Use of small molecule compounds for the preparation of anti-aging medicaments

Abstract

This invention relates to the field of anti-aging drug technology, specifically to the application of small molecule compounds in the preparation of anti-aging drugs. These small molecule compounds competitively block the interaction between KDM6A and the vacuolar ATPase V0 domain subunit ATP6V0C by targeting a specific small molecule binding pocket of the histone demethylase KDM6A, thereby inhibiting the demethylation activity of KDM6A on lysine residue 36 of ATP6V0C. These small molecule compounds can effectively downregulate aging-related β-galactosidase activity, improve cell cycle arrest, and reverse the senescent phenotype of nerve cells induced by the environmental pollutant GenX, making them particularly suitable for combating premature aging of nerve cells induced by perfluorinated and polyfluoroalkyl compounds such as GenX. This technical solution can solve the technical problem of the lack of drugs with clearly defined components, a clear mechanism of action, stable efficacy, and the ability to specifically alleviate aging in existing technologies, and has ideal prospects for widespread application.

Description

Technical Field

[0001] This invention relates to the field of anti-aging drug technology, specifically to the application of small molecule compounds in the preparation of anti-aging drugs. Background Technology

[0002] Aging is a complex and pervasive biological phenomenon that occurs throughout the life processes of cells, tissues, organs, and even the entire organism. At the cellular level, the morphology, structure, and function of aging cells undergo significant changes. At the molecular level, gene expression patterns in aging cells change, with some genes related to cell proliferation being downregulated and aging-related genes being upregulated. Aging is also accompanied by the accumulation of reactive oxygen species (ROS) within cells and a decline in DNA damage repair capabilities. These changes work together to gradually reduce cell proliferation, slow metabolic activity, and ultimately lead to the decline of cellular function. As a vital regulatory system, the nervous system is severely affected by neuroaging. The ultimate manifestations of neuroaging include cognitive decline, such as memory loss and impaired learning ability, as well as motor dysfunction, such as poor coordination and reduced balance, significantly impacting an individual's quality of life.

[0003] The damage of environmental pollutants to the nervous system is an increasingly serious problem, with per- and polyfluoroalkyl compounds (PFAS) receiving particular attention. PFAS are a class of synthetic organic compounds widely used in textiles, leather, papermaking, fire protection, and food packaging due to their excellent thermal stability, chemical stability, and surface activity. However, PFAS are difficult to degrade in the environment, exhibiting strong bioaccumulation and persistence, and can accumulate in organisms through the food chain. Studies have found that PFAS can cross the blood-brain barrier and enter the central nervous system, accumulating in the brain and exerting toxic effects on neurons, promoting neuronal aging, and subsequently triggering neurodegenerative diseases such as Alzheimer's and Parkinson's. Furthermore, the human body lacks the enzymes necessary to metabolize PFAS, making them difficult to degrade in the body and prone to long-term accumulation, further exacerbating their toxic effects. Hexafluoropropylene oxide dimer acid (HFPO-DA; trade name GenX), as a novel alternative to the PFAS family, also exhibits the aforementioned hazards. GenX is widespread in the environment, and humans can be exposed to it through various routes, including drinking water, food, and air. Numerous studies have shown that GenX can persist in water and soil for extended periods and can also be detected in everyday consumer products such as non-stick cookware coatings, waterproof and breathable clothing, and food packaging materials. With the continuous increase in PFAS pollutants like GenX in the environment, the risk of exposure to the population is constantly rising, and the probability of environmental pollutant-induced neurosenescence is gradually increasing, placing a heavy burden on patients and their families and putting enormous pressure on social medical resources. Therefore, there is an urgent need to find effective treatments to address this challenge.

[0004] Chinese Patent CN103800386B discloses an extract of Cordyceps militaris and its application in the preparation of neuroprotective and anti-aging drugs. The extract is the water-soluble fraction of the ethanol extract of Cordyceps militaris; it is obtained by the following method: Cordyceps militaris is pulverized and sieved, extracted with ethanol under reflux, the ethanol extract is dispersed in water, and then fractionally extracted with petroleum ether, ethyl acetate, and n-butanol. The solvent is recovered by vacuum concentration, and the fractions are freeze-dried to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and water fraction, respectively. The water fraction is concentrated and passed through AB-8 macroporous adsorption resin, first washed with water until nearly colorless, then eluted with 80% ethanol. The ethanol eluent is collected, concentrated, and freeze-dried to obtain the water-soluble fraction. This extract can significantly inhibit the aging and damage of nerve cells. However, extracts of traditional Chinese medicine have inherent limitations. Due to the complex sources of Chinese medicinal materials, and the influence of various factors such as origin, climate, and cultivation methods, the composition of extracts is difficult to maintain stably and consistently. Furthermore, extracts of traditional Chinese medicine are often mixtures of various chemical components, and their specific active ingredients and mechanisms of action are not yet clear. This not only poses a significant challenge to quality control but also limits their precise clinical application. More importantly, although Cordyceps militaris extract has some effect in inhibiting glutamate-induced cellular senescence damage, its therapeutic effect on neurosenescence induced by environmental pollutants (such as GenX) remains unknown, lacking relevant experimental verification and clinical research.

[0005] In conclusion, given the increasingly serious problem of neurosenescence induced by environmental pollutants and the many shortcomings of existing treatments, developing a drug or method with clearly defined components, a clear mechanism of action, stable efficacy, and the ability to specifically treat neurosenescence induced by environmental pollutants is of great scientific significance and has broad application prospects. Summary of the Invention

[0006] The present invention aims to provide the application of small molecule compounds in the preparation of anti-aging drugs, so as to solve the technical problem of the lack of drugs with clear components, clear mechanisms of action, stable efficacy and targeted aging relief in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Application of small molecule compounds in the preparation of anti-aging drugs, wherein the small molecule compounds are used to upregulate the methylation level of ATP6V0C protein.

[0009] Furthermore, the small molecule compound is used to inhibit the binding of KDM6A and ATP6V0C proteins.

[0010] Furthermore, the small molecule compound is used to bind to the small molecule binding pocket of KDM6A; the amino acid residues of the small molecule binding pocket of KDM6A include proline at position 924, serine at position 925, serine at position 926, glycine at position 1175, valine at position 1176, aspartic acid at position 1179, phenylalanine at position 1180, lysine at position 1183, and alanine at position 1203.

[0011] Furthermore, the small molecule compound is shown in Formula 1 or Formula 5;

[0012]

[0013] Furthermore, the compound shown in Formula 1 is used to interact with serine at position 926, aspartic acid at position 1179, and alanine at position 1203 of KDM6A; the compound shown in Formula 5 is used to interact with serine at position 926, valine at position 1176, aspartic acid at position 1179, lysine at position 1183, and alanine at position 1203 of KDM6A.

[0014] Furthermore, the drug is used to combat neuroaging.

[0015] Furthermore, the drug is used to combat GenX-induced neuronal senescence. GenX-induced neuronal senescence is a specific type of neuronal senescence (a sub-indication or sub-indication) that is linked to disease progression and is sufficient to guide clinical practice (e.g., guidance value for clinical dosing).

[0016] The differences between GenX-induced neuronal senescence (exogenous pathological senescence) and ordinary neuronal senescence (physiological natural senescence) include:

[0017] The causes are different: the latter is natural aging caused by endogenous factors such as telomere shortening and ROS accumulation, which progresses with age; the former is "premature aging" caused by the direct accumulation and toxic effects of environmental pollutants GenX, which can also induce and promote the process of physiological natural aging.

[0018] The mechanisms are different: the former, in addition to activating classical aging pathways, also has GenX-specific toxicity and progresses rapidly; the latter is mainly characterized by the gradual dysregulation of classical aging pathways and progresses slowly.

[0019] The characteristics are different: the former is a combination of "acute injury + aging"; the latter is mainly "natural functional decline" with no significant damage.

[0020] The intervention focuses on different aspects: the former requires blocking GenX exposure first, and then repairing specific damage; the latter focuses on "delaying the natural process" (such as anti-oxidation and activating autophagy).

[0021] This technical solution also provides the application of KDM6A protein in screening drugs that resist GenX-induced neuronal senescence caused by environmental pollutant.

[0022] Furthermore, the amino acid residues in the small molecule binding pocket of KDM6A are used for computer-aided molecular docking with candidate small molecule drugs; the amino acid residues in the small molecule binding pocket of KDM6A include proline at position 924, serine at position 925, serine at position 926, glycine at position 1175, valine at position 1176, aspartic acid at position 1179, phenylalanine at position 1180, lysine at position 1183, and alanine at position 1203.

