Application of diethyl succinate or its derivatives in the preparation of RIPK1 kinase inhibitors and / or drugs for preventing and treating inflammatory diseases

Abstract

The present invention provides an application of diethyl succinate and its derivatives in the preparation of RIPK1 kinase inhibitors and / or drugs for preventing and treating inflammatory diseases, belonging to the technical field of chemical drugs. The present invention provides an application of diethyl succinate or its derivatives in the preparation of RIPK1 inhibitors. Experiments have shown that the diethyl succinate and / or its derivatives have the effects of inhibiting RIPK1 kinase activity and inhibiting RIPK1-mediated cell necrosis, apoptosis, and pyroptosis, and can be used in the preparation of drugs for preventing and treating RIPK1-related inflammatory diseases (sepsis), and have a wide range of promotion and application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical drugs, and specifically relates to the use of diethyl succinate and its derivatives in the preparation of RIPK1 kinase inhibitors and / or drugs for preventing and treating inflammatory diseases. Background Art

[0002] Receptor interacting serine / threonine protein kinase 1 (RIPK1) is a critical regulator of pro-survival NF-κB signaling and cell death signaling in response to a range of inflammatory and cell death stimuli during human disease pathology. RIPK1 kinase activation has been demonstrated in postmortem human pathology samples of autoimmune and neurodegenerative diseases, and inhibition of RIPK1 kinase activity can effectively ameliorate the phenotypes of animal models of these diseases. Because TNFR1-mediated RIPK1 activation is the most comprehensively characterized paradigm, RIPK1 inhibitors were initially thought to provide a small molecule alternative to anti-TNF antibodies for the treatment of TNF-driven autoimmune diseases (sepsis). However, as the research progressed, researchers found that RIPK1 inhibitors can play an advantage that anti-TNF antibodies do not have. First, RIPK1 inhibitors are safe in the central nervous system (CNS) because RIPK1 kinase does not mediate signals through TNFR2; second, RIPK1 is not only involved in the inflammatory response of TNF, but also in many other inflammatory processes; third, the RIPK1 molecule is regulated by a series of molecules in the cell, and the diseases caused by people with genetic mutations of these molecules can be used in clinical trials of RIPK1 inhibitors.

[0003] Necrostatin-1s (nec1-1s) is the first small molecule inhibitor of RIPK1 kinase to be developed and has been widely used to study the role of RIPK1 in human disease mechanism studies and animal models. Clinical trials are investigating the broad therapeutic application of RIPK1 inhibitors in the treatment of a variety of human diseases. GSK's 772 is an inhibitor used for peripheral immune diseases, mainly for the treatment of diseases such as psoriasis, rheumatoid arthritis (RA) and ulcerative colitis. The clinical trial of the brain penetrant DNL747 for amyotrophic lateral sclerosis (ALS) is in Phase Ib / IIa. These trials lay the foundation for advancing the clinical application of RIPK1 inhibitors.

[0004] There have been reports on RIPK1 inhibitors and the treatment of inflammatory diseases, including peptides or peptidomimetics and small molecule non-peptide compounds. Currently, only a very small number of small molecule non-peptide compounds have entered Phase I / II clinical trials, and no drugs have been approved for marketing. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an application of diethyl succinate and its derivatives in the preparation of RIPK1 kinase inhibitors, expand the types of compounds that inhibit RIPK1 kinase activity, and provide a new means for treating RIPK1-related inflammatory diseases.

[0006] The present invention provides use of diethyl succinate or a derivative thereof in preparing a RIPK1 inhibitor.

[0007] The invention provides an application of diethyl succinate or a derivative thereof in preparing a preparation for inhibiting cell necrosis, apoptosis or pyroptosis.

[0008] Preferably, the cell necrosis, apoptosis or pyroptosis is mediated by RIPK1.

[0009] Preferably, the types of cells include somatic cells, immune cells and cancer cells;

[0010] The cells include those of human and / or animal origin.

