Application of small molecule compound in preparation of pyroptosis inhibitor

Abstract

The invention discloses application of a small molecule compound in preparation of a pyroptosis inhibitor. The chemical name of the small molecule compound is isopropyl-2-(2, 2-dichloroacetamido)-5-(diethylaminoformyl)-4-methylthiophene-3-carboxylic acid diethyl ester, and the short name is C9. According to the invention, the pyroptosis model for highly simulating the in-vivo environment is constructed, and the model can accurately reflect the biological change of the cells in the pyroptosis process. Through multiple rounds of screening and verification, the small molecule compound C9 is excellent in performance in a test and shows excellent pyroptosis inhibition performance. Through an MST experiment, it is clear that good affinity and high specificity exist between the ninth compound and the target protein.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of a small molecule compound in the preparation of a cell pyroptosis inhibitor. Background Art

[0002] Pyroptosis is a form of inflammatory programmed cell death, characterized by the activation of the Gasdermin family (mainly Gasdermin D), the formation of 10-14nm pores in the plasma membrane, and the release of inflammatory factors such as IL-1β and IL-18, which cause an inflammatory cascade reaction. Inflammatory cysteine ​​proteases cut Gasdermin D (GSDMD) into two domains, the N-terminal and C-terminal, of which the N-terminal domain (GSDMD-N) undergoes oligomerization and binds to lipids to form membrane pores.

[0003] A large number of studies have shown that pyroptosis is widely involved in the occurrence and development of infectious diseases, neurodegenerative diseases, cardiovascular diseases, cancer and other diseases, especially playing an important role in Parkinson's syndrome, Alzheimer's disease, atherosclerosis and other diseases. Therefore, the development of an efficient pyroptosis inhibitor is of great significance for the prevention and treatment of related diseases.

[0004] At present, a variety of cell pyroptosis inhibitors have been reported, which mainly inhibit the occurrence of cell pyroptosis by inhibiting the activity of the Gasdermin family (mainly Gasdermin D), blocking upstream signaling pathways or regulating immune responses. However, these inhibitors still face many challenges in clinical application, such as poor affinity and K D (Equilibrium dissociation constant) is generally in the micromolar level; lack of specificity, simultaneous inhibition of multiple cell death modes, leading to increased side effects. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides an application of a small molecule compound in the preparation of a cell pyroptosis inhibitor.

[0006] Our research group has constructed a cell pyroptosis model that highly simulates the in vivo environment. This model can accurately reflect the biological changes of cells during pyroptosis. After multiple rounds of screening and verification, the small molecule compound numbered 9 performed well in the test and showed excellent pyroptosis inhibition performance.

[0007] To further verify the potential of C9, MST (microscale thermophoresis) technology was used to conduct an in-depth analysis of its binding affinity and specificity with specific target proteins. MST technology provides accurate and reliable molecular interaction data with its high sensitivity, no labeling and real-time detection. Through MST experiments, it was confirmed that there is good affinity and high specificity between compound 9 and the target protein.

[0008] The small molecule compound of the present invention has a chemical name of isopropyl-2-(2,2-dichloroacetamido)-5-(diethylcarbamoyl)-4-methylthiothiophene-3-carboxylic acid diethyl ester, abbreviated as C9, and its structure is shown below:

[0009] .

[0010] Application of the small molecule compound of the present invention in the preparation of cell pyroptosis inhibitors.

[0011] The cell pyroptosis inhibitor can target GSDMD and has high affinity and strong specific binding.

[0012] The small molecule compound C9 involved in the present invention has good cell pyroptosis inhibitory activity and high GSDMD protein specificity, and can be used as a superior lead compound of cell pyroptosis inhibitor for further treatment and detection of related drug modification.

[0013] The small molecule compound C9 of the present invention has no biomedical application before. C9 exerts a stronger inhibitory activity against pyroptosis than the reported pyroptosis inhibitor disulfiram. Currently, there is no report that C9 can inhibit cell pyroptosis, and there is a lack of specific cell pyroptosis drugs in clinical practice.

