Small molecule targeting foxc1 protein and application thereof

Abstract

The application belongs to the technical field of medical treatment and specifically relates to a small molecule targeting FOXC1 protein and application thereof. The small molecule targeting FOXC1 protein is N-(4-hydroxy-benzyl)-2-[4-(3-hydroxy-3-phenyl-propenyl)-phenyl]-N-methylacetamide. The small molecule can target FOXC1 protein binding and inhibit migration and invasion of lung cancer cells, and can provide support for preparation of lung cancer metastasis inhibiting drugs and provide a new basis for treatment of lung cancer metastasis.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a small molecule that targets the FOXC1 protein and its applications. Background Technology

[0002] Cancer is seriously threatening human health. Among all cancers, lung cancer is the leading cause of death, and lung cancer metastasis is a major cause of death. The five-year survival rate for lung cancer patients is 16.8%, but for those with metastatic lung cancer, the five-year survival rate is only 4%. Inhibiting metastasis is a pressing challenge in lung cancer treatment.

[0003] The human FOXC1 gene is located at 6p25, is 3500 bp in length, contains a 1600 bp exon, and encodes the FOXC1 protein, which contains 553 amino acid residues. Numerous studies have shown that FOXC1 is an important protein promoting cancer development and progression. The FOXC1 protein can promote the invasion and metastasis of lung cancer cells, making it a potential target for the treatment of lung cancer metastases.

[0004] The inventors discovered a small molecule that targets the FOXC1 protein, which can support the preparation of drugs that inhibit lung cancer metastasis and provide a new basis for the treatment of lung cancer metastasis.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a small molecule that targets the FOXC1 protein and its application, which can provide support for the preparation of drugs that inhibit lung cancer metastasis and provide a new basis for the treatment of lung cancer metastasis.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The first objective of this invention is to provide a small molecule that targets the FOXC1 protein, wherein the small molecule targeting the FOXC1 protein is N-(4-hydroxy-benzyl)-2-[4-(3-hydroxy-3-phenyl-propenyl)-phenyl]-N-methylacetamide, and its structural formula is as follows:

[0009]

[0010] A second object of the present invention is to provide the use of the small molecule targeting the FOXC1 protein in the preparation of a drug for inhibiting lung cancer metastasis.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The small molecule of this invention can target and bind to the FOXC1 protein, and inhibit the migration and invasion of lung cancer cells. This can provide support for the preparation of drugs that inhibit lung cancer metastasis and provide a new basis for the treatment of lung cancer metastasis. Attached Figure Description

[0013] Figure 1 The results are based on molecular dynamics simulations of the small molecules and FOXC1 protein of this invention.

[0014] Figure 2 The results of surface plasmon resonance experiments were used to detect the binding of the small molecules of this invention to FOXC1 protein.

[0015] Figure 3 The results of micro-thermophoresis were used to detect the binding of the small molecule of this invention to the FOXC1 protein;

[0016] Figure 4 The results of Transwell assays were used to detect the effects of the small molecules of this invention on lung cancer cells;

[0017] Figure 5 The results of in vivo imaging in small animals were used to detect the inhibitory effect of the small molecule of this invention on lung cancer cell metastasis. Detailed Implementation

[0018] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0019] Example 1 Name and structure of the small molecule of this invention

[0020] A small molecule targeting the FOXC1 protein, named N-(4-hydroxy-benzyl)-2-[4-(3-hydroxy-3-phenyl-propenyl)-phenyl]-N-methylacetamide, has the following structural formula:

[0021]

[0022] Example 2 Molecular dynamics simulations of small molecules and FOXC1 protein in this invention.

[0023] Molecular dynamics simulations were performed using Gromacs (version 2022.3). For the small molecule in this invention, a GAFF force field was added using AmberTools22, and hydrogen was added using Gaussian 16W with the RESP potential calculated. The simulation conditions were a temperature of 300 K and a pressure of 1 Bar, with the force field being Amber99sb-ildn, and the solvent being water molecules (Tip3p water model). The molecular dynamics simulation system employed the steepest descent method for energy minimization, followed by 100,000 steps of isothermal-isochoric (NVT) ensemble equilibrium and isothermal-isobaric (NPT) ensemble equilibrium, with a coupling constant of 0.1 ps and a duration of 100 ps. Finally, free molecular dynamics simulations were run for 5,000,000 steps at a step size of 2 fs and a duration of 100 ns. The root mean square variance (RMSD), root mean square fluctuation (RMSF), protein radius of gyration (Gyrate), solvent accessible area (SASA), hydrogen bond (HBond), binding free energy (MMGBSA), and free energy morphology of amino acid trajectories were calculated and analyzed. Results are shown in […]. Figure 1 .