[0023] The small molecule binding pocket Site1 of KDM6A contains the amino acids Pro924, Ser925, Ser926, Gly1175, Val1176, Asp1179, Phe1180, Lys1183, and Ala1203, which can bind to small molecule compounds and thus affect the downstream function of proteins, and can serve as a drug designation site.

[0024] Furthermore, the aspartic acid at position 1179 of KDM6A is used for computer-aided molecular docking with candidate small molecule drugs.

[0025] The technical principle of this technical solution is as follows:

[0026] This invention focuses on targeted intervention of GenX-induced neuronal senescence caused by environmental pollutant. Through a full-chain technical approach including mechanism analysis, target screening, drug development, and functional verification, a small molecule drug targeting GenX-induced neurosenescence has been developed.

[0027] Using mouse neuroblastoma cells (Neuro-2a, N2A) as the research subject, we exposed N2A cells to different concentrations (with gradient concentration settings) of GenX in an in vitro culture system. By detecting aging-related biomarkers such as aging-associated β-galactosidase (SA-β-gal), cell cycle distribution, and aging-related genes, we demonstrated that GenX exposure can induce typical aging phenotypes in N2A cells.

[0028] Transcriptome sequencing (RNA-seq) was performed on N2A cells from both the GenX-exposed and control groups. Combined with bioinformatics analysis, the study focused on pathways related to cellular senescence and neurotoxicity, identifying the key regulator—histone demethylase KDM6A—that showed the most significant differential expression after GenX exposure and was closely related to senescence pathways. Protein methylation modification proteomics was performed on KDM6A-regulated cells from both groups to screen for downstream target proteins regulated by KDM6A. It was determined that KDM6A can directly bind to the V0 domain subunit ATP6V0C of vacuolar ATPase, and GenX exposure enhances this binding. Ultimately, ATP6V0C was identified as a key downstream molecule of KDM6A regulating neuronal senescence.

[0029] By analyzing the protein structure, the interaction region between KDM6A and ATP6V0C was determined, and candidate compounds with high affinity for the binding pocket were obtained through virtual screening. The candidate small molecule compounds obtained from the initial screening were applied to GenX-induced senescent N2A cells, and their functions were verified through multidimensional experiments. Finally, small molecule compounds with definite activities were identified (such as compounds shown in Formulas 1 and 5).

[0030] This technical solution reveals for the first time the specific molecular mechanism by which GenX induces neuronal senescence: it breaks through the previous technical bottleneck of unclear neurotoxicity mechanism of GenX, and for the first time confirms that GenX drives neuronal senescence through the pathway of "promoting the binding of KDM6A to ATP6V0C → specifically removing ATP6V0C-K36 trimethylation → upregulating SA-β-gal activity → inducing cell cycle arrest", filling the research gap in the molecular mechanism of neurosenescence induced by environmental pollutant GenX.

[0031] Establishing a precision drug screening system: For the first time, this study identified ATP6V0C-K36 as a key functional residue for the interaction between KDM6A and ATP6V0C, and determined that the small molecule binding pocket on KDM6A is the core region for their binding. This enables highly efficient screening of small molecule compounds, solving the problems of unclear targets and low screening efficiency in traditional drug screening.

[0032] The developed small molecule compounds that can specifically reverse GenX-induced neurosensitivity overcome the limitations of existing technologies that cannot specifically intervene in neurosensitivity induced by environmental pollutants. The screened small molecule compounds (such as Formula 1 and Formula 5) can competitively bind to the small molecule binding pocket of KDM6A, precisely blocking the interaction between KDM6A and ATP6V0C, restoring the ATP6V0C-K36 trimethylation level from the molecular source, and thus reversing the GenX-induced neuronal senescence phenotype, achieving a technological breakthrough in targeted mechanism and precise intervention.

[0033] The beneficial effects of this technical solution are as follows:

[0034] (1) Filling the gap in the study of mechanism of action

[0035] This study elucidates the molecular mechanism by which GenX induces neurosenescence, providing a novel theoretical basis for research on the neurotoxicity of environmental pollutants. Simultaneously, it reveals the regulatory role of KDM6A in ATP6V0C methylation, offering a new perspective for research on the epigenetic regulatory mechanisms of cellular senescence.

[0036] (2) Improve the efficiency and accuracy of drug screening

[0037] The screening system based on key residues and binding pockets can directly screen compounds targeting the core target of KDM6A-ATP6V0C interaction, avoiding the blindness of traditional screening and significantly improving the screening efficiency and activity hit rate of candidate drugs. At the same time, the clear binding site provides a clear direction for subsequent structural optimization of compounds (such as enhancing affinity and reducing toxicity), shortening the drug development cycle.

[0038] (3) To address the shortcomings of existing treatment methods and meet clinical needs.

[0039] Traditional Chinese medicine preparations, including the Cordyceps militaris extract (CN103800386B), primarily exert their anti-aging effects through antioxidant activity. However, this technical solution provides a structurally well-defined small molecule substance that exerts its anti-aging effect by improving the cell cycle. Compared to the complex and unstable composition of traditional Chinese medicine extracts, the small molecule compound of this invention has a well-defined composition and a clear mechanism of action, enabling precise targeted therapy against GenX-induced neurosensitivity. It can effectively improve cell cycle arrest and downregulate the activity of aging markers, providing a practical treatment option for neuroprotection in GenX-exposed individuals and alleviating the current clinical dilemma of lacking effective interventions for environmental pollutant-induced neurological diseases.

[0040] (4) Possesses broad clinical and industrial prospects.

[0041] The small molecule compounds screened can not only be used to treat GenX-induced neurosenescence, but also, through further optimization, can be extended to the intervention of other neurodegenerative diseases (such as age-related cognitive impairment) related to abnormal activation of the KDM6A-ATP6V0C pathway. At the same time, small molecule compounds are easy to synthesize, easy to formulate into various dosage forms (such as tablets and injections), and have high bioavailability, which facilitates industrial production and clinical translation. They are expected to form innovative drugs with independent intellectual property rights and generate significant social and economic benefits. Attached Figure Description

[0042] Figure 1The results of the GenX exposure-induced senescence phenotype in N2A cells in Example 1 are shown below (cell viability test results are shown in ab; SA-β-gal activity test results are shown in cd; transcriptomic analysis results are shown in e after N2A cells were exposed to 250 μM GenX for 72 h; expression levels and semi-quantitative analysis of senescence-related proteins are shown in fg after N2A cells were exposed to 0, 50, 250, and 500 μM GenX for 72 h; scale bar: 100 μm. Data are expressed as mean ± SD, n=3; *p<0.05, **p<0.01; ns: no significance).

[0043] Figure 2 The results of Example 2 on the role of KDM6A in GenX-induced neuronal senescence are as follows: (a) Western blot analysis of changes in total protein methylation levels in cells after treatment with different concentrations of 0, 50, 250, and 500 μM GenX for 72 h; (b) Transcriptomic heatmap showing changes in the expression profile of the demethylase family KDMs; (c) Venn diagram showing the number of intersections between differentially expressed genes and lysine demethylase genes after 72 h of treatment with 250 μM GenX; (d) qPCR quantitative verification of the effect of 250 μM GenX treatment for 72 h.) Kdm3a , Kdm4a and Kdm6a mRNA expression levels; data are expressed as mean ± SD, n = 3; *p < 0.05, **p < 0.01).

[0044] Figure 3 Knockout in Example 2 Kdm6a Results of the study on the effects of GenX exposure on aging phenotypes (a) validation of sgRNA knockdown effect in mixed cell lines; b) Western blot analysis of control group, GenX-exposed group, Kdm6a - Results of KDM6A, P53, P21 and P16 protein expression in the KO group; c is the semi-quantitative analysis result of b, expressed as normalized control mean; data are expressed as mean ± SD, n=3; *p<0.05, **p<0.01).

[0045] Figure 4 The results of the experimental study on KDM6A-specific removal of ATP6V0C-K36me3 in Example 3 (a is the knockout) Kdm6a Differentially methylated proteins with post-methylation changes; heatmap showing differentially methylated proteins after Kdm6a knockout; b represents knockout. Kdm6aKEGG cell pathway enrichment analysis was performed; c shows the mass spectrometry quantitative results of ATP6V0C-K36 methylation changes; d shows the conservation comparison of ATP6V0C protein and the cross-species conserved sequence comparison, indicating that the K36 site is conserved.

