A polypeptide compound, and a preparation method and application thereof

Abstract

The application discloses a polypeptide compound and a preparation method and application thereof, and belongs to the technical field of marine active substances. Simplicillium sp. Two polypeptide compounds are separated from HDN17-843 liquid fermentation products, which can significantly inhibit NLRP3 inflammasome-driven IL-1beta secretion. Research shows that the polypeptide compounds have significant anti-inflammatory activity, indicating that the polypeptide compounds have the potential to be developed into new anti-inflammatory drugs.

Description

Technical Field

[0001] This invention belongs to the field of marine-derived bioactive substances technology, specifically relating to a polypeptide compound, its preparation method, and its application. Background Technology

[0002] The NLRP3 inflammasome is an intracellular multiprotein complex that plays a crucial role in inflammatory responses. Its core components include the NLRP3 protein, apoptosis-associated speckle protein (ASC), and cysteine ​​protease-1 (Caspase-1). Activation of the NLRP3 inflammasome typically requires two signaling steps: first, the cell receives initiation signals via pathways such as Toll-like receptors, placing the cell in a pre-activated state; subsequently, activation signals such as extracellular potassium efflux and lysosomal damage further activate the NLRP3 protein, recruiting ASC and Caspase-1 to form the complex. Activated Caspase-1 can cleave and mature the precursor proteins of interleukin-1β (IL-1β) and interleukin-18 (IL-18), triggering an inflammatory response. Under physiological conditions, the NLRP3 inflammasome helps clear pathogens and maintain immune homeostasis. However, under pathological conditions, its abnormal activation can lead to various diseases, such as rheumatoid arthritis, Alzheimer's disease, diabetes, cancer, and cardiovascular disease.

[0003] Therefore, the development of NLRP3 inhibitors is of great significance for the prevention and treatment of rheumatoid arthritis, Alzheimer's disease, diabetes, cancer, and cardiovascular diseases. Summary of the Invention

[0004] The purpose of this invention is to provide a polypeptide compound, and to provide a method for preparing the polypeptide compound and its specific applications, so as to overcome the shortcomings of the prior art.

[0005] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0006] A polypeptide compound, the structural formula of which is shown in Formula I or Formula II:

[0007] Formula I; Formula II.

[0008] The preparation method of the polypeptide compound involves first obtaining a crude fermentation extract through microbial fermentation culture, and then separating and purifying the crude fermentation extract using methods such as C-18 ODS reversed-phase column chromatography and semi-preparative HPLC.

[0009] The specific microorganism mentioned is: fungus HDN17-843. Simplicillium sp.HDN17-843, Accession Number: CCTCC No.M 2023673, Accession Date: May 4, 2023, Depository Institution: China Center for Type Culture Collection, Address: School of Life Sciences, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China.

[0010] Furthermore, the preparation method specifically includes:

[0011] (1) Take fungi according to the conventional method for culturing microorganisms. Simplicillium sp. An appropriate amount of HDN17-843 was inoculated into fungal liquid culture medium #2 and incubated statically at room temperature to obtain the fermentation product;

[0012] (2) After being cultured at room temperature for several days, the fermentation product was treated. An equal volume of ethyl acetate was added to the fermentation broth, and the extraction was repeated three times. The extract was then concentrated under reduced pressure to obtain an extract. After the mycelium was broken, an equal volume of methanol was added, and the mixture was sonicated and extracted multiple times to obtain an extract. The extracts obtained from the two extractions were combined.

[0013] (3) The extract obtained in step (2) was subjected to C-18 ODS reversed-phase column chromatography with methanol-water as the mobile phase, and after gradient elution, it was subjected to reversed-phase semi-preparative high performance liquid chromatography (acetonitrile:water = 50:50) to obtain polypeptide compounds as shown in Formula I or Formula II.

[0014] The polypeptide compound can significantly inhibit NLRP3 inflammasome-driven IL-1β secretion, and its application in the preparation of products that inhibit NLRP3 inflammasome-driven IL-1β secretion.

[0015] The application of the polypeptide compounds in the preparation of anti-inflammatory drugs.

[0016] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0017] This invention originates from a fungus derived from an Antarctic marine sediment sample. Simplicillium sp. Two polypeptide compounds were isolated from the liquid fermentation products of HDN17-843 that significantly inhibited NLRP3 inflammasome-driven IL-1β secretion. The study found that these polypeptide compounds possess significant anti-inflammatory activity, indicating their potential for further development into novel anti-inflammatory drugs. Attached Figure Description

[0018] Figure 1 The graph shows the inhibition results of compound I on IL-1β at different concentrations.

[0019] Figure 2 The graph shows the inhibition results of compound II on IL-1β at different concentrations. Detailed Implementation

[0020] The present invention will be further explained and illustrated below through specific embodiments and in conjunction with the accompanying drawings.

[0021] Example 1: Fermentation production and purification of compounds I and II

[0022] The preparation method of polypeptide compounds as shown in Formula I or Formula II includes the following steps:

[0023] 1. Fermentation production

[0024] Fermentation culture of the production strain: Following standard methods for culturing microorganisms, fungi were collected... Simplicillium sp. An appropriate amount of HDN17-843 (the fungus was collected by Zhu Tianjiao on January 2, 2017, in the Weddell Sea in Antarctica) was inoculated into fungal liquid culture medium #2 [prepared with seawater, pH adjusted to 6.5]. The specific ingredients are shown in Table 1. The culture was then incubated at room temperature for 30 days.

