A natural small molecule Peli1 inhibitor

Abstract

The present invention discloses the use of [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]diethyl phosphate in the preparation of Peli1 inhibitors. The present invention also discloses the use of [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]diethyl phosphate in the preparation of a drug for inhibiting the activity of Peli1E3 ubiquitin ligase. The compound can interact with Peli1 protein, specifically inhibit its E3 ubiquitin ligase activity, and inhibit the activation of cell pathways in different cell models. The present invention also discloses the use of [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]diethyl phosphate in the preparation of a drug for treating myocardial ischemia-reperfusion injury. For an I / R disease model, the compound can improve the expression of the key protein GSDMD in the disease model, suggesting a certain therapeutic effect.

Description

Technical Field

[0001] The present invention relates to the use of diethyl [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl] phosphate in the preparation of Peli1 inhibitors, the use of diethyl [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl] phosphate in the preparation of drugs for inhibiting Peli1 E3 ubiquitin ligase activity, and the use of diethyl [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl] phosphate in the preparation of drugs for treating myocardial ischemia-reperfusion injury. Background Art

[0002] Peli1 is an evolutionarily conserved scaffold protein that was originally defined as a family of E3 ubiquitin ligases that play an important role in the TLR / IL-1R pathway. The Peli1 protein contains an N-terminal forkhead FHA domain and a C-terminal RING domain. The FHA domain is a phosphothreonine binding module that can bind to phosphothreonine-containing peptides, indicating that the FHA domain may specifically recognize substrate proteins and regulate the function of substrate proteins. The RING domain is composed of a conserved pattern of cysteine ​​and histidine residues, forming a two-zinc atom structure that can coordinate and define the function of E3 ubiquitin ligases.

[0003] At present, there are many reports on the correlation between Peli1 protein and disease. Studies have found that there are no obvious abnormalities in the development of immune organs and cells in Peli1 knockout mice, but the sensitivity to endotoxin shock induced by a lethal dose of LPS is significantly reduced. At the same time, the immune tolerance of Peli1-deficient mice will suffer a certain degree of destruction, leading to the appearance of lupus-like symptoms. In human lung adenocarcinoma tissues, Peli1 participates in the occurrence of lung tumors by inducing overexpression of Snail and Slug and promoting EMT, indicating that Peli1 may become a potential therapeutic target for lung cancer. In clinical samples of human skin melanoma, Peli1 expression is reduced and is associated with its poor overall survival, suggesting that it has diagnostic and therapeutic prospects for skin melanoma. In patients with diffuse large B-cell tumors (DLBCLs), Peli1 expression levels are positively correlated with the expression of the transcriptional repressor BCL6. Increased Peli1 expression and subsequent induction of BCL6 promote the occurrence of lymphomas, and this pathway may be a potential target for the treatment of B cell lymphomas. In addition, the inventors' previous studies have shown that in a mouse model of myocardial infarction, the loss of Peli1 can significantly reduce myocardial infarction-induced cardiac dysfunction and left ventricular remodeling; in a mouse model of myocardial ischemia-reperfusion, Peli1 aggravates the damage of autophagic flux by destroying P62 dimers and inhibiting P62 activity, thereby leading to MI / R injury. These results indicate that Peli1 is also an important therapeutic target in heart disease. It can be seen that Peli1 protein is closely related to the occurrence and development of many diseases. Therefore, screening effective, selective, and low-toxic Peli1 inhibitors has laid a solid foundation for the development of drugs for related diseases. Summary of the invention

