Targeted selenol inhibitor and application thereof

Abstract

The invention belongs to the technical field of medicine, and particularly relates to a targeted selenol inhibitor and application thereof. Through metabonomics and transcriptomics analysis, the redox steady state, the metabolic steady state, the cell death mode and the molecular mechanism of the selenol-inhibited glioma cells are researched. Comprising the steps of ROS (reactive oxygen species) generation, GSH / GSSG (glutathione / reduced glutathione) metabolism, lipid peroxidation level and cell death marker detection, the main cell death mode triggered by inhibition of selenol is further identified, and whether molecular events such as endoplasmic reticulum stress and protein misfolding are involved or not is analyzed. Based on the research results, the combined application potential of selenol targeted therapy and existing chemotherapy, radiotherapy and immunotherapy is further evaluated, so that the treatment strategy is optimized, and the treatment response rate of glioma patients is increased.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a targeted selenol inhibitor and its application. Background Art

[0002] Glioblastoma (GBM) is a highly invasive malignant brain tumor. Due to its highly diffuse growth, rapid proliferation, and strong drug resistance, existing treatment methods are difficult to achieve long-term effective control. The standard treatment regimen includes surgical resection, radiotherapy, and chemotherapy (such as temozolomide, TMZ). However, due to the presence of the blood-brain barrier (BBB), the drug tolerance of glioma cells, and the immunosuppressive characteristics of the tumor microenvironment, the prognosis of patients remains extremely poor.

[0003] In recent years, ferroptosis has been considered a potential direction to break through the bottleneck of glioma treatment due to its unique lipid peroxidation-mediated cell death mechanism. However, glioma cells can maintain the intracellular redox homeostasis by enhancing the antioxidant defense system, such as upregulating antioxidant proteins such as glutathione peroxidase 4 (GPX4) and thioredoxin reductase 1 (TXNRD1), thereby reducing lipid peroxidation damage and decreasing the induction efficiency of ferroptosis. This compensatory mechanism severely limits the application of existing ferroptosis induction strategies.

[0004] Therefore, how to effectively break through the antioxidant defense of glioma cells and improve their sensitivity to ferroptosis is an important research topic in current precision targeted therapy. Summary of the Invention

[0005] The purpose of the present invention is to provide a targeted selenol inhibitor. The inhibitor provided by the present invention targets selenol and simultaneously inhibits the functions of multiple selenoproteins, inducing cell death.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] The present invention provides an inhibitor, and the inhibitor targets selenol; the selenol is a functional group in selenoprotein.

[0008] Preferably, the selenol is related to cellular redox regulation, antioxidant defense, and metabolic adaptation.

[0009] The present invention also provides the application of the above inhibitor in inhibiting the metabolism of tumor cells.

[0010] Preferably, the metabolism of the tumor cells includes GSH / GSSG metabolism, lipid peroxidation level, or ROS generation.

[0011] The present invention also provides the application of the above inhibitor in inducing ferroptosis of tumor cells.

[0012] Preferably, in the application, the inhibitor promotes ferroptosis of tumor cells by enhancing endoplasmic reticulum stress.

[0013] The present invention also provides an application of the above inhibitor in inducing activation of the UPR pathway.

[0014] Preferably, in the application, the inhibitor promotes intracellular oxidative stress and lipid peroxidation.

[0015] The present invention also provides an application of the above inhibitor in preparing a drug targeting ferroptosis.

[0016] The present invention also provides an application of the above inhibitor in preparing an anti-tumor drug.

[0017] Advantages of the present invention:

[0018] The inhibitor of the present invention targets selenols, breaks through the limitations of single-target therapy, can simultaneously inhibit multiple antioxidant selenoproteins, weaken the antioxidant ability of tumor cells, disrupt their redox state, and improve the efficiency of inducing ferroptosis of tumor cells. Brief Description of the Drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0020] Figure 1 It is a result graph of metabolomics analysis of oxidized glutathione and reduced glutathione;

[0021] Figure 2 It is a result graph of live cell confocal ROS detection and its analysis result graph;

[0022] Figure 3 It is a result graph of the detection of the content of lipid peroxidation product MDA;

[0023] Figure 4 It is a result graph of the detection of the intracellular protein carbonylation level;

[0024] Figure 5 It is a result graph of immunofluorescence detection of p-H2A.X level;

[0025] Figure 6 It is a result graph of RNA-seq analysis of the expression of genes related to the ferroptosis pathway;

[0026] Figure 7 It is a mitochondrial structure graph of cells observed by transmission electron microscopy;

[0027] Figure 8 Result graph of cell viability for functional rescue experiments

[0028] Figure 9 (a) Result graph of detecting selenoprotein levels by Western blot; (b) Result heat map of selenoprotein level expression

