A small molecule compound from a marine fungus and application thereof in preparation of a medicine for treating anti-kidney fibrosis

Abstract

This invention discloses a small molecule compound derived from marine fungi and its application in the preparation of drugs for treating renal fibrosis, relating to the field of marine natural products. The compound, Gamahorin A, has the structural formula shown in Formula (I). Gamahorin A exhibits significant anti-fibrotic activity by inhibiting TGF-β1-induced expression of α-smooth muscle actin (α-SMA) and the production of ECM components (type I collagen and fibronectin), thus inhibiting the development of renal fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of natural product application technology, specifically relating to a small molecule compound derived from marine fungi, its preparation method, and its application in the preparation of anti-renal fibrosis drugs. Background Technology

[0002] Chronic kidney disease (CKD) has become a significant global health problem, affecting approximately 700 million people. Chronic inflammation and renal fibrosis are the main driving factors of this disease. Chronic tubular nephritis, diabetic nephropathy, and hypertensive nephropathy share common fibrotic pathological features, ultimately leading to end-stage renal failure. Kidney damage causes nephron atrophy, fibroblast transformation into myofibroblasts, and excessive accumulation of the extracellular matrix (ECM). The ECM consists of a fibrous network and gel-like components, including collagen, elastin, fibronectin, and laminin. Collagen, primarily produced by myofibroblasts, 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 antifibrotic drugs show limited efficacy and significant side effects. Pirfenidone (PFD) showed only transient improvement in a phase II clinical trial, and some patients discontinued the drug due to adverse reactions such as rash and gastrointestinal problems. The pharmacokinetics of PFD are dependent on renal function, limiting its clinical application. Therefore, developing novel, safe, and effective antifibrotic drugs is of great significance. Summary of the Invention

[0003] To address the above-mentioned problems, the technical solution adopted by this invention is as follows:

[0004] The first objective of this invention is to provide a novel small molecule compound, Gamahorin A or its pharmaceutical salt, with anti-renal fibrosis activity, as shown in formula (I):

[0005]

[0006] Formula (I).

[0007] The second objective of this invention is to provide a method for preparing the aforementioned compound Gamahorin A, which is isolated and prepared from the fermentation product of the marine-derived Neopsestalotiopsis sp. SCSIO 41422.

[0008] Preferably, the following steps are included:

[0009] (a) The fermentation product of Neopestalotiopsis sp. SCSIO 41422 was prepared by soaking the fermentation product in ethyl acetate, and the ethyl acetate extract was concentrated by evaporation to obtain the extract.

[0010] (b) The crude extract was fractionated by silica gel medium-pressure liquid chromatography with gradient elution using different volume ratios of petroleum ether and dichloromethane mixtures as eluents at ratios of 1:0, 3:1, 2:1, 1:1, and 0:1. The petroleum ether to dichloromethane elution fractions of 3:1 to 1:1 (Frs. 3–7) were further fractionated by gradient elution using an ODS silica gel column with a methanol-water gradient eluent ranging from 5% to 100%. The 8% methanol-water eluent fraction (Fr. A-3) was collected and then purified to obtain the compound Gamahorin A.

[0011] Preferably, the fermentation product of Neopestalotiopsis sp. SCSIO 41422 is obtained by inoculating Neopestalotiopsis sp. SCSIO 41422 onto MB solid medium, culturing it under static conditions at 28°C until new colonies grow, and then transferring it to MB seed culture medium. The seed culture is then cultured for three days at 28°C on a rotary shaker at 180 rpm to obtain the seed culture. The seed culture is then subjected to large-scale fermentation using rice culture medium, culturing it under static conditions at 26°C for 30 days. The rice culture medium consists of 200g of rice, 0.48g of sea salt, and 250mL of water per bottle. The MB seed culture medium contains 1.5% malt extract powder, 2.4% sea salt, and has a pH of 7.4–7.8 by mass fraction. The MB solid medium contains 1.5% malt extract powder, 1.8% agar powder, 2.4% sea salt, and has a pH of 7.4–7.8 by mass fraction.

[0012] Preferably, the purification is performed by semi-preparative high-performance liquid chromatography (HPLC), with a mobile phase of 50% CH3OH / H2O and a flow rate of 3 mL / min. R = 26 min.

[0013] A third objective of this invention is to provide the use of the aforementioned compound Gamahorin A or its pharmaceutically acceptable salt in the preparation of an anti-renal fibrosis drug. This anti-renal fibrosis drug exhibits significant anti-fibrotic activity by inhibiting TGF-β1-induced expression of α-smooth muscle actin (α-SMA) and the production of ECM components (type I collagen and fibronectin).

[0014] A fourth objective of this invention is an anti-renal fibrosis drug containing the aforementioned compound Gamahorin A or its pharmaceutical salt as an active ingredient.

