Use of compound 1125-0440 in the preparation of btl a inhibitors and lung cancer therapeutic drugs

Abstract

This invention discloses the application of compound 1125-0440 in the preparation of BTLA inhibitors and lung cancer treatment drugs, belonging to the field of biomedicine. This invention discovers that compound 1125-0440 can bind to BTLA and inhibit its activity, thereby inhibiting BTLA transcription and expression, and suppressing the migration of lung cancer cells. This invention provides the pharmaceutical application of compound 1125-0440, offering a novel preventative and therapeutic drug for NSCLC or other BTLA-overexpressing tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the application of compound 1125-0440 in the preparation of BTLA inhibitors and lung cancer treatment drugs. Background Technology

[0002] Lung cancer imposes a heavy disease burden globally and causes numerous deaths. Non-small cell lung cancer (NSCLC) is one of the major types of lung cancer, accounting for 80%-85% of all lung cancer cases.

[0003] The immune system is the body's natural defense system, responsible for recognizing and eliminating pathogens and abnormal cells to prevent disease progression. B and T lymphocyte attenuating factors (BTLA), members of the immunosuppressive receptor family, play a crucial role in tumor immunity. BTLA inhibits T cell responses by binding to herpesvirus entry mediators (HVEMs), while blocking BTLA activates T cells. This mechanism can help tumor cells evade immune surveillance in the tumor microenvironment, thereby promoting tumor growth and metastasis. BTLA expression in non-sclerotic lymphocyte-mediated cancer (NSCLC) affects T cell function, thus influencing tumor immune escape.

[0004] With the deepening research on immune checkpoint inhibitors, BTLA has emerged as a potential therapeutic target. In some studies, BTLA is thought to potentially influence the efficacy of immunotherapy along with other immune checkpoint molecules. Therefore, finding new BTLA inhibitors is crucial for the treatment of NSCLC. Summary of the Invention

[0005] This invention has discovered that compound 1125-0440 can bind to BTLA and inhibit its activity, compound 1125-0440 can inhibit the transcription and expression of BTLA, and compound 1125-0440 can inhibit the migration of lung cancer cells.

[0006] The purpose of this invention is to provide the application of compound 1125-0440, specifically its application in the preparation of BTLA inhibitors and lung cancer treatment drugs.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] Use of compound 1125-0440 or a pharmaceutically acceptable salt thereof in the preparation of BTLA inhibitors.

[0009] The structure of compound 1125-0440 is shown below:

[0010]

[0011] The use of compound 1125-0440 or a pharmaceutically acceptable salt thereof in the preparation of an antitumor drug. The tumor in question is a tumor associated with high BTLA expression.

[0012] The use of compound 1125-0440 or a pharmaceutically acceptable salt thereof in the preparation of a lung cancer therapeutic agent. The lung cancer is non-small cell lung cancer (NSCLC). Further, the lung cancer therapeutic agent is a drug that inhibits the migration of lung cancer cells.

[0013] The present invention has the following advantages and beneficial effects: The present invention has discovered new pharmaceutical value of compound 1125-0440, providing a new drug for the prevention and treatment of NSCLC or other BTLA-overexpressing tumors. Attached Figure Description

[0014] Figure 1 The effect of compound 1125-0440 on the proliferation of HeLa and RAW 264.7 cells.

[0015] Figure 2 This is the result of the effect of compound 1125-0440 on BTLA transcriptional levels.

[0016] Figure 3 This is the result of the effect of compound 1125-0440 on BTLA protein levels.

[0017] Figure 4 This diagram illustrates the molecular docking of compound 1125-0440 with the BTLA protein. A: Docking conformation of BTLA with 1125-0440; B: Highest affinity conformation of 1125-0440 with BTLA; C: Interaction diagram between 1125-0440 and BTLA.

[0018] Figure 5 This is a diagram showing the SPR binding of compound 1125-0440 to the BTLA protein.

[0019] Figure 6 This describes the inhibitory effect of compound 1125-0440 on the migration of lung cancer cell line A549. A: The migration effect of 1125-0440 on A549 cells; B: Quantitative histogram of migration rate. Detailed Implementation

[0020] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0021] Compound 1125-0440 used in the following implementations was purchased from Taoshu Biotechnology; the statistical analysis of the results of the examples was performed using a two-tailed unpaired t-test, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001.

[0022] Example 1

[0023] HeLa cells and RAW 264.7 cells were cultured and seeded into 96-well cell culture plates. After the cells reached confluence, different concentrations of compound 1125-0440 were added to the 96-well plates. Cells with only PBS were used as a negative control. After culturing in a 37°C CO2 incubator for 2 hours, the cells were washed three times with PBS, and then fresh culture medium containing 10% CCK8 solution was added for another 3 hours of culturing. The absorbance at OD590 was read using a spectrophotometer.

