v.1.6.15.0) to process the obtained MS / MS data. The tandem mass spectra are searched in the human SwissProt database (20,422 entries) and linked to a reverse decoy database. Trypsin / P is designated as the cleavage enzyme, allowing up to 2 missed cleavages. The precursor ion first search mass tolerance is 20 ppm, the main search mass tolerance is 5 ppm, and the fragment ion mass tolerance is 0.02 Da. Carbamylation on Cys is designated as a fixed modification, and acetylation at the protein N-terminus and oxidation on Met are designated as variable modifications. The FDR is adjusted to <1%.

[0109] 1.3 Bioinformatics methods

[0110] (1) Analysis method

[0111] Conventional GO analysis, domain analysis (annotate the functional descriptions of the identified protein domains using the sequence analysis software InterProScan), KEGG pathway analysis, and subcellular localization (predict subcellular localization using the Wolfpsort subcellular localization prediction software) were adopted.

[0112] (2) Functional enrichment

[0113] 1) Gene ontology analysis enrichment

[0114] Proteins were classified into three categories: biological process, cellular component, and molecular function through GO annotation.

[0115] 2) Enrichment-based clustering: Further hierarchical clustering based on the functional classification of differentially expressed proteins (such as: GO, Domain, Pathway, Complex).

[0116] 3) Protein-protein interaction network Search for protein-protein interactions in the STRING database version 11.0 by adding all differentially expressed protein databases or sequences.

[0117] 2. Preparation and identification of rabbit anti-RAB7A-succinyl-K31 antibody:

[0118] Immunize healthy New Zealand rabbits by conventional methods of the existing technology to obtain rabbit anti-RAB7A-succinyl-K31 antibody, that is, prepare an antibody against RAB7A modified by succinylation at K31. RAB7A modified by succinylation at K31 is abbreviated as: RAB7AK31 su 。

[0119] Among them, the antigen design is shown in Table 1. According to the protein information, 2 modified polypeptides and 1 unmodified polypeptide were designed and synthesized. The 2 modified polypeptides were conjugated with KLH for rabbit immunization. After immunization, sera were taken for ELISA and Western blotting serum screening, and those with positive results (rabbit-r4 / 5 / 6) were used for purification (Table 2, Table 3, Table 4). ELISA confirmed the purified Ab4 / 5 / 6 antibody (Table 5). Finally, Ab5 was selected for Western blotting to detect RAB7A K31 su 。

[0120] Table 1: Antigen protein information

[0121] 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

[0122] Table 2: ELISA serum screening results (for RAB7A K31 su Peptide1)

[0123]

[0124]

[0125] Table 3: ELISA serum screening results (for RAB7A K31 su Peptide2)

[0126]

[0127] Table 4: ELISA serum screening results (for unmodified Peptide3) Table 5: ELISA detection of rabbit anti-RAB7A-succinyl-K31 antibody

[0128]

[0129] 3. Molecular docking

[0130] Molecular docking analysis was performed using the docking web server (GRAMM) and AutoDockTools-1.5.7 software. The crystal structure of SIRT5 predicted by the AlphaFold protein structure database was docked with the crystal structure of RAB7A obtained from the PubChem protein database (PDBID: 5JRH). Regularized protein identification was used to predict the essential amino acids within the binding site.

[0131] 4. Microscale Thermophoresis (MST)

[0132] To determine the solution equilibrium interaction constant between small molecules and proteins, we used the Microscale Thermophoresis (MST) technique. The small molecule was used as the ligand and the protein as the target. The protein was labeled using the RED-NHS protein labeling kit. The small molecule was serially diluted 16-fold in the reaction buffer (50 mM HEPES buffer [pH = 7.4] containing 0.05% Tween 20) and mixed in equal amounts and incubated at room temperature for 20 minutes. The mixture samples were loaded into the capillary (Monolith NT.115 Capillary) and the thermophoretic signal was measured using the Monolith NT.115 (Nano Temper). The change in the Kd value was analyzed using the Nano Temper analysis software.