[0046] Figure 5 The experimental results of GenX exposure enhancing the interaction between KDM6A and ATP6V0C in Example 3 are shown in Figure 3. (a is a schematic diagram of molecular docking simulation of the interaction between KDM6A and ATP6V0C; b is the co-precipitation of KDM6A and ATP6V0C detected by Western blot after Flag-KDM6A immunoprecipitation IP; c is the co-precipitation of ATP6V0C and KDM6A detected by Western blot after HA-ATP6V0C immunoprecipitation).

[0047] Figure 6 Conservation analysis of KDM6A protein in Example 4 (sequence alignment of human Homo sapiens, NP_066963.2 and mouse Mus musculus, NP_001297373.1).

[0048] Figure 7 This is the prediction of the small molecule binding pocket of the KDM6A protein in Example 4 (prediction of the small molecule binding pocket of KDM6A and key amino acid residues).

[0049] Figure 8 This is the screening process for small molecule compounds that antagonize the binding of KDM6A and ATP6V0C to improve the cellular senescence phenotype, as described in Example 5.

[0050] Figure 9 The results of the virtual screening in Example 5 are shown in Figure 5. (a) shows the quantity-score distribution of compounds from the D3100 compound library that were screened for KDM6A structure; b) shows the PLIF analysis of 556 compounds, with the same amino acid represented by the same color, and the higher the vertical density, the higher the frequency of interaction with the compound.

[0051] Figure 10 The results of the safety range test of the small molecule compound in Example 5 are as follows (N2A cell viability was determined by CCK-8 assay after treatment with different concentrations of 0, 5, 10, 20, and 40 μM small molecule compounds for 72 h; data are expressed as mean ± SD, n=3; *p<0.05, **p<0.01; ns: no significant difference).

[0052] Figure 11The small molecule compound in Example 5 was shown to improve the activity of GenX-treated N2A cells (group data are expressed as mean ± SD, n=3; *p<0.05, **p<0.01; ns: no significant difference).

[0053] Figure 12 The experimental results of Z-KA1 targeting and blocking the binding of KDM6-ATATP6V0C in Example 6 are shown in Figure 6 (a is a 3D and 2D schematic diagram of the binding of small molecule compounds to KDM6; b is the protein immunoprecipitation experiment results of Z-KA1 weakening the binding of KDM6A to ATP6V0C; c is the protein immunoprecipitation experiment results of Z-KA1 upregulating the trimethylation level of ATP6V0C).

[0054] Figure 13 The experimental results of Z-KA1 improving the senescence phenotype of N2A cells in Example 6 are shown in Figure 6. (a) shows the experimental results of Z-KA1 inhibiting SA-β-gal enzyme activity; b) shows the experimental results of Z-KA1 reducing the expression of senescence-related proteins. Data are expressed as mean ± SD, n=3; *p<0.05, **p<0.01; ns: no significant difference).

[0055] Figure 14 The experimental results of Z-KA1 improving cell cycle arrest in Example 6 are shown in Figure 6 (a is a distribution map of cell DNA content; b is a statistical graph of cell cycle ratio; data are expressed as mean ± SD, n=3; **p<0.01 vs Ctrl; ##p<0.01 vs GenX; ns: no significant difference).

[0056] Figure 15 The experimental results of Z-KA5 targeting and blocking the binding of KDM6-ATATP6V0C in Example 6 are shown in Figure 6 (a is a 3D and 2D schematic diagram of the binding of small molecule compounds to KDM6; b is the protein immunoprecipitation experiment results of Z-KA1 weakening the binding of KDM6A to ATP6V0C; c is the protein immunoprecipitation experiment results of Z-KA1 upregulating the trimethylation level of ATP6V0C).

[0057] Figure 16 The experimental results of Z-KA5 improving the senescence phenotype of N2A cells in Example 6 are shown in Figure 6. (a) shows the experimental results of Z-KA1 inhibiting SA-β-gal enzyme activity; b) shows the experimental results of Z-KA1 reducing the expression of senescence-related proteins. Data are expressed as mean ± SD, n=3; *p<0.05, **p<0.01; ns: no significant difference).

[0058] Figure 17The experimental results of Z-KA5 improving cell cycle arrest in Example 6 are shown in Figure 6 (a is a distribution map of cell DNA content; b is a statistical graph of cell cycle ratio; data are expressed as mean ± SD, n=3; **p<0.01 vs Ctrl; ##p<0.01 vs GenX; ns: no significant difference). Detailed Implementation

[0059] 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.

[0060] To facilitate understanding of this technical solution, the relevant technical terms and concepts are explained in detail below:

[0061] Neuroblastoma cells (Neuro-2a cells, N2A): These cells are widely used mouse-derived neuroblastoma cell lines in neuroscience and cell biology research. Due to their combination of tumor cell characteristics and neural cell differentiation potential, they have become an important tool bridging basic research and disease models. N2A cells are widely used in multiple research directions due to their ease of culture, differentiation potential, and high correlation with neural cells: research on neural development and differentiation mechanisms, construction of neurodegenerative disease models, neurotoxicity and drug screening research, and oncology research (related to neuroblastoma).

[0062] By exogenously applying aging-related stress factors, N2A cells can be directly induced to exhibit aging phenotypes, forming a neurocellular aging model. This allows N2A cells to be applied to research on neuroaging. In this technical approach, the exogenous environmental pollutant GenX can induce aging phenotypes in N2A cells. The resulting GenX-induced neurocellular aging model can be used to study neuroaging caused by related environmental pollutants, and further to screen and validate anti-neuroaging drugs / intervention strategies. By detecting indicators such as SA-β-gal activity (a marker of aging cells), cell viability (CCK-8 assay), cell cycle, and aging-related proteins, substances with potential anti-neuroaging activity can be rapidly screened.

[0063] Both N2A cells and human neuroblastoma (SH-SY5Y) are tumor cell lines derived from neuroblastoma that can differentiate into neuron-like cells, and both serve as model cells for related mechanism studies and drug screening. Furthermore, N2A cells significantly outperform human lines in four key indicators: transfection efficiency, ion channel function, cost control, and genetic homogeneity. SH-SY5Y contains triploids and subclones, and phenotypic drift is common, leading to some errors in experimental results. N2A cells are an inbred line and are genetically stable. Moreover, the small molecule drugs involved in this protocol, KDM6A and ATP6V0C, have very high homology between humans and mice; therefore, using N2A cells can effectively simulate human cells in terms of mechanism. Therefore, this technical protocol uses N2A cells as a cell model to construct an in vitro model of GenX-induced senescent nerve cells, which is perfectly feasible for screening and studying drugs that combat GenX-induced neurosenescence. In addition, N2A cells are frequently used as cell models to screen various types of drugs. For example, Chinese patent CN103536593B (use of pyrroloquinoline quinone in the preparation of anti-tumor drugs) uses N2A cells as a cell model to screen drugs for the treatment of neuroblastoma.

[0064] SA-β-gal (Senescence-Associated β-galactosidase) staining is a classic and commonly used cytochemical staining method in biological and medical research for detecting cellular senescence. Its core function is to identify, locate, and quantify senescent cells by specifically labeling their unique biochemical characteristics. Senescent cells exhibit a series of morphological and biochemical changes, one key feature of which is increased lysosomal β-galactosidase activity.

[0065] Aging-related proteins P53, P21, and P16: These three proteins are the core proteins in the cellular senescence regulatory network. They work together to regulate cell cycle arrest and are key molecules that trigger and maintain cellular senescence. They are widely involved in processes such as replicative senescence (e.g., senescence caused by telomere shortening) and stress-induced senescence (e.g., senescence caused by DNA damage and oxidative stress).

[0066] p53, short for tumor suppressor p53, is encoded by the TP53 gene located on human chromosome 17. In aging-related functions, it plays a central role in sensing aging stress signals. Its core function is to trigger cell cycle arrest by regulating downstream genes (such as the gene encoding p21), thereby participating in the initiation of the aging process.

[0067] P21 stands for Cyclin-Dependent Kinase Inhibitor 1A, encoded by the gene CDKN1A located on human chromosome 6. Its core function is as a direct brake on the cell cycle; by inhibiting the activity of the complex formed by cyclin-dependent kinase (CDK) and cyclin, it directly blocks cell proliferation, making it one of the direct executors of aging effects.

