Use of a compound that inhibits phosphorylation of armc10 in the manufacture of a medicament for antagonizing tin-induced nervous system damage

Abstract

The present application relates to the technical field of drugs related to nerve injury caused by heavy metal pollution, and particularly relates to application of a compound for inhibiting ARMC10 phosphorylation in preparation of a drug for antagonizing tin-induced nervous system injury. Exposure of trimethyltin chloride induces mitochondrial dysfunction of nerve cells, and a key mechanism thereof is abnormal phosphorylation of ARMC10 at a serine 43 site. The phosphorylated protein triggers excessive division of mitochondria, collapse of membrane potential and energy metabolism disorder, leading to neuron injury. Based on the above target, a compound for antagonizing tin-induced nervous system injury is screened, the compound can specifically inhibit protein phosphorylation, effectively reverse mitochondrial dynamics imbalance and dysfunction induced by trimethyltin chloride, and reduce neurotoxicity. The technical scheme can solve the technical problem that the prior art lacks an effective drug for antagonizing tin-induced nervous system injury, and the compound has application potential for treating nervous system injury and cognitive dysfunction caused by tin exposure.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology for nerve damage caused by heavy metal pollution, specifically to the application of a compound that inhibits ARMC10 phosphorylation in the preparation of a drug that antagonizes tin-induced nervous system damage. Background Technology

[0002] Trimethyltin chloride (TMT) is a typical toxic organotin compound that was once widely used in industry and agriculture, such as in antifouling coatings for ships, heat stabilizers for polyvinyl chloride (PVC) plastics, and pesticides. Although its use has been restricted in some countries, TMT can still be detected in food, drinking water, daily chemical products, and even human tissues due to its environmental persistence and bioaccumulation. Although the neurotoxic phenotype of TMT has been widely reported, its molecular mechanisms are not fully understood, especially the key signaling nodes mediating mitochondrial dysfunction and neuronal death, which greatly limits the development of targeted intervention strategies.

[0003] TMT exposure disrupts the structural and functional integrity of mitochondria, leading to neurotoxicity. TMT induces excessive reactive oxygen species (ROS) production and disrupts intracellular calcium homeostasis, resulting in mitochondrial dysfunction in neurons. In recent years, increasing evidence suggests that abnormal mitochondrial dynamics is a key pathological mechanism of TMT-induced mitochondrial dysfunction. Studies have shown that TMT damages mitochondrial structure and disrupts the mitochondrial respiratory chain in the mouse hippocampus, leading to decreased learning and memory abilities. Furthermore, artemisinin treatment can reverse TMT-induced neurotoxicity by improving mitochondrial swelling, vacuolation, and other structural abnormalities, increasing mitochondrial content, and reducing hippocampal neuronal apoptosis. Therefore, elucidating the regulatory mechanisms of mitochondrial dynamics in TMT-induced neurotoxicity is crucial for developing targeted neuroprotective strategies.

[0004] In recent years, ARMC10 (Armadillo Repeat-containing Protein 10), as a new member of the armadillo repeat-domain-containing protein family, has been increasingly found to participate in various key cellular processes, including cell signal transduction, mitochondrial dynamics regulation, autophagy, and cell fate determination. Studies have shown that ARMC10 is located on the outer mitochondrial membrane and is an important factor regulating mitochondrial fusion-fission balance; its expression or modification status directly affects mitochondrial morphology, energy metabolism, and neuronal survival. Of particular note is that ARMC10's function can be dynamically regulated through post-translational modifications (such as phosphorylation). However, whether ARMC10 is a key molecular hub in neurodegenerative diseases associated with environmental toxin exposure, and whether it can serve as a target for drugs antagonizing tin-induced nervous system damage, requires further research and verification. Summary of the Invention

[0005] The present invention aims to provide the application of a compound that inhibits ARMC10 phosphorylation in the preparation of a drug that antagonizes tin-induced nervous system damage, in order to solve the technical problem of the lack of effective drugs to antagonize tin-induced nervous system damage in the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The application of a compound in the preparation of a drug antagonizing tin-induced nervous system damage; the structural formula of the compound is shown in Formula III:

[0008]

[0009] Formula III.

[0010] Furthermore, compounds with the structural formula shown in Formula III are used to bind to ARMC10 and inhibit ARMC10 phosphorylation.

[0011] Furthermore, the compound with the structural formula shown in Formula III is used to inhibit the phosphorylation of serine at position 43 of ARMC10.

[0012] Furthermore, tin-induced nervous system injury refers to acute / subacute nervous system injury induced by organotin.

[0013] Furthermore, the organotin is trimethyltin chloride.

[0014] Furthermore, the neurological damage manifests as reduced neuronal activity, impaired neuronal mitochondrial function, and abnormal neuronal mitochondrial morphology.

[0015] This technical solution also provides a drug for antagonizing tin-induced nervous system damage, the structural formula of which is shown in Formula III:

[0016]

[0017] Formula III.

[0018] Furthermore, the compound with the structural formula shown in Formula III is used to inhibit the phosphorylation of serine at position 43 of ARMC10; the compound with the structural formula shown in Formula III has an affinity of -10.3 kcal / mol for ARMC10 protein.

[0019] Furthermore, tin-induced nervous system damage is specifically the nervous system damage induced by trimethyltin chloride.

[0020] This technical solution also provides a compound for antagonizing neuronal damage caused by acute / subacute trimethyltin chloride exposure, characterized in that its structural formula is shown in Formula III:

[0021]

[0022] Formula III.

[0023] This technical solution also provides an ARMC10 phosphorylation inhibitor (ARMC10(S43) phosphorylation inhibitor), the structural formula of which is shown in Formula III:

[0024]

[0025] Formula III;

[0026] It is used to inhibit the phosphorylation of serine at position 43 of ARMC10.