[0133] 5. Surface plasmon resonance (SPR)

[0134] Surface Plasmon Resonance (SPR) measurement: 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. The 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 experiment was carried out on a BIAcoreT200 instrument (v.2.0 GE Healthcare, USA), and the K value was determined using BIAcoreT200 458 evaluation software (v.2.0 GE Healthcare, USA) according to the 1:1 Langmuir binding model.

[0135] Cytotoxicity detection, western blot, RFP-GFP-LC3B transfection, LysoSenor Green DND-189 fluorescence intensity measurement, co-localization of GFP-LC3B and LAMP2, CTSB activity detection, and statistical analysis are shown in Example 1.

[0136] B: Experimental results

[0137] Considering that SIRT5 can induce de-succinylation, de-acrylation or de-glutarylation of acyl-lysine residues, the levels of these post-translational modifications (PTMs) were evaluated in Neuro-2a cells treated with or without Cd for 72 hours. Compared with the control group, after Cd treatment, the level of protein succinylation showed a significant dose-dependent increase, while the levels of malonylation and glutarylation did not change significantly ( Figure 6 ). The experimental results showed that the level of lysine-succinylated proteins in Neuro-2a cells treated with different concentrations (0, 1, 2 or 4 μM) of Cd for 72 hours increased significantly.

[0138] To identify the lysine succinylation (K-Succ) sites in proteins significantly altered by Cd treatment, label-free quantitative proteomic analysis of succinylation was performed on Cd-treated Neuro-2a cells and control cells by LC-MS / MS ( Figure 7 A-D). Most of the modified sites were within the KsuxxxxxxxK motif ( Figure 7 E-F). Quantitative data with a p value < 0.05 and a ratio > 1.5 or < 0.667 were considered differentially succinylated. The succinylation of 559 sites in 233 proteins increased, and the succinylation of 25 sites in 19 proteins decreased ( Figure 7 G-I).

[0139] 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.

[0140] 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 K31RSignificantly reversed the decrease in the co-localization coefficient of Cd-induced RILP and RAB7A, while the presence of RAB7AK31E had no obvious effect, indicating that the de-succinylation of RAB7A K31 promoted its activity( Figure 11 A-B). Notably, RAB7A K31R , mimicking the de-succinylated state, rather than RAB7A K31E , mimicking the succinylated state, significantly reversed the decrease in the fusion between autophagosomes and lysosomes induced by Cd, as indicated by the increased co-localization coefficient between GFP-LC3B and LAMP2( Figure 11 C-D). Similarly, the overexpression of RAB7A K31R significantly promoted more red spots rather than yellow spots, and the increased fluorescence intensity was related to the decreased pH of acidic organelles such as lysosomes and the proteolytic activity of CTSB in Cd-treated Neuro-2a cells( Figure 11 E-H). However, compared with the control vector, the overexpression of RAB7A K31E in Cd-treated Neuro-2a cells showed little change( Figure 11 E-H). WB showed that the level of MAP1LC3B-II changed consistently in Neuro-2a cells transfected with RAB7A K31R , RAB7A K31E or the control vector in the absence or presence of Cd( Figure 11 I). These results indicate that RAB7A K31R restored the autophagy mechanism under Cd exposure. Interestingly, these changes induced by RAB7A K31R rather than RAB7A K31E eliminated the abnormal accumulation of Cd-induced APP and cytotoxicity in Neuro-2a cells( Figure 11 J-L). Overall, these data indicate that de-succinylated RAB7A at the K31 site can eliminate Cd-induced autophagic flux blockade and neurotoxicity.

