Use of inhibitors targeting slc7a7 in the preparation of medicaments for treating lung cancer bone metastasis

Abstract

The application discloses application of an inhibitor targeting SLC7A7 in preparation of a drug for treating lung cancer bone metastasis, and the inhibitor targeting SLC7A7 is a small molecule compound SLCi18. The application firstly verifies high expression of SLC7A7 in lung cancer bone metastasis cells and lung cancer bone metastasis clinical tissues, and silencing the expression of SLC7A7 can inhibit the development of lung cancer bone metastasis; the small molecule compound SLCi18 as the inhibitor targeting SLC7A7 can inhibit the function of SLC7A7 in transporting methionine, and inhibit lung cancer bone metastasis; and the application provides a new target and treatment strategy for lung cancer bone metastasis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of inhibitors targeting SLC7A7 in the preparation of drugs for treating bone metastases from lung cancer. Background Technology

[0002] Because early symptoms of lung cancer are often subtle and the disease progresses rapidly, approximately 40% of patients already have distant metastases by the time they seek medical attention. The development of lung cancer metastases is the leading cause of treatment failure and death in lung cancer patients. Common sites of lung cancer metastasis include the brain, bones, and liver, with bone metastasis occurring in about 40% of cases. Lung cancer bone metastases severely impact patients' quality of life and survival time, accelerating their mortality. Therefore, treatment strategies for lung cancer bone metastases urgently require further research.

[0003] To adapt to their new metastatic environment, metastatic cancers often reprogram their metabolic state, thus exhibiting different metabolic characteristics compared to the primary lesion. Amino acid metabolic reprogramming coordinates energy supply, redox homeostasis maintenance, and other metabolic pathways during tumor metastasis. Increased amino acid uptake is one of the common manifestations of amino acid metabolic reprogramming. Transmembrane transport of amino acids is mediated by various amino acid transporter systems belonging to the solute carrier (SLC) superfamily. The SLC superfamily currently includes 458 transporters from 65 families. These proteins can transport a variety of substances across the membrane. To date, more than 60 SLC members have been identified as amino acid transporters, including the SLC1 family, SLC6 family, SLC7 / SLC3 family, SLC38 family, and SLC43 family.

[0004] Multiple studies have shown that SLC transporters are promising targets for cancer therapy. Many transporter inhibitors are under investigation in preclinical or clinical trials as potential cancer treatments. Among SLC7 family inhibitors, the most researched targets are SLC7A11 and SLC7A5 / 8 (LAT1 / LAT2). SLC7A11 is highly expressed in hepatocellular carcinoma, ovarian cancer, and pancreatic cancer. Targeting and inhibiting SLC7A11 can induce ferroptosis in tumor cells. Current inhibitors include FDA-approved Sulfasalazine and Sorafenib, and experimental compounds erastin and HG106. SLC7A5 transports branched-chain amino acids and activates the mTORC1 pathway. Currently, inhibitors targeting SLC7A5 include BCH and SKN103. However, there are no reports of using SLC as a target for treating bone metastases from lung cancer. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing the application of SLC7A7-targeting inhibitors in the preparation of drugs for treating bone metastases from lung cancer.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This study aims to provide information on the application of SLC7A7-targeting inhibitors in the development of drugs for treating bone metastases from lung cancer.

[0008] Furthermore, the inhibitor targeting SLC7A7 is the small molecule compound SLCi18.

[0009] SLCi18, or Uprosertib (GSK2141795), is a potent and selective broad-spectrum Akt inhibitor that inhibits the activity of Akt1 / Akt2 / Akt3, with IC50 values ​​of 180 / 328 / 38 nM, respectively. Originally developed by GSK Plc, it is currently in Phase I / II clinical trials globally, intended for the treatment of multiple myeloma, melanoma, lymphoma, cutaneous melanoma, solid tumors, and hematological malignancies. The structure of SLCi18 is shown in formula (I) below:

[0010] ;

[0011] Equation (I)

[0012] Furthermore, the SLC7A7-targeting inhibitor inhibits lung cancer bone metastasis by suppressing the function of SLC7A7 in transporting methionine.

[0013] Furthermore, the drug for treating bone metastases from lung cancer includes the small molecule compound SLCi18, as well as pharmaceutically acceptable excipients.