[0011] The present invention provides use of diethyl succinate or a derivative thereof in preparing a medicine for preventing and / or treating inflammatory diseases.

[0012] Preferably, the inflammatory disease comprises sepsis.

[0013] Preferably, the prevention and / or treatment of sepsis includes increasing the survival period of sepsis patients and / or reducing cecal damage and / or inflammatory infiltration in sepsis patients.

[0014] Preferably, the derivative of diethyl succinate includes dimethyl succinate.

[0015] The invention provides a composition for inhibiting RIPK1 activity, comprising diethyl succinate and dimethyl succinate.

[0016] The present invention provides use of the composition in preparing a RIPK1 inhibitor or a drug for preventing and / or treating inflammatory diseases.

[0017] The present invention provides an application of diethyl succinate or its derivatives in the preparation of RIPK1 inhibitors. Experiments have shown that diethyl succinate and its derivatives have the effect of inhibiting the activity of RIPK1 kinase, and their inhibitory activity is equivalent to the inhibitory ability of Nec-1.

[0018] The present invention provides an application of diethyl succinate and its derivatives in the preparation of a preparation for inhibiting cell necrosis, apoptosis or pyroptosis. Experiments show that diethyl succinate can significantly inhibit necrosis of L929 cells induced by TNF, zVAD and TNF+zVAD, and diethyl succinate can significantly inhibit necrosis of HT29 cells induced by TNF+SM164+zVAD, but succinate (or ester) does not have the effect of inhibiting necrosis of the two cells. It can be seen that based on the effect of diethyl succinate or its derivatives on inhibiting the activity of RIPK1 kinase, cell necrosis, apoptosis or pyroptosis can be inhibited.

[0019] The present invention provides an application of diethyl succinate or its derivatives in the preparation of drugs for preventing and / or treating inflammatory diseases. The present invention uses a TNF-mediated sepsis animal model as an experimental subject to evaluate the medicinal effects of diethyl succinate or its derivatives. The results show that diethyl succinate or its derivatives can significantly improve the survival of TNF-mediated sepsis, and the HE staining results show that diethyl succinate or its derivatives can effectively improve the cecal injury and inflammatory infiltration in the sepsis model. It can be seen that the RIPK1 inhibitor diethyl succinate or its derivatives provided by the present invention can be used for the development of related inflammatory disease treatment drugs and have a wide range of uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results show that diethyl succinate inhibits necrosis of L929 cells induced by TNF, zVAD and TNF+zVAD.

[0021] Figure 2 The results show that diethyl succinate inhibits TNF+SM164+zVAD-induced necrosis of HT29 cells.

[0022] Figure 3 The results show that diethyl succinate inhibits necrosis of mouse macrophages;

[0023] Figure 4 The results show that diethyl succinate inhibits the necrosis of mouse BMDM cells;

[0024] Figure 5 The results of the test on the inhibition of RIPK1 autoactivation by diethyl succinate and its derivatives;

[0025] Figure 6 The results showed that diethyl succinate improves survival in TNF-mediated sepsis;

[0026] Figure 7The results show that diethyl succinate improves cecal injury and inflammatory infiltration in a sepsis model, where the upper part of the figure shows the HE histochemical staining result, and the lower part of the figure shows the CD45 staining result; SA in the figure stands for diethyl succinate;

[0027] Figure 8 The results show the effects of succinic acid on the body temperature and improvement of tissue damage (LDH, ALT, AST) in mice, where DSA represents diethylsuccinate. DETAILED DESCRIPTION

[0028] The present invention provides use of diethyl succinate or a derivative thereof in preparing a RIPK1 inhibitor.

[0029] In the present invention, the chemical structural formula of diethylsuccinate is shown in Formula I, and the CAS number is 123-25-1. The derivative of diethylsuccinate preferably includes dimethylsuccinate, and the chemical structural formula is shown in Formula II, and the CAS number is 106-65-0.

[0030] In the present invention, the diethyl succinate and dimethyl succinate are purchased from Sigma.

