A sesquiterpenoid compound, a preparation method thereof and application thereof in preparing a medicine for treating psoriasis

Abstract

The application discloses a sesquiterpenoid compound and a preparation method and application thereof in preparing a medicine for treating psoriasis, and belongs to the technical field of biological medicines. The sesquiterpenoid compound comprises compounds 1-4, and the structural formulae are as follows: the application provides a new molecular template for developing a new medicine for treating psoriasis, and provides a new thought for further developing a new treatment strategy with an immune regulation function.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a sesquiterpenoid compound, a preparation method thereof and application thereof in preparing a medicine for treating psoriasis. BACKGROUND

[0002] Psoriasis is a common chronic, systemic, inflammatory, immune-mediated disease with high incidence, long duration, unpredictable course and great harm, which seriously damages the physical health and quality of life of patients. According to the data of the World Health Organization, about 0.09%-11.43% of the global population suffers from psoriasis, which is one of the most common chronic skin diseases in the world. According to the epidemiological survey in China, the number of patients with psoriasis in China has reached 7 million, and is showing a trend of continuous increase. The typical histological features of psoriasis plaques include excessive proliferation of epidermal keratinocytes, and infiltration of dendritic cells, T lymphocytes, neutrophils and macrophages in the dermis. Traditional topical drugs for treating psoriasis, such as corticosteroids, calcineurin inhibitors, vitamin D analogues, anthralins and tar, are often limited in application due to their poor efficacy, large side effects and poor tolerance; therefore, the development of new psoriasis inhibitors with strong immune tolerance and small adverse reactions has become an urgent problem and research hotspot in clinical practice.

[0003] The exact pathogenesis of psoriasis is not completely clear, but studies have shown that genetic, immune, environmental and lifestyle factors play an important role in its pathogenesis. Current research suggests that the interaction of immune cells keratinocytes through various cytokines is the most important pathogenesis of psoriasis. In the development of psoriasis, abnormally activated dendritic cells secrete a large amount of cytokines such as IL-23 and TNF-α. IL-23 can initiate the formation of IL-23 / IL-17 axis by Th17 cells. Th17 cells play a key role in this process, and the IL-23 / IL-17 axis is considered to be another major immune pathway involved in the pathological process of psoriasis.

[0004] Two pro-inflammatory factors S100A8 (myeloid-related protein 8, MRP8, calgranulin A) S100A9 (MRP14, calgranulin A) are highly expressed in keratinocytes and leukocytes in psoriatic skin, but not in healthy human skin. The traditional view is that the heterodimer S100A8 / S100A9 (calprotectin) is released upon cellular stress or tissue damage and is only to be considered as a damage-associated pattern molecule (DAMP) or alarm molecule. However, recent studies have shown that S100A8 / A9 has a wide range of biological functions, including calcium homeostasis, cytoskeletal dynamics, inflammation regulation, energy metabolism and antibacterial properties. It has been reported that S100A8 / A9 is involved in the development of autoimmune-related diseases such as psoriasis, rheumatoid arthritis, systemic lupus erythematosus, etc. Inhibition of the production or release of S100A8 / A9 has shown significant therapeutic effect, which highlights the application potential of targeting S100A8 / A9 in the treatment of autoimmune diseases.

[0005] Aspergillus fungi are filamentous fungi that exist in different environments in nature and are widely distributed. Aspergillus has a rich variety of enzymes and can produce various types of physiologically active metabolites. For thousands of years, it has gradually become an important strain in many fields such as food fermentation, medicine, and agricultural production. The metabolic products of Aspergillus fungi have complexity, diversity and novelty in chemical structure, mainly including terpenes, alkaloids, polyketides, peptides and the like. These metabolites have various biological activities such as antibacterial, antitumor, antiviral, and antioxidant. Therefore, it is of great significance to find new active lead compounds in Aspergillus fungi. SUMMARY

[0006] The purpose of the present application is to provide a sesquiterpenoid compound and a preparation method thereof and application thereof in preparing a medicine for treating psoriasis, so as to solve the problems existing in the prior art.

[0007] To achieve the above-mentioned purpose, the present application provides the following solutions.

[0008] One of the technical solutions of the present application is a sesquiterpenoid compound, which comprises compounds 1-4, and the structural formulae are as follows:

[0009]

[0010] The second technical solution of the present application is a preparation method of the sesquiterpenoid compound, which comprises the following steps: fermenting rice by using Aspergillus undulatus to obtain a rice ferment; and extracting and separating the rice ferment to obtain the sesquiterpenoid compound.

[0011] The third technical solution of the present application is application of the sesquiterpenoid compound in preparing a medicine for treating psoriasis.

[0012] The fourth aspect of the present application is a medicine for treating psoriasis, comprising the sesquiterpenoid compound.

[0013] The fifth aspect of the present application is the use of the sesquiterpenoid compound in the preparation of an immunosuppressant.

[0014] The sixth aspect of the present application is the use of the sesquiterpenoid compound in the preparation of an S100A8 / A9 targeted inhibitor.

[0015] The seventh aspect of the present application is an S100A8 / A9 targeted inhibitor, comprising the sesquiterpenoid compound.

[0016] Based on the above technical solutions, the present application has the following technical effects:

[0017] (1) Compounds 1-4 have a tightly fused "6 / 5 / 5 / 6 / 5" ring system, a rare nested cyclopentadiene [cd] indene fragment, and the presence of a bridgehead double bond makes one of the five-membered ring atoms almost coplanar.

[0018] (2) Further biological activity evaluation results show that compound 2 can bind to S100A8 / A9 to inhibit its secretion, thereby having a significant therapeutic effect on psoriasis dermatitis.

[0019] (3) This kind of compound is the first natural product that can bind to S100A8 / A9 dimers to inhibit their secretion, and has great significance for the development of S100A8 / A9 targeted inhibitors.

