Application of polyphyllin VII in preparation of Akt signaling pathway inhibitor targeting GRB2

Abstract

The invention provides an application of polyphyllin VII in preparation of an Akt signaling pathway inhibitor targeting GRB2, the invention reveals that PPVII is specifically combined with GRB2 protein and regulates and controls the function of the GRB2 protein for the first time, the Akt pathway inhibition effect is exerted, and the molecular mechanism of PPVII is specifically shown as follows: molecular docking and dynamic simulation confirms that PPVII and an SH3 structural domain of GRB2 form a stable compound, and the stable compound is used for inhibiting the Akt pathway of the GRB2. And the key amino acid binding sites are Ile65, Gln157, Tyr160 and Phe182. Cell thermal transfer experiments (CETSA) show that PPVII can significantly enhance the thermal stability of GRB2 protein, surface plasmon resonance (SPR) determines the direct binding affinity KD = 58.5 [mu] M, functional research proves that PPVII inhibits an Akt signal channel through a GRB2 dependency mode, the polyphyllin VII is used as a GRB2 targeted inhibitor, and by specifically binding with an SH3 structural domain of GRB2 and inhibiting a downstream Akt signal channel, the polyphyllin VII can be used for inhibiting the Akt signal channel in the GRB2 targeted inhibitor. And a new strategy and a candidate compound are provided for intervention of related diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to an application of Paris saponin VII in preparation of a medicine for preventing and / or treating bladder cancer. BACKGROUND

[0002] Growth factor receptor-bound protein 2 (GRB2) is an important member of the adaptor protein family and plays a key role in cell signal transduction. GRB2 interacts with various signaling molecules through its SH2 and SH3 domains, and is involved in the regulation of important signaling pathways such as Ras / MAPK and PI3K / Akt. Studies have shown that abnormal expression or dysfunction of GRB2 is closely related to the occurrence and development of various diseases, but the precise regulatory mechanism still needs to be further explored.

[0003] The development of inhibitors targeting GRB2 still faces challenges: the reported small molecule inhibitors of GRB2 (such as peptide compounds) have poor membrane penetration and low stability, which limits their application; some natural products (such as flavonoids and alkaloids) have been reported to affect GRB2-related signaling pathways, but there is a lack of direct targeting of GRB2 evidence; the binding mechanism of the SH3 domain of GRB2 to ligands and the structure-function relationship have not been fully elucidated, and the role of key amino acid sites still needs to be further verified. The current regulatory strategies for GRB2 have the following problems: the developed GRB2 inhibitors (such as linear peptide analogs) have poor stability and are complex to synthesize, making it difficult to meet clinical needs; there are potential GRB2 modulators in natural products, but there is a lack of clear target validation and mechanism research; the specific small molecule binding mechanism of the GRB2-SH3 domain has not been fully elucidated, which limits the development of highly selective inhibitors.

[0004] Paris saponin VII is a steroidal saponin compound isolated from the traditional Chinese medicine Paris polyphylla, and has various biological activities. Existing studies have shown that PP VII can regulate signaling pathways such as NF-κB and MAPK, and there is currently no related report on Paris saponin VII as an Akt signaling pathway inhibitor targeting GRB2 and its treatment of bladder cancer. SUMMARY

[0005] The application provides a new use of Paris saponin VII (PP VII), i.e., an application of Paris saponin VII in preparation of an Akt signaling pathway inhibitor targeting GRB2 and an application of Paris saponin VII in preparation of a medicine for preventing and / or treating bladder cancer.

[0006] The chemical structural formula of the Paris saponin VII is as follows:

[0007] As an Akt pathway inhibitor targeting GRB2, PPⅦ specifically binds to GRB2 protein and inhibits the activity of downstream protein kinase Akt, thereby achieving the purpose of inhibiting bladder cancer.

[0008] The active ingredient of the drug of the present application is PPⅦ, and one or more pharmaceutically acceptable adjuvants or other active ingredients can be added to the drug to inhibit the effect; the preparation can be prepared into tablets, pills, capsules, granules, oral liquids, injection liquids and other pharmaceutically acceptable forms.

[0009] The present application proves that the tumor volume and weight of the PPⅦ treatment group are significantly reduced compared with the control group through animal experiments (nude mouse xenotransplantation model); in the T24CDDP bladder cancer cell model, PPⅦ inhibits cell proliferation, and Western blot analysis shows that PPⅦ significantly reduces the p-Akt level.

