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. The pyroptosis model capable of highly simulating the in-vivo environment is constructed based on the human cell line and the mouse primary cells, and the model can accurately reflect the biological change of the cells in the pyroptosis process. Through multiple rounds of screening and verification, C9 is excellent in performance in a test and shows excellent pyroptosis inhibition performance. Through MST and SPR experiments, good affinity and high specificity between the C9 and the target protein are determined. Finally, a disease model proves that C9 can effectively relieve key pathological injury and inflammatory response of mouse acute sepsis and mouse colitis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of a small molecule compound in the preparation of pyroptosis inhibitors. Background Technology

[0002] Pyroptosis is a form of inflammatory programmed cell death characterized by the activation of the Gasdermin family (primarily Gasdermin D), forming 10-14 nm pores in the plasma membrane and releasing inflammatory factors, primarily IL-1β and IL-18, triggering an amplified inflammatory cascade. Inflammatory cysteine ​​proteases cleave Gasdermin D (GSDMD) into N-terminal and C-terminal domains. The N-terminal domain (GSDMD-N) oligomerizes and binds to lipids to form membrane pores.

[0003] Numerous studies have shown that pyroptosis is widely involved in the development and progression of infectious diseases, neurodegenerative diseases, cardiovascular diseases, and cancers, playing a particularly important role in diseases such as Parkinson's disease, Alzheimer's disease, and atherosclerosis. Therefore, developing a highly effective pyroptosis inhibitor is of great significance for the prevention and treatment of these diseases.

[0004] Several pyroptosis inhibitors have been reported, primarily inhibiting pyroptosis by suppressing the activity of the Gasdermin family (mainly Gasdermin D), blocking upstream signaling pathways, or modulating immune responses. However, these inhibitors still face many challenges in clinical application, including poor affinity and K... D The equilibrium dissociation constant is generally in the micromolar range; however, it lacks specificity and inhibits multiple cell death pathways, leading to increased side effects. Summary of the Invention

[0005] To address the shortcomings of the prior art, this invention provides an application of small molecule compounds in the preparation of pyroptosis inhibitors.

[0006] Our research group constructed a pyroptosis model that highly simulates the in vivo environment using human cell lines and primary mouse cells. This model can accurately reflect the biological changes in cells during pyroptosis. After multiple rounds of screening and validation, the small molecule compound numbered 9 (hereinafter referred to as C9) performed excellently in the tests, demonstrating outstanding and specific pyroptosis inhibitory properties.

[0007] To further validate the potential of C9, MST (microthermophoresis) and SPR (surface plasmon resonance) techniques were employed to conduct in-depth analyses of its binding affinity and specificity with specific target proteins. SPR is considered the gold standard in molecular interactions, and it was officially included in the Chinese Pharmacopoeia in 2020. MST and SPR techniques provided accurate and reliable molecular interaction data, and experiments clearly demonstrated the excellent affinity and high specificity between C9 and its target proteins.

[0008] To verify the therapeutic potential of C9 at the in vivo level, we evaluated its efficacy in LPS-induced acute sepsis and DSS-induced colitis models in mice. The results showed that C9 effectively alleviated key pathological damage and inflammatory responses in the model animals, thus exerting a significant therapeutic effect on the related diseases.

[0009] The small molecule compound of this invention, chemically named isopropyl-2-(2,2-dichloroacetamido)-5-(diethylcarbamoyl)-4-methylthiothiophene-3-carboxylic acid diethyl ester, abbreviated as C9, has the following structure:

[0010] .

[0011] The application of the small molecule compound of this invention in the preparation of pyroptosis inhibitors.

[0012] The pyroptosis inhibitor can target GSDMD and has high affinity and strong specificity.

[0013] The application of the small molecule compounds of this invention in the preparation of pharmaceutical formulations for treating sepsis.

[0014] The application of the small molecule compound of the present invention in the preparation of pharmaceutical formulations for treating colitis.

[0015] The small molecule C9 involved in this invention has good pyroptosis inhibitory activity and high GSDMD protein specificity, and can be used as a leading compound for pyroptosis inhibitors for further treatment and detection of related drug modifications.

