Use of sm1-71 in the manufacture of a medicament for treating glioma

Abstract

The application provides application of SM1-71 in preparation of a drug for preventing or treating glioma, and through in-vitro cell experiments and in-vivo animal experiments, it is confirmed that SM1-71 can significantly inhibit glioma cell proliferation, induce cell apoptosis, inhibit cell migration, and inhibit glioma transplanted tumor growth in vivo, and meanwhile, the anti-glioma mechanism is determined.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the use of SM1-71 in the preparation of drugs for the treatment of gliomas. Background Technology

[0002] SM1-71 is a preclinical multi-target covalent kinase inhibitor, currently used primarily as a chemical biology tool molecule and lead scaffold, rather than an approved or systematically validated therapeutic in glioma. Existing research indicates that SM1-71 can reversibly bind to the ATP pocket and form covalent bonds with adjacent cysteine ​​residues, simultaneously targeting multiple kinases such as MEK1 / 2 / 3 / 4 / 6 / 7, ERK1 / 2, SRC, YES1, FGFR1, LIMK1, and TAK1, thereby synergistically inhibiting the MAPK and PI3K survival signaling networks. However, to date, direct primary evidence for SM1-71 in glioma remains limited, defining it as a multi-target covalent kinase probe with anti-glioma potential.

[0003] SM1-71 was initially developed as a multi-target covalent kinase inhibitor based on TAK1-centered polypharmacology and subsequent kinome chemoproteomics (TAK1-centered multi-target drug action and subsequent kinaseomics proteomics). Publicly available data shows that it can covalently target 23 kinases, including MKNK2, MAP2K1 / 2 / 3 / 4 / 6 / 7, MAP3K7 (TAK1), MAPK1 / 3, SRC, YES1, FGFR1, LIMK1, and RSK2. Subsequent cell studies suggest that it can influence a broader kinase network at the cellular level. Based on these characteristics, SM1-71 has been used as a tool compound for tumor signaling network research and as a lead scaffold for developing selective SRC, MKK4 / 7, and LIMK1 kinase inhibitors.

[0004] Currently, among the existing technologies related to glioma, selective inhibitors of LIMK1 have been reported to have demonstrated antitumor activity in the LN229 glioblastoma cell model. While compound SM1-71 has provided structural inspiration for the design of such inhibitors due to its structural characteristics, publicly available information does not explicitly state that SM1-71 itself can serve as a direct anti-glioma drug; its direct anti-glioma effect still requires further confirmation. Summary of the Invention

[0005] The purpose of this invention is to provide the application of SM1-71 in the preparation of a drug for treating glioma, which achieves anti-tumor effects by inhibiting glioma cell proliferation, inducing apoptosis, and inhibiting cell migration.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: This invention provides the use of SM1-71, as shown in Formula I, in the preparation of a medicament for the prevention or treatment of gliomas. Formula 1.

[0007] Optionally, in the above-mentioned application of SM1-71 in the preparation of drugs for the prevention or treatment of glioma, the concentration range of SM1-71 is: 285.3 nM to 375.6 nM for LN229 cells; 315.4 nM to 405.6 nM for U251 cells; and 364.85 nM to 476.79 nM for A172 cells.

[0008] Optionally, in the use of the above-mentioned SM1-71 in the preparation of a medicament for the prevention or treatment of glioma, the glioma is glioblastoma.

[0009] Optionally, in the application of SM1-71 in the preparation of a medicament for the prevention or treatment of glioma, the medicament is used to inhibit glioma cell proliferation, induce glioma cell apoptosis, or inhibit glioma cell migration.

[0010] Optionally, in the use of SM1-71 in the preparation of a medicament for the prevention or treatment of glioma, the medicament comprises a therapeutically effective amount of SM1-71 and pharmaceutically acceptable excipients and / or carriers.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention demonstrates through in vitro cell experiments and in vivo animal experiments that SM1-71 can significantly inhibit glioma cell proliferation, induce apoptosis, inhibit cell migration, and inhibit glioma xenograft growth in vivo, while clarifying its anti-glioma mechanism of action.

