Path of sodium overload cell death and application thereof

Abstract

The invention provides a sodium overload cell death pathway and application thereof, and a series of factors having an influence effect on the pathway. The invention also discloses a drug screening model and a screening method for regulating the cell death pathway. The novel drug screening model is based on TRPM4 or a transmembrane domain thereof. The invention also discloses several compounds with regulation and control effects, and the compounds can be used for preparing medicines effective for protecting cells.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and molecular biology. More specifically, this invention relates to the pathway of sodium overload cell death (sodium death, NECSO) and its applications. Background Art

[0002] Cell death has a variety of causes. It is a highly organized and fundamental activity, an event in which a biological cell ceases to perform its functions. Cell death occurs in one of two ways. One is apoptosis, also known as programmed cell death or cell suicide, a physiological form of cell death that is a process of active cell death under genetic control. The other is necrosis, an uncontrolled form of cell death that occurs more frequently in response to events such as ischemia and other forms of injury.

[0003] Necrosis is the premature death of cells, caused by damage, toxins, or nutrient deprivation. Cells die when exposed to environmental factors such as chemical agents (strong acids, strong alkalis, toxic substances), physical agents (heat, radiation), and biological agents (pathogens). Illness can also lead to cell death.

[0004] Some enzymes are involved in the cell death process. If the enzyme originates from the lysosomes of the dying cell itself, it is called autolysis; if the enzyme originates from the lysosomes of leukocytes infiltrating necrotic tissue, it is called heterolysis.

[0005] In the early stages of cell necrosis, mitochondria and endoplasmic reticulum in the cytoplasm swell and disintegrate, structural lipid droplets become free and vacuolated, protein particles increase, and the nucleus undergoes condensation or breakage. As cytoplasmic proteins denature, coagulate, or fragment, and basophilic nucleoproteins degrade, the cytoplasm becomes strongly eosinophilic. Therefore, in hematoxylin / eosin stained sections, the cytoplasm of necrotic tissue or cells appears as a uniform deep eosin color, and the original fine structure disappears.

[0006] Under certain conditions, cells lose functional control, and the plasma membrane and organelle membranes become unstable. Due to increased osmotic pressure, cells and organelles swell, eventually leading to cell rupture. In cells with high water content, the continuous enlargement and dissolution of cytoplasmic vacuoles can cause the complete disappearance of cell structure, ultimately resulting in the rupture of cell membranes and organelles, DNA degradation, and leakage of cell contents, causing an inflammatory response in surrounding tissues.

[0007] Overall, cell death is a complex process, and different factors under different conditions can lead to cell necrosis. The full picture of this process has not yet been revealed in this field. Summary of the Invention

[0008] The purpose of this invention is to provide a pathway for sodium overload-induced cell death and its application.

[0009] In a first aspect of the invention, a method is provided for screening substances (including potential substances, said substances including drugs) that regulate cell death, comprising: using TRPM4 or its transmembrane domain in cells as an observation target, or using TRPM4-mediated cellular Na+ as an observation target. + Overload-induced cell death (NECSO) is the target of observation. The effects of candidate substances on the target are analyzed to obtain substances that regulate cell death. The transmembrane domain includes a binding pocket. The Na+... + Overload-induced cell death in cells leads to the activation of the TRPM4 channel, resulting in Na+. + Excessive influx is characterized by cell necrosis.

[0010] In one or more embodiments, the method includes: (1) contacting a candidate substance with a screening system (screening model), the screening system comprising (e.g. expressing) TRPM4 or its transmembrane domain, the transmembrane domain comprising a binding pocket; (2) adding the candidate substance to the screening system of (1) and observing the interaction between the candidate substance and the binding pocket; if the candidate substance enters the binding pocket, interacts and inhibits TRPM4 channel activity, then the candidate substance is a cell-protective substance; if the candidate substance enters the binding pocket, interacts and activates TRPM4 channel activity, then the candidate substance is a cell-degrading substance.

[0011] In one or more embodiments, the method includes: (a) treating cells with Necrocide 1 (NC1) to induce Na+ + Cells undergoing necrosis by sodium overload (NECSO); preferably, Necrocide 1 enters the binding pocket, interacts, and activates TRPM4 channel activity, thereby inducing cell death; (b) treating cells with a candidate substance; (c) analyzing the necrotic cell death of the cells, indicating that the candidate substance is a cell-protective substance if it reduces necrotic cell death, and that the candidate substance is a cell-promoting substance if it increases necrotic cell death.

[0012] In one or more embodiments, the "transmembrane domain" includes a portion (protein fragment) of TRPM4 containing its transmembrane domain.

[0013] In one or more embodiments, the "transmembrane domain" of the TRPM4 includes six transmembrane helices (S1-S6 helix) and a TRP domain.

[0014] In one or more embodiments, the "transmembrane domain" of TRPM4 is the domain from position 768 (Cys) to position 1094 (Arg) in the amino acid sequence shown in SEQ ID NO:1.

[0015] In one or more embodiments, the TRPM4 includes human TRPM4 (hTRPM4).

[0016] In one or more embodiments, the term "protecting cells" includes "inhibiting cell death".

[0017] In one or more embodiments, in (2), if the candidate substance enters the binding pocket and interacts with it, the method further includes: observing the cell survival status; if the cell viability increases or the proportion of surviving cells increases, then the candidate substance is a cell-protecting substance; otherwise, the candidate substance is a cell-degrading substance.

[0018] In one or more embodiments, in (b), Necrocide 1 is added to the screening system before, simultaneously with or after the addition of the candidate substance.

[0019] In one or more embodiments, the system includes (but is not limited to): cell (culture) system, subcellular (culture) system, solution system, tissue system, organ system, animal system, and protein structure (including crystal structure) simulation system.

[0020] In one or more embodiments, the protein structure simulation system includes (but is not limited to): a lipid bilayer-protein-ligand system constructed based on a membrane construction procedure, and a molecular docking simulation system (such as ligand simulation using ChemDraw software, etc.). Maestro software suite performs molecular docking.

[0021] In one or more embodiments, the cell-protecting (cell-death-inhibiting) substance is Na. + Overload-induced cell death (sodium death) inhibitors (NECSO inhibitors).

[0022] In one or more embodiments, the Na + Overload-induced cell death is caused by excessive Na+. + Influx triggers cell death.

[0023] In one or more embodiments, the cells are cells that are highly sensitive or moderately sensitive to NC1.

[0024] In one or more embodiments, the binding pocket includes a binding cavity (capsaicin binding pocket); preferably, the binding cavity includes: S3 and S4 helices, an S4-S5 connector, and an enclosing structure formed by S5 and S6 helices adjacent to the subunit.

[0025] In one or more embodiments, the binding pocket includes the following sites of TRPM4: S5 helix Met927 (M927), Phe936 (F936); S3 helix Ser863 (S863), Trp864 (W864); S4 helix Val904 (V904), Leu907 (L907), His908 (H908).

[0026] In one or more embodiments, the regulatory effect of candidate substances on cell death is analyzed by observing the interaction between candidate substances and the site; or, the activity of TRPM4 channels and cell death are analyzed by observing the interaction between Necrocide 1 and the site; more preferably, the interaction (binding) with the site includes (but is not limited to): covalent binding or non-covalent binding (such as the formation of hydrogen bonds, π-π stacking, hydrophobic interactions, van der Waals forces, and ionic bonds).

[0027] In one or more embodiments, the inhibition / promotion is statistically significant, such as inhibition / promotion of 2% or more, 5% or more, 10% or more, 20% or more, 30% or more, 50% or more, 60% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%.

[0028] In one or more embodiments, the activation of the TRPM4 channel is a statistically significant activation, such as an increase / upregulation of activity of 2% or more, 5% or more, 10% or more, 20% or more, 30% or more, 50% or more, 60% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% or more.

[0029] In one or more embodiments, the sites in TRPM4 or its domains are counted based on the amino acid sequence of the full-length TRPM4 (e.g., the sequence number NP_060106.2 in the NCBI database).

[0030] In one or more embodiments, the candidate substances include: small molecule compounds, interacting molecules, regulatory molecules designed for signaling pathways or pathway proteins involved in the interaction, or upstream or downstream proteins or genes thereof, and CRISPR constructs; preferably, the small molecule compounds include compounds from a compound library; preferably, the interacting molecules include small molecule compounds, interfering molecules, biomacromolecules, etc.

[0031] In one or more embodiments, the methods for observing the interaction include (but are not limited to): molecular docking analysis, binding energy analysis, pull-down method, SPR method, Western blotting, DNA sequence analysis, and immunoprecipitation.

[0032] In one or more embodiments, the screening system is a molecular docking model (system).

[0033] In one or more embodiments, the interaction between the candidate material and the binding pocket is detected in pairs or between the candidate material, NC1 and the binding pocket.

[0034] In one or more embodiments, the method further includes: conducting further cell experiments and / or animal experiments on the obtained potential substances to further select and identify substances useful for inhibiting cell death from the candidate substances.

[0035] In one or more embodiments, the method for screening substances that inhibit cell death is not a method with disease treatment as its direct purpose.

[0036] In another aspect of the invention, an application of any of the methods described above is provided for: screening substances that regulate cell death (including potential substances, said substances including drugs); or screening drugs to alleviate or treat TRPM4 dysfunction.

[0037] In one or more embodiments, the TRPM4 dysfunction includes: cell or tissue damage; more preferably, the cell or tissue damage includes: heart disease.

[0038] In one or more embodiments, the TRPM4 dysfunction includes: diseases caused by TRPM4 lack of activity or function / deficiency.

[0039] In one or more embodiments, the heart disease includes: heart disease caused by damage to cardiomyocytes.

[0040] In another aspect of the invention, the application of Necrocide 1 (NC1) in the preparation of drug screening models, wherein the drug screening model is a cell model including TRPM4 or its transmembrane domain, and the Necrocide 1 cells induce Na+. + Overload-induced cell death (NECSO); the transmembrane domain includes a binding pocket; the Na + Overload-induced cell death in cells leads to the activation of the TRPM4 channel, resulting in Na+. + Excessive influx is characterized by cell necrosis.

[0041] In another aspect of the invention, a method for preparing a cell model is provided, comprising: treating cells (including cell cultures) with Necrocide 1 (NC1) to obtain a cell model, said cell model exhibiting Na+ + Overload-induced cell death (NECSO); the cells include TRPM4 or its transmembrane domain; the transmembrane domain includes a binding pocket; the Na + Overload-induced cell death in cells leads to the activation of the TRPM4 channel, resulting in Na+. + Excessive influx is characterized by cell necrosis.

[0042] In one or more embodiments, when cells are treated with NC1, the NC1 is added in the range of 1 to 10,000 nM; preferably 10 to 5,000, for example 20, 50, 100, 200, 300, 500, 800, 1200, 1500, 2000, or 3000 nM.

[0043] In another aspect of the invention, a cell model is provided, which is prepared by the aforementioned method for preparing a cell model.

[0044] In another aspect of the invention, the use of clotrimazole or dihydropyridine (DHP) compounds in the preparation of compositions (including pharmaceutical compositions) that inhibit cell death is provided; preferably, the dihydropyridine compound comprises an L-type calcium channel inhibitor; more preferably, the L-type calcium channel inhibitor comprises: cilindipine, lacidipine, benidipine, lercanidipine, nifedipine, amlodipine, and nimodipine.

[0045] In one or more embodiments, the cells include cardiomyocytes.

[0046] In one or more embodiments, the cell death includes necrotic cell death; preferably, the necrotic cell death is Na+. + Necrosis by Sodium Overload (NECSO), the substance that inhibits cell death is Na. + Overload cell death inhibitors (NECSO inhibitors).

[0047] In one or more embodiments, the cells further include: liver cells, skin cells, nerve cells, cardiomyocytes, small intestinal epithelial cells, etc.