[0020] The present inventors isolated and purified secondary metabolites from Aspergillus undulates to obtain four new compounds with a 6 / 5 / 5 / 6 / 5 five-ring ring system sesquiterpenoid compound, the specific structure of which is shown in formula (1). Through the evaluation of the psoriasis treatment activity of compounds 1-4, it was found that compounds 1-4 all have psoriasis treatment effect, especially compound 2. Omics results show that compound 2 can inhibit the TNF and IL-17 signaling pathways in HaCaT cells. PharmMapper prediction shows that the highest ranked inflammation-related target of compound 2 is the S100A8 / A9 protein, and the combination of the two is further confirmed by in vitro CETSA experiment and dynamic simulation. In the imiquimod-induced mouse psoriasis model, compounds 1 and 2 significantly alleviate the psoriasis dermatitis of mice, and immunohistochemistry shows that the expression of IL-17, Ki67 and S100A9 proteins is significantly reduced after administration. The present application provides a new molecular template for the development of new psoriasis treatment drugs, and provides a new idea for the further development of new treatment strategies with immunomodulatory effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 The crystal structure of compound 1.

[0023] Figure 2 The crystal structure of compound 2.

[0024] Figure 3 The crystal structure of compound 3.

[0025] Figure 4 This is a screening of the activity of compounds 1-4. (A): Compounds 1-4 showed inhibitory effects on hyperproliferative HaCaT cells. (B): Compounds 1-4 showed low cytotoxicity against normal HaCaT cells. (C): Compound 2 has a regulatory effect on the immune system.

[0026] Figure 5 Compound 2 inhibits genes associated with the TNF and IL-17 signaling pathways in HaCaT cells. (A): Principal component analysis (PCA) of RNA sequencing data, showing clustering of the control, model, and compound 2-treated groups. (BC): Volcano plots showing upregulated (red) and downregulated (green) genes in the control versus model group, and in the compound 2 (1.25 μM)-treated versus model group. Genes were considered downregulated when log2 (fold change) < -1.0 and -log10 (adjusted P value) > 1.0; genes were considered upregulated when log2 (fold change) > 1.0 and -log10 (adjusted P value) > 1.0. (DE): Comparison of differentially expressed genes (DEGs) between the control, model, and compound 2-treated groups. (F): Top 10 signaling pathways identified by KEGG pathway enrichment analysis for upregulated DEGs between the control and model groups. (G): The number of DEGs related to IL-17 and TNF signaling pathways in the control group, model group, and compound 2 (1.25 μM)-treated group. (H): Heat map showing the expression levels of inflammation-related genes in the control group, model group, and compound 2-treated group.

[0027] Figure 6Compound 1 inhibits the expression of inflammation-related proteins in HaCaT cells and blocks the activation of NF-κB. Among them, (A): Compound 1 inhibits the expression of inflammation-related proteins in HaCaT cells. (B): Standardization and quantification of protein band density and GAPDH band density. Data are presented as mean ± standard deviation. # p<0.05, compared with the blank control group; ***P<0.001, **P<0.01, *p<0.05, compared with the TNF-α model group. (C): Immunofluorescence results of compound 1 (scale bar = 50 μm).

[0028] Figure 7 Compound 2 inhibits the expression of inflammation-related proteins in HaCaT cells and blocks the activation of NF-κB. Among them, (A): Compound 2 inhibits the expression of inflammation-related proteins in HaCaT cells. (B): Standardization and quantification of protein band density and GAPDH band density. Data are presented as mean ± standard deviation. # p<0.05, compared with the blank control group; ***P<0.001, **P<0.01, *p<0.05, compared with the TNF-α model group. (C): Immunofluorescence results of compound 2 (scale bar = 50 μm).

[0029] Figure 8 Effect of compounds 1 and 2 on S100A9 protein. Among them, (A): Western Blotting detection found that compound 2 inhibited the protein secretion of S100A9. Data are presented as mean ± standard deviation. # p<0.05, compared with the blank control group; ***P<0.001, **P<0.01, *p<0.05, compared with the TNF-α model group. (B): TSA analysis of intracellular binding of compounds 1 and 2 with S100A9.

[0030] Figure 9 Molecular docking results of compounds 1-4 with calprotectin. Among them, (A): The overall structure of calprotectin and the location of two hydrophobic pockets. (B): Compounds 1-4 are docked into two hydrophobic pockets in turn, and the docking scores are pocket 1 / pocket 2, respectively.

[0031] Figure 10Compounds 2 bind to both hydrophobic pockets and interfere with the binding of endogenous ligands. Among them, (A): RMSD plots of calprotectin in apo, com and com2 states and trajectories of ligands binding to the protein. (B): Rg plots of calprotectin in apo, com and com2 states. (C): RMSF plots of four chains of calprotectin in apo, com and com2 states. (D): Ion trajectories of the protein (including six blue calcium ions and four gray zinc ions) in apo, com and com2 states. (E): Size and volume of two pockets in docking, apo, com and com2 states. (F): Conformational changes of two ligands (i) and the residues (ii-vii) they cause.

[0032] Figure 11 Compounds 1-4 bind to calprotectin and degrade it. Among them, (A): Affinity between compounds 1-4 and calprotectin. (B): Compounds 2 and 4 reduce the thermal stability of calprotectin. (C): Effect of compound 2 on S100A9 protein expression, tapinarof as a positive control.

[0033] Figure 12 Therapeutic effects of compounds 1 and 2 on IMQ-induced psoriasis-like dermatitis. Among them, (A): Skin lesions of mice. (B): Quantification of CSS of mice in each group. (C): Epidermal thickness of mice in each group. (D): Performance and histological characteristics of skin lesions in each group. DETAILED DESCRIPTION

[0034] A number of exemplary embodiments of the present application are now described in detail below. The following description of certain

[0035] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, for a range of values of or statements of a value, understand that each intervening value, to the upper or lower limit, is also specifically included within the scope of the present application. Each smaller range between any stated value or intervening value in a stated range, and any other stated or intervening value or range in stated values are specifically included within the scope of the present application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also specifically included within the scope of the present application.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0040] The present invention provides a diterpenoid compound, including compounds 1 to 4, whose structural formulas are as follows:

[0041]

[0042] The embodiment of the present invention also provides a method for preparing the diterpenoid compound, comprising the following steps: fermenting rice with Aspergillus undulatus to obtain a rice fermentation product; and extracting and separating the rice fermentation product to obtain the diterpenoid compound.