[0027] The application value of this ARMC10 (S43) phosphorylation inhibitor focuses on the development of targeted therapies and research tools for mitochondrial dysfunction-related diseases, especially in the fields of neurotoxic injury and neurodegenerative diseases, where it has significant translational potential. As a specific tool for studying mitochondrial dynamics mechanisms, phosphorylation at the S43 site of ARMC10 is a key switch regulating mitochondrial fission / fusion balance. This inhibitor can specifically block phosphorylation at this site, thereby affecting downstream ARMC10 pathways and mitochondrial function. This inhibitor can be used to verify the causal relationship of the pathway "ARMC10 phosphorylation → mitochondrial fragmentation → abnormal cell function," overcoming the limitations of long-term reliance on gene knockout / silencing in this field (gene manipulation has off-target effects and cannot achieve reversible regulation), and can help elucidate the role of mitochondrial dynamics in cell metabolism and stress response. As a positive control for drug screening in neurotoxic injury models, this inhibitor can serve as a positive control drug for neurotoxic injury induced by trimethyltin chloride (TMT) and other substances (the core pathology being elevated ARMC10 phosphorylation and mitochondrial dysfunction). It can also be used to evaluate the neuroprotective activity of new candidate compounds (comparing their effects with this inhibitor on improving mitochondrial membrane potential and other levels).

[0028] The technical principle of this technical solution is as follows:

[0029] This invention systematically studies the molecular mechanism of trimethyltin chloride (TMT)-induced neurological damage. First, phenotypic analysis confirms that TMT exposure can induce dual abnormalities in the structure and function of neuronal mitochondria, specifically manifested as increased mitochondrial fragmentation, a significant decrease in membrane potential, a large accumulation of mitochondrial reactive oxygen species (mtROS), and a weakened ATP synthesis capacity, which in turn leads to neuronal death and ultimately induces neurological dysfunction.

[0030] To clarify the core molecular target mediating mitochondrial dysfunction, this invention further examined the expression and phosphorylation status of neural cells after TMT treatment. The study found a significant upregulation of serine residue 43 (S43) of armadillo repeat protein 10 (ARMC10), located on the outer mitochondrial membrane. The increased phosphorylation level of ARMC10 (S43) was further confirmed by using a customized antibody specifically recognizing ARMC10 (S43) phosphorylation and parallel reaction detection technology. The construction of phosphorylated mutant and non-phosphorylated mutant plasmids clarified its crucial role. Therefore, this invention elucidates for the first time that ARMC10 (S43) phosphorylation is a key molecular switch for TMT-induced neuronal injury and a core target mediating TMT neurotoxicity.

[0031] Target-based drug screening, focusing on the key target ARMC10, constructs a multi-level, highly specific drug screening system encompassing virtual screening, in vitro validation, and target binding confirmation. Based on the optimized ARMC10 protein crystal structure, virtual screening was conducted, and the top five candidate compounds (named M1 to M5) were selected through molecular docking analysis and binding affinity ranking. Using Neuro-2a cells with TMT-induced damage as a basic model, a triple screening index was established, comprising ARMC10 (S43) phosphorylation level detection, mitochondrial morphology evaluation, and neuronal viability assay, to validate the in vitro activity of the five candidate compounds. The results showed that compound M3, at a concentration of 10 μM, significantly inhibited ARMC10 (S43) phosphorylation, effectively repaired mitochondrial fragmentation, and enhanced neuronal viability, exhibiting significantly superior overall activity compared to other candidate compounds.

[0032] In a TMT-induced Neuro-2a cell neural injury model, M3 exerts its neuroprotective effect through the following pathways:

[0033] It specifically inhibits ARMC10 (S43) phosphorylation, thereby reversing TMT-induced mitochondrial damage and restoring the normal tubular / reticular morphology of mitochondria. It significantly enhances mitochondrial membrane potential, reduces the generation of mitochondrial reactive oxygen species (mtROS), restores the activity of the mitochondrial respiratory chain complex, promotes ATP synthesis, and improves neuronal energy metabolism homeostasis. It antagonizes the effects of TMT on neuronal activity, ultimately achieving effective antagonism against TMT neurotoxicity.

[0034] The beneficial effects of this technical solution are as follows:

[0035] (1) This invention is the first to discover and confirm that ARMC10 (S43) phosphorylation is the core target of TMT-induced nerve damage, breaking through the limitations of existing technologies in the study of TMT neurotoxicity mechanisms. Compound M3 can specifically bind to ARMC10 and target the phosphorylation process at the S43 site, which has a significant advantage in strong targeting compared with traditional non-specific antioxidants and mitochondrial protectants.

[0036] (2) The virtual screening and in vitro validation screening system constructed in this invention first obtains compound molecules with high affinity for ARMC10 protein from more than 160,000 compounds using computer-aided drug design technology. These compounds can effectively antagonize TMT neurotoxicity, inhibiting target phosphorylation at the molecular level, repairing mitochondrial structure and function at the organelle level, and enhancing neuronal vitality at the cellular level. Their therapeutic effects cover the pathological process of TMT-induced nerve damage, solving the technical pain point of existing technologies lacking effective intervention methods for TMT neurotoxicity.

[0037] (3) It has a wide range of applications and high value in scientific research and clinical translation.

[0038] In clinical treatment: In addition to antagonizing acute / chronic nerve damage induced by organotin compounds such as TMT, this compound also has the potential to be applied to the treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, which are characterized by mitochondrial dynamics imbalance and abnormal ARMC10 phosphorylation, providing a new targeted treatment strategy for these diseases.

[0039] In terms of research tools: This compound can be used as a specific inhibitor to study the mechanism of action of ARMC10 phosphorylation in mitochondrial dynamics regulation, neuronal development and stress response. It overcomes the limitations of gene editing technology (which has off-target effects and cannot achieve reversible regulation) that has long been relied upon in this field, and provides an important tool for mitochondrial biology and neurotoxicology research. Attached Figure Description

[0040] Figure 1The experimental results of TMT reducing cell viability and inducing mitochondrial dysfunction in Example 1 are shown below: (A: Statistical results of cell confluence and typical images after treatment with different concentrations of TMT for 0, 6, 12, 18, and 24 hours; scale bar: 200 μm; B: Statistical results of mitochondrial membrane potential and typical images; sample size n=3; scale bar: 50 μm; mitochondria in a polarized state exhibit red fluorescent aggregates, i.e., polymeric state; depolarized mitochondria emit green monomeric fluorescence, i.e., monomeric state; C: Statistical results of mitochondrial reactive oxygen species (MitoROS) production and flow cytometry images; sample size n=3; D: Statistical results of ATP levels; sample size n=3; Except for confluence, all the above indicators were detected after Neuro-2a cells were exposed to 0, 2, 4, and 8 μM TMT for 24 hours; the bar charts all use the control group (0 μM TMT) value as 1.0; compared with the control group (0 μM TMT)...) Compared with TMT, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001; ns indicates no statistical significance).