[0048] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0049] Figure 1NC1 triggers a unique cell death pathway dependent on TRPM4. a. Chemical structure of NC1. b. Live cell image of MCF7 cells treated with 50 nM NC1 in SytoxGreen medium. Time points after treatment are shown in the figure. Scale bar represents 25 μm. c. Cells were pretreated for 1 hour before treatment with 20 μM Z-VAD-FMK (Z-VAD), 10 μM N ecrostatin-1 (Nec-1), 50 μM chloroquine (CQ), 50 μM 3-methyladenine (3-MA), 1 μM Ferrostatin-1 (Fer-1), 1 μM Liproxstatin-1 (Liprox-1), or 100 μM deferoxamine (DFO), followed by treatment with 100 nM NC1, RSL3, 50 ng / ml TNF-α + 1 μM Smac mimic (TS), or 50 ng / ml TNF-α + 1 μM Smac mimic + 20 μM Z-VAD (TSZ) for 24 hours (average three replicates). d. Scatter plots of β-fractions for NC1-treated and control cells were performed during the CRISPR-Cas9 screening described above. RRA scores were analyzed and normalized using MAGeCKFlute. The horizontal dashed line on the y-axis corresponds to a value of 1.30 (-log10(0.05)). The threshold for the x-axis was set at ±3.03, calculated from the standard normal distribution vector. e. Dose-dependent cytotoxicity of NC1 in MCF7 cells (control and TRPM4 KO cells using three different sgRNAs) after 24 hours of treatment was determined using CellTiter-Glo, and immunoblotting analysis was performed on control and TRPM4 KO cells. Data are expressed as mean ± standard deviation (SD) of triplicate wells. f. Cell death in MCF7 cells (control and TRPM4 KO) after 24 hours of NC1 treatment was determined by LDH release. Data are expressed as mean ± SD of triplicate wells. g. Cell viability of wild-type (WT) and TRPM4 knockout (KO) MCF7 cells with or without TRPM4 re-expression. The right figure shows immunoblotting analysis of MCF7 WT and TRPM4 KO cells with or without TRPM4 re-expression. Data are expressed as mean ± SD from three replicate wells. h. Dose-dependent inhibition of NC1-induced cell death by 9-ph. Data are expressed as mean ± SD from three replicate wells. i,j. Cell thermal transformation assay (CETSA) of NC1 or NC1i in HEK-293T-TRPM4 cells. i. Representative TRPM4 protein blots for each sample.j. The graph shows the relative TRPM4 band intensity ± standard deviation (sd) of the samples shown in three independent experiments, and indicates the statistical significance of the difference between NC1 or NC1i and DMSO. The NC1 or NC1i and DMSO samples were analyzed simultaneously using the same exposure time. k. Purification and size exclusion chromatography (SEC) of hTRPM4 (left panel), surface plasmon resonance (SPR) measurements showed that NC1 binds to the hTRPM4 protein, with an estimated KD value of 5.8 μM (right panel). l. Whole-cell current density-voltage relationship in HEK-293T cells stably expressing hTRPM4. m. NC1 activation of hTRPM4 in HEK293 cells, EC50 306.3 nM. Data are expressed as mean ± standard error (sem). n. Whole-cell current density-voltage relationship in NC1-activated MCF7 and MCF7-TRPM4 KO cells. o. Statistical data of hTRPM4 current density at +100 mV in Figure n. Whole-cell current density-voltage relationship of p.NC1-activated HEK-293T cells stably expressing hTRPM4, recorded over time. Data are expressed as mean ± sem for at least four independent cells.

[0050] Figure 2Sodium ion influx is a key event in NC1-induced cell death. a. Sodium, potassium, calcium, iron, and magnesium levels in MCF7 cells treated with 50 nM NC1 for 1 h, 2.5 h, and 0 h (control cells) were determined by ICP-MS. Data are presented as mean ± standard deviation (SD) of triplet samples. b. MCF7 cells were loaded with sodium indicator CoroNa Green and potassium indicator PBFI, followed by treatment with 50 nM NC1 for 0, 1, or 2 h. Representative confocal images are shown. Scale bars represent 50 μm. c. Comparisons were calculated using a two-tailed Student's t-test. Data are presented as mean ± standard deviation (SD), with each point representing data from a single image in Figure b. d. Representative time-lapse images of MCF7 cells treated with 50 nM NC1 in different extracellular buffers (EBS): normal, KCl concentration increased to 25 mM, no calcium, and no sodium (osmolarity maintained by isostatic NMDG-Cl). Scale bars represent 25 μm. e. Cell viability of MCF7 cells treated with NC1 for 6 hours in different EBSs. Data are expressed as mean ± SD of triplet wells. f. Sodium and potassium content of MCF7 cells treated with 50 nM NC1 for 3 hours in different EBSs (normal, sodium-free, and 25 mM KCl) determined by ICP-MS. Results are expressed as fold change relative to control. Data are expressed as mean ± SD of triplet wells. g. Cell viability of MCF7 cells treated with NC1 for 6 hours in EBSs containing different Na+ concentrations (osmolarity maintained by equal concentrations of NMDG+). Data are expressed as mean ± standard deviation (SD) of triplet wells. h. Cell viability of MCF7 cells treated with NC1 for 6 hours in EBSs containing different monovalent cations (extracellular Na+ was replaced by equal concentrations of K+, Li+, Cs+, or Rb+). Data are expressed as mean ± SD of triplet wells. i. Measure the intracellular sodium concentration in MCF7 WT and TRPM4-KO cells treated with 50 nM NC1 for 0, 1 and 3 hours.

[0051] Figure 3 NC1 specifically targets human TRPM4 through interaction with the transmembrane region. a. TRPM4 protein expression profiles of human (left) or mouse (right) cell lines, and corresponding sensitivity heatmaps to NC1-induced cell death. b. Each graph represents a cell line. The TRPM4 band intensity of the samples shown in a was analyzed using ImageJ software. The IC50 of NC1 was derived from... Figure 7c. Linear regression was fitted in Graphpad Prism 9. c. Dose-dependent cytotoxicity of NC1 in TRPM4 knockout MCF7 cells, re-expressed with human TRPM4 (h-TRPM4) or mouse TRPM4 (m-TRPM4), assessed using CTG 24 hours after treatment (left), and immunoblotting analysis of TRPM4 re-expression levels (right). Data are presented as mean ± standard deviation (SD) of triplet wells. d. CETSA assay of NC1 in HEK-293T-hTRPM4 or -mTRPM4 cells. The figure shows the relative TRPM4 band intensity ± standard deviation for samples shown in three independent experiments, and highlights the significant difference between NC1 and DMSO in hTRPM4. e. Whole-cell current density-voltage relationship in HeLa cells expressing hTRPM4 or mTRPM4. f. Schematic diagram of the validation system to confirm whether mutant or chimeric TRPM4 responds to NC1. g. Phylogenetic tree (protein sequence accession number) of TRPM4 proteins in multiple species constructed using CLUSTALW multiple sequence alignment, including TRPM4 isoforms in humans (Homo Sapiens, NP_060106.2), rhesus monkeys (Macaca Mulatta, XP_028695988.1), dogs (Canis lupus familiaris, XP_541500.3), pigs (Sus Scrofa, XP_020950369.1), mice (Mus Musculus, NP_780339.2), and pikas (Ochotona Princeps, XP_012785770.1) (left panel). Summary of their corresponding sensitivities to NC1-induced channel activation or cell death (right panel). h. Phylogenetic tree of TRPM family proteins and summary of their sensitivities to NC1-induced channel activation or cell death at 1 μM NC1. i,j,k. TRPM4 chimeric constructs and their corresponding sensitivity to NC1-induced channel currents or cell death. Schematic diagram of the chimeric TRPM4 construct. Human and mouse TRPM4 are represented by h and m, respectively. The chimeric constructs were generated by PCR cloning (i). In whole-cell patch-clamp experiments using HeLa cells, the sensitivity of the TRPM4 chimeric constructs to NC1-induced current density at +100 mV was statistically shown. Data are expressed as mean ± standard error of at least four independent cells (j). In cytotoxicity assays performed in MCF7 TRPM4 knockout cells, NC1-induced cell death was observed by re-expression of human, mouse TRPM4 wild-type, or chimeric constructs, respectively. Data are expressed as mean ± standard deviation of triplet wells (k).

[0052] Figure 4Molecular docking and mutation analysis revealed key residues for TRPM4 activation of NC1. a. Surface plot showing the overall structure of the TRPM4-NC1 complex in molecular model. Inset shows a close-up view of the NC1 binding site. Individual TRPM4 subunits are indicated in cyan or gray, while NC1 is highlighted in yellow. The chemical structure of NC1 is shown in the lower right corner. b, c. Specific recognition of human TRPM4 by NC1 binding. 3D plot shows NC1 binding in a deep cavity of TRPM4, located between the S3, S4, S5', and S6' helices, with Ca2+ indicated in purple (b). 2D plot shows NC1 interacting with key residues of TRPM4 through hydrogen bonding, hydrophobic interactions, and π-π stacking (c). d. CETSA experiments on NC1 and TRPM4 mutants of the aforementioned key residues. The figure shows the relative TRPM4 band intensity ± standard deviation of the samples shown in three independent experiments, and marks the significant difference between NC1 and DMSO. e. Current density-voltage relationship of hTRPM4 mutants with key residues binding to NC1 detected in HeLa cells (left panel). f. Summary figure shows the maximum amplitude of current density activated by NC1 in the TRPM4-wt channel and in the mutants described above (e) (n = 5–10). g. Dose-dependent cytotoxicity of NC1 in MCF7 cells reexpressing WT-TRPM4 or the aforementioned TRPM4 mutants, assessed using CTG 24 hours after treatment (left panel). Immunoblot analysis of protein expression levels in the reexpression experiment (right panel). Data are presented as mean ± standard deviation of triplet wells. h. Summary data show the compositional activity of the aforementioned hTRPM4 mutants recorded under an inside-out sheet at -100 mV. Data are presented as mean ± standard error of at least three independent cells.

[0053] Figure 5Energy depletion-induced cell death is characteristic of NECSO. a. Heatmaps show cell death (measured by LDH release) in Cos7 cells stably expressing different hTRPM4 mutants after treatment with 2DG+NaN3 or NC1, as averages of three replicates. Each heatmap's columns from left to right represent the results of 8 hours of treatment with different concentrations of 2DG+NaN3 (0, 5, 10, 20 mM 2DG plus 1 mM NaN3) or NC1 (0, 200, 500, 1000 nM) (left panel). Immunoblot analysis of different hTRPM4 mutant expression levels in Cos7 cells (right panel). b. Heatmaps show cell death in Cos7 cells expressing different NC1-insensitive hTRPM4 mutants after treatment with 2DG+NaN3. c. Whole-cell current density-voltage relationship in Cos7 cells expressing hTRPM4 under 2DG activation. Data are presented as mean ± standard error of at least three independent cells. d. Intracellular sodium ion concentration in Cos7 cells stably expressing hTRPM4 was measured by flow cytometry (FACS) after treatment with 20 mM 2DG and 1 mM NaN3 for 6 hours. The sodium concentration in the extracellular buffer was maintained at physiological concentration (150 mM) or completely replaced with equimolar amounts of NMDG. e. Heatmaps show cell death in Cos7 cells transfected with hTRPM4-WT or -H159A mutants under sodium-containing and sodium-free conditions after treatment with 2DG+NaN3 (measured by LDH release, mean of three replicates). f. Heatmaps show cell death in rat cardiac myoblast H9C2 cells transiently expressing different hTRPM4 enhanced-function (GOF) or loss-of-function (LOF) mutants after treatment with 2DG+NaN3 or NC1 (measured by LDH release, mean of three replicates). Immunoblot analysis of different hTRPM4 mutant expression levels in H9C2 cells. g. The dose-dependent cytotoxic effect of NC1 in AC16-WT or TRPM4 knockout cells (TRPM4KO) was assessed using CTG after 24 hours of treatment, and the expression level of TRPM4 in AC16TRPM4KO cells was analyzed by Western blot. h. Heatmaps show cell death in AC16-WT or TRPM4KO cells under 2DG+NaN3 treatment (mean of three replicates as determined by LDH release). Columns in each heatmap from left to right represent the results after 16 hours of treatment with different concentrations of 2DG+NaN3 (0, 5, 10, 20 mM 2DG plus 1 mM NaN3). i. TRPM4 expression levels in human heart failure as shown by a meta-analysis of multiple studies (saezlab.shinyapps.io / reheat / ). t-values ​​represent adjusted gene expression levels, and each data point represents an independent study. Thick dashed lines represent the mean, and thin dashed lines represent the upper and lower quartiles.

[0054] Figure 6 DHPs and CLT as small molecule inhibitors of NECSO. a. 3D plot showing CLT specifically binding to hTRPM4, recognizing a deep cavity similar to NC1 (left). 2D plot showing CLT interacting with key residues of TRPM4 through hydrogen bonding and hydrophobic interactions. b. Best hits in NECSO inhibitor screening. DHP icons are highlighted in cyan, with its chemical structure shown in the upper right corner. c. DHP exhibits a synergistic effect in preventing NECSO. MCF7 cells were pretreated with different concentrations of DHP before treatment with 100 nM NC1, and cell death was assessed using CTG, with results detected 24 hours post-treatment. Data are presented as mean ± standard deviation (SD) of triplet wells. d. Whole-cell current density-voltage relationship in HEK293T cells stably expressing hTRPM4. Cells were pretreated with 30 μM DHP or CLT before treatment with 1 μM NC1. Data are presented as mean ± standard error (SEM) of at least three independent cells. e. In MCF7 cells, intracellular sodium ion concentration was measured after 1 hour of pretreatment with 30 μM DHP or CLT, followed by exposure to 100 nM NC1 over the next 3 hours, and analyzed using flow cytometry (FACS). f. Heatmaps show cell viability of MCF7 or MB468 cells after pretreatment with different types of calcium channel inhibitors, followed by exposure to NC1 (mean value of three replicates). g. Heatmaps show cell death in Cos7 cells stably expressing hTRPM4 after pretreatment with different inhibitors, followed by exposure to 2DG+NaN3 or NC1 for 16 hours (measured by LDH release, mean value of three replicates). The right panel shows representative bright-field images of cell morphology under each treatment condition. h. Heatmaps show cell death in H9C2 cells expressing the hTRPM4 mutant after treatment with 2DG+NaN3 or NC1, combined with different inhibitors (measured by LDH release, mean value of three replicates).

