Marine fungus-derived small molecule compound and application thereof in preparation of anti-renal fibrosis drugs

Abstract

The invention discloses a marine fungus-derived small molecule compound and application thereof in preparation of anti-renal fibrosis drugs, and relates to the field of marine natural products. The structural formula of the compound Gamahorin A is shown as a formula (I) in the specification. The Gamahorin A shows obvious anti-fibrosis activity by inhibiting the expression of alpha-smooth muscle actin (alpha-SMA) induced by TGF-beta1 and the generation of ECM (extracellular matrix) components (type I collagen and fibronectin), and the development of renal fibrosis is inhibited. # imgabs0 #

Description

Technical Field:

[0001] The present invention belongs to the technical field of the application of natural products, and specifically relates to a small molecule compound derived from a marine fungus, a preparation method thereof, and an application in the preparation of an anti-renal fibrosis drug. Background Art:

[0002] Chronic kidney disease (CKD) has become an important global health problem, affecting approximately 700 million people. Chronic inflammation and renal fibrosis are the main drivers of the disease. Diseases such as chronic tubulointerstitial nephritis, diabetic nephropathy, and hypertensive nephropathy share common fibrotic pathological features, ultimately leading to end-stage renal failure. Kidney injury triggers renal unit atrophy, the transformation of fibroblasts into myofibroblasts, and excessive accumulation of the extracellular matrix (ECM). The extracellular matrix consists of a fibrous network and a gel-like component, including collagen, elastin, fibronectin, and laminin. Collagen is mainly produced by myofibroblasts and plays a central role in fibrosis. The transformation of fibroblasts into myofibroblasts is a key process in renal fibrosis and a therapeutic target. However, current clinical trials of anti-fibrotic drugs have shown limited efficacy and significant side effects. Pirfenidone (PFD) only showed a transient improvement effect in phase II clinical trials, and some patients stopped taking the drug due to adverse reactions (such as rash and gastrointestinal problems). The pharmacokinetics of PFD depends on renal function, limiting its clinical application. Therefore, the development of new, safe, and effective anti-fibrotic drugs is of great significance. Summary of the Invention:

[0003] In view of the above problems, the technical solution adopted by the present invention is as follows:

[0004] The first object of the present invention is to provide a small molecule compound Gamahorin A with a novel structure having anti-renal fibrosis activity or a pharmaceutically acceptable salt thereof, and the chemical structural formula is shown as formula (I):

[0005]

[0006] The second object of the present invention is to provide a preparation method of the above compound Gamahorin A, which is isolated and prepared from the fermentation product of the marine-derived Neopestalotiopsis sp. SCSIO 41422.

[0007] Preferably, it specifically includes the following steps:

[0008] (a) Prepare the fermentation product of Neopestalotiopsis sp. SCSIO 41422, soak and extract the fermentation product with ethyl acetate, and evaporate and concentrate the ethyl acetate extract to obtain an extract;

[0009] (b) The crude extract was fractionated by medium-pressure liquid chromatography on silica gel, using mixtures of petroleum ether and dichloromethane with different volume ratios as eluents, with ratios of 1:0, 3:1, 2:1, 1:1, and 0:1. The petroleum ether and dichloromethane 3:1 - 1:1 elution fractions Frs. 3–7 were further fractionated by gradient elution using an ODS silica gel column, with the eluent being a methanol-water gradient with concentrations varying from 5% to 100%. The methanol-water 8% elution fraction Fr. A-3 was collected and further purified to obtain compound Gamahorin A.

[0010] Preferably, the fermentation product of Neopestalotiopsis sp. SCSIO 41422 is obtained by inoculating Neopestalotiopsis sp. SCSIO 41422 onto an MB solid medium and culturing it statically at 28 °C until new colonies grow. Subsequently, it is transferred to an MB seed liquid medium and cultured on a rotary shaker at 28 °C and 180 rpm for three days to obtain a seed liquid. The seed liquid is subjected to large-scale fermentation using a rice medium and cultured statically at 26 °C for 30 days. The rice medium consists of 200 g of rice per bottle, 0.48 g of sea salt, and 250 mL of water. The MB seed liquid medium contains, by mass fraction, 1.5% malt extract powder, 2.4% sea salt, and has a pH of 7.4 - 7.8. The MB solid medium contains, by mass fraction, 1.5% malt extract powder, 1.8% agar powder, 2.4% sea salt, and has a pH of 7.4 - 7.8.