[0012] SM1-71 has the following advantages in the treatment of gliomas: 1. Glioblastoma exhibits almost universal abnormal kinase signaling, especially persistent activation of the RTK / Ras / Raf / MEK / ERK axis, which is closely associated with invasiveness, radiotherapy and chemotherapy resistance, and recurrence. SM1-71 is not a single-point inhibitor, but can simultaneously target multiple modules such as MEK / ERK, RTK-AKT, SRC / YES1, FGFR1, and LIMK1. This "one drug for multiple axes" characteristic is theoretically more suitable for addressing pathway redundancy and bypass compensation in GBM.

[0013] 2. Several of the targets of SM1-71 are highly correlated with key phenotypes in gliomas. For example, FGFR1 is associated with glioma stem cell maintenance, proliferation, migration, and radiotherapy resistance in GBM; c-Src / Fyn is an important effector molecule of oncogenic EGFR signaling, which can enhance GBM invasion and tumor cell survival; LIMK1 / 2 directly regulates GBM cytoskeleton remodeling and invasive movement. Therefore, SM1-71 theoretically has the potential to inhibit proliferation / survival, inhibit invasion and migration, and intervene in stemness and treatment resistance. Attached Figure Description

[0014] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0015] Figure 1 The dose-response curve and IC50 value of SM1-71 after 48 hours of treatment on LN229 and U251 cells; Figure 2 This describes the SM1-71-induced apoptosis in U251 cells; Figure 3 This describes the SM1-71-induced apoptosis in LN229 cells; Figure 3a This describes the SM1-71-induced apoptosis in A172 cells; Figure 4 These are the results of a scratch assay showing the effect of SM1-71 on the migration of LN229 cells; Figure 5 These are the results of a scratch assay showing the effect of SM1-71 on U251 in cell migration; Figure 6 These are experimental photos of subcutaneous tumor formation in mice during a nude mouse subcutaneous tumor formation experiment. Figure 7 These are photographs of tumors removed during a subcutaneous tumorigenesis experiment in nude mice. Figure 8 This refers to the tumor weights in the experimental and control groups of mice in a nude mouse subcutaneous tumor formation experiment. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0017] The biological materials, pharmaceuticals, and experimental methods used in the following examples are as follows: Drug and cell sources: Human glioblastoma cell lines sourced from Pronosei Corporation: U251, LN229, and A172 cell lines were all derived from Pronosei. The drug SM1-71 (C 24 H 26 CIN7 (purity 96.09%, molecular weight 463.96) is from MCE Company.

[0018] IC50 Measurement Experiment IC50 was determined using the CCK-8 assay. Cells were evenly seeded in 96-well plates and pre-cultured at 37°C with 5% CO2 for 24 hours to allow cell adhesion and logarithmic growth. After 24 hours, the reagent was added, and the plates were incubated for 48 hours. After 48 hours, the plates were removed from the incubator. 10 µL of CCK-8 reagent was added directly to each well (CCK-8 reagent is photosensitive and should be used in the dark). The plates were returned to the 37°C incubator and incubated in the dark for 1–4 hours. After incubation, the plates were removed from the incubator. Ensure there are no air bubbles in the wells; if any are present, they can be punctured with a needle. The plates were then placed in a microplate reader, and the absorbance of each well was measured at 450 nm. The average absorbance values ​​from each replicate were taken. Calculate cell viability: Cell viability (%) = [(OD (experimental group) - OD (blank group)) / (OD (control group) - OD (blank group))] × 100%, where: OD (experimental group) represents the absorbance of wells containing cells, drugs, and CCK-8; OD (control group) represents the absorbance of wells containing cells but without drugs (or with solvent) and CCK-8; and OD (blank group) represents the absorbance of wells containing no cells but only culture medium and CCK-8.