[0055] Figure 7 Different effects of stereoisomers NC1 and NC1i on cell death induction. a. Chemical structures of compounds NC1 and NC1i. b. Cell viability of MCF7 cells 24 hours after treatment with NC1 or NC1i. c. IC50 values ​​of NC1 and NC1i 48 hours after treatment in different cell lines. Data are expressed as mean ± standard deviation (SD) of triplet wells.

[0056] Figure 8Data quality of CRISPR screening. a. Schematic diagram of the whole-genome CRISPR-Cas9 screening strategy. b. Immunoblot analysis of Cas9 expression in MCF7-Cas9 and -WT cells. c. sgRNA count analysis in sgRNA-transfected MCF7-Cas9 cell pools by deep sequencing. d. Assessment of dose-dependent cytotoxicity of NC1 24 hours after treatment in parental MCF7 cells, MCF7-Cas9 cells, and CRISPR-Cas9-selected NC1-resistant MCF7 cells. Data are presented as mean ± standard deviation (SD) of triplet wells.

[0057] Figure 9 NC1, not NC1i, triggers TRPM4 activation. a, b. Equilibrium analysis of NC1(a) and NC1i(b) binding to immobilized hTRPM4. The figures show the equilibrium relative response units versus compound concentration and are fitted. c. Typical whole-cell current trajectories recorded from HEK-293T cells stably expressing the hTRPM4 channel, with the stimulation voltage step used in the experiment at the top (left). The whole-cell current trajectory after 1 μM NC1 application is then recorded (middle), and finally the current trajectory after inhibition with a 50 μM 9-ph inhibitor (right). All three figures are recorded from the same patch. d. Current density-voltage relationship of hTRPM4 steady-state current under vehicle or NC1i treatment. Statistical results of hTRPM4 steady-state whole-cell current density under vehicle, NC1i, or NC1 treatment at +100 mV are shown in ed. fi. Typical current trajectories of the hTRPM4 channel under vehicle or 1 μM NC1 treatment recorded using the outward (f) or inward (h) patch. Statistical results of outward (g) or inward (i) currents of g, i.hTRPM4. Data are expressed as mean ± standard error (sem). Statistical analysis was performed using two-way ANOVA and the Sidak test. j. Time-varying curves of TRPM4 current density in HEK-293T-TRPM4 cells after 420 seconds of activation and washing with bath solution. Whole-cell currents were recorded at -100 and +100 mV.

[0058] Figure 10NECSO accompanied by membrane depolarization and cell edema. a. Dose-dependent cytotoxicity of NC1 in MCF7 cells pretreated with 20 μM NKCC inhibitors (furosemide, bumetanide, and azosylmethionine) or KATP inhibitors (PNU 37883A and gliclazide) was assessed using CTG at 24 h post-treatment. Data are presented as mean ± standard deviation (SD) of triplet wells. b. Typical membrane potentials of MCF7 cells treated with 1 μM NC1. c. Statistical analysis of membrane potential values ​​of MCF7 cells 10 min post-exposure to vehicle or NC1, as recorded in b. d. Representative confocal images of MCF7 cells loaded with 5 μM DiBAC4(3) followed by treatment with 50 nM NC1 for 1.5 h. e. Histograms summarizing fluorescence intensity ratios calculated from DiBAC4(3) staining, illustrating the depolarizing effect of NC1 in d. f. MCF7 cells loaded with chloride indicator MQAE 3 hours after treatment with DMSO, 50 or 100 nM NC1. Representative confocal images are shown. Scale bar indicates 10 μm. g. Statistical analysis of fluorescence intensity. Comparisons were calculated by one-way ANOVA analysis. Data are presented as mean ± standard deviation (SD). Each point represents data from a single image in f. h. Dose-dependent cytotoxicity of NC1 in MCF7 cells pretreated with multiple chloride anion channel inhibitors, assessed using CTG at 24 hours post-treatment. Data are presented as mean ± standard deviation (SD) of triplet wells. i. Transmission electron microscopy (TEM) images of MCF7 cells treated with 100 nM NC1 at different time points. Scale bar indicates 500 nm. Red arrows in the images indicate swollen mitochondria, endoplasmic reticulum, and Golgi apparatus at 0.5, 1.5, and 2.5 hours, respectively. Red arrows in the 4-hour image highlight the swollen nuclear membrane (top) and ruptured cell membrane (bottom). j. Cell death assay of MCF7 cells treated with NC1 for 24 hours in medium containing different doses of the osmotic protectant D-mannitol, measured by LDH release. Data are presented as mean ± standard deviation (SD) of triple-duplicate wells.

[0059] Figure 11Different responses of TRPM4 from multiple species to NC1. a. IC50 values ​​of NC1 were measured in various cell lines of different species after 48 hours of treatment. b. Representative confocal images of HeLa cells expressing TRPM4 from different species after 24 hours of treatment with 1 μM NC1. Dead cells were stained with PI. c. Histograms summarizing the percentage of TRPM4-EGFP transfected cells in b that responded to NC1. Data are presented as mean ± standard deviation (SD) of 50 cells from three independent experiments. d. Representative sensitivity summarizing the sensitivity of different species of TRPM4 to NC1-induced current density at +100 mV in whole-cell patch-clamp experiments. Data are presented as mean ± standard error (SEM) of at least three independent cells. e. Representative confocal images of different TRPM family protein expression in HeLa cells after 24 hours of treatment with 1 μM NC1. Dead cells were stained with PI. f. Histograms summarizing the percentage of TRPM4-EGFP transfected cells in e that responded to NC1. Data are presented as mean ± standard deviation (SD) of 50 cells from three independent experiments. g, h, i. Conductivity-voltage relationships of TRPM5, TRPM2, and TRPM8 in HeLa cells treated with DMSO, 1 μM, or 10 μM NC1. j. Statistics of current density at +100 mV in g, h, i. Data are presented as mean ± standard error (sem) of at least three independent cells. A p-value greater than 0.05 was considered not statistically significant (ns).

[0060] Figure 12 Different responses of human-mouse TRPM4 chimeras to NC1. a. Schematic diagram of the three major domains of hTRPM4 and their corresponding amino acid sequence numbers (top). Each structural component is labeled and color-coded in detail (bottom). b. Representative confocal images of HeLa cells transfected with different TRPM4 chimeras, cells treated with 1 μM NC1 for 24 hours. c. Histograms summarizing the percentage of NC1-responsive cells expressing TRPM4-EGFP in b. Data are presented as mean ± standard deviation (SD) of 50 cells from three independent experiments. d, e, f, g. Whole-cell current density-voltage relationship in NC1-activated HeLa cells overexpressing TRPM4 chimeras. Data are presented as mean ± standard error (SEM) of at least three independent cells.

[0061] Figure 13Model representation of ligand binding sites in the vanillin binding pocket (VBP). The overall structure of the TRP channel is shown, with one subunit highlighted. Ligands are presented as spheres (top). Ligands are represented as rods. Oxygen atoms are red, nitrogen atoms are blue, sulfur atoms are yellow, fluorine atoms are light blue, bromine atoms are dark red, and chlorine atoms are green (bottom).

[0062] Figure 14 Different cross-sections of the NC1 binding pocket in hTRPM4 (PDB: 6BQV). a, b. Overall surface view of TRPM4. The NC1 ligand is represented by a green sphere. Subunits are represented by gray-black or gray-white, respectively. The NC1 ligand binding pocket consists of residues from two adjacent subunits. Views are front (a) and top (b), respectively.

[0063] Figure 15 Molecular dynamics (MD) simulation analysis of NC1 binding to TRPM4. a) Simulated state diagram of the TRPM4-NC1 complex embedded in the cell membrane phospholipid bilayer. b) Summary of structural stability analysis results (root mean square deviation, RMSD) of the channel-ligand complex after NC1 binding to the TRPM4 channel, over a time period of 100 ns. c) Calculation of the binding energy of NC1 to the TRPM4 channel along the trajectory of the converging conformation. d) Calculation of the binding pocket information of NC1 to the TRPM4 channel after MD simulation. e) Distance from NC1 at... The energy contribution of residues within a certain range. The average energy value is represented by a blue bar, and the black line represents the standard error.

[0064] Figure 16 TRPM4 NC1 activation itself is not affected by calcium. a, b, c, Conductivity-voltage relationship of whole-cell hTRPM4 current in HEK293T-TRPM4-EGFP cells after adding different concentrations of EGTA (0, 1, 10 mM) to the patch pipette solution, with control or 1 μM NC1 treatment, respectively. d, Statistical data of whole-cell hTRPM4 current density at +100 mV in a, b, c. Data represent the mean ± standard error of at least 5 independent cells. e, In the presence or absence of 1 μM EGTA in cells... 2In the + case, the current density-voltage relationship of whole-cell hTRPM4 current in HEK293T-TRPM4 cells treated with control or 1 μM NC1. NC1 further increased the TRPM4 current amplitude in the presence of intracellular calcium. Data are presented as mean ± standard error. f, g, dose-dependent cytotoxicity of NC1 in MCF7 cells pretreated with calcium chelators BAPTA-AM (f) or EGTA-AM (g), assessed using CTG after 24 hours. Data are presented as mean ± standard deviation of three-well replicates. h, i, whole-cell current-voltage relationship of hTRPM4 E828K (h) or E1068Q (i) mutants expressed in HeLa cells (right panel). The left panel shows the maximum amplitude statistics of NC1 activation current at +100 mV (n = 5–10). j, dose-dependent cytotoxicity of NC1 after reexpression of TRPM4 WT or the above mutants in TRPM4-KO MCF7 cells, assessed using CTG after 24 hours. The right figure shows the immunoblotting analysis of protein expression levels in the reexpression experiment. Data are presented as mean ± standard deviation of three-well replicates.

[0065] Figure 17 CLT exhibits dose-dependent inhibition of hTRPM4. CLT inhibits 1 μM Ca 2 +Induced hTRPM4 current in HEK293 cells. IC50 was 1.69 μM. Data are presented as mean ± standard error.

[0066] Figure 18 : Working mode diagram. DETAILED DESCRIPTION

[0067] This invention reveals a novel pathway regulating cell death and a series of factors influencing this pathway. Based on these new findings, this invention also reveals a drug screening model and method for regulating this cell death pathway, with the novel drug screening model based on TRPM4 or its transmembrane domain.

[0068] As used in this invention, a protein "binding pocket" (hereinafter referred to as a pocket) refers to a cavity on or inside a protein surface suitable for binding with ligands. The amino acid residues surrounding the pocket determine its shape, location, physicochemical properties, and function. The dynamics of the pocket are crucial for the specific interactions of proteins. The flexibility and mobility of protein structure allow the opening, closing, and adaptation of binding pockets, thereby regulating the ligand binding process and enabling specific protein functions.

[0069] As used in this invention, the terms "sodium overload (induced) cell death", "sodium overload (induced) cell death", and "Na" are used interchangeably. + "Overload cell death" and "Necrosis by Sodium Overload (NECSO)" are interchangeable, referring to cells experiencing excessive sodium overload. + Influx, triggering cell death.

[0070] Novel pathways regulating cell death

[0071] This invention proposes a unique necrotic cell death method, which is Na + Overload-induced cell death (NECSO). Sodium ions are a critical electrolyte, essential for osmotic balance. Imbalanced Na+... + Influx can lead to harmful effects such as organ dysfunction or failure. Whether sodium overload disrupting osmotic balance has a causal relationship with necrotic cell death and the mechanisms involved remain unclear. Through in-depth and extensive analysis, the inventors determined that the transient receptor potential M4 type (TRPM4) is mediated by NC1 in sodium... + Targets of action in influx and NECSO ( Figure 18 In specific embodiments of the present invention, it has been demonstrated that TRPM4-deficient cells are completely resistant to NC1-induced Na+. + Influx and NECSO. NC1 binds to TRPM4 and activates the ion channel activity of TRPM4, allowing Na+ to flow in. + Inflow. The effect of NC1 on NECSO is mediated by human TRPM4. A human species-specific NC1 binding pocket in the transmembrane region of TRPM4 was identified through domain substitution and molecular docking analysis. NECSO inhibitors identified by chemical screening blocked NC1- or energy depletion-induced necrotic cell death, indicating a similarity between the two processes. Gain-of-function mutations in human TRPM4 associated with arrhythmias are more susceptible to NC1- or 2-deoxy-D-glucose-induced NECSO and can be blocked by NECSO inhibitors. These studies elucidate the effect of Na+ on NECSO. + The regulation mechanism of NECSO mediated by influx.