[0014] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0015] This invention is the first to verify that SLC7A7 is highly expressed in lung cancer bone metastasis cells and clinical tissues of lung cancer bone metastasis. Silencing the expression of SLC7A7 can inhibit the development of lung cancer bone metastasis. The small molecule compound SLCi18, as an inhibitor targeting SLC7A7, inhibits lung cancer bone metastasis by inhibiting the function of SLC7A7 in transporting methionine. This provides a new target and treatment strategy for lung cancer bone metastasis. Attached Figure Description

[0016] Figure 1 The transcriptome sequencing results of lung cancer cells A549 and lung cancer bone metastasis cells A549 bone are shown.

[0017] Figure 2The results show the expression levels of SLC7A7 in clinical tissues and bone metastasis cells of lung cancer. Among them, (A) shows the immunohistochemical staining results of adjacent normal tissues, primary lung cancer lesions, and bone metastasis lesions of lung cancer patients, with brown indicating SLC7A7; (B) shows the immunoblotting results of normal lung tissue cells BEAS2B, primary lung cancer cells L0, and lung cancer bone metastasis cells L2 / L6.

[0018] Figure 3 The results show that silencing SLC7A7 inhibits the proliferation and migration of lung cancer bone metastases, while overexpressing SLC7A7 promotes the proliferation and migration of lung cancer bone metastases. Among them, (A) is the immunoblotting result of lung cancer cells A549 and H441 with SLC7A7 silence; (B) is the colony formation result of lung cancer cells with SLC7A7 silence inhibiting proliferation; (C) is the in vitro migration experiment result of lung cancer cells with SLC7A7 silence inhibiting proliferation; (D) is the immunoblotting result of lung cancer cells A549 and H441 with SLC7A7 overexpression; (E) is the colony formation result of lung cancer cells with SLC7A7 overexpression promoting proliferation; and (F) is the in vitro migration experiment result of lung cancer cells with SLC7A7 overexpression promoting proliferation.

[0019] Figure 4 The results show that the culture medium silencing SLC7A7 cells inhibits the differentiation of monocytes into osteoclasts, while the culture medium overexpressing SLC7A7 cells promotes the differentiation of monocytes into osteoclasts; (A) is the TRAP staining result; (B) is the statistical result; (C) is the TRAP staining result; (D) is the statistical result.

[0020] Figure 5 The results show the effect of silencing SLC7A7 expression to inhibit the development of bone metastasis in mouse lung cancer; (A) is a mouse left ventricular injection model; (B) is the in vivo imaging results of Luciferase-labeled H441-shNC and H441-shSLC7A7 lung cancer cells 4 weeks after left ventricular injection, showing the effect of silencing SLC7A7 expression to inhibit the development of bone metastasis in mouse lung cancer; (C) is the biostatistical results of in vivo imaging within 4 weeks.

[0021] Figure 6The results show that SLC7A7 promotes the proliferation and migration of lung cancer bone metastases by transporting methionine; (A) Edu staining results: In A549 bone-shNC cells, the addition of methionine promoted the proliferation of lung cancer bone metastases compared with the absence of methionine; in A549 bone-shSLC7A7 cells, the addition of methionine did not promote the proliferation of lung cancer bone metastases compared with the absence of methionine. The proliferative effect of methionine depends on the transport of SLC7A7; (B) Statistical results; (C) In vitro migration experiment results: In A549 bone-shNC cells, the addition of methionine promoted the migration of lung cancer bone metastases compared with the absence of methionine; in A549 bone-shSLC7A7 cells, the addition of methionine did not promote the migration of lung cancer bone metastases compared with the absence of methionine. The migration-promoting effect of methionine depends on the transport of SLC7A7; (D) Statistical results.

[0022] Figure 7 The results of screening for the small molecule compound SLCi18, which inhibits SLC7A7, are shown. (A) shows the inward open conformation of the L-type amino acid transporter; (B) shows the AlphaFold model; and (C) shows the half-maximal inhibitory concentrations (WMCs) of the small molecule compounds SLCi8 and SLCi18 in A549 bone-shNC and A549 bone-shSLC7A7 cells, respectively.

[0023] Figure 8 The results of the in vitro validation experiment of SLCi18 binding to SLC7A7 are shown; (A) is the result of the cell thermal transfer experiment; (B) is the result of the drug affinity response targeting stability experiment; and (C) is the result of the biotin-labeled small molecule pull-down experiment.