[0011] Preferably, the purification is by semi-preparative high-performance liquid chromatography, with the mobile phase being 50% CH3OH / H2O, the flow rate being 3 mL / min, R t = 26 min.

[0012] The third object of the present invention is to provide the use of the above-mentioned compound Gamahorin A or its pharmaceutically acceptable salt in the preparation of an anti-renal fibrosis drug. The anti-renal fibrosis drug exhibits significant anti-fibrotic activity by inhibiting the expression of TGF-β1-induced α-smooth muscle actin (α-SMA) and the production of ECM components (type I collagen and fibronectin).

[0013] The fourth object of the present invention is an anti-renal fibrosis drug containing the above-mentioned compound Gamahorin A or its pharmaceutically acceptable salt as an active ingredient.

[0014] Preferably, the drug is prepared into a clinically acceptable pharmaceutical preparation with Gamahorin A as the main ingredient, and a pharmaceutically acceptable carrier or excipient, or a pharmaceutically acceptable excipient or auxiliary ingredient, and the content of Gamahorin A is 1-99% (w / w) based on the total weight of the drug.

[0015] Due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0016] In the process of studying the secondary metabolites of sponge-associated fungi Neopestalotiopsis sp.SCSIO 41422, the present invention separated and obtained a novel small molecule compound Gamahorin A, which can significantly inhibit renal fibrosis induced by transforming growth factor-β (TGF-β) and can be used to prepare anti-renal fibrosis drugs. Therefore, the Gamahorin A of the present invention can be used as a supplementary choice or combined treatment drug choice after pirfenidone (PFE) resistance, which is of great significance to the development of China's marine drug resources.

[0017] The fungus Neopestalotiopsis sp.SCSIO41422 of the present invention was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on August 4, 2022, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Postal Code: 510070, and its deposit number is GDMCC NO.62689. Description of the drawings:

[0018] Figure 1 is the chemical structure of the small molecule compound Gamahorin A;

[0019] Figure 2 For Gamahorin A 1 H- 1 H COSY and HMBC key 2D NMR data

[0020] Figure 3 The measured and calculated ECD spectra of Gamahorin A;

[0021] Figure 4Results of Gamahorin A inhibiting transforming growth factor-β (TGF-β). (A) Gamahorin A inhibited the expression of α-smooth muscle actin (α-SMA) induced by TGF-β1 at a concentration of 10 μM; (B) Gamahorin A inhibited the production of type I collagen (collagen I) induced by TGF-β1 at a concentration of 10 μM; (C) Gamahorin A inhibited the production of fibronectin induced by TGF-β1 at a concentration of 10 μM; (D) The data were analyzed by one-way analysis of variance (ANOVA) and subsequent Bonferroni multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (Compound 1 in the figure is Gamahorin A, and compounds 5-8, 11, 12 are gamahorin C (5), epi-pestalotiopyrone I (6), epiclactone C (7), pestalotiopyrone M (8), aspergillol A (11) and 4-hydroxyphenethyl 3-hydroxybenzoate (12), respectively.) Detailed implementation manners:

[0022] In order to make the objectives, technical solutions and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described in this specification are only used to explain the present invention and are not used to limit the present invention.

[0023] Example 1 Fermentation of marine fungus Neopestalotiopsis sp. SCSIO 41422 and isolation of Gamahorin A

[0024] 1.1 Strain isolation and preservation: The sponge-associated fungus Neopestalotiopsis sp. SCSIO41422 collected from near Weizhou Island, Beihai City, Guangxi Zhuang Autonomous Region, China was preserved in the Guangdong Provincial Microbial Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou City, postcode: 510070, and its preservation number is GDMCC NO. 62689.

