Method for screening small molecule compound capable of regulating and controlling brown adipose differentiation from ginseng and application of small molecule compound

Abstract

The invention discloses a method for screening a small molecule compound capable of regulating and controlling brown adipose differentiation from ginsenoside and application of the small molecule compound. The present invention identifies a novel binding pocket in PPAR [gamma]-LBD. Through molecular docking, the Ginsenoside Rg3 is obtained by screening a plurality of ginsenosides, and experiments prove that the Ginsenoside Rg3 has relatively low toxicity to adipose-derived stem cells (ADSC) and can be used for remarkably activating an ADSC browning process at the same time.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of drug screening, in particular to a method for screening small molecule compounds capable of regulating the differentiation of brown adipose tissue from ginseng and application thereof. BACKGROUND

[0002] PPARγ (peroxisome proliferator-activated receptor gamma) is a nuclear receptor transcription factor that plays a crucial role in the regulation of obesity. PPARγ promotes the differentiation of preadipocytes into mature adipocytes by activating lipogenesis-related genes, affecting lipid balance and storage. Activating PPARγ can improve insulin sensitivity, reduce blood glucose levels, improve adipocyte function and insulin signaling, thereby indirectly affecting obesity-related metabolic disorders. Its agonists (such as thiazolidinediones) have been used as drugs for the treatment of type II diabetes. Fat browning refers to the process of white adipose tissue (WAT) transforming into brown adipose tissue (BAT) characteristics. This process has potential importance in improving obesity and metabolic health. In recent years, it has been found that Bardoxolone (CDDO) can effectively agonize PPARγ, promote white fat browning, and thus regulate adipocyte metabolism, improve insulin sensitivity and reduce inflammation, which is of great significance for alleviating obesity. However, the binding mode of CDDO with PPARγ is not clear, and its cytotoxicity is relatively large, which limits its further clinical application.

[0003] Panax ginseng is a traditional Chinese herbal medicine with a long history of use. One of its main active ingredients is ginsenosides, a class of steroidal saponins with a wide range of biological activities. Ginsenosides include a variety of compounds such as Rg1, Rb1, Re, Rd, etc., each with different pharmacological effects. In recent years, the potential of ginsenosides in treating metabolic diseases has attracted widespread attention. Ginsenosides can increase the expression of insulin receptors and insulin signaling, improve insulin resistance, and some ginsenosides (such as Rg1 and Rb1) can stimulate insulin secretion by pancreatic beta cells; ginsenosides can promote lipolysis and energy consumption by activating AMPK (adenosine monophosphate-activated protein kinase) and regulating the expression of lipolysis-related genes.

[0004] Natural products are an important source of discovering new PPARγ ligands. Many compounds targeting PPARγ with agonistic effects have been identified in traditional medicinal plants. However, the complexity of natural products poses challenges for drug development and screening, including technical obstacles in screening, isolation, characterization, and optimization. For example, traditional Chinese medicine and natural products contain a large number of complex components, and only a few key components may exhibit biological activity, which increases the difficulty of identification and screening.

[0005] Traditional methods for discovering and verifying active compounds in natural products are costly and time-consuming. Virtual screening (VS) uses large-scale databases to search and screen potential active compounds from compound libraries, with higher success rates and lower costs, which can improve screening efficiency and accelerate drug development. VS is an important method for finding potential active compounds. It mainly includes structure-based virtual screening (SBVS) and ligand-based virtual screening (LBVS). SBVS uses the three-dimensional structure information of target proteins to predict and screen the affinity and stability of small molecule compounds and targets through molecular docking and molecular dynamics simulation. LBVS is based on the structure and properties of known active ligands, and uses pharmacophore modeling and quantitative structure-activity relationship (QSAR) to screen compounds with similar structures or properties. Typical screening methods include pharmacophore screening and molecular similarity search.

[0006] Molecular docking is an important method in computational chemistry, which is used to predict the interaction and binding mode between molecules. In the process of molecular docking, the pocket provides a binding space for the ligand, so that the ligand can form a stable complex with the protein. The structure and characteristics of the pocket directly affect the affinity between the ligand and the protein. By analyzing the size, shape, charge distribution and other characteristics of the pocket, the strength and specificity of the ligand and protein binding can be predicted. Therefore, the selection of the pocket is of great significance to improve the docking accuracy, screen potential drug molecules with high affinity and guide drug design.

[0007] By comprehensively using Fpoket, molecular docking and molecular dynamics simulation technology, we identified a novel binding pocket of PPARγ in H3 helix and H2' helix.

[0008] By molecularly docking different ginsenoside compounds with a novel pocket of PPARγ, we screened Ginsenoside Rg3 as a PPARγ agonist. Experiments demonstrated that Ginsenoside Rg3 exhibits low toxicity to adipose-derived stem cells (ADSCs) while significantly activating the browning process in ADSCs. This screening method provides a novel strategy for the screening and identification of PPARγ agonists. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the above-mentioned problems and to provide a method for screening small molecule compounds that can regulate the differentiation of brown fats from ginseng and its application.

[0010] The first objective of this invention is to provide a method for identifying novel binding pockets of PPARγ.