[0024] Molecular docking was analyzed at both 3D and 2D levels to determine the binding of the small molecule of this invention to the FOXC1 protein. Figure 1 (A and 1B), RMSD is an important indicator for assessing structural stability, derived from... Figure 1 As shown in C, during the simulation, the RMSD value between the FOXC1 protein and the small molecules was relatively small in the first 40 ns, and then tended to stabilize. Overall, the RMSD was within a small range, indicating that the binding between the protein and the small molecules was stable.

[0025] RMSF can reflect the dynamics of protein structural regions. (By...) Figure 1 As shown in D, the simulation results indicate that RMSF has a smaller value in the binding region of the FOXC1 protein and a larger value in the non-binding region, suggesting that the small molecule of this invention has a certain impact on the stability of the FOXC1 protein.

[0026] HBond reflects the number of hydrogen bonds. (From...) Figure 1 As can be seen from E, multiple hydrogen bonds are formed between the small molecule of the present invention and the FOXC1 protein, and the main bonds between some residues of the protein and the important groups in the small molecule of the present invention are hydrogen bonds.

[0027] SASA can assess protein surface area. Figure 1 As can be seen from F, from the initial 600nm 2 Reduced to less than 400nm 2 This indicates that the binding of small molecules in this invention reduces the surface area of ​​proteins.

[0028] Gyrate reflects the overall compactness of a protein. Figure 1 As can be seen from G, the calculation results show that the binding of the FOXC1 protein with the small molecule of the present invention reduces the Gyrate value, indicating that the binding of the small molecule of the present invention makes the FOXC1 protein structure more compact.

[0029] Free energy morphology diagrams were constructed. RMSD and Gyrate were used to reflect the 2D structure of the FOXC1 protein. Figure 1 H) and 3D Figure 1 I) Energy diagram, with blue representing low-energy regions and red representing high-energy regions. The binding free energy (ΔGMMGBSA) of the small molecule of this invention with FOXC1 protein was calculated. ΔGMMGBSA reflects the binding energy and interaction between the small molecule of this invention and FOXC1 protein. The calculated ΔGMMGBSA is equal to -27.87 KCal / Mol, indicating that the energy of the small molecule of this invention decreases and its state stabilizes after binding with FOXC1 protein.

[0030] Example 3 The binding of the small molecule of this invention to the FOXC1 protein was detected using surface plasmon resonance (SPR) experiments.

[0031] The FOXC1 protein was coupled to a Biacore T200 molecular interaction analyzer using a CM5 sensor chip. The small molecule of this invention was prepared at seven concentrations (25, 12.5, 6.25, 3.125, 1.5625, 0.7813, and 0 μM), with a flow rate of 30 μL / min, a binding time of 90 s, and a dissociation time of 120 s. Samples were loaded and analyzed from low to high concentrations. The binding and dissociation curves are shown below. Figure 2 .

[0032] Depend on Figure 2 It is known that the affinity constant between the small molecule of this invention and FOXC1 is 2.68 μM.

[0033] Example 4: The binding of the above molecules to the FOXC1 protein was detected using microscale thermophoresis (MST) assays.

[0034] NT-647 dye solution (100 nM) and FOXC1 protein solution (200 nM) were prepared using PBS. 100 μL of protein solution was mixed with 100 μL of dye. The mixture was incubated at room temperature for 40 min, then centrifuged at 16000 g for 15 min at 4 °C. The supernatant was collected to obtain the FOXC1 fluorescently labeled solution. Six concentrations of the small molecule solution of this invention were prepared (25, 12.5, 6.25, 3.125, 1.563, and 0.781 μM). 20 μL of the small molecule solution of this invention and 10 μL of FOXC1 fluorescently labeled solution were mixed and loaded into a capillary tube for detection. The data were analyzed using MO Affinity Analysis Software v2.3 to obtain the dose-response curve. The results are shown in [Figure number missing]. Figure 3 .

[0035] Depend on Figure 3 The measured KD value is 3.43 μM.

[0036] Example 5 Transwell assay was used to detect the inhibitory effect of the small molecule of this invention on the migration and invasion of lung cancer cells.