[0025] 1.2 Fermentation: First, the strain preserved in paraffin oil was inoculated onto an MB plate (by mass fraction, malt extract powder 1.5%, agar powder 1.8%, sea salt 2.4%, pH 7.4 - 7.8) and cultured until new colonies grew out. Then it was inoculated into an MB liquid medium (by mass fraction, malt extract powder 1.5%, sea salt 2.4%, pH 7.4 - 7.8), and cultured on a shaker at 28 °C (180 rpm) for 3 days. Then it was inoculated into a sterilized rice medium (200 g of rice, 2.4% of coarse sea salt, water

[0026] 250 mL) for fermentation. There were 200 bottles (about 40 kg of rice), and it was statically fermented at room temperature for 30 days to prepare the rice medium ferment of the fungus Neopestalotiopsis sp. SCSIO 41422.

[0027] 1.3 Extraction: After the fermentation of the rice medium was completed, the medium was soaked with an equal volume of ethyl acetate for 24 hours, then mashed, sonicated for 15 min, and then extracted with ethyl acetate 3 times until colorless. The ethyl acetate extracts were concentrated by rotary evaporation and combined to obtain 288 g of a brown oily crude extract.

[0028] 1.4 Isolation: The brown oily crude extract was fractionated by medium - pressure liquid chromatography (MPLC) on silica gel with gradient elution. Initially, mixtures of petroleum ether (PE) and dichloromethane (DCM) with different volume ratios were used as eluents, with ratios of 1:0, 3:1, 2:1, 1:1, and 0:1. Subsequently, gradient elution with dichloromethane and methanol (DCM / CH3OH) was carried out, and the methanol concentrations were 1%, 3%, 5%, 10%, 50%, and 70% in sequence. The combination of eluents was guided by thin - layer chromatography (TLC) analysis, and finally 16 fractions (named Frs.1–16) were obtained. Fractions Frs.3–7 (elution fractions of PE / DCM 3:1 - 1:1) were further fractionated by gradient elution using an ODS silica column, with the eluent being a methanol - water (CH3OH / H2O) gradient, and the concentration varying from 5% to 100%, to obtain 16 sub - fractions (named Frs.A - 1–A - 16 respectively). Fr.A - 3 (elution fraction of CH3OH / H2O 8%) was purified by semi - preparative high - performance liquid chromatography (HPLC) (Infinity 1260 liquid chromatograph, YMC - pack ODS - A chromatographic column, 10 mm×250 mm, 5 μm, mobile phase was 50% CH3OH / H2O, flow rate was 3 mL / min) to obtain compound Gamahorin A (1.9 mg, t R = 26 min).

[0029] Example 2 Structure Identification of Gamahorin A

[0030] The chemical structure of the small molecule compound Gamahorin A obtained by separation in Example 1 is shown in Chemical Structure 1. The structural derivation process is as follows:

[0031]

[0032] Compound Gamahorin A was isolated as a brown oily liquid, and its molecular formula was determined to be C 12 H 14 O4 by HRESIMS data, indicating six degrees of unsaturation. The 1D NMR and HSQC spectra (Table 1) revealed the following characteristic signals: two methyl groups [δ C / H 19.9 / 1.37 (CH3-9), 15.5 / 2.22 (CH3-11)], two vinylic methylene groups [δ C / H 120.1 / 6.79 (CH-5), 138.4 / 7.39 (CH-6)], one methylene group [δ C / H 64.7 / 3.66 (CH2-10)], two methine groups [δ C / H 77.6 / 5.05 (CH-3), 46.7 / 2.89 (CH-4)], and five quaternary carbons [δ C 170.5 (C-1), 137.9 (C-4a), 126.6 (C-7), 161.3 (C-8), 108.2 (C-8a)]. According to the 1 H- 1 H COSY spectral correlation signals ( Figure 2 ), the proton spin coupling systems of H-3 / H-4, H-3 / H-9, and H-4 / H-10. Combining with the HMBC spectral correlation signals ( Figure 2 ), the carbon-hydrogen correlations from H-4 to C-8a, from H-5 to C-7, C-4, and C-8a, from H-6 to C-4a, C-11, from H-9 to C-4, from H-10 to C-3, C-4a, and from H-11 to C-8 were speculated, and the planar structure of compound Gamahorin A was as shown in Figure 1 .