[0004] Studies have shown that Peli1 mediates the k63-linked ubiquitination of IRF5 in M1 macrophages through E3 ubiquitin ligase activity. The inventors' studies have shown that after mechanical stress, Peli1 increases the ubiquitination of tumor necrosis factor-receptor associated factor-6 (TRAF6) and receptor interacting protein 1 (RIP1) through E3 ligase activity. A recent study showed that Peli1 can also regulate the expression of LC3II by interacting with TSC1, promoting the ubiquitination of the TSC1 / TSC2 complex, thereby inhibiting the activity of mTORC1. In addition, Peli1 can ubiquitinate and activate the NLRP3 inflammasome, thereby inducing the cleavage of the downstream pyroptosis substrate protein GSDMD, which forms an active form of GSMDD-N to induce cell pyroptosis. Therefore, it is shown that the E3 ubiquitin ligase activity of Peli1 can be used as an important indicator for inhibitor screening. Based on this result, the inventors screened out 10 natural small molecule compounds from the high-throughput screening natural product library through a computer-assisted system according to the E3 ubiquitin ligase activity of the Peli1 RING domain, and screened out the compound [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]diethyl phosphate (Compound No. 2) through layer-by-layer experiments, which can interact with the Peli1 protein and specifically inhibit its E3 ubiquitin ligase activity, thereby inhibiting the activation of cellular pathways in different cell models; in addition, for the I / R disease model, [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]diethyl phosphate can improve the expression of the key protein GSDMD in the disease model, suggesting that it has a certain therapeutic effect.

[0005] The structural formula of diethyl [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]phosphite is as follows:

[0006]

[0007] The purpose of the present invention is to provide the use of diethyl [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]phosphate in the preparation of Peli1 inhibitors.

[0008] A Peli1 inhibitor, which uses [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]diethyl phosphate as an effective component or a main effective component.

[0009] The purpose of the present invention is to provide the use of [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]diethyl phosphate in the preparation of a drug for inhibiting the activity of Peli1 E3 ubiquitin ligase.

[0010] The application is the application of [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]diethyl phosphate in the preparation of a drug for inhibiting the expression of pyroptosis substrate protein GSDMD-N and GSDMD-FL protein by inhibiting the activity of Peli1 E3 ubiquitin ligase.

[0011] A drug for inhibiting the activity of Peli1 E3 ubiquitin ligase, which uses [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]diethyl phosphate as an effective component or a main effective component.

[0012] Another object of the present invention is to provide the use of diethyl [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]phosphate in the preparation of a drug for treating myocardial ischemia-reperfusion injury.

[0013] The application is the use of [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]diethyl phosphate in the preparation of a drug for treating myocardial ischemia-reperfusion injury by inhibiting the activation of the NLRP3-mediated pyroptosis pathway, inhibiting the expression of the protein GSDMD-N, and inhibiting the expression of the GSDMD-FL protein by inhibiting the activity of the Peli1 E3 enzyme.

[0014] A medicine for treating myocardial ischemia-reperfusion injury, which uses [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl] diethyl phosphate as an effective component or a main effective component. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the proliferation activity of neonatal rat cardiomyocytes at the compound concentration (10 μM).

[0016] Figure 2 The effect of the compound on the LPS-induced increase in Peli1 E3 ubiquitin ligase activity.

[0017] Figure 3 The effect of compounds on the expression of NLRP3 signaling pathway related proteins in the LPS+ATP-induced RAW cell pyroptosis model.

[0018] Figure 4 The effect of compound No. 2 on the expression of NLRP3 signaling pathway related proteins in the LPS+ATP-induced cardiomyocyte pyroptosis model.

[0019] Figure 5 Figure 2 shows the inhibitory effect of compound 2 on GSDMD-N in the H / R (hypoxia 6h / reoxygenation 18h) model of neonatal rat cardiomyocytes; Figure a shows the effect of different concentrations of compound 2 on GSDMD-N protein expression, and Figure b shows the effect of compound 2 on GSDMD-NIC 50 Dose response curve of .

[0020] Figure 6 The inhibitory effect of compound 2 on GSDMD in the I / R model of C57BL / 6 mice.

[0021] Figure 7 MST was used to detect the interaction between Peli1 and compound 2; Figure a is the laser decay curve, and Figure b is the dose response curve. DETAILED DESCRIPTION

[0022] The technical scheme of the present invention is described in detail below using embodiments so that those skilled in the art can fully understand the present invention, but the present invention is not limited in any way.

[0023] In order to screen specific inhibitors of Peli1 E3 ubiquitin ligase activity, the inventors performed computer virtual docking screening based on the Peli1 RING domain and screened out 10 natural small molecule compounds (Table 1).