[0043] In some specific embodiments, the Aspergillus undulatus is from the China General Microorganism Culture Collection Center with a collection number of CGMCC No. 3.6295.

[0044] In some specific embodiments, the method of fermenting rice using Aspergillus undulatus comprises: mixing rice and water in a mass ratio of 1:1, inoculating the Aspergillus undulatus, and culturing at 28° C. for 45 days to obtain the rice fermentation product.

[0045] In some specific embodiments, the extraction and separation method includes:

[0046] (1) extracting the rice fermentation product with alcohol and extracting with ethyl acetate to obtain a crude extract;

[0047] (2) gradient elution of the crude extract with a mixed solvent of petroleum ether:ethyl acetate:methanol = 15:1:0-5:5:1 to obtain six components Fr.1-Fr.6;

[0048] (3) Component Fr.4 was subjected to reverse C 18 Column chromatography afforded seven components, Fr.4.1–Fr.4.7. Fr.4.4 was purified by silica gel column chromatography using a gradient elution system of petroleum ether: dichloromethane: methanol (2:1:0-0:1:0-0:50:1) to afford eight components, Fr.4.4.1–Fr.4.4.8. Fr.4.4.4 was purified by reverse-phase high-performance liquid chromatography to afford compound 1.

[0049] (4) Component Fr.3 was subjected to reverse C 18 Column chromatography yielded 21 fractions Fr.3.1-Fr.3.21;

[0050] (5) Fr.3.12 was subjected to gel column chromatography and then silica gel column chromatography to obtain 10 components Fr.3.12.1-Fr.3.12.10; Fr.3.12.8 was separated into 4 components Fr.3.12.8.1-Fr.3.12.8.4 by preparative liquid chromatography;

[0051] (6) Fr.3.12.8.3 was separated twice by HPLC to obtain three components, Fr.3.12.8.3.3-Fr.3.12.8.3.3. Fr.3.12.8.3.2 was purified again by HPLC to obtain compound 2;

[0052] (7) Fr.3.12.8.4 was separated and purified twice by HPLC to obtain compound 3;

[0053] (8) Fr.3.14 was subjected to gel column chromatography and then silica gel column chromatography to obtain four components Fr.3.14.1-Fr.3.14.4. Fr.3.14.3 was separated by high performance liquid chromatography to obtain compound 4.

[0054] In some specific embodiments, the Aspergillus undulatus is expanded before inoculation; the expansion culture conditions include: inoculating the Aspergillus undulatus on PDA and culturing for 10-14 days.

[0055] In some specific embodiments, the C 18 The column chromatography conditions were methanol-water, 20%-100%.

[0056] The embodiment of the present invention also provides the use of the diterpenoid compound in preparing a drug for treating psoriasis.

[0057] An embodiment of the present invention further provides a drug for treating psoriasis, comprising the sesquiterpenoid compound.

[0058] The application also provides application of the sesquiterpenoids in preparation of immunosuppressants.

[0059] The application also provides application of the sesquiterpenoids in preparation of S100A8 / A9 targeted inhibitors.

[0060] The application also provides a S100A8 / A9 targeted inhibitor comprising the sesquiterpenoids.

[0061] The application discloses undulanoids A-D (1-4), secondary metabolites of Aspergillus undulates in Aspergillus nidulans, and determines that the undulanoids A-D (1-4) are 6 / 5 / 5 / 6 / 5 ring system sesquiterpenoids new skeleton compounds by comprehensively using a plurality of spectrum analysis methods and other means, and provides the structure, preparation method, structure identification and biological activity evaluation of the compounds. In addition, the compounds have a strong treatment effect on psoriasis, can inhibit the secretion of S100A8 / A9 protein, and promote the degradation of the S100A8 / A9 protein, which is the first natural small molecule inhibitor antagonizing the S100A8 / A9 protein target.

[0062] Preparation and structure identification of compounds 1-4 in the embodiment 1

[0063] (I) Preparation of the compounds 1-4.

[0064] 1, strain information

[0065] The Aspergillus undulates strain is purchased from China General Microbiological Culture Collection Center, is separated from soil in Shennongjia, Hubei, is preserved on December 4, 2001, and has a preservation number of CGMCC No.3.6295. It is identified that the genetic sequence of the strain has a similarity of 99.5% with Aspergillus undulates, and is determined as Aspergillus undulates.

[0066] 2, extraction and separation

[0067] The strain is inoculated on PDA and cultured for 10-14 days, and then the PDA is divided into small pieces and inoculated on sterilized rice (1L conical flask includes 250g of rice, 250ml of water, a total of 70kg of rice, and the inoculation amount of live bacteria is 2cm 2Rice fermentate was obtained by incubating the rice in PDA medium (potato 200 g, glucose 20 g, agar 15 g, distilled water 1 L) at 28℃ for 45 days. The rice fermentate was soaked with anhydrous ethanol for 8 times, and the ethanol extract was concentrated under reduced pressure to obtain the alcohol extract. The alcohol extract was suspended in water and extracted with ethyl acetate for 7 times. The ethyl acetate extract was concentrated under reduced pressure to obtain the crude extract. The crude extract was subjected to gradient elution with petroleum ether: ethyl acetate: methanol (15:1:0-5:5:1) mixed solvent to obtain 6 fractions (Fr.1-Fr.6).

[0068] Fraction Fr. 4 was purified by reverse phase HPLC (C 18 Column chromatography (methanol-water, 20%-100%) yielded 7 fractions (Fr.4.1-Fr.4.7). Fr.4.4 was subjected to silica gel column chromatography and gradient elution with petroleum ether: dichloromethane: methanol (2:1:0-0:1:0-0:50:1) to obtain 8 fractions (Fr.4.4.1-Fr.4.4.8). Compound 1 was obtained by purifying Fr.4.4.4 by reverse phase HPLC.