[0033] Table 1 1 H (500 MHz) and 13 C (125 MHz) NMR data (CD3OD)

[0034]

[0035]

[0036] The NOESY spectrum showed a correlation between H-4 and H-9 ( Figure 2), indicating that H-4 and CH3-9 are on the same side of the molecule. The vicinal coupling constant (J = 5.6 Hz) between the two methine protons (H-3 and H-4) at C-3 supports the threo stereochemistry. Based on these results, the relative configuration of Gamahorin A was determined to be 3S*, 4S* and 3R*, 4R*. The absolute configuration was established by computational electronic circular dichroism (ECD) spectroscopy analysis. The calculated ECD spectrum of 3S, 4S highly matched the experimental ECD spectrum ( Figure 3 ), confirming the absolute configuration as 3S, 4S. In summary, the structure of compound Gamahorin A is shown in Formula 1 and is named Gamahorin A.

[0037] Example 3 Anti-fibrotic activity test of Gamahorin A

[0038] 3.1 TGF-β1-stimulated HK-2 cell model: HK-2 cells were cultured in a 37 °C, 5% CO2 incubator with DMEM / F12 (Gibco) (ExCell, FSP500) supplemented with 10% fetal bovine serum (FBS). Cells were seeded in 6-well plates (2 × 10^5 cells per well) and cultured for 12 hours. To establish a TGF-1-stimulated HK-2 cell model, cells were starved in serum-free medium for 24 hours and then treated with 10 ng / mL recombinant TGF-β1 (240-B-002, R&D Systems), or co-treated with the compound (10 μM) for 48 hours.

[0039] 3.2 Western Blot (WB) analysis: HK-2 cells were lysed in RIPA buffer containing protease inhibitors (Ncmblo, WB3100), then centrifuged at 13,000 g for 15 minutes at 4 °C. The total protein concentration was determined using a BCA kit (Epizyme, ZJ102). The quantified protein was mixed with loading buffer (Epizyme, LT101S) and boiled at 100 °C for 10 minutes, then separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, ISEQ00010). After blocking the membrane for 1 hour at room temperature, it was incubated overnight at 4 °C with specific primary antibodies. Then, the membrane was incubated with secondary antibodies (anti-rabbit or anti-mouse IgG) for 2 hours at room temperature. Western blot bands were detected using an ECL substrate (Fdbio, FD8020) and analyzed using Image-J1.52a software.

[0040] 3.3 Statistical analysis: All experiments were performed at least three times. All data are presented as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA), including independent samples t-test, one-way ANOVA, and two-way ANOVA. P < 0.05 was considered a significant difference (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0041] To investigate the therapeutic potential of Gamahorin A in renal fibrosis, we established a TGF-β1-induced HK-2 cell model to mimic epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition, and screened and evaluated seven marine natural products isolated from the same strain of microorganisms, including Gamahorin A (Compound 1). The results showed that Gamahorin A exhibited significant anti-fibrotic activity by inhibiting TGF-β1-induced α-smooth muscle actin (α-SMA) expression and ECM component (type I collagen and fibronectin) production at a concentration of 10 μM ( Figure 4 ). Among the seven compounds tested, Gamahorin A has been proven to be the most potent and promising renal fibrosis inhibitor, even superior to the current clinical standard fibrotic treatment drug, pirfenidone. Gamahorin A also demonstrated the strongest ECM regulatory effect, significantly inhibiting the accumulation of fibronectin and type I collagen, exceeding all other derivatives.