[0068] P16 stands for Cyclin-Dependent Kinase Inhibitor 2A (also known as INK4a protein), encoded by the CDKN2A gene located on human chromosome 9. Its core function is to maintain senescence arrest stability by specifically inhibiting the activity of CDK4 and CDK6, indirectly blocking the initiation of the cell cycle and ensuring that senescent cells remain in a stable arrested state.

[0069] During cellular and organismal aging, the expression levels of P53, P21, and P16 all show a core trend of significant upregulation with the aging process, and the changes in expression are directly related to the initiation and maintenance of aging.

[0070] The cell cycle refers to the entire process that continuously dividing cells undergo from the end of one mitosis to the end of the next, mainly including the G0 / G1 phase (pre-DNA synthesis), S phase (DNA synthesis phase), G2 phase (post-DNA synthesis phase), and M phase (mitosis phase). There is a close and complex relationship between the cell cycle and cellular senescence. Cell cycle arrest is an important characteristic of cellular senescence. Normal cells undergo a series of preparatory processes in the G0 / G1 phase, such as synthesizing RNA and proteins, to determine whether the cell will enter the S phase for DNA replication. When cells exhibit a senescent phenotype, a series of signaling pathways (such as the p53-p21 and p16-Rb pathways) are activated, causing the cells to arrest in the G1 phase and unable to enter the S phase for proliferation. The proportion of cells in the S phase decreases, thereby suppressing cell proliferation and promoting cellular senescence.

[0071] Kdm3a (Lysine Demethylase 3A): The lysine demethylase 3A gene; the function of this protein is to specifically remove the monomethyl (me1) and dimethyl (me2) modifications on the 9th lysine (H3K9) of histone H3. H3K9me1 / me2 are typical transcriptional repressive histone markers; therefore, this protein, through demethylation, can open up chromatin, thereby activating the transcription of downstream target genes.

[0072] Kdm4a(Lysine Demethylase 4A): The lysine demethylase 4A gene; the function of this protein is mainly to act on the trimethylation (me3) of histone H3 at lysine position 9 (H3K9) and lysine position 36 (H3K36), and it can also remove the dimethylation (me2) at these two sites. H3K9me3 is a strong transcriptional repression marker (often associated with heterochromatin formation), while H3K36me3 is associated with gene transcriptional elongation and mRNA splicing regulation. Therefore, this protein, through demethylation, can both undo H3K9me3-mediated gene silencing and regulate the fine-tuning of H3K36me3-related gene transcription.

[0073] Kdm6a (Lysine Demethylase 6A): Lysine demethylase 6A; this protein specifically removes the trimethyl (me3) and dimethyl (me2) modifications from lysine residue 27 (H3K27) of histone H3. H3K27me3 is a classic transcriptional repression marker. Therefore, this protein can activate the expression of downstream target genes through demethylation. Simultaneously, KDM6A can also directly remove dimethyl / trimethyl modifications from proteins, directly affecting their function. Its target genes are widely involved in processes such as cell differentiation, development, DNA repair, and inflammation regulation. Previous studies have focused on the regulatory role of the KDM family in histone methylation, but this technical approach is the first to discover that KDM6A protein can regulate the K36 trimethylation modification of non-histone ATP6V0C. This discovery is not only the core innovation of this approach compared to existing technologies, but also provides a novel drug target for the development of novel small molecule drugs.

[0074] ATP6V0C is the core proton channel subunit of the V0 region of vacuolar ATPase (V-ATPase), and together with the V1 region, it forms a transmembrane proton pump. The V1 domain hydrolyzes ATP to generate energy, which drives the rotation of the V0 region, thereby enabling proton transmembrane transport and maintaining the acidic microenvironment of organelles such as lysosomes.

[0075] Example 1: GenX exposure induces decreased viability and senescence phenotype in N2A cells

[0076] A: Experimental methods

[0077] Mouse neuroblastoma cells (Neuro-2a cells, N2A) were obtained from the cell bank of the Institute of Biochemistry and Cell Biology (Shanghai, China, TCM29). N2A cells were treated with 0, 50, 250, and 500 μM GenX (Maclean's, A76175; GenX stock solution was prepared with deionized water) for 24, 48, and 72 hours, respectively. N2A 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. To investigate the effect of GenX exposure on cell viability, N2A cells were seeded at a density of 3 × 10³ cells per well in 96-well plates (Essen BioScience, USA), with 5–6 replicates per group. CCK-8 reagent (Taoshu Biotechnology, C0005) was used, and cell viability was assessed according to the kit instructions. Cell images were recorded during the culture process. Cell morphology was analyzed using an induced cell zoom live-cell imaging system (Essen BioScience, USA).

[0078] N2A cells were divided into 1×10 4 Cells were seeded in 24-well plates and treated with GenX for 24, 48, and 72 hours, followed by routine SA-β-gal staining. N2A cells were exposed to 250 μM GenX for 72 hours before routine transcriptomic analysis, as well as routine analysis of aging-related protein expression levels and semi-quantitative analysis.

[0079] Data were analyzed and expressed as mean ± SD. Unpaired two-tailed 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.

[0080] B: Experimental Results

[0081] To elucidate the molecular mechanism of GenX-induced neurotoxicity, cell viability was first assessed using CCK-8 assays. Figure 1 As shown in ab, compared with the control group (0 μM), there were no significant differences in cell viability after 24 and 48 hours of exposure at 50 μM, 250 μM, and 500 μM (P>0.05); compared with the control group (0 μM), cell viability was significantly reduced after 72 hours of exposure at 250 μM and 500 μM, while there was no significant difference after 72 hours of exposure at 50 μM (P>0.05). SA-β-gal staining results showed ( Figure 1Compared with the control group (0 μM), there were no significant differences in SA-β-gal activity in the 50 μM, 250 μM, and 500 μM groups after 24 and 48 hours of exposure (P>0.05). Compared with the control group (0 μM), SA-β-gal activity was significantly increased in cells exposed to 250 μM and 500 μM for 72 hours, while there was no significant difference in activity in cells exposed to 50 μM for 72 hours (P>0.05). Transcriptome analysis revealed that GenX exposure significantly affected cellular senescence-related pathways: cell cycle arrest; activation of the p53 signaling pathway (…). Figure 1 e). Western blot results showed that GenX exposure for 72 h significantly upregulated the expression of senescence-related proteins in N2A cells. Compared with the control group (0 μM), there was no significant difference in P53 protein expression in the 50 μM group (P>0.05), while P53 protein expression was significantly decreased in the 250 μM and 500 μM groups (P<0.05, P<0.01). Compared with the control group (0 μM), P21 protein expression was significantly upregulated in the 50 μM, 250 μM, and 500 μM groups (P<0.05). Compared with the control group (0), there was no significant difference in P16 protein expression in the 50 μM group (P>0.05), while P16 protein expression was significantly decreased in the 250 μM and 500 μM groups (P<0.05).

[0082] Therefore, these results suggest that GenX induces cell cycle arrest by upregulating the P53 / P21 / P16 axis, thereby driving the aging phenotype, and this effect is dose- and time-dependent.

[0083] Example 2: Knockout Kdm6a Reversing GenX exposure-induced aging phenotype

[0084] A: Experimental methods

[0085] Western blotting was used to detect changes in total protein methylation levels in cells after treatment with different concentrations of 0, 50, 250, and 500 μM GenX for 72 h, and transcriptomic heatmap analysis was performed to show changes in the expression profile of the demethylase family KDMs. qPCR was used to quantitatively verify the effects of 250 μM GenX treatment for 72 h. Kdm3a , Kdm4a and Kdm6a mRNA expression levels.

[0086] Knockout Kdm6a After the gene, research knockout Kdm6a The experimental procedure for investigating the effects of GenX exposure on senescence phenotypes included: construction of the CRISPR-Cas9-Kdm6a-KO plasmid, viral packaging and concentration, and screening of monoclonal cell lines.

[0087] download Kdm6aThe longest transcript CDS was used to design three sgRNAs: sgRNA-#1, sgRNA-#2, and sgRNA-#3, and the plasmid pLenti-U6-spgRNAv2.0-CMV-Puro-P2A-3×Flag-spCas9-WPRE was constructed. After transfection with engineered bacteria, plasmid extraction was performed. Virus packaging and concentration were then carried out to obtain viral fluid.