[0025] Table 1. Fungal Liquid Culture Medium #2

[0026] .

[0027] 2. Obtaining the extract

[0028] After static incubation at room temperature for 30 days, the strain was tested. Simplicillium sp. The fermentation products of HDN17-843 were processed by adding an equal volume of ethyl acetate to the fermentation broth, repeating the extraction three times, and then concentrating under reduced pressure to obtain an extract. The mycelium was then broken up, and an equal volume of methanol was added. The mixture was sonicated for 30 min, extracted three times, and then concentrated to obtain another extract. The two extracts were combined to obtain a total extract of 10.0 g.

[0029] 3. Isolation and purification of compounds

[0030] The extract was subjected to C-18 ODS reversed-phase column chromatography with methanol-water as the mobile phase, and eluted in a gradient of methanol:water = 75:15 to 80:20. Then, it was subjected to reversed-phase semi-preparative high performance liquid chromatography (acetonitrile:water = 50:50) to obtain compound I (7.1 mg) and compound II (15.0 mg).

[0031] Compound I is a white solid with the molecular formula C0. 45 H 71 N7O 11 HR-ESI-MS m / z 886.5284 [M + H] + (Calculated value: 886.5290); IR (KBr) n max 3305, 2928, 1653, 1517, 1456, 1264 cm -1 ;1 Hand 13 The C NMR values ​​are shown in Table 2.

[0032] Table 2. Compound I 1 H and 13 C NMR data (400 and 150 MHz, in DMSO- d 6)

[0033] ;

[0034] .

[0035] Compound II is a white solid with the molecular formula C. 44 H 69 N7O 11 HR-ESI-MS m / z 894.4965 [M + Na] + (Calculated value: 894.4947); IR (KBr) n max 3301, 2928, 1654, 1540, 1517, 1456, 1205 cm -1 ; 1 H and 13 The C NMR values ​​are shown in Table 3.

[0036] Table 3. Compound II 1 H and 13 C NMR data (400 and 150 MHz, in DMSO- d 6)

[0037] ;

[0038] .

[0039] The chemical structures of compounds I and II prepared above (the Arabic numerals in the structural formulas are the carbon atom markers in the chemical structures) are as follows:

[0040] Formula I; Formula II.

[0041] Example 2: IL-1β inhibitory activity test of the compound

[0042] 1. Experimental Samples and Methods

[0043] Preparation of the test sample solution: The test sample consists of pure compounds I and II, separated and purified in Step 1 above. Accurately weigh an appropriate amount of sample and prepare a 20 mM stock solution using DMSO. The final concentration of compound I is 25%. μ M, 8.3 μ M, 2.8 μ M, 0.9 μ M, 0.3 μ M; The final concentration of compound II was 30. μ M, 10 μ M, 3.3 μ M, 1.1 μ M, 0.3 μ M; the positive control is 5-chloro-2-methoxy-N-(4-(N-methylsulfonyl)phenethyl)benzamide (JC-124).

[0044] Experimental methods: J774A.1 cells were fixed in DMEM medium in 96-well plates (1.5 × 10⁻⁶). 5 (cells / well), incubate overnight at 37°C with 5% CO2. Use a solution containing 1... μ Cells were pretreated with g / mL 0111:B4 LPS (Sigma-Aldrich) E. coli for 4.5 h, then incubated with different concentrations of the test compound for 30 min. NLRP3 inflammasome activation was then stimulated with ATP (5 mM). After 30 min, the supernatant was collected, and IL-1β levels were detected using the mouse IL-1β ELISA kit (DuoSetELISA, R&D Systems) according to the manufacturer's instructions.

[0045] 2. Experimental Results

[0046] In the anti-inflammatory activity test, the results of the inhibitory activity of compounds I and II against the inflammatory factor IL-1β are shown in Table 4. Figure 1 and Figure 2 As shown, compound I has an IC50 value for the inflammatory factor IL-1β. 50 It is 5.38 μ M, the IC50 of compound II against IL-1β 50 It is 12.15 μ M indicates that compounds I and II have relatively significant inhibitory activity against the inflammatory factor IL-1β.

[0047] Table 4. IC50 of different concentrations of compound I and compound II for IL-1β 50 result

[0048] .

[0049] The above experiments show that the activity screening results of compounds I and II indicate that they have significant inhibitory effects on the inflammatory factor IL-1β, and can be used as potential novel anti-inflammatory drugs for the treatment of inflammation-related diseases.

[0050] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a polypeptide compound in the preparation of anti-inflammatory drugs; the structural formula of the polypeptide compound is shown in Formula I or Formula II: ； The polypeptide compound was prepared by microbial fermentation culture, wherein the microorganisms are specifically: Simplicillium sp . HDN17-843, Accession Number: CCTCC No.M 2023673, Accession Date: May 4, 2023, Depository Institution: China Center for Type Culture Collection.

Images