[0019] The specific operating steps for determining IC50 using the CCK8 experimental method are as follows: 1. Cell plating: Cell suspension preparation and cell plating: U251 and LN229 cells were digested and resuspended. Using a hemocytometer, 5,000 cells were seeded per well (100 μL of culture medium, consisting of 10% FBS + 1% penicillin-streptomycin + DMEM high-glucose medium). The 100 μL cell suspension was evenly seeded into the wells of a 96-well plate. To avoid edge effects, the outermost wells were usually filled with PBS or culture medium and were not used for data analysis. The cell culture plates were incubated at 37°C with 5% CO2 for 12–24 hours to allow the cells to fully adhere and enter the logarithmic growth phase.

[0020] Drug treatment: The experiment set up a blank control group (only culture medium, no cells (used to calibrate the ELISA reader zero point)), a control group (only culture medium and cells, no drug treatment) and an experimental group (cells + gradient concentration of SM1-71, drug treatment for 48h).

[0021] The test drug (i.e., SM1-71) was diluted to a series of concentration gradients using culture medium or serum-free culture medium: 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 800 nm, 1000 nm, 1500 nm and 3000 nm.

[0022] Remove the original culture medium and add fresh culture medium containing different concentrations of the drug. It is recommended to set up 5 replicates for each concentration to ensure data reliability. Place the cell culture plate back into the incubator and continue culturing for a predetermined 48 hours.

[0023] 3. CCK8 detection Add 10 μL CCK-8 solution to each well, return the culture plate to the incubator, and incubate in the dark for 1-4 hours. Use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0024] When adding the sample, avoid touching the well wall with the pipette tip; it is recommended to add the sample while the pipette is suspended in the air to avoid introducing cell errors or contamination. After adding the sample, gently shake the culture plate several times to ensure the reagent and culture medium are thoroughly mixed. The optimal incubation time needs to be determined based on preliminary experimental results. If the time is too short, the color will be too light and the signal will be weak; if the time is too long, the color will be too dark, which may exceed the linear range of the microplate reader, and the cells may die.

[0025] 4. Data Processing and Analysis Calculate the average absorbance value for each replicate group. Calculate cell viability: Cell viability (%) = [(OD (experimental group) - OD (blank group)) / (OD (control group) - OD (blank group))] × 100%. Plot a dose-response curve with drug concentration on the x-axis and cell viability on the y-axis. Calculate the IC50 value (drug concentration that inhibits 50% cell viability) using software such as GraphPadPrism. The dose-response curves for LN229 and U251 cells are shown below. Figure 1 As shown in A and B in the diagram.

[0026] This demonstrates that SM1-71 maintained a relatively high inhibitory concentration (IC50) in LN229, U251, and A172 cells for 48 hours. 50 The concentrations of SM1-71 were 320.3 nM, 365.58 nM, and 416.8 nM, respectively, indicating that SM1-71 significantly inhibited the proliferation of glioma cells.

[0027] The concentration ranges for SM1-71 in inhibiting gliomas are as follows: for LN229 cells, the concentration range is 285.3 nM to 375.6 nM; for U251 cells, the concentration range is 315.4 nM to 405.6 nM; and for A172 cells, the concentration range is 364.85 nM to 476.79 nM.

[0028] Effects of SM1-71 on glioma cell apoptosis A control group and an experimental group were set up. The control group was given 1‰ DMSO, and the drug concentrations in the experimental groups were: 350 nM for U251 cells, 300 nM for LN229 cells, and 400 nM for A172 cells.

