Polypeptide compound as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide compound as well as a preparation method and application thereof, and belongs to the technical field of marine-derived active substances. According to the present invention, two polypeptide compounds capable of significantly inhibiting the secretion of IL-1beta driven by NLRP3 inflammasomes are separated from the liquid fermentation product of a fungus Simplicum sp. HDN17-843 derived from an Antarctic marine sediment sample; research finds that the polypeptide compound has remarkable anti-inflammatory activity, and shows that the polypeptide compound has the potential of being further developed into a novel anti-inflammatory drug.

Description

Technical Field

[0001] The present invention belongs to the technical field of marine-derived active substances, and in particular relates to a polypeptide compound, a preparation method and an application thereof. Background Art

[0002] The NLRP3 inflammasome is an intracellular multiprotein complex that plays a key role in inflammatory responses. Its core components include NLRP3, apoptosis-associated speck protein (ASC), and cysteine ​​protease-1 (Caspase-1). Activation of the NLRP3 inflammasome typically requires two signaling steps: first, cells receive a priming signal through pathways such as Toll-like receptors, placing them in a pre-activated state. Subsequently, activation signals such as extracellular potassium ion efflux and lysosomal damage further activate NLRP3, recruiting ASC and Caspase-1 to form a complex. Activated Caspase-1 cleaves and matures the precursor proteins of interleukin-1β (IL-1β) and interleukin-18 (IL-18), triggering an inflammatory response. Under physiological conditions, the NLRP3 inflammasome contributes to pathogen clearance and immune homeostasis. However, under pathological conditions, its abnormal activation can lead to a variety of diseases, including 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, cardiovascular disease, etc. Summary of the Invention

[0004] The purpose of the present invention is to provide a polypeptide compound, and to provide a preparation method and specific application of the polypeptide compound, so as to make up for the deficiencies of the prior art.

[0005] In order to achieve the above object, the specific technical solution adopted by the present invention is: A polypeptide compound, the structural formula of the compound is shown in Formula I or Formula II: Formula I; Formula II.

[0006] The preparation method of the polypeptide compound comprises the following steps: firstly preparing a fermentation crude extract by microbial fermentation culture, and then separating and purifying the fermentation crude extract by C-18 ODS reverse phase column chromatography and semi-preparative HPLC.

[0007] The microorganism is specifically: fungus HDN17-843 Simplicillium sp.HDN17-843, deposit number: CCTCC No.M 2023673, deposit date: May 4, 2023, depositor: China Center for Type Culture Collection, deposit address: School of Life Sciences, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, Postal Code: 430072.

[0008] Furthermore, the preparation method specifically includes: (1) According to the conventional method of culturing microorganisms, take fungi Simplicillium sp. An appropriate amount of HDN17-843 was inoculated into fungal liquid culture medium No. 2 and cultured at room temperature to obtain a fermentation product; (2) After being cultured at room temperature for several days, the fermented product was processed by adding an equal volume of ethyl acetate to the fermented broth, extracting it three times, and then concentrating it under reduced pressure to obtain an extract; after the mycelium was crushed, an equal volume of methanol was added, ultrasonicated, and extracted several times, and then concentrated to obtain an extract; the extracts obtained from the two extractions were combined; (3) The extract prepared in step (2) was subjected to C-18 ODS reverse phase column chromatography using methanol-water as the mobile phase, and then subjected to reverse phase semi-preparative high performance liquid chromatography (acetonitrile:water = 50:50) after gradient elution to obtain a polypeptide compound as shown in Formula I or Formula II.

[0009] The polypeptide compound can significantly inhibit the IL-1β secretion driven by the NLRP3 inflammasome, and is used in the preparation of a product that inhibits the IL-1β secretion driven by the NLRP3 inflammasome.

[0010] Application of the polypeptide compound in the preparation of anti-inflammatory drugs.

[0011] Compared with the prior art, the advantages and beneficial effects of the present invention are: The present invention is a fungus derived from an Antarctic ocean sediment sample Simplicillium sp. Two peptide compounds were isolated from the liquid fermentation product of HDN17-843 that significantly inhibited NLRP3 inflammasome-driven IL-1β secretion. The study found that these peptide compounds possessed significant anti-inflammatory activity, suggesting that they have the potential to be further developed into novel anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The graph shows the inhibition of IL-1β by compound I at different concentrations.

[0013] Figure 2 This is a graph showing the inhibition of IL-1β by compound II at different concentrations. DETAILED DESCRIPTION

[0014] The present invention is further explained and illustrated below through specific embodiments in conjunction with the accompanying drawings.

[0015] Example 1 Fermentation production and separation and purification of compounds I and II The preparation method of the polypeptide compound represented by Formula I or Formula II comprises the following steps: 1. Fermentation production Fermentation culture of production bacteria: According to the conventional method of culturing microorganisms, take fungi Simplicillium sp. An appropriate amount of HDN17-843 (collected by Zhu Tianjiao in the Weddell Sea, Antarctica on January 2, 2017) was inoculated into fungal liquid culture medium #2 [prepared with seawater, pH adjusted to 6.5]. The specific ingredients are shown in Table 1, and the culture was incubated at room temperature for 30 days.

[0016] Table 1 Fungus 2# liquid culture medium .