[0014] To further verify the potential of C9, MST (microscale thermophoresis) technology was used to conduct an in-depth analysis of its binding affinity and specificity with specific target proteins. MST technology provides accurate and reliable molecular interaction data with its high sensitivity, no labeling and real-time detection. Through MST experiments, it was confirmed that there is good affinity and high specificity between the C9 compound and the target protein.

[0015] The beneficial effects of the present invention are embodied in:

[0016] The present invention provides a C9 small molecule compound that can be used as a cell pyroptosis inhibitor and as a lead compound with high affinity and strong specificity to the GSDMD protein for further drug modification. Specifically, the application of C9 in inhibiting cell pyroptosis and the application of C9 in cell pyroptosis imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1This is a classic cell pyroptosis pathway model constructed using THP-1. THP-1 cells were stimulated with lipopolysaccharide (LPS), incubated with C9 (10 μM) and DIS (10 μM) before nigericin induction, and then the lactate dehydrogenase content in the culture medium was measured to analyze cell death.

[0018] Figure 2 The results of propidium iodide (PI) uptake to assess membrane permeability in the THP-1 cell pyroptosis model. Scale bar, 100 μm.

[0019] Figure 3 This is a necroptosis model constructed by HT-29 cells. HT-29 cells were pretreated with C9 (10 μM) or NSA (10 μM) for 1 h, and then stimulated with TNFα (25 ng / ml) (T), 400 nM SM-164 (S), and 20 μM Z-VAD-FMK (Z) for 24 h, respectively. Cell viability was detected by lactate dehydrogenase method (left) and CTG method to measure ATP (right).

[0020] Figure 4 This is a bright field observation of the necroptosis model constructed by HT-29 cells. Scale bars, 4x, 500 μm; 10x, 200 μm; 20x, 100 μm; 40x, 50 μm.

[0021] Figure 5 This is the MST curve of recombinant human GSDMD protein (20 nM) and C9.

[0022] Figure 6 This is the MST curve of recombinant human GSDMD protein (20 nM) and DMF (dimethyl fumarate).

[0023] Figure 7 is the K of the MST experiment D Comparison of curves (C9, green curve; DMF, blue curve). Bar graphs are means ± SEM, n = 3. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further analyzed and explained below through specific embodiments.

[0025] The C9 used in the following examples was obtained from the SPECS compound library with the catalog number AK-968 / 40370365.

[0026] Example 1: C9 can inhibit cell pyroptosis damage in the classic pyroptosis model of macrophages

[0027] 1. Induce THP-1 cells with 150 nM phorbol methyl ester (PMA) for 18 hours to differentiate them into adherent macrophages.

[0028] 2. Remove the culture medium and wash once with PBS. Induce with fresh culture medium containing 1 μg / mL lipopolysaccharide (LPS) for 4 hours (using low-serum culture medium containing 1% serum), induce with 10 μM Nigericin for 30 minutes, and divide each culture well into the following groups: culture medium wells without cells (background blank control wells), cell wells without drug treatment for subsequent lysis (sample maximum enzyme activity control wells), control cell wells without drug treatment (LPS), pyroptosis group (LPS+Nigericin), drug group (LPS+Nigericin+C9), positive molecule control group (LPS+Nigericin+disulfiram) and mark them.

[0029] Lactate dehydrogenase release assay:

[0030] (1) After the above cell model reaches the predetermined time, centrifuge the cell culture plate at 400g for 3 minutes using a multi-well plate centrifuge. Take 120 μL of the supernatant from each well and add it to the corresponding well of a new 96-well plate.

[0031] (2) Add 60 μL of LDH detection working solution to each well, mix well, and incubate at room temperature (about 25°C) in the dark for 30 minutes. Then measure the absorbance at 490 nm. Use 600 nm as the reference wavelength for dual wavelength measurement.

[0032] (3) Calculation (the absorbance of each group should be subtracted from the absorbance of the background blank control well).