[0024] Table 1. Compounds to be screened

[0025]

[0026]

[0027] Example 1

[0028] 1.1. Isolation of neonatal rat cardiomyocytes

[0029] (1) Moisten gauze with alcohol and wipe the suckling rat;

[0030] (2) Place two curved forceps and two curved scissors on the mouse plate in the clean bench. Use forceps to take out a certain number of alcohol cotton balls and place them in the clean bench. Take out the heart of the suckling rat and place it in a 100 mm cell culture dish filled with 10 mL of pre-cooled PBS buffer.

[0031] (3) Trim the heart: cut off the blood vessels and atrial appendages, and transfer the heart to a new 100 mm cell culture dish containing 10 mL of pre-cooled PBS buffer;

[0032] (4) Transfer the heart into a 5 mL EP tube and cut the heart into pieces about 1 mm in size, resembling gravel;

[0033] (5) Add the PBS buffer in the EP tube and the tissue into the conical flask. After the tissue in the conical flask settles, remove the PBS and add 7 mL of 0.25% trypsin along the inner wall of the conical flask.

[0034] (6) Shake the conical flask in a 37°C water bath (fixed direction). Shake for 3 min in the first round. Discard the liquid after the first round (to wash away blood cells). Start the second round, shake for 5 min each time. Digest for 5 rounds. Divide the digested liquid evenly into 15 mL glass centrifuge tubes.

[0035] (7) Centrifugation: Centrifuge at 600 rcf / min for 6 min, gently remove the supernatant, add 1 mL of complete culture medium (45 mL of high-glucose DMEM medium, 5 mL of FBS, 0.5 mL of 100 μg / mL penicillin and 100 μg / mL streptomycin) and resuspend;

[0036] (8) Transfer the liquid in the centrifuge tube to a 100 mm cell culture dish, mix well, and place in a cell culture incubator at 37°C, 5% CO2, and 95% relative humidity for 4 h;

[0037] (9) Differential separation of cardiomyocytes and fibroblasts: Use a dropper to transfer the liquid in the culture dish to a clean 50 mL centrifuge tube, wash the culture dish twice with complete culture medium, and transfer all the liquid to the centrifuge tube;

[0038] (10) Myocardial cell sieving: The liquid in the centrifuge tube is passed through a 100-mesh sieve (washed once with complete culture medium) to obtain a myocardial cell suspension;

[0039] (11) Add BrdU (to inhibit fibroblast proliferation) according to the volume ratio of BrdU to cardiomyocyte suspension of 1:250, mix well, transfer the mixed cell suspension to a 96-well cell culture plate, and culture it in a cell culture incubator for 24 hours to wait for the cardiomyocytes to adhere to the wall before conducting subsequent experiments.

[0040] 1.2. CCK-8 assay for cell proliferation toxicity

[0041] (1) Take the cells from “1.1 Extraction of neonatal rat cardiomyocytes (11)”, discard the original culture medium, and replace it with 100 μL serum-free culture medium;

[0042] (2) Use DMSO to prepare 10 compounds to be screened into a stock solution with an initial concentration of 10 mM. Add 0.1 μL of the stock solution to the drug-treated group (the final concentration is 10 μM); add 0.1 μL of PBS to the blank control group (con group); and add 0.1 μL of DMSO to the solvent control group (DMSO group).

[0043] (3) Place the culture plate in a cell culture incubator at 37°C, 5% CO2, and 95% relative humidity for 24 h;

[0044] (4) Add 10 μL CCK-8 solution (Cell counting KIT-8 kit purchased from Solarbio LIFESCIENCES, catalog number: CK04) to each well;

[0045] (5) Continue to incubate the culture plate in a cell culture incubator at 37°C, 5% CO2, and 95% relative humidity for 2 h;

[0046] (6) Measure the absorbance at 450 nm using an ELISA reader (Bio-tek, catalog number: 900Hi).