[0031]

[0032] In the present invention, recombinant cells overexpressing RIPK1 were used as the object, and the inhibitory effects of diethyl succinate and dimethyl succinate on the autoactivation of RIPK1 were detected by immunoblotting. The results showed that diethyl succinate and dimethyl succinate inhibited the activation of the S166 site of RIPK1, thereby achieving the effect of inhibiting the kinase activity of RIPK1.

[0033] The invention provides an application of diethyl succinate or a derivative thereof in preparing a preparation for inhibiting cell necrosis, apoptosis or pyroptosis.

[0034] In the present invention, the cell necrosis, apoptosis or pyroptosis is preferably mediated by RIPK1. The types of cells preferably include somatic cells, immune cells and cancer cells. The somatic cells are preferably fibroblasts. The immune cells preferably include macrophages. The cancer cells preferably include colon cancer cells. The cells preferably include human and / or animal sources. The animal source preferably includes mouse sources. The working concentration of diethyl succinate or its derivatives is preferably 5 μM.

[0035] In the examples of the present invention, mouse fibroblast L929, human colon cancer cell HT29, and macrophage PMs were used as the subjects to evaluate the inhibitory effect of diethyl succinate or its derivatives on cell necrosis. The results showed that diethyl succinate could significantly inhibit the necrosis of L929 cells induced by TNF, zVAD, and TNF+zVAD, and inhibit the necrosis of HT29 cells induced by TNF+SM164+zVAD, while succinate (or ester) did not show any inhibitory activity.

[0036] Based on the fact that diethyl succinate or its derivatives have RIPK1 inhibitory activity and the effects of inhibiting cell necrosis, pyroptosis and apoptosis, the present invention provides an application of diethyl succinate or its derivatives in the preparation of a drug for preventing and / or treating inflammatory diseases.

[0037] In the present invention, the inflammatory disease preferably includes sepsis. The sepsis belongs to an inflammatory disease associated with cell necrosis. Since RIPK1 inhibitors are commonly used in the prior art to replace anti-TNF antibodies to treat TNF-driven autoimmune diseases (sepsis), the present invention confirms that diethyl succinate or its derivatives can be used as RIPK1 inhibitors to inhibit cell necrosis, apoptosis and pyroptosis. Therefore, diethyl succinate or its derivatives are used in the prevention and / or treatment of inflammatory diseases. The prevention and / or treatment of sepsis preferably includes increasing the survival period of sepsis and / or reducing cecal damage and / or inflammatory infiltration in patients with sepsis.

[0038] In the present invention, the concentration of diethyl succinate or its derivatives in the drug is 4-6 mM, more preferably 5 mM. The present invention has no special limitation on the dosage form of the drug, and any dosage form known in the art, such as powder or injection, can be used.

[0039] In the present embodiment, a TNF-induced sepsis animal model was used as an experimental subject, and the effects of diethyl succinate administration on the survival period, cecal injury, and inflammatory infiltration of the animal model were evaluated. The results showed that diethyl succinate can significantly improve the survival period of TNF-mediated sepsis. At the same time, diethyl succinate can effectively reduce the degree of cecal injury and inflammatory infiltration in the sepsis animal model.

[0040] In view of the fact that both diethyl succinate and dimethyl succinate have the effect of inhibiting the kinase activity of RIPK1, the present invention provides a composition for inhibiting the activity of RIPK1, comprising diethyl succinate and dimethyl succinate.

[0041] The present invention has no particular limitation on the mass ratio of diethyl succinate to dimethyl succinate, and preferably they are compounded in a mass ratio of 1:1.

[0042] The present invention provides use of the composition in preparing a RIPK1 inhibitor or a drug for preventing and / or treating inflammatory diseases.