[0016] The small molecule compound C9 of this invention has no prior biomedical applications. C9 exhibits stronger pyroptosis inhibitory activity than disulfiram (DIS), a previously reported pyroptosis inhibitor. Currently, there are no reports of C9 inhibiting pyroptosis, and there is a lack of drugs specifically inhibiting pyroptosis in clinical practice.

[0017] The beneficial effects of this invention are reflected in:

[0018] This invention provides a small molecule compound C9 that can be used as a pyroptosis inhibitor and as a lead compound with high affinity and strong specificity for GSDMD protein for further drug modification. It effectively alleviates key pathological damage and inflammatory response in mice with acute sepsis and colitis, thereby exerting a significant therapeutic effect on related diseases. Attached Figure Description

[0019] Figure 1 This study established (A) a classical pyroptosis pathway model using human monocytic leukemia (THP-1) and (B) mouse bone marrow-derived macrophages (BMDM). THP-1 cells treated with BMDM and phorbol ester were stimulated with 1 μg / mL LPS for 4 hours, then incubated with C9 (10 μM) and DIS (10 μM) for 30 minutes. After induction with 10 μM Nigericin for 3 hours, the lactate dehydrogenase content in the culture medium was measured to analyze cell death. Bar charts are presented as Mean ± SEM, n = 3. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (Note: The last sentence appears to be incomplete and may be a fragment from a different context. It has been left as is.)

[0020] Figure 2 This is the result of assessing membrane permeability in a THP-1 cell pyroptosis model using propidium iodide (PI) uptake. Scale bar, 100 μm.

[0021] Figure 3 This study established a necrotizing apoptosis model using human colon cancer cells (HT-29). HT-29 cells were pretreated with C9 (10 μM) or the positive molecule NSA (10 μM) for 1 h, followed by stimulation with TNFα (25 ng / ml) (T), 400 nM SM-164 (S), and 20 μM Z-VAD-fmk (Z) for 24 h. Cell viability was assessed using (A) the lactate dehydrogenase method and (B) the CTG method. Bar charts are presented as Mean ± SEM, n = 3. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (Note: The last sentence appears to be incomplete and possibly contains errors. It can be omitted from the translation.)

[0022] Figure 4 This is a bright-field observation of a necrotizing apoptosis model constructed from HT-29 cells. Scale bars: 4×, 500 μm; 10×, 200 μm; 20×, 100 μm; 40×, 50 μm.

[0023] Figure 5The interaction between the compound and the GSDMD protein was detected using micro-thermophoresis (MST). A is the MST curve of recombinant human GSDMD protein (20 nM) and C9, B is the MST curve of recombinant human GSDMD protein (20 nM) and the positive molecule DMF (dimethyl fumarate), and C is a direct comparison of the equilibrium dissociation constant (KD) curves fitted based on the MST data (C9, green curve; DMF, blue curve).

[0024] Figure 6 This is a SPR sensing image of the interaction between C9 and the protein GSDMD. Figures A and B represent the actual sensing images of the binding of different concentrations of analyte C9 to the immobilized protein GSDMD on the chip surface; Figure C is the result of a global fit using the instrument's accompanying analysis software (such as BiacoreEvaluation Software) based on a 1:1 Langmuir binding model. The equilibrium dissociation constant (KD) calculated through fitting is 3.7 μM.

[0025] Figure 7 This is the survival curve of mice in different treatment groups in an LPS-induced mouse sepsis model. DIS indicates the positive molecule disulfiram.

[0026] Figure 8 The concentrations of inflammatory cytokines TNF-α (A), IL-1β (B), and IL-6 (C) in the spleen of mice in each group of a mouse sepsis model were measured using ELISA. D represents the spleen index (spleen weight to body weight) of each group of mice. The bar chart is presented as Mean ± SEM, n = 5. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (ns, not significant).

[0027] Figure 9 Figure A shows the body weight change curves of mice in each group with DSS-induced experimental colitis throughout the experiment (based on initial body weight); Figure B shows the disease activity index (DAI) scores of each group of mice.