[0024] Figure 9 The results show the effects of SLCi18 on inhibiting the proliferation and migration of lung cancer bone metastases; (A) shows the clone formation results of A549bone-shNC and A549bone-shSLC7A7 cells treated with SLCi18; (B) shows the statistical results; (C) shows the migration results of A549 bone cells treated with SLCi18; (D) shows the statistical results.

[0025] Figure 10 The results of SLCi18 treatment of lung cancer bone metastases organoids are shown.

[0026] Figure 11The results of SLCi18 treatment in a mouse lung cancer bone metastasis model are shown. Among them, (A) is the in vivo imaging results of Luciferase-labeled H441 lung cancer cells injected into the left ventricle for 1 week and SLCi18 injected intraperitoneally for 4 weeks, showing that SLCi18 treatment inhibits the development of lung cancer bone metastasis in mice; (B) is the biostatistical results of in vivo imaging within 4 weeks; and (C) is the mouse weight monitoring results.

[0027] Figure 12 The results show that SLCi18 inhibits lung cancer bone metastasis by acting on SLC7A7 at the cellular and animal levels; (A) shows the colony formation results of A549 bone-shNC, A549 bone-shAKT, and A549 bone-shSLC7A7 cells treated with SLCi18; (B) shows the statistical results; (C) shows the imaging results of A549 bone-shNC and A549 bone-shSLC7A7 cells after left ventricular injection for 1 week, divided into control and treatment groups, at weeks 2 and 6; (D) shows the statistical results.

[0028] Figure 13 The results show that SLCi18 inhibits the proliferation and migration of lung cancer bone metastases by inhibiting methionine transport. Among them, (A) is the Edu staining result. In the DMSO control group, compared with the lack of methionine, the addition of methionine promoted the proliferation of lung cancer bone metastases. In the SLCi18 treatment group, compared with the lack of methionine, the addition of methionine did not promote the proliferation of lung cancer bone metastases. The proliferative effect of methionine was blocked by SLCi18. (B) is the statistical result. (C) is the in vitro migration experiment result. In the DMSO control group, compared with the lack of methionine, the addition of methionine promoted the migration of lung cancer bone metastases. In the SLCi18 treatment group, compared with the lack of methionine, the addition of methionine did not promote the migration of lung cancer bone metastases. The migration-promoting effect of methionine was blocked by SLCi18. (D) is the statistical result. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0030] Example 1: Immunohistochemical staining of tissues from patients with lung cancer bone metastases

[0031] 1.1 Method and Procedure:

[0032] Sections were placed in a 65℃ drying oven for 2 hours; after dewaxing, the sections were successively immersed in xylene II, xylene I, and anhydrous ethanol and xylene in a 1:1 solution for 7 minutes each; after rehydration, the sections were successively immersed in solutions of 100%-95%-85%-75%...

[0033] -50% ethanol solution for 3 min; antigen retrieval: immerse sections in 100℃ EDTA retrieval solution for 20 min; immerse sections in PBS, 3 min / time, wash three times; quenching: immerse sections in PBS 3% H2O2 for 10 min; immerse sections in PBS, 3 min / time, wash three times; blocking solution is added to the tissue surface and blocked at room temperature for 10 min; antibody-antigen reaction: the prepared primary antibody solution (prepared in PBS) is added to the tissue, placed in a humidified chamber, and incubated overnight at 4℃; immerse sections in PBS, 3 min / time, wash three times; secondary antibody: incubate at room temperature for 20 min; horseradish peroxidase (HRP) labeling: room temperature for 10 min; immerse sections in PBS, 3 min / time, wash three times; DAB After staining, the slides were placed in natural water to stop the staining reaction. For nuclear staining, the slides were placed in hematoxylin staining solution and stained for 5 minutes. Then, the staining solution was rinsed off the slide surface with tap water to stop the staining. The slides were then separated by 1% HCl solution, soaked for 1-2 seconds, and then rinsed off with tap water. Finally, the slides were placed in natural water for 30 minutes to indole. The slides were then photographed.

[0034] 1.2 Experimental Results

[0035] refer to Figure 2 A. The expression level of SLC7A7 in bone metastases of lung cancer is higher than that in the original lung cancer lesions, while the expression level of SLC7A7 in the adjacent normal tissue of lung cancer is the lowest.