[0141] To determine whether SIRT5 can de-succinylate the K31 site in RAB7A and regulate the function of RAB7A, we conducted preliminary studies to determine the direct interaction between SIRT5 and RAB7A. First, we performed molecular docking analysis using 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). Multiple sets of residues were identified to be involved in the formation of hydrogen bonds between RAB7A and SIRT5( Figure 12A, Table 6). Notably, a favorable hydrogen bond was observed between Lys31 of RAB7A and Tyr255 of SIRT5 ( Figure 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 ( Figure 12 B). Then, surface plasmon resonance (SPR) experiments confirmed the strong binding of SIRT5 to RAB7A ( Figure 12 C). In addition, immunofluorescence analysis revealed significant co-localization of intracellular SIRT5 and RAB7A, and the co-localization was significantly reduced in Cd-treated Neuro-2a cells compared to control cells ( Figure 12 D-E). To investigate the regulatory effect of SIRT5 on the succinylation of RAB7A at the K31 site, we used an anti-RAB7A Succ-K31 specific antibody to evaluate the level of RAB7A K31. After transfection with SIRT5-WT, SIRT5-H158Y, or empty vector, in the presence or absence of Cd in Neuro-2a cells, overexpression of SIRT5-WT eliminated the Cd-induced increase in RAB7A K31, but the SIRT5-H158Y mutant did not. This suggests that K31 succinylation is a physiological PTM of RAB7A and is directly regulated by Sirt5-dependent desuccinylation ( su F). In addition, overexpression of SIRT5-WT, but not SIRT5-H158Y, induced more binding of RAB7A to RILP, thus alleviating the interference of Cd-induced co-localization of RAB7A and RILP ( su G-H). Overall, these results indicate that SIRT5 desuccinylates the K31 site of RAB7A, thereby regulating the activity of RAB7A in Cd-treated Neuro-2a cells. SIRT5 interacts with RAB7A at the Lys31 site and desuccinylates it, and antagonizes the Cd-inhibited activity of RAB7A. Figure 12 F). In addition, overexpression of SIRT5-WT, but not SIRT5-H158Y, induced more binding of RAB7A to RILP, thus alleviating the interference of Cd-induced co-localization of RAB7A and RILP ( Figure 12 G-H). Overall, these results indicate that SIRT5 desuccinylates the K31 site of RAB7A, thereby regulating the activity of RAB7A in Cd-treated Neuro-2a cells. SIRT5 interacts with RAB7A at the Lys31 site and desuccinylates it, and antagonizes the Cd-inhibited activity of RAB7A.

[0142] Table 6: Hydrogen bond determination by molecular docking

[0143]

[0144]

[0145] Example 4: Screening of small molecule compounds that may improve autophagic flux and antagonize cadmium-induced neurotoxicity based on the binding site of SIRT5 and RAB7A

[0146] Predict the small molecule drug binding pocket of SIRT5 and find that the PLB value at Site1 is significantly higher than other sites. See Figure 13 . Subsequently, consider screening small molecule drugs based on Site1. Table 6 shows that a hydrogen bond can be formed between Lys31 of RAB7A and Tyr255 of SIRT5, and the two proteins can interact through this position to regulate the succinylation of Lys31 of RAB7A. The analysis results of the small molecule drug binding pocket of SIRT5 show that Tyr255 is exactly located at Site1 and is a potential site that can bind to small molecule drugs. That is, Tyr255 may be the key site for the binding of RAB7A and SIRT5 and the regulation of succinylation, as well as the key site for the binding of small molecule drugs to SIRT5. The binding of small molecule drugs to Tyr255 of SIRT5 affects the succinylation effect of the Lys31 site of RAB7A by SIRT5, thereby eliminating the blockage of autophagic flux and reducing the neurotoxicity of cadmium. Therefore, Tyr255 of SIRT5 can be used as a more specific site for small molecule drug screening.

[0147] Use 1.6 million structurally diverse compounds to screen drugs based on the Site1 site of SIRT5. After molecular docking, analyze the affinity data of the screened compounds, and count the interactions between the virtual screened compounds and amino acid residues. Then, determine the effective targeted small molecule compounds according to the docking score and the small molecule binding mode. Each compound is subjected to molecular docking with the Site1 region of SIRT5. The results show that 9050 compounds in the compound library bind to the Site1 region, and their affinities are distributed between -5.99 kcal / mol and -12.10 kcal / mol (see Figure 14 ). Due to the difference in the affinity distribution of the compounds binding to the Site1 region, a total of 930 compounds (red circles) with an affinity value less than -9 kcal / mol binding to the Site1 region are selected. Due to the lack of reports on small molecule inhibitors targeting SIRT5 and the study of the key residues of the interaction between SIRT5 and ligands, in order to further select small molecules, the interaction sites and force types between 930 compounds and the structure of SIRT5 are analyzed, and 929 compounds are screened out. Among the 929 compounds, 212 compounds can interact with the Tyr255 amino acid, and finally 212 compounds with interaction with the Tyr255 amino acid are retained for further analysis. Through the analysis of the drug-likeness properties, finally 114 compounds are selected, and finally 30 compounds with the top scores are selected for verification of subsequent experiments.