[0041] Figure 2 The results of the TMT-induced excessive mitochondrial fragmentation experiment in Example 1 are shown below (A: Electron micrographs of cell sections treated with 8 μM TMT for 24 hours (treatment group) and untreated (control group); scale bars: 5 μm and 1 μm; B: Representative images and quantitative analysis of mitochondrial morphology in cells treated with 8 μM TMT for 24 hours (treatment group) and untreated (control group); sample size n=10; scale bars: 10 μm and 5 μm; bar charts are all based on the control group value as 1.0; compared with the control group, *** p<0.001; ns indicates no statistical significance).

[0042] Figure 3The following are experimental results of the expression of mitochondrial dynamics-related proteins in cells treated with TMT in Example 1: (A, B: Representative immunoblot images and quantitative analysis results of protein levels of optic atrophy protein 1 (OPA1), mitochondrial fusion protein 1 (MFN1), mitochondrial fusion protein 2 (MFN2), dynamics-related protein 1 (DRP1), and fibrillation protein 1 (FIS1) after cells were exposed to 0, 2, 4, and 8 μM TMT for 24 hours; bar charts are set with the 0 μM TMT group (as control group) as 1.0; sample size n=3; ACTB is the internal reference β-actin; ns indicates no statistical significance; C: Representative immunofluorescence staining images of OPA1, MFN1, MFN2, DRP1, and FIS1 protein expression after cells were exposed to 0 (control group) and 8 μM (treatment group) TMT for 24 hours; TOM20 is the mitochondrial marker mitochondrial outer membrane translocase 20; DAPI is the nuclear staining agent; D: Cells exposed to 0, 2, 4, and 8 μM TMT) Quantitative analysis results of OPA1, MFN1, MFN2, DRP1 and FIS1 protein expression after 24 hours; bar charts were performed with the 0 μM TMT group (as control group) value as 1.0; sample size n=3; scale bar: 40 μm; ns indicates no statistical significance).

[0043] Figure 4 The results of TMT-treated cells in Example 2 are shown in the phosphorylated proteomics analysis (A: number of differentially expressed modified peptides in cells of the treatment group (8 μM TMT, 24 hours) compared with the control group (0 μM TMT, 24 hours); B: cluster heatmap of differentially expressed phosphorylated peptides; red: upregulated expression; blue: downregulated expression).

[0044] Figure 5 The results of the study on the effect of TMT treatment on the phosphorylation level of ARMC10 protein at the S43 site in Example 2 (A: -log of quantitatively phosphorylated peptides in the treated group (8 μM TMT, 24 hours) and the untreated group (0 μM TMT, 24 hours)). 10 A: Volcano plot of (P-value) versus log2 (fold change); B: Detailed information on phosphorylation at S43 site of ARMC10 protein; C: Evolutionary conservation of S43 site of ARMC10 protein.

[0045] Figure 6The results of phosphorylated ARMC10 (S43) protein level detection in Example 2 are as follows: (A: Representative immunoblot and quantitative analysis of phosphorylated ARMC10 (S43) protein levels in Neuro-2a cells after treatment with 0, 2, 4, and 8 μM TMT for 24 hours; p-ARMC10 (S43) is phosphorylated ARMC10 (S43) protein; ARMC10 is ARMC10 protein; ACTB is internal reference β-actin; B: Quantitative analysis of A, showing the relative phosphorylation level of phosphorylated ARMC10 (S43) protein; with the control group (0 μM TMT) value as 1.0; compared with the control group (0 μM TMT), ** is p<0.01, *** is p<0.001).

[0046] Figure 7 The phosphorylated ARMC10 (S43) protein level obtained by parallel reaction monitoring in Example 2 (8 μM TMT-treated cells for 24 hours were the experimental group, and 0 μM TMT-treated cells for 24 hours were the control group, with the control group value as 1; sample size n=3; compared with the control group, ***p<0.001).

[0047] Figure 8 The results of Example 2 are as follows: Study on the effects of ARMC10 phosphorylation on TMT-treated Neuro-2a cells and their mitochondrial function (Neuro-2a cells were all transfected with empty plasmid and ARMC10). S43A Plasmid (simulating non-phosphorylated state) or ARMC10 S43D Plasmids (simulating phosphorylation) were then treated with 8 μM TMT or 0 μM TMT for 24 hours before subsequent detection; A: Changes and statistical analysis of Neuro-2a cell confluence; Scale bar: 200 μm; B: Representative images of mitochondrial morphology and quantitative analysis of mitochondrial length; Scale bar: 10 μm and 5 μm; C: Electron micrographs of Neuro-2a cell sections; Scale bar: 1 μm and 5 μm; D-1, D-2: Statistical results and typical images of mitochondrial membrane potential; Sample number n=3; Scale bar: 40 μm; Mitochondria in a polarized state appear as red fluorescent aggregates, i.e., polymeric state; Depolarized mitochondria emit green monomeric fluorescence, i.e., monomeric state; E: ATP level statistical graph; Sample number n=3; F: Statistical results of mitochondrial reactive oxygen species (ROS) production; Sample number n=3; S43A indicates transfection with ARMC10. S43A Experimental group of plasmids; S43D indicates transfection with ARMC10 S43DThe experimental group of plasmids; except for fusion, all bar charts use the empty plasmid (0μm) group (as control group) value as 1.0; compared with the empty plasmid (0μm) group, ** is p<0.01; compared with the empty plasmid (8μm) group, ## is p<0.01).

[0048] Figure 9 Example 3 shows the virtual screening process and the selected compounds M1-M5, along with experimental verification (A: Schematic diagram of the drug virtual screening process; B: Information on the top 5 compounds ranked based on affinity values; C: Compound affinity volcano plot; D: Cell viability of Neuro-2a cells after 24 hours of treatment with 8 μM TMT and any one of the 10 μM M1-M5 candidate compounds or no treatment; with the control group (without M3 and TMT) value as 1.0; E, F: Cell confluence (percentage of cells on the culture surface) of Neuro-2a cells after 24 hours of treatment with 8 μM TMT and any one of the 10 μM M1-M5 candidate compounds or no treatment; Scale bar: 200 μm; Compared with the control group (without candidate compounds and TMT), ** p < 0.01; Compared with the trimethyltin chloride group (only TMT added, no candidate compounds added), ## p < 0.01).