[0088] Target cells were seeded in 6-well plates and cultured with a gradient of 0.5-10 µg / mL puromycin (Beyotime, ST551). After 48 hours, the 100% lethal concentration for N2A cells was determined, which became the subsequent selection concentration. When cells reached 70% confluence, Cas9-sgRNA-puromycin lentivirus + polybrene (Heyuan Biotechnology, OGTR(C)20181002) was added, and the cells were incubated at 37°C for 8 hours. The medium was then replaced with fresh medium, and the cells were cultured for another 24 hours. The medium was then replaced with a PC-containing medium. 90 Complete culture medium containing puromycin at a high concentration was used; the medium was changed every 48 hours. All uninfected control wells died within 3 days; resistant clones appeared in 5-7 days. Monoclonal cells were obtained using limiting dilution, and resistant cells were digested with trypsin, counted, and seeded at 0.5-1 cells / well in 96-well plates (200 µL / well). After 2 weeks, monoclonal cells were labeled under a microscope and picked for amplification in 24-well plates. qPCR / Western blot was used to detect KDM6A expression deficiency; positive clones were cryopreserved.

[0089] B: Experimental Results

[0090] like Figure 2 As shown in figure a, total protein methylation modification in cells was significantly reduced after GenX exposure. Therefore, we continued to screen for differentially altered demethylase family genes based on transcriptomics. Heatmaps and Venn diagrams show (…). Figure 2 (bc), after GenX was exposed, the KDMs family Kdm3a , Kdm4a , Kdm6a Upregulation. Further validation by qRT-PCR revealed ( Figure 2 d) GenX exposure, Kmd6a The upregulation was most significant (P < 0.01). Meanwhile, Western blot results showed similar findings. Figure 2 e), indicating that GenX exposure significantly upregulated KDM6a. This study suggests that KDM6A protein is a key effector molecule mediating the GenX-induced senescence phenotype in N2A cells.

[0091] Therefore, to further explore the role of KDM6A, we constructed using CRISPA-Cas9 technology. Kdm6a-KO N2A cell lines. After infecting with three sgRNA sequences, the knockdown effect of the three sgRNAs was tested in mixed cell lines. The results showed that all three sgRNAs had a good knockdown effect. Figure 3 a) Continue screening for single-clonal cell lines for subsequent experiments. For example... Figure 3 As shown in bc, compared to the control group (NC), the proteins of KDM6A, P53, P21, and P16 were significantly upregulated in the GenX-exposed group (P < 0.01, P < 0.05); compared to the GenX-exposed group, the proteins of GenX+ were significantly upregulated. Kdm6a In the -KO group, KDM6A protein knockout (P < 0.01) and the expression levels of P53, P21, and P16 proteins were significantly reduced (P < 0.01).

[0092] In conclusion, Kdm6a The absence of KDM6A can significantly reverse GenX-induced activation of senescence pathways, suggesting that KDM6A plays a core regulatory role in GenX-driven senescence of N2A cells.

[0093] Example 3: GenX exposure promotes KDM6A-specific removal of ATP6V0C-K36me3, driving cellular senescence.

[0094] A: Experimental methods

[0095] Methylation modification omics analysis

[0096] First, sample preparation was performed. Tissues or cells were washed with PBS, flash-frozen in liquid nitrogen, and stored at -80°C. Then, lysis buffer containing 2 mM EDTA and protease / demethylase inhibitors was used for sonication on ice for 3 × 10⁻⁶ s, followed by centrifugation at 16,000 g, 4°C for 20 min. The supernatant was collected and quantified using the BCA method to a concentration of 2 mg / mL. Next, protein reductive alkylation and enzyme digestion were performed. The peptides were first treated with 10 mM DTT at 56°C for 30 min, then with 2 mM IAA at room temperature in the dark for 20 min. Afterwards, FASP or solution digestion was performed with trypsin at a 1:50 (w / w) ratio at 37°C overnight. The peptides were desalted using C18 chromatography and vacuum dried. The next step was methylated peptide enrichment. Anti-methyllysine antibodies (anti-Kme1 / Kme2 / Kme3) were conjugated with Protein A / G magnetic beads and incubated at 4°C for 3 h for antibody enrichment. Finally, Stage Tip was used for further enrichment. High-pH reverse-phase fractionation was performed using C18 or microcolumn gradient elution, with 6-8 fractions combined. LC-MS / MS analysis was then performed. UPLC was conducted using a 75 µm × 25 cm C18 column with a 5–40% ACN gradient elution for 120 min. Mass spectrometry was performed using an Orbitrap Q-Exactive HF-X in DIA / SWATH or PRM mode, with resolutions of 120,000 (MS1) and 30,000 (MS2), respectively. For data analysis, Max Quant or DIA-NN was used for library search, with "Kme1 / Kme2 / Kme3" variable modifications set and an FDR of 1%. Quantification was performed using label-free or iTRAQ / TMT, and differential results with FC ≥ 1.5 and FDR < 0.05 were screened. GO / KEG enrichment was then performed using DAVID / cluster Profiler. Finally, Western blotting was used to detect the overall trend of changes using anti-methylation site antibodies to validate the results.

[0097] protein docking

[0098] Protein crystal structures were downloaded using AlphaFold3 and Uniport, molecular docking analysis was performed using Auto DockTools-1.5.7 software, and visualization analysis was performed using Pymol.

[0099] Protein immunoprecipitation (Co-IP)

[0100] The longest transcript CDS regions of KDM6A (NM_001403371) and ATP6V0C (NM_001361531.1) were obtained from NCBI and inserted into the Ubc-MCS-3×Flag-SV40-Puromycin vector and the Pcmv-MCS-3×HA-IRES2-Neo vector, respectively. Plasmids expressing Flag-KDM6A and HA-ATP6V0C were obtained using this method. These two plasmids were then co-transfected into N2A cells to obtain N2A cells expressing both Flag-KDM6A and HA-ATP6V0C; or the two plasmids were transfected into N2A cells separately to obtain two types of N2A cells expressing Flag-KDM6A and HA-ATP6V0C, respectively. Protein immunoprecipitation experiments were performed using these three cell types.

[0101] Samples (N2A cells expressing Flag-KDM6A, N2A cells expressing HA-ATP6V0C, or N2A cells expressing both Flag-KDM6A and HA-ATP6V0C) were first treated with RIPA lysis buffer containing a protease / phosphatase inhibitor at 4°C for 30 minutes. Then, the samples were centrifuged at 12000g for 15 minutes, and the supernatant was collected. 800µg of total protein supernatant was taken, and 20µL of Protein A / G magnetic beads conjugated with HA / Flag antibodies were added for immunoprecipitation. The mixture was incubated at 4°C for 3 hours. Next, the magnetic beads were washed three times at 4°C with low-salt buffer (containing 20mM Tris-HCl, 150mM NaCl, 0.1% Tween-20, pH 7.4). After the final wash, the residual liquid was aspirated, and 20µL of 2×SDS loading buffer was added. The mixture was heated at 95°C for 8 minutes to dissociate the proteins. After centrifugation, the supernatant was collected and analyzed by SDS-PAGE electrophoresis and Western spectroscopy. blot experiments were used to verify the target protein and its interacting proteins.

[0102] B: Experimental Results

[0103] Given the significant decrease in protein methylation levels after GenX treatment, we further utilized... Kdm6a -KO model, and analyzed methylation modification omics data. Results showed that Kdm6a After knockout, differentially methylated proteins were significantly enriched in cell cycle arrest and cellular senescence-related pathways. Figure 4 (ab) This suggests that KDM6A-mediated methylation regulation is one of the core mechanisms of GenX-induced cellular senescence. Mass spectrometry results showed that after Kdm6a knockout, the level of trimethylation (me3) of Lys(K36) in ATP6V0C was significantly upregulated. Figure 4 c). Through cross-species protein conservation comparison, the K36 site of ATP6V0C was found to be conserved ( Figure 4 d).