[0047] Figure 1 The effect of the compounds (at a concentration of 10 μM) on the viability of neonatal rat cardiomyocytes showed that compounds 1, 2, 6, and 9 had no significant effect on the viability of neonatal rat cardiomyocytes.

[0048] Example 2

[0049] (1) Cultivation of RAW264.7 cells: Take out the RAW264.7 cells frozen in liquid nitrogen, thaw them quickly in a 37°C water bath to form a cell suspension, transfer them to a 15mL centrifuge tube, centrifuge at 0.5rcf / min for 5min, discard the supernatant, add 5mL PBS to wash twice, then add new complete medium to resuspend, transfer the resuspended cell suspension to a cell culture dish and place it in a cell culture incubator at 37°C, 5% CO2, and a relative humidity of 95% for culture; observe the density of adherent cells under a microscope every two days and find that it is 90%; discard the culture medium, add 3mL of fresh complete medium, blow off the adherent cells by blowing, and then transfer them to a 15mL centrifuge tube; resuspend the cells and transfer them to a six-well cell culture dish and place it in a cell culture incubator at 37°C, 5% CO2, and a relative humidity of 95% for culture for 24h;

[0050] (2) Treatment of RAW264.7 cells: The original culture medium was discarded and replaced with 1 mL of fresh serum-free culture medium for each well. The drug group was given compound solutions 1, 2, 6, and 9 (10 μM, prepared as in Example 1) to pretreat RAW264.7 cells for 2 h, and then treated with LPS (1 μg / mL) for 4 h; the DMSO group was first given 1 μL DMSO pretreatment for 2 h, and then treated with LPS (1 μg / mL) for 4 h; the Con group was given 1 μL PBS;

[0051] (3) Detection of Peli1 E3 ubiquitin ligase activity

[0052] (a) Protein preparation: The culture medium of the RAW264.7 cells treated in step (2) was discarded, and the cells were lysed with protein lysis buffer for 50 minutes, centrifuged at 12,000 rpm at 4°C, and the protein supernatant was collected;

[0053] (b) BCA method for protein concentration detection: 0, 1, 2, 4, 6, and 8 μL of 0.5 mg / ml BSA (standard bovine serum albumin) were added to a 96-well plate in sequence, and then PBS was added to each well to make up to 10 μL, and 3 replicates were made for each well; 2 μL of sample protein was added to a 96-well plate, and 3 replicates were made; 200 μl of BCA working solution (A solution: B solution = 50:1, fully mixed) was added to the protein standard well and sample protein well, respectively, and incubated at 37°C for 30 min. The OD value was measured at 550 nm by an enzyme marker, and the standard curve was drawn with the concentration of the standard as the horizontal axis and the corresponding OD value as the vertical axis. The protein concentration of the unknown sample was calculated based on the measured OD value; the concentration of all samples was adjusted to 3 μg / μL with protein lysis buffer;

[0054] (c) Take 300 μL of protein supernatant, add Peli1 antibody (Abcam, ab199336) at a volume ratio of 1:100 between Peli1 antibody and protein supernatant, set negative reference: take Con group protein, select rabbit IgG (beyotime, A7016) as the antibody Peli1 is rabbit antibody, and combine in a rotating incubator at 4°C for 4 h;

[0055] (d) Pipette 50 μL of Protein A / G-bound beads (cytiva, 17061801); add 600 μL of CO-IP elution buffer (elution buffer formula see Table 2) to wash the beads, wash three times at 4°C and 2500 rpm, each time for 5 minutes;

[0056] Table 2. CO-IP eluent

[0057]

[0058] (e) Take the protein supernatant of step (a), centrifuge at 300g for 10 minutes at 4°C, add it to the Protein A / G binding beads, bind at 4°C for 12 hours, and then centrifuge at 1000g for 10 minutes at 4°C, discard the protein supernatant, and keep the Protein A / G binding beads;

[0059] (f) Add 600 μL of washing solution to wash the Protein A / G-bound beads, centrifuge at 1000 g for 10 min at 4°C, repeat 3 times, and retain the Protein A / G-bound beads;