[0029] Figure 10 Result graph of KEGG enrichment analysis

[0030] Figure 11 Result graph of GO pathway analysis for intracellular lipid peroxidation metabolic pathway and protein misfolding pathway

[0031] Figure 12 Result graph of Gene Set Enrichment Analysis (GSEA)

[0032] Figure 13 Result graph of correlation analysis of ferroptosis and endoplasmic reticulum-related gene expression

[0033] Figure 14 Result graph of Western blot analysis of PERK, IRE1, and ATF6 after DS@Fn treatment; (a) Result graph of Western blot analysis of PERK; (b) Result graph of Western blot analysis of IRE1; (c) Result graph of Western blot analysis of ATF6

[0034] Figure 15 (a) Analysis graph of dynamic distribution of H2O2 (hydrogen peroxide) in glioma cells at different times; (b) Fluorescence intensity data analysis graph

[0035] Figure 16 (a) Result graph of dynamic distribution of lipid peroxidation at different times after selenol inhibition; (b) Fluorescence intensity data analysis graph Detailed implementation manner

[0036] The present invention has systematically studied the molecular mechanism of selenol as a biochemical family target (BFT) in the treatment of glioblastoma, aiming to reveal how selenol-targeted inhibition affects key biological processes such as the redox homeostasis, antioxidant defense system, lipid peroxidation, endoplasmic reticulum stress (ER stress), and ferroptosis of glioma cells. The present invention comprehensively analyzes the action mechanism of selenol-targeted therapy by integrating metabolomics, transcriptomics, proteomics, and functional verification experiments, providing theoretical support for further optimizing the selenol-based targeted therapy strategy for glioblastoma.

[0037] In view of the molecular mechanism of selenol-targeted therapy inducing the death of glioma cells, a systematic cell and molecular biology research platform was constructed to deeply analyze the effects of selenol inhibition on the metabolism, redox balance and cell death mode of glioma cells. The main components of this research system include the following core elements:

[0038] 1. Research models

[0039] Cell model: The human glioma cell line U87 was selected to study the specific effects of selenol-targeted therapy.

[0040] Animal model: Subcutaneous tumor model and orthotopic glioma model were used to verify the biological effects of selenol-targeted therapy in vivo.

[0041] 2. Key detection techniques

[0042] Metabolomics analysis: LC-MS (liquid chromatography-mass spectrometry) was used to detect the intracellular glutathione (GSH) metabolism level and the recruitment of metabolic pathways, and to analyze the effects of selenol inhibition on the cellular redox balance and lipid peroxidation.

[0043] Transcriptomics: RNA-seq technology was used to analyze the dynamic changes of cell signaling pathways after selenol inhibition.

[0044] Detection of cell death mechanism: Western blot, immunofluorescence labeling and live cell imaging were combined to study the mode of cell death induced by selenol inhibition in glioma cells.

[0045] 3. Characteristics of experimental design

[0046] Experiments such as studying the accumulation of reactive oxygen species (ROS) and lipid peroxidation (LPO) after selenol inhibition were carried out to clarify whether it affects the oxidative stress response of glioma cells by regulating GPX4 / TXNRD1.

[0047] The strategy of combining gene knockout and inhibitors was adopted to explore the compensatory mechanism of selenoprotein family members in selenol-targeted therapy, so as to analyze how glioma cells respond to the metabolic stress caused by selenol inhibition.

[0048] Selenoproteins (such as GPX4, TXNRD1) are highly expressed in glioma cells, maintaining redox homeostasis and inhibiting ferroptosis. The research results of the embodiments of the present invention show that selenol inhibition can lead to a significant down-regulation of key antioxidant enzymes such as GPX4 and TXNRD1, thereby depleting the cellular GSH (glutathione) reserve (decreasing to 2% of the normal level), weakening the antioxidant ability; the ROS level increases significantly, and the lipid peroxidation product MDA increases significantly, resulting in membrane lipid damage; the DNA oxidation damage marker p-H2A.X is up-regulated, indicating impaired genomic stability.

[0049] In the embodiments of the present invention, it is demonstrated for the first time that selenol-targeted inhibition can simultaneously inhibit the functions of multiple selenoproteins, completely weakening the antioxidant defense of tumor cells; transcriptome, metabolome and related experimental results show that lipid peroxidation and ferroptosis are strongly activated, confirming that ferroptosis is the main cell death mode.