[0104] Furthermore, through protein docking analysis, we selected the conformation with the highest score to analyze interactions. In this conformation, proteins primarily interact through hydrogen bonds and intermolecular forces. Specifically, there is a hydrogen bond between LYS787 residue of the KDM6A protein and GLY83 residue of the ATP6V0C protein; a hydrogen bond between LYS2 residue of the KDM6A protein and TYR144 residue of the ATP6V0C protein; and a hydrogen bond between ASP1179 residue of the KDM6A protein and LYS36 residue of the ATP6V0C protein. Figure 5 a). This part of the results suggests that KDM6A may regulate cellular senescence by directly binding to and specifically removing ATP6V0C-K36me3. After co-transfecting N2A cells with HA-ATP6V0C and Flag-KDM6A plasmids, a bidirectional immunoprecipitation assay using HA and Flag confirmed a direct interaction between KDM6A and ATP6V0C; further experiments showed that GenX exposure significantly enhanced the binding ability between the two. Figure 5 bc).

[0105] In summary, KDM6A synergistically drives GenX-induced cellular senescence by specifically removing ATP6V0C-K36me3 and directly binding to it.

[0106] Example 4: Screening for small molecule compounds that may improve GenX-induced cellular senescence based on the KDM6A and ATP6V0C binding sites.

[0107] Analysis of small compound ligand binding sites: Based on the interaction between KDM6A and ATP6V0C and the key amino acid D1179 of methylation modification, the MOE was used to find the pockets around D1179 where small molecules bind to proteins.

[0108] Since the murine KDM6A currently only has the AlphaFold3 prediction model, while the human KDM6A (NM_001291415) has had its high-resolution crystal structure resolved, we first compared the sequences of the two species. The full-length structure of the KDM6A_HUMAN protein is 1401 amino acids, and four crystal structures have been resolved. Structural alignment using MOE showed an RMSD of 0.661 Å, indicating relatively small structural differences. This experiment selected the 6FUL structure, which has the lowest resolution (1.65 Å) and the highest clarity.

[0109] The results showed that the overall sequence identity of human-mouse KDM6A was 99%, and the key catalytic domains were completely identical. Figure 6 This indicates that the differences in three-dimensional conformation are negligible. Therefore, subsequent virtual screening used human-derived crystal structures as templates to ensure docking accuracy and repeatability. Figure 7 As shown, the small molecule binding pocket Site1 of KDM6A contains the amino acids Pro924, Ser925, Ser926, Gly1175, Val1176, Asp1179, Phe1180, Lys1183, and Ala1203.

[0110] Example 5: Screening of small molecule compounds that antagonize the binding of KDM6A to ATP6V0C and thereby improve the cellular senescence phenotype

[0111] A: Experimental Section

[0112] Small molecule compound screening process

[0113] (1) KDM6A protein structure optimization: The KDM6A protein crystal structure was optimized by bond order using the Protein Preparation Wizard module in Schrödinger software, including hydrogenation, disulfide bond allocation, and protonation at pH 7.0 using the PROPKA method; confinement energy optimization was performed using the OPLS4 force field to eliminate interatomic conflicts in the structure, so that the RMSD of heavy atoms converged to 0.3, and side chain position optimization was performed to obtain a good side chain structure. The prepared structure was used as the receptor file for subsequent virtual screening.

[0114] (2) Preparation of small molecule compound library: The D3100 (Pre Diversity Compound Library) compound library was selected for this virtual screening. The LigPrep module in Schrödinger software (LigPrep, Schrödinger, LLC, New York, NY, 2021) was used to process the compound library. The Epik method was used to protonate and desalt the compound library under pH 7.0 ± 2.0 conditions to generate tautomers while maintaining the original atomic chirality. To ensure the global conformation of small molecules during the virtual screening process, conformation generation was performed on the small molecules, with a maximum of 32 conformations generated for each small molecule. The prepared compound library was used as a ligand file for subsequent virtual screening.

[0115] (3) Screening process (see Figure 8 The process involves importing prepared receptor and ligand files and employing a step-wise strategy (HTVS [High-Throughput Virtual Screening] → SP [Standard Precision] → XP [Extra Precision]) to perform molecular docking on small molecule compounds from the D3100 database. Energy optimization is performed after docking, and the top 50% of small molecules in each docking score are retained for the next round of screening. Finally, the top 5 small molecule compounds with the highest docking scores in the synthetic pathways are selected for subsequent in vitro functional validation.

[0116] Drug safety experimental verification

[0117] Mouse neuroblastoma cells (N2A) were seeded at 5000 cells / well in 96-well plates and cultured in DMEM / H medium (Gibco, C11995500BT) with 10% fetal bovine serum (AUSGENEX, FBS500-S) and 1% penicillin / streptomycin (Beyotime, C0222) added to the medium, and cultured at 37°C in 5% CO2. After 24 h of culture in untreated medium, one of the following small molecule compounds, Z-KA1, Z-KA2, Z-KA3, Z-KA4, and Z-KA5, was added at concentrations of 0 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively. After 72 h of treatment with the small molecule compounds, routine CCK-8 cell viability was measured.

[0118] Experimental verification of drug efficacy

[0119] Mouse neuroblastoma cells (N2A) were seeded at 5000 cells / well in 96-well plates and cultured in DMEM / H medium (Gibco, C11995500BT) with 10% fetal bovine serum (AUSGENEX, FBS500-S) and 1% penicillin / streptomycin (Beyotime, C0222) added to the medium, and cultured at 37°C in a 5% CO2 environment. After 24 hours of culture in untreated medium, one of the following small molecule compounds, Z-KA1, Z-KA2, Z-KA3, Z-KA4, and Z-KA5, was added, with the working concentration of each small molecule compound controlled at its maximum safe concentration (Z-KA1 10 μM, Z-KA2 40 μM, Z-KA3 10 μM, Z-KA4 10 μM, Z-KA5 20 μM), and 250 μM GenX was added simultaneously. After co-treatment with small molecule compounds and GenX for 72 hours, routine CCK-8 cell viability assays were performed.

[0120] B: Experimental Results

[0121] Schrödinger software was used to perform compound docking on each compound in the compound library, and the affinity scores between the compounds and the 6FUL structure were calculated. The D3100 compound library contained 17302 compounds that bound to KDM6A, with affinity ranging from -6.4633 kcal / mol to -2.4931 kcal / mol. Figure 9a). Due to the varying affinity distributions of the KDM6A_HUMAN protein structure, 557 compounds with affinity values ​​less than -4 kcal / mol binding to the KDM6A structure were selected. Protein-ligand interaction fingerprinting (PLIF) analysis was performed on the D3100 compound library based on its binding to the KDM6A structure. Figure 9 (b) In the statistical results of amino acid interaction frequencies, 556 out of 557 compounds bound to the KDM6A structure D3100 compound library exhibit protein interactions (hydrogen bonds, ionic bonds, aromatic ring-aromatic ring stacking, aromatic ring-hydrogen stacking, aromatic ring-cation stacking, excluding van der Waals interactions). Except for D1179, which has a relatively high frequency, the interaction frequencies of the remaining amino acids are similar to uniform. Further analysis of the specific drug-like properties of the 556 compounds bound to the KDM6A structure D3100 compound library included the following criteria: relative molecular mass (WMS500), hydrogen bond donors and acceptors (OH's and NH's ≤ 5; N's and O's ≤ 10, number of rotatable bonds ≤ 11); polar surface area (TPSAS140); water solubility (-4 ≤ logS ≤ 0.5); logarithm of the n-octanol / water partition coefficient (0 ≤ logP3)). The first step in drug absorption is the disintegration of the tablet or capsule, followed by the dissolution of the active drug. Low solubility is detrimental to good and complete oral absorption. 1ogP has a significant impact on membrane permeability and hydrophobic binding to macromolecules, including target receptors and other proteins such as plasma proteins, transport proteins, or metabolic enzymes. Based on these properties, we eliminated some compounds with undesirable properties, ultimately identifying 251 compounds from the D3100 compound library that conform to druggability. Structural diversity analysis was performed on the 251 compounds from the D3100 compound library that conform to KDM6A. Structure-based clustering was conducted using MOE software, with the Jarvis-Patrick fingerprint clustering algorithm set at 70%. The 251 compounds from the D3100 compound library that conform to KDM6A were divided into 208 classes. Compounds with the best affinity in each class were retained for subsequent experiments. For the 208 compounds bound to KDM6A in the D3100 compound library, the binding conformation between the compound and the protein was examined by visualization. Compounds that could not interact with D1179 were removed, leaving 162 compounds in the D3100 compound library bound to KDM6A. We selected the top 5 compounds for subsequent experimental validation and named them: Z-KA1 (Equation 1), Z-KA2 (Equation 2), Z-KA3 (Equation 3), Z-KA4 (Equation 4), and Z-KA5 (Equation 5). Detailed information is shown in Table 1.