[0037] 5.1 Inhibitory effect of the small molecule of this invention on the migration of lung cancer cells

[0038] Resuspend the logarithmic growth phase cells in culture medium containing 1% serum, and take 0.2 mL (containing 2 × 10⁶ cells / mL) of the medium. 5 Cells were seeded in the upper chamber of a 24-well Transwell. 500 μL of complete culture medium containing the small molecule of this invention (low-dose group: 2 μM, medium-dose group: 4 μM, high-dose group: 8 μM) was added to the lower chamber of the Transwell. The cells were incubated for 8 hours. The upper chamber was removed, the culture medium was aspirated, and unmigrated cells were wiped off with cotton swabs. The cells were washed three times with PBS, allowed to evaporate at room temperature, and then soaked in 700 μL of crystal violet for 40 minutes. Residual crystal violet was washed away with PBS, and the cells were allowed to evaporate at room temperature. Microscopic images were taken, and the number of migrating cells in each group was compared. Results are shown in [Figure number missing]. Figure 4 A.

[0039] Depend on Figure 4 As shown in A, the number of migrating lung cancer cells gradually decreases as the drug concentration increases.

[0040] 5.2 Inhibitory effect of the small molecule of this invention on lung cancer cell invasion

[0041] Adjust the Matrigel concentration to 220 μg / mL using pre-cooled serum-free medium. Add 120 μL of Matrigel to the upper chamber of a 24-well Transwell, transfer to an incubator, and incubate for 2 hours. Remove 70 μL of serum-free medium and resuspend the logarithmic growth phase cells in culture medium containing 1% serum. Take 0.2 mL (containing 2 × 10⁻⁶ cells / mL) of this medium.5 Cells were seeded in the upper chamber of a 24-well Transwell apparatus. 500 μL of the complete culture medium containing the small molecule of this invention (low-dose group: 2 μM, medium-dose group: 4 μM, high-dose group: 8 μM) was added to the lower chamber of the Transwell apparatus. The mixture was incubated for 12 h. The upper chamber was removed, the culture medium was aspirated, and unmigrated cells were wiped off with cotton swabs. The cells were washed three times with PBS, allowed to evaporate at room temperature, and then soaked in 700 μL of crystal violet for 40 min. Residual crystal violet was washed away with PBS, and the mixture was allowed to evaporate at room temperature. Microscopic images were taken, and the number of migrating cells in each group was compared. Results are shown in [Figure number missing]. Figure 4 B.

[0042] Depend on Figure 4 As shown in B, the number of invasive lung cancer cells gradually decreases as the drug concentration increases.

[0043] Example 6 In vivo imaging of small animals was used to detect the inhibitory effect of the small molecule of this invention on lung cancer cell metastasis.

[0044] Luc-NCI-H1299 cells, a stable cell line expressing luciferase, were constructed for lung cancer. Luc-NCI-H1299 cells in the logarithmic growth phase were collected, resuspended three times in PBS to remove serum, and the cell concentration was adjusted to 1×10⁻⁶. 7 A lung cancer metastasis model was established by injecting 100 μL / mouse into the bare left ventricle. Mice were then anesthetized with the drug via tail vein injection (low-dose group: 2.5 mg / kg, medium-dose group: 5 mg / kg, high-dose group: 10 mg / kg), followed by intraperitoneal injection of sodium pentobarbital saline solution (0.5 mg / 10 g) and tail vein injection of fluorescein potassium saline solution (1.5 mg / 10 g). Fluorescence signals were detected using an IVIS LuminaLT small animal in vivo imaging system after 30 minutes. Results are shown in the table below. Figure 5 .

[0045] Depend on Figure 5 It can be seen that the fluorescence signal in the treatment group was significantly lower than that in the control group, and the higher the dosage, the weaker the fluorescence signal in the group, indicating that the compound can inhibit the metastasis of lung cancer cells.

[0046] In summary, the small molecule of this invention can target and bind to the FOXC1 protein, and inhibit the migration and invasion of lung cancer cells, which can provide support for the preparation of drugs to inhibit lung cancer metastasis and provide a new foundation for the treatment of lung cancer metastasis.

[0047] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A small molecule targeting the FOXC1 protein, characterized in that, The small molecule targeting the FOXC1 protein is N-(4-hydroxy-benzyl)-2-[4-(3-hydroxy-3-phenyl-propenyl)-phenyl]-N-methylacetamide, with the following structural formula:

2. Use of the small molecule targeting FOXC1 protein as described in claim 1 in the preparation of a drug to inhibit lung cancer metastasis.

Images