[0043] The following is a detailed description of the use of diethyl succinate and its derivatives provided by the present invention in the preparation of RIPK1 kinase inhibitors and / or drugs for preventing and treating inflammatory diseases in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] The small molecule compound diethylsuccinate inhibits the necrosis of mouse L929 cells and human HT29 cells

[0046] 1. Cell culture: L929 cells and TH29 cells were both derived from ATCC; cells were cultured in RPMI1640 medium mixed with 10% fetal bovine serum (Gibco), 100 U / mL penicillin and 100 μg / mL streptomycin. All cells were cultured in a saturated humidity cell culture incubator at 37°C and 5% CO2.

[0047] 2. Compound treatment: The concentration of succinate and diethylsuccinate in treating cells was 5 μM; the concentration of Nec-1 compound (purchased from Selleck) in treating cells was 20 μM.

[0048] 3. Induction of cell necrosis: In L929 cells, cell necrosis was induced by TNF (10 ng / mL), zVAD (20 μM), and TNF (10 ng / mL) + zVAD (20 μM), and the cells were treated for 12 h. In HT29 cells, cell necrosis was induced by TNF (30 ng / mL) + SM164 (1 μM) + zVAD (20 μM), and the cells were treated for 12 h.

[0049] 4. Cell necrosis detection: The amount of LDH released into the culture medium was detected by the LDH kit (purchased from Promega) to determine the cell death. The simple operation is as follows: after cell treatment, 30 μL of cell supernatant was collected, 30 μL of LDH substrate was added to a 96-well flat-bottom plate, and the reaction was allowed to proceed for 20 minutes. The reaction was terminated by adding 30 μL of stop solution, and the OD was detected. 490nm The numerical value of .

[0050] HT29 cells or L929 cells were seeded in a 96-well plate. After overnight, the complete medium was replaced with OPTI medium without phenol red. The cells were pretreated with compounds succinate (or ester), diethyl succinate, and Nec-1 for 1 hour, and then necrosis was induced in the cells. Then, the necrosis of the cells was detected 12 hours later.

[0051] The results are as follows Figure 1 As shown, in L929 cells, diethyl succinate can significantly inhibit cell necrosis induced by TNF, zVAD and TNF+zVAD, but succinate (or ester) cannot. The positive control Nec-1 can also completely inhibit cell necrosis.

[0052] The results are as follows Figure 2 As shown, in HT29 cells, diethyl succinate can significantly inhibit TNF+SM164+zVAD-induced cell necrosis, but succinate (or ester) cannot, and the positive control Nec-1 can also completely inhibit cell necrosis.

[0053] In summary: The small molecule compound diethyl succinate can significantly inhibit cell necrosis in humans and mice.

[0054] Example 2

[0055] The small molecule compound diethylsuccinate inhibits the necrosis of mouse macrophages and BMDM cells

[0056] Wild-type C57 mice were injected with 3% FTG, and primary mouse peritoneal macrophages (PMs) were isolated three days later. The cells were pretreated with diethylsuccinate and Nec-1 for 3 h, respectively, and then cell necrosis was induced by LPS, polyI:C and TNF, respectively, and cell death was detected.

[0057] Results Figure 3 The results showed that diethylsuccinate could significantly inhibit cell necrosis in PMs cells.

[0058] Primary BMDM cells were differentiated by M-CSF induction. After being treated with SA succinate, DSA diethyl succinate, and DMSA dimethylsuccinate for 3 h, the cells were induced to undergo pyroptosis by LPS + 5z7. The secretion of cytokine IL-1b in the cell supernatant and cell death were detected.

[0059] Results Figure 4 The results showed that DSA and DMSA could significantly inhibit cell pyroptosis and the secretion of inflammatory factor IL-1b.

[0060] Example 3

[0061] Small molecule compounds diethyl succinate and dimethyl succinate inhibit RIPK1 kinase activity

[0062] 1 Cell culture: 293T cells were obtained from ATCC and cultured in DMEM medium mixed with 10% fetal bovine serum (Gibco), 100 U / mL penicillin and 100 μg / mL streptomycin. All cells were cultured in a saturated humidity cell culture incubator at 37°C and 5% CO2.