[0072] In addition to disclosing the binding pocket, this invention also discloses key sites involved in the above process, including: calcium-binding (related) sites: E828, E1068; Ca2+ +Sites near the binding site: Phe936 (from the S5 helix of the neighboring subunit), Ser863, Trp864 (from the S3 helix), Val904, Leu907, His908 (from the S4 helix); Sites near the binding pocket (regulating the binding pocket): Met927. In a preferred embodiment, the major residues that are stably bound include: Ser863, Trp864, Val904, L907, His908, and Phe936. The regulatory effect of the candidate substance on cell death is analyzed by observing the interaction between the candidate substance and the said sites; preferably, it also includes observing the interaction between Necrocide 1 and the said sites; preferably, the interaction (binding) with the said sites includes (but is not limited to): covalent binding or non-covalent binding (such as the formation of hydrogen bonds, π-π stacking, hydrophobic interactions, van der Waals forces, and ionic bonds, etc.).

[0073] It should be understood that, once the binding pocket or the key sites therein and the mechanisms involved are known, various methods well known to those skilled in the art can be used to conduct targeted studies on the sites or to screen drugs, and these methods are covered within the scope of this invention.

[0074] When used as targets for artificial regulation or in the creation of artificial screening systems, the proteins or encoding genes described in this invention can be naturally occurring, such as those purified and isolated from mammals; or they can be recombinantly prepared, for example, recombinant proteins produced using conventional gene recombination techniques. Furthermore, any variations that do not affect the biological activity of these proteins are acceptable, such as derivatives or variants whose function remains unchanged.

[0075] Drug screening

[0076] Based on the site and mode of action disclosed in this invention, a drug screening model can be established to screen or design drugs suitable for targeted regulation, including screening drugs that inhibit cell death (protect cells) or drugs that alleviate or treat TRPM4 dysfunction. TRPM4 dysfunction includes cell or tissue damage, including heart disease.

[0077] In a preferred embodiment of the present invention, a virtual molecular docking model can be obtained by combining the identified key structural domains, binding pockets, and key amino acid sites.

[0078] In a preferred embodiment of the invention, a cell model can be obtained by combining the identified key structural domains, binding pockets, and key amino acid sites for cell-based screening. This includes establishing recombinant cells that express TRPM4 or its transmembrane domains to form a binding pocket. Based on this cell model, whether a candidate drug is a potential drug of interest can be determined by observing whether changes in cell activity or cell number occur before and after treatment with the candidate substance.

[0079] In this invention, the types of cells can be diverse, including prokaryotic cells or eukaryotic cells, with eukaryotic cells being preferred. The cells can be normal cells or diseased cells, such as cardiomyocytes or tumor cells.

[0080] Based on the inventors' new discoveries, the corresponding amino acid residue sites, site combinations, and drug screening models constructed as described above have multiple applications, including screening for substances that inhibit cell death in order to find effective drugs that have a protective effect on cells.

[0081] This invention provides a method for screening substances that inhibit cell death (or screen for substances that alleviate or treat TRPM4 dysfunction), comprising: (1) contacting a candidate substance with a screening system (screening model), the screening system containing (e.g., expressing) TRPM4 or its transmembrane domain to form a binding pocket; (2) adding the candidate substance to the screening system of (1) and observing the interaction between the candidate substance and the binding pocket; if the candidate substance enters the binding pocket and interacts, then the candidate substance is a substance that activates the TRPM4 channel to promote cell death or inhibits the TRPM4 channel to inhibit cell death.

[0082] The systems described may include, but are not limited to, cell systems (or cell culture systems), subcellular systems (or subcellular culture systems), solution systems, animal systems, or tissue systems (or tissue culture systems). It should be understood that, after understanding the specific sites of the target proteins and the characteristics of the interacting proteins indicated in this invention, those skilled in the art can design a variety of screening systems based on this understanding, all of which are covered within the scope of protection of this invention.

[0083] The system can be a virtual / simulated system, such as a molecular docking model, which is a simulation performed on a real spatial structure and can be applied to virtual screening, design, and optimization of small molecule compounds.

[0084] As a preferred embodiment of the present invention, the method further includes: conducting further cell experiments and / or animal experiments on the obtained potential substances to further select and identify substances useful for inhibiting cell death (protecting cells) from the candidate substances.

[0085] When screening, various techniques well known in the art can be used to determine the variations and interactions of proteins or their encoding genes.

[0086] A variety of conventional techniques can be used to identify gene transcription or expression in a system. These techniques include, but are not limited to, oligonucleotide hybridization techniques (such as probes), polymerase chain reaction (PCR), and polyacrylamide gel electrophoresis. Protein-protein interactions and their strength can be detected using various techniques well-known to those skilled in the art, such as SPR, immunoprecipitation, GST precipitation, phage display, or yeast two-hybrid systems.

[0087] The substances initially screened using the above methods can form a screening library, which will allow people to eventually screen out substances that are truly useful in inhibiting cell damage.

[0088] The present invention also provides potential substances obtained using the screening method described above that can be used to inhibit cell damage.

[0089] The present invention also provides a method for preparing a drug that inhibits cell damage, the method comprising: synthesizing and / or purifying substances useful for inhibiting cell damage obtained by the method of the present invention, as drugs for inhibiting cell damage. Further, the obtained substances useful for inhibiting cell damage can be used to prepare pharmaceutical compositions.

[0090] Methods for screening substances that act on proteins or genes or specific regions thereof as targets are well known to those skilled in the art, and these methods can all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art understand how to select an appropriate screening method.

[0091] drug

[0092] In this invention, using the aforementioned screening model and method, a series of drugs effective in inhibiting cell death were obtained, including clotrimazole (CLT) or dihydropyridine (DHP) compounds. The dihydropyridine compounds include L-type calcium channel inhibitors such as clinidipine, lacidipine, benidipine, and lercanidipine.

[0093] The present invention may also include isomers, solvates, precursors, or salts thereof of the above-mentioned compounds.

[0094] In this invention, the small molecule compound may be a compound existing in pure form, or a compound with a purity greater than 85% (preferably greater than 90%, for example greater than 95%, 98%, 99%).

[0095] Knowing its chemical structure, the small molecule compound can be obtained through chemical synthesis. The invention also includes a precursor of the compound, which refers to a precursor of the compound that, when taken by an appropriate method, undergoes metabolism or chemical reaction in the patient's body to transform into the active compound.

[0096] This invention provides the use of the above-mentioned small molecule compounds for preparing pharmaceutical compositions or kits that inhibit cell death and alleviate or treat TRPM4 dysfunction.

[0097] The present invention also provides a composition (such as a pharmaceutical composition) comprising: (a) an effective amount of the pharmaceutical composition; and (b) a pharmaceutically acceptable carrier or excipient.

[0098] In this invention, the term "containing" indicates that various ingredients may be used together in the mixtures or compositions of this invention. Therefore, the terms "consistent with..." and "composed of..." are included in the term "containing," but they indicate that in the pharmaceutical composition or mixture, the active ingredient is limited to the small molecule compound described herein.

[0099] In this invention, a "pharmaceuticalally acceptable" ingredient is a substance that is suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., has a reasonable benefit / risk ratio.

[0100] In this invention, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent, or excipient used to deliver the active ingredient of this invention to animals or humans. A "pharmaceutically acceptable carrier" can be a liquid or a solid.

[0101] The pharmaceutical compositions or mixtures of the present invention can be formulated into any conventional dosage form using conventional methods. Dosage forms can be diverse, as long as they enable the active ingredient to effectively reach the mammalian body. Examples include: injections, infusions, tablets, capsules, and pills. The active ingredient can be contained in a suitable solid or liquid carrier or diluent.

[0102] The small molecule compound or pharmaceutical composition containing it may also be stored in a sterile instrument suitable for injection or infusion. Typically, in the pharmaceutical compositions of the present invention, the small molecule compound, as the active ingredient, may account for 0.0001-20% of the total weight of the pharmaceutical composition, with the remainder being a pharmaceutically acceptable carrier.

[0103] The effective dose of the small molecule compound may vary depending on the administration method and the severity of the disease being treated. When necessary, the small molecule compound may also be administered in combination with other active ingredients or drugs.

[0104] The present invention also provides a kit for inhibiting cell death, the kit comprising: a container, and the small molecule compound or a pharmaceutical composition containing the small molecule compound disposed in the container.

[0105] In addition, the medicine box may also contain some auxiliary medication materials, such as syringes for injection.

[0106] In addition, the medicine box may also contain instructions for use, explaining the method of inhibiting cell death using the combined drug method of the present invention.

[0107] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0108] Materials and methods

[0109] Cell lines and culture conditions

[0110] MCF7, MB468, HeLa, H9c2, HEK293T, SW620, C127, CHO, Cos7, and MEF cells (ATCC) were cultured in DMEM medium (Sigma Aldrich) containing 4.5 g / L glucose and L-glutamine. PC3, HCC1143, 4T-1, EMT-6, CT26WT, B16, and MC38 cells (ATCC) were cultured in RPMI 1640 medium (Sigma Aldrich) containing high glucose, L-glutamine, and HEPES. U-2OS cells were cultured in McCoy's 5A medium (ATCC). A549 cells were cultured in Ham's F-12K medium (BaseMedia). Neuro-2a cells were cultured in EMEM medium (ATCC). All media were supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin. Cells were cultured at 37°C in a humid 5% CO2 atmosphere. All cell lines were tested for mold, and the results were negative.

[0111] TRPM4 amino acid sequence (SEQ ID NO:1) (the underlined portion is its transmembrane domain (768-1094aa)):

[0112] MVVPEKEQSWIPKIFKKKTCTTFIVDSTDPGGTLCQCGRPRTAHPAVAMEDAFGAAVVTVWDSDAHTTEKPTDAYGELDFTGAGRKHSNFLRLSDRTDPAAVYSLVTRTWGFRAPNLVVSVLGGSGGPVLQTWLQDLLRRGLVRAAQSTGAWIVTGGLHTGIGRHVGVAVRDHQMASTGGTK VVAMGVAPWGVVRNRDTLINPKGSFPARYRWRGDPEDGVQFPLDYNYSAFFLVDDGTHGCLGGENRFRLRLESYISQQKTGVGGTGIDIPVLLLLIDGDEKMLTRIENATQAQLPCLLVAGSGGAADCLAETLEDTLAPGSGGARQGEARDRIRRFFPKGDLEVLQAQVERIMTRKELLTVY

[0113]

[0114] plasmid

[0115] Plasmid transfection was performed in HeLa or HEK293T cells using PEI (Polysciences, Inc.) transfection reagent. Virus was generated by co-transfecting HEK293T cells with GAG, POL, and pLenti expression plasmids at a 1:1:1 weight ratio using PEI transfection reagent. After 48 hours, the culture medium containing the secreted virus was collected and sterile filtered. A CRISPR guide RNA (sgRNA) sequence targeting TRPM4 was designed. The oligonucleotide sequence preceding the prespacer sequence was:

[0116] 5'-GAACCGCTTCCGCTTGCGCC-3' (SEQ ID NO: 2);

[0117] 5'-CCTAGGGCTCGTTCCCTGCG-3' (SEQ ID NO: 3);

[0118] 5'-GTCAACTATGAACGTCGTGC-3' (SEQ ID NO: 4).

[0119] Western Imprint

[0120] Proteins were extracted from cells using RIPA lysis buffer containing protease and phosphatase inhibitors (APEX BIO). Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal loading volumes of protein (20 μg) were separated by electrophoresis on a 10% SDS-PAGE gel (Bio-Rad Laboratories, Inc.) and then transferred to a PVDF membrane (Bio-Rad Laboratories, Inc.). The membrane was subsequently blocked with 5% skim milk at room temperature for 1 hour and then incubated overnight at 4°C with a primary antibody against the target protein. After washing, the membrane was incubated with a secondary antibody conjugated with horseradish peroxidase at room temperature for 1 hour. Protein bands were visualized using an enhanced chemiluminescent substrate and detected using a chemiluminescent imaging system (Bio-Rad Laboratories, Inc.). The following antibodies were used: anti-α-tubulin (Sigma Aldrich), anti-β-actin (Sigma Aldrich), anti-TRPM4 (Abclonal), anti-Na / K ATPase (Abclonal), and anti-Cas9 (CST).

