Application of A-674563 in killing neuroblastoma cells

Abstract

The invention relates to the technical field of medical biology, in particular to application of A-674563 in killing neuroblastoma cells, and finds for the first time that A-674563 can efficiently kill various neuroblastoma cells in a concentration-dependent manner, the IC50 value is as low as 0.1508 mu M, and proliferation, migration and clone formation of the neuroblastoma cells can be remarkably inhibited. The study on the mechanism of action shows that A-674563 accurately targets anaplastic lymphoma kinase (ALK) and is combined with a key residue through a hydrogen bond to down-regulate the expression of ALK protein and activate a downstream apoptosis pathway. Molecular docking and dynamics simulation prove that the two are stably and strongly combined. The invention provides an efficient novel candidate drug and a precise treatment strategy for solving the problems of limited curative effect, insufficient synergism and poor metastasis control of the existing treatment of neuroblastoma, and has a remarkable clinical transformation prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to an application of A-674563 in killing neuroblastoma cells. BACKGROUND

[0002] As a malignant tumor with high incidence in childhood, neuroblastoma has high malignancy, rapid progression, and is prone to distant metastasis in the early stage of the disease, which seriously threatens the life and health of children. At present, the treatment of neuroblastoma in clinical practice is constantly improving, covering surgical resection combined with postoperative adjuvant chemotherapy, radiotherapy, and salvage therapy for relapsed or refractory cases, but still faces many severe challenges in practical application.

[0003] The existing treatment drugs have obvious limitations in efficacy: even the commonly used first-line chemotherapy drugs (such as vincristine and etoposide) have low response rates for advanced or high-risk neuroblastoma, and often cannot effectively control tumor progression, leading to poor prognosis of patients. The combined treatment regimen has the problem of insufficient synergy: the current combined treatment of multiple drugs attempts to improve efficacy, but due to the dispersion of drug target points, it is difficult to form a synergistic effect of killing tumor cells, and may increase the tolerance burden of patients due to the superimposed toxicity, affecting the sustainability of treatment. The control means for tumor metastasis is scarce: neuroblastoma cells have strong invasion and metastasis ability, and the existing drugs have limited inhibitory effect on circulating tumor cells and metastatic lesions, resulting in a high recurrence rate after treatment, which is a major obstacle to improving the long-term survival rate of patients. Therefore, the development of a new drug with high killing activity, synergistic effect with existing treatment methods, and effective inhibition of tumor metastasis is a key requirement in the field of neuroblastoma treatment. The present application is committed to exploring the application of A-674563 in killing neuroblastoma cells in order to solve the above problems in the prior art and provide a new idea and basis for improving the treatment effect of neuroblastoma. SUMMARY

[0004] The present application aims to provide an application of A-674563 in killing neuroblastoma cells, which has high killing activity, synergistic effect with existing treatment methods, and can effectively inhibit tumor metastasis.

[0005] To achieve the above-mentioned purpose, in a first aspect, the present application provides an application of A-674563 in preparing a drug for treating neuroblastoma.

[0006] 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.

[0007] 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.

[0008] The effective concentration of the drug is 0.01 μM to 20 μM, and the IC 50 value of the drug is 0.1508 μM to 4.758 μM.

[0009] The A-674563 acts by targeting anaplastic lymphoma kinase protein, which is ALK protein.

[0010] The A-674563 binds to the ALK protein, and the binding includes forming a hydrogen bond interaction with ARG-1253 and / or GLU-1197 residues of the ALK protein.

[0011] The A-674563 down-regulates the expression of the ALK protein and / or regulates a downstream apoptosis pathway, and the regulating the downstream apoptosis pathway includes down-regulating the expression of Bcl-2 protein and / or up-regulating the expression of Cleaved PARP-N protein.

[0012] In a second aspect, the present application provides a method for evaluating the anti-neuroblastoma activity of A-674563 in vitro, for evaluating the use as described in the first aspect, comprising the following steps: Co-culturing neuroblastoma cells with different concentrations of A-674563; Detecting the effect of A-674563 on killing, proliferation, migration, colony formation ability or related protein expression of the cells by at least one method selected from CCK-8 method, cell morphology observation experiment, EdU incorporation experiment, Transwell migration experiment, colony formation experiment, Western blot detection of apoptosis-related protein expression, molecular docking experiment detection of ALK protein expression, molecular dynamics simulation of ALK protein binding, stretching molecular dynamics simulation of dissociation energy of ALK protein or Western blot detection of ALK protein expression; The molecular dynamics simulation of ALK protein binding includes analyzing at least one parameter of the complex formed by the A-674563 and the ALK protein, such as root mean square deviation, radius of gyration, solvent accessible surface area, free energy landscape, binding free energy or dissociation energy.