[0010] The present application uses AlphaFold3 software to predict the three-dimensional binding conformation of the active ingredient to the key target; Western blot experiment is used to detect the expression level of the key target protein or gene; CETSA is used to detect the influence of the active ingredient on the thermal stability of the key target; surface plasmon resonance (SPR) is used to determine the binding kinetics of the active ingredient and the key target; molecular dynamics simulation is used to evaluate the stability of the complex and analyze the number of key interaction residues and hydrogen bonds; 1. Through AlphaFold3 structure analysis and molecular dynamics simulation verification, PPⅦ specifically binds to GRB2 protein, and the average value of the binding free energy is-54.6469±6.0130 kcal / mol; 2. Through CETSA experiment verification, the relative protein level of GRB2 is significantly improved at 57℃-82℃, and PPⅦ enhances the thermal stability of GRB2 protein; 3. Through Western Blot verification, GRB2 knockdown or PPⅦ treatment significantly reduces the expression level of p-Akt and inhibits Akt phosphorylation; 4. The SPR experiment verifies that PPⅦ directly binds to GRB2, and the binding affinity KD is 58.5 μM; 5. In the molecular dynamics simulation, a stable GRB2-PPⅦ complex is formed, and the number of hydrogen bonds is greater than or equal to 2.

[0011] Compared with existing technologies, this invention offers the advantages of strong targeting: First, strong targeting. PPVII has been demonstrated for the first time to specifically bind to the SH3 domain of the GRB2 protein (key binding sites: Ile65, Gln157, Tyr160, and Phe182). Surface plasmon resonance (SPR) has been used to precisely measure the binding affinity (KD = 58.5 μM), demonstrating higher target selectivity than traditional signaling pathway modulators. Second, its mechanism of action is well-defined. AlphaFold3 was used to accurately predict the three-dimensional structure of the PPVII-GRB2 complex. Cellular thermal shift assays (CETSA) demonstrated that PPVII significantly enhanced the thermal stability of GRB2. 200 ns molecular dynamics simulations verified the stability of the complex. This invention, for the first time, elucidates the molecular mechanism by which a natural small molecule regulates GRB2 protein function. This invention innovatively reveals the specific interaction mechanism of the natural small molecule PPVII with the GRB2 protein, overcoming key challenges of existing technologies such as insufficient targeting and unclear mechanism of action. This provides a new tool molecule and research approach for signaling pathway regulation research, and offers new strategies and candidate compounds for the prevention and treatment of bladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a schematic diagram of the molecular docking simulation between PPVII and GRB2 based on alphafold3; Figure 2 The molecular dynamics simulation and system stability analysis results of the interaction between PPVII and GRB2 are shown in the figure above, where the RMSD result is shown, and the RMSF result is shown in the figure below. Figure 3 It is a schematic diagram of Rogyr of GRB2 and PPVII; Figure 4 is a schematic diagram of the hydrogen bond counting analysis between GRB2 and PPVII; Figure 5 is a schematic diagram of GRB2-PPVII binding free energy; Figure 6 This is a schematic diagram of the analysis of key amino acid residues for GRB2-PPVII binding; Figure 7 This is a diagram of the binding pattern between GRB2 and PPVII; Figure 8 The results of CETSA analysis of the effect of PPVII on the thermal stability of GRB2 (left panel) and GAPDH (right panel) are shown; Figure 9 is a schematic diagram showing the interaction between PPVII and GRB2 as shown by SPR experiments; Figure 10 This is a schematic diagram of the p-Akt expression results after knocking down the GRB2 protein expression level in bladder cancer cells. The upper figure is the protein immunoblotting result, and the lower figure is the statistical result of p-Akt expression; Figure 11 Schematic diagram of protein expression results after PPVII treatment of T24CDDP cells, where the upper figure is the Western blotting result, and the lower figure is the statistical result of protein expression; Figure 12 IC values ​​of PPVII (left) and cisplatin (right) in MTT cell proliferation assay on T24CDDP cells 50 Value result; Figure 13 This is a graph showing the inhibitory effect of PPVII and cisplatin treatment on the volume and weight of T24CDDP transplanted tumors. The left figure is a schematic diagram of tumor size, the middle figure is a statistical graph of tumor volume, and the right figure is a statistical graph of tumor weight. DETAILED DESCRIPTION

[0013] The technical solutions of the present invention are further described below in conjunction with the accompanying drawings and specific embodiments. However, these embodiments should not be used to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available unless otherwise specified. After reading the description of the present invention, various equivalent changes, modifications, and modifications made by technicians in this field are within the scope defined by the claims of the present invention.

[0014] In the embodiment, paridis saponin VII (PPVII) is derived from Paridis yunnanensis ( Paris polyphylla ) of the isolated extract.