[0050] The present invention discovers a new mechanism by which selenol inhibition promotes ferroptosis through endoplasmic reticulum stress (ER stress), and for the first time discovers the key role of endoplasmic reticulum stress in inhibiting selenol-induced ferroptosis: KEGG analysis and gene set enrichment analysis (GSEA) show that the unfolded protein response (UPR) signaling pathway is significantly enriched after selenol inhibition; Western blot confirms that the three major UPR signaling pathways (PERK, IRE1, ATF6) are all activated; H2O2 accumulates in the ER and diffuses to the cell membrane, triggering a lipid peroxidation cascade reaction, ultimately promoting ferroptosis.

[0051] In summary, the research of the present invention shows that inhibiting selenol targets can induce oxidative stress in glioma cells, leading to imbalance of the cellular oxidation balance; and can weaken the antioxidant capacity of glioma cells and induce ferroptosis of glioma cells by inhibiting the activities of selenoproteins such as GPX4 and TXNRD1. Further studying the molecular mechanism of selenol-targeted ferroptosis in glioma cells, targeting selenol leads to protein misfolding in the ER, activating the UPR (PERK, IRE1, ATF6) signaling pathway, thereby inducing the accumulation of H2O2 in the ER and its diffusion to the cell membrane, which exacerbates the lipid peroxidation cascade reaction and ultimately leads to ferroptosis. This research not only clarifies the molecular mechanism of selenol inhibition-induced ferroptosis in glioma cells, but also provides a new strategy for selenol-targeted ferroptosis therapy.

[0052] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] The production processes, experimental methods or detection methods involved in the embodiments of the present invention are all conventional methods in the prior art without special instructions, and their names and / or abbreviations are all conventional names in the art, which are very clear and definite in the relevant application fields. Those skilled in the art can understand the conventional process steps according to the names and apply the corresponding equipment, and implement them under conventional conditions or conditions recommended by the manufacturer.

[0054] There are no special restrictions on the sources of various instruments, equipment, raw materials or reagents used in the embodiments of the present invention. They are all conventional products that can be obtained through regular commercial channels, and can also be prepared according to the conventional methods well-known to those skilled in the art.

[0055] Example 1: Effects of selenol inhibition on the redox homeostasis of glioma cells

[0056] Selenol is a key functional group for maintaining cellular redox balance. Members of the selenoprotein family (such as GPX4, TXNRD1) are highly expressed in glioma cells and are responsible for regulating intracellular antioxidant capacity. In this study, we first explored whether selenol-targeted therapy disrupts the redox homeostasis of glioma cells and thereby affects their survival.

[0057] After treating glioma cells (U87) with the selenol-targeted nanodrug DS@Fn, the cellular redox homeostasis was significantly disrupted. DS@Fn is a brain glioma-targeted nanodrug for selenol, prepared by using human heavy chain ferritin (Fn) as a nanocarrier to encapsulate the small molecule drug N-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-2,4-dinitrobenzenesulfonamide (DS) that specifically responds to selenocysteine (Sec).

[0058] The results of metabolomics analysis, as Figure 1 shown, after treatment with DS@Fn, both oxidized glutathione and reduced glutathione in the cells decreased extremely significantly (P < 0.0001), indicating that the antioxidant capacity of the cells was severely weakened. At the same time, the level of reactive oxygen species (ROS) increased extremely significantly compared with the control group (P < 0.0001) ( Figure 2 ), showing an enhancement of oxidative stress.

[0059] In addition, the content of the lipid peroxidation product MDA increased significantly ( Figure 3 ), suggesting severe damage to membrane lipids, while the increase in the level of protein carbonylation ( Figure 4 ) further indicated that intracellular proteins had suffered oxidative damage. The increase in the level of the oxidative DNA damage marker p-H2A.X ( Figure 5 ) indicated that the cell nucleus had suffered oxidative damage, which might affect genomic stability.

[0060] In summary, the above data indicate that DS@Fn can effectively induce oxidative stress, severely disrupting the oxidative balance of glioma cells and laying the foundation for subsequent cell death processes.

[0061] Example 2: Mechanistic analysis of selenol inhibition-induced ferroptosis in glioma cells

[0062] Explore the specific mechanism of DS@Fn-induced glioma cell death and analyze the expression changes of ferroptosis-related genes.

[0063] The results of transcriptome analysis ( Figure 6 ) showed that after treatment with DS@Fn, ferroptosis pathway-related genes (such as SLC7A11, SAT1, etc.) were significantly upregulated, suggesting that the ferroptosis mechanism was activated. The observation results of transmission electron microscopy (TEM) (Figure 7 ) showed that the mitochondrial membrane density of cells in the DS@Fn treatment group increased, and the cristae structure decreased, presenting typical mitochondrial pathological changes of ferroptosis.