[0013] The application of A-674563 for killing neuroblastoma cells of the present application first discovers that A-674563 can efficiently and in a concentration-dependent manner kill a plurality of neuroblastoma cells, and the IC 50The value is as low as 0.1508 muM, and can significantly inhibit the proliferation, migration and clone formation. Mechanism research shows that: A-674563 precisely targets anaplastic lymphoma kinase (ALK), binds to key residues through hydrogen bonds, down-regulates the expression of ALK protein and activates the downstream apoptosis pathway. Molecular docking and dynamics simulation confirm that the combination of the two is stable and strong. The application provides an efficient new candidate drug and precise treatment strategy for solving the limitations of existing treatment, lack of synergy and poor metastasis control of neuroblastoma, and has a significant clinical conversion prospect. BRIEF DESCRIPTION OF DRAWINGS

[0014] 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 as follows.

[0015] Figure 1 is a result schematic diagram of CCK-8 method for detecting the influence of different concentrations of A-674563 on the activity of human neuroblastoma cell lines treated for 24 hours.

[0016] Figure 2 is a result schematic diagram of inverted microscope observation of the influence of A-674563 on the morphology of human neuroblastoma cells (SH-SY5Y) treated for 24 hours.

[0017] Figure 3 is a result schematic diagram of EdU incorporation experiment for detecting the influence of A-674563 on the proliferation of SH-SY5Y cells.

[0018] Figure 4 is a structure schematic diagram of Transwell experiment for detecting the influence of A-674563 on the migration of SH-SY5Y cells.

[0019] Figure 5 is a result schematic diagram of clone formation experiment for detecting the influence of A-674563 on the clone formation ability of cells.

[0020] Figure 6 is a result schematic diagram of Western blot for detecting the influence of A-674563 on the expression of apoptosis-related proteins of cells.

[0021] Figure 7 is a result schematic diagram of molecular docking simulation of the interaction between A-674563 and ALK protein.

[0022] Figure 8 is a RMSD change graph of ALK and A-674563 complex in molecular dynamics simulation.

[0023] Figure 9 is a change graph of radius of gyration (Rg) of ALK and A-674563 complex in molecular dynamics simulation.

[0024] Figure 10 is a plot of the solvent accessible surface area (SASA) change of the ALK-A-674563 complex in a molecular dynamics simulation.

[0025] Figure 11 is a plot of the free energy landscape of the ALK-A-674563 complex.

[0026] Figure 12 is a plot of the potential of mean force (PMF) analysis of the ALK-A-674563 complex.

[0027] Figure 13 is a plot of the results of a Western blot to detect the effect of A-674563 on ALK protein expression. DETAILED DESCRIPTION

[0028] Reference will now be made in detail to the exemplary embodiments, examples of which are illustrated in the accompanying drawings. The following description refers to the accompanying drawings in which the same numbers represent the same entities or the like throughout the several figures. The implementations described in this detailed description with respect to the following exemplary embodiments do not represent all or the only implementations consistent with the application.

[0029] The terminology used in this application, and by those of ordinary skill in the art, is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this application, 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.

[0030] 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 solely to distinguish one from another only. For example, a first information can be termed a second information, and, similarly, a second information can be termed a first information, without departing from the scope of the present application. As used herein, the word "if' can be construed to mean "when" or "in response to determining" or "in response to a determination" depending on the context.

[0031] The application provides an application of A-674563 in killing neuroblastoma cells, and mainly an application of A-674563 in preparation of a drug for treating neuroblastoma. The purpose is to solve the problems of limited drug efficacy, insufficient synergy of combined treatment, poor tumor metastasis control and the like in existing neuroblastoma treatment, and to provide the application of A-674563 in killing neuroblastoma cells. Specifically, the purpose is to prove that A-674563 can kill neuroblastoma cells specifically by targeting ALK (anaplastic lymphoma kinase) and inhibit the malignant biological behaviors (proliferation, migration, clonal formation and the like) of the neuroblastoma cells, so as to provide a scientific basis and a new treatment strategy for precise treatment of neuroblastoma.

[0032] A-674563 (CAS No. 552325-73-2) 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: Experimental materials Drug: A-674563 (purity ≥ 98%), dissolved with DMSO to prepare working solutions (0.1 μM, 1 μM, 5 μM, 10 μM and 20 μM) of different concentrations.

[0033] Cell model: Human neuroblastoma cell lines SH-SY5Y and SK-N-AS are cultured in DMEM culture medium containing 10% fetal bovine serum; human neuroblastoma cell lines SK-N-SH and IMR-32 are cultured in MEM culture medium containing 10% fetal bovine serum and NEAA (non-essential amino acids); human neuroblastoma cell line SK-N-BE2 is cultured in DMEM / F12 culture medium containing 10% fetal bovine serum; all the cells are cultured in a 37°C, 5% CO2 incubator, and the liquid is changed regularly to maintain the logarithmic growth state of the cells.