[0042] In summary, Gamahorin A exhibited significant anti-fibrotic activity by inhibiting TGF-β1-induced α-smooth muscle actin (α-SMA) expression and ECM component (type I collagen and fibronectin) production, and inhibited the development of renal fibrosis. The application of the small molecule compound Gamahorin A in the preparation of anti-renal fibrosis drugs suggests that Gamahorin A has potential therapeutic effects on renal fibrosis and has good clinical application prospects. This invention is of great significance for the development of new drugs with independent intellectual property rights in China.

[0043] The above description is a detailed description of the preferred and feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications made under the technical concept disclosed by the present invention shall fall within the scope of the patent covered by the present invention.

Claims

1. The compound Gamahorin A or a pharmaceutically acceptable salt thereof, the chemical structure of which is shown in formula (I):

2. Neopestalotiopsis sp.SCSIO41422, whose deposit number is GDMCC NO.62689.

3. The method for preparing the compound Gamahorin A according to claim 1, characterized in that: The invention is separated and prepared from the fermentation product of Neopestalotiopsis sp.SCSIO 41422 described in claim 2.

4. The preparation method according to claim 3, characterized in that: The specific steps include: (a) preparing a fermentation product of Neopestalotiopsis sp.SCSIO 41422, wherein the fermentation product is extracted by soaking with ethyl acetate, and the ethyl acetate extract is evaporated and concentrated to obtain an extract; (b) The extract was subjected to stepwise gradient elution by silica gel medium pressure liquid chromatography using a mixture of petroleum ether and dichloromethane in different volume ratios as the eluent, the ratios being 1:0, 3:1, 2:1, 1:1 and 0:

1. The petroleum ether and dichloromethane 3:1-1:1 elution fraction Frs.3–7 was further fractionated by stepwise gradient elution using an ODS silica gel column with methanol-water in a concentration varying from 5% to 100%. The methanol-water 8% elution fraction Fr.A-3 was collected and purified to obtain compound Gamahorin A.

5. The preparation method according to claim 4, characterized in that: The fermentation product of Neopestalotiopsis sp.SCSIO 41422 is prepared by inoculating Neopestalotiopsis sp.SCSIO 41422 onto an MB solid culture medium, culturing the culture medium under a static condition of 28°C until new colonies grow out, then transferring the culture medium to an MB seed liquid culture medium, culturing the culture medium on a rotary shaker at a rotation speed of 180 rpm at 28°C for three days to obtain a seed liquid, inoculating the seed liquid into a rice culture medium, and culturing the culture medium at 26°C for 30 days under a static condition. The rice culture medium is composed of 0.48 g of sea salt and 250 mL of water per 200 g of rice. The MB seed liquid culture medium contains, by mass fraction, 1.5% of malt extract powder and 2.4% of sea salt, with a pH value of 7.4-7.

8. The MB solid culture medium contains, by mass fraction, 1.5% of malt extract powder, 1.8% of agar powder, and 2.4% of sea salt, with a pH value of 7.4-7.

8.

6. The preparation method according to claim 4, characterized in that: The purification was performed by semi-preparative high performance liquid chromatography, with a mobile phase of 50% CH3OH / H2O, a flow rate of 3 mL / min, and a t R =26min.

7. Use of the compound Gamahorin A or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-renal fibrosis drug.

8. An anti-renal fibrosis drug, characterized in that: The invention contains the compound Gamahorin A or a pharmaceutically acceptable salt thereof as claimed in claim 1 as an active ingredient.

9. The anti-renal fibrosis drug according to claim 8, characterized in that: The drug is prepared into a clinically acceptable drug preparation by taking Gamahorin A as the main component and adding a pharmaceutically acceptable carrier or excipient, or adding pharmaceutically acceptable auxiliary materials or auxiliary components.

10. The anti-renal fibrosis drug according to claim 8 or 9, characterized in that: The content of Gamahorin A is 1-99% w / w based on the total weight of the drug.

Images