[0121] Measurement of cell viability / death

[0122] Cell viability / death was assessed using the CellTiter-Glo (CTG) kit or the LDH release assay. Cells were cultured at 1 x 10⁻⁶ cells per cell. 4 Cells were seeded at a density of cells / well in 96-well plates and allowed to adhere overnight in culture dishes. After treatment under different experimental conditions, the culture medium was aspirated. For the CTG assay, the assay was performed using the CTG luminescence kit (Promega, G7570) according to the manufacturer's instructions. Briefly, cells were washed with phosphate-buffered saline (PBS), and then 100 μL of CTG reagent was added to each well. The plate was gently shaken to lyse the cells and release intracellular ATP. Cell viability was characterized by measuring the luminescence signal proportional to the amount of ATP using a microplate reader. For the LDH release assay, the reaction mixture containing culture supernatant and LDH assay solution (Promega) was incubated at room temperature for 30 minutes, followed by the addition of a stop reagent. The absorbance was measured at 490 nm using a microplate reader. A standard curve was prepared using gradually diluted LDH control samples to calculate the percentage of LDH released by treated cells. Experiments were performed in triplicate, and results are expressed as mean ± standard deviation.

[0123] CRISPR-Cas9 screening and data processing

[0124] A diagram illustrating CRISPR screening is shown below. Figure 8a. Specifically, firstly, stably Cas9-expressing MCF7 cells were generated (selected using blastcin), and then infected with a genome-wide targeted CRISPR / Cas9 sgRNA lentiviral library (human library, GeCKO v2.0), which targets 19,050 protein-coding genes (6 targeting gRNA sequences per gene) with 122,411 gRNA sequences. After treatment with 2 μg / ml puromycin for 3 days, the surviving cells were designated as the "Day 0" group. Subsequently, during a total of 14 days of culture (labeled as Day 14 group cells), cells were passaged every 2 days while maintaining a total cell count of 500-fold coverage. At the end of 14 days, the cells were divided into two groups and cultured in media containing either DMSO or 2 nM NC1, respectively. Samples were collected from the Day 0, DMSO, and NC1 groups, and genomic DNA was extracted using the QIAamp DNA blood midi kit (Qiagen). Single gRNA inserts were amplified using the NEBNext Q5 hot start HiFi PCR master mix (New England Biolabs). In the second round of PCR, primers with the i5 and i7 adapter sequences from TruSeq were added, and the PCR products were then subjected to next-generation sequencing. The resulting sequencing results were subsequently used for further analysis. Differences in gRNA enrichment between the DMSO and NC1 groups were calculated and analyzed using MAGeCKFlute.

[0125] Electrophysiology

[0126] All electrophysiological recordings were performed using patch clamp: (1) macroscopic currents in HEK293T, HeLa, or Cos7 cells overexpressing TRPM4 and its mutants; (2) membrane potentials in MCF7 cells. For transfected cells, patch clamp recordings were performed within 24–48 hours post-transfection. The bath solution contained 165 mM NaCl, 10 mM HEPES, 1 mM CaCl2, pH 7.3. The electrode tube solution contained 140 mM KCl, 10 mM HEPES, pH 7.2. The electrode tubes were drawn from PC-10 (NARISHIGE) and thermally polished to a resistance of 3–10 MΩ. Patch clamp recordings were performed using an Axopatch-200B amplifier and an Axon Digidata 1550A driven by Clampex10 software (Molecular Devices). Current signals were filtered at 1 kHz and digitized at a sampling frequency of 10 kHz. Whole-cell current recordings were performed in 20mV increments from -100mV to +100mV, then down to -100mV. All values ​​were measured using Clampfit 10.6 software.

[0127] Flow cytometry

[0128] To measure intracellular sodium concentration, cells were isolated from the culture plate using 1X PBS+EDTA. Single-cell suspensions were prepared in 1X PBS containing 4% FBS. Cells were then stained with 2 μM CoroNa-AM dye at 37°C for 30 min, followed by washing twice with 1X PBS and 4% FBS. Analysis of stained cells was performed using a CytoFlex S (Beckman Coulter) instrument, and subsequent data analysis was performed using FlowJo software. Sodium concentration was measured in the FITC (525 / 50 505LP) channel. To assess intracellular sodium concentration under NC1 or 2DG treatment, cells were subjected to the above treatment times and concentrations before being extracted from the culture plate.

[0129] Fluorescence microscopy

[0130] For fluorescence microscopy, HeLa cells transiently transfected with the hTRPM4-EGFP fusion protein were seeded on glass coverslips and cultured to a density of 70-80%. After appropriate NC1 treatment, the cells were stained with 2.5 μg / ml propiodoform (PI) at room temperature for 20 minutes to stain the nuclei. After washing three times with PBS, the coverslips were mounted onto the slides, ready for fluorescence imaging.

[0131] For live-cell microscopy, cells were grown in a 4-well Lab-Tek II Chambered Coverglass (Thermo Fisher Scientific) imaging chamber. To observe changes in cell membrane potential, cells were incubated in 5 μM DiBAC4(3) (Dojindo) for 2 hours and then treated with 50 nM NC1 before imaging. To observe the effects of ions on NECSO, MCF7 cells were incubated in SytoxGreen (Thermo Fisher Scientific) at room temperature for 30 minutes, then cultured with a medium that replaced the medium with other isotonic solutions containing different ions (as shown in the previous figure) before imaging.

[0132] Transmission electron microscope

[0133] Transmission electron microscopy (TEM) analysis was performed according to a previously reported protocol. Briefly, MCF7 cells were treated with 100 nM NC1 at different time points and then fixed with a fixative containing 2% formaldehyde, 3% glutaraldehyde, and 0.1 M metachromate buffer (pH 7.3). Cells were subsequently washed with 0.1 M metachromate buffer and treated with 0.1% filtered metachromate-buffered tannic acid. They were then fixed with 1% buffered sodium ruthenate and stained with 1% filtered uranilate. After dehydration, samples were subjected to a series of Epon treatments at 37°C for 6 hours until 100% concentrated. They were then embedded in purified Epon and incubated overnight at 60°C. Thin sections were obtained using a Leica EMUC7 ultramicrotome (Wetzlar, Germany), placed on coated grids (Gilder Grids, AG100N), and stained with 3% uranilate and lead citrate. Finally, the samples were observed under a PHILIPS CM-120 transmission electron microscope at 80 or 120 kV.

[0134] Protein expression and purification

[0135] Human TRPM4 (NCBI accession number NM_017636), containing a C-terminal 3×Flag tag, was cloned into the PCDNA4.1 vector and heterologously expressed in HEK293S cells (Life Technologies) via the PEI transfection system (Thermo Fisher Scientific). Cells were cultured in suspension at 37°C for 48 hours and harvested by centrifugation at 1500 rpm for 15 minutes. The cell pellet was resuspended in buffer A (20 mM HEPES, pH 7.4, 150 mM NaCl), and a protease inhibitor mixture (containing 2 μg / ml DNase I, 0.5 μg / ml pepstatin, 2 μg / ml leupeptin, 1 μg / ml aprotinin, and 0.1 mM PMSF) was added. The cell membrane was then ruptured by sonication on ice. TRPM4 protein was extracted for 3 hours at 4°C with gentle shaking using a mixed solvent of 2% (w / v) N-dodecyl-β-d-pyranoside (DDM, Goldbio) and 0.2% (w / v) cholesterol hemisuccinate (CHS, Sigma Aldrich). After extraction, the supernatant was collected by centrifugation at 48,000g for 40 minutes and gently shaken overnight in a buffer containing 3% Flag-Beads resin (Sigma Aldrich). The Flag-Beads were then collected by centrifugation at 300g for 5 minutes and washed three times with buffer B (buffer A + 0.1% DDM + 0.02% CHS). TRPM4 protein was eluted with buffer B containing 2 mg / ml Flag peptide and finally purified on a pre-equilibrated Superose 6 10 / 300GL column (GE Healthcare). The target protein peak was collected and concentrated for subsequent use.

[0136] Protein-ligand interactions

[0137] For the Cell Thermal Stability Assay (CETSA), HEK-293T cells stably expressing TRPM4 were lysed and incubated at room temperature for 1 hour with 100 μM NC1 or NC1i added to the lysis buffer. After compound treatment, cells were divided into aliquots and incubated for 3 minutes at different temperatures ranging from 37°C to 70°C. Cells were then immediately cooled on ice and centrifuged to obtain the soluble fraction. The TRPM4 protein content in the soluble fraction was assessed using Western blotting. The grayscale of the TRPM4 protein band was quantified using image analysis software (Fiji Image J, v1.53a), and protein thermal stability curves were generated by plotting the relationship between protein band grayscale and thermal shock temperature. Baseline thermal stability curves were established using untreated control samples. The shift in the thermal stability temperature (Tm) of the target protein in the presence of the compound indicates the binding and stabilization of the protein. Statistical analysis was performed using GraphPad Prism 9 software, and the significance of Tm shifts was determined using one-way ANOVA followed by Dunnett's test.

[0138] For surface plasmon resonance (SPR)-based binding experiments, a Biacore 8K instrument (GE Healthcare) was used. Experiments were performed at 25°C in HBS-EP flow buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, and 0.2% (v / v) surfactant DDM, 2% DMSO). Wild-type TRPM4 protein was immobilized on a CM7 sensor chip using a standard amino coupling procedure and 10 mM sodium acetate (pH 4.5) fixative. Compounds were serially diluted and injected into the sensor chip at a flow rate of 30 μl / min for a 120-second contact phase, followed by a 120-second buffer flow (dissociation phase). KD values ​​were determined by analyzing equilibrium data using Biacore 8k evaluation software (GE Healthcare, v3.0.12).

[0139] Molecular docking simulation

[0140] The structure of TRPM4 is derived from PDB (PDB ID: 6BQV), while the two-dimensional structures of NC1 or CLT were constructed using ChemDraw software (CambridgeSoft, v18.0). Molecular docking was performed using... Maestro software suite Performed in 2020-3). Prior to docking, the ligands were optimized using the OPLS3e force field in the LigPrep module. The protein was pretreated using the protein preparation wizard, which included adding missing residues, removing water molecules, and optimizing hydrogen bonding and energy using the OPLS3e force field. The protonation state of the protein and ligands was determined using Epik at pH 7.4 ± 0.2. A single protonation was performed on a selected residue using a... Docking was performed using a docking lattice. Extra-precision docking (Glide XP) with flexible ligand sampling was employed, generating up to 10 ligand conformations. The docking conformations with the highest scores were analyzed to identify key molecular interactions between the ligands and amino acid residues in the binding pocket, guiding subsequent experimental validation of site-directed mutagenesis.

[0141] Molecular dynamics (MD) simulations

[0142] For MD simulations, only the transmembrane domain of TRPM4 was selected. The optimal orientation of the docked TRPM4-NC1 complex was chosen, and a ligand-protein-lipid bilayer system was constructed using the CHARMM-GUI (https: / / charmm-gui.org) membrane construction program. The complex was inserted into a layer consisting of 180 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) molecules with a thickness of [missing information]. The entire system was situated in a lipid bilayer within an aqueous solution containing 150 mM NaCl. To assess the stability of the ligand-protein conformation, a 50 ns simulation was performed. After 25,000 simulation steps, the system underwent a six-step equilibration process based on the default settings of the CHARMM-GUI lipid bilayer output, followed by a 50 ns simulation run. All simulations were performed using GROMACS2020.3 software. Proteins were modeled using the WYF parameters of the CHARMM36m force field, ligands using the CHARMM General Force Field (CGenFF) force field, lipids using the CHARMM36 force field, and water using the TIP3P model. The simulations were conducted under isothermal-isobaric (NPT) parameters at a constant temperature of 303.15 K. Finally, structural stability analysis of the simulated trajectory was performed using root mean square deviation (RMSD) calculations. The MD simulation trajectory was also calculated using the gmx_mmpbsa bash script for MM-PBSA, where the solvent accessible surface area (SASA) was used as a model for the nonpolar solubility energy. The Fpocket 2.0 software was used for analysis combining pocket size and other properties. The dpocket program was used to generate pocket parameter information with default settings.

[0143] Quantitative and statistical analysis

[0144] Statistical analyses, including two-tailed Student's t-test and one-way ANOVA, were performed using GraphPad Prism software. All statistics are reported as mean ± standard deviation (sd), except for patch-clamp data, which are presented as mean ± standard error of mean (sem). Experiments were independently repeated at least three times with similar results. The means of the control and experimental groups were compared to determine significance. Data distributions were assumed to be normal, although no formal tests were performed. A p-value less than 0.05 was considered statistically significant. Sample size was not determined by statistical methods. No data were excluded from the analysis. Illustrations were created using Adobe Illustrator and BioRender (BioRender.com). Experiments were not randomized, and researchers did not blind treatment assignments or outcome assessments unless otherwise stated.

[0145] Example 1: RPM4 determines the sensitivity of cells to NC1-induced necrotic cell death.