[0069] Fraction Fr. 3 was purified by reverse phase HPLC (C 18 Column chromatography (methanol-water, 20%-100%) yielded 21 fractions (Fr.3.1-Fr.3.21). Fr.3.12 was subjected to gel column chromatography and then silica gel column chromatography to obtain 10 fractions (Fr.3.12.1-Fr.3.12.10). Fr.3.12.8 was divided into 4 fractions (Fr.3.12.8.1-Fr.3.12.8.4) by preparative liquid chromatography. Fr.3.12.8.3 was separated by HPLC twice to obtain 3 fractions (Fr.3.12.8.3.3-Fr.3.12.8.3.3). Fr.3.12.8.3.2 was purified by HPLC again to obtain compound 2. Fr.3.12.8.4 was purified by HPLC twice to obtain compound 3. Fr.3.14 was subjected to gel column chromatography and then silica gel column chromatography to obtain 4 fractions (Fr.3.14.1-Fr.3.14.4). Fr.3.14.3 was separated by HPLC to obtain compound 4.

[0070] Structure identification of compounds 1-4

[0071] The structure of compounds 1-4 was determined by comprehensive analysis of high resolution mass spectrometry, nuclear magnetic resonance signals, circular dichroism spectrum, ultraviolet spectrum, infrared spectrum, specific optical rotation, and X-ray single crystal diffraction data of compounds 1-4.

[0072] Compound 1: colorless crystal; melting point 185.5-185.8℃; (c 1.0, MeOH); UV (MeOH) λ max (logε) 202(4.04) nm; ECD (MeOH) λmax (Δε) = 211 (+ 17.2) nm; IR v max 3363, 2954, 1633, 1458, and 1385 cm -1 ; 1 H and 13 C NMR data, Table 1; HRESIMS m / z 395.2925 [M + Na] + (calcd. for C 25 H 40 NaO2, 395.2926). The nuclear magnetic resonance (NMR) data of compound 1 are shown in Table 1.

[0073] Compound 2: colorless crystal; melting point 185.3-180.2 °C; (c 1.0, MeOH); UV (MeOH) λ max (log ε) 196 (4.03) nm; ECD (MeOH) λ max (Δε) = 200 (+ 6.8) nm; IR v max 3363, 2926, 1668, 1461, and 1380 cm -1 ; 1 H and 13 C NMR data, Table 1; HRESIMS m / z 395.2913 [M + Na] + (calcd. for C 25 H 40 NaO2, 395.2926). The nuclear magnetic resonance (NMR) data of compound 2 are shown in Table 1.

[0074] Compound 3: colorless crystal; melting point 176.1-177.6.2 °C; (c 1.0, MeOH); UV (MeOH) λ max (log ε) 203 (4.08) nm; ECD (MeOH) λ max (Δε) = 206 (+ 6.1) nm; IR v max 3436, 2954, 1634, 1460, and 1384 cm -1 ; 1 H and 13 C NMR data, Table 1; HRESIMS m / z 395.2922 [M + Na] + (calcd. for C 25 H 40NaO2, 395.2926). The nuclear magnetic resonance (NMR) data of compound 3 is shown in Table 1.

[0075] Compound 4: colorless oil; (c 1.0, MeOH); UV (MeOH) λ max (log ε) 202 (3.76) nm; ECD (MeOH) λ max (Δε) = 209 (+18.0) nm; IR v max 3415, 2929, 1668, 1462, and 1380 cm -1 ; 1 H and 13 C NMR data, Table 1; HRESIMS m / z 395.2912 [M+Na] + (calcd. for C 25 H 40 NaO2, 395.2926). The nuclear magnetic resonance (NMR) data of compound 4 is shown in Table 1.

[0076] Table 1 Hydrogen spectrum and carbon spectrum data of compounds 1-4

[0077]

[0078]

[0079] Example 2 Therapeutic effect of compounds 1-4 in the present application on psoriasis at cell level and animal level

[0080] (I) In vitro cell activity

[0081] 1. Resuscitation, subculture and cryopreservation of HaCaT cells

[0082] (1) Resuscitation: The human immortalized keratinocyte (HaCaT) cell line was obtained from Procell (Wuhan, China). After being taken out from the -80°C freezer, the cells were quickly thawed, transferred to a 15 ml centrifuge tube, resuspended with an appropriate amount of culture medium, and then added to a cell culture bottle. The bottle was placed in a carbon dioxide incubator at 37°C and 5% carbon dioxide concentration. The next day, after the cells adhered, the old culture medium was discarded, and the preheated culture medium was added to the incubator. The cell growth was observed under a microscope.

[0083] (2) Passage: when the cells grow to 80% full, discard the old culture medium, rinse with 4 mL of PBS for 1-2 times and then pour it out, add 4 mL of preheated trypsin and put it in the incubator for digestion, take it out after 3 minutes, observe the cells under a microscope, and then gently blow them down with a pipette gun, centrifuge to collect the cells, add complete culture medium to resuspend them, and then divide them into two culture dishes and incubate them in the incubator.

[0084] (3) Freezing: after the cells grow to cover the culture dish, discard the old culture medium, collect the cells, resuspend them with 1 mL of freezing solution, and then put them in a freezing tube, label the cell name and freezing time, and store them in a -80 °C refrigerator.

[0085] 2. Preliminary screening of the compound's inhibition of cell proliferation activity

[0086] HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) in a humidified environment (5% CO2, 37°C). To simulate psoriasis-like inflammation, HaCaT cells were treated with TNF-α (10 ng / mL) for 24 hours. Compound 1 or 2 was administered at 0.625, 1.25, 2.5, 5, 10, and 20 μM to unstimulated and TNF-α-stimulated cells, respectively. After 24 hours, the absorbance was measured at a wavelength of 450 nm using a CCK-8 kit (KGA317; KeyGen, Nanjing, China).