[0034] Experimental method and step: (1) CCK-8 method for detecting cell activity Purpose: The inhibitory effect of different concentrations of A-674563 on the activity of neuroblastoma cells is detected, and the half inhibitory concentration (IC 50 ) is calculated.

[0035] Step: Cell inoculation: the logarithmic growth phase human neuroblastoma cell lines (SK-N-BE2, SK-N-AS, IMR-32, SH-SY5Y and SK-N-SH) are taken out, the concentration is adjusted to 5×10³ cells per hole with the corresponding culture medium (according to the requirements of the cell model) containing 10% fetal bovine serum, inoculated in a 96-well plate, the volume of each hole is 100 μL, and 3 replicate holes are set in each group.

[0036] Drug treatment: After 24h incubation, the original medium was discarded, and fresh medium containing different concentrations of A-674563 (concentration gradient: 0, 0.01, 0.1, 1, 5, 10, 20 μmol / L, final concentration of solvent DMSO ≤0.1%) was added, 100 μL per well, and the control group was added with medium containing the same amount of DMSO.

[0037] Incubation and detection: After 24h incubation at 37℃ in a 5% CO2 incubator, 10 μL of CCK-8 reagent was added to each well, and incubation was continued for 2h; the absorbance (OD value) of each well was measured at 450nm wavelength using a microplate reader.

[0038] Data calculation: Cell survival rate (%) = (treated group OD value / control group OD value) x 100%; the IC 50 value and 95% confidence interval of each cell line were calculated by fitting the dose-effect curve using GraphPad Prism 9 software.

[0039] (2) Cell morphology observation experiment Objective: To visually observe the effect of A-674563 on the morphology of neuroblastoma cells.

[0040] Steps: Cell inoculation: SH-SY5Y cells (logarithmic growth phase) were taken and adjusted to a concentration of 2 x 10 4 cells / well, inoculated in a 6-well plate with 2 mL of DMEM medium containing 10% fetal bovine serum per well, and incubated at 37℃ with 5% CO2 for 24h until the cells adhered.

[0041] Drug treatment: The original medium was discarded, and fresh medium containing 1 μmol / L A-674563 was added to the experimental group, and medium containing the same amount of DMSO was added to the control group, and incubation was continued for 24h.

[0042] Observation and photography: An inverted phase contrast microscope (model: Olympus IX73) was used to observe the cell morphology at 100x magnification, and 3 different fields were selected to take images (ruler = 1000 μm), and changes in cell density, morphology (such as rounding and dispersion) were recorded.

[0043] (3) EdU incorporation experiment to detect cell proliferation Objective: To label proliferating cells with EdU and quantify the inhibitory effect of A-674563 on the proliferation of neuroblastoma cells.

[0044] Steps: Cell inoculation: SH-SY5Y cells were adjusted to a concentration of 1 x 10 4Cell culture: SH-SY5Y cells were seeded at 5×10

[0045] Drug treatment: Experimental group was added with 1 μmol / L A-674563, and control group was added with equal amount of DMSO, and incubated for 24 h.

[0046] EdU labeling: The culture medium was removed, and 500 μL of culture medium containing 50 μmol / L EdU was added to each well, and incubated at 37°C for 2 h.

[0047] Fixation and permeation: The EdU culture medium was removed, and washed twice with PBS; 4% paraformaldehyde (prepared with PBS) was added for fixation for 30 min, and washed twice with PBS; 0.5% Triton X-100 (prepared with PBS) was added for permeation for 15 min, and washed twice with PBS.

[0048] Fluorescent staining: According to the EdU detection kit (RiboBio) instructions, 500 μL of Click reaction solution (containing Alexa Fluor 594-azide) was added to each well, and incubated in the dark for 30 min; after washing with PBS for 3 times, DAPI staining solution (1 μg / mL) was added for incubation in the dark for 10 min, and washed with PBS for 3 times.

[0049] Observation and statistics: The coverslips were removed, and mounted with anti-fluorescence quenching mounting medium; observed under a fluorescence microscope (200x magnification), EdU positive cells (red) were proliferative cells, and DAPI (blue) labeled cell nuclei; 5 fields of view were randomly selected from each group, and the proportion of EdU positive cells in total cells was counted, and statistical analysis was performed using GraphPad Prism9 (t test, P<0.01).

[0050] (4) Transwell experiment to detect cell migration ability Objective: To evaluate the inhibitory effect of A-674563 on the migration ability of neuroblastoma cells.