[0064] In addition, the results of the functional rescue experiment ( Figure 8 ) showed that the addition of the ferroptosis inhibitor Ferrostatin-1 (Ferr-1) to the cells in the DS@Fn treatment group could significantly restore cell viability, while the apoptosis inhibitor Z-VAD-FMK failed to provide a similar protective effect, further confirming that the cell death induced by DS@Fn mainly occurred through the ferroptosis mechanism.

[0065] The results of Western blot analysis ( Figure 9 ) showed that the expression levels of GPX4 and TXNRD1 proteins and all other selenoproteins decreased significantly, indicating that DS@Fn could effectively inhibit the functions of these key antioxidant selenoproteins, and the supplementation of exogenous selenocysteine could partially restore their expression.

[0066] Collectively, the above experimental results showed that DS@Fn could induce ferroptosis in glioma cells by inhibiting the activities of selenoproteins such as GPX4 and TXNRD1, weakening the antioxidant ability of cells.

[0067] Example 3 Molecular mechanism study on the inhibition of selenol targets inducing ferroptosis in glioma cells

[0068] To further analyze how the inhibition of selenol targets induces ferroptosis in glioma cells, we performed multi-omics analysis and verified its molecular mechanism by combining cell experiments.

[0069] First, the results of KEGG and GO pathway analysis showed that after treatment with DS@Fn, the unfolded protein response (UPR) and lipid metabolism pathways in cells were significantly enriched ( Figure 10 , Figure 11 ), indicating that endoplasmic reticulum (ER) stress might play a key role in the process of ferroptosis.

[0070] Further gene set enrichment analysis (GSEA) showed that among the 13 significantly upregulated signaling pathways, 9 were directly involved in the process of protein misfolding ( Figure 12 ), suggesting that selenol inhibition might mediate cell death through the UPR pathway.

[0071] The correlation between the expression of ferroptosis-related genes and endoplasmic reticulum-related genes was further verified. The results showed that there was a positive correlation between the expression of endoplasmic reticulum stress-related molecules and ferroptosis marker molecules ( Figure 13 ).

[0072] The Western blot experiment further confirmed that after treatment with DS@Fn, the phosphorylation levels of PERK, IRE1, and ATF6 were significantly increased ( Figure 14 ), indicating that the UPR pathway was activated, which might promote an increase in intracellular oxidative stress levels, leading to an increase in intracellular oxidative stress levels and promoting the occurrence of ferroptosis.

[0073] In addition, by tracking the dynamic changes of intracellular H2O2 (hydrogen peroxide) at different times in glioma cells, the results showed that after treatment with DS@Fn, the level of H2O2 rapidly accumulated in the endoplasmic reticulum (ER) and spread to the plasma membrane (PM) after 4 hours ( Figure 15 ), showing the dynamic propagation of H2O2 from the endoplasmic reticulum to the plasma membrane after treatment.

[0074] Since the ER is rich in polyunsaturated fatty acids (PUFAs) and is extremely prone to lipid peroxidation, the time-space distribution of intracellular lipid peroxidation was further detected ( Figure 16 ). The results showed that after treatment with DS@Fn, lipid peroxidation first occurred in the ER, then spread to the PM, and reached a peak in the late stage of ferroptosis. This change pattern was highly consistent with the accumulation dynamics of H2O2.

[0075] Based on the above results, the inhibition of selenol targets would lead to protein misfolding in the ER, activate the UPR signaling pathway, thereby inducing the accumulation of H2O2 in the ER and its diffusion to the cell membrane, triggering lipid peroxidation, and ultimately resulting in ferroptosis. This study not only clarified the molecular mechanism of selenol inhibition-induced ferroptosis in glioma cells but also provided a new strategy for selenol-targeted ferroptosis therapy.

[0076] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An inhibitor, characterized in that, The inhibitor targets selenol; the selenol is a functional group in selenoprotein.

2. The inhibitor according to claim 1, wherein The selenol is related to cellular redox regulation, antioxidant defense, and metabolic adaptation.

3. Use of the inhibitor according to claim 1 in inhibiting the metabolism of tumor cells.

4. The application according to claim 3, characterized in that The metabolism of the tumor cells includes GSH / GSSG metabolism, lipid peroxidation level, or ROS generation.

5. Use of the inhibitor according to claim 1 in inducing ferroptosis of tumor cells.

6. The application according to claim 5, characterized in that, In the said use, the inhibitor promotes ferroptosis of tumor cells by enhancing endoplasmic reticulum stress.

7. Use of the inhibitor according to claim 1 in inducing activation of the UPR pathway.

8. The application according to claim 7, characterized in that In the said use, the inhibitor promotes intracellular oxidative stress and lipid peroxidation.

9. Use of the inhibitor according to claim 1 in the preparation of a drug targeting ferroptosis.

10. Use of the inhibitor according to claim 1 in the preparation of an anti-tumor drug.

Images