[0087] 3. Flow cytometry detection of CD4, CD8, and NK cell proportion

[0088] (1) Preparation of mouse spleen lymphocyte suspension: After the mouse is decapitated, it is immediately soaked in 75% alcohol for 10 minutes, fixed on a dissection table, and the abdominal cavity is opened using sterile surgical instruments. The spleen is removed and rinsed repeatedly with PBS until there are no other attachments on the surface. A nylon membrane is placed on the mouth of a 50 mL centrifuge tube, and the rinsed spleen is placed on the nylon membrane and ground thoroughly with a 1 mL syringe tip until it is white. After grinding, the nylon membrane is rinsed with PBS for two to three times, centrifuged at 400 g for 5 minutes to collect, 2-3 mL of red blood cell lysis solution is added, and after lysis at room temperature for five minutes, centrifuged at 300 g for 5 minutes to discard the supernatant, and then rinsed with PBS for 2-3 times. Add RPMI 1640 complete culture medium to prepare a spleen cell suspension, and then place it in the incubator for 4 hours before aspirating the lymphocytes in the supernatant.

[0089] (2) The cell suspension was evenly spread into a six-well plate, and concanavalin A (5 μg / mL) and the corresponding concentration of compound were added, and the plate was cultured in an incubator for 48 hours. The six-well plate was gently blown with culture medium to fully collect the cells, and centrifuged at 300g for 10 minutes. After washing the cells with culture medium containing serum, they were resuspended in buffer, and antigen CD3 / CD4 / CD8 / CD49b antibodies were added and incubated at 4℃ in the dark for 30 minutes. The cells were resuspended in 500 μL PBS, vortexed to mix, and detected by the instrument.

[0090] 4. Western Blotting to detect changes in related protein levels in HaCaT cells

[0091] (1) HaCaT cell protein extraction: According to the experimental grouping, HaCaT cells were evenly plated in a six-well plate for culture. Cells from different groups were collected into 1.5 mL centrifuge tubes, washed 2-3 times with pre-cooled PBS, and the supernatant was discarded after centrifugation. Lysis buffer was prepared on ice at a ratio of phenylmethylsulfonyl fluoride: RIPA lysis buffer = 1:100. After preparation, 30-50 μL of lysis buffer was added to each centrifuge tube, and the cells were placed on ice for 30 minutes, with gentle shaking every 10 minutes. After the lysis was completed, the cells were centrifuged in an ultra-low temperature high-speed centrifuge for 10-15 minutes (speed: 12000 rpm; temperature: 4°C), the precipitate was discarded, the supernatant was retained, and then transferred to a new 1.5 mL EP tube and placed at -80°C for use.

[0092] (2) Determination of protein concentration by BCA method: Add 1 μL of the sample to be tested or a protein standard with a concentration gradient to a 96-well plate, then add 19 μL of PBS, and prepare 200 μL of BCA working solution (A:B = 50:1) according to the BCA operating instructions. After mixing, place the plate in a 37°C incubator and incubate for about 30 minutes. Use a microplate reader to detect the absorbance of each well at 562 nm, and calculate the protein concentration based on the standard curve.

[0093] (3) The experiment used 10% and 15% SDS-PAGE gels. The upper and lower gels were prepared using the Yazyme PAGE Gel Rapid Preparation Kit according to the instructions. Then, electrophoresis, transfer, blocking, incubation with primary antibody, incubation with secondary antibody in the dark, development and imaging were performed.

[0094] 5. Immunofluorescence detection of changes in NF-κB-P65 and S100A9 in HaCaT cells

[0095] Place sterile cell slides of appropriate size in a six-well cell culture plate (1 slide / well), collect HaCaT cells in good growth condition and dilute them to a density of 1.25×10 6Compound 1 (2.5, 5.0 μM), Compound 2 (1.25, 2.5 μM) were given to cells respectively, 2 hours later, TNF-α (10 ng / mL) was added to the model and compound groups, 24 hours later, the supernatant was removed. Pour out the culture medium, wash with PBS for 3 times. 4% paraformaldehyde fixation for 20 minutes, wash with PBS for 3 times. Circle with histo pen, prevent the incubation liquid from flowing away in the following process, wash with PBS. Dilute the antibody with 5% BSA (Biofroxx, 4240GR250) at a certain ratio, add a certain amount of primary antibody (NF-κB-P65 or NLRP3, 1:1000, Cell Signaling Technology, USA) working solution, 4°C overnight. Warm up, wash with PBS for 3 times, 5 minutes each time. Add the corresponding species of secondary antibody (SA00013-2 / AS-1111 / SA00013-1 / SA00009-3 / SA00003-3 / SA00007-7, Three Eagle) working solution in the circle, 37°C water bath, avoid light incubation for 40 minutes, wash with PBS for 3 times, 5 minutes each time. Add DAPI (Sigma, D8417-1MG) to stain the nucleus, incubate at room temperature for 20-30 minutes, wash with PBS. Use anti-fluorescence quenching mounting medium (Sigma, V900155-25G) to mount the slide, after mounting, store the slide in a dark box at 4°C. Observe and take pictures under a microscope (OLYMPUS, IX51).

[0096] 6. Cell thermal shift assay (CETSA) experiment

[0097] (1) Collect and process cells: HaCaT cells in 15 cm culture dishes, when they grow to about 80%, treat them with compounds or DMSO for 1 hour, collect the cells.

[0098] (2) Extract protein and detect binding force: wash with PBS once, then suspend in 1 mL of PBS added with protease and phosphatase inhibitors, and keep the same dose of compound or DMSO as the initial treatment.

[0099] The cell suspension was distributed into 7-10 0.2 mL PCR tubes with a volume of 100 μL (about 1 million cells) per tube, each tube assigned a temperature point. The samples were heated in a metal bath at the assigned temperature for 2 minutes. After heating, the tubes were immediately removed and incubated at room temperature for 3 minutes before being flash frozen in liquid nitrogen and stored at -80°C. To lyse the cells, three freeze-thaw cycles were performed in liquid nitrogen. After each thaw, the tubes were briefly spun. The cell lysate was collected and centrifuged at 15000 rpm for 15 minutes at 4°C to remove cell debris and precipitated and aggregated proteins. The cell lysate samples were boiled in loading buffer at 95°C for 5 minutes before being removed and stored in a -80°C freezer. Western Blotting was used to detect the binding of the target protein to HaCaT cells at different temperatures, and the protein fluorescence density and relative expression amount were detected using Image J software.