[0146] The inventors discovered a compound with a stereoisomer structure and named it Necrocide1(NC1). Figure 1 a) It can induce strong immunogenic cell death in a range of specific cancer cell lines, but the mechanism of this process is currently unclear. Using a chemical regulation analysis strategy, the inventors investigated the effects of various small molecule inhibitors on NC1-induced cell death in three cell lines: MCF7, MB468, and PC3. Treatment with inhibitors of known cell death pathways, including apoptosis (Z-VAD), programmed necrosis (Nec-1), autophagy (CQ, 3-MA), and ferroptosis (Fer-1, Liprox-1, DFO), failed to inhibit NC1-induced cell death, suggesting that NC1 induces a novel cell death pathway different from currently known pathways.

[0147] To investigate the molecular mechanism of NC1-induced cell necrosis, unbiased whole-genome screening was performed in MCF7 cells that were sensitive to NC1 and stably expressed Cas9 using CRISPR-Cas9 technology. Deep sequencing was used to assess the quality of the sgRNA-transfected MCF7-Cas9 cell pools; sgRNA counting analysis showed that 99.9% of sgRNAs were successfully transfected. Subsequently, cell pools tolerant to NC1 were screened and subjected to deep sequencing to identify key genes involved in NC1-induced necrosis. The CRISPR screening data were analyzed using the MAGeCKFlute algorithm. Based on the enrichment of sgRNAs in the sequencing results, RRA (Robust Rank Aggregation) scores were generated for corresponding gene loci. Trpm4 was identified as the only gene with high enrichment that was repeatedly identified. Figure 1 ).

[0148] To validate TPRM4 function, TRPM4 knockout (KO) MCF7 cells were obtained using three different gRNAs. Based on cell viability and LDH release analysis, it was found that TRPM4 deficiency completely prevented NC1-induced cell death, while re-expression of human TRPM4 protein restored its sensitivity to NC1-induced necrosis. Furthermore, the TRPM4-specific inhibitor 9-phenanthrol (9-ph) alleviated NC1-induced cell death in a dose-dependent manner. Figure 1 , Figure 8 ).

[0149] This demonstrates that TRPM4 plays a crucial role in NC1-induced cell death.

[0150] Example 2: NC1 directly binds to and activates TRPM4

[0151] Since TRPM4 is the only gene with a high RRA score in the aforementioned CRISPR screening, the inventors hypothesized that TRPM4 is a direct target of NC1. First, CETSA (Cellular Thermal Shift Assay) was performed on lysates of HEK293T cells stably expressing TRPM4, using the stereoisomer NC1i, which lacks cell death-inducing activity, as a negative control. Upon temperature treatment, NC1 stabilized TRPM4, while DMSO or NC1i showed no stabilizing effect, indicating that NC1 can bind to TRPM4, but its inactive isoform cannot. Biacore experiments using purified TRPM4 protein also confirmed that NC1 can directly bind to TRPM4, with a binding affinity K0. D The value is 880nM.

[0152] To further investigate the effect of NC1 on TRPM4, whole-cell patch-clamp recordings were performed in HEK293T cells stably expressing TRPM4-EGFP to analyze TRPM4 ion channel activity. NC1 treatment induced a strong TRPM4-dependent current, which could be inhibited by the TRPM4 inhibitor 9-ph, indicating that NC1 can induce TRPM4-specific currents. Furthermore, NC1-induced currents were recorded in wild-type (WT) and TRPM4 KO MCF7 cells, respectively. WT cells produced a higher current, while TRPM4 gene knockout blocked current generation. Conversely, the inactive stereoisomer NC1i failed to activate TRPM4 ion channel activity. Subsequently, single TRPM4 ion channels were recorded using an outside-to-outside ex vivo patch-clamp technique. When the outer side of the channel came into contact with NC1-containing medium, TRPM4 was gated, suggesting that NC1 is a direct agonist of TRPM4. Furthermore, NC1 triggers a sustained and time-dependently increasing current that does not decrease even after NC1 is removed, indicating that TRPM4 is continuously and irreversibly activated when exposed to NC1. These results demonstrate that NC1 directly binds to TRPM4 and triggers its sustained activation. Figure 9 ).

[0153] Example 3: NC1-mediated TRPM4-dependent cell necrosis is characterized by sodium ion influx.

[0154] TRPM4 is a calcium-activated, non-selective monovalent cation channel protein. Similarly, in GO enrichment analysis, genes encoding ion-binding proteins, particularly metal ion-binding proteins, were highly enriched in NC1-treated cells. Figure 2 a) This suggests that NC1-induced necrosis is related to intracellular ion homeostasis. Therefore, the inventors used inductively coupled plasma-mass spectrometry (ICP-MS) to analyze the content of major intracellular metal ions, including sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), and iron (Fe). They found that the overall intracellular Ca / Mg / Fe levels remained basically unchanged after NC1 treatment, but Na content increased and K content decreased. Figure 2 b) This suggests that NC1 induces Na + Inflow and K + Efflux. Simultaneously, intracellular Na+ in MCF7 cells was measured using the sodium indicator CoroNa Green and the potassium indicator PBFI. + and K + At the level, CoroNaGreen and PBFI are respectively at the level of Na + and K +After binding, the fluorescence emission intensity increased. Treatment with NC1 significantly enhanced intracellular CoroNaGreen signal and weakened PBFI signal. Figure 2 c, d). These results indicate that Na+ occurs during NC1-induced cell necrosis. + Inflow and K + outflow.

[0155] Next, the determining factor of NC1-induced necrosis was investigated, specifically Na. + Inflow and K + Effervescence. First, to rule out the potential influence of amino acids or other bioactive components in serum, the cytotoxic activity of NC1 was tested in isotonic extracellular buffer solution (EBS, 150 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES, 2 mM CaCl2, 1 mM MgCl2) free of serum or amino acids, with the treatment time limited to 6 hours to avoid potential effects of starvation on cell death. The results showed that the cytotoxic effect of NC1 in EBS was the same as that in normal culture medium. Figure 2 e, f). In removing Ca 2+ When cells were cultured in EBS, NC1-induced cell death was not affected. Figure 2 e, f). When cells were cultured using the same concentration of non-permeable organic cation NMDG-Cl instead of NaCl in EBS to maintain osmotic balance, it was found that Na... + Removal of [specific substance] significantly inhibited NC1-induced cell death. Figure 2 e, f).

[0156] At the same time, by using extracellular K + Increasing the concentration from 5 mM to 25 mM to delay K + The outflow process, thus analyzing K + Importance ( Figure 2 g). However, 25 mM KCl did not interfere with the cell death process, suggesting that K... + Efflux is a concomitant phenomenon and does not determine NC1-induced cell death. Figure 2 e, f). Detection of intracellular Na+ using ICP-MS + and K + The concentration of Na was confirmed under the above treatment conditions. + Inflow and K + The outflow process was suppressed.

[0157] To further confirm Na + The dependence on inflow gradually reduces Na in EBS. + Concentration was found to reduce the cytotoxic activity of NC1. Figure 2 h). Replace Na with other monovalent cations. + It can inhibit NC1-induced cell death at moderate or minimum levels. Figure 2 h).

[0158] These data indicate that Na + Influx is the main cause of NC1-induced cell death. To confirm that NC1 treatment leads to Na+ ionization, we need to investigate whether TRPM4 activation is the cause of Na+ ionization. + Influx was observed in NC1-treated MCF7 WT and TRPM4 KO cells stained with CoroNa. Flow cytometry analysis revealed that knockout of the TRPM4 gene eliminated NC1-induced Na+ influx. + internal flow ( Figure 2 i). Furthermore, TRPM4 lacks ion permeability. D984A The mutant could not rescue the NC1-induced death phenotype in TRPM4 KO cells. Figure 2 i) indicates that NC1-induced cell death requires TRPM4 ion channel activity.

[0159] In summary, NC1 induces activation of the TRPM4 channel, leading to Na+ activation. + Excessive influx triggers cell necrosis, hence the inventors named this type of cell necrosis Na+. + Necrosis by Sodium Overload (NECSO)

[0160] extracellular Na + It can maintain ion balance, thereby maintaining the cell's resting potential and osmotic balance. Therefore, the inventors hypothesize that NC1-induced Na+ + Influx may lead to cell membrane depolarization. Using whole-cell patch-clamp (…) Figure 10 a, b) and membrane potential fluorescent probe DiBAC4(3) Figure 10 c) The potential of cells after NC1 treatment was detected, and rapid membrane depolarization was observed. Figure 10 ac). Na + Overload typically increases intracellular osmotic pressure, leading to water influx and cell edema. Accordingly, transmission electron microscopy (TEM) revealed severe organelle swelling in NC1-treated cells, including mitochondria, endoplasmic reticulum, and Golgi apparatus, along with cell edema and membrane rupture. Figure 10 d). These data support the view that NC1-induced cell death is caused by cell swelling due to osmotic imbalance. Subsequently, D-mannitol was added to the culture medium to increase extracellular osmotic pressure, and the cytotoxic activity of NC1 was examined. It was found that the increase in extracellular osmotic pressure delayed cell death. Figure 10e). These results indicate that cell membrane depolarization and cell edema are key characteristics of NECSO.

[0161] Example 4: NC1-specific targeting of human TRPM4

[0162] So, is TRPM4 expression related to cellular sensitivity to NECSO? The inventors found that among nine types of human cancer cells, MCF7, MB468, HCC1143, and PC cells showed high intracellular TRPM4 expression levels and were highly sensitive to NC1; HEK293T, U2OS, and A549 cells showed relatively low intracellular TRPM4 expression levels and were moderately sensitive to NC1; SW620 and HeLa cells showed almost undetectable TRPM4 expression and the lowest sensitivity to NC1. Figure 3 a and Figure 7 c). Compared to human cancer cells, all mouse or hamster-derived cells, including normal and cancer cells, were resistant to NC1 (>1000-fold), although TRPM4 expression levels in several mouse cancer cells were comparable to those in U2OS and A549 cells. Figure 3 a and Figure 11 a) suggests that the difference in TRPM4 in human and mouse cells leads to different cellular sensitivities to NC1 treatment.

[0163] Therefore, the inventors hypothesized that NC1 could only target human TRPM4 and not mouse TRPM4, and verified this by ectopically expressing human or mouse TRPM4 in TRPM4KO human cells. Re-expression of human TRPM4 (hTRPM4) in TRPM4KO MCF7 cells restored cell sensitivity to NC1, while re-expression of mouse TRPM4 (mTRPM4) left cells insensitive to NC1. Figure 3 (b) Although the expression levels of the two TRPM4 types were similar. Similarly, thermal transfer assays showed that NC1 binds to hTRPM4 but not to mTRPM4. Figure 3 c). Furthermore, NC1 can activate the plasma membrane ion channel activity of hTRPM4 but not that of mTRPM4, indicating that NC1 can specifically activate hTRPM4 (…). Figure 3 d).

[0164] Based on the above results, the inventors hypothesized that sequence differences between human and mouse-derived TRPM4 cells determine a significant difference in their sensitivity to NC1. To verify this hypothesis, HeLa cell lines that do not express TRPM4 and are resistant to NC1-induced cell death were selected for experiments. Transient expression of hTRPM4 in HeLa cells followed by NC1 treatment restored the NECSO process, resulting in a "balloon-like" necrotic morphology. Figure 3e). Because this system provides a rapid, convenient, and highly specific way to analyze the requirements of different types of TRPM4 for NC1-induced cell death, it also investigated and compared the functions of TRPM4 homologs from different species and other members of the TRPM family in cell death. Figure 3 f, g and Figure 11 As expected, TRPM4 in other species was largely insensitive to NC1-induced cell death. Figure 3 f and Figure 11 bd), other TRPM family members, including the closest homolog TRPM5, are also insensitive to NC1. Figure 3 g and Figure 11 e.g.

[0165] These data indicate that NC1 is a highly specific agonist targeting human TRPM4.

[0166] Example 5: The transmembrane region of TRPM4 determines the differences in the response of different species to NC1.

[0167] In existing studies, TRP family proteins from different species have shown varying sensitivities to physiological stimuli. This characteristic has been used to classify receptor domains that promote ligand binding or activation. The overall sequence similarity between hTRPM4 and mTRPM4 proteins is 88.4%, and they possess similar tetrameric structures on the cell membrane. Furthermore, protein regulatory modifications include phosphorylation, SUMOylation, and glycosylation, with the relevant sites being conserved between the two proteins.