[0033] (4) Cell mortality rate (%) = (absorbance of treated sample - absorbance of sample control well) / (absorbance of maximum enzyme activity of cells - absorbance of sample control well) × 100.

[0034] Propidium iodide (PI) uptake to assess cell membrane permeability:

[0035] 50 μg / mL propidium iodide (PI) was added to the 96-well plate from which the supernatant was taken for staining, and the images were recorded using an inverted fluorescence microscope OLYMPUS IX73.

[0036] The results of lactate dehydrogenase assay showed that ( Figure 1), C9 group and DIS group (disulfiram) significantly reduced the release of lactate dehydrogenase in THP-1 cells induced by PMA, and C9 had a better inhibitory effect than disulfiram. Propidium iodide uptake experiment ( Figure 2 ) also proved that C9 can significantly inhibit the damage of cell membrane caused by cell pyroptosis.

[0037] Example 2: This example explores the specificity of C9 on cell pyroptosis

[0038] A necroptosis model was constructed using human colon cancer HT-29 cells. After pretreatment with C9 (10 μM) or NSA (10 μM) for 1 h, HT-29 cells were stimulated with TNF-α (25 ng / ml), SM-164 (400 nM), and Z-VAD-FMK (20 μM) for 24 h. Necrosulfonamide, which has an inhibitory effect on necroptosis, was used as a reference to measure the release of lactate dehydrogenase and the amount of intracellular ATP, and the cell state was recorded in bright field using an inverted fluorescence microscope OLYMPUS IX73.

[0039] Lactate dehydrogenase ( Figure 3 Left) and ATP ( Figure 3 Right) The results showed that there was no difference between the C9 group (T+Z+S+C9) and the apoptosis group (T+Z+S), while the NSA group (T+Z+S+NSA) significantly inhibited necroptosis and cell status ( Figure 4 ) showed that the cell membranes of the apoptosis group (T+Z+S) and the C9 group (T+Z+S+C9) were broken and incomplete, while the cell boundaries of the NSA group (T+Z+S+NSA) were clear and the cell membranes were transparent, indicating that they were in a good state. Ultimately, it was shown that C9 had no inhibitory effect on necroptosis, reflecting the specificity of inhibiting cell pyroptosis.

[0040] Example 3: This example explores the high affinity between C9 and GSDMD protein

[0041] The binding affinity of C9 to GSDMD was evaluated by microscale thermophoresis (MST). First, the protein was labeled using the RED-NHS protein fluorescent labeling kit, and the labeled GSDMD protein with a concentration of 2 μM was diluted with PBST buffer to a working concentration of 20 nM. The 10 mM DMSO solution of C9 was diluted with PBST buffer to prepare a series of concentration gradients of C9 and positive molecule dimethyl fumarate samples (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625, 0.048828125, 0.0244140625, 0.01220703125, 0.006103515625 μM). C9 was diluted to the specified concentration and incubated with the labeled GSDMD protein in PBST assay buffer (containing 300 mM NaCl, 0.5‰ Tween 20) at pH 7.4 for 5 min. The samples were loaded into capillaries and MST was performed using Monolith NT.115. K was calculated using the mass action equation using MO Affinity Analysis V2.1.3 software. D value.

[0042] According to the relevant data analysis of MST experiment (C9 and dimethyl fumarate are Figure 5 , Figure 6 , Figure 7 The green curve on the left is C9. Figure 7 The blue curve on the right is dimethyl fumarate. C9 binds to recombinant human GSDMD protein with a high affinity (K D =0.737± 0.329μM), which was superior to the GSDMD inhibitor dimethyl fumarate (21.75±14.83μM).

Claims

1. Use of a small molecule compound in the preparation of a cell pyroptosis inhibitor, characterized in that: The chemical name of the small molecule compound is isopropyl-2-(2,2-dichloroacetamido)-5-(diethylcarbamoyl)-4-methylthiothiophene-3-carboxylic acid diethyl ester, abbreviated as C9, and its structure is shown below: 。 2. The use according to claim 1, characterized in that: The pyroptosis inhibitor specifically targets GSDMD.

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