[0017] 2. Obtaining the extract After 30 days of static culture at room temperature, the strain Simplicillium sp. The fermentation product of HDN17-843 was processed. An equal volume of ethyl acetate was added to the fermentation broth, extracted three times, and then concentrated under reduced pressure to obtain an extract. The mycelium was then disrupted and an equal volume of methanol was added. Ultrasonication was performed for 30 minutes, and the extract was extracted three times before concentration. The two extracts were combined to obtain a total extract of 10.0 g.

[0018] 3. Isolation and purification of compounds The extract was subjected to C-18 ODS reverse phase column chromatography with methanol-water as the mobile phase, with a gradient elution of methanol:water = 75:15 to 80:20, and then to reverse phase semi-preparative high performance liquid chromatography (acetonitrile:water = 50:50) to obtain compound I (7.1 mg) and compound II (15.0 mg).

[0019] Compound I is a white solid with the molecular formula C 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 C NMR is shown in Table 2.

[0020] Table 2 Compound Ⅰ 1 H and 13 C NMR data (400 and 150 MHz, in DMSO-d 6) ; .

[0021] 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 C NMR is shown in Table 3.

[0022] Table 3 Compound II 1 H and 13 C NMR data (400 and 150 MHz, in DMSO- d 6) ; .

[0023] The chemical structures of the compounds I and II prepared above (the Arabic numerals in the structural formula represent the positions of carbon atoms in the chemical structure) are: Formula I; Formula II.

[0024] Example 2 IL-1β inhibitory activity test of compounds 1. Experimental samples and methods Preparation of test sample solution: The test samples were the pure products of Compound I and Compound II separated and purified in Example 1. Accurately weigh an appropriate amount of sample and prepare it into a 20 mM stock solution with DMSO. The final concentration of Compound I was 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 μM; the positive drug control was 5-chloro-2-methoxy-N-(4-(N-methylsulfonyl)phenethyl)benzamide (JC-124).

[0025] Experimental method: J774A.1 cells were fixed in 96-well plates (1.5×10 5 cells / well) and incubated at 37°C in 5% CO2 overnight. μ Escherichia coli cells were pretreated with 100 μg / mL 0111:B4 LPS (Sigma-Aldrich) for 4.5 h, and then different concentrations of the test compounds were added and incubated for 30 min. ATP (5 mM) was then used to stimulate NLRP3 inflammasome activation. After 30 min, the supernatant was collected and the IL-1β level was detected using a mouse IL-1β ELISA kit (DuoSet ELISA, R&D Systems) according to the manufacturer's instructions.

[0026] 2. Experimental Results In the anti-inflammatory activity test, the results of the inhibitory activity of compound I and compound II on the inflammatory factor IL-1β are shown in Table 4. Figure 1 and Figure 2 As shown, the IC of compound I against inflammatory factor IL-1β 50 5.38 μ M, IC of compound II against IL-1β 50 12.15 μ M, indicating that compounds Ⅰ and Ⅱ have relatively obvious inhibitory activity against the inflammatory factor IL-1β.

[0027] Table 4 IC of different concentrations of compound I and compound II against IL-1β 50 result .

[0028] From the above experiments, the activity screening results of Compound I and Compound II showed that Compound I and Compound II had significant inhibitory effects on the inflammatory factor IL-1β, and can be used as potential new anti-inflammatory drugs for the treatment of inflammation-related diseases.

[0029] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects disclosed in the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A polypeptide compound, characterized in that: The structural formula of the compound is shown in Formula I or Formula II: Formula I; Formula II.

2. The method for preparing the polypeptide compound according to claim 1, characterized in that: Firstly, a fermentation crude extract is prepared by microbial fermentation culture, and then the fermentation crude extract is separated and purified by methods such as C-18 ODS reverse phase column chromatography and semi-preparative HPLC.

3. The preparation method according to claim 2, wherein The microorganism is specifically: fungus HDN17-843 Simplicillium sp. HDN17-843, deposit number: CCTCC No.M 2023673, deposit date: May 4, 2023, depositor: China Center for Type Culture Collection, deposit address: School of Life Sciences, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, Postal Code: 430072.

4. The preparation method according to claim 2, wherein The preparation method specifically comprises: (1) According to the conventional method of culturing microorganisms, take fungi Simplicillium sp. An appropriate amount of HDN17-843 was inoculated into fungal liquid culture medium No. 2 and cultured at room temperature to obtain a fermentation product; (2) After being cultured at room temperature for several days, the fermented product was processed by adding an equal volume of ethyl acetate to the fermented broth, extracting it three times, and then concentrating it under reduced pressure to obtain an extract; after the mycelium was crushed, an equal volume of methanol was added, ultrasonicated, and extracted several times, and then concentrated to obtain an extract; the extracts obtained from the two extractions were combined; (3) The extract prepared in step (2) is subjected to C-18 ODS reverse phase column chromatography using methanol-water as the mobile phase, and then subjected to reverse phase semi-preparative high performance liquid chromatography after gradient elution to obtain a polypeptide compound as shown in Formula I or Formula II.

5. The preparation method according to claim 4, wherein In the C-18 ODS reverse phase column chromatography, the mobile phase was methanol:water = 75:15 to 80:20 gradient elution; in the reverse phase semi-preparative high performance liquid chromatography, the mobile phase was acetonitrile:water = 50:

50.

6. Use of the polypeptide compound according to claim 1 in the preparation of a product that inhibits IL-1β secretion driven by NLRP3 inflammasome.

7. Use of the polypeptide compound according to claim 1 in the preparation of anti-inflammatory drugs.

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