[0036] Example 2: Immunoblotting to show the expression level of SLC7A7 in cells

[0037] 2.1 Method and Procedure:

[0038] (1) Protein lysis: Add RIPA to the cell sample and sonicate to lyse; centrifuge at 4°C, 12000 rpm, 5 min, and transfer the supernatant to a new ep tube; after BCA quantification, add 6× loading buffer and boil in a metal bath for 15 min.

[0039] (2) Western blot experiment:

[0040] 1) Gel preparation: Clean the gel plate, clamp it on the gel preparation frame, add double distilled water, and check for leaks; prepare the lower separating gel according to the formula, pour it into the gel plate, quickly add 1 mL of anhydrous ethanol blocking solution, and let it stand for 1 hour to gel; discard the anhydrous ethanol, prepare the upper stacking gel according to the formula, pour it into the gel plate, quickly insert the comb, and let it stand for 1 hour to gel; remove the comb, and remove the polyacrylamide gel from the gel preparation frame for electrophoresis.

[0041] 2) SDS-PAGE: Prepare 1L of 1×Running Buffer solution using dd H2O. Mount the polyacrylamide gel on the gel bath and pour in the 1×Running Buffer solution. Add the protein samples sequentially to the wells. Add 1μL of protein marker to both sides of the protein samples and fill with 1×Loading Buffer. Connect the electrophoresis apparatus and perform electrophoresis at a constant voltage of 80V. After the protein markers separate, increase the voltage to 120V and continue electrophoresis until the bromophenol blue indicator reaches the bottom of the gel bath, at which point stop the gel run.

[0042] (3) Transfer: Prepare filter paper and NC membrane, and prepare 1L of 1×Transfer Buffer solution using dd H2O; remove the protein gel from the gel bath and cut it; add the following to the transfer clamp in sequence: sponge, filter paper, protein gel, NC membrane, filter paper, sponge; install the transfer clamp in the transfer bath, place it in an ice box, and pour in 1×Transfer Buffer solution; connect the electrophoresis apparatus, transfer the membrane at a constant current of 200mA in an ice water bath, and the transfer time should be slightly longer than the molecular weight of the target protein.

[0043] (4) Blocking: Prepare blocking solution: 7% skim milk, with PBS buffer solution as the solvent; remove the NC membrane from the transfer tank and transfer clamp, add PBS buffer solution, place it on a shaker and wash for 3 min, repeat the washing 3 times; discard the PBS buffer solution, add blocking solution, place it on a shaker and block at room temperature for 1 h.

[0044] (5) Primary antibody incubation: Prepare antibody dilution buffer: 3% BSA, solvent is sterile PBS solution, filter with 0.45μM filter and syringe; dilute primary antibody with antibody dilution buffer; discard blocking solution, add PBS buffer solution, place on shaker and wash for 3 min, repeat washing 3 times; cut membrane according to the molecular weight of target protein and protein marker, retain NC membrane containing target protein; place NC membrane in dark box, add corresponding diluted primary antibody; place on shaker at 4℃ and incubate overnight.

[0045] (6) Secondary antibody incubation: Dilute the secondary antibody with antibody diluent; recover the primary antibody, add PBST buffer solution, wash on a shaker for 3 min, repeat washing 3 times; discard the PBST buffer solution, add the corresponding secondary antibody; incubate on a shaker at 4℃ for 1 h; recover the secondary antibody, add PBST buffer solution, wash on a shaker for 3 min, repeat washing 3 times; discard the PBST buffer solution, add PBS buffer solution; scan the membrane with a membrane scanner for analysis.

[0046] 2.2 Experimental Results

[0047] refer to Figure 2 B. The expression level of SLC7A7 in lung cancer bone metastasis cells L2 / L6 was higher than that in lung cancer orthotopic cells L0, while the expression level of SLC7A7 in normal lung cells BEAS2B was the lowest.

[0048] Example 3: Cloning Experiment

[0049] 3.1 Method and Procedure:

[0050] (1) Cell seeding: Digest and count the cells to ensure that each well of the 12-well plate contains 2000 cells and calculate the required cell suspension volume for each well; add 1 mL of complete DMEM medium to each well of the 12-well plate, add the cell suspension, shake the 12-well plate to ensure that the cells are uniform; place it in a 37℃ cell incubator for culture.

