Application of Skepinone-L in killing neuroblastoma cells

Abstract

The invention relates to the technical field of medical biology, in particular to application of Skepinone-L in killing neuroblastoma cells, and finds that the Skepinone-L can specifically kill the neuroblastoma cells and significantly inhibit proliferation, migration and clone formation capabilities of the neuroblastoma cells for the first time. An action mechanism research shows that the Skepinone-L accurately targets ubiquitin specific protease 7, and is combined with a key residue through a hydrogen bond to down-regulate USP7 expression and activate a downstream apoptosis pathway. Molecular dynamics simulation proves that the compound is high in stability. The invention provides a new candidate drug with high efficiency and low toxicity for neuroblastoma, and has remarkable clinical transformation potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to an application of Skepinone-L for killing neuroblastoma cells. BACKGROUND

[0002] Neuroblastoma is one of the most common extracranial malignant solid tumors in children, which originates from neural crest cells of the sympathetic nervous system and has high invasiveness and heterogeneity. Although the current clinical comprehensive treatment scheme of surgical resection, chemotherapy, radiotherapy, stem cell transplantation and immunotherapy (such as anti-GD2 monoclonal antibody) is adopted, the long-term survival rate of high-risk neuroblastoma patients is still less than 50%, and there are many problems to be solved: Existing treatment drugs have poor specificity: traditional chemotherapy drugs (such as cyclophosphamide, cisplatin) can kill tumor cells, but have strong cytotoxicity to normal tissue cells, easily leading to bone marrow suppression, gastrointestinal reactions and other serious side effects, and affecting the quality of life of patients.

[0003] Drug resistance is a major problem: tumor cells are prone to primary or secondary drug resistance to chemotherapy drugs, leading to treatment failure and recurrence, which is one of the main difficulties in clinical treatment.

[0004] The mechanism is not clear: the selection of target points and molecular mechanism of some new targeted drugs are insufficient, which makes it difficult to achieve precise treatment and limits the further improvement of curative effect.

[0005] Therefore, it is an urgent need in the current field to find a new anti-neuroblastoma drug with high specificity, low toxicity and clear mechanism. SUMMARY

[0006] The purpose of the present application is to provide an application of Skepinone-L for killing neuroblastoma cells, which provides a new candidate drug with high efficiency and low toxicity for neuroblastoma, and has significant clinical transformation potential.

[0007] To achieve the above purpose, in a first aspect, the present application provides an application of Skepinone-L in the preparation of a drug for treating neuroblastoma.

[0008] The drug is used for at least one of killing neuroblastoma cells, inhibiting the proliferation of neuroblastoma cells, inhibiting the migration of neuroblastoma cells or inhibiting the clonal formation of neuroblastoma cells.

[0009] The neuroblastoma cells are selected from one or more of SH-SY5Y, SK-N-SH, SK-N-BE2, IMR-32 or SK-N-AS cell strains.

[0010] The effective concentration of the drug is 0.1 μM to 20 μM, and the IC 50 value of the drug is 3.862 μM to 10.74 μM.

[0011] The Skepinone-L acts by targeting ubiquitin-specific protease 7 protein, and the targeted ubiquitin-specific protease 7 is USP7.

[0012] The Skepinone-L binds to the USP7 protein, and the binding includes forming a hydrogen bond interaction with the TYR-465 and / or HIS-461 residues of the USP7 protein, and the distance of the hydrogen bond interaction is at least one of 1.7 Å, 2.2 Å or 3.1 Å.

[0013] The Skepinone-L down-regulates the expression of the USP7 protein and / or regulates the downstream apoptosis pathway, and the regulation of the downstream apoptosis pathway includes down-regulating the expression of Bcl-2 protein and / or up-regulating the expression of CleavedPARP-N protein.

[0014] In a second aspect, the present application provides a method for evaluating the anti-neuroblastoma activity of Skepinone-L in vitro, for evaluating the use as described in the first aspect, comprising the following steps: co-culturing neuroblastoma cells with different concentrations of Skepinone-L; detecting the effect of Skepinone-L on the killing, proliferation, migration, clonogenic ability or related protein expression of the cells by at least one method selected from CCK-8 method, cell morphology experiment, EdU incorporation experiment, Transwell migration experiment, clonogenic experiment, Western blot detection of apoptosis-related protein expression, molecular docking experiment detection of USP7 protein expression, molecular dynamics simulation of USP7 protein binding, stretching molecular dynamics simulation of the dissociation energy of USP7 protein or Western blot detection of USP7 protein expression; The molecular dynamics simulation of USP7 protein binding includes analyzing at least one parameter of the complex formed by the Skepinone-L and the USP7 protein, such as root mean square deviation, radius of gyration, solvent accessible surface area, free energy landscape, binding free energy or dissociation energy.

[0015] The application of a Skepinone-L for killing neuroblastoma cells, for the first time, it is found that Skepinone-L can specifically kill neuroblastoma cells, significantly inhibit the proliferation, migration and clonogenic ability of the neuroblastoma cells. Mechanism research shows that: Skepinone-L precisely targets ubiquitin-specific protease 7 (USP7), binds to the key residues through hydrogen bonds, down-regulates the expression of USP7 and activates the downstream apoptosis pathway. Molecular dynamics simulation confirms that the complex has high stability. The application provides a new candidate drug with high efficiency and low toxicity for neuroblastoma, and has significant clinical transformation potential. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiment or prior art description will be briefly introduced below.

[0017] Figure 1 is a result schematic diagram of CCK-8 method for detecting the influence of different concentrations of Skepinone-L on the activity of human neuroblastoma cell lines treated for 24 hours.

[0018] Figure 2 is a result schematic diagram of inverted microscope observation of the influence of Skepinone-L on the morphology of human neuroblastoma cells (SH-SY5Y) treated for 24 hours.

[0019] Figure 3 is a result schematic diagram of EdU incorporation experiment for detecting the influence of Skepinone-L on the proliferation of SH-SY5Y cells.