[0029] Apoptosis was detected using the Annexin-VAPC / 7-AAD double staining method. The specific steps were as follows: 1) Digest, count, and prepare cells (LN229 and U251) to a concentration of 2×10⁻⁶. 5 A cell suspension of 1 cell / mL was seeded into 2 mL of the solution into a six-well plate. The next day, after the cells adhered to the plate, the appropriate drug-containing culture medium was added according to the group settings. 2) After 48 hours of drug treatment, cells were digested and collected using 0.25% trypsin (without EDTA); 3) Wash cells twice with PBS (centrifuge at 1000 rpm for 5 min), and collect 5 × 10⁶ cells. 5 cell; 4) Add 500 μL of binding buffer to suspend the cells; 5) Add 5 μL of Annexin V-Allophytin Complex (Annexin V-APC) and mix well, then add 5 μL of 7-AAD and mix well; 6) React at room temperature, in the dark, for 5-15 minutes; use flow cytometry to detect cell apoptosis. The apoptosis status of U251 cells is as follows: Figure 2 A and B in the figure and Table 1 are shown; the apoptosis status of LN229 cells is as follows: Figure 3 The apoptosis of A172 cells is shown in Tables A and B and Table 2; Figure 3a A and B are shown in Table 3.

[0030] Table 1: Figure 2 The quantitative results of the effect of SM1-71 on apoptosis in U251 cells are shown. Among them, UL (top left): percentage of mechanically damaged / necrotic cells; UR (top right): percentage of late apoptosis + necrotic cells; LL (bottom left): percentage of normal living cells; LR (bottom right): percentage of early apoptotic cells; total apoptosis rate = early apoptosis (LR) + late apoptosis (UR).

[0031] Table 2: Figure 3 The quantitative results of the effect of SM1-71 on apoptosis in LN229 cells are shown. Among them, UL (top left): percentage of mechanically damaged / necrotic cells; UR (top right): percentage of late apoptosis + necrotic cells; LL (bottom left): percentage of normal living cells; LR (bottom right): percentage of early apoptotic cells; total apoptosis rate = early apoptosis (LR) + late apoptosis (UR).

[0032] Table 3: Figure 3a The quantitative results of the effect of SM1-71 on apoptosis in A172 cells are shown. Among them, UL (top left): percentage of mechanically damaged / necrotic cells; UR (top right): percentage of late apoptosis + necrotic cells; LL (bottom left): percentage of normal living cells; LR (bottom right): percentage of early apoptotic cells; total apoptosis rate = early apoptosis (LR) + late apoptosis (UR).

[0033] The results of the apoptosis experiment showed that the apoptosis rate in the experimental group was significantly higher than that in the control group, indicating that SM1-71 can effectively induce apoptosis in glioma cells.

[0034] Effects of SM1-71 on glioma cell migration (cell scratch assay) The steps are as follows: 1. Plate preparation: Inoculate 5×10⁶ cells per well of a six-well plate. 5 Cells were collected and the well plate was placed in an incubator at 37°C and 5% CO2 for about 18-24 hours until a dense monolayer was formed.

[0035] Using a marker and ruler, draw several parallel lines on the back of the well plate along the predetermined direction of the scratches. These lines will serve as fixed reference points for subsequent photography, ensuring that photos are taken from the same location each time. Seed the cell suspension into the well plate. The amount of cells seeded is crucial; the goal is for the cells to grow into a 100% dense monolayer by the next day, without becoming excessively thick or overlapping.

[0036] 2. Creating a scratch: Using a sterile 200μL pipette tip (ensuring the tip is flat), make a vertical scratch and gently rinse 1-2 times with sterile PBS to remove any cell debris detached from the scratch. Specifically, hold the pipette tip perpendicular to the marking line on the back of the well plate and apply even pressure across the cell layer. Key points: The movement should be steady, fast, and straight, with consistent pressure. After scratching, some cells will detach from the well. Gently rinse 1-2 times with sterile PBS to remove any cell debris detached from the scratch.

[0037] 3. Group treatment: Add culture medium containing 2% serum. Add DMSO to the control group and add SM1-71 to the experimental group. The drug concentration is 300 nM for LN229 cell line and 400 nM for U251 cell line.

[0038] 4. Observation and testing: Take 0-hour images; place the well plate on an inverted microscope. Go to the previously marked line and take pictures at multiple predetermined points (e.g., 3-5 points on each line).

[0039] Key points: Be sure to record the location of each point so that you can take photos at the same location later. When taking photos, it is best to use a low-magnification objective lens to ensure that the full width of the scratch is captured. After taking the 0-hour photos, put the plate back into the incubator to continue culturing.