[0028] Figure 10 Figure A shows the statistical results of colon length (in cm) for each group of mice at the end of the experiment; Figure BD shows the levels of key pro-inflammatory cytokines (B, TNF-α; C, IL-1β; D, IL-6) in colon tissue detected by ELISA. The bar charts represent Mean ± SEM, n = 6. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (ns, not significant).

[0029] Figure 11 These are gross photographs of the colons of representative mice from each group.

[0030] Figure 12 These are the results of hematoxylin and eosin (HE) staining of the colons of mice in each group. Detailed Implementation

[0031] The technical solution of the present invention will be further analyzed and explained through specific embodiments below.

[0032] The C9 used in the following examples was obtained from the SPECS compound library, catalog number AK-968 / 40370365.

[0033] Example 1: C9 can inhibit pyroptosis damage in a classic macrophage pyroptosis model.

[0034] 1. THP-1 cells were induced to differentiate into adherent macrophages by 150 nM phorbol ester (PMA) for 18 hours.

[0035] 2. Aspirate the culture medium and wash once with PBS. Induce for 4 hours with fresh culture medium containing lipopolysaccharide LPS (1 μg / mL) (using low-serum culture medium containing 1% serum), then induce for 30 minutes with 10 μM Nigericin. Divide the culture wells into the following groups: cell-free culture medium wells (background blank control wells), untreated cell wells for subsequent lysis (sample maximum enzyme activity control wells), untreated control cell wells (LPS), pyroptosis group (LPS+Nigericin), drug-treated group (LPS+Nigericin+C9), and positive molecule control group (LPS+Nigericin+disulfiram), and label them accordingly.

[0036] Lactate dehydrogenase release assay:

[0037] (1) After the cell model reaches the predetermined time, the cell culture plate is centrifuged 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.

[0038] (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 wavelength as the reference wavelength for dual-wavelength measurement.

[0039] (3) Calculation (the absorbance of each group should be subtracted from the absorbance of the background blank control well).

[0040] (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.

[0041] Assessing cell membrane permeability using propidium iodide (PI) uptake:

[0042] Add 50 μg / mL propidium iodide (PI) to the 96-well plate containing the supernatant and stain. Record the staining using an inverted fluorescence microscope OLYMPUS IX73.

[0043] The results of the lactate dehydrogenase (LDH) release assay showed that ( Figure 1 In PMA-induced differentiated THP-1 and BMDM cells, both the C9 treatment group and the positive control disulfiram (DIS) treatment group significantly inhibited LDH release, and C9 showed stronger inhibitory activity than disulfiram. Further propidium iodide (PI) uptake experiments showed… Figure 2 This indicates that C9 can effectively maintain cell membrane integrity and significantly inhibit the increase in membrane permeability caused by pyroptosis.

[0044] Example 2: This example investigates the specificity of C9 on pyroptosis.

[0045] A necroptosis model was established using human colon cancer HT-29 cells. HT-29 cells were pretreated with C9 (10 μM) or NSA (10 μM) for 1 h, and then stimulated with TNF-α (25 ng / ml), SM-164 (400 nM), and Z-VAD-FMK (20 μM) for 24 h. Necrosulfonamide, which inhibits necroptosis, was used as a control. The release of lactate dehydrogenase and the amount of intracellular ATP were measured, and the cell state was recorded using an inverted fluorescence microscope OLYMPUS IX73 in bright field.

[0046] lactate dehydrogenase (LDH) release ( Figure 3 A) and intracellular ATP levels ( Figure 3 B) The test results showed that there was no significant difference between the C9 treatment group (T+Z+S+C9) and the necrosis-apoptosis model group (T+Z+S), while the necrosis-apoptosis-specific inhibitor NSA treatment group (T+Z+S+NSA) significantly inhibited LDH release and maintained ATP levels. Cell morphology observation results ( Figure 4 Further analysis showed that the cell membranes in the model group and the C9-treated group were ruptured and morphologically incomplete, while the cells in the NSA-treated group had clear boundaries, intact membrane structures, and were in significantly better condition. These results collectively indicate that C9 has no inhibitory effect on the necroptotic pathway, further demonstrating its specificity in inhibiting pyroptosis.