[0020] Figure 4 is a structure schematic diagram of Transwell experiment for detecting the influence of Skepinone-L on the migration of SH-SY5Y cells.

[0021] Figure 5 is a result schematic diagram of clonogenic experiment for detecting the influence of Skepinone-L on the clonogenic ability of cells.

[0022] Figure 6 is a result schematic diagram of Western blot for detecting the influence of Skepinone-L on the expression of apoptosis-related proteins.

[0023] Figure 7 is a result schematic diagram of molecular docking simulation of the interaction between Skepinone-L and USP7 protein.

[0024] Figure 8 is a RMSD change graph of USP7-Skepinone-L complex in molecular dynamics simulation.

[0025] Figure 9 is a plot of the change in radius of gyration (Rg) of the USP7-Skepinone-L complex in a molecular dynamics simulation.

[0026] Figure 10 is a plot of the change in solvent accessible surface area (SASA) of the USP7-Skepinone-L complex in a molecular dynamics simulation.

[0027] Figure 11 is a plot of the free energy landscape analysis of the USP7-Skepinone-L complex with root mean square deviation (RMSD) and radius of gyration (Rg) as two-dimensional variables.

[0028] Figure 12 is a plot of the potential of mean force (PMF) analysis of the USP7-Skepinone-L complex.

[0029] Figure 13 is a schematic representation of the results of Western blot detection of the effect of Skepinone-L on the expression of USP7 protein. DETAILED DESCRIPTION

[0030] The exemplary embodiments will be described in detail herein with reference to the attached drawings. The description of the exemplary embodiments is intended to apply to all alternative embodiments, as would be understood by persons skilled in the art. The following exemplary embodiments are described in order to provide a thorough understanding of the present application.

[0031] The terminology used in the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used in the present application and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0032] It will be understood that, although the terms first, second, third, etc. can be used herein to describe various information, these terms are not intended to denote a temporal or chronological order. Rather, these terms are used only as a shorthand notation to distinguish one piece of information from another. For example, a first piece of information can be termed a second piece of information without departing from the scope of the present application. Similarly, a second piece of information can be termed a first piece of information. As used herein, the word "if' can be interpreted to mean "when" or "upon" or "in response to determining" taking into account the context in which the term is used.

[0033] The application provides an application of Skepinone-L in killing neuroblastoma cells, and mainly an application of Skepinone-L in preparing a drug for treating neuroblastoma. The purpose is to solve the problems of poor drug specificity, unclear action mechanism, obvious toxic and side effects and the like in existing neuroblastoma treatment, and to provide a new treatment candidate drug and an action mechanism thereof. Specifically, the purpose is to prove the effect of Skepinone-L on specific killing of neuroblastoma cells and inhibition of malignant biological behaviors (proliferation, migration, clonal formation and the like) of the neuroblastoma cells, and to clarify the molecular mechanism of the action of Skepinone-L on the neuroblastoma cells by targeting USP7 protein and regulating a downstream apoptosis pathway, so as to provide a scientific basis and a new treatment strategy for precise treatment of neuroblastoma.

[0034] Skepinone-L (CAS No. 1221485-83-1) is found to have a significant killing effect on neuroblastoma cells for the first time, and the effect and mechanism thereof are verified through a series of experiments, and the specific technical scheme is as follows: 1. Experimental materials Drug: Skepinone-L (purity greater than or equal to 98%), dissolved with DMSO to prepare working solutions (0.1 μM, 1 μM, 5 μM, 10 μM and 20 μM) of different concentrations.

[0035] 2. Cell model Human neuroblastoma cell lines SH-SY5Y and SK-N-AS are cultured in DMEM medium containing 10% fetal bovine serum; human neuroblastoma cell lines SK-N-SH and IMR-32 are cultured in MEM medium containing 10% fetal bovine serum and NEAA (non-essential amino acids); human neuroblastoma cell line SK-N-BE2 is cultured in DMEM / F12 medium containing 10% fetal bovine serum; all cells are cultured in a 37°C, 5% CO2 incubator, and the liquid is changed regularly to maintain the logarithmic growth phase of the cells.

[0036] 3. Experimental method and steps (1) CCK-8 method for detecting cell viability 1) Cells in the logarithmic growth phase of human neuroblastoma cell lines (SK-N-BE2, SK-N-AS, SH-SY5Y, IMR-32 and SK-N-SH) are inoculated, and single-cell suspensions are prepared with corresponding culture media (for example, DMEM / F12 + 10% fetal bovine serum for SK-N-BE2, DMEM + 10% fetal bovine serum for SH-SY5Y, etc.), and the cell concentration is adjusted to 1×10 5 cells / mL. 100 μL of cell suspension is added to each well of a 96-well plate (i.e. 1×10 4Each group was set with 3 duplicate holes, and the edge holes were filled with sterile PBS to reduce the evaporation effect. The 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours, and the cells were allowed to adhere.

[0037] 2) Drug treatment Skepinone-L working solution was prepared: Skepinone-L (DMSO dissolved stock solution) was diluted with the corresponding medium to 0 μM (control group, containing equal amount of DMSO), 0.1 μM, 1 μM, 5 μM, 10 μM, 20 μM, ensuring that the final DMSO concentration in each well was ≤0.1% (to avoid solvent toxicity). The original culture medium in the 96-well plate was aspirated, and 100 μL of fresh culture medium containing different concentrations of Skepinone-L was added to each well, and the culture was continued for 24 hours.

[0038] 3) CCK-8 detection 10 μL of CCK-8 reagent was added to each well (operation in the dark), and the 96-well plate was gently shaken to mix the reagent with the medium. After incubation in the incubator for 2 hours, the absorbance (OD value) of each well was measured at 450 nm wavelength using a microplate reader.

[0039] 4) Data calculation Cell survival rate (%) = (experimental group OD value / control group OD value) x 100%. The concentration-effect curve was fitted using GraphPad Prism software, and the half inhibitory concentration (IC 50 ) and 95% confidence interval, goodness of fit R 2 of each cell line were calculated.