[0040] Timed photo taking: At predetermined time points (6h, 12h, 24h, 48h), remove the orifice plate and place it in the exact same position. Take another photo. Note: To minimize the impact of the external environment on the cells, each operation should be performed as quickly as possible.

[0041] Data Analysis The migration ability of cells can be quantified by measuring changes in the area of ​​scratches at different time points.

[0042] Image processing: Analysis was performed using image analysis software (such as ImageJ). The results for LN229 cells and U251 cells are as follows: Figure 4 and Figure 5 As shown.

[0043] The results of this experiment show that SM1-71 significantly inhibits scratch healing in LN229 and U251 cells and inhibits cell migration.

[0044] Nude mouse subcutaneous tumor formation experiment The steps are as follows: 1. Eight 4-week-old BALB / cNude nude mice were selected, and 10 million LN229 cells were subcutaneously implanted on one side of each mouse. The size of the subcutaneous tumor was observed, and the tumor volume was reduced to 60-70 mm. 3 Mice were randomly divided into control group (4 mice) and experimental group (4 mice). 2. Drug administration: The experimental group was administered the drug via intraperitoneal injection at a dose of 10 mg / kg, every 3 days. The control group received an equal volume of DMSO. After 5 administrations, a significant difference in subcutaneous tumor volume was observed in the mice. Figure 6 As shown; the tumor was removed and photographed for documentation, such as... Figure 7 As shown; tumor weight changes are as follows Figure 8 As shown, each dot represents the tumor weight of a mouse.

[0045] The results showed that the tumor volume in the SM1-71 group was significantly smaller than that in the control group, and SM1-71 effectively inhibited the growth of gliomas in vivo.

[0046] The above experiments show that SM1-71 can inhibit glioma cell proliferation, induce apoptosis, and inhibit migration in vitro, and can be used to prepare drugs for the treatment of glioma.

[0047] Mechanism of action of SM1-71 in inhibiting glioma SSM1-71 is a 2,4-disubstituted pyrimidine covalent kinase inhibitor with an acrylamide warhead. It exhibits both reversible binding via its pyrimidine backbone to the kinase hinge region and covalent bonding via acrylamide to a reactive cysteine ​​residue near the ATP pocket, thus possessing both reversible binding and irreversible covalent locking mechanisms. Structurally, the publicly available structure has resolved the SM1-71–TAK1-TAB1 complex (PDB5J7S), while the Src-SM1-71 crystal structure (PDB6ATE) in the Src system shows that this compound induces p-loop kinked conformation in Src, indicating that its inhibition is not merely due to ordinary ATP competition but also involves characteristic conformational remodeling.

[0048] The second mechanism involves broad-spectrum covalent targeting of multiple oncogenic kinase pathways. Currently confirmed covalent targets include MEK1 / 2 / 3 / 4 / 6 / 7, ERK1 / 2, TAK1, SRC, YES1, FGFR1, LIMK1, MKNK2, GSK3A / B, and RSK2. In other words, SM1-71 does not simply inhibit a single driver gene, but simultaneously suppresses the MAPK cascade, downstream RTK survival signals, cytoskeleton / migration signals, and stress-related kinase networks. This multi-target characteristic is the core difference between it and conventional single-target inhibitors.

[0049] The third mechanism is that it can directly and simultaneously suppress both the MAPK and PI3K trunk pathways in cells. Functional studies in JBC showed that SM1-71 significantly inhibited p-AKT (S473) and p-ERK1 / 2 (T202 / Y204) in sensitive cancer cells; more importantly, in the elution assay, SM1-71 showed sustained inhibition of ERK, while its non-covalent control compound SM1-71-R mainly showed only transient p-AKT inhibition, indicating that the cellular effect of SM1-71 does indeed depend on the sustained blockade caused by covalent binding.