[0047] Example 3: Quantitative assessment of the non-covalent binding affinity between C9 and GSDMD using microscale thermophoresis (MST).

[0048] The binding affinity of C9 to GSDMD was assessed using microthermophoresis (MST). First, the protein was labeled using the RED-NHS protein fluorescent labeling kit. The labeled 2 μM GSDMD protein was diluted with PBST buffer to a working concentration of 20 nM. A series of concentration gradients of C9 and the positive molecule dimethyl fumarate were prepared using PBST buffer to dilute the 10 mM DMSO solution. These concentrations were 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, and 0.006103515625 μM. C9 was diluted to the specified concentration and incubated with labeled GSDMD protein in PBST experimental buffer (containing 300 mM NaCl, 0.5‰ Tween 20) at pH 7.4 for 5 minutes. The sample was loaded into capillary tubes and MST was detected using a Monolith NT.115. K was calculated using the mass action equation with MO Affinity Analysis V2.1.3 software. D value.

[0049] Based on the analysis of relevant data from the MST experiment (C9 and dimethyl fumarate were respectively...), Figure 5 A, 5B; 5C (the green curve on the left is C9, and the blue curve on the right is dimethyl fumarate). C9 interacts with recombinant human GSDMD protein with a high affinity (K... D The binding of the drug (0.737 ± 0.329 μM) was superior to that of the GSDMD inhibitor dimethyl fumarate (21.75 ± 14.83 μM).

[0050] Example 4: Quantitative assessment of the non-covalent binding affinity between C9 and GSDMD using surface plasmon resonance (SPR).

[0051] Surface plasmon resonance (SPR) experiments were performed using the Biacore 8K molecular interaction analysis system (Cytiva). Recombinant full-length human GSDMD biotinylated protein was immobilized on the channels of a Series S Sensor Chip SA chip according to the biotin-streptavidin capture principle and the manufacturer's instructions.

[0052] The analyte, small molecule C9, was serially diluted using run buffer (containing 5% DMSO) at concentration gradients from 1.5625 µM to 100 µM. Binding (60 sec) and dissociation (180 sec) processes were monitored in real-time using a multi-cycle kinetic assay. The chip surface was regenerated after each cycle to prepare for the next concentration determination.

[0053] After reference channel subtraction and baseline correction, the sensor map was globally fitted using a 1:1 Langmuir combined model to calculate the equilibrium dissociation constant (KD value) of the interaction between analyte C9 and GSDMD.

[0054] When the C9 solution flowed across the chip surface, typical real-time binding and dissociation processes were observed. For example... Figure 6 As shown in Figure AB, the sensor plots exhibit a clear concentration-dependent binding signal; that is, the binding response value (RU) increases with increasing analyte concentration, and the dissociation phase is smooth, indicating good compound binding specificity. After reference subtraction and baseline correction, the obtained sensor plots were globally fitted using a 1:1 Langmuir binding model in Biacore Evaluation Software to calculate kinetic parameters. Figure 6 As shown in Figure C, the equilibrium dissociation constant (KD) of C9 is 3.7 μM. This KD value indicates that C9 has a moderate affinity for the GSDMD protein.

[0055] Example 5: This example explores the therapeutic effect of C9 on sepsis.

[0056] Male C57BL / 6J mice weighing 18–22 g were intraperitoneally injected with lipopolysaccharide (LPS) (10 mg / kg) and administered C9 (5 mg / kg, 10 mg / kg) or disulfiram (50 mg / kg). In the survival study, 10 mice were in each group, and monitoring was performed every 12 hours after administration, with the endpoint at 96 hours. Cytokine detection: In a separate group of 5 mice, spleens were harvested 5 hours after LPS stimulation, and cytokine concentrations were measured using ELISA. The spleen index was calculated using the formula: Spleen index (g / g) = Spleen mass / Body weight × 100%.