[0051] Steps: Cell pretreatment: SH-SY5Y cells were starved with serum-free DMEM medium for 12 h, and the concentration was adjusted to 5×10 4 per mL.

[0052] Transwell chamber preparation: Take the Transwell chamber (pore size 8 μm) matched with the 24-well plate, add 200 μL of cell suspension (containing 1×10 4 per mL) and 1 μmol / L A-674563 (experimental group) or equal amount of DMSO (control group) to the upper chamber; add 600 μL of DMEM medium containing 20% fetal bovine serum (chemotactic factor) to the lower chamber.

[0053] Incubation and fixation: After 24 h incubation at 37℃ in 5% CO2 incubator, remove the chamber and gently wipe off the non-migrated cells on the upper chamber with a wet cotton swab; fix the lower surface of the chamber membrane with 4% paraformaldehyde for 30 min, and wash twice with PBS.

[0054] Staining and counting: Stain with 0.1% crystal violet solution (prepared in PBS) for 20 min, and wash off the excess stain with PBS; observe under an inverted microscope (200x magnification) and count the migrated cells (darkly stained cells) in 5 randomly selected fields for each group; analyze the data using GraphPad Prism 9 (t-test, P < 0.001).

[0055] (5) Clonogenic assay to detect cell clonogenicity Objective: To detect the effects of A-674563 on the long-term proliferation and clonogenicity of neuroblastoma cells.

[0056] Procedure: Cell seeding: Adjust the concentration of SH-SY5Y cells to 200 cells / well, and seed them in a 6-well plate with 2 mL of DMEM medium containing 10% fetal bovine serum per well, and incubate for 24 h for adhesion.

[0057] Drug treatment: Add fresh medium containing 0, 0.01, 0.1, and 1 μmol / L of A-674563 (DMSO final concentration ≤ 0.1%) to each well, with 3 replicate wells for each group.

[0058] Culture and staining: Incubate at 37℃ in a 5% CO2 incubator for 14 days (replace the medium containing the drug every 3 days); after the clones form (visible to the naked eye), aspirate the medium and wash twice with PBS; fix with 4% paraformaldehyde for 30 min, and then stain with 0.1% crystal violet solution for 20 min; wash off the excess stain with running water, and air dry.

[0059] Counting and statistics: Count the number of clones with ≥ 50 cells per well under a microscope; calculate the clonogenicity rate (%) = (number of clones in the treatment group / number of clones in the control group) x 100%; analyze the concentration-dependent differences using GraphPad Prism 9 (one-way ANOVA, P < 0.05).

[0060] (6) Western blot to detect the expression levels of apoptosis-related proteins Objective: To detect the regulation of A-674563 on the expression of apoptosis-related proteins (Bcl-2, PARP, and Cleaved PARP-N) in neuroblastoma cells.

[0061] Procedure: Cell treatment and protein extraction: Seed SH-SY5Y cells in a 6-cm culture dish (5 x 105 After 24 h of culture, 1 μmol / L A-674563 was added to the experimental group, and the same amount of DMSO was added to the control group, and then incubated for 24 h. The culture medium was discarded, and the cells were washed twice with PBS. RIPA lysis buffer (100 μL / dish) containing PMSF was added, and the cells were lysed on ice for 30 min. The supernatant (total protein) was obtained by centrifugation at 12,000 rpm for 15 min at 4 °C.

[0062] Protein quantification: The protein concentration was determined using a BCA protein quantification kit, and the protein concentration of each group was adjusted to be consistent (2 μg / μL). 5x loading buffer was added (final concentration 1x), and denaturation was performed at 95 °C for 10 min.

[0063] Electrophoresis and membrane transfer: 30 μg of protein was loaded into each well, and 10% SDS-PAGE gel electrophoresis was performed (concentrated gel 80 V for 30 min, and separation gel 120 V for 90 min). Wet transfer (300 mA constant current, 120 min) was performed to PVDF membrane (pre-activated with methanol).

[0064] Blocking and incubation: 5% skim milk powder (TBST configuration) was used for room temperature blocking for 2 h. TBST was washed for 3 times (10 min each time). The primary antibody (Bcl-2: 1:1000, PARP: 1:1000, Cleaved PARP-N: 1:1000, GAPDH: 1:5000) was added, and incubated at 4 °C overnight. TBST was washed for 3 times, and the HRP-labeled secondary antibody (1:5000) was added, and incubated at room temperature for 1 h. TBST was washed for 3 times.

[0065] Development and analysis: ECL chemiluminescence kit was used for development, and ImageQuant LAS4000 imaging was used. ImageJ software was used to analyze the gray value of the band. GAPDH was used as the internal reference, and the relative expression of the target protein (treatment group / control group) and the Cleaved PARP-N / PARP ratio were calculated. Statistical analysis (t test, P<0.05) was performed.