[0122]

[0123] Table 1: Basic Information on Small Molecule Compounds

[0124]

[0125] The effect of Z-KA1-5 on cell viability was verified by the CCK-8 assay. Figure 10 The results showed that compared with the control group (0 μM), Z-KA1 showed no significant difference in cell viability at 5 μM and 10 μM (P>0.05); cell viability was significantly reduced at 20 μM and 40 μM (P<0.01); Z-KA2 showed no significant difference at 5 μM, 10 μM, 20 μM, and 40 μM (P>0.05); Z-KA3 showed no significant difference in cell viability at 5 μM and 10 μM (P>0.05); cell viability was significantly reduced at 20 μM and 40 μM (P<0.01); Z-KA4 showed no significant difference in cell viability at 5 μM and 10 μM (P>0.05); cell viability was significantly reduced at 20 μM and 40 μM (P<0.01); Z-KA5 showed no significant difference in cell viability at 5 μM, 10 μM, and 20 μM (P>0.05), but cell viability was significantly reduced at 40 μM (P<0.05).

[0126] Cell viability experiments such as Figure 11 As shown, compared to the control group (Ctrl), cell viability was significantly reduced in the GenX group (P<0.01); compared to the GenX-exposed group, cell viability was significantly upregulated in the GenX+Z-KA1 and GenX+Z-KA5 groups (P<0.05); while there was no significant difference in the GenX+Z-KA2, GenX+Z-KA3, and GenX+Z-KA4 groups (P>0.05). Experimental data showed that, through virtual screening, the small molecule compounds Z-KA1, Z-KA2, Z-KA3, Z-KA4, and Z-KA5 all had the ability to inhibit the binding of KDM6A to ATP6V0C, thereby antagonizing the decrease in ATP6V0C methylation levels caused by GenX exposure and ultimately delaying GenX-induced neuronal senescence. However, actual experimental studies showed that only Z-KA1 and Z-KA5 could reverse the decrease in cell viability caused by GenX exposure to a certain extent; among them, Z-KA1 not only had a lower effective concentration but also a slightly better effect than Z-KA5.

[0127] Example 6: Small molecule compound Z-KA1 targets and blocks the binding of KDM6A-ATP6V0C, thereby improving the cellular senescence phenotype.

[0128] (1) Molecular docking

[0129] Molecular docking analysis was conducted to examine the interaction between Z-KA1 and KDM6A binding sites. The results showed ( Figure 12 a) Z-KA1 interacts with the S926, D1179, and A1203 amino acid residues of KDM6A (3D diagram: red dashed lines represent hydrogen bond interactions; 2D diagram: arrows represent hydrogen bond interactions).

[0130] (2) Immunoprecipitation

[0131] N2A cells co-expressing Flag-KDM6A and HA-ATP6V0C proteins were harvested and processed at a rate of 4 × 10⁻⁶. 5 Cells / well were seeded into 10 cm dishes and cultured in DMEM / H medium (Gibco, C11995500BT) with 10% fetal bovine serum (AUSGENEX, FBS500-S) and 1% penicillin / streptomycin (Beyotime, C0222) added. The culture was then incubated at 37°C in 5% CO2. After 24 hours of incubation in untreated medium, 10 μM of the small molecule compound Z-KA1 was added, and the treatment lasted 72 hours before protein immunoprecipitation. Alternatively, the following method was used: 10 μM of the small molecule compound Z-KA1 and 250 μM of M GenX were added, and the treatment lasted 72 hours before protein immunoprecipitation.

[0132] N2A cells expressing HA-ATP6V0C protein were harvested and processed at a rate of 4 × 10⁻⁶. 5 Cells / well were seeded into 10 cm dishes and cultured in DMEM / H medium (Gibco, C11995500BT) with 10% fetal bovine serum (AUSGENEX, FBS500-S) and 1% penicillin / streptomycin (Beyotime, C0222) added. The culture was then incubated at 37°C in 5% CO2. After 24 hours of incubation in untreated medium, 10 μM of the small molecule compound Z-KA1, or 250 μM of GenX, or both Z-KA1 and GenX were added. After 72 hours of treatment, protein immunoprecipitation was performed.

[0133] Co-transfection with Flag-KDM6A and HA-ATP6V0C revealed that Z-KA1 could weaken the binding of KDM6A to ATP6V0C. Figure 12 b). Simultaneously, the results showed that Z-KA1 could upregulate the methylation level of ATP6V0C ( Figure 12 c).

[0134] More specifically, in Figure 12In the immunoprecipitation experiment shown in b, after co-transfection with Flag-KDM6A and HA-ATP6V0C, precipitation was performed using Flag antibody and detection was performed using HA antibody. A clear HA signal was observed in the GenX-treated group, indicating that HA-ATP6V0C was effectively captured by Flag-KDM6A. The HA signal was significantly reduced in the Z-KA1-treated group, suggesting that Z-KA1 weakened the interaction between Flag-KDM6A and HA-ATP6V0C. Simultaneously, the IP:Flag+IB:Flag results showed consistent Flag-KDM6A precipitation efficiency across all groups, ruling out bias in the precipitation system; there was no difference in HA and Flag protein levels in WCL (total protein) between groups, confirming that the weakened interaction was not due to insufficient protein expression.

[0135] More specifically, in Figure 12 In step b, for IP:Flag+IB:HA, when Flag-KDM6A and HA-ATP6V0C were simultaneously transfected, GenX treatment resulted in a detectable HA signal (indicating that HA-ATRP6VC was precipitated by Flag-Kdm6a); however, the HA signal was significantly weakened after the addition of Z-KA1. This indicates that Z-KA1 inhibits the interaction between Flag-Kdm6a and HA-ATRP6VC. For IP:Flag+IB:Flag, the successful and stable precipitation of Flag-Kdm6a was verified. In WCL (total protein), the results for IB:HA and IB:Flag confirmed that all proteins were normally expressed in the cells, ruling out interference from weak IP signals due to protein non-expression.

[0136] exist Figure 12 In step c, the effects of GenX and Z-KA1 on methylation modification of HA-ATP6V0C (Pan-me3, a non-histone pan-trimethylation modification antibody) were investigated. For IP:HA+IB:Pan-me3, when HA-ATP6V0C was transfected and treated with GenX, the Pan-me3 signal (representing the methylation level of HA-ATP6V0C) was significantly lower than that of the experimental group transfected with HA-ATP6V0C alone. However, when cells transfected with HA-ATP6V0C were treated with both Z-KA1 and GenX, the Pan-me3 signal was increased to some extent. This indicates that GenX can reduce the methylation level of HA-ATP6V0C, while Z-KA1 can reverse the effect of GenX to some extent. For IP:HA+IB:HA, the precipitation of HA-ATRP6VC was verified, indicating stable precipitation efficiency. IB:HA and IB:GAPDH in WCL were used to verify that HA-ATRP6VC was expressed consistently in all groups. GAPDH was used as an internal control to prove that the total protein loading was equal, thus eliminating the interference of methylation signal changes caused by differences in protein expression.

[0137] SA-β-gal staining assay, senescence-related protein detection, and cell cycle analysis

[0138] Mouse neuroblastoma cells (N2A) were seeded at 20,000 cells / well in 24-well plates and cultured in DMEM / H medium (Gibco, C11995500BT) with 10% fetal bovine serum (AUSGENEX, FBS500-S) and 1% penicillin / streptomycin (Beyotime, C0222) added. The plates were then cultured at 37°C in 5% CO2. After 24 h of untreated culture, either 10 μM of the small molecule compound Z-KA1 was added along with 250 μM GenX, or only 250 μM GenX was added, or no treatment was used as a blank control. After 72 h of drug treatment, routine SA-β-gal staining was performed, and the percentage of SA-β-gal-positive cells was counted (cell positivity rate was recorded in 3 wells for each group). In addition, after the above treatment, senescence-related protein detection and cell cycle analysis were performed using flow cytometry.

[0139] SA-β-gal staining showed that Z-KA1 could significantly reverse GenX-induced cell senescence, SA-β-gal activity decreased, and staining became lighter ( Figure 13 a). Western blot results showed ( Figure 13 (b) Z-KA1 significantly inhibited the expression of aging-related proteins. Compared with the control group, the expression of P53, P21, and P16 proteins was significantly upregulated in the GenX group (P<0.01), while the small molecule compound Z-KA1 significantly inhibited the expression of P53, P21, and P16 proteins (P<0.05), thereby inhibiting the aging process. Therefore, Z-KA1 can significantly reverse the GenX-induced cellular senescence phenotype.