[0051] (2) Cell proliferation:

[0052] Change the medium every two days until a cluster of cell clones that are visible to the naked eye forms.

[0053] (3) Plate staining:

[0054] 1) Discard the original culture medium, add 500 μL of PBS buffer solution to each well, let stand and wash for 3 min, and repeat the washing 3 times;

[0055] 2) Discard the PBS buffer solution, add 500 μL of 4% paraformaldehyde to each well, and let it stand for 20 min to fix;

[0056] 3) Discard the 4% paraformaldehyde solution, add 500 μL of PBS buffer to each well, let stand and wash for 3 min, and repeat the washing 3 times;

[0057] 4) Discard the PBS buffer solution, add 500 μL of 0.9% crystal violet solution to each well, and let it stand for 20 min to stain;

[0058] 5) Discard the 0.9% crystal violet solution, add 500 μL of PBS buffer to each well, let stand and wash for 3 min, and repeat the washing 3 times;

[0059] 6) After letting it stand and dry, take photos and make statistics.

[0060] 3.2 Experimental Results

[0061] like Figure 3 As shown in B and 3E, lung cancer cells with silenced SLC7A7 showed a significantly reduced ability to form colonies, while those with overexpressed SLC7A7 showed a significantly enhanced ability to form colonies. Figure 9As shown in Figure A, treatment of A549bone-shNC lung cancer bone metastasis cells with SLCi18 significantly reduced the cell colony formation ability in a concentration gradient-dependent manner; however, treatment of A549bone-shSLC7A7 cells with SLCi18 showed no concentration gradient dependence, indicating that the ability of SLCi18 to inhibit the proliferation of bone metastasis cells is dependent on SLC7A7. Figure 12 As shown in Figure A, SLCi18 treatment of A549 bone-shNC cells inhibited colony formation, treatment of A549 bone-shAKT cells inhibited colony formation, and treatment of A549 bone-shSLC7A7 cells showed no inhibitory effect. This indicates that the ability of SLCi18 to inhibit the proliferation of bone metastases depends on SLC7A7 cells, rather than AKT cells.

[0062] Example 4 In vitro migration experiment

[0063] 4.1 Method and Procedure:

[0064] (1) Cell digestion and plating:

[0065] 1) Add 700 μL of serum-containing DMEM medium to a 12-well plate;

[0066] 2) Slowly add the Transwell cells into the holes, avoiding the formation of air bubbles;

[0067] 3) After cell digestion, wash once with PBS and resuspend in serum-free culture medium, count the cells, and ensure that each well of the 12-well plate contains 100,000 cells;

[0068] 4) Add 500 μL of serum-free DMEM medium containing cell suspension to the Transwell chamber;

[0069] 5) Place in a 37℃ cell culture incubator and incubate for 24 hours.

[0070] (2) Chamber dyeing

[0071] 1) Discard the original culture medium, add 500 μL of PBS buffer solution to the top and bottom of each Transwell chamber, let stand and wash for 3 min, and repeat the washing 3 times;

[0072] 2) Discard the PBS buffer solution, add 500 μL of 4% paraformaldehyde solution to the top and bottom of each Transwell chamber respectively, and let it stand for 20 min to fix;

[0073] 3) Discard the 4% paraformaldehyde solution, add 500 μL of PBS solution to the top and bottom of each Transwell chamber, let stand and wash for 3 min, and repeat the washing 3 times;

[0074] 4) Discard the PBS buffer solution, add 0.9% crystal violet solution to the top and bottom of each Transwell chamber respectively, and let it stand for 20 min to stain;

[0075] 5) Discard the 0.9% crystal violet solution, add 500 μL of PBS buffer solution to the top and bottom of each Transwell chamber, let stand and wash for 3 min, and repeat the washing 3 times;

[0076] 6) Wipe away the cells on the upper layer of the Transwell chamber with a cotton swab, leaving only the cells that have migrated through the chamber;

[0077] 7) After letting it stand and dry, photograph the cell migration.

[0078] 4.2 Experimental Results:

[0079] refer to Figure 3 C and 3F: Silencing SLC7A7 significantly reduced the number of lung cancer cells passing through the Transwell chamber; overexpressing SLC7A7 significantly increased the number of lung cancer cells passing through the Transwell chamber.