[0049] Figure 10 The results of Western blot analysis of Neuro-2a cells treated with TMT and M3 compounds in Example 3 are shown in the figure. (A: Western blot image; p-ARMC10(S43) is phosphorylated ARMC10(S43) protein; ARMC10 is ARMC10 protein; ACTIN is internal reference β-actin; B: Statistical analysis results of A, showing the relative phosphorylation level of phosphorylated ARMC10(S43) protein; the control group (without M3 and TMT) value is 1.0; compared with the control group (without M3 and TMT), ** is p<0.01; compared with the trimethyltin chloride group (only TMT added, no M3 added), ## is p<0.01).

[0050] Figure 11The results of the experimental study on the antagonistic effect of M3 on TMT-induced neurotoxicity, mitochondrial fragmentation, and dysfunction in Example 3 are as follows: (A: Statistical results and typical images of mitochondrial membrane potential; scale bar: 20 μm; mitochondria in a polarized state exhibit red fluorescent aggregates, i.e., polymer state; depolarized mitochondria emit green monomeric fluorescence, i.e., monomeric state; B: Statistical results of mitochondrial reactive oxygen species (ROS) production; C: Statistical results of ATP levels; D, E: Representative images and quantitative analysis of mitochondrial morphology; quantitative analysis combines 3 data points, totaling 9 independent cells; scale bars: 10 μm and 5 μm; all the above indicators were obtained in Neuro-2a cells after treatment with 8 μM TMT and 10 μM TMT.) M3 treatment or no treatment 24 hours later; all bar charts were calculated with the control group (no M3 and TMT added) as 1.0; sample size n=3; compared with the control group (no M3 and TMT added), **p<0.01; compared with the trimethyltin chloride group (only TMT added, no M3 added), ##p<0.01; ns: no statistical significance). Detailed Implementation

[0051] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0052] The key technical terms used in this solution are explained as follows:

[0053] Tin-induced nervous system damage (tin exposure leading to nervous system damage): This refers to the toxic process by which tin (especially organotin compounds) exposure causes pathological changes and functional abnormalities in the nervous system by interfering with cell metabolism and damaging neural structure and function. The core issue is the direct damage of organotin to neurons, mitochondria, etc. Further accumulation of tin-induced nervous system damage can lead to various subsequent harms. Tin (organotin)-induced nervous system damage can trigger a variety of further acute and chronic diseases, including:

[0054] (1) Chronic neurodegenerative disease

[0055] Long-term low-dose exposure can accelerate neuronal degeneration and increase the risk of developing neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease (the mechanism is related to oxidative stress and mitochondrial dysfunction).

[0056] (2) Neurasthenia syndrome

[0057] Symptoms include chronic headaches, insomnia, memory loss, and difficulty concentrating, which affect daily work and quality of life.

[0058] (3) Peripheral neuropathy

[0059] Organotin can damage peripheral nerves, leading to symptoms such as numbness, decreased sensation, and weakness in the limbs.

[0060] In summary, tin-induced nervous system injury (tin exposure leading to nervous system damage) is a type of poisoning disease with clinical significance, its core manifestation being nervous system damage. Therefore, nervous system damage is both a "symptom" of poisoning and a "major clinical manifestation" or "diagnostic criterion" of this poisoning disease. This protocol uses Neuro-2a cells as a research model to study and screen tin-induced nervous system injury (tin exposure leading to nervous system damage) and related drugs.

[0061] Neuro-2a cells:

[0062] The mouse neuroblastoma cell line, derived from neuroblastoma in the adrenal medulla of mice, is a commonly used in vitro model in neuroscience research. It possesses strong proliferative capacity and neuronal functional characteristics, and is frequently used in studies of neurotoxicity, neurodevelopment, neurodegenerative diseases, and drug screening. Compared to primary neurons (which are difficult to culture and have short survival periods), Neuro-2a has simpler culture conditions, stronger resistance to interference, and facilitates experimental procedures such as toxicity treatment and drug intervention. It can simulate the neuronal response to toxins (such as trimethyltin chloride), reflecting the damage of toxicity to nerve cells (mitochondrial function, morphology, etc.); it allows for rapid assessment of the protective / repairing effects of drugs on nerve cells; and it facilitates experiments such as gene editing and protein detection, elucidating neuronal molecular pathways.

[0063] According to literature reports, trimethyltin chloride (TMT) exhibits unique hippocampal selective neurotoxicity, particularly affecting the IC50 of Neuro-2a cells. 50Typically, 6-10 μM (24h) and 8 μM can induce significant damage while preserving some cell viability. This protocol used 8 μM TMT to treat Neuro-2a cells for 24h to establish an in vitro model of TMT-induced neuronal injury in Neuro-2a cells. This model stably exhibited a TMT-specific neurotoxic phenotype; more specifically, it was an in vitro model of neuronal injury caused by acute / subacute TMT exposure, reflecting the state of nerve cells during acute / subacute organotin (TMT) poisoning. Its core principle is to simulate neuronal damage caused by TMT exposure (characterized by moderate to high toxicity and reproducible mitochondrial / signaling pathway abnormalities), suitable for neurotoxicological mechanism research, drug screening, and biomarker validation. This model directly corresponds to the core pathological aspects of neuronal injury caused by acute / subacute TMT (occupational, environmental) exposure or accidental poisoning, providing cellular-level experimental support for the mechanism analysis of acute poisoning and the validation of therapeutic targets. Without intervention in cases of acute / subacute organotin (TMT) poisoning, it may develop into neurological diseases such as Alzheimer's and Parkinson's.

[0064] ARMC10 protein (Armadillo repeat containing 10; UniProt database unique identifier UniProtID: Q9D0L7): ARMC10, or Armadillo repeat-containing protein 10, is a conserved protein expressed in eukaryotes. Its core characteristic is the presence of multiple armadillo repeat (ARM) domains in its amino acid sequence. These domains, typically composed of 40-45 amino acid residues, mediate protein-protein interactions and are key functional domains for intracellular signal transduction and structural support. ARMC10 proteins are highly conserved across species, especially at key functional sites such as mitochondrial localization signals. This characteristic is a crucial structural basis for maintaining the consistency of its core biological functions across species.

[0065] Phosphorylated ARMC10 (S43) protein: can be abbreviated as p-ARMC10 (S43); a phosphorylated protein formed by phosphorylation of serine at position 43 of the ARMC10 protein.