[0011] A second objective of this invention is to provide a method for using virtual screening for PPARγ agonist screening.

[0012] A third objective of this invention is to provide PPARγ agonist candidates obtained by the method described above.

[0013] A fourth objective of this invention is to provide an inducing compound that promotes the browning of fatty acids.

[0014] The above-mentioned objective of this invention is achieved through the following technical solution:

[0015] This invention provides a method for screening small molecule compounds from ginseng that can regulate the differentiation of brown fats and its application, specifically including the following steps:

[0016] 1. Identification of a novel binding pocket for PPARγ

[0017] Using F-pocket and molecular docking, the binding pockets in the PPARγ-LBD region were analyzed. Pocket 1 was ultimately found to be the most ideal binding pocket.

[0018] 2. Virtual Filtering

[0019] Eighteen known ginsenoside compounds were screened, and molecular docking was performed using Pocket1 as the binding pocket.

[0020] 3. Cytotoxicity assessment

[0021] The compound Ginsenoside Rg3, which had the highest docking score, was screened using the above method. The cytotoxicity of ADSC was detected by CCK-8 and apoptosis flow cytometry, and it was found that Ginsenoside Rg3 had extremely low toxicity to ADSC.

[0022] 4. Assessment of adipogenic differentiation capacity

[0023] The morphology and lipid droplet formation of ADSCs were observed using fiber photography and Oil Red staining. The expression levels of ADSC adipogenesis-related genes were detected by qPCR, and it was found that Ginsenoside Rg3 can enhance the adipogenic differentiation ability of ADSCs.

[0024] 5. Assessment of browning ability

[0025] qPCR analysis of the expression of adipocyte browning-related genes revealed that Ginsenoside Rg3 can enhance the browning ability of adipocytes (ADSCs).

[0026] This invention offers the following advantages: it identifies a novel PPARγ binding pocket, providing valuable information for the efficient screening of selective PPARγ modulators. The method saves time and cost, and improves screening efficiency and hit rate. Through this virtual screening method, highly effective selective modulators targeting PPARγ can be obtained, promoting adipose tissue browning, which is significant for regulating adipocyte metabolism and improving insulin sensitivity. Attached Figure Description

[0027] Figure 1 To identify a novel binding pocket for PPARγ in Example 1;

[0028] Figure 2 Example 1 shows the molecular docking binding mode of Ginsenoside Rg3 and PPARγ.

[0029] Figure 3 CCK-8 assay of Ginsenoside Rg3 on ADSC cells in Example 3;

[0030] Figure 4 Apoptosis of ADSC cells at different concentrations of Ginsenoside Rg3 was detected by flow cytometry in Example 3.

[0031] Figure 5 In Example 3 Figure 4 Statistical graph of apoptosis in ADSC cells;

[0032] Figure 6 Microscopic images of ADSC at different time points in Example 4;

[0033] Figure 7 Oil Red staining micrograph of ADSC on day 8 in Example 4;

[0034] Figure 8 In Example 4 Figure 7 Statistical chart of the proportion of cinnabar staining;

[0035] Figure 9 Statistical graph of qPCR results for the expression of genes related to lipid droplet formation in Example 4;

[0036] Figure 10 Statistical graph of qPCR results for browning-related gene expression in Example 5. Detailed Implementation

[0037] The principles and features of this invention are described below with reference to examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field.

[0038] Example 1. Screening of compounds from ginsenosides that can bind PPARγ

[0039] 1. Identification of a novel binding pocket for PPARγ

[0040] Using the Fpocket website (https: / / fpocket.sourceforge.net / ), a deep learning algorithm for predicting protein pockets, the volume and druggability of binding pockets were evaluated, generating a total of 5 binding pockets (Table 1). Next, the CDDO small molecule was docked to each of these pockets, with Pocket 1 achieving the highest molecular docking score. Figure 1 Molecular dynamics simulations of the PPARγ-CDDO complex formed at Pocket 1 showed that the RMSD value of the complex fluctuated around 0.45 Å, indicating that the energy system of the protein complex tends to be stable under the binding mode. Table 1The characteristics of five pockets predicted by Fpocket

[0041] 2. Virtual Filtering

[0042] Eighteen known ginsenoside compounds were screened, and molecular docking was performed using Pocket 1 as the binding pocket. The results showed that Ginsenoside Rg3 had the highest molecular docking score, reaching 7.56. The binding mode of Ginsenoside Rg3 to PPARγ is as follows: Figure 2 As shown. Table 2 Molecular docking scoring information

[0043] Example 2. Extraction and Induction of Differentiation of ADSCs (Adipose-Derived Stem Cells)

[0044] 1. Extraction of ADSC

[0045] Mice were euthanized, and fresh adipose tissue was thoroughly washed with PBS to remove blood, residual tissue, and hair. The tissue was then minced. Krebs-Ringerbicarbonate digestion buffer was prepared, and the adipose tissue was immersed in the buffer. 5% BSA and 0.5% type I collagenase were added, and the mixture was incubated at 37°C with shaking at 150 rpm for 90 min. The mixture was then filtered through a 100 μm filter and centrifuged at 1500 rpm for 10 min at room temperature. Three times the volume of red blood cell lysis buffer was added, and the cells were lysed at room temperature for 2 min. The mixture was then centrifuged at 4°C and 500g for 5 min, and the red supernatant was discarded. The cells were washed twice with PBS and cultured in DMEM / F12 medium with 15% FBS and 5% CO2 at 37°C for 48 h.