[0015] Reagents: RPMI 1640 medium (Gibco, USA), fetal bovine serum (Guangzhou Saiku Biotechnology Co., Ltd.), trypsin (Gibco, USA), Opti-MEM medium (Gibco, USA), Lipofectamine 2000 (Invitrogen, USA), h-GRB2 siRNA nucleic acid (Guangzhou Jidan Biotechnology Co., Ltd.).

[0016] Cell line: Bladder cancer cell T24, purchased from Guangzhou Saiku Biotechnology Co., Ltd.

[0017] Example 1: Molecular docking and dynamics simulation 1. Use AlphaFold3 software (https: / / alphafoldserver.com / ) to perform preliminary binding mode prediction for the full-length amino acid sequence of GRB2_HUMAN and Paris polyphylla saponin VII, and output the results. The structure superposition is as follows: Figure 1 As shown in the figure, Paris polyphylla saponin VII is bound to the same position in all the predicted results.

[0018] 2. Molecular docking was performed using AutoDock Vina (version 1.1.2). The docking space was defined as a 20 Å × 20 Å × 20 Å cubic box, the binding mode clustering energy range was set to 4 kcal / mol, and the global search exhaustive parameter was set to 16.

[0019] 3. For molecular dynamics simulations, the initial conformation of the complex was dissolved in a TIP3P water box, and Na⁺ or Cl⁻ ions were added to neutralize the system charge. Simulations were performed at 300 K and 1 atm using Amber software (version 2022). The SHAKE algorithm was used to constrain hydrogen bonds, with an integration step of 2 fs and a cutoff radius of 10 Å. The total simulation time was 200 ns. All calculations were performed using the pmemd.cuda GPU-accelerated module.

[0020] See the results Figure 1-7 ,from Figure 1 As can be seen in the figure, the structural analysis of PPVII and GRB2 based on alphafold3 shows that PPVII and GRB2 can bind to each other. RMSD statistical analysis of the molecular dynamics simulation results showed that the system did not begin to converge until 25 ns, and tended to a stable state after 100 ns ( Figure 2 In the RMSF results, the amino acids of the protein system show large fluctuations near some loop regions. From the fluctuation range, it can be seen that the fluctuation of the SH31 domain at the N-terminus is larger ( Figure 2 Further molecular dynamics simulations showed that the protein's radius of rotation remained stable at 19-20 Å, while the ligand's radius of rotation remained at 8 Å, indicating a stable conformation ( Figure 3 ). Hydrogen bond analysis showed that during most of the simulation process, PPVII maintained 2-3 stable hydrogen bond interactions with GRB2 ( Figure 4 The calculated average value of binding free energy is -54.6469±6.0130 kcal / mol ( Figure 5 ), among which the four key residues (Ile65, Gln157, Tyr160 and Phe182) contributed most significantly, at -2.64, -2.53, -2.74 and -2.12 kcal / mol, respectively ( Figure 6 Structural analysis showed that these residues formed a structure similar to a "molecular latch", achieving optimal spatial complementarity and hydrophobic interactions with PPVII to ensure stable binding ( Figure 7 ).

[0021] Example 2: Identifying GRB2 as a hub target through a cell-based thermal shift assay (CETSA) 1. Preparation of cisplatin (CDDP)-resistant strain of human bladder cancer cell T24 Human bladder cancer T24 cells were inoculated into T25 culture flasks containing high-glucose DMEM medium containing 10% fetal bovine serum, placed in a cell culture incubator, and cultured at 37°C and 5% CO2. Cisplatin-resistant cells were induced by gradually increasing the drug dose. 0.5 μM / L CDDP was used as the initial concentration. After culturing until they could grow stably and continued to be cultured for 3 passages, the CDDP concentration was gradually increased until the cells could grow stably in the culture medium with a CDDP concentration of 10 μM / L and were successfully passaged 10 times. A cisplatin-resistant (CDDP) human bladder cancer cell model was obtained and named cisplatin-resistant human bladder cancer cell (T24CDDP cell).

[0022] 2. Cellular Thermal Shift Assay (CETSA) T24CDDP cells were harvested and lysed on ice for 20 minutes using a non-denaturing lysis buffer (Cat. #9803; Cell Signaling Technology, Boston, MA, USA). The lysate was centrifuged at 12,000 rpm for 15 minutes at 4°C to obtain the supernatant, which was then mixed with the test drug (PPVII: 200 μM) and stored at 4°C. The mixed sample was incubated at different temperatures (57, 62, 67, 72, 77, and 82°C) in a PCR instrument for 10 minutes at each temperature, immediately cooled on ice for 3 minutes, and centrifuged again at 12,000 rpm at 4°C for 15 minutes. The supernatant was then collected and analyzed by Western blot for GRB2 protein levels. A blank control without PPVII was also included. See the results Figure 8 The experimental results showed that PPVII treatment significantly enhanced the thermal stability of GRB2 without affecting GAPDH.