[0080] Example 5: Osteoclast Differentiation Experiment

[0081] 5.1 Method and Procedure:

[0082] (1) Collect the culture medium of cells under different treatment conditions and concentrate it tenfold.

[0083] (2) Extraction of mouse BMM:

[0084] 1) Sacrifice the mice, disinfect them with alcohol, and collect the tibia and femur of the mice;

[0085] 2) Cut open both ends of the tibia or femur, use a syringe filled with PBS to blow out the cells from the bone marrow, and collect them in a centrifuge tube;

[0086] 3) Centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in a 6 cm culture dish;

[0087] 4) The next day, collect the floating cells into centrifuge tubes, centrifuge at 1000 rpm for 5 min, and then re-coat them in a new 6 cm culture dish and add M-CSF;

[0088] (3) Osteoclast induction:

[0089] :1) On the third day, digest the cells, count them, resuspend them in α-MEM medium and add M-CSF and RANKL, and seed them in 96-well plates at a density of 8000-10000 / well;

[0090] 2) On the fifth day, the concentrated cell culture medium was mixed with α-MEM medium (1:40), and M-CSF and RANKL were added. The medium was changed, and then the medium was changed every other day until osteoclast differentiation.

[0091] 3) TRAP staining and photographing.

[0092] 5.2 Experimental Results:

[0093] refer to Figure 4 A, 4C, and the culture medium of lung cancer cells A549 and H441 that silence SLC7A7 inhibited the differentiation of monocytes into osteoclasts; the culture medium of cells that overexpress SLC7A7 promoted the differentiation of monocytes into osteoclasts.

[0094] Example 6: Mouse left ventricle model

[0095] 6.1 Method and Procedure:

[0096] (1) Construct stable cell lines of H441-luc, H441-shNC-luc, and H441-shSLC7A7-luc;

[0097] (2) Inject 1×10 into the left ventricle of mice 6 100 μL H441-luc cells;

[0098] (3) If medication is needed after the tumor has grown for one week, the mice will be randomly divided into groups.

[0099] (4) Administer the drug intraperitoneally daily, weigh the mice every three days, and perform in vivo imaging weekly to record the development of lung cancer bone metastasis.

[0100] 6.2 Experimental Results:

[0101] refer to Figure 5 B. Silencing SLC7A7 expression inhibits bone metastasis development in mouse lung cancer. (Reference) Figure 11 A, SLCi18 effectively inhibited the development of bone metastases in mouse lung cancer at experimental doses of 5 mg / kg and 10 mg / kg. (Reference) Figure 12 In the model of left ventricular injection of H441-shNC cells, SLCi18 inhibited the development of bone metastasis in mouse lung cancer. However, in the model of left ventricular injection of H441-shSLC7A7 cells, SLCi18 did not inhibit the development of bone metastasis in mouse lung cancer, indicating that SLCi18 inhibits the development of bone metastasis in mouse lung cancer through SLC7A7.

[0102] Example 7: Drug half-inhibitory concentration experiment

[0103] 7.1 Method and Procedure:

[0104] (1) Cell seeding: The cells were seeded in a 96-well plate at a density of 3000 cells / well.

[0105] (2) Medication preparation and administration:

[0106] 1) Dissolve the drug in DMSO to prepare a stock solution with a concentration of 50 mM;

[0107] 2) Dilute the 50mM drug stock solution to different concentrations using DMEM medium containing serum: DMSO, 0.01μM, 0.03μM, 0.1μM, 0.3μM, 1μM, 3μM, 10μM, 30μM, and 100μM, ensuring that each well of the 96-well plate contains 100μL of drug solution, and that each drug concentration treatment group has 3 replicates.

[0108] 3) Discard the original culture medium and add culture medium solutions containing different concentrations of drugs to the 96 plates;

[0109] 4) Place the cells back into a 37°C cell incubator and incubate for 48 hours.

[0110] (3) Cellular IC50 assay:

[0111] 1) Observe the degree of cell death after drug-induced cell killing under an optical microscope;

[0112] 2) Prepare sufficient CCK-8 solution using serum-free DMEM medium at a ratio of CCK-8:DMEM=1:10, ensuring that each well contains 100μL of solution, and keep the entire process in the dark.