[0100] (ii) Dynamic simulation

[0101] 1. Molecular docking

[0102] Molecular docking was performed in . The protein model 4GGF was downloaded from the PDB crystal database and pre-processed using the protein processing tool Protein Preparation Workflow. The structures of compounds 1-4 were pre-processed by the LigPrep tool. The receptor grid was centered on the entire protein and covered the entire protein. Ligand docking was performed using default settings. Interactions were provided by Pymol software.

[0103] 2. Molecular dynamics simulation

[0104] Molecular dynamics simulations were performed in the CUDA-accelerated GROMACS-2022.2 program, with the protein AMBER99SB-ILDN force field and the ligand GAFF force field (whose topology files were generated by Acpype). In the MD simulation, the complex structure of the protein and ligand was obtained from the molecular docking results and used as the original coordinate file. The complex was dissolved in a cubic periodic box using the TIP3P water molecule model, and the system charge was neutralized by sodium and chloride ions, with a final ion strength of 0.15 M. Energy minimization was performed using the gradient descent method for 5000 steps to eliminate undesirable contacts in the system. Then, the temperature and pressure of the system were kept constant at 310 K and 1 bar, respectively, using the microcanonical (NVT) and isothermal-isobaric (NPT) ensembles. Finally, the 100 ns MD simulation was performed on the equilibrated system. Subsequent MD analysis was performed using the built-in modules of GROMACS, and the data was processed using Origin 2021, pymol, and VMD. Binding free energy calculation and decomposition analysis were performed using the gmx_MMPBSA program. From the start of system equilibration, 1 frame was taken at 20 ps intervals. Other parameters were kept at their default values. The volume of the pocket was calculated by the SiteMap module of Schrodinger software (evaluating single binding site regions).

[0105] 3. Protein production and purification

[0106] (1) The sequences of S100A8 (UniProt: P05109) and S100A9 (UniProt: P06702) were synthesized by GENEWIZ according to the literature and inserted into the vector pET21a (NdeI / XhoI) to construct the plasmid.

[0107] (2) For protein expression, the recombinant plasmid was transformed into E. coli BL21 (DE3) cells, cultured in LB medium containing antibiotics at 37°C until the OD 600between 0.6-0.8. Then the inducer IPTG (final concentration: 0.4 mM) was added and the culture was further incubated at 18°C for 16-20 h. Equal amounts of cell pellets containing S100A8 and S100A9 were collected by centrifugation and resuspended in buffer A (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM beta-mercaptoethanol). The inclusion bodies were lysed by sonication and further ultracentrifuged at 10000 g, 4°C for 1 h, after which the inclusion bodies were resuspended in buffer A containing 0.5% (v / v) Triton X-100 and sonicated 3 times, followed by 3 washes in buffer A. The washed inclusion bodies were solubilized in 6 M guanidine hydrochloride and dialyzed 3 times against buffer B (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM CaCl2, 2 mM ZnSO4). The resolubilized supernatant containing calprotectin was filtered and further purified by size exclusion chromatography (HiLoad 16 / 60 Superdex 75, Cytiva) in buffer B. TM 16 / 60 Superdex 75, Cytiva).

[0108] 4. TSA experiment

[0109] Purified calprotectin was diluted to 0.5 mg / mL in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM CaCl2, and 2 mM ZnSO4and incubated with compound or DMSO for 30 min at room temperature. The reactions were performed in 384-well plates in a system of 10 μL per well, containing 9 μL of the mixed solution and 1 μL SYPRO Orange protein gel dye (Invitrogen). After centrifugation, the melting curves were monitored in an Applied Biosystems QuantStudio 6 Pro Real-Time PCR System (Thermos Fisher Scientific) with a first step of incubation at 25°C for 10 min, followed by a gradient heating from 25°C to 95°C at a rate of 0.075°C / s. The Tm values were fitted and calculated by the Quantstudio™ Design and Analysis Software v1.5.2. The experiment was repeated three times. TM Orange protein gel dye (Invitrogen). After centrifugation, the melting curves were monitored in an Applied Biosystems QuantStudio 6 Pro Real-Time PCR System (Thermos Fisher Scientific) with a first step of incubation at 25°C for 10 min, followed by a gradient heating from 25°C to 95°C at a rate of 0.075°C / s. The Tm values were fitted and calculated by the Quantstudio™ Design and Analysis Software v1.5.2. The experiment was repeated three times. QuantStudio TM 6 Pro Real-Time PCR System (Thermos Fisher Scientific) with a first step of incubation at 25°C for 10 min, followed by a gradient heating from 25°C to 95°C at a rate of 0.075°C / s. The Tm values were fitted and calculated by the Quantstudio™ Design and Analysis Software v1.5.2. The experiment was repeated three times.

[0110] 5. Surface plasmon resonance (SPR) assay of compound affinity

[0111] The affinity of calprotectin and compounds was determined by Biacore 1K (Cytiva). Recombinant proteins were immobilized on a Series S sensor chip CM5. Binding or dissociation was performed under a predefined program (Multi-cycle kinetics / affinity) with 5 dilution concentrations of compounds in 10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% v / v Surfactant P20, 5% v / v DMSO. Results were analyzed using a predefined program (Multi-cycle kinetics) to calculate Ka, Kd and KD values.

[0112] (III) Animal Experiments

[0113] 1. Modeling and treatment of animals

[0114] (1) Male 8-week-old Balb / C mice weighing 20-25 g were adaptively fed in a SPF barrier environment for one week, and then randomly divided into 5 groups: blank control group (Con), imiquimod model group (IMQ), positive drug group (tapinarof), compound 1 group (10 mg / kg), and compound 2 group (10 mg / kg). One day before administration, the hair on the middle back of the mice was removed with a mouse hair clipper.