[0023] Example 3: Surface plasmon resonance (SPR) further confirmed the direct binding of PPVII to GRB2 Recombinant human GRB2 protein was purchased from Wuhan Aitaikang Biotechnology Co., Ltd., China. GRB2 protein was dissolved in sodium acetate buffer (10 mM, pH 4.5) to a final concentration of 100 µg / mL. Following the standard OpenSPR™ instrument protocol, a COOH sensor chip was mounted and activated with EDC / NHS solution (1:1 molar ratio). After rinsing with running buffer until the baseline stabilized, assay buffer (PBST containing 1% DMSO, pH 7.4) was injected. After monitoring the baseline signal for 5 minutes to ensure system equilibrium, a gradient of saponin solution was injected onto the GRB2-immobilized COOH sensor chip at a flow rate of 20 µL / min. The association phase was set for 240 seconds, followed by a 360-second dissociation phase with buffer. The equilibrium dissociation constant (KD) was calculated using TraceDrawer software (Ridgeview Instruments AB) using a one-to-one binding model.

[0024] See the results Figure 9 , SPR further verified the direct binding of PPVII-GRB2 with a KD of 58.5 μM.

[0025] Example 4: Knockdown of GRB2 protein expression in bladder cancer cells confirmed the key role of GRB2 in mediating PPVII inhibition of Akt signaling pathway 1. T24CDDP bladder cancer cells in the logarithmic growth phase were evenly seeded into 6-well plates and placed in a cell culture incubator. They were cultured at 37°C and 5% CO2 for 24 hours. Transfection was performed after the cells were fully attached. Tube A: 125µL Opti-MEM + 3µL Lipofectamine 2000 (gently mix and let stand at room temperature for 5 minutes). Tube B: 125µL Opti-MEM + 2.5µL siRNA (stock concentration: 20µM). Tubes A and B were mixed (total volume 250µL), gently mixed, and incubated at room temperature for 15-20 minutes. 750µL Opti-MEM was added and gently mixed. The old cell culture medium was aspirated and the siRNA-liposome complex was added dropwise to the wells. The culture medium was gently shaken to mix. After 6 hours, the culture medium was replaced with complete culture medium and cultured for 36 hours. After 1 h, total cell protein was extracted and western blotting was performed to detect the expression level of p-Akt protein (GRB2 siRNA cell group); the above experiment was repeated three times according to three different GRB2 siRNA sequences (GRB2 siRNA sequence 1, GRB2 siRNA sequence 2, GRB2 siRNA sequence 3) to avoid off-target effects.

[0026] Meanwhile, blank control group (T24CDDP cisplatin-resistant bladder cancer), PPⅦ group (T24CDDP cisplatin-resistant bladder cancer + 2 µM PPⅦ), GRB2 siRNA group (T24CDDP cisplatin-resistant bladder cancer with GRB2 protein knockdown), and PPⅦ + GRB2 siRNA group (T24CDDP cisplatin-resistant bladder cancer with GRB2 protein knockdown + 2 µM PPⅦ) were set up. The siRNA sequence targeting GRB2 is as follows, which is synthesized by Guangzhou Qidun Biotechnology Co., Ltd., China: GRB2 siRNA sequence 1: sense strand 5'-CCAUCGCCAAAUAUGACUUTT-3', antisense strand 5'-AAGUCAUAUUUGGCGAUGGTT-3'; GRB2 siRNA sequence 2: sense strand 5'-CCCAAGAACUACAUAGAAATT-3', antisense strand 5'-UUUCUAUGUAGUUCUUGGGTT-3'; GRB2 siRNA sequence 3: sense strand 5'-CCAGAAACCAGCAGAUAUUTT-3', antisense strand 5'-AAUAUCUGCUGGUUUCUGGTT-3'; The results are shown in Figure 10 As can be seen from the figure, compared with the blank control group, after adding PPⅦ in the PPⅦ group, PPⅦ inhibited the expression of p-Akt in T24CDDP cells; after knocking down GRB2 protein in T24CDDP cells, the expression of p-Akt was lower than that of the blank, indicating that GRB2 knockdown was successful; after knocking down GRB2 protein in T24CDDP cells and administering PPⅦ, the expression of p-Akt was lower than that of the PPⅦ group; this indicates that GRB2 is essential for PPⅦ to completely inhibit Akt signaling in bladder cancer, confirming that GRB2 is a therapeutic target of PPⅦ, i.e., PPⅦ directly targets GRB2 to inhibit the Akt pathway.