[0066] (7) Molecular docking experiment to predict the interaction mode of A-674563 and target protein ALK Objective: To predict the binding site and interaction mode of A-674563 and ALK protein.

[0067] Steps: Protein preparation: ALK protein crystal structure (PDB ID: 2XP2) was obtained from PDB database. PyMOL was used to remove crystal water and ligand. AutoDockTools1.5.6 was used to add Gasteiger charge and define the active pocket (center coordinates: x=50.1, y=112.3, z=88.5; size: 30x30x30 Å).

[0068] Ligand preparation: SMILES structure of A-674563 (CAS 552325-73-2) was converted to 3D structure by ChemDraw, energy minimized at DFT / B3LYP / 6-31G level by Gaussian09, saved as pdbqt format.

[0069] Molecular docking: Docking was performed by AutoDock Vina with exhaustiveness=32, other parameters default; the lowest energy conformation (-8.2 kcal / mol) was selected, and the binding mode was visualized by PyMOL, focusing on the hydrogen bond interactions (e.g. with ARG-1253, GLU-1197) and distances.

[0070] (8) Analysis of the binding affinity and stability of A-674563 to the target protein ALK by molecular dynamics simulation Objective: To verify the structural stability and thermodynamic characteristics of the A-674563-ALK complex.

[0071] Steps: System construction: The initial structure was the optimal conformation from molecular docking, and the complex system was constructed by GROMACS2021.4; the CHARMM36 force field parameterized the protein and ligand, and the TIP3P water model filled the water box (10 Å away from the protein surface) and added Na⁺ to neutralize the charge.

[0072] Energy minimization: The steepest descent method (steepest descent) was used to optimize the system, with a maximum of 50,000 steps until the potential energy converged (<1000 kJ / mol•nm).

[0073] Equilibrium simulation: NVT equilibrium (300 K, V-rescale temperature control, 100 ps) → NPT equilibrium (1 atm, Parrinello-Rahman pressure control, 100 ps), with the protein backbone atoms constrained.

[0074] Production simulation: 100 ns of molecular dynamics simulation was performed under unconstrained conditions, with one frame saved every 10 ps, and five independent simulations were repeated (ALK-A-674563-1 to 5).

[0075] Trajectory analysis: GROMACS tools were used to calculate the back bone RMSD (to evaluate the overall structural stability), the radius of gyration Rg (to evaluate the structural compactness), and the solvent accessible surface area SASA (to evaluate the interaction with the solvent); PyMOL was used to plot the free energy landscape (RMSD and Rg as variables, gmx sham calculation).

[0076] (9) Steered molecular dynamics simulation and umbrella sampling simulation to analyze the dissociation energy of A-674563 from target protein ALK Objective: Calculate the energy required for A-674563 to dissociate from the ALK protein to evaluate the binding strength.

[0077] Steps: Steered molecular dynamics (SMD): Take the complex equilibrated by molecular dynamics as the initial structure, define the reaction coordinate (ξ) along the direction from the ALK active pocket to the solvent, apply a constant stretching force (1000 kJ / mol•nm 2 , rate 0.01 nm / ps), simulate the dissociation process of A-674563, and record the force-distance curve.

[0078] Umbrella sampling (Umbrella Sampling): Set 30 windows (ξ=0 to 5 nm, interval 0.17 nm) on the key dissociation path determined by SMD, apply a harmonic potential (force constant 1000 kJ / mol•nm 2 ) to each window, and perform 10 ns simulation; process the trajectory using the WHAM method to calculate the potential energy surface (PMF), and the dissociation energy is the peak value of the PMF curve (ΔG≈32.22 kJ / mol).

[0079] (10) Western blot to detect the expression level of target protein ALK Objective: Detect the effect of A-674563 on the expression of ALK protein in neuroblastoma cells.

[0080] Steps: Cell treatment and protein extraction: Follow step 6 (apoptosis-related protein detection), the treatment group is 1 μmol / L A-674563, and the control group is DMSO.

[0081] Electrophoresis and antibody incubation: 10% SDS-PAGE electrophoresis (30 μg protein / well), transfer to PVDF membrane; after blocking with 5% skim milk powder for 2 h, add ALK primary antibody (1:1000) and GAPDH primary antibody (1:5000), incubate overnight at 4°C; HRP secondary antibody (1:5000) incubate at room temperature for 1 h.

[0082] Development and analysis: ECL development, ImageJ analysis of band gray value, GAPDH normalization of ALK relative expression, statistical analysis (t test, P<0.01).