[0046] 2. Induction of differentiation of ADSC

[0047] Replace the DMEM / F12 medium in the above culture system with DMEM / F12 induction medium (containing 0.5 mM isobutyl methylxanthine (IBMX), 125 nM indomethacin, 5 μM dexamethasone, 850 nM Minsulin, and 1 nM T3), and harvest differentiated and mature adipocytes on day 8.

[0048] Example 3. Toxicity assessment of Ginsenoside Rg3 compound

[0049] Different concentrations of Ginsenoside Rg3 were added to the DMEM / F12 induction medium in the ADSC induction culture system of Example 2. On day 8 of induced differentiation, the toxicity of Ginsenoside Rg3 was assessed by CCK-8 cell viability assay and flow cytometry apoptosis assay. Throughout the tested concentration range (10 nM–20 μM), ADSC cell viability did not significantly decrease. Figure 3 Apoptosis levels showed no significant difference below 10 μM, but were slightly elevated above 10 μM. Figure 4 and Figure 5 This indicates that Ginsenoside Rg3 exhibits low toxicity during ADSC-induced differentiation.

[0050] Example 4. Effect of Ginsenoside Rg3 compound on the adipogenic differentiation ability of ADSCs

[0051] 1. Effects of Ginsenoside Rg3 on ADSC morphological changes

[0052] ADSCs were extracted according to the method described in Example 2 above. During differentiation induction, 200 nM Ginsenoside Rg3 was added to the culture system. Microscopic photographs were taken daily to observe morphological changes. Figure 6 As shown, compared with the control induction culture medium group, the Ginsenoside Rg3 group had more mature adipocytes in the field of view, indicating that Ginsenoside Rg3 can promote adipogenic differentiation of ADSCs.

[0053] 2. Effect of Ginsenoside Rg3 on ADSC lipid droplet formation

[0054] ADSCs were extracted according to the method described in Example 2 above. During differentiation induction, 200 nM Ginsenoside Rg3 was added to the culture system. Cells were collected on day 8 of induction and fixed with 4% paraformaldehyde solution for 1 hour; washed three times with PBS; stained with 0.5% Oil Red staining solution prepared with 1,2-propanediol; incubated at 37°C for 4 hours; the Oil Red solution was removed; residual Oil Red dye was repeatedly washed with PBS at least three times; the cells were then covered with PBS and observed and photographed under a microscope. Figure 7 As shown, the staining results revealed a significant increase in the number of cells containing red lipid droplets in the Ginsenoside Rg3 group. Figure 7 and Figure 8 We further extracted RNA from induced mature adipocytes and evaluated their lipid droplet formation capacity using qPCR experiments. Figure 9 As shown, Ginsenoside Rg3 significantly upregulated the expression levels of fatty acid transporter CD36, fatty acid binding protein Fapp4, fatty acid synthase Fasn, and lipoprotein lipase Lpl, indicating that Ginsenoside Rg3 has the ability to promote the formation of lipid droplets from ADSC.

[0055] Example 5. Effect of Ginsenoside Rg3 on browning of ADSC

[0056] ADSCs were extracted according to the method described in Example 2 above. During differentiation induction, 200 nM Ginsenoside Rg3 was added to the culture system. Cells were collected on day 8 of induction, and the expression of adipocyte browning-related genes, including thermogenic genes Ucp1, Prdm16, Cox8b, Dio2, and Pgcla, was analyzed using qPCR. Figure 10 As shown, Ginsenoside Rg3 can significantly upregulate the expression of browning genes in adipocytes, indicating that Ginsenoside Rg3 can successfully induce primary ADSCs to turn into brown adipose tissue.

[0057] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for screening small molecule compounds from ginseng that can regulate the differentiation of browning lipids, characterized in that, Includes the following steps: S1: Using computer chemistry techniques, a novel binding pocket was identified from PPARγ; S2: Using virtual screening technology, candidate compounds with high binding affinity to PPARγ were screened from the ginsenoside library; S3: Through cell experiments, verify the toxicity of the candidate compound to adipose-derived stem cells (ADSC) and its ability to promote browning differentiation.

2. The method according to claim 1, characterized in that, The computer chemistry techniques mentioned are Fpooket, molecular docking, and molecular dynamics simulation.

3. The method according to claim 1, characterized in that, The virtual screening technology is a structure-based molecular docking.

4. The method according to claim 1, characterized in that, The cell experiments include CCK-8 cell viability detection, flow cytometry apoptosis detection, Oil Red staining detection, and qPCR detection of browning-related gene expression.

5. The method according to claim 1, characterized in that, The small molecule compound that can regulate the differentiation of browning fats is Ginsenoside Rg3.

6. Use of the Ginsenoside Rg3 as described in claim 5 in the preparation of a medicament for regulating lipid metabolism.

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