[0027] 2. The T24CDDP cells in the logarithmic growth phase were uniformly inoculated in a 6-well plate and placed in a cell culture box for incubation at 37°C and 5% CO2 for 24 hours. After the cells were completely adherent, the cells were treated with different concentrations (2 µM / L, 4 µM / L) of PPⅦ for 24 hours, and then total protein was extracted for Western blotting to detect the expression levels of c-PARP, c-cas-7, P21, p-PI3K, p-Akt, p-mTOR, p-GSK3β, p-P70S6K, SREBP1, ACC, FASN, and SCD1 proteins. The results are shown in Figure 11PPVII upregulated the expression of c-PARP, c-cas-7, and P21 in T24CDDP cells, while downregulated the levels of p-PI3K, p-Akt, p-mTOR, and p-GSK3β. Collectively, these findings suggest that PPVII targets GRB2 to inhibit the Akt signaling pathway.

[0028] Example 5: Inhibitory experiment of cisplatin CDDP and PPVII on T24CDDP cells T24CDDP cells were evenly plated into a 96-well plate (4000 cells / well, 100 μL per well), placed in a cell culture incubator, and cultured at 37°C, 5% CO2 for 24 hours. After the cells were completely attached, they were treated with different concentrations of Paris polyphylla saponin VII and cisplatin (CDDP) for 48 hours. Then, 30 μL of MTT solution (5 mg / mL) was added to the 96-well plate and placed in an incubator for reaction at 37°C, 5% CO2 for 5 hours. The liquid in the plate was then discarded and 100 μL of DMSO solution (99.99% purity) was added. The cells were shaken on a shaker for 10 minutes (200 rpm). The absorbance was measured at 570 nm, and the cell viability was calculated and a dose-effect curve was drawn. See the results Figure 12 As can be seen from the figure, under the action of PPVII and CDDP, the proliferation of T24CDDP cells was inhibited, and PPVII and CDDP inhibited the proliferation of cancer cells in a concentration-dependent manner. The IC50 of PPVII and CDDP on T24CDDP cells were 0.6848μM / L and 9.492μM / L, respectively. The inhibitory effect of PPVII was better than that of cisplatin.

[0029] Example 6: Evaluation of the anti-tumor effect of PPVII on T24CDDP transplanted tumors in vivo BALB / c nu / nu male mice (13–17 g; 4–5 weeks) were purchased from the Animal Research and Resource Center of Yunnan University (Kunming, Yunnan).

[0030] Mice were housed at 25°C with a 12 / 12 h light / dark cycle and acclimated to a standard rodent diet for one week before being randomly divided into three groups: a blank control group, a CDDP group, and a PPⅦ treatment group; T24CDDP cells were resuspended in PBS solution containing 50% Corning Matrigel (Corning, New York, USA) and then injected subcutaneously into the right inguinal region of each mouse (3 × 10 6 The long diameter (L; mm) and short diameter (W; mm) of xenograft tumors were measured regularly and the expression of the formula (V = 0.5 × L × W 2 ) to calculate tumor volume (V; mm 3 When the transplanted tumor volume reaches 50-100 mm 3At 4 dpi, mice were treated with intraperitoneal injections of the drug (50% Corning Matrigel in PBS; blank control, once every two days), CDDP (2 mg / kg, once every two days), and PPVII (2 mg / kg, once). On day 14, mice were sacrificed (under carbon dioxide anesthesia), and tumors were removed, measured, and weighed.

[0031] See the results Figure 13 Animal experiments showed that, as can be seen from the figure, in the T24CDDP transplant tumor model, PPVII treatment showed a significant tumor growth inhibitory effect, which was better than cisplatin.

[0032] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with this technical field can easily think of changes or replacements within the technical scope disclosed by the present invention, which should be covered by the present invention.

Claims

1. Application of Paris polyphylla saponin VII in the preparation of an Akt signaling pathway inhibitor targeting GRB2.

2. Use of Paris polyphylla saponin VII in the preparation of drugs for preventing and / or treating bladder cancer.

3. The use according to claim 2, characterized in that: Paris polyphylla saponin VII specifically binds to GRB2 protein, inhibiting the activity of downstream protein kinase Akt, thereby achieving the purpose of inhibiting bladder cancer.

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