[0083] Experimental results Cell level action verification (1) Inhibition of A-674563 on neuroblastoma cell viability, corresponding to CCK-8 detection chart, asFigure 1 As shown.

[0084] Different concentrations of A-674563 (0, 0.01, 0.1, 1, 5, 10, 20 μmol / L) were detected by CCK-8 assay after 24 h of treatment with SK-N-BE2 (… Figure 1 A), SK-N-AS ( Figure 1 B), IMR-32 Figure 1 C), SH-SY5Y Figure 1 D), SK-N-SH Figure 1 E) Effects on the viability of neuroblastoma cell lines. The x-axis represents the concentration of A-674563 (μmol / L), and the y-axis represents cell viability (%). Data are presented as mean ± standard deviation. The results showed that the viability of each cell line decreased significantly in a concentration-dependent manner with increasing drug concentration. Compared with the control group (0 μmol / L), the difference in cell viability was statistically significant when the drug concentration reached a certain value (P<0.001). This indicates that A-674563 can effectively inhibit the viability of neuroblastoma cells, and the inhibitory effect is positively correlated with concentration. The IC50 of A-674563 on different neuroblastoma cell lines. 50 The values ​​(μM) are shown in Table 1.

[0085] Table 1. Killing effect of A-674563 on different neuroblastoma cell lines (IC50) 50 Value (μM) (2) Effects of A-674563 on the morphology of neuroblastoma cells, corresponding to inverted microscope observations, as shown in the attached figure. Figure 2 As shown.

[0086] Morphological changes in human neuroblastoma cells (taking SH-SY5Y as an example) after treatment with 1 μmol / LA-674563 for 24 h were observed using an inverted microscope. Figure 2 Control group () Figure 2 A) Cells are densely distributed and grow regularly; treatment group ( Figure 2 B) Decreased cell density, and rounded and dispersed cell morphology. This visually demonstrates that A-674563 disrupts cell growth, resulting in decreased cell density and rounded and dispersed cell morphology, indicating a significant effect of the drug on cell morphology.

[0087] (3) The inhibitory effect of A-674563 on the proliferation of neuroblastoma cells, corresponding to the EdU incorporation experiment diagram, as shown in the figure. Figure 3 As shown.

[0088] The effect of A-674563 on the proliferation of SH-SY5Y cells was detected using an EdU incorporation assay. Figure 3 Control group ()Figure 3 A) EdU staining (red) showed a high number of proliferating cells, indicating active cell proliferation; the group treated with 1 μmol / LA-674563 for 24 h ( Figure 3 (Under A), EdU-positive cells were significantly reduced. Statistical analysis ( Figure 3 B) showed that the proportion of EdU-positive cells in the treatment group was significantly different from that in the control group (**, P<0.01), confirming that A-674563 can inhibit the proliferation of SH-SY5Y cells, thus explaining its inhibitory effect on tumor cell growth from the perspective of proliferation.

[0089] (4) The inhibitory effect of A-674563 on the migration of neuroblastoma cells, corresponding to the Transwell assay diagram, as shown in the figure. Figure 4 As shown.

[0090] The effect of A-674563 on the migration ability of SH-SY5Y cells was evaluated using a Transwell assay. Figure 4 Control group () Figure 4 (Left) A large number of deeply stained cells have completed migration. EdU staining (red) marks proliferating cells, and DAPI staining (blue) marks cell nuclei. Merge is an overlay image; 1 μmol / LA-674563 treatment group ( Figure 4 (A right) The number of migrating cells was significantly reduced. Statistical analysis ( Figure 4 B) indicates that the number of cells migrating in the treatment group was significantly different from that in the control group (P<0.001), proving that A-674563 can inhibit the migration of SH-SY5Y cells and weaken the invasive and metastatic ability of tumor cells.

[0091] (5) The inhibitory effect of A-674563 on the colony formation ability of neuroblastoma cells, corresponding to the plate colony formation experiment diagram, as shown in the figure. Figure 5 As shown.

[0092] The effect of A-674563 on cell colony formation ability was detected by plate colony formation assay. Figure 5 After staining, the number of blue clonal clusters in the control group and the treatment groups at different concentrations (0.01, 0.1, 1 μmol / L) decreased with increasing drug concentration. Figure 5 A); Statistical analysis ( Figure 5 B) shows that the number of clones in each treatment group was significantly different from that in the control group (P<0.05). This indicates that A-674563 can inhibit cell colony formation in a concentration-dependent manner, further supporting its inhibitory effect on the long-term growth and self-renewal ability of tumor cells.

[0093] Molecular mechanism exploration (1) Regulation of apoptosis-related protein expression in neuroblastoma cells by A-674563, corresponding to the Western blot analysis, as shown in the figure. Figure 6 As shown.