[0040] (2) Cell morphology observation experiment 1) Cell inoculation and treatment 4 SH-SY5Y cells were taken and a single cell suspension was prepared with DMEM + 10% fetal bovine serum, the concentration was adjusted to 5 x 10 5 cells / mL, inoculated in a 6-well plate (2 mL per well, i.e. 1 x 10 5 cells), and cultured for 24 hours to adhere. It was divided into a control group (added with an equal amount of DMSO-containing medium) and a 1 μM Skepinone-L treatment group, each group was set with 3 duplicate holes, and the culture was continued for 24 hours.

[0041] 2) Morphological observation and recording The 6-well plate was taken out and placed under an inverted microscope, and the growth state (density, morphology, adhesion, etc.) of the cells in each group was observed under 40x magnification.

[0042] (3) EdU incorporation experiment to detect cell proliferation ability 1) Cell culture and inoculation Selecting the logarithmic growth phase of SH-SY5Y cells, trypsin digestion, and then using complete medium to resuspend the cells, adjust the cell density to 2 x 10 5 Preparation of 48-well plates, each well inoculated with 200 μL of cell suspension (i.e. each well inoculation amount of 4 x 10 4 The inoculated 48-well plate was placed in a 37°C, 5% CO2 incubator for overnight culture to allow the cells to fully adhere.

[0043] 2) EdU labeling According to the kit instructions, dilute the EdU solution (reagent A) with complete medium at a ratio of 1000:1 to prepare 50 μM EdU medium. After the cells adhere, add 200 μL of prepared 50 μM EdU medium to each well to ensure a final concentration of 50 μM EdU, and continue to incubate in a 37°C, 5% CO2 incubator for 6h. After incubation, carefully discard the medium and gently wash the cells with 200 μL of PBS for 1-2 times, 5 min each time, to remove the unincorporated EdU.

[0044] 3) Cell fixation Add 100 μL of cell fixation solution (i.e. PBS containing 4% paraformaldehyde) to each well and incubate at room temperature for 30 min to fix the cell morphology. After fixation, discard the fixation solution and add 100 μL of 2 mg / mL glycine to each well and incubate on a decolorizing shaker for 5 min to terminate the fixation reaction, then discard the glycine solution. Next, add 200 μL of PBS to each well and wash for 5 min on a decolorizing shaker, then discard the PBS; add 200 μL of permeabilization agent (0.5% Triton X-100 in PBS) to each well and incubate for 10 min on a decolorizing shaker to enhance cell membrane permeability, then wash with PBS for 5 min.

[0045] 4) Apollo staining Prepare 1 x Apollo staining reaction solution according to the kit instructions, add 200 μL of the staining reaction solution to each well, and note that it needs to be in the dark, place the plate on a decolorizing shaker and incubate at room temperature for 30 min. After incubation, discard the staining reaction solution and add 200 μL of permeabilization agent (0.5% Triton X-100 in PBS) and wash 2-3 times on a decolorizing shaker for 10 min each time to remove unbound Apollo dye. To further wash impurities, add 200 μL of methanol to each well for 1-2 times, 5 min each time, and finally wash with PBS for 5 min.

[0046] 5) DNA staining Dilute DAPI with deionized water at a ratio of 2500:1 to prepare an appropriate amount of 1 x DAPI reaction solution, and note to store in the dark. Add 200 μL of 1 x DAPI reaction solution to each well, and incubate the culture plate on a decolorizing shaker under light protection at room temperature for 10 min to stain the cell nucleus. After staining, discard the staining reaction solution, and add 200 μL of PBS to each well for 1-3 washes to remove unbound DAPI. Image acquisition and analysis: After staining is complete, use a fluorescence microscope to observe and take pictures. Randomly select 5 fields of view per well to take pictures, and take pictures of EdU (red fluorescence, representing proliferating cells) and DAPI (blue fluorescence, marking the cell nucleus). Then use image analysis software (such as Image-ProPlus 6.0) to merge the images and count EdU-positive cells (i.e., cells with overlapping red fluorescence and blue cell nucleus) to calculate the proportion of EdU-positive cells and evaluate the cell proliferation capacity. The experiment is repeated independently 3 times to ensure the reliability of the data.

[0047] (4) Transwell experiment to detect cell migration ability 1) Cell pretreatment Take SH-SY5Y cells and prepare a single-cell suspension with serum-free DMEM, and adjust the concentration to 5 x 10 5 Divide into a control group (add an equal amount of DMSO to serum-free medium) and a 1 μM Skepinone-L treatment group, with 3 replicate wells in each group, and incubate at 37°C for 24 h.

[0048] 2) Transwell inoculation Take out the Transwell chamber (8 μm pore size), and add 600 μL of DMEM medium containing 10% fetal bovine serum to the lower chamber (as a chemotactic factor). Add 200 μL of pretreated cell suspension (i.e., 1 x 10 5 Place the chamber in the incubator at 37°C and 5% CO2 for 24 h.

[0049] 3) Staining and counting Take out the chamber, gently wipe off the cells in the upper chamber that have not migrated with a cotton swab, and fix the migrated cells on the lower chamber side with 4% PFA for 30 min. Stain with 0.1% crystal violet staining solution for 20 min, rinse with tap water to remove excess staining solution, and air dry. Observe under an inverted microscope at 100x magnification, select 3 different fields of view to take pictures (ruler = 500 μm), and count the number of migrated cells.

[0050] (5) Clonogenic assay to detect cell clonogenicity 1) Cell inoculation and drug treatment Take the logarithmic growth phase of neuroblastoma cells (take SH-SY5Y as an example), prepare a single cell suspension with the corresponding medium, adjust the concentration to 50 / mL. Add 1 mL of cell suspension to each well of a 6-well plate (i.e. 50 cells per well), divide into control group (containing equal amount of DMSO), 1 μM, 5 μM, 10 μM Skepinone-L treatment group, each group is set with 3 replicate wells. Shake the 6-well plate gently to make the cells evenly distributed, and place it in the incubator for 14 days (replace the fresh medium containing the corresponding concentration of drug every 3 days during the period).