[0060] Washing solution: 50mM Tris-HCl (pH 7.5) + 1% (v / v) Triton-X-100 + 0.05% β-mercaptoethanol + 0.2MNaCl; for example, preparing 10mL washing solution: 9mL Tris-HCl + 100μL Triton-X-100 + 5μL β-mercaptoethanol + 1mLNaCl;

[0061] (g) Add 2 mL Tris-HCl (pH 7.5) + 2 μL 1000×MgCl2 to the Protein A / G-bound beads obtained in step (f), centrifuge at 1000 g for 10 min at 4°C, repeat twice, and retain the Protein A / G-bound beads;

[0062] (h) Add 20 μL of incubation solution and incubate at 30°C for 30 min; add 5 μL of 5× protein buffer and denature at 99°C for 5 min, and perform Western Blot experiment to detect enzyme activity.

[0063] Table 3. Incubation medium

[0064]

[0065] (4) Western Blot assay to detect enzyme activity

[0066] (a) SDS-PAGE (polyacrylamide gel) electrophoresis: prepare 12% separation gel and 3% stacking gel. Take the sample from "(3) Peli1 E3 ubiquitin ligase activity detection step (h)", and load about 20 μL per well; perform electrophoresis at 110V constant voltage for about 90 minutes;

[0067] (b) Transfer: After electrophoresis, cut off the concentrated gel and immerse the gel in protein transfer buffer (3.6g / L Tris, 200mL / L methanol, 17.3g / L glycine) for 10-20min. Transfer the protein bands to the PVDF membrane using the wet transfer method and perform electrophoresis at a constant current of 0.3A for 80min.

[0068] (c) Blocking: After the transfer, remove the PVDF membrane, soak it in PBS for 5 min, and then soak it in PBS containing 5% skimmed milk powder and incubate it at room temperature for 1 h;

[0069] (d) Primary antibody incubation: After blocking, place the membrane in a hybridization bag, add ub (Santcruz, A-5) antibody, and shake at 4°C overnight;

[0070] (e) Secondary antibody binding: Wash the membrane with PBS-T for 10 min × 3 times; add horseradish peroxidase-labeled antibody and incubate at room temperature for 1 h. Wash the membrane with PBS-T for 10 min × 3 times;

[0071] (g) ECL color development: Mix ECL color development solution A and B, evenly add them onto the membrane surface, expose the membrane in a dark place, and observe the results.

[0072] Figure 2It is the effect of the compounds on the LPS-induced increase in Peli1 E3 ubiquitin ligase activity. It can be seen that compound No. 2 has a significant inhibitory effect on the LPS-induced increase in Peli1 E3 ubiquitin ligase activity, while other compounds have no significant effect.

[0073] Example 3

[0074] (1) Cultivation of RAW264.7 cells is the same as step (1) “Cultivation of RAW264.7 cells” in Example 2;

[0075] (2) Treatment of RAW264.7 cells: Discard the original culture medium and add 1 ml of fresh serum-free culture medium to each well. The drug group was pretreated with 1 μL of compound 1, 2, 6, 9 solution (10 μM, prepared as in Example 1) for 2 h, then treated with LPS (1 μg / ml) for 4 h, and finally treated with 5 mM ATP for 30 min; the LPS+ATP group was pretreated with 1 μL DMSO for 2 h, then treated with LPS and ATP (same as the drug group); the LPS group was treated with LPS (1 μg / ml) without adding compounds and ATP; the Con group was treated with 1 μL PBS.

[0076] (3) Total cell protein extraction

[0077] (a), taking the RAW264.7 cells treated in step (2), discarding the culture medium, lysing them with protein lysis buffer for 50 minutes, centrifuging them at 12000 rpm at 4°C, and taking the protein supernatant;

[0078] (b) Detection of protein concentration by BCA method: the method is the same as step (3) "Peli1 E3 ubiquitin ligase activity detection step (b)" in Example 2;

[0079] (4) Western Blot

[0080] (a) SDS-PAGE (polyacrylamide gel) electrophoresis: prepare 12% separation gel and 3% concentration gel; take 200 μL of the protein supernatant in "step (3) and total cell protein extraction step (b)", add 50 μL of 5× protein buffer, and denature at 99°C for 5 min; the sample volume per well is about 20 μL, and the electrophoresis is carried out at 110V constant voltage for about 90 min; the remaining denatured sample is stored at -20°C;