[0148] The above computer-aided virtual screening processes can all be entrusted to relevant biotechnology companies to complete, that is: for the Tyr255 site of SIRT5, through molecular docking, affinity data analysis, and druggability analysis, the process of screening 114 compounds from 1.6 million compounds is a conventional method of the prior art and can be entrusted to complete. This solution uses the SIRT5 protein of mice for molecular docking and small molecule drug screening. The key points for realizing drug screening mainly lie in the confirmation of the drug screening target, that is, determining the small molecule drug binding pocket of SIRT5 (formed by amino acids 58-293 of the SIRT5 protein) and the Tyr255 site as the key target for drug screening. Through the experimental studies in Examples 1-4 of this solution, it is confirmed that the succinylation of the Lys31 site of RAB7A by SIRT5 has a key impact on cadmium exposure-induced neurotoxicity, APP accumulation, and autophagy flux blockade, etc., and through analysis, the binding relationship between the Tyr255 site of SIRT5 and the Lys31 site of RAB7A is found, and the Tyr255 site of SIRT5 is located in the pocket Site1 of SIRT5 with a high possibility of binding to small molecule drugs. It is precisely due to the discovery of the drug screening target that subsequent drug screening can be carried out, and thus the potential drugs in Example 5 are obtained.

[0149] Example 5: Experimental screening of small molecule drugs that act on the binding site of SIRT5 and RAB7A to improve autophagy flux

[0150] A: Experimental part

[0151] 1. Cell culture: Mouse neuroblastoma cells (Neuro-2a cells) were treated with 4 μM CdCl 2 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% CO 2 environment. The stock solution of CdCl 2 (Sigma-Aldrich, 202908) was prepared with distilled deionized water and diluted with the culture medium.

[0152] 2. Cell viability detection and drug screening: The CCK-8 (Cell Counting Kit-8) cell proliferation-toxicity detection kit from Tokyo Chemical Industry was used to detect cell viability. This is based on the fact that the orange-yellow formazan dye generated after the oxidation-reduction of WST-8 by intracellular dehydrogenase can dissolve in the culture medium, and the amount of formazan generated is proportional to the number of live cells. The concentration of the small molecule compound was 10 μM.

[0153] B: Experimental Results: For the above results, we conducted experimental verification on small molecule drugs that can antagonize Cd-induced neurotoxicity. In the previously established Cd-exposed cell model, 30 small molecule drugs were added respectively to observe the antagonistic effect of small molecule drugs on Cd-exposed induced neurotoxicity. The screening results showed that the antagonistic effects of drugs No. 10 and No. 19 on Cd toxicity were the most significant, and the cell survival rates of the remaining 28 compounds were significantly lower than those of No. 10 or No. 19 in terms of statistical significance. Now, the CCK8 experimental results of No. 7, No. 10, No. 14, No. 18, No. 19, No. 21, No. 26, and No. 28 are listed in Table 7 and Figure 15 as shown below.

[0154] Table 7: Compound Information

[0155]

[0156] We named drug No. 10 as SR10 (Supplier: Chemdiv; Catalog No.: 6158-0051; https: / / www.chemdiv.com / ), and its structural formula is shown in Formula (1). The mass spectrum is shown in Figure 16 . We named drug No. 19 as SR19 (Supplier: Chemdiv; Catalog No.: D576-0016; https: / / www.chemdiv.com / ), and its structural formula is shown in Formula (2). The mass spectrum is shown in Figure 17 , and the chromatogram is shown in Figure 18 .