[0051] 2) Clone staining and counting Discard the culture medium, wash with PBS for 2 times, fix with 4% PFA for 30 min, and wash with PBS for 2 times. Add 1 mL of 0.1% crystal violet staining solution to each well, stain for 30 min, rinse with tap water until the background is clear, and dry. Observe and count the clones with a diameter greater than or equal to 50 μm (or cell number greater than or equal to 50) under a microscope, and calculate the clone formation rate (%) = (clone number / seeded cell number) x 100%.

[0052] (6) Western blot detection of the expression level of apoptosis-related proteins 1) Protein extraction Take the control group (0 μM) and 1 μM Skepinone-L treated SH-SY5Y cells for 24 h, discard the culture medium, and wash with PBS for 2 times. Add 200 μL of RIPA lysis buffer (containing 1% PMSF and 1% phosphatase inhibitor) to each well, lyse on ice for 30 min, and scrape the cells with a cell scraper during the period. Transfer the lysate to a 1.5 mL centrifuge tube, centrifuge at 4°C, 12000 rpm for 15 min, and take the supernatant (total protein).

[0053] 2) Protein quantification and electrophoresis Determine the protein concentration with BCA protein quantification kit, adjust the loading amount according to the concentration (30 μg protein per well), add 5x SDS loading buffer, and denature at 100°C for 10 min. Prepare 10% SDS-PAGE gel, load, and electrophorese at constant voltage of 80V for 30 min (concentration gel), and then at 120V for 90 min (separation gel).

[0054] 3) Membrane transfer and blocking Wet transfer method: transfer the protein from the gel to the PVDF membrane (constant current 300 mA, 90 min), and block with 5% skim milk powder (dissolved in TBST) at room temperature for 2 h after the transfer is completed.

[0055] 4) Antibody incubation Primary antibody dilution with TBST: Bcl-2 (1:1000), PARP (1:1000), Cleaved PARP-N (1:1000), GAPDH (1:5000), incubate PVDF membrane at 4°C overnight. Wash the membrane with TBST for 3 times (10 min each time), add HRP-labeled secondary antibody (1:5000, dilute with TBST), incubate at room temperature for 1 h. Wash the membrane with TBST for 3 times (10 min each time), develop with ECL chemiluminescence kit, and expose to X-ray film or gel imaging system.

[0056] 4) Gray scale analysis Analyze the gray value of the target protein band with ImageJ software, use GAPDH as the internal reference, calculate the relative expression (target protein gray value / internal reference gray value), and express Cleaved PARP-N as "Cleaved PARP-N / PARP" ratio.

[0057] (7) Molecular docking experiment to predict the interaction mode of Skepinone-L and target protein USP7 To explore the binding mode of Skepinone-L and USP7, molecular docking experiment was performed. The crystal structure of USP7 was obtained from the protein database (https: / / www.rcsb.org / ), and the ligand Skepinone-L was prepared using standard procedures. Maestro 13.5 software was used for docking calculation and analysis of the key binding sites and interaction forces (such as hydrogen bonds, hydrophobic interactions, etc.) between the two.

[0058] (8) Molecular dynamics simulation to analyze the binding affinity and stability of Skepinone-L and target protein USP7 Molecular dynamics simulation was performed on Ubuntu 20.04.01 platform using GROMACS 2023.1 (single precision version), and the experimental platform was DELL T3680 workstation equipped with Intel Core i9-14900k CPU and GeForce RTX 4070Ti Super GPU. The heavy atoms of USP7 and Skepinone-L were modified using SPDBV4.10 software, and the topology of USP7 was calculated using AMBER99SB-ILDN force field, and the ligand topology of Skepinone-L was determined by combining AMBER force field and Acpype online tool (https: / / bio2byte.be / acpype / ). The USP7-Skepinone-L complex was dissolved in TIP3P water model and placed in a cubic box with a distance of at least 1 nm from the complex in each direction. Na + and Cl -The neutralization system was supplemented with 0.15 M NaCl to mimic the physiological environment. The steepest descent algorithm was used for 5000 steps of energy minimization (maximum force less than 1000 kJ / mol / nm), followed by 100 ps of NVT equilibration and 100 ps of NPT equilibration in turn, to stabilize the system at 310 K and 1 bar. The production dynamics simulation lasted for 100 ns (5 x 10 7 steps), with a time step of 2 fs, using the Verlet truncation scheme and Leap-frog integrator, and trajectory data was saved every 10 ps. Periodic boundary conditions were introduced during the simulation to eliminate boundary effects. Key parameter analysis is as follows: root mean square deviation (RMSD): calculated using the "gmxrms" command, selecting the backbone atoms, reflecting the spatial conformation fluctuation of the complex. Rotational radius (Rg): calculated by the "gmxgyrate" command, selecting the protein backbone atoms, to evaluate the overall structural compactness of the complex. Free energy landscape (FEL): based on 50-100 ns trajectory (corrected for translation and rotation by gmxtrjconv module), selecting RMSD and Rg as characteristic variables, calculated using gmxsham module, and plotted using Origin2021. Solvent accessible surface area (SASA): calculated using the "gmxsasa" command, selecting the entire complex as the research object, using a 1.4 Å standard probe radius to simulate the size of solvent molecules, to evaluate the interaction area between the complex and the solvent. MM / PBSA binding free energy: calculated using gmx-MM / PBSA1.6.1 software, extracting 5000 conformations from the 50-100 ns trajectory at 2 fs intervals, only considering enthalpy change, and ignoring conformation entropy change (ΔS) (due to high calculation cost and low prediction accuracy).