[0081] (b) The Western Blot step is the same as Example 2 "(4) Western Blot experiment to detect enzyme activity steps (b)-(g)", and the antibodies used are Peli1 (abcam, ab199336), NLRP3 (cell signaling technology, D4D8T), ASC (bioss, bs-1155R), cleaved-caspase-1 (cell signaling technology, E2G2I), and GSDMD (abcam, ab219800).

[0082] Figure 3 The effects of compounds on the expression of NLRP3, cleaved-caspase-1, GSDMD-N and other proteins in the LPS+ATP-induced cell pyroptosis model in RAW264.7 cells show that compound No. 2 can significantly inhibit the expression of pyroptosis substrate protein GSDMD-N and other proteins in the pathway, indicating that compound No. 2 can inhibit LPS+ATP-induced cell pyroptosis.

[0083] Example 4

[0084] (1) The extraction steps of myocardial cells of suckling rats are the same as those in Example 1, “1.1. Extraction of myocardial cells of suckling rats”;

[0085] (2) Discard the original culture medium and replace each well with 1 ml of fresh serum-free culture medium. The cardiomyocytes of neonatal rats in the drug group were pretreated with Compound No. 2 (10 μM, prepared as in Example 1) for 2 h, then treated with LPS (1 μg / ml) for 4 h, and then treated with 5 mM ATP for 30 min; the LPS+ATP group was pretreated with 1 μL DMSO for 2 h, and then treated with LPS and ATP according to the drug group method; the LPS group was treated with LPS (1 μg / ml) without adding compounds and ATP; the Con group was treated with 1 μL PBS;

[0086] (3) Extraction of total protein from neonatal rat cardiomyocytes: Take the neonatal rat cardiomyocytes treated in step (2), discard the culture medium, lyse with protein lysis buffer for 50 minutes, and centrifuge at 12000 rpm at 4°C; detect protein concentration by BCA method: the method is the same as step (3) "Peli1 E3 ubiquitin ligase activity detection step (b)" in Example 2;

[0087] (5) Western Blot was used to detect the protein expressions of Peli1, NLRP3, ASC, Cl-caspase-1, GSDMD-FL, and GSDMD-N.

[0088] (a) SDS-PAGE (polyacrylamide gel) electrophoresis: prepare 12% separation gel and 3% concentration gel; take 100 μL of the protein supernatant from "step (3), total protein extraction of neonatal rat cardiomyocytes", add 25 μL of 5× protein buffer, and denature at 99°C for 5 min; the sample volume per well is about 20 μL, and the electrophoresis is carried out at a constant voltage of 110 V for about 90 min; the remaining denatured sample is stored at -20°C;

[0089] (b) Western Blot steps are the same as those in Example 2 “(4) Western Blot assay for detecting enzyme activity steps (b)-(g)”.

[0090] Figure 4 Compound No. 2 has an effect on the protein expression of NLRP3, cleaved-caspase-1, and GSDMD-N in the LPS+ATP-induced cell pyroptosis model. It can be seen that compound No. 2 can significantly inhibit the expression of pyroptosis substrate protein GSDMD-N and the expression of other proteins in the pathway, indicating that compound No. 2 can inhibit LPS+ATP-induced pyroptosis of neonatal rat cardiomyocytes.

[0091] Example 5

[0092] (1) Extraction of myocardial cells from suckling rats Example 1 "1.1. Extraction of myocardial cells from suckling rats";

[0093] (2) Discard the original culture medium and replace it with serum-free culture medium with different concentrations of compounds. The compound No. 2 with an initial concentration of 1 mM in Example 1 was diluted with serum-free culture medium to different concentrations (0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM), and 1 mL was added for pretreatment for 2 h, and then H / R (hypoxia for 6 h / reoxygenation for 18 h) was performed; the solvent control group (H / R+DMSO) was replaced with 1 mL of serum-free culture medium and then given 1 μL of DMSO; the H / R group was only treated with H / R without adding compounds and DMSO; the Con group was added with 1 μL of PBS.