[0168] Because human and mouse TRPM4 exhibit significant differences in NC1 sensitivity, domain exchange experiments were performed to analyze the domains essential for TRPM4's response to NC1. Both human and mouse TRPM4 are divided into three segments: the N-terminus (amino acids 1-767 of hTRPM4 and 1-763 of mTRPM4), the transmembrane region (TM, amino acids 768-1093 of hTRPM4 and 764-1089 of mTRPM4), and the C-terminus (amino acids 1094-1214 of hTRPM4 and 1090-1213 of mTRPM4). Figure 3 h and Figure 12 a). The TM region includes six core transmembrane domains: pre-S1 (S0), S1-S6, and a TRP domain partially embedded in the phospholipid layer. Figure 12 a).

[0169] Chimeras obtained by replacing the human TRPM4™ region with the mouse TRPM4™ region (hN-mTM-hC) completely lost sensitivity to NC1, while chimeras obtained by replacing the N-terminus (mN-hTM-hC) and C-terminus (hN-hTM-mC) retained NC1 sensitivity. Figure 3 h, i and Figure 12 b, c).

[0170] Replacing the mouse TRPM4™ region (mN-h™-mC) with the human TRPM4™ region restored the chimera's sensitivity to NC1. Figure 3 h, i and Figure 12 (b, c). Furthermore, the aforementioned NC1-sensitive chimera, when expressed in TRPM4 KO MCF7 cells, was able to restore the NC1-induced death phenotype. Figure 3 h, j and Figure 12 b, c).

[0171] These results indicate that the transmembrane region of hTRPM4 is crucial for cellular sensitivity to NC1.

[0172] Example 6: Molecular docking and mutation analysis revealed the activation effect of key residues in TRPM4 on NC1.

[0173] Domain exchange analysis highlighted the importance of the transmembrane (TM) region of human TRPM4 in responding to NC1 treatment. Therefore, molecular docking was performed within the TM region of TRPM4 (PDB code: 6BQV). Molecular docking revealed that NC1 resides within a cavity in the TM region, surrounded by an S3-S4 helix and an S4-S5 linker of one subunit, and S5-S6 helices of adjacent subunits. This cavity is termed the vanillin-binding pocket (VBP) because it is the specific region where vanillin compounds (such as capsaicin) bind and regulate the TRPV1 channel. The VBP is a conserved region within the TRP family and is considered a hotspot for ligand regulation in TRP channels. Figure 13 Although no TRPM4 ligands have been reported to bind to this region to date, the inventors' molecular docking analysis predicted a potential NC1 binding pocket surrounded by amino acid residues from different subregions—Phe936 (F936) from the adjacent S5 helix, Trp864 (W864) from S3, and Val904 (V904), Leu907 (L907), and His908 (H908) from the S4 helix—near Ca 2 Aggregates at the interface cavity of the binding site ( Figure 4 a). Specifically, His908 forms hydrogen bonds and π-π stacking (T-shape) with the indole and hydroxyphenyl groups of NC1, while Trp864 forms hydrogen bonds with the hydroxyphenyl moiety of NC1. Furthermore, the residues with bulky side chains in Val904, Leu907, and Phe936 stabilize the methylindole group of NC1 through hydrophobic interactions. Figure 4 b,c, Figure 13-15To verify the predicted key function of the pocket in the NC1 effect, mutations were performed on residues involved in the pocket. Replacing W864, H908, or F936 with nonpolar alanine successfully disrupted the binding of TRPM4 to NC1, resulting in the failure of the response to NC1-induced TRPM4 channel activation and cell death. Figure 4 Furthermore, mutating the Met927 residue (M927) near the predicted pocket and predictively controlling the opening and closing of the binding pocket to an arginine residue (M927R) with a long side chain that prevents NC1 entry completely disrupts this interaction, while the M927A mutant remains responsive to NC1. Figure 4 e). Meanwhile, the constitutive channel activity of these mutants (W864A, H908A, and F936A) was assessed. Electrophysiological results revealed that these mutants were unresponsive to NC1 but still exhibited varying degrees of ion permeability compared to the channel-inactive mutant D984A ( Figure 4 g).

[0174] In addition, the inventors performed molecular dynamics (MD) simulations to simulate the state transitions of the NC1-TRPM4 complex embedded in the phospholipid bilayer. Figure 15 a) Within the first 100 ns after complex formation, the root mean square deviation (RMSD) converges to less than The range. Furthermore, statistical analysis of the interaction energies of the convergence trajectory further verifies that NC1 can form a stable complex with TRPM4 ( Figure 15 bd). The contributions of different residues to the binding energy indicate that the main residues stabilizing the binding of NC1 to TRPM4 are Ser863, Trp864, His908, and Phe936. Figure 15 e) This is consistent with the results of molecular docking analysis. In summary, these findings indicate that NC1 binds to TRPM4 within a VBP-like cavity.

[0175] The transmembrane (TM) region of TRPM4 consists of six transmembrane helices and a TRP domain. The transmembrane helices S5 and S6, plus a p-ring, form an ion-conducting pore domain surrounded by the S1-S4 domain. The pore domain is connected to the S1-S4 domain via an S4-S5 connector, which is believed to play a crucial role in the gating of the TRP channel. The S1-S4 domain of the TRPM4 channel contains a calcium ion binding site. The TRPM4 channel is impermeable to calcium ions but can be activated by intracellular calcium ions. The inventors tested whether NC1 enhances channel activity in the presence of intracellular calcium ions. In the presence of different concentrations of EGTA (a calcium ion chelator), NC1 exhibited similar effects on TRPM4, indicating that NC1 can activate TRPM4 channel activity independently of calcium ions. Figure 16(ad). Furthermore, NC1 and calcium ion-induced TRPM4 activation were compared. (e.g.) Figure 16 As shown in Figure e, NC1 significantly enhanced the channel current, more significantly than the effect of calcium ions. The presence of intracellular calcium ions did not further enhance the NC1-induced TRPM4 current. Figure 16 e). Consistently, the depletion of calcium ions using chelating agents did not inhibit NC1-induced NECSO ( Figure 16 f,g).

[0176] Furthermore, the inventors introduced point mutations near the calcium binding site that are known to block (E828K) or reduce (E1068Q) TRPM4 sensitivity to calcium ions. The inventors' research found that the E828K-mutated TRPM4 channel no longer responds to NC1, while the E1068Q-mutated channel still responds, but the activation level is reduced. Figure 16 Furthermore, these two calcium-related mutations, when reintroduced into TRPM4 knockout (KO) cells, exhibited partial or no NECSO-induced effects. Figure 16 j).

[0177] These findings also suggest that NC1 and calcium have similar mechanisms of action in activating TRPM4 channel activity.

[0178] Example 7: Necrotic cell death caused by energy depletion has NECSO characteristics.

[0179] Disruption of TRPM4 regulation is associated with severe ATP depletion under pathological conditions, such as ischemia or spinal cord injury. The inventors hypothesized that NC1-induced NECSO is analogous to necrosis induced by energy depletion. Cos7 cells were generated that stably expressed wild-type TRPM4 or with functional mutations including H159A31 (reduced ATP inhibition), D984A (channel activity defect), and E1068Q (reduced calcium sensitivity). Cell death was measured by energy depletion induced by NC1 exposure or NaN3 (a mitochondrial electron respiratory chain blocker) plus 2-deoxyglucose (2DG, a glycolysis inhibitor) (NaN3+2DG). In cells expressing WT TRPM4, but not in control cells, both NC1 and energy depletion induced dose-dependent cell death, while cells expressing the ATP-binding pocket mutant H159A were more vulnerable to NC1-induced cell death. Figure 5 a). Conversely, the channel-deficient mutant D984A and the calcium-sensing-deficient mutant E1068Q elicited marginal effects on NC1 or energy-depletion-induced cell death ( Figure 5 a). These results indicate that cell death caused by energy depletion is similar to that of individuals with NECSO genetic traits.

[0180] Next, several characteristics of NECSO were tested under energy depletion conditions. In the patch-clamping experiment, 2DG treatment activated TRPM4 channel activity in Cos7 cells expressing hTRPM4. Figure 5 b). Furthermore, energy depletion leads to the influx of sodium ions. This sodium influx and the resulting necrotic cell death are effectively counteracted by sodium depletion through the use of NMDG ( Figure 5 c, d).

[0181] These data suggest that energy depletion could also lead to NECSO.

[0182] Example 8: Enhancer function (GOF) mutations in human TRPM4 show susceptibility to NECSO.

[0183] TRPM4 is known to be associated with cardiac arrhythmias. Multiple human TRPM4 mutations are associated with cardiac conduction disorders such as progressive familial heart block type 1 (PFHB1), Brugada syndrome (BrS), and long QT syndrome (LQTS), some of which predispose patients to sudden cardiac death (SCD) due to ventricular arrhythmias or syncope. Abnormal energy supply or stress may be one of the precipitating factors for SCD in these patients. To date, more than 25 mutations have been reported linking TRPM4 to familial heart disease, some characterized as enhancing function (GOF) mutations, while others are described as losing function (LOF).

[0184] TRPM4 has been reported to be more abundant in Purkinje fibers, which play an important role in ventricular arrhythmias. The inventors hypothesized that disease-associated mutations could cause cardiac cells to exhibit different sensitivities to NECSO, leading to cardiac damage. Several well-defined GOF and LOF TRPM4 mutations were expressed in rat H9C2 cardiomyocytes, which exhibited low endogenous TRPM4 expression. Cells were then exposed to NC1 or NaN3+2DG stimulation to induce NECSO. GOF mutation expression showed greater sensitivity to NECSO compared to WT TRPM4, while LOF mutations showed no response. Figure 5 e). These data suggest that, under energy depletion-related pathological conditions, TRPM4 GOF mutations lead to increased cell death and damage to cardiac cells because they induce NECSO.

[0185] While TRPM4 GOF mutations may be a contributing factor to cardiac conduction block, TRPM4 overexpression may also lead to increased NECSO and cardiac damage. In fact, in 16 public transcriptome studies, TRPM4 expression, rather than that of other TRPM family members, was upregulated compared to left ventricular samples from heart failure patients. Figure 5 f).

[0186] Example 9: Identification of dihydropyridine calcium channel blocker (DHP-CCB) and clotrimazole (CLT) as small molecule inhibitors of NECSO

[0187] To explore treatment strategies and screen drugs for NECSO, the inventors utilized the aforementioned discovered mechanism of action and action pocket to identify specific small-molecule inhibitors of this process. Two strategies were employed to find effective NECSO inhibitors.

[0188] 1. Strategy One

[0189] MCF7 cells exhibit high intracellular TRPM4 expression levels and are highly sensitive to NC1. Using NC1-sensitive MCF7 cells as a screening model, we observed the inhibitory effect of candidate drugs (added before NC1) on NC1-induced cell death. If a candidate drug could reduce cell death, it would indicate that it could block NC1-induced cell death and thus have a protective effect on cells.

[0190] A library of FDA-approved drugs containing 1376 small molecules was used as the candidate drug library. The results showed that the nonspecific TRPM4 antagonist glimepiride (an analogue of glibenclamide) and the mitochondrial membrane permeability pore (mPTP) inhibitor cyclosporine A (CsA) were both selected as inhibitors. They have been reported to block NC1-induced cell death. Therefore, this screening strategy is reliable. Figure 6 a).

[0191] The inventors discovered that a class of compounds classified as dihydropyridines (DHPs) exhibited remarkable inhibitory effects on cell death in MCF7, with an EC50 of approximately 10 μM. Figure 6 c). The compounds are: cilinidipine, lacidipine, benidipine, lercanidipine, and clotrimazole (CLT).

[0192] Using this screening strategy, substances that protect cells (reduce cell death) can be obtained.

[0193] 2. Strategy Two

[0194] Using the newly identified NC1 binding pocket from Example 6 as a 3D docking template, in vitro computational simulation screening of a drug library was performed. During screening, the interaction between the binding pocket and candidate drugs was observed. Candidate drugs that could enter the binding pocket and fit the binding cavity were of interest, as they were potential substances that could regulate NECSO by modulating TRPM4.

[0195] Based on molecular docking, the antifungal drug clotrimazole (CLT) can bind within the binding pocket and is stabilized through extensive hydrophobic interactions, van der Waals forces, and hydrogen bonds. Within the cavity, the CLT molecule is surrounded by hydrophobic or aromatic residues, including Met927 in S5 and Phe931 in S6. Furthermore, the inventors observed a significant side-chain interaction between the residue His908 in S4 and the imidazole group of CLT. The side chains of Phe936 and Val904, pointing towards the center of the cavity, interact with CLT. This suggests that CLT can inhibit NECSO; it also suggests that CLT exerts its effect by competitively binding TRPM4 with NC1.

[0196] Structural analysis showed that the antifungal drug clotrimazole (CLT) has a similar chemical structure to NC1 and can enter and adapt to the NC1 binding cavity of TRPM4. Figure 6 b).

[0197] Further experimental analysis showed that CLT had an EC50 of 4 μM in MCF7 cells, thus validating its efficacy. Figure 6 c).