[0059] (9) Tensile molecular dynamics simulation and umbrella sampling simulation to analyze the dissociation energy of Skepinone-L and target protein USP7 Combined with tensile molecular dynamics (SMD) and umbrella sampling (US) simulation, GROMACS was used to explore the dissociation process and energy change of USP7-Skepinone-L complex. In SMD simulation, the ligand was moved by the stretching command of GROMACS, a harmonic force of 500 kJ / mol•nm 2 was applied to the ligand centroid, and the system coordinates were recorded every 0.1 ps. When the ligand moved along the positive direction of the Z axis, the complex structure was recorded once every 0.2 nm of displacement as the initial conformation for US evaluation. In US simulation, a series of complex conformations from the bound state to the unbound state were selected, and 0.1 ns of NPT simulation was performed to relax the system, followed by 10 ns of US simulation. Finally, the weighted histogram analysis method was used to integrate the simulation results and calculate the dissociation energy during stretching.

[0060] (10) Western blot detection of target protein USP7 expression level The experimental method of Western blot detection of target protein USP7 expression level is consistent with the whole process of (6), only the difference is in the antibody incubation link, as follows: dilute the primary antibody with TBST: USP7 (1:1000), GAPDH (1:5000), 4℃ incubate PVDF membrane overnight.

[0061] 4. Experimental results (1) Inhibition of Skepinone-L on the viability of neuroblastoma cells The CCK-8 method was used to systematically detect the effect of different concentrations of Skepinone-L on the viability of human neuroblastoma cell lines SK-N-BE2, SK-N-AS, SH-SY5Y, IMR-32 and SK-N-SH after 24h treatment. The results clearly showed that the survival rate of each cell line showed a significant downward trend with the increase of Skepinone-L concentration, showing a typical concentration-dependent inhibitory effect (A-E). Figure 1 Figure 1 The survival rates of SK-N-BE2 (A), SK-N-AS (B), SH-SY5Y (C), IMR-32 (D), and SK-N-SH (E) cells are shown in the figure. The abscissa is the concentration of Skepinone-L (μmol / L), and the ordinate is the cell survival rate (%). The data are expressed as mean ± standard deviation. Taking the control group (0 μmol / L) as the reference, when the concentration of Skepinone-L reached 1 μmol / L, the survival rate of SK-N-BE2 cells decreased rapidly from nearly 100% in the control group to about 75%, the survival rate of SK-N-AS cells decreased to about 70%, the survival rate of SH-SY5Y cells decreased to about 65%, the survival rate of IMR-32 cells decreased to about 72%, and the survival rate of SK-N-SH cells decreased to about 80%. As the concentration continued to rise to 10 μmol / L, the survival rate of SK-N-BE2 cells was only about 20%, the survival rate of SK-N-AS cells was about 15%, the survival rate of SH-SY5Y cells was as low as about 10%, the survival rate of IMR-32 cells was about 18%, and the survival rate of SK-N-SH cells was about 25%. Among them, the difference was statistically significant compared with the control group (0 μmol / L) (P < 0.001).

[0062] Further accurate calculation of the half inhibitory concentration (IC 50 ), the results showed that the killing effect of Skepinone-L on each cell line was different to some extent, as shown in Table 1. The IC 50 ​The lowest concentration was 3.862 μM, meaning that at this concentration, approximately 50% of SH-SY5Y cell viability was inhibited; the IC50 concentration for SK-N-SH cells was also low. 50 The highest concentration was 10.74 μM. It is noteworthy that the IC50 values ​​for each cell line were... 50 The values ​​were all within a relatively low concentration range (3.862-10.74 μM), and the 95% confidence interval was narrow, indicating a goodness of fit R. 2 All values ​​were greater than 0.84, which fully demonstrates that the experimental data have high reliability and stability, and that Skepinone-L has a definite inhibitory effect on the viability of neuroblastoma cells.

[0063] Table 1. Killing effect of Skepinone-L on different neuroblastoma cell lines (IC50) 50 Value (μM) (2) Effects of Skepinone-L on the morphology of neuroblastoma cells The effect of Skepinone-L treatment for 24 hours on the morphology of human neuroblastoma SH-SY5Y cells was observed directly using an inverted microscope. The SH-SY5Y cells in the control group (without treatment) exhibited a typical dense, regular adherent growth pattern, with clear cell outlines, intact morphology, and tight interconnections, forming a uniform and orderly cell monolayer. Figure 2 A). However, after treatment with 1 μS kepinone-L for 24 h, the cell morphology changed significantly. Cell density decreased significantly, by about 60%-70% compared to the control group, and some cells detached from the adherent state and became suspended in the culture medium. At the same time, the cell morphology became noticeably rounder, losing its original extended shape, and the intercellular connections became loose and dispersed, exhibiting typical characteristics of cell damage morphology. Figure 2 B). These morphological changes clearly suggest that Skepinone-L can disrupt the normal growth state of neuroblastoma cells and interfere with their adhesion and extension functions.

[0064] (3) Inhibitory effect of Skepinone-L on the proliferation of neuroblastoma cells The effect of Skepinone-L on the proliferation of SH-SY5Y cells was investigated using an EdU incorporation assay. In the control group, a large number of EdU-positive cells (red) were observed under a fluorescence microscope, with a positive cell rate as high as 40%-45%, indicating that the cells were in a highly active proliferative state. Figure 3A is the result of fluorescent staining: EdU staining (red) marks proliferative cells, DAPI staining (blue) marks cell nucleus, and Merge is the superimposed image; the control group (Control) has a large number of EdU-positive cells, and the 1 μmol / L Skepinone-L treatment group for 24 h has a reduced number of positive cells. After treatment with 1 μmol / L Skepinone-L for 24 h, the proportion of EdU-positive cells significantly decreased. Statistical analysis showed that Figure 3 B, the vertical coordinate is the proportion of EdU-positive cells (%), and the proportion of EdU-positive cells in the treatment group decreased to 15%-20%, which was significantly lower than that in the control group (n=3, indicates that the difference is statistically significant compared with the control group, P<0.05). This data change clearly confirms that Skepinone-L can effectively inhibit the proliferation of SH-SY5Y cells, and reveals the mechanism of its inhibitory effect on the growth of neuroblastoma cells from the aspect of cell proliferation.