[0094] (3) Extracting total cell protein: Take the neonatal rat cardiomyocytes treated in step (2), discard the culture medium, lyse with protein lysis buffer for 50 minutes, and centrifuge at 12000 rpm at 4°C; detect protein concentration by BCA method: the method is the same as step (3) "Peli1 E3 ubiquitin ligase activity detection step (b)" in Example 2;

[0095] (4) Western blot detection of the expression of pyroptosis substrate protein GSDMD-N:

[0096] (a) SDS-PAGE (polyacrylamide gel) electrophoresis: prepare 12% separation gel and 3% concentration gel; take 100 μL of the protein supernatant from "step (3), total cell protein extraction", add 25 μL of 5× protein buffer, and denature at 99°C for 5 min; the sample volume per well is about 20 μL, and the electrophoresis is carried out at a constant voltage of 110 V for about 90 min; the remaining denatured sample is stored at -20°C;

[0097] Step (b) is the same as that in Example 2, "(4), Western Blot assay for detecting enzyme activity, steps (b) to (g)".

[0098] Figure 5 The inhibitory effect of different concentrations of compound 2 on GSDMD-N in the H / R (hypoxia 6h / reoxygenation 18h) model of neonatal rat cardiomyocytes. Figure 5 a is the effect of different concentrations of compound 2 on the expression of GSDMD-N protein. Figure 5 b is the IC of compound 2 against GSDMD-N 50 Dose-response curve. It can be seen that compound 2 can inhibit the protein expression of GSDMD-N in a dose-dependent manner, and the IC 50 The results showed that compound 2 may inhibit the activation of NLRP3-mediated pyroptosis pathway by inhibiting Peli1 E3 enzyme activity, and its inhibitory effect on GSDMD-N, a key protein in the signaling pathway, was well dose-dependent.

[0099] Example 6

[0100] 1. Myocardial ischemia-reperfusion (I / R) model in C57BL / 6 mice

[0101] (1) Mice were 8-10 weeks old and weighed 20-25 g. They were anesthetized with isoflurane inhalation anesthesia at a flow rate of 2 L / min.

[0102] (2) After anesthesia, the mouse was intubated orally and connected to a small animal ventilator to maintain breathing. The mouse's breathing fluctuations were observed to be consistent with the ventilator frequency. The mouse was placed in a supine position with its limbs fixed on a circulating heating operating table and connected to the limb conduction electrodes of a small animal electrocardiogram to record the ECG changes during the operation.

[0103] (3) 2 h before surgery, different doses of compound 2 (0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1 mg / kg) were intraperitoneally injected. The control group underwent sham surgery, and the solvent control group (I / R+DMSO) was intraperitoneally injected with 1% DMSO.

[0104] (4) Ischemia-reperfusion model surgical operation: The surgical area was routinely prepared and disinfected with iodine tincture. The skin was cut open between the 3rd and 4th intercostal spaces on the left side of the sternum. The subcutaneous tissue, pectoralis major and pectoralis minor were bluntly separated layer by layer. The intercostal space was opened with a thoracotomy device to fully expose the heart. The pericardium was cut open. A pink blood vessel was seen at the lower edge or left side of the atrial appendage, which was the anterior descending branch of the left coronary artery (LAD). A 6-0 suture needle with thread was used to penetrate the myocardial surface 1 mm below the atrial appendage, and the LAD was ligated under pressure. Before ligation, a 0.2-0.3 cm No. 10 polyethylene tube was placed under the thread. After ligation of the LAD, the myocardium of the anterior wall of the left ventricle turned white. The local contraction movement was limited, the left atrial appendage was filled and swollen, and the electrocardiogram showed that the ST segment was continuously arched and elevated, indicating that myocardial ischemia was successful. The chest cavity was closed with simple sutures. After 45 minutes of ischemia, the chest cavity was opened again, the polyethylene tube was removed, and the ligature was cut. The heart surface changed from pale to red. The ST segment elevation of the electrocardiogram fell back, indicating successful reperfusion. The chest cavity and skin were sutured. After 24 hours, the heart tissue protein was extracted.