[0066] Example 1: Trimethyltin chloride (TMT) exposure promotes mitochondrial fragmentation and dysfunction in Neuro-2a cells

[0067] Mouse neuroblastoma cells (Neuro-2a cells) were obtained from the cell bank of the Institute of Biochemistry and Cell Biology (Shanghai, China, TCM29). Cells were treated with 0, 2, 4, or 8 μM TMT for 24 hours (or other specified treatment durations) before subsequent assays. 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 and 5% CO2. The TMT stock solution (Sigma-Aldrich, 146,498) was prepared with distilled deionized water and diluted with culture medium.

[0068] After treating cells according to the above TMT treatment method, cell confluence, mitochondrial membrane potential, mitochondrial reactive oxygen species (MitoROS) production, mitochondrial morphology, and mitochondrial dynamics-related proteins were measured. The detection methods were standard existing techniques, and are roughly described as follows: For cytotoxicity detection (detecting cell proliferation), Neuro-2a cells were used at a density of 1 × 10⁻⁶ cells per well. 4 Cells were seeded at density in 96-well plates. Cell images were recorded after different treatment times. Cell confluence was analyzed using the Induced Cell Zoom live cell analysis system (EssenBioScience, USA) (characterized by confluence: percentage of cells covering the culture dish surface). For cell membrane potential detection, JC-1 staining was performed on cells treated with different TMT concentrations for 24 hours according to the manufacturer's instructions (Beyotime, C2006). Cellular ATP levels were quantified using an ATP assay kit (Thermo Fisher, A22066). MitoSOX was used to analyze mitochondrial ROS. TM Mitochondrial reactive oxygen species (mtROS) were quantified using a red fluorescent probe (ThermoFisher Scientific, M36008). Mitochondrial morphology was analyzed using the MitoTracker Deep Red FM fluorescent probe (Invitrogen, M22426). Neuro-2a cells were cultured at a density of 1×10⁻⁶. 4Cells were seeded at a density of 100 cells / well, treated with TMT for 24 h, and then fixed, dehydrated, embedded, sectioned, and observed under an electron microscope. Total protein was extracted from cells after TMT treatment and analyzed by Western blotting (WB) to determine the expression of proteins related to mitochondrial dynamics. Immunocytochemistry was also performed on cells after TMT treatment to determine the expression of these proteins. Statistical analysis was performed on the results, expressed as mean ± SEM. Unpaired two-sided t-tests were used for comparisons between two experimental groups, and one-way ANOVA was used for comparisons among multiple groups. Each experiment was repeated at least three times, and *p < 0.05 was defined as statistically significant.

[0069] The experimental results are as follows: This study used a live-cell analysis system to assess the neurotoxicity of TMT. Specifically, after Neuro-2a cells were exposed to TMT at concentrations of 0, 2, 4, and 8 μM for 24 hours, their survival rate decreased in a dose- and time-dependent manner. Figure 1 A). Given that mitochondrial dysfunction is considered to play a key role in TMT-induced neurotoxicity, we further investigated whether TMT exposure impaired mitochondrial function in Neuro-2a cells. This was determined by measuring mitochondrial membrane potential (Δψ). m The functional status of mitochondria was assessed by measuring mitochondrial reactive oxygen species (mtROS) levels and adenosine triphosphate (ATP) production. Results showed that after 24 hours of treatment with 8 μM TMT, the mitochondrial membrane potential of Neuro-2a cells significantly decreased. Figure 1 B), mtROS levels were significantly elevated ( Figure 1 C) and intracellular ATP levels were significantly reduced ( Figure 1 D). In summary, these results indicate that TMT exposure is cytotoxic to Neuro-2a cells and impairs their mitochondrial function. Mitochondrial dynamics dysregulation is a key pathogenic mechanism in a range of diseases characterized by mitochondrial dysfunction. To clarify whether TMT affects mitochondrial dynamics, we evaluated mitochondrial morphology after TMT treatment. MitoTracker staining results showed that after 24 hours of exposure to 8 μM TMT in Neuro-2a cells, mitochondria exhibited significant fragmentation and a significant reduction in mitochondrial length. Figure 2 A). Electron microscopy revealed structural damage in the mitochondria of Neuro-2a cells in the TMT-treated group, including division, swelling, cristae breakage, and mitochondrial membrane rupture. Figure 2 B).

[0070] Mitochondrial dynamics are precisely regulated by a set of key proteins: dyskinetic-associated protein 1 (DRP1) and fission protein 1 (FIS1) dominate mitochondrial fission, while mitochondrial fusion proteins (MFN1 and MFN2) and optic atrophy protein 1 (OPA1) regulate mitochondrial fusion. Notably, although we demonstrated impaired mitochondrial dynamics, the expression levels of the key proteins OPA1, MFN1, MFN2, DRP1, and FIS1 remained unchanged. Figure 3 A and B). Immunofluorescence staining results further validated this finding, showing no statistically significant difference in the overall fluorescence signal intensity of these proteins in the cell population (A and B). Figure 3 (C and D). In summary, these results strongly demonstrate that TMT impairs mitochondrial dynamics; furthermore, the fact that the expression levels of key mitochondrial proteins remained unchanged suggests the existence of other mechanisms regulating TMT-induced neurotoxicity.

[0071] Example 2: Study on the effect of phosphorylation at serine site 43 (S43) of ARMC10

[0072] Neuro-2a cells were treated with 8 μM TMT for 24 hours. Then, phosphorylation modification proteomics analysis based on tandem mass tag was performed on the treated cells (treated group) and untreated cells (control group) (three replicates were set for each group). The analysis process is a routine procedure of existing technology and can be outsourced to a biotechnology company. The detection process will not be described in detail here.

[0073] Mitochondrial homeostasis and functional status are highly dependent on the regulation of protein phosphorylation. Phosphoryptiform proteomics analysis detected 922 upregulated phosphorylation sites and 949 downregulated phosphorylation sites (fold change >1.2 or <0.83, P<0.05). For details on the number of differentially expressed modified peptides in Neuro-2a cells of the treatment group compared to the control group, please refer to [link to relevant documentation]. Figure 4 A, For a detailed clustering heatmap of differentially expressed phosphorylated peptides, see [link to heatmap]. Figure 4 B. Analysis showed that TMT treatment significantly upregulated the phosphorylation level of a single amino acid site, serine 43 (S43), in the ARMC10 protein in Neuro-2a cells. Figure 5 A and B). ARMC10 is an anchoring protein located on the outer mitochondrial membrane, containing a classic ankyrin repeat domain that mediates protein-protein interactions. Sequence homology analysis showed that the S43 site is highly conserved across multiple species, suggesting its important role in the function of ARMC10. Figure 5 C).