[0094] Western blot was used to detect the effect of A-674563 on apoptosis-related proteins in neuroblastoma cells. Figure 6 Using GAPDH as an internal control, the proteins PARP, CleavedPARP-N, and Bcl-2 were detected. Figure 6 A); Statistical analysis showed that, compared with the control group, the relative expression level of Bcl-2 protein in the 1 μmol / L treatment group was decreased ( Figure 6 B, P<0.05), the grayscale ratio of CleavedPARP-N to PARP increased ( Figure 6 C, P<0.05). This indicates that A-674563 can regulate the expression of apoptosis-related proteins and promote cell apoptosis, providing a basis for its apoptosis mechanism in inhibiting tumor cell growth.

[0095] (2) Molecular docking between A-674563 and ALK protein, corresponding to the molecular docking simulation diagram, as shown in the figure. Figure 7 As shown.

[0096] The binding mode of A-674563 to ALK protein was simulated by molecular docking. Figure 7 Overall assembly diagram () Figure 7 A) Showing the binding site of A-674563 (green) in the active pocket of the ALK protein (red 3D structure); details of the local interaction ( Figure 7 B) Demonstrates the hydrogen bonding between A-674563 and specific amino acid residues (such as ARG-1253 and GLU-1197) (yellow dashed lines, indicating distances). This reveals the molecular binding mechanism of A-674563 targeting ALK, laying the foundation for further investigation into the pathway of action, as shown in Table 2, which displays the MM / PBSA binding free energy of the ALK-A-674563 complex.

[0097] Table 2. MM / PBSA binding free energy of the ALK-A-674563 complex (kcal / mol) Thermodynamic and structural stability analysis of the ALK-A-674563 complex (1) Combining free energy analysis with the corresponding potential energy surface analysis diagram, such as Figure 13 As shown.

[0098] Binding free energy analysis of the ALK-A-674563 complex was performed (based on stretching molecular dynamics simulations and umbrella sampling simulation data). Figure 13), the free energy difference (such as ΔG≈32.22 kJ / mol) reflects the thermodynamic trend of the combination of the two, indicating that the complex formation has an energy advantage, supporting the thermodynamic stability of the combination of A-674563 and ALK, and providing a basis for the mechanism study.

[0099] (2) RMSD change analysis, corresponding to the RMSD change curve as shown in Figure 9 .

[0100] In the molecular dynamics simulation, the backbone RMSD change of the ALK-A-674563 complex was monitored Figure 9 . The abscissa is the simulation time (ns), and the ordinate is the root mean square deviation (RMSD, nm) of the backbone atoms; the overall fluctuation of the multiple independent simulation curves (ALK-A-674563-1 to 5) is small and tends to be stable, indicating that the structure of the complex is stable during the simulation, verifying the stability of the combination of the two, and providing data for analyzing the relationship between structure and function.

[0101] (3) Change analysis of radius of gyration (Rg), corresponding to the Rg change curve as shown in Figure 10 .

[0102] The change of the radius of gyration (Rg) of the ALK-A-674563 complex in the molecular dynamics simulation was analyzed Figure 10 . The abscissa is the simulation time (ns), and the ordinate is the radius of gyration (Rg, nm) of the complex; the multiple independent simulation curves fluctuate little and the values are stable, reflecting the good compactness of the structure of the complex during the simulation, reflecting the stability from the structural dimension, and helping to understand the spatial conformation and functional influence after the combination of A-674563 and ALK.

[0103] (4) Solvent accessible surface area (SASA) change analysis, corresponding to the SASA change curve as shown in Figure 11 .

[0104] The change of the solvent accessible surface area (SASA) of the ALK-A-674563 complex in the molecular dynamics simulation was explored Figure 11 . The abscissa is the simulation time (ns), and the ordinate is the SASA (nm²); the multiple independent simulation curves fluctuate little and the values are stable, indicating that the surface and solvent interaction area of the complex is relatively constant during the simulation period, reflecting the structural stability, and creating conditions for analyzing the behavior of the complex in the solution environment and the interaction with surrounding molecules.

[0105] (5) Free energy landscape analysis, corresponding to the free energy landscape graph as shown in Figure 12 .

[0106] The free energy landscape graph of the ALK-A-674563 complex was constructedFigure 12 ), with RMSD (nm) and Rg (nm) as two-dimensional variables, to present the free energy distribution (kJ / mol) in simulation; the landscape map is funnel-shaped in energy, with low-energy states concentrated at the bottom, reflecting that the complex tends to be stable in a specific conformation, and clarifying the dominant conformation and thermodynamic stability characteristics of the complex after binding.