[0113] 3) Discard the original drug-containing culture medium in the 96-well plate, add the prepared CCK-8 solution, and place it in a 37℃ cell incubator for static reaction for 1 hour, avoiding light throughout the process;

[0114] 4) Remove the 96-well plate, place it in a microplate reader, and measure its absorbance at 450 nM. Keep the entire process dark.

[0115] 5) Normalize the obtained absorbance values, use Graphpad software to fit the IC50 value of the drug, and observe whether the value is consistent with the cell death observed under an optical microscope. If they are consistent, the value is the IC50 value of the drug; otherwise, change the algorithm until they are consistent.

[0116] 7.2 Experimental Results:

[0117] refer to Figure 7 C. SLCi8 has similar inhibitory effects on both cell types, while SLCi18 is more than ten times more effective at killing A549 bone-shNC cells than A549 bone-shSLC7A7 cells.

[0118] Example 8 Cell heat transfer experiment

[0119] 8.1 Method and Procedure:

[0120] (1) Cell lysis: Collect 90% density A549 bone cells from a 10cm culture dish, resuspend them in 240μL of ice-bathed PBS lysis buffer and mix well. Add protein inhibitor, NaF, Na3VO4 and other proteasome inhibitors to the lysis buffer, place in an ultrasonic cleaner and sonicate on ice for 10min; centrifuge at 12000 rcf for 15-20min at 4℃, and transfer the protein supernatant to a centrifuge tube;

[0121] (2) Divide the supernatant after lysis into two groups, each with 120 μL of protein supernatant. Group 1 is the control group, and Group 2 is incubated with SLCi18 at room temperature using a rotary mixer for 3 h.

[0122] (3) Each of the two groups was divided into six temperature groups and heated in a PCR instrument for 3 minutes;

[0123] (4) Add 5×SDS Loading buffer for lysis, lyse in a 100℃ metal bath for 5-10 min, and detect the binding of the compound to the target protein by Western blot.

[0124] 8.2 Experimental Results

[0125] refer to Figure 8 A. As the temperature increases, SLC7A7 is gradually degraded. Under a certain temperature gradient, SLCi18 combines with SLC7A7 to prevent SLC7A7 from being degraded.

[0126] Example 9 Drug affinity response targeting stability experiment

[0127] 9.1 Method and Procedure:

[0128] (1) Obtaining protein supernatant:

[0129] 1) Collect A549 bone cells from a 10cm culture dish, wash twice with PBS, collect the cells into a clean 1.5mL centrifuge tube with a spatula, incubate at 4°C, 5000rpm for 5min, and discard the supernatant.

[0130] 2) Add 300 μL of lysis buffer (M-PER reagent, containing protease inhibitors such as PI, NaF, and Na3VO4), resuspend and mix well, then lyse on ice for 10 min.

[0131] 3) Centrifuge at 18000 rcf for 10 min at 4℃, transfer the protein supernatant to a new 1.5 mL centrifuge tube, and add 33.3 μL of 10×TNC buffer to the protein supernatant;

[0132] 4) Protein quantification (BCA quantification method);

[0133] (2) Incubation

[0134] 1) Dilute the test compound to 100× storage stock solution according to the final concentration to be used during the incubation process (20uM / 50uM);

[0135] 2) Divide the quantified protein supernatant into 99 μL portions and place each portion into 3 2 mL centrifuge tubes;

[0136] 3) Add 1 μL LDMSO to sample I, and add 1 μL of 100× test compound to samples II and III. Incubate at room temperature on a rotary mixer for 45 min.

[0137] (3) Pronase digestion

[0138] 1) Dilute Pronase (10 μg / μL) immediately before use, using 1×TNC buffer;

[0139] 2) After incubation, take 3 portions from each of samples I, II and III, add Pronase according to the protein concentration, and digest at 37°C for 5 minutes;

[0140] 3) After digestion, add 20×protease inhibitor cocktail to each sample to stop the digestion of protease, and incubate on ice for 10 min;

[0141] (4) Add 5×SDS-PAGE loading buffer to the final system, vortex to mix, place in a 100℃ metal bath for 10 min, and use Western blot to detect the binding of the compound to the target protein.

[0142] 9.2 Experimental Results

[0143] refer to Figure 8 B. Streptomycin degrades SLC7A7, and SLCi18 binds to SLC7A7 to prevent SLC7A7 from being degraded.