[0024] The calculation formula is: Cell viability = [(Absorbance of experimental wells - Absorbance of blank wells) / (Absorbance of control wells - Absorbance of blank wells)] × 100%.

[0025] The results are as follows Figure 1 As shown, the addition of different concentrations of compound 1125-0440 had no effect on the proliferation of HeLa cells and RAW 264.7 cells.

[0026] Example 2

[0027] Construction of shRNA recombinant plasmids: DNA oligos were dissolved in TE buffer (pH = 8.0) to a concentration of 100 μmol / L. The corresponding sense and antisense oligo solutions (sequences shown in Table 1) were annealed. Annealing conditions: 95℃ for 5 min, 85℃ for 5 min, 75℃ for 5 min, 70℃ for 5 min, and stored at 4℃. The annealed shRNA template was diluted to a final concentration of 200 nmol / L for the ligation reaction.

[0028] First, the pcDNA3.1(+) vector was double-digested with BamHI and KpnI. Then, shDNA template was added, and ligation was performed using ligase to construct the recombinant plasmid. The recombinant plasmid was transformed into *E. coli* DH5α competent cells. The transformed *E. coli* DH5α cells were plated on LB agar plates containing ampicillin and incubated at 37°C for 16 h. Single colonies were then picked and incubated overnight in LB medium containing ampicillin. The plasmid was extracted and identified by sequencing.

[0029] Table 1 BTLA-shRNA primer sequences

[0030]

[0031] Note: "" indicates an enzyme cleavage site.

[0032] Construction of the BTLA silencing cell line (sh-BTLA): A549 non-small cell lung cancer cells in good logarithmic growth phase were uniformly seeded in 6-well plates and cultured until the cell adhesion density reached approximately 60-80%. The old culture medium was discarded, and the cells were washed 2-3 times with sterile PBS to remove cell metabolites and poorly functioning cells. Then, 1.75 mL / well of serum-free culture medium was added. The transfection reagent was prepared according to the Thermo Fisher Lipofectamine 3000 manufacturer's instructions. 125 μL of serum-free culture medium was gently mixed with 3.75 μL of Lipo3000. 2.5 μg of the above-mentioned shRNA recombinant plasmid was gently mixed with 125 μL of serum-free culture medium and 5 μL of Lipo3000. The mixture was then gently stirred and incubated at room temperature for 5 minutes before being added to the 6-well plates. After 6 hours of culture, the culture medium in the 6-well plates was discarded, and the cells were washed 1-2 times with sterile PBS. Fresh complete culture medium was then added. After 24 hours of culture, the cells were observed under a microscope for green fluorescence to determine successful transfection.

[0033] Overnight cultured sh-BTLA cells and A549 cells (5 × 10⁶ cells) treated for 12 h with 5, 10, and 15 μM compound 1125-0440, respectively, were compared. 5 The culture medium was washed off with PBS, and total RNA was extracted from the cell line using TRIzol reagent (Invitrogen). RNA was reverse transcribed, and cDNA was prepared using a first-strand cDNA synthesis kit (Thermo Fisher Scientific). Next, gene expression was measured using SYBR Select Master Mix primers and a Bio-Rad real-time PCR system. mRNA expression was normalized to β-actin using the formula (ΔCt = Ct target gene - Ct internal reference) and expressed as relative mRNA expression (ΔCt = 2). -(ΔCt样品-ΔCt对照) Each expression analysis was performed in at least three independent biological replicates. All experimental procedures were performed according to the manufacturer's (Thermo Fisher Scientific) instructions. Primer sequences are as follows:

[0034] BTLA Forward Primer: 5'-CATCTTAGCAGGAGATCCCTTTG-3',

[0035] BTLA Reverse Primer: 5'-GACCCATTGTCATTAGGAAGCA-3';

[0036] GAPDH Forward Primer: 5'-GCACCGTCAAGGCTGAGAAC-3',

[0037] GAPDH Reverse Primer: 5'-TGGTGAAGACCGCCAGTGGA-3'.

[0038] The results are visible. Figure 2 With increasing concentrations of compound 1125-0440, the transcriptional level of BTLA decreased significantly.