[0065] (4) Inhibitory effect of Skepinone-L on the migration of neuroblastoma cells The Transwell experiment was used to evaluate the effect of Skepinone-L on the migration ability of SH-SY5Y cells. In the control group, a large number of cells successfully migrated to the lower chamber of the Transwell, and after crystal violet staining, the lower chamber showed a dense purple cell mass ( Figure 4 A is the result of crystal violet staining (scale = 500 μm), and a large number of migrating cells can be seen in the control group (Control). After treatment with 1 μmol / L Skepinone-L for 24 h, the number of cells migrating to the lower chamber significantly decreased. Statistical analysis showed that Figure 4 B, the vertical coordinate is the number of SH-SY5Y cells migrating, and the number of migrating cells in the treatment group was more than 70% lower than that in the control group (n=3, indicates that the difference is statistically significant compared with the control group, P<0.001). Specifically, the average number of migrating cells in the lower chamber of the control group was 300-350 per field, while that in the lower chamber of the treatment group was only 80-100 per field. This significant difference in data suggests that Skepinone-L can significantly inhibit the migration ability of SH-SY5Y cells, which is of great significance for inhibiting the invasion and metastasis of neuroblastoma cells.

[0066] (5) Inhibitory effect of Skepinone-L on the clonogenic ability of neuroblastoma cells The clonogenic experiment was used to comprehensively evaluate the effect of Skepinone-L on the clonogenic ability of cells. In the control group, after 14 days of culture, a large number of purple clones with a diameter of greater than or equal to 50 μm were formed, and the number of clones was large and evenly distributed ( Figure 5A). With the increasing concentration of Skepinone-L (1 μmol / L, 5 μmol / L, 10 μmol / L), the number of clones showed a trend of gradual decrease. Statistical analysis showed that (P<0.05, n=3) Figure 5 B), the number of clones in the 1 μmol / L treatment group was significantly lower than that in the control group (P<0.05, n=3), and the clone formation rate decreased from 30%-40% in the control group to 10%-15%; the number of clones in the 5 μmol / L and 10 μmol / L treatment groups decreased more significantly (P<0.01, n=3), and the clone formation rate in the 10 μmol / L treatment group decreased to less than 5%. This fully shows that Skepinone-L can inhibit the clonogenicity of cells in a concentration-dependent manner, further supporting its inhibitory effect on the long-term growth and proliferation of neuroblastoma cells.

[0067] (6) Regulation of Skepinone-L on the expression of apoptosis-related proteins in neuroblastoma cells The effect of Skepinone-L on the expression of apoptosis-related proteins was analyzed by Western blot. The results showed that (P<0.05, n=3) Figure 6 A is the protein band, showing the expression of PARP, Cleaved PARP-N, Bcl-2 and internal reference GAPDH; the control group (Control) and the 1 μmol / L Skepinone-L treatment group showed different bands), compared with the control group, the expression band of anti-apoptotic protein Bcl-2 was obviously lighter after 1 μmol / L Skepinone-L treatment, indicating that its expression level was significantly down-regulated; while the expression band of pro-apoptotic related Cleaved PARP-N (PARP cleavage) was obviously deeper, suggesting that its expression was up-regulated. Statistical analysis showed that (P<0.05, n=3) Figure 6 B is the statistical analysis of the relative expression of Bcl-2 protein (normalized by GAPDH), Figure 6 C is the statistical analysis of the relative expression of Cleaved PARP-N / PARP), the relative expression of Bcl-2 in the treatment group was reduced by about 50%-60% compared with the control group, and the ratio of Cleaved PARP-N / PARP was increased by 2-3 times compared with the control group (P<0.05, n=3). These data clearly indicate that Skepinone-L can down-regulate the expression of Bcl-2 and up-regulate the expression of Cleaved PARP-N, break the balance of apoptosis regulation in cells, and promote the apoptosis of neuroblastoma cells, thus explaining the molecular mechanism of its cell death induction pathway.

[0068] (7) Molecular docking of Skepinone-L and USP7 protein The interaction mechanism between Skepinone-L and USP7 protein was further explored by molecular docking simulation. The results showed that (Fig. 1A), Skepinone-L (red ball-stick model) could accurately bind to the active pocket region of USP7 protein (green surface model) with good spatial matching, indicating that Skepinone-L could specifically target USP7 protein. Further analysis of the binding mode details (Fig. 1B) found that Skepinone-L formed multiple hydrogen bond interactions with key residues of USP7 protein (such as TYR-465, HIS-461, etc.), with hydrogen bond distances of 1.7 Å, 2.2 Å, and 3.1 Å, respectively. These short-distance hydrogen bond interactions enhanced the stability of the binding of Skepinone-L to USP7 protein and were an important molecular basis for their specific binding, revealing the molecular action mechanism of Skepinone-L targeting USP7. Figure 7 A), Skepinone-L (red ball-stick model) could accurately bind to the active pocket region of USP7 protein (green surface model) with good spatial matching, indicating that Skepinone-L could specifically target USP7 protein. Further analysis of the binding mode details (Fig. 1B) found that Skepinone-L formed multiple hydrogen bond interactions with key residues of USP7 protein (such as TYR-465, HIS-461, etc.), with hydrogen bond distances of 1.7 Å, 2.2 Å, and 3.1 Å, respectively. These short-distance hydrogen bond interactions enhanced the stability of the binding of Skepinone-L to USP7 protein and were an important molecular basis for their specific binding, revealing the molecular action mechanism of Skepinone-L targeting USP7. Figure 7 A), Skepinone-L (red ball-stick model) could accurately bind to the active pocket region of USP7 protein (green surface model) with good spatial matching, indicating that Skepinone-L could specifically target USP7 protein. Further analysis of the binding mode details (Fig. 1B) found that Skepinone-L formed multiple hydrogen bond interactions with key residues of USP7 protein (such as TYR-465, HIS-461, etc.), with hydrogen bond distances of 1.7 Å, 2.2 Å, and 3.1 Å, respectively. These short-distance hydrogen bond interactions enhanced the stability of the binding of Skepinone-L to USP7 protein and were an important molecular basis for their specific binding, revealing the molecular action mechanism of Skepinone-L targeting USP7.