[0115] The method for constructing a psoriasis-like mouse model is as follows: for the imiquimod model group, the positive drug group, the compound 1 group, and the compound 2 group, 62.5 mg of imiquimod cream was applied daily. When the mice showed obvious symptoms of dermatitis, such as redness, scaling, thickening of the epidermis, and desquamation, it was considered that the psoriasis-like mouse model was successfully constructed.

[0116] The blank control group was given the corresponding amount of petrolatum for seven consecutive days.

[0117] Method of administration:

[0118] The compound 1 group and the compound 2 group were each given 0.2 mL of compound (10 mg / kg) prepared in normal saline by intraperitoneal injection daily;

[0119] The positive drug was a mixture of 60% anhydrous ethanol and 40% glycerol dissolved to a mass concentration of 1% and applied to the back of the mouse;

[0120] The imiquimod model group was administered by intraperitoneal injection of 0.2 mL of normal saline daily.

[0121] The blank control group was administered by intraperitoneal injection of 0.2 mL of normal saline daily.

[0122] The dynamic changes in the skin lesions on the back of the mice were observed and recorded daily before administration. On the seventh day, the mice were sacrificed by cervical dislocation after blood was collected by enucleation, and the skin and spleen were removed.

[0123] 2. PASI score

[0124] The mice skin was observed and photographed, and the PASI score was performed every day. These factors included erythema, scaling, thickness of 0-4, including: 0: none; 1: mild; 2: moderate; 3: severe; 4: very severe. The score was performed every day for 7 consecutive days.

[0125] 3. HE staining and epidermal thickness measurement

[0126] Most of the mouse skin lesion tissues were stored in liquid nitrogen or at -80°C, and a small part of the skin lesion tissues were fixed with 4% paraformaldehyde. Paraffin-embedded sections were prepared as follows: first, the sections were dehydrated from low concentration to high concentration, i.e. the sections were soaked in xylene for 20 minutes, repeated once, then the xylene was discarded, anhydrous ethanol was added for 5 minutes, repeated once, then 75% alcohol was added for gradient deparaffinization for 5 minutes, and then washed with water. Then hematoxylin staining was performed: the hydrated sections were stained with hematoxylin dye for cell nucleus staining for several minutes to several tens of minutes, then acid washing was performed, such as 1% hydrochloric acid aqueous solution to remove excess dye and differentiation, and finally alkaline solution was used for blue returning treatment to make the cell nucleus blue. Then eosin staining was performed: the sections after hematoxylin staining were stained with eosin dye for cytoplasmic staining, the paraffin sections were dehydrated with gradient alcohol (85% ethanol→95% ethanol), and then soaked in eosin staining solution for several minutes. Finally, dehydration and mounting were performed: the paraffin sections were soaked in anhydrous ethanol for several minutes and then discarded, and then soaked in transparent agent (usually xylene) for several minutes and repeated once, then the sections were naturally dried, and the tissues on the sections were photographed with a fluorescence microscope, in which the cell nucleus was stained blue and the cytoplasm was red.

[0127] 4. Detection of changes of psoriasis-related indicators in mouse skin tissues by immunohistochemical method

[0128] Sample pretreatment was the same as for HE staining. The immunohistochemistry procedure for paraffin sections was as follows: tissue sections were placed in a retrieval box filled with EDTA (pH 9.0) antigen retrieval solution. Microwave oven heat was used to bring the solution to a boil on medium heat for 8 minutes, followed by 8 minutes on medium-low heat and 7 minutes on a shaker. After cooling to room temperature, the slides were washed with PBS buffer (pH 7.4) on a destaining shaker. Slides were soaked in 3% hydrogen peroxide solution and incubated in the dark at room temperature for 25 minutes. The slides were then washed with PBS buffer. 3% BSA or rabbit serum was added dropwise to the histochemical staining ring to evenly cover the tissue, and the staining was blocked at room temperature for 30 minutes. The blocking solution was gently shaken off, and the primary antibody (diluted in PBS) was applied directly to the sections. The sections were incubated at 4°C overnight. The next day, the sections were rewarmed in a 37°C water bath for 30 minutes. After washing with PBS buffer, the solution was shaken dry. A secondary antibody (HRP-conjugated) of the species corresponding to the primary antibody was added dropwise to the sections to cover the tissue, and the sections were incubated at room temperature for 50 minutes. After washing with PBS, add freshly prepared DAB colorimetric solution. Observe the slides under a microscope for color development. Rinse the sections with tap water to terminate color development. Then, soak the sections in hematoxylin stain for 3 minutes. Rinse with tap water to remove excess stain. Differentiate with hematoxylin differentiation solution for several seconds, rinse with tap water, and soak in hematoxylin bluing solution to return the sections to blue. Rinse with running water. Dehydrate, mount, and photograph 3-5 random fields of view. Cell nuclei appear blue, while positive markers appear brownish-yellow.

[0129] (IV) Results Analysis

[0130] 1. Initial screening of activity: Figure 4 As shown, TNF-α was used to stimulate the hyperproliferation of HaCaT cells, and the anti-hyperproliferation effects of compounds 1-4 were evaluated. Compounds 1-4 showed significant inhibitory activity, and their IC 50 The values ​​were 2.47, 1.25, 6.72 and 8.54 μM, respectively, and the cytotoxicity to normal HaCaT cells was low. Then, the most effective compound 2 was selected to further evaluate its effectiveness and explore its mechanism of action. Flow cytometry was used to detect the ratio of CD4, CD8 and NK cells in Con A-induced mouse primary splenocytes. After Con A stimulation, the CD4 / CD8 value increased significantly (>2.5), while the NK cell level decreased slightly. This disorder is common in autoimmune diseases. The proportion of immune cells can be restored after administration, indicating that compounds 1 and 2 have immunomodulatory effects. Compounds 3 and 4 have immunomodulatory effects comparable to compound 1.