[0039] Example 3

[0040] A549 cells (5 × 10) 5 Cells were treated with 0, 1, 5, 10, and 15 μM compound 1125-0440 for 12 h, respectively. The culture medium was washed off with PBS, and the cells were then lysed in RIPA buffer (MACKLIN) containing a protease phosphatase inhibitor. The total protein concentration of each sample was measured and quantified using a BCA protein quantification kit (Beyotime). 15 μg of total protein from each sample was separated onto a 10% SDS-polyacrylamide electrophoresis gel and electroblotted onto a polyvinylidene fluoride membrane (Millipore). The proteins were incubated overnight with a 1:1000 or 1:2000 diluted primary antibody, washed, and incubated for two hours with goat anti-rabbit HRP antibody (1:5000, Beyotime). Developing was then performed using a Bio-Rad integrated gel imaging system. Anti-BTLA antibody (ab181406) was purchased from Abcam, and Anti-β-tubulin antibody (66240-1-Ig) was purchased from Proteintech.

[0041] The results are as follows Figure 3 As shown, the expression level of BTLA protein decreased significantly with the increase of the concentration of compound 1125-0440, indicating that 1125-0440 has a direct inhibitory effect on BTLA protein.

[0042] Example 4

[0043] The molecular docking mode of BTLA with compound 1125-0440 was analyzed using Autodoc 4.2. The BTLA structure ID is 2AW2, which is the BTLA-HVEM complex obtained from the RCSB database. The HVEM protein was removed in MOE software (v.2019.0102). The 2D and 3D structures of 1125-0440 were plotted using ChemBioDraw Ultra 14.0 and ChemBio3DUltra 14.0, respectively. Preprocessing of BTLA and 1125-0440 included hydrogenation, dehydration, protonation, and energy minimization. The predicted docking conformations and highest affinity conformations of BTLA with 1125-0440 were obtained through computational simulations. Figure 4 As shown in A and 4B, the docking score of BTLA with 1125-0440 is -9.1082, indicating a relatively strong binding force. The interaction was assessed using the PLIP online service (https: / / plip-tool.biotec.tu-dresden.de / plip-web / plip / index), and the interaction diagram between 1125-0440 and BTLA is shown below. Figure 4 As shown in C.

[0044] Example 5

[0045] The equilibrium dissociation constants of BTLA and compound 1125-0440 were determined using surface plasmon resonance (LSPR) technology, further demonstrating the interaction between BTLA protein and 1125-0440. BTLA protein was immobilized on a COOH sensor chip (Nicoya, Canada). The chip was washed with PBS (pH 7.4) to obtain a stable detection baseline. Incremental concentrations of 1125-0440 (0.09375 μM, 0.375 μM, 0.75 μM, 1.5 μM, 3 μM, 12 μM) were injected into the chip. PBS was used as a negative control. The rate of measurement was set at 20 μL / min per cycle. The obtained data were analyzed using Trace Drawer software. The interaction between BTLA and 1125-0440 was evaluated using binding reaction kinetic parameters. Figure 5 As shown, the equilibrium dissociation constant (Kd) of BTLA and 1125-0440, calculated using Trace Drawer software, is 3.25 × 10⁻⁶. -6 M further indicates that BTLA has a strong binding affinity with 1125-0440.

[0046] Example 6

[0047] Cell migration: (1) Cell plating: Draw 5 even horizontal lines on the back of a six-well plate with a marker, and add A549 cells or sh-BTLA cell suspension that has been pipetted and mixed into the wells, about 7 × 10⁶ cells per well. 5 Use a figure-eight motion to shake the well plate to evenly distribute the cells, and incubate overnight in a 37°C CO2 incubator.

[0048] (2) Scratching: The next day, use a 200 μL pipette tip to scratch the cells using a sterilized ruler. Ensure the pipette tip is vertical and the scratching action is quick and continuous. Try to use the same pipette tip for each well. Slowly add 1 mL of PBS per well, gently shake the plate, wash the cells twice, and after washing away the detached cells, add serum-free medium. Add 20 μM 1125-0440 to the 1125-0440 treatment group and incubate at 37°C.

[0049] (3) Observation and photography: Samples were taken at 0h and 24h respectively, and the migration of cells was observed under a microscope and photographed and recorded.

[0050] (4) Results Analysis: ImageJ software was used to measure the pixels in the scratched area and calculate the inter-cell distance to compare cell migration speed. Migration rate = (initial inter-cell distance - subsequent inter-cell distance) / initial inter-cell distance × 100%.

[0051] The results are visible. Figure 6 Compared with the control group, the migration rate of BTLA-silenced cell lines was significantly reduced, and the inhibition of cell migration was more pronounced in the 1125-0440 addition group.

[0052] The above embodiments are only used to help illustrate the present invention. The implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered as equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. The use of compound 1125-0440 or a pharmaceutically acceptable salt thereof in the preparation of a lung cancer treatment medicament, characterized in that: The structure of compound 1125-0440 is shown in the following formula: ； The lung cancer mentioned is non-small cell lung cancer.

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