[0107] Effect of A-674563 on ALK protein expression (corresponding to the Western blot detection figure, as shown in Figure 6B) Figure 13 ) Effect of A-674563 on ALK protein expression in neuroblastoma cells was detected by Western blot (Figure 6B) Figure 13 , and ALK (180 kDa) protein was detected (Figure 6B) with GAPDH as an internal reference. Figure 13 A); statistical analysis showed that the relative expression of ALK protein in the 1 μmol / L treatment group was reduced, and the difference with the control group was statistically significant (P<0.01, Figure 13 B). It is shown that A-674563 can down-regulate the ALK protein level in tumor cells, and reveals its regulation effect on the ALK-related signal pathway from the protein expression level.

[0108] In summary, A-674563 can target ALK protein to inhibit the viability, proliferation, migration and clonogenicity of neuroblastoma cells, regulate the expression of apoptosis-related proteins to promote cell apoptosis; molecular docking and dynamics simulation verify the thermodynamic and structural stability of its binding with ALK, which provides a new strategy and scientific basis for precise treatment of neuroblastoma.

[0109] Advantages of the present application: 1. Significant killing effect with concentration dependence: A-674563 can effectively inhibit the viability of various neuroblastoma cell lines, and the inhibition effect is significantly enhanced with the increase of concentration, and the half-inhibitory concentration (IC 50 ) of different cell lines is at a low level, indicating that it has high killing activity.

[0110] 2. Multidimensional inhibition of tumor cell malignant behavior: not only can reduce cell viability, but also can significantly inhibit the proliferation, migration and clonogenicity of neuroblastoma cells, block the growth and development of tumor cells from multiple key links, and comprehensively suppress tumor progression.

[0111] 3. Promote tumor cell apoptosis: by regulating apoptosis-related proteins (such as reducing Bcl-2 expression and increasing CleavedPARP-N / PARP ratio), effectively promote neuroblastoma cell apoptosis, and provide clear mechanism support for its killing tumor cells.

[0112] 4. Strong targeting and stable binding: clear targeting of ALK protein, molecular docking and dynamics simulation confirmed that the two have good thermodynamic and structural stability, which guarantees the specificity and effectiveness of the action, and helps to achieve precise treatment.

[0113] 5. Provide new strategies for clinical treatment: solve the problems of drug efficacy limitation, insufficient synergy of combination therapy, poor control of tumor metastasis and other problems in existing treatment, provide new ideas and scientific basis for the treatment of neuroblastoma, and are expected to improve the prognosis of patients.

[0114] 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 cover any and all variations of the application that come within the scope of the general inventive concepts and that include customarily known or customary technical features not specifically shown or described herein.

[0115] It should be understood that the application is not limited to the precise construction that has been described above and shown in the accompanying drawings, and that various modifications and changes can be made without departing from the scope thereof.

Claims

1. Use of A-674563 in the manufacture of a medicament for treating neuroblastoma.

2. Use of A-674563 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 A-674563 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 A-674563 for killing neuroblastoma cells according to claim 1, wherein the neuroblastoma cells are SH-SY5Y cells.

5. Use of A-674563 for killing neuroblastoma cells according to claim 1, wherein the A-674563 functions by targeting anaplastic lymphoma kinase protein, which is ALK protein.

6. Use of A-674563 for killing neuroblastoma cells according to claim 1, wherein the A-674563 binds to ALK protein, and the binding comprises hydrogen bond interaction with ARG-1253 and / or GLU-1197 residues of ALK protein. The effective concentration of the drug is 0.01 μM to 20 μM, and the IC 50 value of the drug is 0.1508 μM to 4.758 μM.

7. Use of A-674563 for killing neuroblastoma cells according to claim 1, wherein the A-674563 down-regulates expression of ALK protein and / or modulates downstream apoptosis pathway, and the modulating downstream apoptosis pathway comprises down-regulating Bcl-2 protein expression and / or up-regulating Cleaved PARP-N protein expression. comprising the steps of: co-culturing neuroblastoma cells with different concentrations of A-674563; detecting the effect of A-674563 on killing, proliferation, migration, clonogenicity, or related protein expression of the cells by at least one method selected from CCK-8 method, cell morphology observation experiment, EdU incorporation experiment, Transwell migration experiment, clonogenicity experiment, Western blot detection of apoptosis-related protein expression, molecular docking experiment detection of ALK protein expression, molecular dynamics simulation of ALK protein binding, stretching molecular dynamics simulation of dissociation energy of ALK protein, or Western blot detection of ALK protein expression; wherein the molecular dynamics simulation of ALK protein binding comprises analyzing at least one parameter of the complex formed by the A-674563 and ALK protein, including 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 the anti-neuroblastoma activity of A-674563 in vitro for use in the method of any one of claims 1 to 7, characterized in that, ​ ​ ​ ​

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