[0015] Preferably, the drug is prepared into a clinically acceptable pharmaceutical formulation with Gamahorin A as the main component, plus a pharmaceutically acceptable carrier or excipient, or with pharmaceutically acceptable excipients or auxiliary components, and the content of Gamahorin A is 1-99% (w / w) based on the total weight of the drug.

[0016] By adopting the above technical solution, the present invention has the following beneficial effects:

[0017] In the course of studying the secondary metabolites of the sponge-symbiotic fungus *Neopestalotiopsis* sp. SCSIO 41422, this invention isolated a novel small molecule compound, Gamahorin A, which can significantly inhibit transforming growth factor-β (TGF-β)-induced renal fibrosis and can be used to prepare anti-renal fibrosis drugs. Therefore, Gamahorin A described in this invention can serve as a supplementary or combination therapy option after pirfenidone (PFE) resistance, which is of great significance for the development of marine drug resources in China.

[0018] The fungus Neopestalotiopsis sp. SCSIO41422 of this invention was deposited on August 4, 2022 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, with accession number GDMCC NO. 62689. Attached Figure Description

[0019] Figure 1 The chemical structure of the small molecule compound Gamahorin A is shown.

[0020] Figure 2 For Gamahorin A 1 H- 1 Key 2D NMR data from H COSY and HMBC

[0021] Figure 3 Measured and calculated ECD spectra of Gamahorin A;

[0022] Figure 4Results of Gamahorin A inhibiting transforming growth factor-β (TGF-β). (A) Gamahorin A at a concentration of 10 µM inhibited TGF-β1-induced α-smooth muscle actin (α-SMA) expression; (B) Gamahorin A at a concentration of 10 µM inhibited TGF-β1-induced collagen I production; (C) Gamahorin A at a concentration of 10 µM inhibited TGF-β1-induced fibronectin production; (D) Data were analyzed by one-way ANOVA and subsequent Bonferroni multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (In the figure, compound 1 is Gamahorin A, and compounds 5-8, 11, and 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

[0023] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the invention and are not intended to limit the invention.

[0024] Example 1: Fermentation of marine fungus Neopestalotiopsis sp. SCSIO 41422 and isolation of Gamahorin A

[0025] 1.1 Isolation and Preservation of Strains: Neopestalotiopsis sp. SCSIO41422, a sponge symbiotic fungus collected near Weizhou Island, Beihai City, Guangxi Zhuang Autonomous Region, China, is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, with accession number GDMCC NO. 62689.

[0026] 1.2 Fermentation: First, the strain preserved in paraffin oil was inoculated into MB plates (1.5% malt extract, 1.8% agar powder, 2.4% sea salt, pH 7.4–7.8 by mass fraction) and cultured until new colonies grew. Then, it was inoculated into MB liquid medium (1.5% malt extract, 2.4% sea salt, pH 7.4–7.8 by mass fraction) and cultured on a shaker (180 rpm) at 28 °C for 3 days. Then, it was inoculated into sterilized rice medium (200 g rice, 2.4% coarse sea salt, 250 mL water) for fermentation. 200 bottles (about 40 kg rice) were allowed to ferment statically at room temperature for 30 days to prepare rice culture product of the fungus Neopestalotiopsis sp. SCSIO41422.

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

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

[0029] Example 2: Structural Identification of Gamahorin A

[0030] The chemical structure of the small molecule compound Gamahorin A isolated in Example 1 is shown in Figure 1. The structural derivation process is as follows:

[0031]

[0032] Formula 1 Chemical structure of the small molecule compound Gamahorin A

[0033] Compound Gamahorin A was isolated as a brown oily liquid, and its molecular formula was determined to be C using HRESIMS data. 12 H 14 O4 indicates six degrees of unsaturation. 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 alkenyl groups [δ C / H 120.1 / 6.79 (CH-5), 138.4 / 7.39 (CH-6)], one methylene [δ C / H 64.7 / 3.66 (CH2-10)], two methines [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 1 H- 1 H COSY spectrum correlation signal ( Figure 2 The proton spin-coupled systems in H-3 / H-4, H-3 / H-9, and H-4 / H-10. Combined with the correlation signals in the HMBC spectrum ( Figure 2 Based on the C-H 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, the planar structure of compound Gamahorin A is inferred as follows: Figure 1 As shown.

[0034] Table 1. Compound Gamahorin A 1 H (500 MHz) and 13 C (125 MHz) NMR data (CD3OD)

[0035]

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

[0037] Example 3: Test of the anti-renal fibrosis activity 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 in 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, followed by treatment 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) and then centrifuged at 13,000g for 15 min at 4°C. Total protein concentration was determined using a BCA kit (Epizyme, ZJ102). Quantitative proteins were mixed with loading buffer (Epizyme, LT101S) and boiled at 100°C for 10 min, then separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, ISEQ00010). After blocking the membrane at room temperature for 1 hour, it was incubated overnight at 4°C with a specific primary antibody. The membrane was then incubated with a secondary antibody (anti-rabbit or anti-mouse IgG) at room temperature for 2 hours. Western blot bands were detected by ECL substrate (Fdbio, FD8020) and analyzed using Image-J 1.52a software.