[0140] Cell cycle detection by flow cytometry Figure 14 (ab) The results showed that compared with the control group (Ctrl), the cell DNA content in the GenX-exposed group was significantly reduced, and the S phase was significantly decreased (P<0.01); compared with the GenX group, the cell DNA content in the GenX+Z-KA1 group was significantly upregulated, and the S phase was significantly upregulated (P<0.01). This indicates that Z-KA1 can improve cell cycle arrest induced by GenX exposure.

[0141] More specifically, regarding cellular DNA content ( Figure 4a. Each group is shown in 3 replicates. The vertical axis (Count) represents the number of cells with that fluorescence intensity; the horizontal axis represents DNA content. The peaks from left to right represent G0, S, and G2 / M phases, respectively. It can be seen that the S phase in the GenX-treated group is significantly reduced, indicating cell cycle arrest. The addition of the small molecule compound Z-KA1 can improve the S phase ratio. At different stages of the cell cycle: G0 / G1 phase (G0 phase) cells have not replicated DNA and are diploid, with lower fluorescence intensity, corresponding to the peak on the left side of the figure; S phase cells are undergoing DNA replication, with DNA content between diploid and tetraploid, and fluorescence intensity in the middle range; G2 / M phase cells have completed DNA replication, are tetraploid, and have higher fluorescence intensity, corresponding to the peak on the right side of the figure. Utilizing the binding characteristics of fluorescent dyes to DNA, the relative DNA content is reflected by detecting fluorescence intensity. The experimental results show that GenX treatment reduces the DNA content in cells compared to the control group, while in the GenX+Z-KA1 group, Z-KA1 can reverse the decreasing trend of DNA content caused by GenX. Figure 4 b is a bar chart showing the proportions of different cell cycle phases (G0 / G1, S, G2 / M) in different treatment groups (Ctrl, GenX, GenX+Z-KA1, 20,000 cells per group). GenX treatment (relative to the control group) resulted in a decrease in the proportion of cells in the S phase and an increase in the proportion of cells in the G0 / G1 phase. The additional application of Z-KA1 reversed the effects of GenX treatment; the GenX+Z-KA1 group showed an increase in the proportion of cells in the S phase compared to the GenX group. The cell cycle reflects senescence because the normality of the cell cycle progression is closely related to cell proliferation and viability. Under normal circumstances, cells sequentially go through the G0 / G1, S, and G2 / M phases, completing DNA replication and cell division to maintain cell renewal and function. When cells age, cell cycle arrest often occurs, such as more cells remaining in the G0 / G1 phase and a decrease in the proportion of cells in the S phase. This means that cell DNA replication is inhibited and proliferation capacity decreases. Experiments showed that GenX treatment reduced the proportion of neurons in the S phase and increased the proportion in the G0 / G1 phase, indicating that GenX induced cell cycle arrest, affecting normal cell proliferation and promoting neuronal senescence. Compound Z-KA1 antagonized the GenX-induced cell cycle arrest effect, significantly increased the proportion of cells in the S phase, restored DNA replication and cell proliferation capacity, thereby alleviating GenX-related neuronal senescence caused by cell cycle abnormalities.

[0142] In summary, Z-KA1 can antagonize the interaction between KDM6A and ATP6V0C, thereby upregulating the methylation level of ATP6V0C and improving GenX-induced aging phenotype and neurotoxicity.

[0143] Example 7: Small molecule compound Z-KA5 targets and blocks the binding of KDM6A-ATP6V0C, thereby improving the cellular senescence phenotype.

[0144] The experimental method in this embodiment is the same as in embodiment 6, except that Z-KA1 is replaced with Z-KA5, and the working concentration of Z-KA5 is its maximum safe concentration of 20 μM.

[0145] Molecular docking analysis was performed on the binding sites of Z-KA5 and KDM6A. The results showed that ( Figure 15 a) Z-KA5 interacts with amino acid residues S926, V1176, D1179, K1183, and A1203 of KDM6A (3D diagram: red dashed lines represent hydrogen bond interactions; 2D diagram: arrows represent hydrogen bond interactions). Co-transfection of Flag-KDM6A and HA-ATP6V0C revealed that Z-KA5 could weaken the binding of KDM6A to ATP6V0C. Figure 15 b). Simultaneously, the results showed that Z-KA5 could upregulate the methylation level of ATP6V0C ( Figure 15 c).

[0146] SA-β-gal staining showed that Z-KA5 could significantly reverse GenX-induced cell senescence, SA-β-gal activity decreased, and staining became lighter ( Figure 16 a). Western blot results showed ( Figure 16 (b) Z-KA5 significantly inhibited the expression of aging-related proteins. Compared with the control group, the expression of P53, P21, and P16 proteins was significantly upregulated in the GenX group (P<0.01), while the small molecule compound Z-KA1 significantly inhibited the expression of P53, P21, and P16 proteins (P<0.05).

[0147] Cell cycle detection by flow cytometry Figure 17 (ab) We found that, compared with the control group (Ctrl), the cell DNA content in the GenX-exposed group was significantly reduced, and the S phase was significantly decreased (P<0.01); compared with the GenX group, the cell DNA content in the GenX+Z-KA5 group was significantly upregulated, and the S phase was significantly upregulated (P<0.01). This indicates that Z-KA5 can improve cell cycle arrest induced by GenX exposure.

[0148] In summary, Z-KA5 antagonizes the interaction between KDM6A and ATP6V0C, thereby upregulating the methylation level of ATP6V0C and improving GenX-induced aging phenotypes and neurotoxicity.

[0149] In addition, considering the experimental data of Z-KA1 and Z-KA5, Z-KA1 showed better overall performance than Z-KA5, specifically in that Z-KA1 exhibited lower cytotoxicity at concentrations of 10 μM and below. Figure 10 Furthermore, at a concentration of 10 μM, Z-KA1 has demonstrated a significant effect in reversing GenX (…). Figure 11 Z-KA1 is more effective than Z-KA5 in weakening the interaction between Flag-KDM6A and HA-ATP6V0C. Figure 12 , Figure 15 Z-KA1 is more effective at inhibiting the binding of KDM6A to ATP6V0C, which is more conducive to increasing the methylation level of ATP6V0C and thus inhibiting the cellular senescence process.

[0150] 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 application of small molecule compounds in the preparation of anti-aging drugs, characterized in that, The anti-aging drug is a drug that combats the senescence of nerve cells induced by the environmental pollutant GenX; the small molecule compound is shown in Formula 1 or Formula 5. 。 2. The application of the small molecule compound according to claim 1 in the preparation of anti-aging drugs, characterized in that: The small molecule compound is used to upregulate the methylation level of the ATP6V0C protein.

3. The application of the small molecule compound according to claim 2 in the preparation of anti-aging drugs, characterized in that, The small molecule compound is used to inhibit the binding of KDM6A and ATP6V0C proteins.

4. The application of the small molecule compound according to claim 3 in the preparation of anti-aging drugs, characterized in that, The small molecule compound is used to bind to the small molecule binding pocket of KDM6A; the amino acid residues in the small molecule binding pocket of KDM6A include proline at position 924, serine at position 925, serine at position 926, glycine at position 1175, valine at position 1176, aspartic acid at position 1179, phenylalanine at position 1180, lysine at position 1183, and alanine at position 1203.

5. The application of the small molecule compound according to claim 4 in the preparation of anti-aging drugs, characterized in that, The compound shown in Formula 1 is used to interact with serine at position 926, aspartic acid at position 1179, and alanine at position 1203 of KDM6A; the compound shown in Formula 5 is used to interact with serine at position 926, valine at position 1176, aspartic acid at position 1179, lysine at position 1183, and alanine at position 1203 of KDM6A.

6. The application of KDM6A protein in screening drugs to resist GenX-induced neuronal senescence, characterized by: The application is for non-diagnostic and non-therapeutic purposes outside the body; The amino acid residues in the small molecule binding pocket of the KDM6A protein are used for computer-aided molecular docking with candidate small molecule drugs. The candidate small molecule drug is designed to bind to the small molecule binding pocket of the KDM6A protein and inhibit the binding of KDM6A and ATP6V0C proteins. The amino acid residue in the small molecule binding pocket of KDM6A is aspartic acid at position 1179.

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