[0069] (8) Binding free energy analysis of USP7-Skepinone-L complex The MM / PBSA method was used to calculate the binding free energy and related energy terms of the USP7-Skepinone-L complex, and the results are shown in Table 2. The binding free energy (∆Gbind) of the 5 independent complexes (USP7-Skepinone-L-1 to -5) was negative, ranging from -24.05±4.61 kcal / mol to -39.89±3.81 kcal / mol, indicating that the binding process of Skepinone-L to USP7 protein was spontaneous and had strong binding ability. In terms of energy terms, the van der Waals interaction energy (∆EvdW) was negative and had a large absolute value, ranging from -32.81±5.14 kcal / mol to -48.94±2.70 kcal / mol, which was the main driving force for the binding of the two. The electrostatic interaction energy (∆Eelec) was also mostly negative and played a certain auxiliary role in the binding. The total gas energy (∆Ggas), which was the sum of the van der Waals interaction energy and the electrostatic interaction energy, was also negative, reflecting the mutual attraction between the two in the gas phase. The solvation free energy (∆Gsolv) was positive, ranging from 13.02±4.92 kcal / mol to 32.72±7.89 kcal / mol, which had a certain hindering effect on the binding, but overall its absolute value was smaller than the total gas energy, making the binding free energy still negative and ensuring the stable formation of the complex.

[0070] Table 2 MM / PBSA binding free energy (kcal / mol) of USP7-Skepinone-L complex (9) RMSD variation analysis of the USP7-Skepinone-L complex The root mean square deviation (RMSD) of the USP7-Skepinone-L complex in molecular dynamics simulations is as follows: Figure 8 As shown, the RMSD values ​​of the five independent simulations (USP7-Skepinone-L-1 to -5) fluctuated somewhat in the initial stage of the simulation, but all tended to stabilize after 20 ns, with the fluctuation range remaining between 0.2 and 0.4 nm. The stability of the RMSD values ​​indicates that the complex formed by USP7 and Skepinone-L undergoes minimal structural changes during molecular dynamics and can maintain a relatively stable conformation, further confirming the reliability of their binding interaction (n=5).

[0071] (10) Analysis of the radius of gyration (Rg) of the USP7-Skepinone-L complex Radius of gyration (Rg) is an important parameter reflecting the compactness of protein molecules. The Rg variation of the USP7-Skepinone-L complex is shown in the figure below. Figure 9 As shown in the figure, in five independent simulations (USP7-Skepinone-L-1 to -5), the Rg value remained stable within the range of 2.2-2.3 nm throughout the simulation, with minimal fluctuations. This result suggests that after USP7 binds to Skepinone-L, the overall conformation of the protein does not change significantly, and the complex structure remains compact, further demonstrating the good stability of the complex (n=5).

[0072] (11) Analysis of changes in solvent-accessible surface area (SASA) of the USP7-Skepinone-L complex Solvent-accessible surface area (SASA) reflects the area of ​​a protein surface in contact with a solvent. The SASA changes of the USP7-Skepinone-L complex are shown below. Figure 10 As shown, in five independent simulations (USP7-Skepinone-L-1 to -5), the SASA value remained stable at 180-200 nm during the simulation. 2 Within the specified range, no significant fluctuations were observed. This indicates that after USP7 binds to Skepinone-L, the surface area of ​​the protein exposed to the solvent remains relatively stable, and the complex maintains a stable structure in the solvent environment, exhibiting good solvent stability (n=5).

[0073] (12) Free energy landscape analysis of USP7-Skepinone-L complex Using root mean square deviation (RMSD) and radius of gyration (Rg) as two-dimensional variables, a free energy landscape analysis was performed on the USP7-Skepinone-L complex. The results are as follows: Figure 11The color gradient corresponds to the free energy value, and the deep blue region in the center is the low free energy stable conformation region. The existence of this region indicates that after the binding of USP7 and Skepinone-L, the complex has an energy advantage in a specific RMSD and Rg range, and can form a stable conformation. Based on the analysis results of 100 ns molecular dynamics simulation data, the stability of the structure of the USP7-Skepinone-L complex after binding is further verified.

[0074] (13) PMF analysis of the USP7-Skepinone-L complex PMF analysis can be used to evaluate the thermodynamic stability of intermolecular binding. The PMF analysis results of the USP7-Skepinone-L complex are shown in Figure 12 . The curve shows the energy change of the dissociation of Skepinone-L from the USP7 protein, and the results show that the dissociation of the complex requires an energy barrier of about 26.39 kJ / mol (free energy difference). This high energy barrier indicates that the binding between Skepinone-L and USP7 protein is strong, and the complex has high thermodynamic stability, further confirming the tightness of the combination from the energy point of view.

[0075] (14) Effect of Skepinone-L on the expression of USP7 protein The effect of Skepinone-L on the expression of USP7 protein was detected by Western blot technology, and the results are shown in Figure 13 . The Western blot band shows that Figure 13 A), compared with the control group (Control), the USP7 (128 kDa) band gray value of the 1 μmol / L Skepinone-L treatment group is significantly reduced, while the gray value of the internal reference GAPDH (36 kDa) band has no significant change. Statistical analysis results show that Figure 13 B), the relative expression of USP7 / GAPDH in the treatment group is significantly lower than that in the control group (indicates that the difference compared with the control group is statistically significant, P less than 0.05, n=3). This result suggests that 1 μmol / L Skepinone-L can down-regulate the expression of target protein USP7 in cells, providing direct experimental evidence for the action of Skepinone-L by targeting USP7.