[0063] 2. Compound treatment: The concentration of succinate (or ester), diethyl succinate and dimethyl succinate in treating cells was 5 μM; the concentration of Nec-1 compound (purchased from selleck) in treating cells was 20 μM.

[0064] 3. 293T cells were transfected with pcDNA-RIPK1-HA plasmid (NCBI accession number of RIPK1 gene: NP_001341859.1) using lipo2000. After 24 hours, the cells were treated with compounds (succinate (or ester), diethyl succinate, dimethyl succinate and Nec-1) for 2 hours. A blank control group (cells without any treatment) was set up at the same time. RIPK1 activity was analyzed by Western blot. The specific method is as follows:

[0065] Total protein was extracted using RIPA buffer (1% TritonX-100, 50mMTris-HCl pH 7.2, 0.1% SDS and 1mMEDTA, 150mMNaCl, 1% sodium deoxycholate). Quantification was performed using the BCA method, and 20μg to 25μg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the protein was transferred to a 0.45μM nitrocellulose membrane. The membrane was soaked in TBST buffer containing 5% skim milk for about 1h and incubated with antibodies against pRIPK1 (S166) (CST) or RIPK1 (BD)) at 4°C overnight. After washing with TBST, the membrane was then incubated with horseradish peroxidase (HRP) goat anti-rabbit IgG antibody or goat anti-mouse IgG antibody at room temperature and developed in a dark room.

[0066] The results are as follows Figure 5 As shown, compared with the blank control group, diethyl succinate and dimethyl succinate can significantly inhibit the self-activation of RIPK1, among which Nec-1 as a positive control can also inhibit the self-activation of RIPK1.

[0067] Example 4

[0068] Diethyl succinate improves TNF-mediated sepsis phenotype in animals

[0069] 1TNF-mediated sepsis animal model:

[0070] Model group: Select 6-8 weeks old SPF wild-type C57BL / 6 female mice (purchased from Jicui Pharmaceutical Company), and inject PBS at a dose of 360 mg / kg intraperitoneally. After 30 minutes, inject TNF recombinant protein intravenously, and detect the survival of mice every 6 hours. When the body temperature of mice drops significantly, the mice are in a depressed state, move slowly, and die in severe cases. By testing the blood liver function indicators ALT and AST, it can be seen that the blood inflammatory factors are upregulated, as well as cecal damage and inflammatory infiltration, which indicates that the animal model of sepsis has been successfully established.

[0071] Experimental group: SPF wild-type C57BL / 6 female mice aged 6-8 weeks were selected and intraperitoneally injected with 360 mg / kg of diethyl succinate. 30 minutes later, 5 μg / mouse mTNF recombinant protein was injected through the tail vein. The survival of the mice was monitored every 6 hours.

[0072] 2HE staining: 12 hours after the TNF-mediated sepsis model was established, the cecum of the mice was collected for tissue section staining. The general process was dewaxing to water - hematoxylin staining - eosin staining - dehydration and sealing - microscopic examination.

[0073] The TNF-mediated sepsis model was established by injecting diethyl succinate into mice and observing the survival of the mice; the cecum of the mice was collected 12 hours later to analyze its tissue damage and inflammatory infiltration. Aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and alanine aminotransferase (ALT) were detected using the kit method.

[0074] Depend on Figure 6 It can be seen that diethyl succinate can significantly improve the survival of TNF-mediated sepsis.

[0075] Depend on Figure 7 It can be seen that diethyl succinate can improve cecal damage and inflammatory infiltration in the sepsis model.

[0076] Depend on Figure 8 As shown, diethyl succinate can alleviate the decrease in body temperature and improve tissue damage (LDH, ALT, AST) in mice.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of diethyl succinate in the preparation of a medicament for preventing and / or treating sepsis, wherein the prevention and / or treatment of sepsis comprises inhibiting RIPK1 activity and / or inhibiting cell necrosis, apoptosis or pyroptosis.

2. The application according to claim 1, characterized in that: The types of cells include somatic cells, immune cells and cancer cells; The cells include those of human and / or animal origin.

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