[0198] Dihydropyridines (DHPs) have been identified as calcium channel blockers that specifically target L-type voltage-gated calcium channels (LTCCs) on the cell membrane. Therefore, the inventors hypothesized that DHPs and CLTs might block NECSO by acting indirectly (DHPs) and directly (CLTs) on the inhibition of NC1-mediated TRPM4 channel activation. Indeed, electrophysiological results showed that none of the four DHPs that inhibit NECSO had any effect on TRPM4 channels, but CLTs directly inhibited TRPM4 channel activity. Figure 6 d), although both DHP and CLT blocked sodium influx and cell death after NC1 treatment ( Figure 6 e).

[0199] In addition, the inventors tested whether other types of calcium channels were involved in NECSO. The results showed that the LTCC blocker most effectively inhibited NECSO in MCF7 and MB468 cells. Figure 6 f). Since LTCC has been found to be selectively activated by TRPM4-dependent membrane depolarization, and TRPM4 has been identified as a calcium-activated nonselective (CAN) cation channel, these two ion channels on the cell membrane may interact and form a feedback loop regulating NECSO.

[0200] CLT exhibits dose-dependent inhibition of hTRPM4; CLT inhibits 1 μM Ca 2+Induced hTRPM4 current in HEK293 cells ( Figure 17 ).

[0201] The data above indicate that DHPs negate TRPM4 activation by targeting LTCC, while CLTs block NECSO by directly competing for the binding pocket between NC1 and TRPM4.

[0202] The aforementioned DHP-CCBs and CLTs have been identified as potent NECSO blockers. Therefore, the inventors tested whether these inhibitors were effective against energy depletion-induced cell death in cardiac cells. Indeed, these four well-defined DHPs and CLTs reduced NC1 or energy depletion-induced cell death in Cos7 cells with superior efficiency compared to glimepiride (…). Figure 6 g).

[0203] Further experimental analysis examined the protective effect of the NECSO inhibitor on cardiomyocytes (cells exposed to NC1 or NaN3+2DG stimulation to induce NECSO). The results showed that the NECSO inhibitor (10 μM) exhibited excellent protective effects against cardiomyocytes expressing the TRPM4-GoF mutation under energy depletion conditions. Figure 6 h).

[0204] These data indicate that NECSO inhibitors can block cell death caused by energy depletion in cardiac cells, and therefore have a therapeutic effect under pathological conditions of energy depletion, especially for patients with TRPM4 enhancing mutations, and can protect cardiomyocytes.

[0205] discuss

[0206] In this invention, a previously undefined necrotic cell death, NECSO, characterized by sodium overload, is reported, dependent on the monovalent cation channel TRPM4. The small molecule compound NC1, as a high-affinity agonist of TRPM4, functions through a ligand-binding regulatory pocket similar to that of other TRP family proteins, specifically binding to a structure on TRPM4. NC1 is the first ligand shown to bind to this pocket in TRPM4 and critically regulate TRPM4 channel activity. NC1 sustainably activates TRPM4 activity on the cell membrane by opening the TRPM4 channel and retaining TRPM4 on the cell membrane. Therefore, uncontrolled TRPM4 channel opening leads to massive sodium influx and cell membrane depolarization, resulting in specific cell death. NECSO can be indirectly blocked by calcium channel blockers and by the antifungal drug CLT as a direct competitor. Energy depletion can activate NECSO given the shared spectrum of cellular morphological changes, genetic and chemical interventions. TRPM4-enhancing mutations associated with arrhythmias are more susceptible to NECSO and can be blocked by its inhibitors.

[0207] TRPM4 has been identified as involved in cell death in various cellular environments, including neurons, astrocytes, and endothelial cells. This cell death has long been classified as oncosis. However, the classification of oncosis is based on morphological changes, such as cell swelling and vesicle formation, typically resulting from altered cell membrane permeability caused by cellular stress or injury. Many factors can lead to altered cell membrane permeability; in this invention, NECSO is described as a unique category where necrotic cell death is specifically caused by a surge of sodium ions driven by TRPM4 activation. The inventors' research highlights sodium ion influx as a key driver of cell death, as it leads to the most significant cell death response. Furthermore, the influx of sodium ions mediated by TRPM4, rather than other cations, may dominate the major cellular response of NC1 in vivo, considering the limited physiological concentrations of other monovalent cations in the extracellular fluid.

[0208] NECSO's genetic and biochemical composition distinguishes it significantly from apoptosis, autophagy, and various well-defined programmed necrosis pathways. However, some shared features may overlap with different cell death pathways. Similar to necrotizing apoptosis and pyroptosis, NECSO involves the translocation of key executive factors across the cell membrane. However, NECSO exhibits more restricted membrane permeability, selectively allowing ion exchange prior to widespread membrane rupture. While the exact mechanism by which sodium influx triggers specific cell death remains unclear, membrane depolarization is considered significant. The sodium pump (Na,K-ATPase) is thought to be involved in this process. Excessive sodium influx drives the hyperactivation of the sodium pump, which continuously hydrolyzes ATP to expel sodium from the cell, thereby maintaining membrane potential homeostasis. This highly energy-intensive process may require more than 70% of cellular energy, particularly in some neural tissues. Continuous sodium pump hyperactivation depletes the cell's ATP reserves, leading to energy depletion. Supporting this view are the inventors' observations highlighting the influence of mitochondria on NECSO, where cyclosporine A (CsA), a mitochondrial permeation modulator, effectively inhibits NC1-triggered cell death.

[0209] The function and regulatory mechanism of TRPM4 remain largely a mystery, as no natural or physiological ligands for TRPM4 have been reported to date. Several compounds, such as intracellular calcium, decavanadium, tissue plasminogen activator (tPA), U7312275, and Diazoxide, have been documented as activating TRPM4, but their effects may differ from those of NC1. NC1 strongly and persistently activates TRPM4 channel activity and maintains its membrane localization, enabling a rapid influx of large amounts of sodium ions. Molecular docking analysis revealed that NC1 binds to a cavity in which the S3 and S4 helices around one subunit in the TM region, an S4–S5 connector of one subunit, and the S5 and S6 helices of adjacent subunits are bonded. This binding pocket of NC1-hTRPM4 exhibits a degree of conservation in TRP channels and is termed the VBP pocket for ligand binding. For example, ligand-receptor pairs including capsaicin-TRPV1 (PDB number: 5IRX), ECN-TRPV5 (PDB number: 6B5V), and GNE551-TRPA1 (PDB number: 6X2J) all bind via VBPs. Figure 14 NC1 is the first chemical ligand shown to bind to the VBP region of TRPM4, which may explain its strong activation of this channel.

[0210] Although human and mouse TRPM4 share 88% sequence homology, NC1 specifically activates human TRPM4 but not mouse TRPM4. NC1 fails to bind to mouse TRPM4, and mouse TRPM4 cannot rescue the defects caused by human TRPM4 KO. This invention demonstrates the role of the TM domain in the different sensitivities of human and mouse TRPM4 to NC1. Furthermore, NC1 may stabilize and activate TRPM4 in an isomeric manner, which may help to capture dynamic channel activation processes. Structural studies of TRPV1 with double-knotted spider toxin (DkTx) successfully revealed the dynamic regulation of TRPV1. Until now, the structure of the open state of TRPM4 has remained unclear, and future studies on the structure of NC1-hTRPM4 may help to address this issue.

[0211] Therefore, the NC1 binding pocket is a valuable target for drug design of TRPM4 agonists and antagonists. Clotrimazole (CLT), an antifungal drug according to the present invention, is an example that shares structural similarity with NC1 and fits into the NC1-hTRPM4 pocket. CLT, as an antagonist, restricts NC1-triggered NECSO. CLT is a ring-open analog of NC1 with a similar 3D structure, containing a unique combination of hydrophobicity, π-π interactions, and a spherical shape, with a full-carbon all-season stereocenter. Notably, the neuroprotective effect of CLT against spinal cord ischemia / reperfusion injury may also stem from inhibition of TRPM4.

[0212] Under physiological conditions, the tendency of TRPM4 to open is influenced by cytoplasmic [Ca] 2+ The effects of increased and decreased ATP concentrations. In pathological settings, cytoplasmic calcium... 2+ Elevated levels or severe ATP depletion typically trigger TRPM4-dependent cell death. Consistently, energy depletion-induced necrotic cell death has been observed to have significant overlap in morphological and chemical characteristics with NECSO, suggesting that this specific type of cell death is involved in this cell death process. In this invention, it was observed that TRPM4-enhancing mutations associated with heart disease are more susceptible to NECSO, while its inhibitors can block cell death. Therefore, the development of TRPM4 antagonists is of great significance for protecting cells from cellular and tissue damage associated with sodium overload.

[0213] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for screening a substance that regulates cell death, comprising: Using TRPM4 or its transmembrane domain in cells as the observation target, or using TRPM4-involved cellular Na + Overload cell death is the observation target, and the effect of candidate substances on the target is analyzed to obtain substances that regulate cell death; Wherein, the transmembrane domain includes a binding pocket; Among them, the Na + Overload-type cell death occurs through activation of TRPM4 channels leading to Na + Excessive influx is characterized by cell necrosis.

2. The method according to claim 1, characterized in that The method comprises: (1) contacting a candidate substance with a screening system, wherein the screening system comprises TRPM4 or a transmembrane domain thereof, wherein the transmembrane domain comprises a binding pocket; (2) Adding a candidate substance to the screening system of (1) and observing the interaction between the candidate substance and the binding pocket; if the candidate substance enters the binding pocket, interacts with the candidate substance and activates the TRPM4 channel activity, the candidate substance is a substance that promotes cell death; if the candidate substance enters the binding pocket, interacts with the candidate substance and inhibits the TRPM4 channel activity, the candidate substance is a substance that protects cells.

3. The method according to claim 1, characterized in that The method comprises: (a) Cells were treated with Necrocide 1 to induce Na + Cells with overload-type cell death; preferably, the Necrocide1 enters the binding pocket, interacts and activates TRPM4 channel activity, thereby inducing cell death; (b) treating cells with a candidate substance; (c) analyzing the necrotic cell death of the cells. If the candidate substance causes a decrease in necrotic cell death, it indicates that the candidate substance is a cell-protecting substance; if the necrotic cell death increases, it indicates that the candidate substance is a cell-promoting substance.

4. The method according to any one of claims 1 to 3, characterized in that: The binding pocket comprises a binding cavity; preferably, the binding cavity comprises: S3, S4 helices, S4-S5 connector, and an enclosing structure formed by S5 and S6 helices of adjacent subunits; Preferably, the binding pocket includes the following sites of TRPM4: Met927 and Phe936 of the S5 helix; Ser863 and Trp864 of the S3 helix; Val904, Leu907, His908 of the S4 helix; Preferably, the regulatory effect of the candidate substance on cell death is analyzed by observing the interaction between the candidate substance and the site; or, the TRPM4 channel activity and cell death are analyzed by observing the interaction between Necrocide 1 and the site; more preferably, the interaction with the site includes: covalent binding or non-covalent binding.

5. The method according to claim 2 or 3, characterized in that: Methods for observing the interaction include: molecular docking analysis, binding energy analysis, PULL DOWN method, SPR method, Western blotting method, DNA sequence analysis, and immunoprecipitation method.

6. Use of the method according to any one of claims 1 to 5 for: Screening for substances that regulate cell death; or Screening for drugs to alleviate or treat diseases with TRPM4 dysfunction; Preferably, the TRPM4 dysfunction disease includes: cell or tissue damage; more preferably, the cell or tissue damage includes: heart disease.

7. Application of Necrocide 1 in preparing a drug screening model, wherein the drug screening model is a cell model, including TRPM4 or its transmembrane domain, wherein the Necrocide 1 cell induces Na + Overload cell death; the transmembrane domain includes a binding pocket; the Na + Overload-type cell death occurs through activation of TRPM4 channels leading to Na + Excessive influx is characterized by cell necrosis.

8. A method for preparing a cell model, comprising: Cells were treated with Necrocide 1 to obtain a cell model that exhibited Na + Overload cell death; the cell includes TRPM4 or its transmembrane domain; the transmembrane domain includes a binding pocket; the Na + Overload-type cell death occurs through activation of TRPM4 channels leading to Na + Excessive influx is characterized by cell necrosis.

9. Use of clotrimazole or a dihydropyridine compound in the preparation of a composition for inhibiting cell death; preferably, the dihydropyridine compound comprises an L-type calcium channel inhibitor; more preferably, the L-type calcium channel inhibitor comprises: Cilnidipine, lacidipine, benidipine, lercanidipine, nifedipine, amlodipine, nimodipine.

10. The use according to claim 9, characterized in that The cells include: cardiomyocytes; or The cell death includes necrotic cell death; preferably, the necrotic cell death is Na + Overload cell death, the substance that inhibits cell death is Na + Overload inhibitor of cell death.

Images