[0057] like Figure 7 As shown, compared with the untreated model group, C9 combined with disulfiram treatment significantly improved the survival rate of septicemia mice. Furthermore, as... Figure 8 As shown in the AC, after C9 treatment, the levels of key pro-inflammatory factors in the spleen changed significantly: the levels of TNF-α, IL-1β, and IL-6 were significantly reduced, indicating that C9 can effectively inhibit the inflammatory response induced by sepsis.

[0058] Analysis results of spleen index ( Figure 8 D) showed that C9 treatment significantly alleviated splenomegaly caused by LPS, further confirming the ameliorative effect of C9 on sepsis-related pathological changes.

[0059] In conclusion, in the LPS-induced mouse sepsis model, C9 treatment effectively improved the survival rate of mice and significantly inhibited systemic inflammatory response and alleviated key pathological features such as splenomegaly, indicating that it has significant therapeutic effects.

[0060] Example 6: This example explores the therapeutic effect of C9 on colitis.

[0061] Male C57BL / 6J mice weighing 18–22 g were randomly divided into 5 groups (n = 6). The control group had free access to water, while the other 4 groups received 3% sodium dextran sulfate (DSS, 36,000–50,000 MW) in drinking water for 6 days, followed by 5 days of normal drinking water. During the 5 days of normal drinking water, the 4 groups receiving DSS received daily intraperitoneal injections of the corresponding drugs (DSS group as control, C9 5 mg / kg group, C9 10 mg / kg group, and DIS 50 mg / kg group). Daily weight loss was recorded, and disease progression (DAI) was monitored throughout the experiment based on weight, fecal characteristics, and blood occult blood. Mice were sacrificed on day 11, and colons were collected for colon length measurement, HE staining analysis, cytokine analysis, and Western blotting.

[0062] like Figure 9 As shown in Figure A, the model group mice treated with DSS experienced a significant decrease in body weight, while C9 treatment alleviated this trend, indicating that it effectively improves the overall health of the animals. Simultaneously, the Disease Activity Index (DAI), calculated based on a combination of body weight, fecal characteristics, and fecal blood loss, showed (…). Figure 9 (B) The C9 treatment group scored lower than the model group, confirming that it has a certain effect on improving the clinical symptoms of colitis.

[0063] At the experimental endpoint, we assessed the pathological changes in the colon. For example... Figure 10 A and Figure 11 As shown, mice in the DSS model group exhibited a typical significant shortening of the colon, which was alleviated by C9 treatment. Furthermore, the results of detecting inflammatory factors in the colonic tissue ( Figure 10 (BD) results showed that C9 significantly inhibited the increase of key pro-inflammatory factors (TNF-α, IL-1β, IL-6) induced by DSS. The decrease in their levels suggests that C9 has an anti-inflammatory effect. HE staining results are as follows... Figure 12As shown, in the normal group, the mucosa and submucosa had only a small number of scattered inflammatory cells, the crypts (intestinal glands) were neatly and densely arranged with regular shapes, and the goblet cells were abundant, plump, and filled with mucus. The mucosal structure was intact, the epithelium was continuous, and there were no defects. In the 3% DSS colitis group, there was diffuse infiltration of a large number of inflammatory cells, the crypts were twisted, the number of goblet cells was significantly reduced, mucosal epithelial ulceration was formed, and the submucosa was widened due to edema. In the C9 5mg / kg group, a large number of inflammatory cells were observed, but the number of goblet cells was increased compared with the colitis group, and the mucosa was relatively intact, indicating that it has a certain therapeutic effect on the disease. The C9 10mg / kg group and the DIS 50mg / kg group significantly reduced the number of inflammatory cells, the crypts were neatly arranged, the number of goblet cells was abundant, and the mucosal structure was intact, indicating that it has a significant therapeutic effect on the disease.

[0064] The above results indicate that C9 has a significant therapeutic effect on the DSS-induced colitis model, and its effects are reflected in reversing weight loss, reducing the disease activity index, improving colonic shortening, and effectively inhibiting intestinal inflammatory response and repairing key damage such as crypt structure at the histopathological level.

Claims

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

Images