[0157]

[0158] Example 6: Experimental Screening of Small Molecule Compounds (SR10) Acting on the Binding Site of SIRT5 and RAB7A to Improve Autophagic Flux

[0159] Molecular docking was used to analyze the interaction between SR10 and the binding site of SIRI5. The results showed that Tyr255 was the effective binding site of 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 RAB7A by binding to the Tyr255 site of Site1 ( Figure 19 ). Subsequently, we treated cells with different concentrations of SR10 to detect the effective concentration range of SR10 in cells. CCK8 detection found that when the concentration of SR10 was 10 - 40 μM, it did not cause cytotoxicity ( Figure 20A). To determine the optimal concentration of SR10 for antagonizing Cd-induced neurotoxicity, Cd-exposed cell models were treated with SR10 at concentrations of 5, 10, 20, and 40 μM. We found that SR10 at 10, 20, and 40 μM could effectively antagonize Cd-induced neurotoxicity, and the effect of 40 μM SR10 was the best. Figure 20 B).

[0160] In previous studies, we found that Cd exposure blocked autophagy in cells, induced increased APP expression, and inhibited cell activity. To investigate the specific mechanism by which SR10 affects Cd toxicity, we used Western blot to detect the expression of autophagy-related proteins and APP in Neuro-2a cells with or without Cd (4 μM, 72 h) exposure before and after treatment with SR10 (40 μM, 72 h). The results showed that Cd exposure significantly increased APP expression, while treatment with SR10 effectively inhibited the increase in APP caused by Cd exposure. Figure 21 ); Cd exposure significantly increased the protein expression levels of MAP1LC3B and SQSTM1 and inhibited the expression of CTSB, indicating impaired autophagy, while treatment with SR10 effectively inhibited the autophagy inhibition caused by Cd exposure. Figure 22 Therefore, we believe that SR10 can antagonize Cd neurotoxicity by inhibiting autophagy blockade caused by Cd exposure.

[0161] Example 7: Experimental screening of small molecule compounds (SR19) that act on the binding site of SIRT5 and RAB7A to improve autophagic flux

[0162] Molecular docking was used to analyze the interaction of SR19 with the binding site of SIRI5. The results showed that SR19 could bind to the Tyr255 site of SIRT5 through hydrogen bonds. Figure 23 ) In previous studies, we found that the K31 site (succinylation site) of RAB7A binds to the Tyr255 site of SIRT5. Therefore, SR19 can exert an effect on RAB7A by binding to the Tyr255 site of SIRT5. Subsequently, we treated cells with different concentrations of SR19 to detect the effective concentration range of SR19 in cells. CCK8 assay showed that when the concentration of SR19 was 10 - 40 μM, it did not produce cytotoxicity. Figure 24 A). To determine the optimal concentration of SR19 for antagonizing Cd-induced neurotoxicity, Cd-exposed cell models were treated with SR19 at concentrations of 5, 10, 20, and 40 μM. We found that SR19 at 5 and 10 μM could effectively antagonize Cd-induced neurotoxicity, and the antagonistic effect decreased at 20 and 40 μM. The effect of 10 μM SR19 was the best. Figure 24 B).

[0163] In previous studies, we found that Cd exposure blocked autophagy to inhibit cell viability. To investigate the specific mechanism by which SR19 affects the toxic effect of Cd, we used Western blot to detect the expression of autophagy-related proteins and APP in Neuro-2a cells with or without Cd (4 μM, 72 h) exposure before and after treatment with SR19 (10 μM, 72 h). The results showed that Cd exposure significantly increased APP expression, while treatment with SR19 effectively inhibited the increase in APP caused by Cd exposure ( Figure 25 ); Cd exposure significantly increased the protein expression levels of MAP1LC3B and SQSTM1 and inhibited the expression of CTSB, suggesting impaired autophagy, while treatment with SR19 effectively inhibited the autophagy inhibition caused by Cd exposure ( Figure 26 ). Therefore, we believe that SR19 can antagonize the neurotoxicity of Cd by inhibiting the autophagy blockade caused by Cd exposure.

[0164] The above are only embodiments of the present invention, and specific technical solutions and / or common knowledge such as characteristics well known in the art are not described in detail herein. It should be noted 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 implementation effect of the present invention and the practicality of the patent. The protection scope required by this application shall be subject to the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.

Claims

1. The use of SR19 in the preparation of a drug for treating Alzheimer's disease, characterized in that: The structural formula of SR19 is:

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