[0074] Based on the above research results, a rabbit anti-phosphorylated ARMC10 (S43) antibody was prepared using conventional methods. This antibody can specifically recognize the aforementioned phosphorylated protein and can be used for subsequent research. Experimental results showed that the prepared rabbit anti-phosphorylated ARMC10 (S43) specific antibody selectively binds to phosphorylated peptides containing the S43 site, without reacting with the non-phosphorylated control peptide, confirming the antibody's high specificity. Western blot (WB) experiments using this site-specific antibody revealed that the phosphorylation level of ARMC10 at the S43 site in Neuro-2a cells increased in a concentration-dependent manner after TMT treatment. Figure 6 A and B). The specific protein level of phosphorylated ARMC10 (S43) is determined by... Figure 6 The relative phosphorylation level of phosphorylated ARMC10 (S43) protein in B was quantified. The calculation method was as follows: First, the relative phosphorylation level of ARMC10 (S43) protein in B was... Figure 6 The gray values ​​of the p-ARMC10(S43) band and the ACTB band in protein A are calculated as follows: the ratio represents the relative content of p-ARMC10(S43) per unit of total protein; the gray values ​​of the ARMC10 band and the ACTB band are then calculated as follows: the ratio represents the relative content of total ARMC10 per unit of total protein. Finally, the two ratios are divided again, and the result is used for statistical analysis of the relative phosphorylation level of phosphorylated ARMC10(S43) protein.

[0075] The level of p-ARMC10 (S43) in Neuro-2a cells treated with 8 μM TMT for 24 hours was quantified using parallel reaction monitoring (PRM) technology. Detailed results can be found in [link to results]. Figure 7 Further evidence confirms that TMT treatment increases phosphorylation at the S43 site of ARMC10. These results indicate that serine 43 (S43) is a key phosphorylation site in the ARMC10 protein, and its phosphorylation is involved in the TMT-mediated neurotoxicity process.

[0076] To confirm the role of the S43 phosphorylation site of ARMC10 in TMT-induced neurotoxicity, two mutant plasmids of the ARMC10 protein were constructed using conventional methods: ARMC10 S43A (The serine (S) at position 43 is replaced with alanine (A), and the corresponding codon is mutated from TCC to GCC to simulate the non-phosphorylated state) and ARMC10 S43D(Serine at position 43 (S) is replaced with aspartic acid (D), and the corresponding codon is mutated from TCC to GAC to simulate phosphorylation.) The empty plasmid is the commercially available pcDNA3.1-C-3×FLAG (a commonly used mammalian expression vector for efficient expression of recombinant proteins with a C-terminal 3×FLAG tag in eukaryotic cells). The expression plasmid is constructed by inserting the target gene sequence into the corresponding multiple cloning site. The plasmids corresponding to the two mutants are ARMC10. S43A Plasmid (simulating non-phosphorylated state) or ARMC10 S43D The preparation process of the plasmid (simulating phosphorylation) will not be detailed here. The mutant plasmid and the empty plasmid were transfected into Neuro-2a cells using standard techniques. The cells were then treated with 8 μM TMT for 24 hours or without TMT treatment. Cell confluence statistics and mitochondrial membrane potential Δψ were then analyzed. m Analysis included ATP level analysis, mitochondrial reactive oxygen species (MitoROS) production analysis, cell section preparation and electron microscopy analysis, and mitochondrial observation.

[0077] The results showed that the non-phosphorylated mimic mutant ARMC10 S43A It can significantly reverse the decrease in cell viability caused by TMT treatment, while the phosphorylation mimic mutant ARMC10 S43D Then it will not have this effect. Figure 8 A). Mitochondrial tracking probe staining and electron microscopy results indicate that overexpression of ARMC10... S43A (instead of ARMC10) S43D It can significantly improve mitochondrial dynamics and reduce mitochondrial fragmentation. Figure 8 (B and C). Furthermore, overexpression of ARMC10 S43A It can significantly reverse TMT-induced mitochondrial dysfunction, manifested as reduced mitochondrial reactive oxygen species production (ROS). Figure 8 F), as well as ATP levels and mitochondrial membrane potential (Δψ). m ) rise ( Figure 8 D-1 Figure 8 D-2 and E). In summary, these results suggest that dephosphorylation of ARMC10 at the S43 site can mitigate TMT-induced neurotoxicity by improving mitochondrial dynamics and function.

[0078] Example 3: Screening for drugs that may inhibit ARMC10 phosphorylation and antagonize TMT-induced neurotoxicity based on ARMC10 protein structure.

[0079] (1) Virtual Filtering

[0080] Virtual screening was performed using the Computer-Aided Drug Design Library (CADD, product number L6030) of Natural Product Derivatives from TargetMol, Inc., a small molecule compound library containing more than 160,000 molecules.

[0081] First, the structure of mouse ARMC10 protein was optimized using the Protein Preparation Wizard module (with OPLS4 force field) in Schrödinger software, with energy minimization of confinement (root mean square deviation of heavy atoms ≤ 0.3 Å). Compounds from the library were processed using the LigPrep program to preserve their chiral characteristics, generating a maximum of 32 conformations per molecule to ensure conformational diversity during the virtual screening process. High-throughput primary screening, precise virtual screening, and fine screening were performed sequentially, with the top 10% of compounds selected at each stage.

[0082] Based on the crystal structure of the ARMC10 protein, a structure-based virtual screening was performed on a compound library containing more than 160,000 small molecules. Figure 9 A). Based on molecular docking analysis and the predicted binding affinity of small molecules to ARMC10, the top five candidate compounds (named M1 to M5) were screened and preliminarily evaluated. Figure 9 (B, C) Virtual screening is a standard technique in the field of biopharmaceutical development and can be outsourced to qualified biotechnology companies. The core technical points of the virtual screening described in this plan are: predicting the crystal structure of the ARMC10 protein; subsequently, screening small molecule compounds from a database of over 160,000 compounds that can interact with the ARMC10 protein with high affinity and inhibit S43 phosphorylation of the ARMC10 protein, and using these as candidate drugs to antagonize organotin neurotoxicity. The chemical formulas of M1, M2, M3, M4, and M5 are shown in Formulas I, II, III, IV, and V, respectively. These compounds can be purchased from ChemDiv; compound ID numbers are detailed in [link to ChemDiv]. Figure 9 B. Conduct subsequent experimental verification studies.