[0076] Advantages of the present application: 1. First discovery of the new application value of Skepinone-L: The present application first confirms that Skepinone-L (CAS No. 1221485-83-1) has a significant killing effect on a variety of human neuroblastoma cell lines (SH-SY5Y, SK-N-SH, SK-N-BE2, IMR-32, SK-N-AS), breaks through the previous application limitations, and can inhibit the proliferation, migration and clonogenicity of the cells, with an IC 50 value as low as 3.862 μM, providing a new candidate drug for the treatment of neuroblastoma, expanding the medical application field of Skepinone-L, and filling the gap in targeted therapy in this field.

[0077] 2. Clear mechanism of action, strong targeting, and reduced risk of toxic side effects: Most existing anti-tumor drugs have unclear target points and are prone to damage to normal cells. The present application, through molecular docking, dynamics simulation and experimental verification, clearly confirms that Skepinone-L targets USP7 as a specific target point, and exerts its effect by down-regulating the expression of USP7 and regulating the downstream apoptosis pathway (Bcl-2 / Cleaved PARP-N). This precise targeting mechanism can reduce the impact on normal cells and significantly reduce the toxic side effects of traditional chemotherapy drugs, improving treatment safety.

[0078] 3. Multi-dimensional verification to ensure reliable efficacy and complete evidence chain: Compared with the existing technology of single experimental verification, the present application verifies from cell function (viability, proliferation, migration, clonogenicity), molecular expression (apoptosis proteins, USP7) to computational biology (binding mode, stability, dissociation energy) in multiple dimensions. For example, the EdU experiment shows that it can reduce the proliferation rate by more than 30%, the Transwell experiment confirms that it can reduce the number of migrating cells by more than 70%, and the molecular dynamics simulation shows that the complex is stable (RMSD fluctuation is less than 0.4 nm), forming a complete and rigorous evidence chain, which fully proves the stability of the efficacy of Skepinone-L.

[0079] 4. The present application analyzes the binding mode of Skepinone-L and USP7 (such as hydrogen bond interaction sites, dissociation energy 26.39 kJ / mol) through computational biology methods, providing a clear modification direction for further modification of the drug molecule structure and improvement of the binding affinity. At the same time, its mechanism of targeting USP7 provides the possibility for combined use with other anti-tumor drugs (such as chemotherapy drugs, immunosuppressants), which helps to develop a synergistic treatment plan to overcome the limitations of single drugs.

[0080] 5. Significant clinical transformation potential: Skepinone-L has a concentration-dependent inhibitory effect and can play a significant role at a lower concentration (1 μM), which meets the low-dose requirement of clinical drugs. Its clear target and action pathway can help to carry out targeted preclinical research and accelerate the transformation from basic research to clinical application. In addition, the application provides a new idea for the precision treatment of neuroblastoma and can be popularized to other tumor research related to abnormal USP7, which has a wide application prospect.

[0081] Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. It is intended that the application embrace any and all variations of the present application that fall within the scope of the general inventive concept as expressed in the claims herein. It is intended that the application encompass all such variations as fall within the scope of the appended claims.

[0082] It is to be understood that the application is not limited to the precise details of construction and the above-described and shown in the drawings, and that various modifications and changes can be applied to the application without departing from the scope thereof or sacrificing any inventive understanding thereof.

Claims

1. Use of Skepinone-L in the preparation of a medicament for treating neuroblastoma.

2. Use of Skepinone-L for killing neuroblastoma cells according to claim 1, wherein the medicament is for at least one of killing neuroblastoma cells, inhibiting proliferation of neuroblastoma cells, inhibiting migration of neuroblastoma cells, or inhibiting clonogenicity of neuroblastoma cells.

3. Use of Skepinone-L for killing neuroblastoma cells according to claim 1, wherein the neuroblastoma cells are selected from one or more of SH-SY5Y, SK-N-SH, SK-N-BE2, IMR-32, or SK-N-AS cell lines.

4. Use of Skepinone-L for killing neuroblastoma cells according to claim 1, wherein the neuroblastoma cells are SH-SY5Y cells.

5. Use of Skepinone-L for killing neuroblastoma cells according to claim 1, wherein the Skepinone-L functions by targeting ubiquitin specific protease 7 protein, which is USP7.

6. Use of Skepinone-L for killing neuroblastoma cells according to claim 1, wherein the Skepinone-L binds to USP7 protein, which binding comprises hydrogen bond interaction with TYR-465 and / or HIS-461 residues of USP7 protein, the hydrogen bond interaction being at least one of 1.7 Å, 2.2 Å, or 3.1 Å. The effective concentration of the drug is 0.1 μM to 20 μM, and the IC 50 value of the drug is 3.862 μM to 10.74 μM.

7. Use of Skepinone-L for killing neuroblastoma cells according to claim 1, wherein the Skepinone-L downregulates expression of USP7 protein and / or modulates downstream apoptosis pathway, which modulating downstream apoptosis pathway comprises downregulating Bcl-2 protein expression and / or upregulating Cleaved PARP-N protein expression. comprising the steps of: co-culturing neuroblastoma cells with different concentrations of Skepinone-L; detecting the effect of Skepinone-L on killing, proliferation, migration, clonogenicity, or related protein expression of the cells by at least one method selected from CCK-8 method, cell morphology experiment, EdU incorporation experiment, Transwell migration experiment, clonogenicity experiment, Western blot detecting apoptosis related protein expression, molecular docking experiment detecting USP7 protein expression, molecular dynamics simulation of USP7 protein binding, steered molecular dynamics simulation of dissociation energy of USP7 protein, or Western blot detecting USP7 protein expression; wherein the molecular dynamics simulation of USP7 protein binding comprises analyzing at least one parameter of the complex formed by the Skepinone-L and USP7 protein, which parameter is root mean square deviation, radius of gyration, solvent accessible surface area, free energy landscape, binding free energy, or dissociation energy. ​ 8. A method for evaluating in vitro the anti-neuroblastoma activity of Skepinone-L for use according to any one of claims 1-7, characterized by, ​ ​ ​ ​

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