[0131] 2. Transcriptomics analysis: Figure 5Figure 6 shows the volcano plot of the differentially expressed genes in HaCaT cells treated with TNF-a and compound 2. The PCA shows that the data is well clustered between groups. Compared with the control group, there are 280 up-regulated genes and 317 down-regulated genes in the model group, while compared with the model group, there are 1709 up-regulated genes and 2704 down-regulated genes after treatment with compound 2. As shown in the volcano plot, the up-regulated genes are represented in red and the down-regulated genes are represented in blue. To further explore the effects of compound 2, the enrichment of up-regulated and down-regulated genes was analyzed, and it was found that 194 up-regulated genes and 241 down-regulated genes returned to baseline levels. In the pathway analysis, the TNF-a and IL-17 signaling pathways were enriched, which are key pathways in the pathogenesis of psoriasis. In addition, 29 genes related to IL-17 signaling and 18 genes related to TNF signaling were found, of which 5 and 7 overlapped, respectively. In addition, the heatmap highlights genes involved in inflammatory pathways (e.g., CXCL1, IL1B, MMP9).

[0132] 3. Analysis of the expression results of related inflammatory pathway proteins: As shown in Figure 6 and Figure 7 According to the results of omics, the expression of proteins related to TNF-a and IL-17 signaling pathways was studied. WB detected that the levels of related proteins p-IKB, NLRP3, and COX-2 decreased after administration. At the same time, immunofluorescence showed that administration of compounds 1-4 can reduce the phosphorylation of NF-kB p65 in the cytoplasm, thereby blocking the nuclear translocation of p65. Compound 2 has the most superior effect.

[0133] 4. Target speculation: As shown in Figure 8 The structures of compounds 1-4 were introduced into PharmMapper (http: / / www.lilab-ecust.cn / pharmmapper / ) for target screening, and S00A8 / A9 was predicted as the top target related to inflammatory response. By detecting the level of S100A9 in HaCaT cells by immunoblotting, it was found that administration of compound 2 can dose-dependently reduce the expression of S100A9. In addition, in the cell thermal shift (CETSA) experiment, S100A9 treated with compounds 1-4 all showed relative stability when the temperature was raised, especially compound 2, which all indicated that S100A9 protein is the direct target of this type of compound.

[0134] 5. Molecular docking: Because there are two hydrophobic binding sites available for endogenous ligand binding, docking was performed at a protein: ligand = 1:2 ratio. 4GGF was used as a protein model. As shown in Figure 9 Compounds 1-4 can all be docked into the hydrophobic pocket of each aP heterodimer.

[0135] 6. Molecular dynamics simulation: In combination with pharmacological experiments, further simulation of the binding trajectory of compound 2 with two hydrophobic pockets showed that compound 2 can occupy the molecular pockets of calprotectin, limiting the interaction of endogenous ligands with calprotectin. The specific results are shown in Figure 10 .

[0136] 7. SPR result analysis: As shown in A and B of Figure 11 , compounds 1-4 can all bind to calprotectin, and the binding constants (KD values) are 9.59, 1.49, 5.07 and 8.10 μM, respectively. Further study of their effects on the stability of calprotectin showed that 2 and 4 reduced its thermal stability (Tm value decreased), especially compound 2. In combination with the results of molecular dynamics simulation, 1-4 can all bind to two hydrophobic pockets, destroying the stable structure of calprotectin and interfering with the binding of endogenous ligands.

[0137] 8. Immunofluorescence result analysis: As shown in C of Figure 11 , the effect of compound 2 on the stability of S100A9 (chain B and chain D of calprotectin) was evaluated in cells. After incubation with TNF-α, the expression of S100A9 was up-regulated, and the process was reversed after administration, even better than the positive control bengamides. It is shown that compound 2 can bind to the hydrophobic pockets of calprotectin, induce its dissociation and make it unstable, thereby effectively inhibiting the inflammation caused by TNF-α, and helping to reduce the expression level of S100A9 or calprotectin.

[0138] 9. Animal experiment result analysis: As shown in Figure 12 , after 7 days of continuous local application of imidazole mox (IMQ), the mouse skin showed obvious symptoms of psoriasis-like skin inflammation, such as skin erythema, thickening and scaling, etc. After treatment with 1 or 2, the above symptoms improved, and the clinical severity score (CSS) and epidermal thickness were also significantly reduced. At the same time, the immunohistochemical results showed that the expression of S100A9, IL-17 and Ki67 was significantly reduced. This all shows that compounds 1 and 2 can alleviate the psoriasis-like skin inflammation of mice by inhibiting the secretion of S100A9 and the proliferation of keratinocytes.

[0139] In summary, four novel sesquiterpenes with 6 / 5 / 5 / 6 / 5 ring system were isolated from Aspergillus undulatus, which have eight chiral centers (5 quaternary carbons, of which 2 quaternary carbons are located at the bridgehead) and a cyclopentadiene [cd] indene fragment. In the pharmacological activity exploration, compounds 1-4 can all block the activation of inflammatory signaling pathways, thereby inhibiting immune response and contributing to the regulation of the immune system. Reverse target prediction, CETSA analysis and molecular dynamics simulation confirmed that S100A8 / A9 is the direct target of the compounds. In addition, compounds 1 and 2 can inhibit S100A9 expression and keratinocyte hyperproliferation to alleviate mouse psoriasis-like dermatitis. This class of compounds is the first natural product that can inhibit the secretion of S100A8 / A9 protein dimers, providing a new lead compound for the drug discovery of psoriasis and even autoimmune diseases, and opening up a new way. However, further research and clinical experiments are needed to verify its safety and efficacy, and to provide a more solid foundation for its clinical application.

[0140] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. For ordinary skilled users in the art, other different forms of changes or variations can also be made on the basis of the above description. Here, it is not necessary and also impossible to exhaust all the embodiments. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A sesquiterpene compound, characterized by, The structural formula is as follows: 。 2. Use of the sesquiterpenoid compound of claim 1 in the preparation of a medicament for treating psoriasis.

3. A medicament for treating psoriasis, characterized by, The sesquiterpenoid compound of claim 1.

4. Use of the sesquiterpenoid compound of claim 1 in the preparation of an S100A9 targeting inhibitor.

5. A S100A9-targeted inhibitor, characterized in that, The sesquiterpenoid compound of claim 1.

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