[0083]

[0084]

[0085] (2) Experimental screening

[0086] (2.1) Cell viability experiment

[0087] Cell viability was assessed using the CCK-8 (Cell Counting Kit-8) cell proliferation-toxicity assay kit from Tokyo Dojin. Neuro-2a cells were cultured at 1×10⁻⁶ cells / year. 4Cells were seeded in 96-well plates and incubated overnight. Three replicates were made for each experimental group. When cells reached 80% confluence, the test compounds (10 μM, represented by formulas I, II, III, IV, and V) were added to each well, with or without TMT (8 μM), and the cells were treated for 24 hours. After treatment, the culture medium was removed, and 100 μL of pre-mixed medium containing 10% CCK-8 reagent was added to each well. Three additional wells were added as blank wells to avoid air bubbles interfering with readings. The 96-well plates were incubated for 30–45 min. Absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated using the formula: Cell viability = [(At-Ab) / (Ac-Ab)] × 100%. At represents the absorbance of the treatment wells (medium containing CCK-8, cells under different treatments); Ac represents the absorbance of the control wells (medium containing CCK-8, control group cells); Ab represents the absorbance of the blank wells (medium containing CCK-8, cell-free).

[0088] For detailed experimental results, please refer to Figure 9 D. Since no TMT treatment was used in the control group, cell viability was not negatively affected by TMT, and the average cell viability in the control group was 1. After the addition of TMT, cell viability decreased significantly; see the experimental group treated only with TMT and without the test compound for details. Of the five candidate compounds in this protocol added to the TMT treatment, only M3 showed resistance to TMT toxicity. Although, according to the virtual screening results, all five candidate compounds have the potential to bind to the ARMC10 protein and antagonize TMT neurotoxicity by inhibiting ARMC10 protein phosphorylation, only M3 showed a significant resistance to TMT toxicity in the cell viability assay. The application of M3 alleviated the inhibitory effect of TMT on Neuro-2a cell activity.

[0089] (2.2) Cytotoxicity detection

[0090] Neuro-2a cells were loaded at a rate of 1 × 10⁻⁶ 4 Cells were seeded in 96-well plates and incubated overnight. Three replicates were performed per experimental group. When cells reached 80% confluence, the test compounds (10 μM, represented by formulas I, II, III, IV, and V) were added, with or without TMT (8 μM). The plates were placed in a cell induction Zoom system, and cell images were recorded for 24 hours. Cell confluence was analyzed using a Zoom live-cell analysis system (EssenBioScience, USA).

[0091] For detailed experimental results, please refer to Figure 9E and 9F, M3 can significantly reduce the toxic effects of TMT and can be used as drugs to antagonize TMT neurotoxicity, while other candidate compounds do not have the above effects.

[0092] Among these candidate compounds, M3 exhibited the strongest protective effect, and its systematic name is:

[0093] 1-(3,5-dimethylbenzenesulfonyl)-8',9'-dimethyl-5'H-spiro[piperidine-4,4'-pyrrolo[1,2-a]quinoxaline];

[0094] (1-(3,5-dimethylbenzenesulfonyl)-8',9'-dimethyl-5'H-spiro[piperidine-4,4'-pyrrolo[1,2-a]quinoxaline];

[0095] The linear chemical structure of SMILES of M3 is expressed as follows:

[0096] CC1=CC(=CC(C)=C1)S(=O)(=O)N1CCC2(CC1)NC1=C(N3C=CC=C23)C(C)=C(C)C=C1.

[0097] Further research was conducted on M3, including cell membrane potential detection, cell ATP detection, mitochondrial morphology detection, and its effect on ARMC10 protein (S43) phosphorylation (p-ARMC10(S43)).

[0098] (2.3) Results of Western blot analysis of proteins

[0099] Neuro-2a cells were loaded at a rate of 1 × 10⁻⁶ 4 Cells were seeded in 96-well plates and incubated overnight. Three replicates were prepared for each experimental group. When cells reached 80% confluence, the test compounds (10 μM, represented by formulas I, II, III, IV, and V) were added, with or without TMT (8 μM), and the cells were treated for 24 h. Total cellular protein was then extracted for Western blot analysis, using β-actin as an internal control. The levels of phosphorylated ARMC10 protein (S43) (p-ARMC10(S43)) and ARMC10 protein were measured, and the relative phosphorylation level of ARMC10 protein (S43) was used to represent the phosphorylation level of ARMC10 protein (S43). Figure 10 The relative phosphorylation level of phosphorylated ARMC10 (S43) protein in the right figure is calculated as follows: First, the relative phosphorylation level of phosphorylated ARMC10 (S43) protein is calculated as follows: Figure 10In the left figure, the ratio of the gray value of the p-ARMC10(S43) band to the gray value of the ACTIN band is calculated. This ratio represents the relative content of p-ARMC10(S43) per unit of total protein. Then, the ratio of the gray value of the ARMC10 band to the gray value of the ACTIN band is calculated. This ratio represents the relative content of total ARMC10 per unit of total protein. Finally, the two ratios are divided again to obtain the relative phosphorylation level of phosphorylated ARMC10(S43) protein used for statistical analysis.

[0100] For detailed experimental results, please refer to Figure 10 Under consistent internal control expression levels, TMT treatment increased intracellular p-ARMC10(S43) levels; however, concurrent application of M3 decreased p-ARMC10(S43) levels. Applying M3 alone did not significantly affect p-ARMC10(S43) levels. Therefore, M3 can be used to reduce TMT-induced ARMC10(S43) phosphorylation levels, antagonizing TMT-induced toxicity.

[0101] (2.4) Detection of mitochondrial membrane potential

[0102] JC-1 staining was used to distinguish between polarized and depolarized mitochondria. The JC-1 staining method was used for detection according to the product instructions (Beyotime, C2006). Polarized mitochondria exhibited red fluorescent aggregates with excitation / emission (Ex / Em) wavelengths of 585 / 590 nm; while depolarized mitochondria emitted green monomeric fluorescence with excitation / emission (Ex / Em) wavelengths of 514 / 529 nm. Neuro-2a cells were seeded in confocal culture dishes and treated with M3 (10 μM) with or without trimethyltin chloride for 24 hours. The culture medium was then removed, and the cells were washed three times with PBS. After treatment, JC-1 staining working solution to a final concentration of 1 μg / mL was added to the cells under light-protected conditions at 37°C and 5% CO2, and incubated for 20 minutes. After incubation, the cells were washed twice with Hank's balanced salt solution (HBSS) to remove residual staining reagent. Finally, the red and green fluorescence signals of JC-1 were observed and captured using a Leica TCS SP8 laser confocal scanning microscope (Leica Microsystems, Germany).