[0105] 2. Extraction of Mouse Heart Tissue Protein

[0106] (1) Add 3 grinding beads to the tissue grinding tube, add 200 μL of protein lysis buffer to each tube, and grind at 60 Hz for 60 seconds. Lyse on ice for 50 minutes, centrifuge at 12,000 rpm at 4°C, and take the protein supernatant;

[0107] (2) Detection of protein concentration by BCA method: the steps are the same as those in Example 2, step (3) "Peli1 E3 ubiquitin ligase activity detection step (b)".

[0108] 3. Western Blot Detection of GSDMD-FL and GSDMD-N Protein Expression

[0109] (a) SDS-PAGE (polyacrylamide gel) electrophoresis: prepare 12% separation gel and 3% concentration gel; take 300 μL of the protein supernatant from "Step 2, mouse heart tissue total protein extraction step (2)", add 75 μL of 5× protein buffer, and denature at 99°C for 5 min; the sample volume per well is about 20 μL, and the electrophoresis is carried out at a constant voltage of 110 V for about 90 min; the remaining denatured sample is stored at -20°C;

[0110] (b) The western blot steps are the same as those in Example 2 “(4), Western Blot assay for detecting enzyme activity steps (b)-(g)”.

[0111] Figure 6 The inhibitory effect of compound No. 2 on GSDMD in the I / R model of C57BL / 6 mice shows that compound No. 2 can inhibit the protein expression of GSDMD-FL and GSDMD-N in a dose-dependent manner.

[0112] Example 7

[0113] Micro-surge thermal (MST)

[0114] (1) Take 100 μL of Peli1 protein solution, add NHS-647 (Thermofisher, A20006) dye, shake well and protect from light, and incubate at 4°C for 30 min.

[0115] (2) Elute the NHS-647 dye using a magnetic adsorption column, collect the labeled protein using a microcentrifuge tube, and store it at -20°C.

[0116] (3) Eight gradient concentrations of compound 2 solution (0.002 μM, 0.005 μM, 0.02 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM) were prepared using PBS, and 10 μL of each solution was taken and mixed with an equal volume of Peli1 labeled protein.

[0117] (4) Use a capillary to absorb the mixed sample and place the capillary in a microcalorimeter to detect laser decay.

[0118] (5) The data acquisition software MO.Control directly processes the data to obtain the dissociation constant.

[0119] Figure 7 It is the binding activity of compound 2 with Peli1 protein. Figure 7 a is the laser decay curve, Figure 7 b is a dose response curve. The results show that Peli1 has good binding activity with compound No. 2, and the binding constant is 37.1nM.

[0120] In view of the above results, [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]diethyl phosphate (Compound No. 2) can interact with Peli1 protein and specifically inhibit its E3 ubiquitin ligase activity, thereby inhibiting the activation of cellular pathways in different cell models. In addition, for I / R disease models, [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]diethyl phosphate can improve the expression of the key protein GSDMD in the disease model, suggesting a certain therapeutic effect. For the development of many diseases related to Peli1 (diffuse large B-cell tumor, lung cancer, sepsis, etc.), its E3 enzyme activity plays an important role, so it can be used to prepare drugs targeting Peli1 E3 ubiquitin ligase activity, and further structural optimization has good application prospects.

Claims

1. Use of diethyl[(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]phosphate in the preparation of drugs for treating myocardial ischemia-reperfusion injury.

2. The use according to claim 1, characterized in that: The application is the use of [(4-nitrophenyl)({4-propan-2-yl)phenyl]amino}methyl]diethyl phosphate in the preparation of a drug for treating myocardial ischemia-reperfusion injury by inhibiting the activation of the NLRP3-mediated pyroptosis pathway, inhibiting the expression of the protein GSDMD-N, and inhibiting the expression of the GSDMD-FL protein by inhibiting the activity of the Peli1 E3 enzyme.

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