[0050] The fourth mechanism is that its cytotoxicity stems from the combined shutdown of key pathways, rather than the inhibition of a single node. Further phenotypic simulation experiments showed that in KRAS-driven cells, neither MET inhibition alone nor IGF1R / INSR inhibition alone was sufficient to replicate the effects of SM1-71; however, the combined inhibition of MEK1 / 2 + IGF1R / INSR more closely reproduced the SM1-71-induced dual downregulation of MAPK and PI3K, proliferation inhibition, and cell death. This indicates that the antitumor activity of SM1-71 essentially derives from a "multi-pathway synergistic attack" mode.

[0051] Projecting this mechanism onto gliomas provides a more targeted explanation. This is because aberrant RTK signaling is almost universally present in GBM, and excessive activation of RTK / Ras / Raf / MEK / ERK is closely associated with malignant proliferation, treatment resistance, cell migration, and metabolic adaptation.

[0052] Therefore, if SM1-71 is used for glioma, its most likely first-line effect will be to inhibit the proliferation drivers and transcriptional programs of glioma cells by covalently attacking the MEK / ERK module. This will affect cell cycle progression, expression of proliferation-related genes, and adaptive responses to therapeutic stress.

[0053] The potential anti-glioma mechanism of SM1-71 is to reduce tumor cell viability through inhibition of the RTK-AKT survival axis. SM1-71 has been shown to suppress p-AKT in non-glioma systems, while the AKT pathway in gliomas is a crucial link in maintaining survival, anti-apoptosis, and treatment tolerance. Therefore, SM1-71 could theoretically weaken GBM's adaptation to nutritional, stress, and therapeutic impacts by inhibiting upstream IGF1R / INSR / MET and downstream AKT signaling.

[0054] Another potential mechanism of SM1-71 is the inhibition of glioma invasion and migration. This is the most noteworthy aspect of SM1-71 in the glioma context, as both SRC / YES1 and LIMK1, which it targets, are key invasion-related nodes. Publicly available research indicates that c-Src / Fyn is an important effector molecule in EGFR oncogenic signaling, enhancing glioma invasion and survival; while the LIMK1 / 2-cofilin axis controls actin remodeling, lamellar pseudopodia formation, and GBM cell motility. Since SM1-71 covers both of these modules, its potential role in gliomas is not only "killing" but may also include reducing cytoskeleton remodeling, inhibiting migration and invasion, and limiting invasive spread.

[0055] Another potential mechanism of SM1-71 is its intervention in glioma stemness and radioresistance. This is because FGFR1 in GBM is not only involved in tumor cell migration but also closely related to GSC maintenance, tumorigenesis, and radioresistance. SM1-71 has been confirmed to covalently target FGFR1; therefore, in the context of glioma, it may also weaken relapse-associated cell populations by intervening in the FGFR1-driven stemness / radio resistance program.

[0056] The above embodiments are merely specific implementations of the present invention, used to illustrate the technical solutions of the present invention, and are not intended to limit it. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or improve the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features, within the scope of the technology disclosed in the present invention; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The use of SM1-71, as shown in Formula I, in the preparation of medicaments for the prevention or treatment of gliomas. Formula 1.

2. The use of SM1-71 according to claim 1 in the preparation of a medicament for the prevention or treatment of glioma, characterized in that, The concentration range for SM1-71 is as follows: 285.3 nM to 375.6 nM for LN229 cells; 315.4 nM to 405.6 nM for U251 cells; and 364.85 nM to 476.79 nM for A172 cells.

3. The use of SM1-71 according to claim 1 in the preparation of a medicament for the prevention or treatment of glioma, characterized in that, The glioma is a glioblastoma.

4. The use of SM1-71 according to claim 1 in the preparation of a medicament for the prevention or treatment of glioma, characterized in that, The drug is used to inhibit glioma cell proliferation, induce glioma cell apoptosis, or inhibit glioma cell migration.

5. The use of SM1-71 according to claim 1 in the preparation of a medicament for the prevention or treatment of glioma, characterized in that, The drug comprises a therapeutically effective amount of SM1-71 and pharmaceutically acceptable excipients and / or carriers.

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