[0103] (2.5) Detection of cellular ATP

[0104] Quantitative analysis of cellular ATP levels was performed using an ATP assay kit (Thermo Fisher, A22066). Neuro-2a cells were seeded in white-walled 96-well plates and treated with M3 (10 μM) with or without TMT for 24 hours, after which the culture medium was removed. 100 µL of the reaction mixture provided in the kit was added to each well, and the microplate was incubated under standard cell culture conditions (37°C, 5% CO2) for 15 minutes to initiate the luminescence reaction. Immediately after incubation, the luminescence intensity of each well was measured using a microplate reader. A known concentration of ATP stock solution was prepared; this stock solution was serially logarithmically diluted; 100 µL of each gradient of ATP dilution standards was added to individual wells in the 96-well plate (measured simultaneously with the sample wells). The measured luminescence intensity values ​​from each cell sample well were then substituted into the plotted ATP standard curve.

[0105] (2.6) Detection of mitochondrial ROS:

[0106] Using MitoSOX TM A red fluorescent probe (Thermo Fisher Scientific, M36008) was used to quantify mitochondrial reactive oxygen species (mtROS). Neuro-2a cells were divided into groups of 1×10⁻⁶. 4 Cells were seeded at a density of 10 μM / well in 6-well plates, treated with or without M3 (10 μM) and TMT for 24 h, and washed once with PBS. Trypsin digestion was performed for 1 min, followed by addition of culture medium to terminate the process. Cells were collected, centrifuged, and washed three times with PBS. MitoSOX was then added, diluted to a final concentration of 5 μM with serum-free DMEM medium. TM Cells were treated with Red probe working solution and incubated for 30 minutes under standard cell culture conditions (37°C, 5% CO2) in the dark. After incubation, cells were washed three times with PBS to remove unbound probes. Immediately after elution, cells were analyzed by flow cytometry using an Accuri C6 flow cytometer (BD Biosciences, USA) to quantify mitochondrial reactive oxygen species (mtROS) levels.

[0107] (2.7) Mitochondrial morphology detection:

[0108] Mitochondrial morphology was analyzed using the MitoTracker Deep Red FM fluorescent probe (Invitrogen, M22426). Neuro-2a cells were seeded in confocal culture dishes and treated with M3 (10 μM) with or without TMT for 24 h. The culture medium was then removed, and the cells were washed three times with PBS. After treatment, the cells were incubated with 200 nM MitoTracker Deep Red FM fluorescent probe staining working solution at 37°C and 5% CO2 in the dark for 20 min. After incubation, the cells were washed twice with PBS to remove residual staining reagent. Finally, images were acquired using a Leica TCS SP8 laser confocal scanning microscope (Leica Microsystems, Germany).

[0109] For the experimental results of (2.4)-(2.7), please refer to [link to relevant documentation]. Figure 11 . Figure 11 A demonstrates the effect of M3 on mitochondrial membrane potential (Δψ) m The improvement of mitochondrial membrane potential (TMT) was observed in JC-1 staining. Red aggregates represented normally polarized mitochondria, while green monomers represented depolarized mitochondria (decreased membrane potential). The TMT-treated group (trimethyltin chloride "+" and M3 "-") showed a significant increase in green monomers and a decrease in red aggregates, indicating a substantial decrease in mitochondrial membrane potential. Conversely, the group with added M3 (trimethyltin chloride "+" and M3 "+") showed a significant recovery of red aggregates and a decrease in green monomers, demonstrating that M3 effectively improved the mitochondrial membrane potential of TMT-damaged cells. Quantitative statistical results also confirmed this, showing that the membrane potential of the "trimethyltin chloride + M3" group was significantly higher than that of the trimethyltin chloride-only treatment group. Figure 11 B shows the changes in mitochondrial reactive oxygen species (mtROS) and MitoSOX. TM Red probes label mtROS, with higher fluorescence intensity indicating more mtROS. After TMT treatment, the fluorescence intensity of mtROS in cells increased significantly; after the addition of M3, the fluorescence intensity of mtROS decreased significantly, indicating that M3 inhibited TMT-induced mitochondrial oxidative stress.

[0110] Figure 11 C shows the ATP levels under different treatments. ATP is the core product of mitochondrial energy supply. After TMT treatment, the cellular ATP level decreased significantly (representing impaired mitochondrial function); after the addition of M3, the ATP level rebounded significantly, and M3 improved the mitochondrial energy metabolism function of TMT-damaged cells.

[0111] Figure 11Figures D and E demonstrate the effect of M3 on mitochondrial morphology repair, with mitochondrial morphology observed through staining. In the TMT-treated group (trimethyltin chloride "+" and M3 "-"), mitochondria exhibited fragmentation (small, scattered dot-like structures); in the group with added M3 (trimethyltin chloride "+" and M3 "+"), mitochondria recovered to a continuous tubular / reticular structure. M3 alleviated TMT-induced mitochondrial fragmentation, significantly increased mitochondrial length, and improved mitochondrial dynamic homeostasis.

[0112] In summary, M3 improves TMT-induced mitochondrial dysfunction and kinetic abnormalities by increasing mitochondrial membrane potential, reducing mtROS production, restoring ATP levels, and repairing mitochondrial morphology. Its effects are related to the inhibition of ARMC10 (S43) phosphorylation. We confirm that M3 is a natural product compound that inhibits ARMC10 (S43) phosphorylation levels, improves mitochondrial kinetics and function, and can alleviate trimethyltin chloride-induced mitochondrial fragmentation and dysfunction.

[0113] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. The use of a compound in the preparation of a drug antagonizing tin-induced nervous system damage, characterized in that: The structural formula of the compound is shown in Formula III: Formula III.

2. The use of the compound according to claim 1 in the preparation of a drug antagonizing tin-induced nervous system damage, characterized in that: Tin-induced nervous system injury refers to acute / subacute nervous system injury induced by organotin.

3. The use of the compound according to claim 2 in the preparation of a drug antagonizing tin-induced nervous system damage, characterized in that: The organotin is trimethyltin chloride.

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