[0040] 3.3 Statistical Analysis: All experiments were performed at least three times. All data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA), including independent samples t-tests, one-way ANOVA, and two-way ANOVA. P < 0.05 was considered statistically significant (*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 simulate epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition. We screened and evaluated seven marine natural products isolated from the same microbial strain, including Gamahorin A (compound 1). The results showed that Gamahorin A at a concentration of 10 µM exhibited significant anti-fibrotic activity by inhibiting TGF-β1-induced expression of α-smooth muscle actin (α-SMA) and the production of ECM components (type I collagen and fibronectin). Figure 4 Of the seven compounds tested, Gamahorin A has proven to be the most promising and potent inhibitor of renal fibrosis, even outperforming pirfenidone, the current clinical standard of care for fibrosis. Gamahorin A also demonstrated the strongest ECM modulation, significantly inhibiting the accumulation of fibronectin and type I collagen, surpassing all other derivatives.

[0042] In summary, Gamahorin A exhibits significant anti-fibrotic activity by inhibiting TGF-β1-induced expression of α-smooth muscle actin (α-SMA) and the production of ECM components (type I collagen and fibronectin), thus inhibiting 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 shows great promise for clinical application. This invention is of great significance to the development of new drugs with independent intellectual property rights in China.

[0043] The above description is a detailed explanation of preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical concept presented in the present invention should fall within the patent scope covered by the present invention.

Claims

1. The chemical structural formula of compound Gamahorin A or its medicinal salt is shown in formula (I): Formula (I).

2. A method for preparing the compound Gamahorin A according to claim 1, characterized in that, It was isolated and prepared from the fermentation products of Neopestalotiopsis sp. SCSIO 41422, and includes the following steps: (a) Neopesteralotiopsis sp. SCSIO 41422 was inoculated onto MB solid medium and cultured at 28°C under static conditions until new colonies appeared. It was then transferred to MB seed culture medium and cultured for three days at 28°C on a rotary shaker at 180 rpm to obtain seed culture. The seed culture was inoculated onto rice medium and cultured at 26°C under static conditions for 30 days to obtain fermentation product. The fermentation product was extracted with ethyl acetate, and the ethyl acetate extract was evaporated and concentrated to obtain the extract. The Neopesteralotiopsis sp. SCSIO 41422 has the preservation number GDMCC NO. 62689. The rice medium consisted of 200g rice, 0.48g sea salt, and 250mL water. The MB seed culture medium contained 1.5% malt extract powder and 2.4% sea salt by mass fraction, with a pH of [missing value]. 7.4–7.8, the MB solid culture medium is composed of 1.5% malt extract powder, 1.8% agar powder, 2.4% sea salt, and pH 7.4–7.8 by mass fraction; (b) The extract was fractionated by silica gel medium-pressure liquid chromatography with gradient elution using petroleum ether and dichloromethane mixtures at different volume ratios of 1:0, 3:1, 2:1, 1:1, and 0:

1. The petroleum ether to dichloromethane elution fractions of 3:1 to 1:1 were eluted with a gradient of dichloromethane and methanol at methanol concentrations of 1%, 3%, 5%, 10%, 50%, and 70%, respectively. Thin-layer chromatography (TLC) guided the eluent combination, ultimately yielding 16 fractions, named Frs. 1–16. Fractions Frs. 3–7 were further fractionated using an ODS silica gel column with gradient elution using methanol-water at concentrations ranging from 5% to 100%. The 8% methanol-water elution fraction Fr. A–3 was collected and purified to obtain compound Gamahorin A. The purification was performed by semi-preparative high-performance liquid chromatography (SHPLC) with a mobile phase of 50% CH3OH / H2O and a flow rate of 3 mL / min. R = 26 min.

3. The use of the compound Gamahorin A of claim 1 or its pharmaceutical salt in the preparation of an anti-renal fibrosis drug.

4. An anti-renal fibrosis drug, characterized in that, It contains the compound Gamahorin A of claim 1 or its pharmaceutical salt as an active ingredient.

5. The anti-renal fibrosis drug according to claim 4, characterized in that, The drug is a clinically acceptable pharmaceutical preparation made by adding Gamahorin A as the active ingredient, along with a pharmaceutically acceptable carrier or excipient, or with pharmaceutically acceptable excipients.

6. The anti-renal fibrosis drug according to claim 4 or 5, characterized in that, The content of Gamahorin A is 1-99% w / w, based on the total weight of the drug.

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