[0144] Example 10 Biotin-labeled small molecule pull-down experiment

[0145] 10.1 Method and Procedure:

[0146] (1) Cell lysis: Collect A549 bone cells at 90% density in a 10 cm culture dish, resuspend them in 400 μL of CHAPS lysis buffer after ice bath and mix well. Add protein inhibitor, NaF, Na3VO4 and other proteasome inhibitors to the lysis buffer, place in an ultrasonic cleaner and sonicate on ice for 10 min; centrifuge at 12000 rcf for 15-20 min at 4℃, and transfer the protein supernatant to a centrifuge tube;

[0147] (2) Divide the supernatant after lysis into 3 groups, each with 100 μL of protein supernatant. At the same time, add 500 μL of washing buffer to each group to make up to 550 μL of incubation system. Add a certain concentration of biotin to group 1, and add different concentration gradients of biotin-SLCi18 to groups 2 and 3. Incubate at room temperature with a rotary mixer for 3 h.

[0148] (3) Add 50 μL of beads suspension to each tube and incubate at room temperature using a rotary mixer for 1 h;

[0149] (4) Centrifuge at 1000 rcf for 2 min at 4℃, let stand for 2 min, and discard the supernatant;

[0150] (5) Clean the incubated beans;

[0151] (6) Add 2×SDS Loading buffer to the beads for lysis, lyse in a 100℃ metal bath for 5-10 min, and detect the binding of the compound to the target protein by Western blot.

[0152] 10.2 Experimental Results

[0153] refer to Figure 8 C, Biotin-labeled SLCi18 binds to SLC7A7 and pulls down SLC7A7.

[0154] Example 11 Organoid drug sensitivity monitoring

[0155] 11.1 Method and Procedure:

[0156] (1) Pretreatment:

[0157] 1) Wash the fresh sample several times with the washing solution to remove the transport preservation solution (collect the transport solution and washing solution);

[0158] 2) Take a portion of the tissue for T2 for pathological testing. Cut the tissue into a paste in a 5ml centrifuge tube. Take a portion of the cut tissue for T1 and freeze it with 1ml of cryopreservation solution.

[0159] 3) Add 4-5 ml of digestion solution (DB) to a 5 ml centrifuge tube to resuspend the tissue fragments, seal the tube with sealing film, and place it on a shaker at 37°C for 30 min (observe whether the digestion solution is cloudy and whether single cells are digested).

[0160] 4) Grind the tissue and filter the cell suspension using the syringe plunger and filter screen, then centrifuge at 1500 rpm for 5 min;

[0161] 5) Discard the supernatant, add 1 ml of lysinogen lysate to resuspend the cells, place on ice for 1-2 min for lysinogen lysate, and centrifuge at 1500 rpm for 5 min;

[0162] 6) Discard the supernatant, add 1 ml of washing buffer to resuspend the cells, count the cells using AO / PI staining, and centrifuge at 1500 rpm for 5 min.

[0163] 7) Resuspend the cells using matrix gel (10⁴ / 10 μl), dispense the gel into a 48-well plate, incubate at 37°C for 10-15 min, and then add (AEBL) medium.

[0164] (2) Drug susceptibility testing:

[0165] 1) After the 96-well plates are plated, add the drug once the organoids have grown to 30-100 μm;

[0166] 2) After 96 hours, the drug was collected, and CTG activity was detected by Calcein-AM / PI fluorescence staining (Calcein-AM:PBS=1:1000, PI:PBS=1:40), followed by light microscopy to detect organoid viability.

[0167] 11.2 Experimental Results

[0168] refer to Figure 10 A, 10B, and SLCi18 effectively inhibit the activity of organoids from lung cancer bone metastases, exhibiting concentration gradient dependence.

[0169] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of the small molecule compound SLCi18, an inhibitor targeting SLC7A7, in the preparation of a drug for treating bone metastases from lung cancer, characterized in that, The structure of the small molecule compound SLCi18 is shown in formula (Ⅰ): ； Equation (Ⅰ).

2. The application according to claim 1, characterized in that, The inhibitor targeting SLC7A7 inhibits lung cancer bone metastasis by suppressing the function of SLC7A7 in transporting methionine.

3. The application according to claim 1, characterized in that, The drug for treating bone metastases from lung cancer also includes pharmaceutically acceptable excipients.

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